SIMPÓSIO INTERNACIONAL SÔBRE VENENOS ANIMAIS

INTERNATIONAL SYMPOSIUM ON ANIMAL VENOMS

INSTITUTO BUTANTAN

17 a 23 de julho de 1966

FASCÍCULO 2

ÍNDICE — INDEX

VI. Bioquímica — Biochemistry Pág.

39. Enzymes of Snake Veiioms as Tools in Biochemkal Research

— E. A. Zei.ler (E.U.A.) . 349

40. Proteolytic Eiizymes of Hothrops Venom — O. B. Henri¬

ques, F. R. Mandelbaum and S. B. Henriques (Brasil) .... 359

-iL41 . Enzymes el Toxines des Venins de Serpents. Recherclies Bio-

chimiques et Immunologiqnes sur le Venin de Naja nigricolUs

— P. Boquet, Y. Izard, M. Jouannet et J. .Meaume (França) 371

• 42. Some Se])aralion Methods Based on Molecular Size and Charge

and lheir A])|)lication to Purification of Polypeptides and Pro-

teins in Snake Venoms — J. Poratii (Suécia) . 379

43. Separation Methods of Animal Venoms Constitiients — T.

Suzuki (Ja])ão) . 389

.44. Some Aclions of Snake Venom on Mitochondria — V. B.

Eueiott, J. M. Augustyn' and C. Gans (E.lkA.) . 411

45. The Venoms of Amphihians — J. W. Dai.y and B. Witkoi'

(E.U.A.) . 425

46. Cardiotoxic Steroids froni Toads — K. Meyer (Suíça) . 433

47. Wasp Kinin — J. J. Pisano (E.U.A.) . 441

48. Purification des Xeurotoxines du Scorpion Androctonus aus-

tralis — C. Rochat, H. Rochat, F. Miranda et S. Lissitzky

(França) . 447

49. Chemical Properties and Biological Activity of T i t y ii s Venom

— C. R. Diniz, M. V. Gomez, A. Antonio, A. P. Corrado

( Brasil j . 453

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10 11 12 13 14 15

ÍNDICE

1 VII. Farmacologia — Phakmacoi.ogy

f>ãg.

1 ^ 50.

Pharinacolojiy of Venoms — Iiilroductory llcmaiks — M. Ro¬

cha E Silva (Rrasil) .

457

1 .51.

Lilieration of Pharmacologically Aclive Siilislanees from Ma.«l

Cells liy Animal Venoms — E. Kaiser and W. Kaar (Áustria)

461

1 < 52.

Meclianism of Hislamine Release by Animal Venoms — A. M.

Rothschii.i) (Brasil) .

467

53.

Use of Venoms in Stiidies on Aerve Excitation — P. Rosen-

BERG (E.U.A.) .

477

54.

Serotonin and Relaled Try])tamine Derivatives in Snake Venoms

— J. H. Welsh (E.U.A.j .

509

55.

Pharmacologically and Riochemically Active Compcnents of Ja-

])anese Ophidian Venoms — T. Suzuki (Japão) .

519

56.

Sjiecific Sites of Action of Snake Venoms in tbe Central Ner-

vous System — 11. 1. Bicher (Israel) .

523

. 57.

Action Neuromnsciilaire des Venins de cjuelcpies CROTALIDAE,

ELAPIDAE et HYDROPHIIDAE — J. CíIEYMOL. F. BoURILl.ET

and M. Roch (França) .

541

58.

Modes of Aclions of Purified Toxins from Elapid Venoms on

Neuromuscnlar Transmission — C. Y. Lee and C. C. Chang

(F ormosa) .

555 1

59.

Cardiotoxic and Cardiostimnlating Faclors in Cobra Venom —

A. Devi and N. K. Sarkar (índia) .

573

60.

Cardiovascular Res|)onses to Snake Venoms and their Fraclions

— F. E. Russell (E.U.A.) .

583

61.

Pharmacology of lhe Com|)onenls of Toad Venom and Allied

Snlislances — M. Okada (Ja|)ão) .

589 1

62.

Some Aspects of lhe Pharmacology of lhe Venoms of African

Scorpions — K. R. Adam and Cu. Weiss (Sudão e Alemanha)

603 1

63.

Pharmacology of the Venoms of Mexican C e n 1 r ii r o i d c s

— E. C. DEL Pozo (México) .

615 1

^ 61.

Pharmacology of lhe Polyjiejilides from the Venom of the

Spider Clionriilria fera — S. Sciie.míerg and I'. A. Pereira

Lima (Brasil) .

627 1

65.

S[)ider Glands and Psycliolrojiics — P. N. W itt (E.U.A.) ...

639 1

Sjiider Wells and Psycholrojiics — C. F. Reed ( E.U.A.) .

615 1

67.

The Effect of the Poison of Spider — and Diggerwasjis on

their Prey (IIY.MENOPTEKA: POMPILIDAE, SPHECIDAE) _

W. Rathmaykr (Alemanha) .

651 1

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BIOQUÍMICA

BIOCHEMISTRY

Mem. Inst. Butantan

Slmp. Internac.

33(2);349-35S, 1966

E. ALBERT ZELLER

349

39. ENZYMES OE SXAKE VEXÍBIS AS TOOLS EX 1?I()CHEMICAE

RESEARCH

E. ALBERT ZELLER

Department of Biochemistry, Northwestern University Medicai Scliool,

Chicago, Illinois, U.S.A.

Althoiigh Riochemists have l)eeii slow in recognizing the remarkalile ])roperlie.«

of snake venonis, they have recently takeii so imich advantage of this material

tliat only a few typical examples of its use as a tool can he preseuted here.

Some of the reasons for this later develo])ment are the followiug: (a) Snake

venoms helong to the most eoncentrated enzyme soiirces in nalure; ih) tlieir

com])osition is very simple in comparison with a liver homogenate; while only

One oxidoreductase is found in snake venoms, it is impossible at the ])resent to

say how inany occur in the liver eell; (c) they display a remarkahle stahilily

when they are properly protected against light and moistnre; Id) many exeel-

lent preparations of snake venoms are now commercially availahle; (e) snake

venoms tend to attack key suhstances and key reactions in the animal hody;

it is. therefore, j)rohahly not accidental that ive find theni of |)ractical value in

the stndy of vitally important metaholic |)rocesses.

t have selected three areas to demonstrate the usefulness of snake venoms

as a part of analytical and preparative jtroeediires and in the stndy of certain

enzyme mechanisrns.

Analytical methods

a. The o|)liidian E-amino acid oxidase (L-AAO) has heen applied to the

determination of L-amino acids in D-aniino acid ])re])arations. Since the oxidative

deamination of L-amino acids ])roceeds at |)ractically the same velocity in lhe

ahsence and presence of high D-amino acid concentrations (1, 2), the occiirrence

of 0.01 ])er cent of the L-form can he detected in D-amino acid prei)arations

with the hei]) of L-AAO. There exists no more sensitive qiialitative or qiian-

titative j)rocedure to lest the optical ])urity of D-amino acids. J. P. Greenstein

and his co-workers have sharpened this tool to a high degree (3).

The same enzyme has heen a])i)lied to the analysis of phenylalanine in the

hlood and urine of infants suffering from phenylketonnria. This precise. ra])id

and specific micromethod is hased on the measurement of ahsorptioTi of the

enol-horate complex of j)henyli)yruvic acid generated from the amino acid hy

the L-AAO of Crotalm adamantem [ A, o ). A specimen as small as 0.02 ml of

plasma ultrafiltrate is sufficient for the test (6). W ith aj)propriate modifications,

tyrosme (4, 5), tryptophan (4), and histidine (7) can he determined hy the same

principie.

2 3

5 6

11 12 13 14 15

350 ENZYMES OF SNAKE VENOMS AS TOOLS IN BIOCHEMICAL RESEARCH

Reccillly, L-AAO lias lieen a|)[)lie(l lo lhe idenlificalion of L-amino acids

separaled hy pajier ehromalograjihy. The elironiatograni is Irealed with a veiiom

solulion iC. (ídumunteus), plienazine raelhosiilfale, and a lelrazoliiim comiioiind.

The laller acls as an eiectron a(a'eplor wilh |)henazine nielhosiilfale serviiig as

an eleelroTi carrier. As lillle as 1 /xg of ihose amino acids, which are rajiidly

deaminaled in lhe presence of lhe L-AAO, can lie reeognized liy lhe appearance

of fonnazans (8).

h. L-AAO can he coiipled vvilh amino acid releasing enzymes ( reaclion I).

If lhe condilions are adequalely chosen, lhe former enzyme hecomes lhe rale

limiling

(peptldase) (L-AAO)

Amino acid donor - >■ L-amino acid - >• a-kelo acid + NH3 (I)

faclor of lhe syslem. On ihis hasis, a qnanlitalive melhod for peplidase aclivily

has heen developed (9,10). Peplides (9, 10, 11), proleins (8), or amino acid es-

lers may lie iised as amino acid donors; oxidalion of lhe lilieraled amino acid

may lhen he followed manomelrically, jiholomelrically, lilrimelrically, or polaro-

gra])hically. Very small aclivilies can ihiis he delermined. lí several differenl

amino acids are lilieraled liy lhe peplidase, ihcse amino acids wili compele foi-

lhe L-AAO. In order lo avoid lhe formalion of siich complex syslems, jieplidase

syslems which release only one amino acid are preferahle. The simidtaneous

produclion of glycine and L-alanine wilh olher íree amino acids does nol inlerfere

wilh lhe measiirement liecanse lhe former Iwo amino acids disjilay very low af-

finily for lhe enzyme and ihus can he neglecled (3). Amino acid eslers serve

as excellent suhslrales hecaiise only one amino acid is involved. The lens amino

peplidase, in conlrasl lo all olher amino pejilidases, acls vigorously on eslers

such as L-phenylalanine elhylesler, or L-tryplojihan methylesler, and can he

delecled wilh lhe help of lhe L-AAO as shown hy Palmherg (12). Ilecently,

ihis principie has heen applied lo lhe delerminalion of peplidase aclivily of lhe

complex formed hetween |)ancreas carhoxypejilidase and ils anlihody(8). The

comjilex, localized wilh lhe hel[) of immimoeleclrophoresis, is Irealed wilh hip-

puryl-L-arginine and lhe reagenls menlioncd ahove. The pejilidase aclivily of

lhe antigen-anlihody comjilex is reeognized hy lhe ap])earance of lhe hlne

formazan.

c. There is a large hody of jnihlicalions dealing with lhe successfiil iise

of [)hos[)hodiesterases and pyro|)hosphalases in lhe ehicidalion of lhe slrnctiire

of niicleic acids and diniicleolide coenzymes. To single out one recent exaniple,

lhe work of Pfleiderer and Woenckhans jiertaining to lhe conformalion of adenine

pyridinc diniicleotide (NAD) is hrieíly summarized here(13). The aiilhors siic-

ceeded in separating «-NAD from lhe ji-iorm of lhe diniicleotide from yeast NAD

hy chromatography on Dowex 1 (formale form). In lhe a-isomer, which is

ealalytically inactive, lhe rihosyl residue is connected lo lhe nicotinamide moiely

hy an a-glycosidic linkage. Cleavage of «-NAD wilh pyrojihosphalase, jmrified

from lhe venom of Naja nivea, residted in an increase in lhe e,„ax at 260 mp,

from 17,900 to 19,900. This increase was of lhe same order of magnilude as

jirevioiisly ohtained for lhe hydrolysis of jnire /?-NAD hy snake venom (14).

This was indicalive of a similar folding of lhe moleciiles and of an inleraction

hetween lhe nicotinamide and adenine rings of holh «- and ^-forms.

1 SciELO

Mem. Inst. Butantan

Simp. Internac.

33(2):349-358, 1966

E. ALBERT ZELLER

351

Preparative procederes

a. The Work on the enzyniatic hydrolysis of «-NAD culminaled iii lhe

isolation of llie «-iiicotinamide mononucleotide (aXMN). The tojdcal and chemical

properlies of the new mononucleotide were thoroughly stndied. It differed very

liltle spectroscopically from the ^-isoiner. hiit chemically the hydrolvsis of the

a-isomer was less easily achieved than that of the y3-isomer. Without the o|)hidian

enzyme, it would have l)een difficnit indeed to carry out this interesting in-

vestigation on the relationship hetween the chemical slnictiire and certain properties

of NAD.

h. Parikh et al., with the help of dialyzed vtnom of C. adamunteus, com-

pletelv destroyed the L-isomer of the racemic form of more than a dozen alijihatic

and aromatic amino acids and isolated the piire D-isomers hy alcohol precipita-

tion(15). This method j)ermits the rapid production of certain D-amino acid

on a small scale hasis. Since the reaction is carried out in the presence of

catalase, a-keto acids are formed which remain in the aqiieous solution diiring

the precipitation.

c. A similar idea to the resolution of racemic mixtures of amino acids has

heen successfully applied to the preparation of the first pure native j)lasmalogen,

phosphatidal cholinetlól. The essential step consisted in treating the crude

plasmalogen with ophidian phospholi])ase A in order to destroy the accompany-

ing lecithins.

d. Since many L-amino acids are cpiantitatively converted into the cor-

responding a-keto acids hy the comhined action of L-AAO and catalase, we have

here the hasis for a simple and efficient procedure to prepare these compounds

as suhstrates for enzymatic experiments (17). An example is foimd in lhe

preparation of a-ketoglutaramic acid from glutaniine for studies in which the

keto acids serve as a receptor in certain transaminase reactions (18).

Mechamsms oe exzyme

reactioxs

As a fairly ])ure and easily accessihle flavoprotein, the L-AAO hecame the

ohject of many studies concerning the mode of action of vellow enzymes.

a. In the very first pajrer on this enzyme. it was noticed that at high

leucine concentration the reaction rate was reduced(l). These rcsults were first

explained hy J. B. S. Haldane’s hypothesis of a sid)strate having a two-point

attachment with the enzyme, the excess suhstrate compeling with lhe initiall)'

hound suhstrate for the second point (19). Douhts concerning this concept arose

when it was found (20) that the pheuomenon was more marked with suhstrates

undergoing rapid oxidation in the presence of L-AAO than with homologous

suhstrates sluggishly degraded (Fig. 1) and that the inhihition caused hy high

suhstrate concentration decreased with iucreasing oxygen density (21. 22). Meis-

ter and Vi ellner, on the hasis of detailed analysis of lhe steady State hehavior of

the enzyme, proposed another inter|)retation of this ])henomenon (23). These

authors had succeeded in crystallizing L-AAO from the venom of C. adarnantcus.

The enzyme, with a molectdar weight of approximately 130,000, contains two

moles of flavin adeniue dinucleotide (FAD). The new hy])othesis takes into

consideratiou the following two points: Both FAD molecides participate in the

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10 11 12 13 14 15

352 ENZYMES OF SNAKE VENOMS as TOOLS IN BIOCHEMICAL RESEARCH

enzymalic ])rocess, and each FAD molecide is first converted inlo a lialf-reducod

State; at high suhstrate tonceiitratioii, lhe FAD is further converted into the

fully rediiced enzynie. FoIIowing lliis sclieine, it would he siifficient to postulate

that the lialf-reduced form is reoxidized more rajjidly l)y molecular oxygen than

the fully reduced enzynie. This couce])l was su])|K)rted hy recenlly jnihlished

ohservatious made hy Desa and (^ilsou (24).

Concentration

Fig. 1

Ju order to ohtain more information ahoul the sinmitaueous parlicipation

of holh FAD molecules iu the amiuo acid degradaliou and lluis ahoiit the validity

of the hypolhesis lo ex])laiu the inhihitiou hy high snhstrale concentration, Well-

ner compared native wilh reversihly inaclivated enzyme(25). The latler was

ohlained hy heating at 38‘’C for 60 minutes. After lhe inactivation, no changes

in electrophorelic mohilily, in sedimenl coefficienls, or in inleraclion wilh anti-

hodies were ohserved. ITom lhese ohservalions, it can he concluded that the

inactivation is nol caused hy lhe loss of FAD, nor hy dissocialion, aggregalion,

or denatiiration of lhe enzyme molecule. A shifl of the ahsorption maxima of

the FAD, however, indicaled an alteration in lhe mode of hinding of lhe FAD

hy the |)rotein. These and other ohservatious sn|)porl the hypolhesis that con-

formalional alterations in lhe environrnenl of lhe FAD molecules had laken ])lace

dnring the inactivation. The jiositions of the Iwo flavin moieties relative lo each

other ajiparently are changed, lluis prevenling their concerted action. AIlhough

lhe conchisions are well snpjiorted hy the data, a direct ]rroof of the simultaneous

action of the two FAD molecules remains lo he presented. It shoiild he men-

tioned here that Massey proposed a mechanism which requires lhe action of

only one FAD molecule (26).

1 SciELO

Mem. Inst. Butantan

Simp. Internac.

33(2);349-358, 1966

E. ALBERT ZELLER

353

I). As the last example, it can he seeii how stuclies carried out vvith L-AAO

have led to the introduction of a new concept iiito the field of enzynie kinetics.

After niany years of investigatioii of the sid)strate and inhihitor patteni of

monoamine oxidase I MAC)), \ve were led to the assiimption that some substrales

can forni two types of complexes with the active site of certain enzymes. Since

the siihstrate pattern of the L-AAO, as known at that time, seemed to indicate

the existence of phenomena similar to that encoimtered in the MAO reaction,

and since the L-AAO could he readily crystallized from the venom of C. ada-

manieus, it seemed to he a convenient tool for fiirther analysis.

Our first approach consisted of determining the constants of the Michaelis-

Menten relationship for more than 60 different aromatic amino acids and their

ring-substituted derivativas 12). Some of data are presented grajihically in Fig. 2.

N.M.

OMP

F Cl

NOz OH CH3

The maximal velocities for substituted phenylalanines and tryptojihans are given

for the L-AAO of crude venom of Vipera russellii, C. adamanteus, and Naja me-

Umoleuca as comjuited from manometric measiirements. It can he easily recognized

that meta-suhstitution in phenylalanine and 5-snhstitiition in tryptojihan, in general,

yield lietter snhstrates than other suhstitiitions. Jt seems as if the locas, size,

and shape of the suhstituents rather than their effects on electron distrihiition

affect the maximal rate.

In trying to interpret these and related data. \ve started from an earlier

observation which led us to believe that the aromatic ring of certain snhstrates

of MAO and L-AAO contrihutes the major part of the binding energy between

the active site and the siihstrate (21. If an aromatic system exists in the active

site (27), so the henzene or heterocyclic ring of the suhstrate can he attached

to it. presnmahly through 7r-orhitals and hydrophohic honding. The symmetries

of the aromatic rings woiild allow the formation of more than one complex. One

type of complex, called eutopic (28) or prodiictive (29), is an intermediate in

the conversion of the suhstrate to the product. while the other type. named dys-

topic or non-productive, is not an intermediate and, therefore, does not lead to

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354 ENZYMES OF SNAKE VENOMS AS TOOLS IN BIOCHEMICAL RESEARCH

{)ro{iucl fornialion. Tliree cases may he etivisaged: (a) The occiirreiice of only

eulopic complexes, llierehy ])erniiltiiig the higliesl reaction rale of a given enzynie

System; (h) the oeciirrenee of dyslopie complexes otdy, lherehy prevenling sidi-

strate degradalioii; (e) lhe occiirrence of hotli productive and noii-productive

complexes. Thiis, in case (c) lhe reaction velocily is a fiinclion of lhe ratio

of lhe occiirrence of lhe Iwo types of complexes, ihis ratio heing delermined

jiossihly hy lhe shajie of the snhstrale molecnles. If we derive in the nsiial way

the relationship hetween snhslrate concenlralion and reaction rale and if we lake

into consideration lhe aiipearance of dyslopie complexes, lhen we arrive at equa-

tion 11. If no olher additional faclors

V — V . s/[Ke + s (1 + K,,/K,i) I (II)

are involved, lhen wilh increasing dyslopie character of the enzyme-siihslrate

complex (i.e., as lhe ratio of occiirrence of dyslopie lo eulopic complexes in-

creases), the Lineweaver-Burk plol is shifted toward increasingly higher ordinate

values without a change in slope. Parallel shifts of the Lineweaver-Burk jilot

have actually heen ohserved on several oceasions (2,28). More general expres-

sions than thal given in equation II are fonnd in lhe papers hy Niemann (29)

and hy Zeller et al. (2). Niemann independently conceived the idea of two dif-

ferent types of comjilexes from his analysis of lhe suhstrate paltern of chymo-

trypsin.

In order to gain further informalion ahonl lhe range of ajiplicahility of

the new concepl, we carried oiil additional experimental investigations. From

lhe outset, we wanted lo replace lhe manometric ])rocedure of measiiring L-AA()

activity with a more suitahle method hecanse the former is franght wilh several

serious shorleomings. Since the measuring of oxygen consumption with the aid

of lhe oxygen electrode avoids the severesl limitations of lhe manometric melhod,

we adopled the polarogra|)hic melhod. Allhoiigh lhe resuits remained essentially

lhe same, we feel miich more secure with oiir new data.

While the interaction hetween suhstrate and flavoprotein is a rather complex

process, and lhe inlerpretation of lhe kinetic data leaves many queslions un-

answered, the hinding of an inhihilor hy a given enzyme is in general a more

straighforward event. We attempted, therefore, to determine whether the same

snhstitution rides are valid for inhihitors as well as suhstrales. Since ring-

suhstiUited henzoic acids hlock L-AAO compclilively, lhey appeared lo he siiilahle

ohjects for tesling this idea. For 8 sets of ortho-, rneta- and para- monosuhslituted

henzoic acids, Clauss delermined lhe concenlralion which inhihiled L-AAO hv 50

per cent. In all hut one series, lhe meta-snhstiluted henzoic acids appeared lo

he lhe slrongest inhihitors. In the series of hydroxylated compounds, the mela- and

the para-derivatives were of eqnal inhihilory ])ower(30).

We summarize onr residis in lhe following way. Il would seem as if lhe

geometry of the suhstrate molecule rather than olher faclors is responaihle for

the reaction rate. If this idea were correcl, the electron density at lhe íeaction

cenler of the suhslrale would nol he imjiortanl as lhe rale delermining , factor.

Since a qiianlilalive relalionshi|) hetween eleclron densily, as determined hy ring

suhslilulion, and reaclion rale/k (or reaclion equilihria) exists in lhe fqrm of

lhe Hamrnetl equalion (111), this poinl can he snhjecled to experimenlal ve-

rification. The constant p is characleristic for a given reaction, while the

log k — log k„ = (T . p (111)

1 SciELO

Mem. Inst. Butantan

E. ALBERT ZELLER

355

Slmp. Internac.

33(2) :349-35S, 1966

constanl u is defined as a measure of lhe electroii densily at lhe reaction cenler.

If, afler plolling lhe left side of eqiialion III againsl o-, \ve ohlain a straight

line, we consider lhe Hammelt relalionship as fulfilled. If lhe sIo])e is positive,

a low electron densily favors lhe reaction at lhe rate deterinining slep, whereas

a negative slope indicates that an electron shift toward lhe reaction cenler jiermits

lhe enzymatic ])rocess to proceed rapidly. In a numher of enzyniatic systeins.

lhe Haminett eqnation has heen found to descrihe lhe relationshij) hetween ring

suhstitution and velocity adequately. To tesl L-AAO in a similar way. Clauss (30)

determined polargraphically lhe reaction rates prodnced hy a niimher of ring

suhstituted phenylalanines and hy the L-AAO froni C. adamunteus. The data

are presented in Fig. 3. The logarithm of lhe maxinial velocity is plotted

SO2NH2

2.0

1.6

SOCH,

• ^

SCH,

• ^

1.2

-.6

Fig. 3 — Hammet plot for ophidian L-amino acid oxidase (see text).

against o-. While a straight line niay he drawn throiigh the ])oints represent-

ing several meta-snhstitiited aniino acids (open circies), it is ohvionsly iinjiossihle

lo estahiish a linear relationshij) for the |)oints rejnesenting i)ara-siihstiliited |)henyl-

alanines (hlack circies; the donhle circle indicates the imsuhstiluted phenylalanine).

This is tantamoiint lo saying that, for the ])ara-suhstituled ])henylaianines, factors

other than electron density at lhe reaction center of the snhstrate are rate de-

termining. Geometric properties, therefore, may he taken into consideration

instead.

If lhese considerations ajtpear too hypolhetical lo he ])rescnled at this sym-

posium, they may slill i)rovide iis wilh a new insight into the hiological function

of L-AAO. This enzyme originally was thought lo play a role in lhe digestive

action of snake venoms(31)- The low LD 50 , as determined for lhe pure enzyme

hy Riissell et al. (32) suggest that it is intimately involved in the toxic action

of lhe venom. This conclusion appears to he untenahle in the light of lhe ex-

2 3

5 6

11 12 13 14 15

356 ENZYMES OF SNAKE VENOMS as TOOLS IN BIOCIIEMICAL RESEARCH

])eriments rcporled liy Zvvisler vvliü foiiiul tlial monos])ecilic antiscra against

L-AAO ara not ahie to reduce llu> toxicity of lhe wliole veiiom (83). However,

the complex formed I)y the L-AA() atid its anlihody lias heen foimd lo he as

active as the free enzyinet25). It seems eonceivahle lhat the L-AAO may

destroy lhe loeal pools of |)heiiylalanine and lyrosiiie, lhe jireeiirsors of the eate-

cholamines. Sinee lhe latler play an iin[)orlanl role in lhe ergolropie reaetion

of the prey, this removal of lhe aromatie amino aeids should weaken lhe defense

meehanism. From Fig. T and froni incomplete data iiresented in several jia-

pers (34,3.8), one can see high ralios for lhe niaxinial velocities of phenylalanine

lo leiicine (Vph,./Vieu) for elapid venoms as eompared with venoms of the olher

two classes of snakes. If lhe aclivily loward leiicine ajipears to he representalive

of the anionnl of L-AAO jiresenl (2), then low L-AAO eoncentrations often fonnd

in elapid venoms are partially or entirely com|)ensaled hy a high (Vphp/Vie„)

value. Aeeording to onr hypothesis, lhe relalive higher povver of phenylalanine

destruclion hy elajiid venoms is due to a higher degree of enlopic eom[)lex

formation hetween L-AAO and aromatie amino aeids.

CONCLUSION

These remarks give only a disaiipointingly small and hy necessity one-sided

seetion of lhe range of studies earried oiit with venoms as an enzyme soiirce.

But I do ho[)e that they demonstrate lhe tremendoiis polential of this lool. Vital

Brazil, in whose honor \ve meet here, used lhe venoms to overeome their fatal

effeets. Today, we are ahle lo go heyond Brazil and to iise snake venoms for

the invesligalion of hiochemieal reaetions of theorelieal and jiraclical imjiortance

as testificei hy many papers jiresented at this Symiiosiiim. In the future, we may

add a deeper meaning to the eurious fact that the snake in the form of the

caduceus is the symhol of lhe medicai profession.

Befere.xces

1. ZELLER, E. A., and MARITZ, A., Helv. chim. Acta, 27, 1888, 1944.

2. ZELLER, E. A., RAMACHANDER, G., FLEISHER, G. A., ISHIMARU, T., and

ZELLER, V., Biochem. J 95, 262, 1965.

3. MEISTER, A., LEVINTOW, L., KINGSLEY, R. B., and GREENSTEIN, J. B.,

J. biol. Chem., 192, 535, 1951.

4. LADU, B. N., and MICHAEL, P. J., J. Lab. clin. Med., 55, 491, 1960.

5. LADU, B. N., HOWELL, R. R., MICHAEL, P. J., and SOBER, E. K., Pedia-

tries, 31, 39, 1963.

6. WOOLF, L. I., and GOODWIN, B. L., Clin. Chem., 10, 146, 1964.

7. BALDRIDGE, R. C., and GREENBERG. N., J. Lnb. clin. Med., 61, 700, 1963.

8. AVRAMEAS, S., and URIEL, J., Compt. Rend., 261, 584, 1965.

9. ZELLER, E. A., and MARITZ, A., Helv. physiol. Acta., 3, C 6, 1945.

10. ZELLER, E. A., Abstracts of the XVII Int. Physiol. Congr., Oxford, 1947,

p. 258.

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

E. ALBERT ZELLER

357

Simp. Internac.

33(2):3-49-35S, 1966

11. ZELLER, E. A., and DEVI, A., Anier. J. Ophthal., 44, Part II, 281, 1957.

12. PALMBERG, P. F., unpublished data.

13. PFLEIDERER, G., and WOENCKHAUS. C., Ann. Cheyn., 690, 170, 1965.

14. SIEGEL, J. M., MONTGOMERY, G. A., and BOCK, R. M., Arcli. Biochem.

Biophys., 82. 288, 1959.

15. PARIKH, J. R., GREENSTEIN, J. P., WINITZ, M., and. BIRNBAUM, S. M.,

J. Amer. chem. Soc., 80, 953, 1958.

16. GOTTFRIED, E. L., and RAPPORT, M. M., J. bioj. Chem., 237, 329, 1962.

17. ROULET, F., and ZELLER, E. A., Verh. Schweiz. Naturforscliges., 127, 112,

' 1947.

18. MEISTER, A., Science, 120, 43, 1954.

19. ZELLER, E. A., Advanc. Enzymol., 8. 459, 1948.

20. FLEISHER, G. A., and ZELLER, E. A., Abstracts of the llSth. Meeting Amer.

chem. Soc., San Francisco, 1949, p. 20 C.

21. SINGER, T. P., and KEARNEY, E. B., Arch. Biochem. Biophys., 29, 190, 1950.

22. MARCUS, A., and FEELEY, J., Biochim. Biophys. Acta, 46, 600, 1961.

23. MEISTER, A., and WELLNER, D., in P. D. BOYER, and K. MYRBÃCK (Edi-

tors), The Eiizymes, Vol. VIII, Ed. 2, Academic Press, Inc., New York, 1962,

p. 615.

24. DESA, R. J., and GILSON, Q. H., Fed. Proc., 25, 649, 1966.

25. WELLNER, D., Biochemistry , 5, 1585, 1966.

26. MASSEY, V., GANTHER, H., BRUMBY, P. E., and CURTI, B., in Proc. Symp.

Amherst, Mass., 1964, p. 335.

27. ZELLER, E. A., Pharmacol. Rev., 11, 387, 1959.

28. ZELLER, E. A., Biochem. Z., 339, 13, 1963.

29. NIEMANN, C., Science, 143, 1287, 1964.

30. CLAUSS, L. M., unpublished data.

31. ZELLER, E. A., in J. B. SUMNER, and K. MYRBÃCK (Editors), The Enzymes,

Vol. 1, Academic Press, Inc., New York, 1951, p. 986.

32. RUSSELL, F. E., BUES, F. W., and WOO, M. Y., Toxicon, 1, 99, 1963.

33. ZWISLER, O., Abstracts of the Int. Syynp. Animal Venoms, São Paulo, July

1966, p. 96.

34. ZELLER, E. A., and MARITZ, A., Helv. chhn. Acta, 28. 365, 1945.

35. SUZUKI, T., and IWANAGA, S., Yakugaku Zasshi, 80, 1002, 1960 and Chem.

Abstr., 55, 3824, 1961.

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(2): 359-370, 1966

OLGA B. HENRIQUES, FAJGA R. MANDELBAUM and

S. B. HENRIQUES

359

40. PROTEOLYTIC EXZVMES OF fWTHROPS VE.\0M

OLGA B. HENRIQUES *, FAJGA R. MANDELBAUM and S. B. HENRIQUES *

Laboratório de Bioquímica, Instituto Butantan, São Paulo, Brasil

Observations on the proteolytic activity of snake venoms had aiready lieen

made at the end of the last century. Studies oii this interesting hiological

material, however, develojied rather slowly and purification of enzymes from

snake venoms was accomplished only recently. The value of proteolytic enzymes

as tools in protein chemistry has induced many scientists to search for new

proteinases, and a dose study of their specificities can only he reached with

purified enzymes.

We are presenting the results of purification and properties of three proteo¬

lytic enzymes from the venom of Bolhrops jararaca-, one vvilh caseinase activity.

a second showing hlood-clotting activity and a third with hydrolyzing activitv

on arginine synthetic suhstrates. In the crnde venom. these activities differ in

sensitivity to metal ions, anions and metal-hinding agents. Thus, in ])resence

of sulphide and cyanide ions, the caseinase activity is strongly iidiihited whereas

the henzovlarginine amidase (BAAase I and the hlood-clotting activities are nn-

altered. Calcium ions increase the caseinase activity, have no action on amidase

activity and slightly increase the hlood-clotting activity when assayed on oxalated

plasma. The hlood-clotting activity, however, is inhihited in presence of Ca^ +

ions when assayed on fihrinogen. The caseinase activity is almost completely

inhihited hy Cd-+ ions. the BAAase is slightly decreased, whereas the hlood-

clotting enzyme is not affected hy the same concentration of Cd-+ ions (Tahle ll.

Also the effect of metal-hinding agents is different on these activities (Tahle 11).

It can he seen that reduced glutathione inhihits the caseinase aiid hlood-clotting

enzymes, and slightly inhihits the BAAase activity of the venom. The caseinase

and hlood-clotting activities are very much decreased in the ])resence of ethyl-

enediaminetetra-acetic acid. whereas the B.AAase activity is imaffected (1,2).

The first attem])t to pnrify these enzymes was made hy fractional ])recipita-

tion with ammoninm suli)hate and showed (1,2) already a preliminary separation

of these activities (Tahle III). Fraction no. 5 had the strongest activity on

casein and gelatin. The ])rotein fraction no. 6 ])resente<l the highest hlood-

clotting activity, showing an amidase activity twice as great as the initial venom;

and fraction no. 7 had the highest BAAase specific activity ahout 5 times greater

than that of criide venom and its hydrolytic activity on casein was less than

half of the activity ])resented hy the starting material. Sinndtaneonsly Holtz

and Raiidonat(8j reported the se[)aration of two fractions from the venom of

B. jararaca, hy precijiitation with ammoninm siilphate; one, named "protease”,

Present Address — The Gamaleya Instltute for EpUiemiology and Microblology, Moscow,

U.S.S.R.

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PROTEOLYTIC ENZYMES OF B O T H R O P S VENOM

had slrotig jjioteolyüc aclivily and the otlier, “koaguliii”, sho\vi‘d rnairdy lilood-

flotliiig activity wilh wi-ak |)rol('olytic aclivity. Ví e further oliserved lhal one

■Jír 1 KrYilif»

minute heating in a hoiling-vvater lialh was aiready sufficieni lo deslroy all

TABLE I — EFFECT OF lONS ON PROTEINASE, AMIDASE AND BLOOD-CLOTTING

ACTIVITIES OF THE VENOM OF BOTUROPS JARARACA

Venom solutlon was incubated for 1 h. at 35® with test substances (final concn. 2 mM)

before addition of substrato. Reactlon mixture for the determinatlon of caselnase activity:

1 ml. of venom-ion mixture and 1 ml. of 1% (w/v) casein in 0.05 M-tris buffer, pH 8.8.

For amldase activity: 0.2 ml. of venom-ion mixture, 0.4 ml. of 0.05 M-benzoyl-L-arginine

amide (BAA) and 0.2 ml. of 0.3 M-tris buffer, pll 8.8. For blood-clotting activity: 0.1 ml.

of venom-ion mixture and 0.2 ml. of oxalated horse plasma.

lon

RELATIVE ACTIVITY

Substrate

Clottlng time

(sec)

Casein

BAA

None

100

Ca 2 +

136

100

Fe 2 4-

19.8

Mg 2 4-

19.5

Mn 2 4-

100

19.5

Zn 2 4-

19.5

Co 2 4-

22.5

Cd 2 4-

19.2

Hg 24-

—

39.5

S 2-

CN “

100

F “

100

ICHj-COO -

100

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Mem. Inst. Butantan

Simp. Internac.

33(2):3õ9-370, 1966

OLGA B. HENRIQUES, FAJGA R. MANDELBAUM and

S. B. HENRIQUES

361

TABLE II — EFFECT OF METAL-BINDING AGENTS ON PROTEINASE, AMIDASE AND

BLOOD-CLOTTING ACTIVITIES OF THE VENOM OF BOTHROPS JARARACA.

Test substances were incubated for 1 hr. at 35“ vvith venom solutlon before addition of

correspondlng substrates. Venom-test substance mixture uas Incubated with substrate

as In Table I.

Added substance

Final con-

centration

RELATIVE ACTIVITY

Clotting

time

(mM)

Casein

Gelatine

BAA

(sec)

None

100

DL-Cystelne

_*•

Reduced giutathlone

Ethylenediaminetetra-

acetic acid

100

240***

8-Hydroxyquinoline

_*♦

Histamine

_ ♦*

• The effect of cysteine could not

acid oxidase in the venom.

*• Not tested.

••• Partlally clotted.

be determined

because of

the presence

of L-amlno

-TABLE III — COMPARISON OF

HYDROLYTIC

ACTIVITY

ON CASEIN,

GELATIN,

BENZOYL-L-ARGININE AMIDE AND OXALATED PLASMA OF THE VARIOUS

FRACTIONS PRECIPITATED WITH AMMONIUM SULPHATE.

Caseinase specific actlvity is expressed as the increase in optical density of the trichloro-

acetlc acid filtrate at 275 m/i/mg of protein in the sample. The gelatinase specific

-actlvity is expressed as v (flow-time index)/íig of protein in the sample, Benzoyl-L-

arginlne amidase specific actlvity is expressed as iig of NR, liberated from benzoyl-L-

arglnlne amlde/mg of protein in the sample. Specific blood-ciotting actlvity is expressed

as 30 (mg. of protein/ml. of the sample that clots the plasma in 30 sec.).

SPECIFIC ACTIVITIES

Fraction

No.

aaturaiion

with

(NH,).SO^

Caseinase

Gelatinase

Amidase

Clotting

Ca^-l- ions

absent

Ca=+ ions

present

None

1.53

2.03

3.15

35.6

248

0 -0.40

2.09

2.92

2.24

11.3

77.5

0.40-0.45

1.33

2.52

2.76

19.7

162

0.45-0.50

1.60

2.86

6.28

27.7

219

0.50-0.55

T.59

3.43

7.15

33.1

318

0.55-0.70

0.61

0.88

1.61

69.0

501

0.70-0.80

0.66

0.94

164.0

0,80-1.00

0.34

0.24

0.46

45.0

♦ Undetectable

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PROTEOLYTIC ENZYMES OF B OT H RO PS VENOM

casciliase wliicli vvas aclivaled liy (^a""*" ions aiul lhe amidase S|)ecific activily

of Üie veiiom was not affeeted hy heating ii]) lo 5 mimiles (Tahle IV). Mcan-

while vvas verified llie presence of a heal-resisUuit caseinase, which is iiol af-

fecled hy Ca-+ ioiis. Tliese residis eorroliorate lhe finding of flamherg and

Roeha e Silva (4) of a heal-resistani henzoylarginine melliyl eslerase activily in

lhe venom of Holhrops jararaca.

TABLE IV

EFFECT OF HEATING ON THE CASEINASE AND BENZOYL-L-ARGININE

AMIDASE SPECIFIC ACTIVITIES OF THE VENOM.

A 2% (w/v) .solution of Bolhrops jararaca venom in saline was heated in a boiiing-water

bath for different periods. At the end of each period the tube containing the sample

was immediately transferred to an ice-cold water bath. Ca.seinase and benzoyl-L-arginine

amidase speclfic activities are expressed as in Table III.

Time (min.)

SPECIFIC ACTIVITIES

Caseinase

2 +

Ca ions

absent

Ca ions

present

Benzoyi arginine

amidase

1.54

2.06

45.2

0.5

0.74

1.11

41.8

0.43

0.44

54.2

0.44

0.41

55.4

0.47

54.3

0.44

0.46

53.1

0.36

—

39.3

0.39

—

27.1

ll was foiind ihaL heating lhe venom in presence of caeodylate huffer np

to 6.4“ desiroyed 75 jier eenl of lhe hlood-cloiling activily and 50 ])cr ceni of

lhe caseinase activily. So, the heal Irealmenl fnrlher dislingnishes lhe amida.se

from caseinase and hlood-clotting activities.

When the arnida.se fraction, parlially pnrified hy heating followed hy pre-

ci[)italion helween 70 and 80 (ler cent satiiration wilh ammoninm sidphate, free

from caseinase activily and stili jiresenting a lovv hlood-clotting activily, was

analysed hy zone electrojihoresis on slarch cohimn, evidence was given of Iwo'

inde[)en(lent entities (hig. 1). These activities were sejiarated into two dislingnish-

ahle peaks (5).

llahermann (61, hy fraclional (irecipitalion with acelone followed hy |)a])er

clecirophoresis, showed a hlood-clotting fraction in the li. jararaca venom.

lhe hlood-clotting enzyme was [tnrified hy Fichman and Henritpies (7) from

the crude venom hy step-wise eliilion afler chroinalogra|)hy on a DEAE-cellnlose

colurnn. The peak of hlood-clotting activily vvas eliited with 0.3 M sodiiim caco

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Slmp. Internac.

33(2): 359-370, 1966

OLGA B. HENRIQUES, FAJGA R. MANDELBAUM and

S. B. HENRIQUES

363

150 -

ÒO -

Fig. 1 — Electrophoresis on a starch column of a crude preparation of Bothrops

protease A (proteln fractlon precipitated between 70 and 80 per cent saturation with

ammonlum sulphate). Tris buffer, pH 7.2, I 0.05. ^ , protein concentration; O, benzoyl-

L-arginlne amidase speciíic activity; , blood-clottlng activity.

tlylate hiiffer, pH G.5 (Fig. 2). After rechromatography of this fractioii under

lhe same comlitions, a protein peak was ohtained which showed a Idood-clotting

activity 9 to 10 times higher than that of the crude venom, an amidase activity

alioiit three times greater than that of the starting material, and no caseinase

activity. The yield of this partially piirified enzyme corresponded to 26 per

cent of the total hlood-clotting activity of the venom (7). This partially purified

hlood-clotting enzyme liherales a pe|)tide from fihrinogen (8). This fact, also

reported hy Hlomhiick and Vestermark (9), shows that the hlood-clotting activity

is diie to the proteolytic action of the enzyme. When the partially purified hlood-

clotting enzyme was compared to thromhin, it showed a potency corresponding

to approximately 153 ATH thromhin units/mg of protein enzyme (7).

SciELO

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364

PROTEOLYTIC ENZYMES OF li O T li K O P S VENOM

Fig. 2 — Chromatography of venom of Bothroys jararaca on DEAE-cellulose column.

• , protein concentration (optical density at 280 m/í); O. blood-clotting specific

activity.

The heat-resislant eiizynie, vvliicli hydrolyzes l)enzolarginine amide and vvas

demonstraled lo he different from lhe hlood-cloUing enzyme, was ])urified hy

chromalography on a DEAE-cellnlose column. The enzyme vvas eluted wilh O.ò M

Tris-HCl l)uffer, pH 7.6 and was eompletely free from lhe hlood-elotling enzyme,

which was elnled wilh 0.2 M Tris-HCl hiiffer (Eig. 3)- The liighesl HAAase

and TAMEase I loluenesnlfonyl melhyl eslerase) aelivilies were found in llie foiirlh

elnled ])eak(10). This enzyme, denominaled B o i h r o p s prolease A, when re-

chromalographed imder lhe

eondilions reaehed a HAAase S])eeific aelivily

68 limes as greal as lhal of lhe erude e.xlrael, wilh a eorresponding 6.3 per

eenl yield of lhe lolal HAAase aelivily of lhe slarling venom. The eapacily of

lhe elnled fraelions lo liherale kinin (11) was al.so sludied. The ])eak of Bo-

throps ])rolease A had ahonl lhe same kinin-releasing aelivily as lhe slarling

healed venom. This finding shows lhal lhe enzyme Bo t hrops ])rolease A,

having lhe highesl hydrolyzing aelivily on arginine synlhelic suhslrales, is in-

depcndenl from lhe kitun liheraling enzyme.

The TAMEase as well as lhe HAAase aelivilies of Bothrops jvrolease A

are not iidiihiled hy Irypsin ÍTihihilors as ovomiieoid and soyhean inhihilor, on

agreemenl wilh lhe ohservalions made hy Deulseh and Diniz on lhe erude ve¬

nom (12), while diisopropylfluorophos])hale inhihils lryi)sin and Bothrops

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(2):3 d9-370, 1966

OLGA B. HENRIQUES, FAJGA R. MANDELBAUM and QCc:

S. B. HENRIQUES

•RIS-M Cf

7,6 I

a M

P" R*A C T lONJ NJUK1BER

Fig. 3 — Chromatography on DEAE-cellulose column of Bothrops protease A

precipitated betvveen 70 and 80 per cent saturation with ammonium sulphate. Step-

vvise gradient elution with tris buffer, pH 7.6. The broken line A presents the ex-

tinction of fractions (vol. 20 ml) at 280 m/í (1 cm light path); O. relatíve specific

benzoyl-L-arginine amidase activity; •, relative specific p-toluene sulfonyl-L-arginine

methyl esterase activity; relative specific blood-clotting activity.

])rotease A. The similarity hetweeii this eiizyme and trypsin. when liydrolyzing

arginiiie synthetic siilistrates. lead iis to compare the hydrolvtic jiower of B o -

throps protease A to twice crystallized try])sin oii several synthetic sid)strates

as BAA, TAME, LEE (lysine ethyl esteri and protainine. The relative potency

of Bothrops protease A on these snhstrates was fonnd to he, respectively.

One eighth, one half. one hundredth and one ninth as greal as that of twice

crystallized tryj)sin iTahle V).

Trypsin, as it is well known. is nnstahie at its pH o])limnm. By contrast,

Bothrops protease A is not affected hy hydrogen ion concentration. In

Tahle VI resnlts concerning the stahility (at room temperatnre) of Bothrops

protease A, hetween pH 3 and 9 are shown. There was no decay of BAAase or

TAMEase activities measiired, iip to 26 honrs (10).

To confirm the previons observations on the hydrolysis of synthetic snh¬

strates and also to characterize the proteolytic activity of B o t h r o p s ])rotease

A on a peptide chain, the action of this enzyme on insniin and on its B chain

was stiidied. Four peptides were ohtained hy the hydrolytic activity of Bo¬

throps protease A on the B chain of oxidized insidin, which were separaled

by high-voltage paper electrophoresis at pH 3.9 (Fig. 4), and denominated

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366

PROTEOI.YTIC ENZYMES OF B OT H ROP 8 VENOM

TABLE V — COMPAHISON OF IIYDROLYTIC ACTIVITIES OF TRYPSIN AND

B OT H RO P 8 PROTEASE A ON SYNTHETIC SUBSTRATES AND PROTAMINE

Benzoyl-L-arglnine amUlase specltic activity is expressed as /tmoles of BAA hy(irolyzc(i/mg.

or protein enzyme in the sample. Toluenesulfonyl-L-arginine methyl esterase speeiflc

activity is expressed as /irnoies ot TAME hydrolyzed/min./mg. of protein enzyme. E-lysine

ethyi esterase specific activity is expressed as ^moies of L-lysine ethyi ester hyíiroiyzed/

min./mg. of protein enzyme in the sample. Protaminase specific activity is expressed as

iig of free-amino-Nj iiberated/min./mg. of protein enzyme in the sample.

Hydrolytic activity on

SPECIFIC ACTIVITIES

Trypsin

Bothrops protease A

Benzoyl-L-arginine amide

9434

1118

p-Toluenesulfonyl>L-arginine methyl

ester

504

230

L-Lysine ethyi ester

0.18

Protamine

2260

254

TABLE VI

THE pll EFFECT ON THE STABILITY OF B O T H R O P 8 PROTEASE A.

Each reaction mixture kept at 25° contained 1 ml. ot enzyme soiution in saline and 1 mi.

of one of the foliovving soiutions: 0.005 M-tris buffer, pH 9.0; 0.005 M-tris butfer, pH

8.0; 0.002 N-HCi, 0.2 N-HCi and 2 N-HCl, to give, respectively, the toliowing pH vaiues:

9.0, 8.0, 3.0, 1.0 and 0.2. Samples of 0.2 mi. were withdrawn at the end ot 0, 2, and

26 hours. Benzoyi-L-arginine amidase and toiuene sulfonyi-L-arginine esterase specific

activities are expressed as described in Tabie III and Tabie V, respectiveiy.

Time

(hours)

SPECIFIC

ACTIVITIES

TAMEase

BAAase

930

9.0

880

850

930

8.0

840

790

880

3.0

880

820

830

1.0

740

690

700

2:; 0.2 (IN-HCi)

1.5

690

480

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Mem. Inst. Butantan

Simp. Internac.

33(2) :359-370, 1966

OLGA B. HENRIQUES, FAJGA R. MANDELBAUM and

S. B. HENRIQUES

367

peptides 0, 1. 2 and 3. Peptide 0 remained on the initial line and peptide 1,

2 and 3 moved towards the calhode. B o t h r o p s protease A has no hvdro-

Ivtic aclion on the A chain of insidin(13), a concliision supported Ity the de-

monstration that the same three peptides moving to the cathode were ohserved

on high voltage paper electro])horesis of incuhating mixture of H o t h r o p s

protease A vvith oxidized insulin or vvilh its H chain ( Fig. 5).

Fig. 4 — Paper electrophoresis of oxidized B chain of insulin incubated with B o -

throps protease A. Pyridine-acetic acid-water buffer, pH 3.9. Electrophoresis for

4 h. at 1000 V. The direction of migration is from left to right towards the cathode.

ta)

oriáln

(b)

Fig- 5 — Paper electrophoresis of oxidized insulin incubated with li o t li r o p s pro¬

tease A. Pyridine-acetic acid-water buffer, pH 3.9. Electropboresis for 2.30 h. at

1000 V. (a). 0.xidlzed insulin as control (b).

Investigations of the amino acid composition and N-terniinal amino acids

of these peptide fragments are siimmarized in Tafile VII. Uniess proline, lysine

and ihreonine, all the other amino acids of the oxidized R chain of insulin

were foimd hy acid hydrolysis of peptide 0. Since it contained only DNP-phenyl-

alanine as N-tenninal amino acid, it can he concluded that this peptide resulted

from hydrolysis hetween amino acids 22 (arginine) and 23 (glycine). Glycine

and phenylalanine were the two amino acids foimd in peptide 1, and the DNP-

derivative of this peptide proved to he DNP-glycine. After dinitrophenilation,

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PROTEOLYTIC ENZYMES OF BOTHROPS V^ENOM

TABLE VII — AMINO ACID COMPOSITION OF PEPTIDES OBTAINED BY THE ACTION

OF BOTHROPS PROTEASE A ON OXIDIZED B CHAIN OF INSULIN

Peptide

Amino acids

DNP-amino acid

ether-soluhle

AIl

found in oxidized B chain of insulin excepting

Pro, Lys and Thr

DNP — Phe

Giy,

Phe

DNP — Gly

Lys,

Thr, Ala, Tyr, Tyr X, Pro

dl DNP — Tyr

Lys,

Thr, Ala, Pro

DNP — Thr

tyrosilie was fourul as the N-terminal amiiio acid of pejitide 2. Hy acid liydro-

lysis il tiirned oul to ho a ])eiilajM‘[)tide of llireoniiie, i)roIine, lysiiie, alaiiiiie

and tyrosine (as well as a small amount of tyrosiiie X, tlie formation of whicli

coidd not he entirely prevenled). Conseqiiently ])t‘plide 2 inust have heeii formed

due lo lhe hydrolysis hetweeii amino acids 25 (phenylalanine) and 26 (tyrosine)

of lhe T5 chain of oxidized insniin. The fastesl electro[)horelic moving peptide

(peptide 3) contained lhe foiir last amino acids (Thr, Pro, Lys and Ala) of B

chain of insniin and DXP-Thr was niade evident as its DNP-derivative.

Il can he concluded frorn lhese data lhat tlie sites of action of Bothrops

j)iotease A on oxidized B chain are ihose indicaled in Fig. 6.

B CHAIN OF OXIDIZED INSULIN

Phe—Vai—Asp—Glu—Hls—Leu- CySOjH—-'dy—Ser—His—Leu—Vai—Glu—Ala—Leu—

1 2 3 4 5 0 7 8 9 10 11 12 13 14 15

-Tyr—Leu—Vai—CySO.H—Gly—Glu—Arg—Gly—Phe—Phe—Tyr—Thr—Pro—Lys—Ala

10 17 18 19 20 21 22 23 24 25 20 27 28 29 30

Fig. 6 — Summary of the speclllclty of B o t h r o p n protcase A on oxltilzed B chain

of insulin. The arrows Indicate the site.s spllt hy the enzyme.

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10 11 12 13 14 15

Mem. Inst. Butantan OLGA B. HENRIQUES, FAJGA R. MANDELBAUM and QCQ

Simp. Internac. S. B. HENRIQUES

.S3(2):359-37ü, 1966

The action of B o t h r o p s |)rolease A oii tyrosiiie honcls must l)e associated

to a very special ])eptide configuration. as oídy lhe 26-27 tyrosine liond (Tyr-

Thrl was split. The olher tyrosine honds, 16-17 (Tyr-Leul in R chain as well

as the 14-15 (Tyr-Ghi l and 19-20 (Tyr-CysSOsH I in A chain, were not hydro-

lyzed. This selective action is also shown towards lhe l)asic amino acid honds,

22-23 (Arg-Gly) and 29-30 iLys-Ala); only Arg-Gly hond was split hy the en-

zyme and confirms the jirevioiis results on lysine and arginine synthetic suhsirates.

References

1. HENRIQUES, O, B., LAVRAS, A. A, C., FICHMAN, M., MANDELBAUM, F.

R„ and HENRIQUES, S. B„ Biochem. J., 68, 597, 1958.

2. HENRIQUES, O. B., FICHMAN, M., and HENRIQUES, S. B., Biochem. J.,

75, 551, 1960.

3. HOLTZ, P., and RAUDONAT, H. W.. Arch. exp. Path. Phurmakoh, 229, 113,

1956.

4. HAMBERG, U.. and ROCHA E SILVA, M., Arch. int. Pharmacodyn., 110, 222,

1957.

5. HENRIQUES, O. B., MANDELBAUM, F. R.. and HENRIQUES, S. B., Nature,

183, 114, 1959.

6. HABERMANN, E., Arch. exp. Path. Pharmakol., 231, 291, 1958.

7. FICHMAN, M., and HENRIQUES, O. B., A7'ch. Biochem. Biophys., 98, 95, 1962.

8. HENRIQUES, O. B., and FICHMAN, M., Personal informations.

9. BLOMBãCK, B., and VESTERMARK, A., Arkiv. for Kemi, 12, 173, 1958.

10. MANDELBAUM, F. R., and HENRIQUES, O. B., Arch. Biochem, Biophys

101. 369, 1964.

11. ROCHA E SILVA, M., BERALDO, W. T., and ROSENFELD, G., Amer. J

Physiol., 156, 261, 1949.

12. DEUTSCH, H. F., and DINIZ. C. R., J. Biol, Che^n., 216, 17, 1955.

13. MANDELBAUM, F. R., CARRILLO, M., and HENRIQUES, S. B., Biochim.

Biophys. Acta, 132, 508, 1967.

Discussio.x

T. Sueuki: “It is interesting that Bothrops protease A differs completely

from the three proteinases of A. halys blomhoffii venom in their hydrolytic actions

on insulin B chain”.

F. R. Mandeibatwi: “In fact, the proteinases purified from the A. bjomhoffii

venom hydrolyze mostiy peptide honds involving the amino groups of leucine and

phenylalanine and the enzyme Bothrops protease A has no activity on these

honds”.

H. Michl: “Are the proteolytic enzymes of B. jararaca venom serin-enzymes?”

F. R. Mandelbamn: “Yes, as trypsin, Bothrops protease A is inhihited hy

diisopropylfluorophosphate (DFP), howev'er, the complete inhihition of that en¬

zyme is only reached with a 10 times higher concentration of DFP”.

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33(2):371-378. 1966

P. BOQUer, Y. IZARD, M. JOUANNET et J. MEAUME

371

41. ENZYMES ET TOXINES DES VEMNS DE SERPENTS. RECHERCHES

BIOCHIMIQUES ET IMMUXOLOGIQUES SER LE VENIX DE

NAJA NIGRICOLLIS

P. BOQUET, Y. IZARD, M. JOUANNET et J. MEAUME

Institui Pasteur, Paris, France

Cet exposé ne constitue pas une revue des recherches publiées sur les en-

zymes et les toxines des venins de serpents. I"ne telle entreprise dépasserait les

limites dTin bref entretien en raison du nombre considérable des ])ublications

parues sur ce sujetll, 2). Xotre but est de vous présenter les résultats d'ex-

périences réceutes entreprises dans le dessein d établir une relatiou de causalité

tntre les antigènes décelés dans un veniu par les niéthodes dJmmuno-diffusion

et dlmmuno-électrophorèse et les facteurs enzymatiques et toxiques de ce mê-

me veniu.

Disposant de quantités relativenient importantes de venin de Naja nigricol-

Us, un ELAPIDAE africain, nos recherches on été orientées vers rétude de ce

poisou.

D’une manière générale, on constate que les pro])riétés nocives des ve¬

nins d’ ELAPIDAE et celles des venins de VIPERIDAE ou de CROTALIDAE diffe-

rent. LVxpérience nous enseigne, par exemple que la jdupart des venins neu-

rotoxiques des ELAPIDAE cFAfrique contiennent ])eu ou ne contiennent pas d'en-

zymes protéolytiques, alors que ceux des CROTALIDAE américains. objet de re-

therclies a])profondies de la part des expérimentateurs brésiliens. dégradeut les

protéines.

Xotre choix étant dicté par les seules raisons matérielles, nous avous procé-

dé de la manière que voici.

Méthodes

Dans des expériences préliminaires, le veniu de Naja nigricollis est filtré

à travers un gel de dextrane, le “Sephadex C/ioo fine” choisi en raison de ses

pro])riétés dVxclusion. Six fractions sont obteuues par ce procédé. Elles sont

lyoj)hilisées et conservées à basse température.

La dernière fraction ou “fraction VI” contient un facteur crune grande no-

civité. que nous appelons “toxine «” (3) et des protéines de masses nioléculaires

voisines. Pour jjuriiier cette toxine a. la fraction VI a été de nouveau filtrée

.à travers un gel de “Sephadex” plus sélectif, le “Sejthadex Gso fine”. Les

techniques de ces filtrations sont décrites “iii extenso” dans une précédente J)U-

blication (4).

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372

ENZYMES ET TOXINES DES VENINS DE SERPENTS. RECIIERCHES

KIOCHIMIQUES ET IMMUNOLOGIQUES SUR LE VENIN DE

NAJA NIGRICOLLIS

(7ia(|iic fraction esl soiiinise à iiiie analyse ininuiiiologiqui- suivant Ics mé-

thodes (l’imnuiiio-(liffuíion douhle cii milieii gélifié(5) et d'imnnmo-éIeclio|)ho-

rèse(r)). Par ce deniier |)ro(édé oii ohtienl la sé|)aralion des aiitigèiies coiitemis

daiis des éclianlillons de 5 à 10 /ig des |)rodinls exaniiiiés. IJii eouranl de

15 v/em esl maiiitemi ])eiidaiil 2 heiires et le taitipon iitilisé esl la soliitioii

nsiielle de véronal sodi(jue/H(d 0,025 M (])H 8,2). Ilans les deiix séries d’ex-

{.érienees on |)réci|)ile le.s aiiligèiies ])ar rimniimo-séruin 984 qiii [irovieiit (Piin

cheval liyperimnumisé jiar des injeelioiis réj)élées de veniii de Naja nigricolUs.

ÜÉSUI.TATS

1 — Le Iracé de la figure l. illustre le résidtat de la fillration de 500 ing

de veiiin de Naja nigricollis à travers le “Sepliadex Gioo fine”. II esl défini

par une mesure al)Soi|)liométrique de la teneur en protéines de eliaque éelianlil-

lun de íillrat reciieüli (A = 2.800 A). Ces éelianlillons sont réi)arlis en 6 lots

corresj)ondanl aux fraelious I à VI.

Le traeé de la figure 2 correspond à la fillration de la fraction VI à tra¬

vers le “Se])l)adex G 50 fine”. Les sous-fraetious Va et V la ainsi oOtenues sont

définies par le second pie de ee gra|)hique. Soumis à une filtratioii de eoutrôle

sur “Sepliadex G^o fine”, le produil terminal semhle homogèue.

2 — La fi gure 3 re])résenle une préj)aration dans laqiielle 25 pg de venin

de Naja nigricoUis ont élé analysés |)ar immuno-électro|)liorèse et à tilre com-

paralif, la re])résenlaliou seliémali(|ue des antigènes dissoeiées j)ar le même procédé.

Cliaipie aniigènc préeipilé étanl désigné par un syrahole, on constate que

ranligène eorrespondaut au premier arc a se déplaee vers Tanode. 11 apparait

seulement dans les préparatious les ])lus riches en venin. Un second antigène

que uous désignons |'ai ia lettre A est sensihlemciit iso-électriqiie à pli 8,2.

I’lusieurs ares eonstituent eusuite le grou])e des antigènes B. Peu disliucts dans

le itnmuuo-éleetro])liortses du venin total, ils ont élé Pobjel d’une analyse ap-

rrofondie dont les lésullals sont ex])osés dans les paragraphes réservés aux frac-

tions 1 et III.

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P. BOQUET, IZARD, M. JOUANNET et J. MEA UME

373

Un antigène mal cléfini. h est ohservé clans les préparations qiii contiennent

L.ne forte quantité de venin.

L'arc de précipilé C correspond à iin antigène hasique. Cet arc est caracté-

risc par son amplitude et son a])latisspment. Enfin. trois antigènes cathodiques

piécipitent selon les ares Dj, D, et D 3 . Onze parmi les treize antigènes dé-

nombrés sont repartis dans les fractions I. 111 et VI.

La fraction 1 est essentiellement eom[)osée de 1 antigènes du groupe B et

la fraction 111 eontient Tantigène A et 3 antigènes du même groupe R.

Les exiiérieuces d immuuo-diffusion selon Oucbterlony démontrent que parmi

ces antigènes un seul est commun aux fractions 1 et 111. Cette oliservation est

confirmé par Texperience que voici. Lorsqidon fait reagir suivant la même techni-

que la fraction III sur un échantillon de sérum 984 épuisé par la fraction I ou

la fraction 1 sur un échantillon du même sérum épuisé par la fraction III, trois

lignes de précipité se forment dans chaque |)réparation. Au contraire le sérum

981 tel qiiel fait a])paraitre quatre lignes de précipité dans les |)réparations té-

moins. Cette ohservation nous a conduit à adoj)ter la nomenclature sinvantc

j)our définir les antigènes du groupe B.

Fraction 1

]R, = 3R, B,

Fraction 111

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374

ENZYMES F:T TOXINES DES VENINS DE SEREENTS. RECIIERCIIES

mOCniMIQUES et immunologiques sur le venin de

NAJA NIGRICOLLÍS

Li's aiiligèiies 115.. iT étaiil ideiilitjues, tious les désigiioiis ])ar lo syni-

l)olc

La fraclion VI coniprend les aiiligèiies forleiiietil hasiijues C. D,, el D^,.

Q)iiant aiix fraelioiis II, IV et V, elles se eomporleiil eotnrne des itiélanges des

fraetions voisiiies. Daiis la fraetion IV, eependant, sont assoeiés aiix aniigèiies

A et C les deiix aiiligènes a el l> doiil la |)roporlion semhie faihie dans le veiiiti

lel (jiiel.

La fraclion «, ohlenue par deux filiralions sueeessives de la fraetion VI à

travers le “Sejdiadex |)réeipile suivanl nn are eorrespondant à D-j lorsqn’on

Ia soumel, à la dose de 25 à 50 /x à riinmiino-éleetro[)Iiorèse. Dans les inênies

eonditions, ec))endant, des doses plus fortes de eelle fraclion soit 100 à 500 p,g,

non seidement fonl apparaílre Tare de précipilation D-,, mais encore des ares

eorrespondant à D^, D, el C dont la ])osilion an voisinage de Taxe de la ])ré-

|)aration indicpie (pie ces aniigènes sont en faihie pro])ortion.

I5ie étiide de Taction dénalurante de Ia clialeur snr les anligèiu-s qiii vien-

nenl trêtre énnrnérés complete ces oOservalions. A cet effet, on chanffe à 96"

dans im Oain-rnarie (2) ime soliition contenani 10 mg de venin jrar cm^ irean

j)nre * et on prélève, à intervalles régniiers, des cclianlillons de eelle soinlion

qni sont aiissilôt soiimis à Téprenve de rimmuno-éleclrophorèse.

Dans ces eonditions en inoins (rim miniile rantigène IB, est privé de sa

])ro|)riétc de jirécipiter en [irésence de Tanticoips spccifiqne.

A Texception de Fantigène 1?^, les autres antigènes dn gronpe B ne prcci-

pitent pins après 5 minutes. Les délais de la perte de leur poiivoir de |)réeipiter

sont de 20 minutes pour rantigène A, de 30 minutes pour rantigène Da, de 45

minutes pour Fantigène C, de 55 minutes pour Fantigène Ba el de 60 minutes

pour Fantigène D^.

Parallèletnent à ces recherches, nous avons entrejiris de definir les |)roprié-

tés enzyrnatiques el toxiques du venin de Naja nigricolli.s et à titre comparalif,

celles des fraetions ohtenues à |)arlir de ce venin.

Les leclmiques utilisées dans ces essais sont rapportées dans une jmhlicaliou

séparée (4).

Si on admet qne la dissocialion des jiroléiues jiar la rnétliode de filtralion

sur les “Se|)liadex” est fondée sur leur exclusioii dans Fordre décroissaul de

leurs masses molécidaires. on est eonduit à jienser (pie la fraclion I est consti-

luée ])ar les jiroléines dn venin dont la masse moléeulaire i'st plus élevée.

Celle fraclion I lirise les liai.sons carhoxy-ester de Facelylcliolinc mais elle

('St sans effet sur les acides aminés estérifiés. Une correlation a été élaldie par

Hamlierg et Bocha e Silva (7) entre Fajililude (pie possède le venin de Bollirops

jararucd k hydrolyser le B.A.E.E et sa propriéié de lihérer de la hradykinine

dans Forganisme animal.

Dans cet ordre de fail, ni le venin de Nitja uigricoUis ni aucnne fraetion

examinée iFonl une aclion sur le B.A.E.E, le B.A.M.E. le T.A.M.E et FA.T.E.E.

La même fraclion I hydrolyse la llaison [leptidiipie de la glycyldencine, el

une des deux liaisons de la glycyl-glycyl-glycine el de la glycyldeucyl-tyrosine

mais elle est saus effet sur Fliémoglohiue el la caséine.

• Eau cleslonlsée.

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I’. FOQUET, Y. IZARD, M. JOUANNET et J. MEAUME

375

Au contraire, elle ronipt très aisément les liaisons phospho-ester du jihospha-

te de paranitro])heiiol, de l'AMF. du lii-phosphate de paranitropheiiol. de l'ADP

et de rATP.

Son action dépolyniérisaiite à Pégard de laeide liyaluroniqiie est faihle.

Klle possède. eiifin. la propriété de [trovoquer in vitro la coagulatioii du saiig.

La fraction III eontient non seideineiit la |)hos|)holipase A [troduetrice de

lysoeithine aux dé[)ens de la lécithine mais eueore uu facteur lytique direct.

Klle délruit le eomjtlémeiit du sérum de cohaye. diminue la vilesse de la sédi-

mentalioii gloliulaire. dé])olymérise Paeide hyaluronicjue et inhilie le phénomène

de Ia eoagulatiou. Knfin. celte íraetion III est légèremeut toxi(|ue, sa Ldsn pour

la souris de 18-20 g. éprouvée par la voie veineuse est de 16 31 p,g alors que

dans les mêmes conditions celle du venin est de 12.36 /xg.

La fraction VI exerce une action anticholinestérasique sur racétyl-cholines-

tcrase des glohules rouges du cheval mais elle est sans effet sur Ia eholinestérase

que nous avons décelée dans la fraction 1.

D’autre part, cette fraction VI est toxique et provoque des jraralysies chez

Ia souris et le lapin.

Le produit le [jlus ]mr que nous avons ohtenu après deux filtrations de

cette fraction à travers un gel de "Sephadex Lrio”. exerce encore une action

anticholinestérasique mais sa Ld.-,,, pour la souris est faihle. Elle est de 2 pg

dans les conditions qui viennent (Pêtre définies. Nous 1’avons désignée sous le

nom de toxine a. et nous avons conclu trime étnde préliminaire (4) que cette

loxine est une j)rotéine de faihle jíoids moléculaire. Elle franchit, en effet, les

memhranes d’acétate de cellulose et son coefficient de sédimenlation exprime en

unités Svedherg est 0,84 (centrifugeuse Spinco; cellule standard de 12 m/m,

angle 2“; concentration du produit 10 mg/cm*; vitesse de rotation 59.780 l/rn;

lemirs de centrifugation 96 min; température amhiante 20" ±; 0,1" I.

I ne ])remière analyse de ses acides aminés ré\èle qi/elle eontient de fortes

proj)orlions d’arginine, de glycine. de lysine, (Paeide aspartique, (Paeide gluta-

mique et de threonine (3).

l’ne collahoration a été étahiie avec J. Porath, E. Karlsson et D. Eaker de

PLniversité d l ppsala qui ont hien voulu consacrer une |)artie de leur activité

à Péliide de la toxine a et entreprendre une analyse de la strueture de ce ])oly-

peplide.

Ces résnitats aequis, il importait (Pétahiir nne relation entre les antigènes

definis ])ar les méihodes immunologiques fon(lé(‘s snr la précipitation mutuelle

des antigèn(*s et des anticorps dans un gel et les diverses pro])riétés (]ui viennent

(I tire énumérées.

Des glohules de cheval additionnés de lécithine puis versés à la surface (Pune

])réparation de la fraction I ou du venin tel quel. soumis au préalahle à une

immimo-électrojdiorèse, sont lysés entre les ares de précipité de Pantigène A qui

peul ainsi ctre considéré comine étant la phospholipase. En Pahsence de léci-

ihine et de la solulion tam|)onnée dont le jihosphate se comporte comine un agent

inhihiteur, on ohserve une hémolyse entre les ares de Pantigène précipité 3IL,

homologue vraisemhlahlement du facteur lytique direct décrit par Hahermann et

Neumann l8) à propos (Pautres venins.

L’em])Ioi de la technique dT riel t9) adaptée à Phvdrolyse des dérivés phos-

phorés du nitrojihenol perinet de siiivre Paction des six fractions sur les liaisons

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QTfi ENZYMKS ET TOXINES DES VENINS DE SERPENTS. RECHERCIIES

BIOCIIIMIQUES ET IMMUNODOGIQUES SUR DE VENIN DE

NAJA NIGEICOLLIS

jjhosplio-eslpr. L’hy(lrolyse du l)i|)lios[)hatp <le [)aranitro|)liénol se (lévelo])])e entre

les ares de j)réci|)ilé de ranligèiie 115i.

Les expérieiices sur la ihermo-sensiliilité des divers anligènes (|iii vieimenl

(rêtre dénomhrés nons enseignenl (|ii’à la lem|)éraliire de 96" raniigène 115, ])erd

en quelqiies secondes son a])tiliide à fornier iin |)réeii)ilé an eonlaet de {'antieorps

spécifiqiie. Or dans les mêmes eondilions la fraction I perd également sa jrro-

priété de eoagider le sang. Cette ohservalion ineite à idenlifier raniigène llier-

molaliile ll?,i facleur accéléralenr de la eoagnialion sangiiine. Privée de ee fac-

lenr, la inême fraction I exerce, an eonlraire. une aetion anlieoagnlanle. On

est eondnil à eonelure. (rnne part. que dans les eondilions hahilnelles (rex])éri-

mentation le facteur eoagulant de la fraction 1 exerce une aetion dominante, et,

d'autre |)art, que les jiropriétés anlicoagulantes eommunes des fractions 1 et III

soul vraisemhlahlemenl sous la dé|)endance d’un rnênie facleur. Les fraetions [

et Hl eontenant un anligène commuu IL, ou est autorisé à penser que cel an-

tigène est un facteur antieoagulaut. A Taiipui de cette liypothèse, Texpórience

démonire que le facteur antieoagulaut des fractions I et III et raniigène R 2 ont

la même sensihilité iherniique.

On constate, enfin, que la neurotoxine a thermorésistanle est représeulée

|>ar raniigène D;,, (resl à cel anligène (pie correspoud le |)olype[)lide récenunent

isolè dans un élal voisin de la ])urelé par Karlssou, Kaker et Poralli (10). Le

talileau I résume ces oOservalious.

TABLEAü I — ANTIGENES DU VENIN

DE NAJA NIGRICOLLIS

Cocte

Funotlon pre.sumce

Phosphollpase

IB,

Ester hydrolase

3B,

Facteur nntlcoagulant

3B.,

Facteur hémolytique dlrcct

IB,

F^acteur eoagulant

Anti enzyme?

D 3

Toxine a

On peut considérer, poiir concinre, que raniigène A iso-électrique à pH 8,2

et raniigène fortemeni hasique 1);, reiirésenteni les proléines responsaldes, la pre-

mière, de raclion liydrolysantc du venin sur les lecilliines et la seeonde de son

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P. BOQUET, Y. IZARD, M. JOUANNET et J. MEAUME

377

action neurotoxique. On est coiiduit à admettre, d’aiitre part. qirun certain

iionihre d’argumenls jtlaident eii faveiir des relations de caiisalilé que noiis avons

tenlé d’étal)lir entre divers aiitigènes et certaines activités enzyniatiqiies exercées.

II apjtarait cependant. que les projtriétés physiologiques et hiochimiques de plu-

sieurs antigènes demeurent indétermiuées et que le nombre des autigènes révélés

par le pliénonièue immunologique de la précipitatiou est inférieur au nombre des

activités actuellemeut eounues du veuiu de Naja nigricollis. LVxpérienee uous

enseignera daus quelle mesure une jtrotéine est capable dVxercer jdusieurs aeti-

vités, dans un venin.

La question stí [)Ose également de savoir quelles relations ])euvent être éta-

blies entre les antigènes caractérisés dans le venin de Naja nigricollis et les an¬

tigènes des autres venins de serpeuts. Des reeherebes immunologiques en voie

de dévelopjtement montrent que les venins de deiix ELAPIDAE, Naja liaje et

Naja naja et eelui d'un VIPERIDAE, Echis carinatus contiennent des protéines

nocives dont la eoiistitution est vraisemblablement voisine de celle de la toxine a.

Dans cet ordre de faits. Tétude de la structure de cette "toxine” entreprise à

Upj)sala par Karlsson, Eaker et Porath. apportera l élément essentiel d’une solu-

tion à ce problème.

Bibliographie

1. BOQUET, P., Toxicou, 1966, à paraitre.

2. BOQUET, P., IZARD, Y., JOUANNET, M. et MEAUME, J., Mem. Inst. Bu-

tantan, SimtJ. Internac., 33, 1966.

3. BOQUET, P., IZARD, Y., JOUANNET, M, et MEAUME, J„ C. R. Acad. Sei.,

2H2, 1134, 1966.

4. BOQUET, P., IZARD, Y., MEAUME, J. et .TOUANNET, M., Mem. Inst. BtiUin-

tan, Simp. Internac., 33, 1966.

5. OUCHTERLONY, O., Actu Microbiol. scand., 32, 231, 1953.

6. GRABAR, P. et WILLIAMS, C. A., Biochim. Biophys. Actn, 17, 67, 1955,

7. HAMBERG, U., ROCHA E SILVA, M., Experientia, 13, 489, 1957.

8. HABERMANN, E. et NEUMANN, W., Arcii. exp. path. Phnrmak., 223, 388,

1954,

9. URIEL, J., An)i. Inst. Pusteur, 101, 104, 1961,

10. KARLSSON, E., EAKER, D. et PORATH, J., Biochim. Biophys. Acta, 1966,

à paraitre.

DlSCl SSIO.N

C. Y. Lee: “Is there any cardiotoxic component in the venom of Naja nUjri-

collisl What is the molecular size of your neurotoxin?”

P. Boquet: “La présence de cardioto.xine n’a pas été recherchée. En ce qui

concerne le poids moléculaire de la toxine, nous avons donné seulement le coeffi-

cient de sédimentation en unités Svedberg. Le Professeur Porath vous donnera

de plus amples informations sur ce point.”

SciELO

10 11 12 13 14 15

Q7S ENZYMES P:T TOXINES DES VENINS DE SERPENTS. RECHERCMES

BIOCHIMIQUES ET IMMUNOLOGIQUES SUR LE VENIN DE

NAJA NIGRICOLLIS

S. Minton: ‘‘In which fraction of Naja nigricoUis venom is the antigen shared

with the venom of Echis carmatus?"

P. Boquet: “Une communante antigcniquo a été mise en évidence entre le

venin de Naja nigricoUis et réchantillon de venin (i’Echis carinatus qui nous a été

adressé d’Ethiopie. Parmi les antigènes communs au venin de Naja nigricoUis et

au venin d’Ecliis carinatus étudiés, il convienl de citer un antigène basique cor-

respondant à l’antigène neurotoxique.”

F. Kornalík: “How liave you tested the procoagulant and anticoagulant pro-

perties of the Naja nigricoUis venom?”

P. Boquet: “L’action du venin de Naja nigricoUis sur la coagulation du sang

a été mesurée selon les techniques usuelles (détermination du temps de prothrom-

bine, etc...) au moyen du plasma humain.”

10 11 12 13 14 15

Mem. Inst. Butantpn

Simp. Internac.

33(2):379-388, 1966

JERKER PORATII

379

12. SOME SEPAHATIOX METHODS HASED OX MOLECELAH SIZE AXl)

CHARGE AXD THEIR APPLICATIOX TO PERIEICATIOX OE

POLYPEPTIDES AXD PROTEIXS IX SXAKE VEXOMS

JERKER PORATH

Institute of Biochemistry, University of UppsaUi. Uppsala, Sweden

Intkoduction

A inalhematical jirohlem can often he solved in many ways. One of iheni

niighl he tlie mo?[ effective. another the most informative, while perhaps all

olhers niight appear disgraceful to the mathematicians. An analogous sitiiation

often arises when lhe hioehemist is faced with the proldem of isolating a particular

snh.stance froni a eomplieated mixlure siich as, for exam])le, an animal venoni.

Many sej)aration melhods are availahle hut a ])ro])er choice among theni is dif-

ficult. Another question coneerns the selection of order when several metliods

are required. I eannot give general answers hut only express some persoiial

opinions in the ho|)e to guide those with limited ex])erienee in these matlers.

íl is a commonplaee misunderstanding that a method which most effeclively

resolves a eomplieated mixture in a single ste]) of neeessity also is superior to

those of less separation power. Although ihis is usually true when the purpose

is entirely analytieal it is not neeessarily the case in preparative fractionation. 1

can mention gel electrophoresis as an example. Analytieal electro))horesls in thin

layers of stareh gel or ])olyacrylamide has heen extremely usefui for studies of

genetically delermined variations in protein patterns of hiological fluids and ex-

tracts. Xevertheless. in spite of the impressivo resolving ])ower of this lechniqne

it is nsnally far more practical to use other methods for large seale se])arations

hecause of the difficnlties involved in the transfer of gel electrophoresis lo a

preparative seale.

Gel electrophoresis is hased on at least three |)hysieal faclors or ])arameters.

viz., eharge, molecular size and shape. Eractionation in two or more sejiarate

])rocesses often gives hetter resnits and affords more information ahonl the physieal

or Chemical properties of the snhstance lo he i)nrified. Actnal ap])lication of

these methods may he fonnd in references 11,2). Provided lhal the gel filtration

step is carried ont in a colnmn calihrated with respect to the molecular size —

retention relationships a rongh estimation is possihie of lhe molecular dimensions

of the snhstance under sludy. Electro|)horesis ])erformed in an adsor|)tion-free

médium permits conclusions ahout elecirical eharge of the migrant species. Since

electrophoresis and gel filtration are highly reliahie and reproducihle methods,

holh can he em|)loyed easily on any seale retpiired in lahoratory work.

Moi.ECUl.AR SIEVING

Several kinds of gel materiais are availahle that provide se])aration hased

on molecular size. Sephadex and Riogel. cross-linked dextran and polyacrylamide,

respeclively, are lhe most importani commercially availahle gel snhstance ai)plieahle

Sephadex and Riogel. cross-linked dextran and polyacrylamide.

2 3

5 6

11 12 13 14 15

Qon SOMK SEPARATION METIIODS BASED ON MOLECULAR SIZE AND

CHARGE AND THEIR APPLICATION TO PURIFICATION OF POLY-

PEPTIDES AND PROTEINS IN SNAKE VENOMS

lo fraclionatioii in aqucous soliilions. IJolli of llifse gcis are availahlo iii dif-

fercnl degree of crossdiiikage, tluis providitig for inolcciilar wcighls froin a fcw

huiidred iip lo several lumdred tfioiisaiids. l'or siil)slaiices of cvcn liiglicr iiiole-

fular sizc and for paiiicles agarose gel can lie used. I'or snake venom fractioii-

alion Sepliadox (ílOO, CTS, (',50 and (;25 luive lieen fouiul lo Oe exireniely

useful.

An almost complete list of referenees lo lhe gel filtraiion nuTiiod ean he

ohtained from Pharmacia Fine Chemicals, U|)pfala, Sweden.

Ellctuoimiouesis

The nnex[)cricnced rnay feei discomfori when faced vvilh lhe jirohlem of

ehoosing among lhe very many versions of eleetro|)horeli(' eipiipinenl and tech-

niqnes now availahie. Yet eleelrophoresis is simple in jiractice ailhough not in

theoretical delails. This is certaiidy trne even for column eleelrophoresis lhe

melhod I [jrefer myself.

Among lhe numerons ap|)aratuses for eleetro|)horetic fractionation al my

dis])osai al lhe Instilnie of Pioehemistry in U|)|)sala I vvonld seleel lhe ai)])araliis

of Steilan Hjerlén |8) for orienting and analylieal purposes and lhe cohimns de-

scrihed hy myself for preparations on any scale in lhe range of 25 milligrarns (4)

to 25 grams |5).

The lechnique developed hy Hjerlén is a kind of free zone eleelrophoresis

hased on a principie first descrihed hy Alexander Kolin (6) and inde])endently

somevvhat laler hy Hjerlén (7,8). The fractionalion chamhcr consisis of a qnartz

luhe a fevv millimeter in diameter placed helvveen two eleclrode chamhers and

sejjaraled from lhem hy memhranes. Dnring lhe rnn, lhe luhe is rotated con-

linually ahonl its longitudinal axis. Samples can he transferred to or wilhdrawn

from lhe luhe hy a capiliary tuhing. FfficienI cooling is achieved hy immersion

of lhe luhe iu a water hath. The [irogressing separation eau he followed hy a

scanning arrangeinent which measnres ahsorption al 280 ni/j,.

The salient fealure of lhe melhod is lhe revolving luhe. P>y sleady rolalion

of lhe luhe al a conslanl speed conveclion is virtualiy eliminaled. A sinall sample

projierly inlroduced (vvhile luhe is revolving) gives a slahie slarling zone. II

no voltage is a|)|)lied, lhe zone vvill hroaden oídy very slovvly, ehiefly hy molecular

diffiision. A prolein zone remains sharp for honrs. When an eleelrie field is

ajiplied lhe varioiis componenls slari lo move aeeording lo lheir eleclrophorelic

mohilities. Al iniervals lhe se])aralion pallern is cheeked hy scanning. Al any

time can he renioved from lhe rotating luhe and analyzed for hiological aclivily,

Chemical |)ro|)erties, etc. Sam|)les rangi ng in size from a fevv /xg lo a fevv mg

ean thus he analyzed.

Unforltmalely ihis apiiaralus is noi yel commereially prodiieed. Iu eerlain

ap|)liealions some of lhe ihindayer eleclro])horesis teehniípies are effeelive

suhstilules.

The Hjerlén eleelrophoresis inelhod unforiunalely cannol yel he sealed iqi

lo allovv fractionalion of lhe hundreds of milligrarns and more required in mosi

|)i'eparalion |rroccdnres. Hovvever, eleelrophoresis in free soluliou (i.e. eonlaining

no solid su|)porl I can he performed on a larger scale lhan is afforded hy lhe

SciELO

10 11 12 13 14 15

i«£er.í. Itist. Butantan

JERKER PORATU

381

Simp. Internae.

33(2):379-388, 1966

*•

Hjeríéii ajiproach. This is acconiplished in flowing-filni eleclroplioresis. a coii-

tir.uous separatiüii process (9). Hecause sample can he inlroduccd (■oiitimiously.

the lailer melhod can piovide separations on any desired scale if lhe componeiits

difíer widely in inohility. Since this niethod is mainly useful for group separa-

tion and does not have lhe resolving |)Ower reqnired for se|)araling very elosely

related suhstances it wili nol l)e fiirlher (hscnssed here. Instead reference is

tnadc' lo lhe lhesis of HannigllO).

In slationary tolumn eleclroplioresis lhe advantage of operating in a uniform.

free solution must he sacrificed. A ])oroiis povvder is introduced inlo tlie hnffer

or a density gradient is jirodiiced ( 11 I lo snppress niacroconvection. Only lhe

first technique wilI he descrihed here in some delail. It wilI suhseqnenlly lie

referred to siinply as “colunin electro|)horesis'’.

Column eleclroplioresis is a discontiniious melhod. As in lhe case of re-

volving tiilie eleclroplioresis the sample is introduced in the cohinin hefore an

electric ficld is applied. The rini may he continued for several days at a safe

temperature provided lhat the tuhe is surrounded with a cooling jacket and the

evoliition of joule heat is kejit low enongh to avoid the creation of a large radial

temperature gradient. Evidently, column eleclroplioresis permits the fractionation

of suhstances with similar iiiohilities.

In my opinion the potentialities of column eleclroplioresis have heeii iinder-

estimated. I should üke to discuss hriefly some of the most important drawhacks

and supjiosed inconveniences inherent or adhereiil lo the method.

The first and niosl important prohlem is lhe interference hy the supiiort.

Adsorptioii has soinetimes heen considered to play a suhstantial role in lhe frac¬

tionation as indicated hy the term ■TIectrochromatography”. If charged suhstances

adhere to the supporl hy adsorption or precipitation the dattiage can he disastroiis.

for this not only increases electroosmosis hut aiso promotes fiirther adsorption.

Fortnnately. siqiports with very high degrees of "inertness'’ are now availahie,

siicli as specially puriíied cellulose I from Grycksho Pap|)ershruk. (irycksho,

Svvedenj and Sephadex.

Another ohjeclion to column eleclroplioresis concerns lhe difficully of irionilor-

ing lhe advaucemenl of lhe migraling zones. Unless lhey are colorcd, suhstances

Iravelling in lhe noii-lransparenl column caiinol he ohserved during a run. How-

ever, suhstances that have reached lhe end of the column can he removed con-

linuously hy a washing lechnique and analysed hy a rccorder hefore collection (12).

riiis jirocedure allows monilored withdrawal of the fast-nioving suhstances after

which lhe eleclroplioresis can he proloiiged to separate lhe componenls with low

uiohilities. Hy |)ro|)er seleclion of column length lhe eleclroplioresis may he pcr-

foriiied very efficientiy. I ncharged and charged colorcd markers may of iour.se

he introduced lo serve as indices of electroosmosis and lo facililalc esliniations ol

lhe stage of fractionation.

■■(ichiniu eleclroplioresis is complicalcd ' is an oficn heard slalcment. 1 hose

who once have tried lhe melhod in oiir institute never use lhat argunienl. It is

an extremelv simple melhod whether employed on a moderate (4) or a large

scale (5). The LKB column for preparative eleclroplioresis on a large scale is

indeed so simple that I can operate it myself when my tcchnician is ill, although

I admit that reading the descriptioii in the manual demands intellectual effoii

I Manual and a|)|)aralus are availahie from LKB Instrumenl AB. Bromma 1.

Svveden I.

2 3

5 6

11 12 13 14 15

382

SOMp; SKPARATION METHODS RASED ON MOLECULAR SIZE AND

CIIARGE AND THEIR APPLICATION TO PURIFICATION OF I>OLY-

PEPTIDES AND PROTEINS IN SNAKE VENOMS

l().\ EXCHANGE ClIliOMATOCliAPIIY

'riie grcat l)n*ak-lliroiin;h for cliromalograjiliy of proleiiis caiiio vvilli lhe iii-

trodtictioii of lhe eelhilose ion excliangers hy Soher and I’elerson |13). Dielhyl-

aniinoelhyl ethers of eelhdose I DKAE-eellulose) liave proved lo he ])arlicidarly

iiseful. More reeenlly, aiialogoiis ioii-exehangers have heen |)roduced wilh Se-

phadex as nialrix for exainple, lhe DKAh]-. CM-(earhoxytnelhyl), and SE-(snl])ho-

elhyl) derivalives. The Sephadex excliangers have lhe advanlage over llie cor-

responding celluloses in iieing more insoinhie. This is parlicniarly evideni wilh

lhe ealionic types. The large volume changes lhal accompany allerations in lhe

ionic slrength of lhe mediurn oflen com|)licale ehromalography on long ion ex-

change Sephadex eolnmns.

The pojmlarily lhal prolein ehromalogra])hy has enjoyed lhe lasl deeade is

dne lo lhe easy lechnical o|)eralions and lhe simple ir.px])ensive eqnipmenl reqnired

— at leasl for crnde gronp separalions. The more effeclive elulion ehromalo-

graphic leclmiques, enqiloying an eluanl of conslanl or conlinnonsly ehanging

eomposilion, are more dernanding however, hecanse small varialions in lhe experi-

menlal condilions oflen ehange lhe elulion |)allern heyond reeognilion. Lack of

re[)roducihilily is oflen experienced nnless exlreme eare is laken in lhe preparalion

of Imffers, in mainlaining a conslanl s])eed of elulion, lemiieralure, elc.

Occasionally, lhe lime and efforl expended in searching for ojilimnm con¬

dilions for elulion ehromalography of prolein mixlures nsing an ion exehanger

is amply rewarded. 1’olycarhoxylic ion excliangers such as Amherlile IHC-5()

have heen used wilh greal success lo fraclionale hasic [lolyjieplides and proleins

of small molecular size.

Application of gel filtration and ion exchance ciihomatogiiapiiy

TO THE PLIlilEICATION OE SNAKE NELUÍOTOXINS

Isolulion procrdurex

1 like lo examplify lhe melhods descrihed ahove wilh some cxperimenls

reeenlly riiade hy l)r. Kverl Karisson and Dr. David Eaker in my lahoralory.

More delailed deseriplions of lhe procediires wili ajipear laler (1-1 ). (iohmin

eleclrojilioresis has heen a|)plied only for orienlalion [iiirposes, sinee in ihis par-

licnlar case, lhe isolalion of iieuroloxins, ion exehange ehromalography has a

higher resolving power.

Hnl eleclrophoresis of crnde venoni gives a good groiiii sejiaralion of iieiiro-

loxins and phospholipases (Fig. Ij-

ophcol dfnsity 280 m/j

Fig. 1 -- Column: Celluto.se, MunktelTs,

103X3 cm. Huffer: O.OIS M Acelute buí-

for, 1.50 M Glycine'-- pll 5.0. Sample: 150

mg cruíle venom. Run: 21 hours, 1200

volt, 35 mA. Fiotd strength 8 volt/cm.

Fractions: 5 ml per 30 min. N and P

Indicate the fractions vvith the highest

neuroloxic and phospholipase activiiies.

(Run made by Dr. Kvert Karlsson).

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

JERKER PORATH

383

Simp. Internac.

33(2):379-38S, 1966

The procedure of isolatioii of the neurotoxin « of Naja nigricoilis, the loxiii

most extensively studied so far, was in short the following:

A sample of 1.1 grani of dry crude Naja nigricoilis venoni dissolved in

10 nil of sodiuni phosphate huffer of pH 7.3, 0.03 M in sodinm ion. This

solution was suhjected to gel filtration in a Sephadex G75 column (3X80 cm I

with elulion S])eed kept at a rale of ahoiit 18 ml jier hoiir. 10 ml fractions wcre

collected. Fig. 2 shows a diagram of the same general paltern ohtained from

an analytical experiment.

Fig. 2 — Gel filtration of 150 mg crutle

venom on a 3.2X80 cm column of Sepha¬

dex G-75 in 0.03 M .sodium-phosphate buf-

fer, pH 7.3. Flow rate: 17.5 ml/hr, 3.5

ml/traction. Tn and T/5 indicate the areas

with highest toxic activity.

Pooled fractions containing T* (see the shaded area in the diagram) were

transferred to a 3.2X45 cm column of Amherlite lRC-50 equilihrated with 0.28 M

sodium phosphate huffer of pH 7.3. Elution was performed at a rale of 30 ml

per hour. Fig. 3 shows the elution pattern ohtained in this experiment. Ahout

25 mg of jiure neurotoxin Tj was ohtained.

Fig. 3 — IRC-50 chromatography of Ta

from gel filtration (Fig. 2). Sample ap-

plied to 3.2X45 cm column in 250 ml 0.03

M fiodium-phosphate huffer. Elution at .30

ml/hr with 0.28 M sodium-phosphate buf-

fer, 6 ml/traction. Fraction 0 = 150 ml.

30 30

r'acUon No

Karlsson and Eaker have adopted this technique for fractionation of other

elapidian venoms. Eig. -1 shows the gel filtration diagram of the venom of

Hemachatus haemachatus. The neurotoxins have ahout the same elution volume

as those of Naja nigricoilis. When chromatograiihed on Amherlite IH(i-50 with

2 3

5 6

11 12 13 14 15

384

SOME SEPAKATION METIIODS BASED ON MOLECULAR SIZE AND

CHARGE AND THEIR APPLICATION TO PURIFICATION OF POLY-

PEPTIDES AND i>ROTEINS IN SNAKE VENOMS

gradieni elulion llu- neiiroloxiii fractioii was n-solved inio a large iniiiiher oí

ilislinclly differeiil eomponeiiLs. iiiaiiy of vvliicli were showii lo possess neiiroloxie

activity (Fig. 5).

*254

EFFLUENT, ml

Fig. 4 — Gel flltration of 1.3 g of erude Hemachatus haemachatus venom on a

3.2X74.5 cm column of Sephadex G-75 In 0.06 M sodium-phosphate buffer, pll 7.3.

Flovv rate: 18.8 ml/hour. Effluent monitored contlnuou.sly at 254 m/j with an

LKB Uvicord. The last peak ha.s Aj,, hlgher than Aj„.

Fig. 5 — Chromatography on Amberlite IRC-50 of fraction “Neurotoxin.s" (Fig. 4).

Column: 3.2X21 cm equilibrated with 0.20 M sodium-phosphate buffer, pll 7.3, and

eluted with 50 ml 0.06 molar buffer before applicatlon of samplo. Run: Performed

with a Beckman Model 130 Spectrochrom Analyzer whlch permits a contlnuous

monitoring of absorbancy, pll and conductivity. After applicatlon of sample elutlon

with 0.06 M sodium-buffer was continued until no more unadsorbed material carne

out. Exponential concave gradient from 0.15 M to 1.50 M sodium. Flow rate

40 ml/hour.

SoiiK’ propcrlirs of lho nvuroloxiiis

Neiiroloxiii Ta of Naja iii^rivollis lias a molecular weiglil of 6737. It coii-

si.sts of 61 amino aeid residiies liiied iip iii a single jieplide ehain eross-linked

liy foiir disulfide liridges. The neurotoxin Ta is liiglily liasie, as are all lhe

snake nenroloxins sludied so far iii Up|)sala. Thiis il moves rajiidly lo lhe ealhode

al |)il 8.7 in lhe HjerUhi eleelrophoresis (l'’ig. 6). The IJ),,io has lieen fonnd

lo he 1.8 /j.g, as delermined in miee of 18-20 g hody weighl.

In Tahie I are eom|)iled amino aeid analysis dala for IVaja nisrrwoUis Ta , for

llcniarlialii.s huornacliiiliis eomponenis .'5, 5 and 12 (Fig. .SI, and for lhe erahii-

loxins a and h i.solaled hy 'Faniiya and Arai (16|. Peaks 3 and ,3 represeni

highly aclive nenroloxins (I^l),,,,, 1..5 and 2.0 /tg). lhe similarily in amino aeid

eomposilion is remarkahie. Fnriher fnel for exeiled speenlalion comes from lhe

very similar fignres for lhe Naja naja atra loxin of Yang and indeed aiso for

lhe seorpion loxin de.serihed al lhe Symposinm liy Dr. Li.ssitzky. All of ihese

dala seern lo indieale lhal a single sjieeifie (or a fevv elosely similar) rnoleenlar

pallerns evolved in widely differeni rpeeies is ea|)ahle of hloeking some fnnda-

menlal physiologie.al proeess.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(2):379-388, 1966

JERKER PORATH

385

• y '

9 ~

-- -

• i

. « i

•

B e i

. .

. '

■i

• <

•

A \

V,8

-£-r

B B i

Fig. 6 — Free zone electrophoresis showing electrophoretic homogeneity. Sample: 10

of Tit in 0.03 M buffer, pH 7.3, optlcal density at 279 mii 1.5. 0.1 M Tris-acetic acicl

buller. Run: pH 8.7 (measured at 22»), 3 mA, 1,520 V and 1.2°. Exposures: 0.60, and

132 min. atter the start. Distance of migratlon: 17.5 cm. Arrow Indlcates the .starting zone.

SciELO

10 11 12 13 14 15

386

SOME SEPARATION METIIODS BASED ON MOLECULAR SIZE AND

CIIARGE AND THEIR APPLICATION TO PURIFICATION OF POLY-

PEI>TIDES AND PROTEINS IN SNAKE VENOMS

TABLE I

N.n*

Il.h**

H.h

Il.h

E.a»**

E.b*»*»

Amino acid residues

Lysine

Histidine

Amkie

Arginlne

Cy.stelc acid

—

Aspartic acid

Methlonine Sulfonc

—

Threonine

Serine

Glutamic acid

Proline

Glycinc

Alanine

Vi Cystlne

Valine

Methionine

Isoleucine

Leucine

•1

Tyrosine

Phenylalanine

Tryptophan

1 or 2

Total number of amino aci(i

residues

* Naja nigricoltis

♦* Hemachatus haemachatnn

*»• Erabutoxin a

«»»» Erabutoxin b

Min. mol. WGight (i787 6828 6823 6707

LD 100, iig (intrnvenousiy in mice, 18-20 g) 1.8 1.5 2 50

Acknowledgements — Dr. Paul Boquet directed my interest to lhe snake venoms.

I am most thankful to him and his associates for fruitful collaboration over some

years, for the supply of venom and their help in the assays of our fractions vvith

respect to their neurotoxic activity.

The Swedish Natural Science Research Foundation spon.sored the snake venom

research and paid my travelling expenses.

SciELO

Mem. Inst. Butantan

Simp. Internac.

33(2):379-38S, 1966

JERKER PORATH

387

Kefekenciís

1 .

2 .

6 .

8 .

10 .

11 .

12 .

13.

14.

15.

16 .

DELIN, S., SQUIRE, P. G., and PORATH, J., Bioclüm. Biophys. Acta, 89,

398, 1964.

MAKONNEN, B., PETTERSSON, C., and PORATH, J., to be published in

Biochim. Biophys. Acta.

HJERTÉN, S., Fr-ee Zone Electrophoresis, Thesis, Uppsala, 1967.

PORATH, J., Methods in immunology and hnmunocheniistry, Vol. I, in press.

PORATH, J., Sei. Tools, 11, no. 2, 21, 1964.

KOLIN, A., J. Appl. Physics, 25, 1442, 1954.

HJERTÉN, S., Arkiv fôr Kemi, 13, 151, 1958.

HJERTÉN, S., in Protides Biol. Fluids, Proc. 7th. Colloqu., Bruges, 1959, Else-

vier, Amsterdam, 1960, p. 28.

BARROLIER, J., WATZKA, E., and GIBIAN, H., Z. Naturforseh., 13b, 754,

1958.

HANNIG, K., Eine Neuentwicklung der tràgerfreien Ablenkungselektrophorese

iind ihre Anwendung auf zytologische Probleme, 1964.

SVENSSON, H., HAGDAHL, L., and LERNER, K. D., Sei. Tools, 4, 1, 1957.

PORATH, J., LINDNER, E. B., and JERSTEDT, S., Nature, 182, 144, 1958.

PETERSON, E. A., and SOBER, H. A., J. Amer. Chem. Soc., 78, 751, 1956.

KARLSSON, E., EAKER, D. L., and PORATH, J., Biochim. Biophys. Acta,

in press.

PORATH, J., and UI, N., Biochim. Biophys. Acta, 90. 324, 1964.

TAMIYA, N., and ARAI, H., Biochem. J., 99, 624. 1966.

1 .

2 .

6 .

8 .

10 .

11 .

12 .

13.

14.

15.

16 .

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(2):389-410, 1966

TOMOJI SUZUKI

389

13. SEPARATIO-X METHODS OE AMiMAL VENOMS CONSTITUENTS

TOMOJI SUZUKI

Institute for Protein Research, Osaka University, Joancho, Kitaku, Osaka, Japan

Al)oiit twentv vears ago, the qiiestion was raisecl in oiir laEoratory of whether

zinc, which was l)ecoming the ohject of attentioii as a metal of hiological im-

portance, was present in varioiis snake venoms. At that time, Dr. Delezene re-

ported that many snake venoms contain a considerahle amount of zinc. On the

other hand, Japanese workers had reported that zinc could not he detected in

the venoms of Japanese and Formosan snakes. W’e re-examined this in thirteen

kinds of venom of Japanese and Formosan snakes hy emission spectro])hotometry,

and it was foimd that all the venoms examined contained considerahle amounts

of zinc and calcium, a small amount of magnesium, and traces of manganese

and other metais. Afterwards. when the effects of cations on the purified en-

zymes were studied. it was shown that zinc ions markedly inhihit the activities

of alkaline phosphatases of the venoms. such as 5’-micleotidase, phosphomono-

esterase, and that calcium ion is an essential component for maintaining the

tertial structures of some physiologically active proteinases and toxic proteins in

the venoms.

In this way, step hy step, our efforts were concentrated on separating the

different enzymes in snake venoms, and the prohlems arising from studies on

the pharmacological actions of snake venoms stimulated our interest to elucidate

the homeostatic mechanisms which were disturhed hy the injection of snake venom

enzymes. At present, we are making systematic studies on the purification of

enzymes in the venoms of snakes in connection with their physiological actions.

At the heginning of these studies, we applied the venoms to paper electro-

phoresis(l). We found that ELAPIDAE snake venoms generally contain hasic

jiroteins while CROTALIDAE snake venoms contain acidic proteins (Fig. 1).

The purification of the phosphodiesterase in these venoms was our next suh-

ject. It was essential to separate phosphodiesterase from 5’-nucleotidase to study

the structure of nucleic acid and to elucidate the structure of new nucleotides

which were isolated from various hiological materiais (2,3,4. 5), and this se-

jiaration was also necessary for development of fundamental studies on the pro-

duction of inosinic acid in Japan. Ratch tests were carried out to select suitahle

adsorhents for chromatographic separation of snake venom phosphodiesterases,

with reference to the results of [laper electroiihoresis. Results of two experiments

using 2 mg. of each venom can he seen in Figure 2. The enzyme of the venom

of the Formosan Cohra, Naja naja atra as we had expected, was readily adsorhed

on alumina C-y-gel. calcium phosphate gel, CM-cellulose or Amberlite CC-50.

On the other hand. the enzyme of the venom of the CROTALIDAE snake Agkistro-

don halys blomhofjii (“Mamushi” in Japanese), was readily adsorhed on calcium

[ihosphate gel, alumina C-y-gel and DEAE-cellulose (6). Therefore, ELAPIDAE

2 3

5 6

11 12 13 14 15

390

SEPARATION METÜODS OF ANIMAL VENOMS CONSTITUENTS

Naja naja atra

Naja naja naja

Naja hannah

Bun(jarus multicinctus

Vipera 7'usselUi forínosensis

Trimeresurns mucrosquaynatns

Trimeresiirus stejnegeri

Trimeresurns flavoviridis

Trimeresurns o7ciíiaa;e«sis

Agkistroãoyi acutus

Agkistrodon halys bloynhof-

fii

0 ^

Flg, 1 _ Paper electrophoresis ot snake venoms. Conditions: Phosphate buffer

pH 6.0 fi = 0.1, Toyo Roshi No. 50.

FlR. 2 — Adsorptlon of Mamushi and Cobra venom Phosphodlesterase on variou.s

adsorbents. 1. CM-Cellulo.se, 2. Cellulose powder, 3. Amborlite CG50. 4. Cal-

cium phosphate gel, 5. Alumina C-,gel, 6. DEAE-Cellulose (Mamushi).

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TOMOJI SUZUKI

391

5 nake venoms wert* appüecl to Ci\I-colliilo?e. alumina C-y-gol and calciiini pliosjjliate

gel. Therefore. any of these ion exchangers could lie used to olitain ])hos])ho-

diesteiase free froni 5’-nucleotidase. On the other hand. “Mamushi” venom was

chromatographed on DEAE-cellulose. Mlien the venom was chromatogra|)hed on

DEAE-cellulose using gradient elution, the ])hos])hodiesterase was sejjarated into

three entities. These partially pnrified enzymes were purified fnrther hy chroma-

togra])hy on CM-cellulose. or Sephadex-gel. Thus the three kinds of phosi)ho-

diesterases were isolated free from 5 -nncleotidases (7, 8, 9). Tahle I shows the

pnrified enzymes which were olitained dnring our stiidies in the last twelve years.

TABLE I

SNAKE VENOM ENZYMES

Phospholipase A

Hyaluronidase

5’-Nucleotidase

Endonuclease

ATPase

Enzymes fountl in all venoms

L-Amlno acid oxldase

Phosphodiesterase

Phosphomonoesterase

Glycerophosphatase

Enzymes found especiaíly in CROTALIDAE venoms

Proteinase

Enzymes connected with physiologica! activities of CROTALIDAE venoms

Arginlne ester hydrolase

Enzymes found especiaíly in ELAPIDAE venoms

Acetylchollnestera.se Peptidase NADase

\\ hen the venom of Agkisírodon halys blomlwHii was applied to a DE.AE-

cellnlose coltimn, jihospliolijiase .A was separated into two entities. Similarly other

snake venoms contain two kinds of jiliospliolipase A. Eor example, phosjiholipase

A in Cohra {Naja naja atra) venom was also sejiarated into two entities liy

chromatography on CM-cellnlose (101. Moreover, the proteinase and arginine

ester hydrolase of ‘Alamuslii*’ venom were hoth separated chromatographieally

into three entities 111.12 ).

In the cotirse of these sttidies. it was foimd that the nucleotide jiyrojihos-

phatases. which had heen reported to hydrolyze the ])yro])hos])hate linkage of

AAD. EAD and coenzvme A. were identical with phos[)hodiesterases (9). and so.

nucleotide |)vrophos|)halase is not shown in the Tahle 1. The two activities in

the “jVIamiishi” venom were always eluted in the same fraction in chromatographic

procednres. So. it is thought that ATPase in this venom corresponds lo jdiospho-

diesterase. N.ADase which decomposes NAD to form nicotinamide and adenosine

diphosphate rihose was |)resent in the venom of Bungaras rmilticinctas and Tri-

meresiirus graniineus. The NADase in the venom of Trinieresurus gramineiis was

purified hy columu chromatographies ou CM-cellulose and DEAE-cellulose 113).

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392

SEPARATION METUODS OF ANIMAL VENOMS CONSTITUENTS

In this paper, 1 will rejiorl lhe procedures íor piirificalion of proteiiiases

and arginine ester hydrolases, and also some hioehemical pro])erties of these

])urified enzymes. The most eharaeteristic j)hysiological sym])tonis prodnced hy

lhe injeclion of lhe vcnoms of A. halys hlomhofjii are connecled vvilh proleinases

and arginine eslerases.

The dislriljiilion of ])roleinases and arginine eslerases in Formosan and

Japanese snake venoms can he seen in Tahie II. It was found lhat proleinase

aelivity is present oídy in venoms of lhe CROTALIDAE and lhal lhe venoms of

ELAPIDAE do not exhihil ap])recial)le proleinase aclivily. And, il was also found

lhal lhe hydrolytic aclivilies for henzoylarginine-elhylesler (HAEE) are ])resent

wilh lhe proteitiase aclivily. When lhe venom was Irealed wilh EDTA, lhe pro¬

leinase aclivily was comj)lelely losl while lhe esterolylic aclivily remained un-

changed. Therefore lhe enzyme responsil)le for eslerolylic aclivily musl he

dislinguished from lhe jjroleinase aclivily. And conversely, on Irealmenl wilh

diisopropyl flnorophosjihale (DFP), esterolylic aclivily was completely losl while

proleinase aclivily remained unchanged. The venom of A. halys hlomhojjii,

containing considerahie amounts of proleinases and arginine eslerases iTahIe III,

was used in furlher sludies.

TABLE II — PROTEINASE AND ARGININE ESTERASE ACTIVITIES IN SNAKE VENOMS

VENOMS

Ca.sein

(PU) cas. FR,

m- tyr.

AzocolI

BAEE

/X mole/min

CROTALIDAE

A. halyH hlomhoffii

20.2

0.96

13.3

A. acutns

27.0

3.38

8.8

T. flavoviriílis

19.2

1.14

4.3

T. imicrosqnarnatus

26.9

1.4

128

T. okÍ7i(ive7isis

13.2

0.98

11.8

T. oramlneuH

9.0

0.14

ELAPIDAE

N. najd atra

0.12

0,02

N, naja naja

0,02

N. hannah

0.05

li. inulticinctus

0.8

All the activities were converted to the value for 1 mg. venom.

A lypical elulion jiallerti of proleinases of ihis venom from a DEAE-celluIose

coliimn is shown in lhe Figure 3. Most of the caseinolytic aclivily was distrihuted

into ihree fraclions and designated as jiroleinase a, h and c. The recovery of

tolal protein from the coinmn was 98 jjer cent and approximately 85 per cent

of lhe total caseinolytic aclivily was recovered in the ehiale. The contents of

proleinases a, h and c in the crude venom were estimaled lo he ahout 0.5, 8.0

and 7.5 per cent, respectively.

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Mem. Inst. Butantan

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TOMOJI SUZUKI

393

Fig. 3 — Separatlon of three Proteinases írom the Venom of A. haliis blornhoffii

by Chromatography on DEAE-cellulose Column.

Tube Number (10 ml/tube)

Column size : 2.5X70 cm

Buffer : 0.005 M ~ 0.1 M ~ 0.2 M ~ 0.5 M CH^COONa (pH 7.0)

Sample applled : 3 g of lyophillzed venom

Among the three fractions with proteinase activity. only the proteinase h

fraction caiised hemorrhage wlien injected intracutaneousiy iii alhino raldiits (14).

As the hemorrhagic activity of proteinase h could he estimated quantitatively l)y

the niethod of Kondo and co-workers (151. \ve attempted to piirify proteinase li

to verify the correlation of the hemorrhagic and the ])roteinase activities. After

gel-filtration on a Sephadex G-25 column for desalting, the proteinase h fraction

was rechromatographed on DEAE-cellulose iising gradient elution, and this chro¬

matography gave a single imiform peak of hemorrhagic and proteinase activity

(Eig. 4). However, as shown in Figure 4. the curve of ahsorhancy at 280 mp

did not coincide with the curve of hemorrhagic or proteinase activity. After

lyo])hilization, the sample was ajiplied to a hydroxylapatite column and the hemor¬

rhagic activity was also eluted together with the caseinolytic activity (Fig. 5l. To

purify the resulting proteinase h pre])aration. chromatograjihy on DEAE-Sephadex

A-25 column was used (Fig. 6). I5y this procedure, some im])urities were removed.

The ])otencies of proteinase h at each purification step are summarized in the

Talde III. The hemorrhagic and caseinolytic activities of |)roteinase h were not

separated hy these purification procedures. The increase in potency of the he¬

morrhagic activity in each ste]) was essentially in parallel with that of the

caseinolytic activity of jiroteinase h. The purified proteinase h thus ohtained

was ])hysicochemically homogeneous. Tahle III shows that the s])eeific activity

of the purified preparation is 2.5-fold that of the proteinase h preparation ohtained

from the first DEAE-cellulose column. The recovery of the proteinase h activity

from the crude venom was only 30 to 35 per cent. Therefore. with reference

to the chromatographic patterns in Tahle III, we attempted to simplify the purifica-

lion method to increase the yield. The proteinase h fraction eluted from the first

DEAE-cellulose column was applied to a Se])hadex G-lOO column (Fig. 7). By

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10 11 12 13 14 15

Fig. 4 — Rechromatography of Proteinase b (HR-II) on DEAE-celluIose.

Tube Number (6 ml/tube)

O m

Column size

Buffer

Sample appiied

1.5X25 cm

0.005 M~ 0.2 M CH,COONa (pH 7.)

IIR-II fraction separateci on DEAE-cellulo.se

at 280 m;ii = 127)

(total absorbancy

Flg. 5

Purlficatlon ot Proteinase b (IlR-II) on Hydroxylapatlte Column.

Tube Number (9.3 ml/tube)

Column size ; 2.5X50 cm

Buffer : 0.001 M~ 0.1 M phosphate (pll 7.5)

Sample appiied : IIR-II fraction eluted from DEAE-cellulose (total ab.sorbancy

at 280 míi = 253)

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10 11 12 13 14 15

-ABSORBANCY AT 280 m>J ABSORBANCY (at 280 fn;j)

Mem. Inst. Butantan

Simp. Internac.

33(2):3S9-410. 1966

TOMOJI SUZUKI

395

Fig. 6 — Purification of Proteinase b on DEAE-Sephadex A-23 Column.

Tube Number (3 ml/tube)

Zolumn size : 1.5X20 cm

Buffer : 0.005 M~0.4 M CH,COONa (pH 7.0)

sample applied : HR-II purified on hydroxylapatite (total absorbancy at 280

m/i = 127)

ÜJ

Flg. 7 — Gel Filtration oí Proteinase b Fraction on Sephadex G-lOO.

Tube Number (5.0 ml/tube)

Ten mllliliter of 3.48% proteinase b fraction was applied to a column of Sephadex

G-lOO (3.2X88 cm), equllibrated wlth 0.05 M AcONa (pH 7.0), and eluted with same

buffer. Flow rate was 60 ml per hour at 40C.

2 3 4

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SEPARATION METHODS OF ANIMAL VENOMS CONSTITUENTS

TABLE III — IIEMORRHAGIC AND ENZYME ACTIVITIES DURING PURIFICATION

OF PROTEINASE b

FRACTION FROM

Ilemorrhagic actlvity

Proteinase activity

MHD* and Its íldu- Specific

ciai llmlts (//g) activity

(PU) .

cas. FR

)ig. tyr.

/mg protein

First DEAE-cellulose column

Second DEAE-cellulose column

Hydroxylapatlte column

DEAE-Sephadex column

0.48

(0.42-0.57)

2.10

20.2

0.42

(0.39-0.49)

2.38

33.0

0.26

(0.22-0.30)

3.84

40.6

0.19

(0.17-0.23)

5.25

70.5

•MIID: Minlmum Hemorrhage Dose when injected In the skin oí the back of albino rabbit.

this procedure, proteinase b was eliited in a symmetrical peak. When lhe pro-

leinase h fraction separated from Üie column was applied to DEAE-Sej)hadex

A-50, lhe al)sorl)ancy curve at 280 m/i completely coincided wilh the proteinase

activity (Eig. 8). Thus, a simple pnrification melhod was achieved increasing

the yield of lhe purified proteinase 1» (Tahie IV) and lhe recovery of proteinase

1) activity from the criide venom was ahout 90 per cent. Tahie V shows the

analytical data of the purified proteinase h. Calcium ion is the component of

this metal-protein.

Fig. 8 — Concave Gradient Chromatography of the purified Proteinase b (IIR-II) from

Sephadex G-lOO on DEAE-Sephadex A-50.

Tube Number (3 ml/tube)

Column slze

Mlxing chamber

Reservolr

Sample applied

1.5X29 cm

250 ml of 0.005 M

0.4 M CIIjCOONa

112.5 mg

CII.COONA (pfl 7.0)

SciELO

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ôimp. Internac.

33(2):3S9-410, 1966

TOMOJI SUZUKI

397

TABLE IV _ SUMMARY OF PURIFICATION PROCEDURES OF PROTEINASE b

FRACTION

Protein (g.)

Total units

Specific activity

Crude venom

38.4

DEAE — cellulose eiuate

6.238

212,700

34.1

Sephadex G-lOO eiuate

2.856

197,500

69.2

TABLE V — AMINO ACID COMPOSITION AND CARBOHYDRATE CONTENT OF

PROTEINASE b (HR-II)

Per

cent of

amino acid residue

Per cent of carbohydrate

residue

Gly

2.58

Tyr

3.86

Galactose

6.0

Ala

2.82

Try

1.83

Ser

2.74

Cys/2

4.54

Mannose

2.0

Thr

Pro

3.95

3.25

Met

Asp

8.00

11.79

Fucose

trace

V'al

Ileu

4.10

4.55

Glu

Amido N

8.43

2.06

Glucosamine

6.5

Leu

5.44

Arg

4.12

Sialic acid

3.0

Phe

2.67

His

2.37

Lys

3.99

Calcium

0.32

Total A.A.

residue 77.69

Total sugar residue

and calcium

17.82

The hemorrhagic and caseinolytic activities of proteinase h was inhihited by

EDTA (Tal)le VI), and the decrease in biological activity was parallel wilh the

aniount of EDTA added. The proteinase activity decreased as the calcium ions

were removed hy electrodialysis ÍEig. 9), and consequentiy a conformational

change of proteinase h was ohserved hv measurement of the difference spectriini

of the protein. The activity was not regained even wlien calcium ion was added

to this dialyzed deionized protein.

TABLE VT

EFFECT OF EDTA ON PROTEINASE b (HR-II)

EDTA (Final M)

HEMORRHAGIC

ACTIVITY

Proteolytic

activity

Relative value

(%)

MHD//ig. protein Relative value (%)

None

3.6

100

1X10-*

2.4

5 X 10-^

0.8

1X10-3

0.5

5 X 10-3

0.1

1X 10-=

130 íig of proteinase b were treated with the indicated amounts of

EDTA for 30 minutes at 37°C in a totai volume of 1.0 ml.

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398

SEPARATIOM M?:TnODS OF ANIMAR VENOMS CONSTITUENTS

100

Electrociialysis Time (hours)

Protein concn: 0.44%, Voltage: 400 V, Cunent 1.9 ~ 0.9 mA

Flovv rate of cielonized water was 25 ml/hr at 40C

\Ve aiso altempled to purify proteiiiase c. The coolent of proleiiiase c in

tlie enide venom of A. Iialys hlomhojjii was al>oiil 7.5 ])er cent. Similar ])ro-

cediires lo tliose iised iii the |)iirifi(atioii of |)roleiiiase I) were ap|)lied. The

residis can he seen in Tahie VII. The (ireparalion ohtained froiti a Sephadex

TABLE VII

PURIFICATION METIIOD OF PROTEINASE

FRACTION

Protein (g.)

Total units *

Specilic activity **

Crude venom

30.0

DEAE-cellulose eluate

6.93

163,548

23.6

Sephadex G-lOO eluate

2.346

174,308

74.3

* One unit w-a.s ciefined as the amount oI enzymc whith yields a color equivalent

to 1.0 ítg oI tyroslne per minute using casein as substrate.

•• Specilic activity is expres.sed as unlts per mg protein.

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Mem. Inst. Butantan

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33(2):3S9-410, 1966

TOMOJI SUZUKI

399

(MOO columii was eleítro|)lioreticaIly and ultraceiitrifugally honiogeneous and so

proteinase c was also purified by this simple nielhod. When 10 /ig of proteinase

c were injected subcutaneously into lhe skin of a depilated albino ral)bit. a marked

edema was observed. Injection of cqual amounts of l)romelain, papain, plasmin,

a-chymotrypsin or bacterial proteinase obtained from Bacillus suhtilis, did not

cause edema. It is not clear by vvhat mechanism proteinase c causes this edema.

Only 0.5 per cent of the crude venom of A. Iialys hlomhojjü corres])onds to

lhe proteinase a.

Phospholipase A, L-amino acid oxidase, hyaluronidase and various olher pro-

teins were found as contaminants of protease a in the eluate of lhe first DEAE-

cellulose column (Fig. 3). To remove these contaminants, this fraction was sub-

mitted to a purification procedure (Table VII1). The recovery of proteinase a

TABLE VIII

PURIFICATION PROCEDURES OF PROTEINASE a FROM THE VENOM

OF A. HALYS BLOMHOFFII

1. DEAE-Cellulose Chromatography oí Crucie Venom, Proteinase a Fraction

2. Phospho-Ceilulose Chromatography

3. Sephadex G-25 Gel Filtration

4. Seconci DEAE (pH 8.5) Chromatography

5. Thirci DEAE (pH 7.0) Chromatography

6. Sephadex G-lOO Ge) Filtration

Puriíied Proteinase a

activity was about 70 per cent of lhat in the crude venom. The jihysiological

activity of jiroteinase a is not yet known. The electrophoretic and ultracentrifugal

])atterns showed that each purified ])roteinase appears as a single protein (Fig.

10 and 111. In addition to these criteria of |)urily, the homogeneity of each

])roteinase was also given by lhe overlapping curves of absorbancy at 280 m/x

and proteinase activity (Fig. 12). Moreover. these purified proteinases were

honiogeneous on polyaerylamide gel electrojilioresis.

The optimum jill values of each [noteinase can he seen in Figure 13.

The sidistrate specificities of purified proteinases a, b and c were examined

using synthetic glucagon and B chain of oxidized insulin from bovine origin

(Fig. 14). These proteinases are considered as digestive enzymes of the snake,

however, they differ from the usual mammalian digestive enzymes. trypsin and

chymotrypsin in their hydrolylic actions. Allhough the venom jiroteinases have

S])ecificities similar to ihose of bacterial proteinases, their hydrolytic sites are

restricted within narrow limits. Proteinase b is the hradvkinin destroving en-

zyme in the venom. and it readily hydrolyzes the glycylphenylalanyl linkage of

bradykinin and of the B chain of insulin. Although proteinase c hydrolyzes

the glycylphenylalanyl linkage of the B chain easily, it did not hvdrolvze the

glycylphenylalanyl linkage of bradykinin.

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10 11 12 13 14 15

400

SEPARATION METHODS OF ANIMAL VENOMS CONSTITUENTS

JU^

d b c

Fig. 10 — Electrophoretic Patterns of Purified Proteinase a, b and c.

Electrophoresis was carricd out at condltions described belovv; proteinase a: 1.0%, pH 8.GO,

= 0.1, G.14 mA, 3 . 30 C; proteinase b: 0.92%, pH 8.51, = 0.1, 6.10 mA, 4.C0C; proteinase

c: 0.80%, pH 6.00, fi ~ 0.1, 6.00 mA, 6 .O 0 C. The pictures were taken after 150, 120 and

125 minutes on proteinase a, b and c, respectively.

abc

Flg. 11 — Ultracentrifugal patterns of purified proteinase a, b, and c.

A soiution of sampie in sodium phosphate buffer (pH 7.0, /i = 0.1) was run at

conditions described beiow; proteinase a: 0.751, 55,430 rpm, 25.7°C; proteinase b;

0.65%, 56,100 rpm, 16.6°C; proteinase c: 0.77%, 56,100 rpm, 16.6“C. The pictures

were taken after 48, 64 and 64 minutes on proteinase a, b and c, respectively.

Fig. 12 — Linear Gradient Elution of Purified Proteina.se a, b and c by DEAE-

cellulo.se Chromntography.

Column size

Buffer

Sampie appiied

1.5X24 cm

a 0.001 M Tris (pH 8.5) to 0.06 M CH,COONa (pH 7.0)

b 0.01 M CHjCOONa (pH 6.2) to 0.2 M CII,COONa (pH 6.2)

c 0.05 M CII^COONa (pH 6.2) to 0.5 M CH^COONa (pH 6.2)

a - 80.4 mg. b = 99.0 mg, c = 69.7 mg

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10 11 12 13 14 15

Per cent activity

Mem. Inst. Butantan

Simp. Internac.

33(2):389-410, 1966

TOMOJI SUZUKI

401

Fig. 13 — Optimum pH of Venom protelnases.

NHj NHj SOjH

Phe-Val -Àsp-Glu -His -Leu-Cys-Gly-Ser-His - Leu-Val-Glu - Ala - Leu

Mamushi

Ha bu

SO,H

Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-LyS'Ala

Ha bu

Fig. 14 — Amlno Acid Sequence of Oxidized Bovine Insulin B-chaln and the Sites

of Hydrolysis by Snake Venom Proteinases.

2 3 4 5 6 SciELO ;lo 11 12 13 14 15

402

SEPARATION METIIODS OF ANIMAL VENOMS CONSTITUENTS

Fig. 13 — Chromatography of the Venom of A. lialys blovüioffii on DEAE-cellulose.

Tube Number (20 ml/tube)

Column size: 5.0X33 cm. Burter, CH^COONa pH 7.0. Venom applled, 6 g.

Wo also aUt'm|)te(l lo piirify llie “cloUiog eiizyiiie” and the "‘('ai)illary-|)er-

meahility-inereasing eiizyme” wliicli vvere present in eonsiderahle aniounts in lhe

eluate of the DEAK-cellulose eohmiii (Fig. 15). The hlood clottiiig eiizyme frac-

tion free from lhe hexnorrhagie proteiiis and ollier sniall iminirities was ohlained

after reehromalograjxhy on a DFAF-celIuIose eolnnin (Talile IX) followed l)y

fractionation on a hydroxyla|)alile eoluinn (Fig. 16). Afler eoneenlralion and

desailitig hy Se|)hadex (1-25 fillialion, lhe hlood elotling enzyine fraetion was

ap|)Iied to a eohiinn of DFAF-Se|)iiadex A-25, and elnled hy eoneave gradienl

elntion (Fig. 17). Ily ihis |)rocednre llie elotling enzyine was purified lo a

TABLE IX — SUMMARY OF PUlUFICATION PROCEDURES USED FOR THE

“CLOTTING ENZYME”

FRACTION FROM

Total protein A.

nt 280 mu

Enzyme unils *

Speciflc activlty

First DEAE-cellulose column

1,242

1,849

1.5

Seconti DEAE-cellulose column

106

1,541

14.5

Hydroxylapatite column

1,392

33.2

DEAE-Sephadex column

22.3

788

33.3

• Tosylarginlne mcthylestcr

was used as substrato

SciELO

10 11 12 13 14 15

ABSORBANCY (at 280

Tube Number (5.5 ml/tube)

Column size : 1.5X20 cm

Buffer : 0.005 M 0.C5 M ~ 0.1 M phosphate (pH 6.8)

Sample appiled : Clotting enzyme fraction (total absorbancy at 280 mu = loS)

Column slze

Sample appiled

Bufíer

Tube Number (5 ml/tube)

1.8X30 cm

0.005 M ^ 0.4 M CHjCOONa (pH 7.0)

Purified clotting enzyme (total absorbancy at 280 m/i = 42)

SciELO

10 11 12 13 14 15

404

SEPARATION METIIODS OF ANIMAL VENOMS CONSTITUENTS

physicofiiemirally lioniogeneous slate and Kig. 18 shows thf ullracenlrifiigal

pattern.

As menlioned hcfora, among the arginine ester liydrolases in lhe eluale froni

lhe firsl DEAE-cellidose coluinn (Eig. 8), an enzynie vvas foiitid which lias a

hypotensive aetion and increases tapillary permeahility. The arginine esler liydro-

lytic activity of ihis enzyme amounted 80 lo 40 per eenl of llie tolal arginine

esler hydrolytic aclivity of lhe venom. This physiological aclive arginine hydro-

lase was jiurified liy a similar inelhod lo thal used for lhe clotling enzyme. The

results are summarizeil in Tahle X. The sjiecific activity of this enzyme increased

TABLE X

SUMMARY OF PURIFICATION PROCEDERES USED FOR THE “CAPILLARY

PERMEABILITY INCREASING ENZYME”

FRACTION FROM

Total protein A.

at 280 m/i

Enzyme unlt.s •

Specific activity

First DEAE-cellulose column

1,030

2,440

2.37

Second DEAE-cellulose column

314

1,916

6.1

Hydroxylapatlte column

65.8

1,363

20.7

DEAE-Sephadex column

29.0

837

28.9

• Tosylarginlne methylester was used as substrate.

markedly and lhe jmrified jireparalion was homogencoiis on iillracenlrifugation

(Eig. 19), and aiso on cyanogum eleclro|)horesis at varions jiH valnes. When

3 /ag of ihis purified preparalion wcre injecled inlo lhe skin of an alliino ralihit,

capillary permeahilily was distinctly increased, as jiulged hy lhe Evans Illue lesl.

When this preparation was incuhated with [lurified liradykininogen, no release

of hradykinin was detected liy assay on gninea-jiig ilenm, and so it is not clear

hy whal mechanism the permeahility of the ca|)illaries is increased. In olher

experiments, we foimd lhat a consideralde amoiml of this ca[)illary permeahilily

incrcasing enzyme was also jiresenl in Crolalua udumunteus venom and Trimere-

siirus jlavoviridis venom.

The fractions in lhe eluate from the first DEAli-celhilose cohimn (Eig 8)

which contained “hradykinin releasing”, and “clotling” activities were collected,

and rcchromatographed. Then the fraclion of the ehiate wilh “hradykinin re¬

leasing activity” was jmrified further on a CM-celhilose cohimn (Tdg. 20). In

this way the “hradykinin releasing enzyme” was ohtained free from clotting en¬

zyme, hiit oniy 5 jier cent of the lolal nnits of arginine esler hydrolytic activity

of lhe venom were recovered in this jiartially jmrified enzyme jirejiaralion, and

no furlher jmrification was altemjited. Althongh, the arginine esler hydrolytic

activity of ihis jmrified enzyme was imexjiectedly low, lhe aclivity in this jire-

paration seems to he linked lo lhe hradykinin releasing activity (16). On heat-

treatment, the hradykinin releasing aclivity decrcased jiarallcl with the arginine

esler hydrolytic activity. Also, in DEP-inhihition exjierimenls, lhe hradykinin

releasing aclivity decrcased jiarallel wilh lhe arginine esler hydrolytic activity,

with increase in lhe concenlration of DEI’.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(2):389-410, 1966

TOMOJI SUZUKI

405

Fig. 18 — Ultracentrlfugal Pattern of

the “Clotting Enzyme”.

A solution of the purified “clotting

enzyme” at a concentration of 1.0 per

eent In sodlum phosphate buffer, pH

7.5, ionic strength, 0.1 ii was run at

58,100 rpm at 19.4°C and the plcture

'vas taken after 52 minutes centriíu-

gatlon.

Fig. 19 — Uitracentrifugai Pattern

of the “Capiliary Permeabiiity In-

creasing Enzyme”.

A soiution of the purified “capiliary

permeabiiity increasing enzyme” at

a concentration of 1.0 per cent in

sodium phosphate buffer, pH 7.5,

ionic strength, 0.1 ii was run at

57,800 rpm at 21.2<>C, and the picture

was taken alter 55 minutes centri-

fugation.

Column

Buffer

Protein applied

Tube Number (5 ml/tube)

1.5X32.5 cm

CHjCOONa, pH 6.0

total absorbancy at 280 m/i = 134

SciELO

10 11 12 13 14 15

ARGININE ESTER HYDROLYTIC

ACTIVITY (unit8/ml.)

406

SEPARATION METHODS OF ANIMAL VENOMS CONSTITUENTS

The effecls of synllietic siihstrale.s caii he seeii in Tal)le XI. The inliil)ition

eauFcd hy henzoylarginiiie elhylesler depeiuled oii its conceiitialion, and lhe lirady-

kinin releasing aeliviiy was com|)leleIy inhihited when a suffieienl amounl ol

henzoylargininc elhylesler was added. Lysine elhylesler and henzoylargininaniide,

which were nol hydrolyzed hy lhe hradykinin releasing enzyine of lhe venoni,

did not itdiihil ils activily.

TABLE XI — EFFECT OF SYNTIIETIC SUBSTRATE ON THE PURIFIED

BRADYKININ RELEASING ENZYME

Synthetic substrate

Final concentration of

Per cent of bradykinin

added

substrate (M)

releasing activity

None

100 *

BAEE

2.5

10 -’

100

2.5

10 -»

2.5

10 -'

2.5

10 -*

2.5

10 -*

LysEE

2.5

10 -»

100

BAA

2.5

10 -’

100

When inhlbitor was not acUied, the amount of hradykinin was estlmated

to be 0.8 lig upon guinea-pig ileum. '25 /ig of the enzyme was used.

Trasylol, which is a polent itdiihilor of nrinary and ])ancreatic kallikreins,

caused dislinel inhihition of lhe aeliviiy of lhe hradykinin releasing enzyme of

lhe venoin. However, il inhihiled lhe arginine esler hydrolylic aeliviiy of lhe

crude venorn only slighlly. We lhoughl lhal Irasylol woiild prohahly nol inhihil

lhe aelivilies of lhe “elolling” and “eajiillary jiermeahilily inereasing” enzynies

of lhe venorn, and ihis was nexl exaniined and foinid lo he lhe ease. Thns, lhe

inhihilory aelion of Irasylol is speeifie for lhe hradykinin releasing enzyme.

From lhese residis and lhe sjieeifieilies of lhe enzymes for varioiis synlhelic

suhslrales, il seems lhal lhe hradykinin releasing enzyme in lhe venorn is a

salivary kallikrein of lhe snake.

I wonld also like lo menlion two Iri-pyroghilamyl peplides which were re-

cenlly isolalcd from lhe vcnoms of CROTALIDAE (17).

\V'hen lhe venorn of A. halys hlomhojjii was applied lo a DEAK-eelhilose

eohimn and eluled wilh 0.00.5 M lo 0.1 M aeelale hnffer al pH 7.0 a ihird

main ])eak wilh ahsorplion al 280 m/a was ehiled. This fraelion eonlained jteplides

of low molecular weighl and ils ahsorhancy al 280 m/i was ahoul 10 [ler cenl

of lhal of lhe linfraetionaled venorn. The llV-sjieelriím of lhe pejrlide fraelion

was similar lo lhal of lry|)lophan. Tahie XII, shows lhe |)roeedure used lo purify

lhe pe|)lides. Afler removing |)rolein eonlaminanls hy gel-fillralion on a eohimn

of Sejihadex G-25. lhe eluled [leplide fraelion was applied lo a eohimn of DFAE-

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

3S(2):389-410, 1966

TOMOJI SUZUKI

407

TABLE XII — PURIFICATION PROCEDURES OF TRYPTOPIIAN-

CONTAINING PEPTIDES FROM THE VENOM OF A. HALYS

HLOMHOFFII

Step

Total

Dry Weight

Grude Venom

40875

32.72 g

DEAE-cellulose

Column

5040

Sephadex G-25

3090

DEAE-Sephadex

A-25

2200

DEAE-cellulose

Column

2010

DEAE-Sephade.x

A-25

1696

160.00 mg

Sephadex A-25, and al)sorl)e(l materiais were eluted hy 0.5 M pyridine acetate

huffer at pH 5.0. Fiiial piirificalion was achieved l)y rechromatograpliies of tlie

jiartially purified jieptide fraction on DEAE-cellulose and DEAE-Se|)hadex A-25.

Al lhe heginniiig of this work, as only a single spot was seen on pajier or

ihin layer chromatography or ])aper electrojihoresis of this pe[)tide fraction, whicli

stained vvith Ehrlich’s reagent, we thouglit there was oídy one jieptide. Acid

hydrolysis of the sample in conslant hoiling HCl in an evacnated sealed tnhe.

yielded as])artic and glntamic acids, ammonia and traces of tryptophan. Sanger’s

teclmiqiie failed to reveal a free N-terininal residne, and C-terminal analysis hv

hydrazinolysis gave only tryptophan. Pyroghitamic acid was identified in jrarlial

hydrolyzates prepared with N-NaOH or 0.1 N-HCl. Afler digestion with carhoxy-

jreptidase A, tryptojrhan and two fragments, ])yroghitamylasparagine and pyro-

glntaniylglntarnine, weie separated froni the reaclion mixture, and these dipejrtides

were separated froni each other hy high voltage eleclrojrhoresis (Fig. 21). They

were identified hy compari.son with synthetic saniples.

O 1

9 Z

0 3

\ __j

'Try

Fig. 21 — Compari.son with CPa.se Digest of Tiyptophan-contain-

ing Peptides and Synthetic Samples.

High voltage electrophoresia was carried out at pH 3.5 at 3000 V

per 60 cm íor 120 min

1. Synthetic Pyroglu-

Asp(NHj) -l-Try

2. CPase Digest of the

Sample

3. Synthetic Pyroglu-

GlutNHJ -l-Try

The ahove restdts snggesl thal there aie two [leptides of similar electrophorelie

mohilily in lhe |)e|)lide fraction. These two com|)onents were se])arated from the

peplide fraction hy high voltage electro|)horesis (Fig. 22). Using ninhvdrin and

SciELO

10 11 12 13 14 15

408

SEPARATiON METHODS OF ANIMAL VENOMS CONSTITUENTS

1. Synthetic

PyrgIu-AspíNHJ — Try

2. Natural Tryptophan-

containing peptides

3. Synthetic

Pyrglu-GluíNH^) -Try

Fig. 22 — Comparlson vvith Synthetic and Natural Peptides.

High voltage electrophoresis vvas carrled out at pH 3.5

at 4000 V per 60 cm for 240 min.

microhiological methocls, lhe molar ratio of amino ackls iii lhe acid hydrolyzate

of One of these two jieplides vvas estalilished as L-Tryploplian :L-Giutaniic acid >=

1:2 and thal of lhe olher as L-Tryj)tophan :L-Glntamic acid :L-As])artic = 1:1:1.

Tluis, lhe seqiiences of lhe two peptides were deduced lo he Pyro-Ghi (NH2)-Try

and Fyro-As[) (NH2)-Try, respectively.

Next, the distrilnilion of these pe])tides in various snake venoms was examined

(Tahle XIII). The venoms of Crolaliis adanianteus, Bothrops jararaca and Tri-

meresiirus jlavoviridis containeti hoth peptides, vvhile the venom of Vipera russellii

contained only one of them, and in the venom of Naja naja atra neither of the

two peptides was foimd. The significance of these pejitides in the salivary gland

of poisonotis snakes is ohscure. It is templing to speeulate that these peptides

are originated from [ireciirsors of enzymes or liiologically active peptides, which

are present in especially high concentrations in CROTALIDAE and VIPERIDAE

venoms, dnring activating processes.

TABLE XIII — DISTRIBUTION OF TRYPTOPHAN-CONTAINING PEPTIDES IN VARIOUS

SNAKE VENOMS

SNAKE

PyrgIu-Glu(NHj)-Try

Pyrglu-AspINHjl-Try

A. halys hlomhoffii

(CROTALIDAE)

A. piscivorus piscivorus

(CROTALIDAE)

T. flavoviridis

(CROTALIDAE)

C. adarncijiteus

(CROTALIDAE)

“T

B. jararaca

(CROTALIDAE)

V. russeUii

(VIPERIDAE)

—

N. naja naja

(ELAPIDAE)

—

lhe Work I have reporled in ihis pajier is maiidy coimected wilh lhe jiliar-

macological and enzymatic aclivities of Jajianese snake venoms. I feel thal phar-

macological investigations on snake venoms are eomplicaled hy the facl thal these

venoms are what yoii mighl eall Solutions of toxins in saliva, and snake venom

SciELO

10 11 12 13 14 15

Mcm. Tnst. Butantan

TOMOJI SUZUKI

Simp. Internac.

33(2):389-410, 1966

foiitains niany com])oneiUs with physiological activities. Therefore, I l)elit‘ve that

for lhe elucidatioii of tlie jihysiological aclions of snake veiioms on a molecular

l)asis, it is importaiil lo piirify each coinjionent from each siiake venom.

We now liave al hand several lechniqiies for this purpose. Cliromatography

on ion-exchaiigers and gel-filtration are excellent melhods for lhe piirification of

jiliarinacologically active proteins. All these tnethods have their limitations, hiil

fortunatelly lhe limitations are not the same for the various materiais. By com-

liining appropriate cliromatographical and gel-filtration procedures and other tech-

niqiies, it may he possiMe to piirify many active components. We hope that

fiirther research will he planned in future to give much clearer results than ihose

which wcre possihle hefore this International Symposium ou Animal Venoms.

Heferences

1. SUZUKI, T., HAGIWARA, H., and TAKAGI, Y., J. Pharmacol. Soc. Jap., 74,

167, 1954.

2. SUGINO, Y., J. Amer. Chem. Soc., 7», 5Ü74. 1957.

3. OKAZAKI, R., Biocheni. Biaphys. Res. Conim., 1, 34, 1959.

4. SUZUKI, T., J. Biol. Chem., 237, 1393, 1962.

5. YOSHIKAWA, H., Bioche7n. Biophys. Res. Connn., 5, 71, 1961.

6. SUZUKI, T., and IWANAGA, S., J. Phnrmacol. Soc. Jap., 78, 354, 1958.

7. SUZUKI, T., and IWANAGA, S., J. Pharmacol. Soc. Jap., 78, 368, 1958.

8. SUZUKI, T., IWANAGA, S., and SATAKE, M., ,/. Pharmacol. Soc. Jap., 80,

857, 1960.

9. SUZUKI, T., IWANAGA, S., and SATAKE, M., J. Pharmacol. Soc. Jap., 80,

861, 1960.

10. WAKUI, K.. and KAWACHI, S., J. Phanimcol. Soc. Jap., 81, 1394, 1961.

11. SATAKE, M., MURATA, Y., and SUZUKI, T., J. Biochem., .53, 438, 1963.

12. SATO, T., IWANAGA, S., -WIZUSHIMA, Y., and SUZUKI, T., ./. Bioche^n., 57,

380, 1965.

13. SUZUKI, T., IIZUKA, K., and MURATA, Y., J. Plumiiacol. Soc. Jap., 80, 868,

1960.

14. IWANAGA, S.. OMORI, T., OSHIMA, G., and SUZUKI, T., J. Biochem., 57,

392, 1965.

15. KONDO, H., KONDO, S., IKEZAWA, H., MURATA, R., and OHSAKA, A.,

Jap. J. med. Sei. Biol., 13, 43, 1960.

16. IWANAGA, S., SATO, T., MIZUSHIMA, Y., and SUZUKI, T., J. Bioche^n., 58,

123, 1965.

17. KATO, H., IWANAGA, S., and SUZUKI, T., Experientia, 22, 49, 1966.

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Internac.

33(2):411-42-4, 1966

W. B. ELLIOTT, JOAN M. AUGUSTYN, and

CARL GANS

411

11. S()!\IE ACTIO.NS OF SNAKE VENOM ON iMlTOCHONDHIA

W. B. ELLIOTT, JOAN M. AUGUSTYN, and CARL GANS

Department of Biochemistry and Bioloçjy, State University of New York at Buffalo,

Buffalo, New York, U.S.A.

l\TKOI)UCTION

I lie fiiPt indicalion of lipolytic aclivity of Piiake veiioni came froni the work

df Eüdecke (1) wlio fhowed lhat vciioni rcacted w ith lecilliin of lilood jdasma

•o iHodiico a lipiiiolyíic agont. TIk> first dcmonslration of lhe sirong inhihition

df eleetion tians])ort caine from lhe work of Ghosh and Chalterjee (2) wlio showed

dii inhihilion willi very diliite snake venoms aeling on jrigeotehrain eells over a

11/^ hoiir jreriod prior to testing for electron transport ea|)ahilit\'.

Nygaard and Siiinner (3) showed lhat inhil)ition hy leeilhinase A nnist he

oecurring al more than one site heeause itdiil)ition inereased with snhstrates eiiler-

'iig al leveis furlher away from oxygen.

Edwards and Hall (4) showed thal ex])osiire of a suecinoxidase |)re])aralioti

•o l\aja naja venom produced an inhihition of snceinale oxidation whieh was

proportional lo lhe fally aeid released hv the venom and thal nnsalurated fatty

‘toid added as lhe sodium sall caiised a similar inhihition. However. apparently

heeause of lhe inhihition produced hy CJostridiu.m ucicliii toxin (whieh conlains

d phosj)holipase lhal splits phos|)horyl( holine from lecithÍTi) lhey eoiu luded lhal

phospholipid was required for eleciron lrans|)orl. They showed lhat added Ivso-

locilhins did noi inhihil.

Aravindakshan and Hragançal,5) showed thal inlra|)eril()neal injections of

í^iija naja venom led to the isolation of parliallv uneouj)led ral liver and hrain

niitochondria.

Peirushka, Quastel aiid Scholefield (6) showed uncoupling aclivity of hoiled

i^aja naja venom was reversihie hy |)hospholii)id. Since Marsden and Heid (7)

fiad re|)orted the app('arance of myoglohin in lhe urine of jjeople envení)mated

l'y hnhydrina sclii.stosa, we decided lo Iry lo release lhe cylochromes from milo-

1'hondria hy use of A', schislosa venom (8). Allhough a slight release oecurred,

've hecame more inierested in lhe actions on mitochondrial swelling and energy

liansformation. In a study made wilh Húngaras jasciutus venom (9), we de-

nionslraled 1| thal low leveis of the venom (5-10 /ig/mg protein) juoduced

"iiconpling of oxidalive phos])horylation after 1-2 minutes ex])osure and 2) that

lhe reduclion of ADH*/() ratios (an index of lhe efficiency of the transformalion

of the energy in reduced coenzymes inlo terminal i)hosphate hond energy of ATI’)

paralleled lhe increase in venom (1-3 /tg/mg protein) used lo Ireal rat liver

niilochondria.

SciELO

10 11 12 13 14 15

412

SOME ACTIONS OF SNAKE VENOM ON MITOCHONDRIA

Jf larger amoiiiils of venoin vvcrf used, lhe aíided ADP iidiihited respiralion

rather lhan stimulated res|)iralion. A similar inhihitory a(-tioii of ADI’ in aged

mitochoiidrial pre|)arations has hecn termed reversc acceptor control l)y Leiiitiger

and Gregg(lO). The itdiil>ition of DPNH linked suhstrates was stronger lhan

lhat for succinate oxidalion (9). However, reversal of eleclron transpori was

possilile in the presence of reversc arce|)lor control (9).

B. fasciatus venom hoiled for 20 minutes at pH 5.9 to produce a phosplio-

lipase A j)reparation (11) caiised imcoupling hiit not reverse acceptor control (9).

Since senim alhumin (1% final concentration) reversed the imcoiipling

activity (9) and .seriim alhumin had shovvn a similar reversal of the iincou])ling

activity caused hy fatty acâds present in mitochrome prej)arations (12), it was

considered prohahie thal the release of fatty acids was responsihle for the venom

action. In fact, the addition of neutral triglycerides to mitochondria during venom

treatment greatly eidianced the inhihition of mitochondrial energy transformations,

while addition of triglyceride to control mitochondria did not affect tlie energy

transformations.

Several workers have reported thal fatty acids cause either inhihition of

respiralion (13) or uncoupling of oxidative phos])horylation (12, 14).

A study of the venoms from a numher of elapids showed that Buiigarus fa.s-

ciatus, Micrurus fiãvius. Naja naja and Walterinncsia aegyptia venoms all showed

uncoupling and reverse acceptor control activities, while Uenisonia pallidiceps

venom did not sliow either activity. None of three VIPEIIINAE tested had

either activity. Of ten CHOTALINAE tested, Agkislrodon piscivorus was highly

active in holh activities sludied and A. bilincatiis and Botiirops jararaca produced

moderate uncoupling of mitochondria (15).

At lhe jtresenl time we are attempting to isolale the factor(s) in Bungariis

jasclutus venom responsihle for lhe jjroduction of lhe uncoupling and/or reverse

acceptor (control activity ou incuhalion with mitochondria.

Mktiiods

highest

])urily

foi lo ws:

.Ml reagents were ohtained cornmercially and were of the

availahle. B. jasciatus venom. collected in our lahoratory, was slored as

unpooled, freshiy collected venom was frozcn iinmediately in an alcohol-dry ice

mixture and either stored in a de.ssicator at —20"C (hereafter designated as

frozen venom) or lyophilized in a Virtis Centrifugai Bio-Dryer and stored in a

dessicator at —20'’C (hereafter designated as lyophilized venom). Allernatively,

freshiy collected venom was stored in a dessicator at room ternperature (here¬

after designated as dessicated venom). Eresh venom (hereafter designated as

fresh venom) was collected and used immediately. Commercial krail venom

(Pure Toxin, liereafter designated as commercial venom) purchased as a lyo-

j)hilized powder from Miami Ser])entarium was stored at —20"C and reconstituted

lo a 10% solulion in 0.05 M potassium phos])hate, |)H 7.4.

Horizontal starch gel electro])horcsis was carried oiit according lo the ()ro-

cedure of Smithies(16) using the. discontinuous Iris-citrate huffer system of

Poulik(17). Gels were stained with Amidoschwarz lOB (National Biological

Slain Color Index 20470). Concenlraled staining solulion conlained 0.5 grams

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Slmp. Internac.

■ 33 ( 2 ): 411 - 424 , 1966

W. B. ELLIOTT, JOAN M. AUGUSTYN, and

CARL GANS

413

Amidoschwarz in 100 ml of aii acetic acid-ethanoi-water solulion 1:4:5. Diliite

staiiiing solution consisled of one part concentrated staining solulion diluled willi

two parts acetic acid-ethanol-water solution 1 :4:5.

In fractionation experiments, niilking was carried out at 4'’C. Elution of

the components of fresh B. jasciatus vcnom after starcli gel electroplioresis was

according to the method of Master and Hao (18) wilh the following modification :

after electrophoresis the starch gel was cut lengthwise into two halves. One half

was stained with Amidoschwarz to determine the degree of migration of each

of the components. A record of each pattern was made wilh a Polariod MP-3

camera. Photographs were takcn ou Polariod Land Film Packets, Type 55 P/N

(Polariod Corp., Camhridge, Mass.) using a Tiffen Photar Filler (No. 8, Yel-

low 2).

The second half of the starch gel was then cut into strips according to the

pattern indicated in the stained half. Isolated fractions were tested for uncoup-

ling and reverse acceptor control activities with rat liver homogenates pref)ared

in a mannitol-sucrose-EDTA médium as descrihed hy Ziegler et al. (19). Oxygen

consumption was measiired with a sealed oxygen electrode (20). Polarization

of the Clark electrode and recording of the changes in oxygen tension was

provided hy a polarograph (Gilson Medicai Electronics, Middleton, Wisc., Mo-

■dei K).

Protease activity was determined hy the degree of clot lysis in a system

consisting of 0.15 ml Bovine fihrinogen, 5 mg/ml (Armour), 0.05 ml of the

venom fraction to he tested and 0.05 ml thromhin (citrate thromhin approximate-

ly 1.2 units). Protein concentration was determined using Haas’ modification (21)

of Lowry’s method (22).

Kesui.ts and discussion

In earlier work with Butigarus fasciatus venom (23), we had exj)erienced

difficulty with the stahility of lyophilized venom, reconstituted with distilled water,

when held at 2‘’C or when held in the frozen State at —ló^C. Loss of production

of reverse acceptor control activity occurred more ra])idly than loss of uncou[)ling

activity. Forrnation of a white preci()itate accompanied the loss of activity (24).

Because of the instahility of the factors heing studied in whole venom, we have

avoided pooling of venom, preferring to use the venom from a single snake in

■ a given experiment and/or storage ])rocedure. We have repeated each experimeni

and/or storage ])rocedure a numher of times using venom from different snakes

to insiire that the ty])ical results presented are not the result of individual variation.

In the initial studies of the separation of B. ju.sciatius venom hy starch gel

’electro|)horesis, either frozen or lyo|)hilized venom was used to test the procedures

for electro[)horcsis and staining. Since the patterns for the two storage forms

differed, a systematic stiidy of the effect of storage conditions on the electro-

phoretic patterns was undertaken.

When cornpared to fresh venom, frozen venom consistently showed a loss of

all or a major portion of the fourth hand from the cathode end atui the |)resence

of a lightly staining hand ahead of the first hand from the cathode end (the

latter hand is not always visihle in photographs of lhe |)atterns).

Venom dessicated over silica gel ,al rooni temperalure gave a pattern in

which the protein hands jtresent are shifted in relation to the patterns of fresh

'venom (Fig. 1).

2 3

5 6

11 12 13 14 15

414

SOME ACTIONS OF SNAKE VENOM ON MITOCHONDRIA

The nallerii of lyo])hilize(l vciiorn ajipears to he closely n'late(l lo lhat of fresh

venom exeept for lhe ahsence of tlie firsl hand migraliiig tovvards lhe ealhodc

(Fig. 1).

12 3 4

IA I A

12 3 4

B I ^ I _A.

0 time

2 hours

16 hours

Frozen

Lyophilized

Dessicated

Fresh

Flg, 1 —■ Varlation In starch gel electrophoresis pattorn oT Biinriaruíi fuschitun venom

with different methods of storage. Eleclrophoresls was carried out at 4oC, for 9

hours, 300 volts. Starch gel was stained with concentrated Amldoschwarz lOB,

prepared as described in Metiiods, and washed in 951» ethanol-.'5% acetic aci<l, 1:1,

and/or distiiled water for time periods indicated.

A |)roniiiieiil featiire of llie venom eleclroiihorelie ])atterns is lhe occurrenee

of “while S[)Ols”, i.e., areas which originally stain wilh eilher dilule or coiicen-

traled dye hiit which wasli free of dye rajiiílly in lhe jtallerns stained wilh dilule

dye and more slowly in (latterns stained wilh concentrated dye. These areas ap-

SciELO

10 11 12 13 14 15

Mem. Inst. liutantan

Simp. Internac.

33(2):-411-424, 1966

W. B. ELI.IOTT, JOAN M. AUGUSTYN, and

CARL GANS

415

])ear wliile liecniisc dye is l)oiin(l it-ss slronj^ly liian liy the starcli gel. In stareh

gel eleclrophoresis patterns of fractions iii lhe ])reparalion of l)ovine piothronil)in,

similar “while spots” were showii to he diie lo lhe ])reseiice of sidfated jroly-

.saccharides (25). However in this stiidy lhe “while spots” occiirred on the anode

side of llie |)oint of ajtjdicalion and were stainahle with Alcian Ifhie and Miiei-

earmin. In oiir study lhe “while S|)ots” are noi stainahle with either Miicicarrnin

or Alcian Bine and are on the cathode side of lhe ])oint of a[)plication.

A sample of commercial H. jasciatu.s venom (Mianii Serpentariuin) was

tolally inaclive in lhe milochondrial test system and gave an alypical eleclro-

l)horetic (jaltern in which several hands were fainl or missing (Fig. 2).

CONCENTRATED

16 IIOURS

Flg. 2 — Stareh gel eleetrophoresi.s pattern of commercial liiiníjaruK JnKCittIvx venom,

10% solution In (1.05 M pota.ssium phosphatc, pll 7.4. Frozen and lyophillzed venom

pattern.s .shown for compari.son. Eleetrophoresi.s and stainlng conditlons same as In

Fig. 1. Destaining time (in distilled water); 16 hours.

Frozen 4 — Blank

1 — Commerciai 2 — I.yophilized 3 —

SciELO

10 11 12 13 14 15

CONCENTRATED

1 — Fresh 2 — Frozen 3 — Lyophillzed 4 — Dessicated

Figs. 3a, b c — Variatlon ot starch gel electrophoresis pattern of Bungarus fasciatun venom, stored as indicated, with either concentrated or dllute

Amidoschwarz lOB (prepared as described in Methods). Electrophoresis conditions' same as In Fig. 1. Destaining times (in distllled water) as indicated.

Note appearance ot “vvhtte spots” whtch mtgrate toward the cathode and become more prominent vvlth Increased destaining time.

Mem. Inst. Butantan

Simp. Internac.

3S(2):411-424, 19GG

W. B. EBLIOTT, JOAN M. AUGUSTYN, and 410

CARL GANS

|! i Ui 111 1

'-•ncoupling

^everse Acceptor Control

^'"otease

^'"otein (mg/ml)

^‘g. 4 _

U .1 . ^

—

0.58

0.90

1.44

I.IO

1.89

0.36

0.35

0.39

/as,

Typical starch gel electrophoresis pattern obtalned in fractionation experlment wlth Bungarus

■aiaÍKs venom. Mllking was carried out at 40 C and electrophoresis and staining condltions were the same

■'loubie

■b Fig. 1 . The unstained half ot the starch gel was cut as Indicated by the single headed arrovvs;

headed arrovv marks the point of sample application. Uncoupling, reverse acceptor control, pro-

tease and proteln were determined as described in Metiiods.

SciELO

11 12 13 14 15 16 17

\D.45

Fig. 5 — Effect of an inactive fraction (Fraction 1) oI Bungarus fasciatns

venom on a rat liver homogenate system. Top curve, a control experiment,

shovvs stimulation of succinate oxidation by ADP in a rat liver homogenate,

temperature 250C, pH 7.4. The numbers above the curves indicate respiratory

activity represented as fiM O^/sec. Concentrations of reagents added are

indicated below the curves. The respirator^y vessel contained a total volume

of 2.5 ml. Bottom curve shows respiratory activity following 5 minutes-

incubation of rat liver homogenate with Fraction 1.

Mem. Inst. Butantan

Slmp. Internac.

33(2):411-424, 1966

W. B. ELLIOTT, JOAN M. AUGUSTYN, and

CARL GANS

421

reverse acceptor control.

We have founcl the use of holli coiicentrated atid diliile dye, one on eacli

lialf of llie same slarch gel pattern, useful iii dislinguishing fine delails of llie

patlerns. The follovviiig of the chauges iii inlcnsity of lhe haiids duritig wash-

ing wilh elhaiiol-acelic aeid and/or distilled water is aiso useful ( Fig. 8 a, h, c).

There is a differeuee in the a])])earance of lhe “white spots” after 18 hours of

washing iii patterns staiued wilh dilule aud coneenlrated dye and a disappearance

of the faslest moving hands iu the frozen venom ])attern with washing of the

SciELO

10 11 12 13 14 15 16

422

SOME ACTIONS OF SNAKE VENOM ON MITOCIIONDRIA

paücTii slaiiiod wilh dilute dyc. Siiice lhe pallein is reslored hy reslaining, lhe

wasliiiig oiiL reflects a differeiiee iii dye hinding of lhe ])ioleins of lhe differeiil

l)ands.

Iii view of lhe varialioii in paltenis willi slorage, in venoni fraclionalioii

experimeiils, snakes were milked iii a 4'’C eold room wliere lhe eleelro[)horesis

apparalus was preassemhied and ready for a|)|)licalion of lhe venom. Fig. 4

shows lhe lypical residis of a fractionation exjierimenl. Uiieoupling activity oc-

ciirred in all hut lhe leading fonr liands. Reverse acceptor conlrol aclivily oe-

cnrred in lhe sevenlli and eighlh hands frorn lhe leading edge of lhe eathode

])ortion of lhe |)allern. Il shonld he noled lhal lhe inlensity of staining varies

consideralily in relation lo tlie jirolein eoneenlralion as delerinined hy llie modified

Lowry jiroeedure.

Protease aclivily, as ineasiired hy degree of lysis of elotiing syslein eons-

liluenls, was limited lo lhe firsl fonr liands. In one experimenl, ihose isolaled

fraelions which originally showed good imeonpling and reverse aeceplor conlrol

aclivilies relained lhese aclivilies afler more lhan d8 hoiirs al 2‘’C in conlrasl lo

whüle venom which showed tolai loss of aclivily in 24- honrs or less.

Kleclrode Iracings are shown for fraelions having no a.clivily (Fraclion 1)

and for fraelions having imeonpling and reverse aecejilor conlrol aclivilies (Frac-

lions 6 and 7 resiieclively) on inciihalion wilh milocliondria (Figs. 5 and 6).

Hepelilion of lhe fraclionalion experimenl has occasionally shown lhe ajijiearance

of uncoiipling and reverse acce|)lor conlrol aclivilies in hands adjacenl I loward

lhe calhode) lo ihose shown in Fig. 1. Snch overlap]iing of aclivilies was al-

Irihiiled lo lhe imeven migralion pallern which makes il difficnll lo acciiralely

measiire and ciil individual hands for isolalion.

The qiieslion of whelher lhe reverse aeceplor conlrol and imcoiijiling aclivilies

are diie lo one and lhe same enzyme or lo a groiip of enzymes is heing examined

hy heal denaluralion and snhslrale specificily sludies.

RliFiailCNCKS

1. LUDECKE, K., in J. B. SUMNER, and G. F. SUMERS (Editors), Clieviistiy

and Methods of Enzyme, Ed. 3, Academic Press, Inc., New York, 1953, p. 77.

2. GHOSH, B. N., and CHATTERJEE, A. K., J. Indum Chem. Soc., 2.5, 359, 1948.

3. NYGAARD, A. P., and SUMNER, J. B., J. Biol. Chem., 200, 723, 1953.

4. EDWARDS, S. W., and BALL, E. G., J. Biol. Chem., 20!), 619, 1954.

5. ARAVINDAKSHAN, 1., and BRAGANÇA, B. M., Biochhn, Biophys. Acto, 31,

463, 1959.

6. PETRUSHKA, E., QUASTEL, J, H., and SCIIOLEFIELD, P. G., Cmnid. J.

Biochem. Physiol., 37, 989, 1959.

7. MARSDEN, A. T. H., and REID, H. A., Brit. Med. J., 1, 1290, 1961.

8. TAUB, A., and ELLIOTT, W. B., Toxicou, 2, 87, 1964.

9. ZIEGLER, F. D., VÁZQUEZ-COLÓN, L., ELLIOTT, W. B., TAUB, A., and

GANS, C., Biochemintry, 4, 555, 1965.

10. LENINGER, A. L., and GREGG, C. T., Biochim. Biophys. Actu, 78, 12, 1963.

2 3

5 6

11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(2):411-424, 1966

W. B. ELLIOTT, JOAN M. AUGUSTYN, and

CARL GANS

423

11. HAYAISHI, O., in S. P. COLOWICK, and N. O. KAPLAN (Editors), Methods

in Enzymology, Vol. 1, Academic Press, Inc., New York, 1955, p. 660, and

HUGHES, A., Biochem. J., 29, 437, 1935.

12. HÜLSMANN, W. C., ELLIOTT, W. B., and SLATER, E. C., Bioclúm. Bio-

phys. Acta, 39, 267, 1960.

13. LENINGER, A. L., J. Biol. Cheni., 157, 363, 1945.

14. PRESSMAN, B. C., and LARDY, H. A., Bioclúm. Biophys. Acta, 21, 458, 1956.

15. ZIEGLER, F. D., VÁZQUEZ-COLÓN, L., ELLIOTT, W. B., GANS, C., and

TAUB, A., Proceedings of the Symposium on Animal Toxins, Atlantic City,

April 1966.

16. SMITHIES, O., Biochem. J., 61, 629, 1955.

17. POULIK, M. D., Nature, 180, 1477, 1957.

18. MASTER, R. W. F., and RAO, S. S., Bioclúm. Biophys. Acta, 71, 416, 1963.

19. ZIEGLER, F. D., STRICKLAND, E. H., and ANTHONY, A., U.S. Army Med.

Res. Lab., Fort Knox, Ky., Rept. 51/9, 1962.

20. STRICKLAND, E. H., ZIEGLER, F. D., and ANTHONY, A., Nature, 191, 969,

1961.

21. HAAS, D. W., Personal communication.

22. LOWRY, O. H., ROSENBROUGH, N. J., FARR, A. L., and RANDALL, R. J.,

J. Biol. Chem., 193, 265, 1951.

23. VÁZQUEZ-COLÓN, L., ZIEGLER, F. D., and ELLIOTT, W. B., Biochemistry,

5, 1134, 1966.

24. ZIEGLER, F. D., and VÁZQUEZ-COLÓN, L., unpublished data.

25. MAGNUSSON, S., Arkiv. for Keini, 24, 211, 1965.

Discussion

E. A. Zeller: "1 stress the importance of clear-cut demonstration that there

are very labile components present in snake venom. Do mitochondria obtained

from both sexes show any difference in response to snake venoms?”

W. B. Elliott: “We have tried the venoms of four true vipers (VIPERINAE),

Echis carinatus, Bitis gabonica, Bitis lachesis and Eristocoplús macmalionii, none

of which caused uncoupiing of mitochondriai oxidative phosphoryiation or reverse

acceptor control. Of several CROTALINAE venoms studied, only Agkistrodon pis-

civorus showed high activity of both types.”

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10 11 12 13 14 15 16

Mem. ItTBt. Butantan

Slmp. Internac.

33(2):425-432, 1966

JOHN W. DALY and BERNARD WITKOF

425

15. THK VENOMS OF AMPHII5ÍANS

JOHN W. DALY and BERNARD WITKOP

National Institute of Arlhritis and Metabolic Diseases

National Institute of Health, Bethesda, Maryland, U.S.A.

The ciilaneous secrelion.s of ami)liil)ians conlain an amazing variely of com-

poimds of (liverse |)harmacological activilies (Tahie I and Fig. 1). These incliide

sirnple hiogenic aniines, i)e|)tides, sleroids and steioidal alkaloids. Their phar-

niacological aclivily enconipasses cardio-, myo-, and neurotoxins, cholinoinimetic

and symj)alliomirnetic agents, local vasoconslrictors and hyi)otpnsive agents and

even one of the most potent halliicinogens known, O-melhylhufotenine. Among

lhese comj)ounds are some of the most powerfid venoms known.

TABLE I

TOXIC SUBSTANCES ISOLATED FROM AMPUIBIANS •

Substances

MLD

í/£g/kg)

Source

Clas.s of

Compound

Pharmacological

activity

Batracliotoxln

Frog: Phyllohaies

bicolor

.Steroidal Alka-

Joid

Cardio- and neuro-

toxin

Tarichatoxin

(Tetrodotoxin)

Newt: Taricha

torosa

Guanidine Dcri-

vative

Neurotoxin

*Sfí?M (i7ularhw

300

Salamander: Sala-

mandra maculam

steroidal Alka-

loid

Central Convulsant

liufotoxin

400

Toad: Bufo vuJya-

ris

Steroid

Cardiotoxin

De}iy(ir()h\(f()ien}ve

ÍÍOOO

Toad: Bufo marinns

índole

Convulsant

Leptodactylhie

7500

Frog: Leptodactylus

penfodactylus

Phenolle amine

Cholinomimetie

Agent

0‘M elhylhufotenine

75000

Toad: Bufo alvarius

índole

Hallucinogen

For comparlson the MLD of stryehnine i.s .600, of d-Tubocurarine Is 1500 and of

mpsearin Is 750.

The fmiction of these com[)ounds in amphihians may involve active defenso

or |)assive protection. In certain cases, as in toads of the gemis B ii j o, the se-

cretions are actively ejected as a defense against certain enemies, siich as dogs

and other carnivores. Other am|)hihians, for exam|)le, the Fiiropean Salamandra

inacalosa, make passive iise of skin secretions as a defense against jtredators.

The high toxicily of these .secretions need noi he viewed in terms of defense only.

Some of these compounds may have [thysiological fnnctions in the skin of the

amphihian.

SciELO

10 11 12 13 14 15 16

Mom. Inst. Butantan

Simp. Internac.

3 :í(2):<125-432, 19(56

JOHN W. DALY and BERNARD WITKOR

427

i hido vasocoiislriclors, cliolitiomimelics and liallucinogcns. Tlie novid hiosynlhclic

corivorsioii of Inifoloidnc to llie Iricyclic dehydrobiijolcninc in liujo marinus is ciir-

ronlly imder study in onr lal)oralori(*s (S. Senoli, J. Daly and 15. Wilkop). AIso

prest-nl in llie glands art' varioiis syinpallioniinielic aniines siich as dopt/niiiie,

noropinephrinc, and cpinephrim' [2).

Fiogs of lhe tropical American genns, L c p I o d a c I y I ti s contain llm'e types

of l)iogcnic amincs (H): llic indídcalkylamirirs, siich as scrotonin and hujolrnidine,

llu^ plicnolic aniines, snch as /ynnuinc, caiidicinc and l(‘plodaclylin(‘, and lhe

imidazolc(dkyl(nnincs, snch as hi.slaininc and spinccamine. Candicine and lepto-

ilactyline are nicotinedike in action, while histamine is a local irrilani. S])ince-

ainine lias no pronoiinced pliarniacological activity.

Kxaiiiination of 7 species of PiiPrto Rican frogs of lhe related genns E leu-

I li fí r o d a c t y I u s did not reveal significani qnaniilies of any of lhese hiogenic

aniines. A large aniounl (0.2-1.2 mg/g skin) of an ainino acid which gave a

Paiili positive color reaction tyjiical of an imidazole was fonnd in all these frogs

(J. W. Daly and H. Heathvvole, in press). This amino acid was not histidine

and was at first thoiight to he spinacine, the ainino acid preciir.sor of the amine,

spinccamine.

Chemical studies proved, however, that the material was the dipeptidi’, car-

nosinc [li-alanylhislidine] previously known only from the miiscle of certain

vertehrates. Carnosine has no known jihysiologieal or pharmacological activity

and certainly is not present as a jirotective poisou in these frogs.

Other prpudcs o/ highcr molecular wciglil have heen fonnd in a variety of

amphihians (4, 5) : Rradykinin and physalacmin, are polcnt hyixilru.sivc agcn/s,

whih' the Iryptokiniiis have no known activity.

Complctcly different in sirncinre is Iclrodotoxiu {lítriclialoxin ), lhe cxíremely

poiriil iiruroloxin from newis of the gemis TaricliaiG). This snhstance is

re])orted only in newts and in piiffer fish of the genera S p h o c r o i d c s and

A r o t h r o n, an imnsnal case of hiochemical disjnnction.

Amphihians aiso contain lipid soinhie poisons in their skin. These inchide

lhe greal variely of bujogcuins which occnr only in loads of lhe genns H u j o

and which are cxlrcmcly aclive cardioloxiii.s and local anent/ielics. Varions xle-

roidal alkídoids are aiso known from amphihians. Samandarine, sainandarone

and related eompoimds occnr in lhe skin of salamanders of the genns Sala-

maiidrail) along vvilh hemolylic proleins. Samandarine has aiso heen re-

ported recenlly from an Ansiralian amiran of the genns R s e ii d o p h r y n e (H).

Samandarine is a cetilrally aclive convulsunl of high toxicily.

.Sleroidal alkaioids have now heen fonnd lo he present in the skins of lro])ical

American dendrohalid frogs of lhe genera, l‘h y 11 o b a t e s and I) e n d r o b a-

/C.S'. In particniar the skin of the poisou arroiv jrog oj Colombiu, known as

Kokoi’ to lhe natives of lhe Choco’ of that coimiry, contains an exiremely poisonons

alkaioid. It has heen iised for cenluries to poisou hlow gun daris for himling

sinall game hy Indians of ihis region.

The frog, which has heen |)rovisionally idenlified as Phyllobules bicolor, is

tiny and contains only mimile amoimts of lhe venom which was named balraclio-

loxiii (9, ]{)). A single frog contains only 40-<5() micrograms of halrachotoxin

which is, however, snfficient to kili 2-1,000 mice on iniravenoiis injeclion. On

oral adminisiralion, halrachotoxin is 60 to 100 times less toxic.

SciELO

10 11 12 13 14 15 16

428

THE VENOMS OF AMPIIIBIANS

Three exj)eclitions to lhe Choco’ Jungle of western Colomliia in 1961, 1964

and 1966, iinder lhe al)le leadership of explorer-zoologisl Mrs. Martè Latham,

have netted over 7,000 frogs.

Two color varieties of the Kokoi’ are present in this region of Colomhia.

The main difference is in the width and color of the dorsolateral stripes. These

are narrow and l)right yellow in lhe frog from lower elevalions ahove Pluya dr.

Oro 011 llie Rio Sun Jiiaii, wliile al higher elevations in lhe sarne watershed, a

slightly larger frog wilh yellow-orange lo red-orange slrijics is fonnd. Often

the dorsolateral stripes nierge and cover lhe enlire hack of this inoiinlain variety.

llolh varieties contain hatrachotoxin in coin[)arahle (piantities. The jro^s uppcur

to l)P quite resistunt to large doses of their own venom.

The melhod of piirification of hatracholoxin has now heen simjilified as

follows: To a methanol extract of the skins is added 1 volume of water; the alka-

loids are then extracted into chloroform. The hasic alkaloids are removed from

lhe chloroform hy extraclion iiilo 0.1 N hydrochloric acid.

The aqueous acid is adjiisted to pH 8.5 and reextracted with chloroform.

The chloroform extract is coneenlrated in vacno and the final piirification makes

use of ihindayer chromatography as descrihed jireviously (9). All steps must

lie carried out at 5"C lo jirevent large losses of aclivity. Sliidies on lhe slrucTiire

of hatrachotoxin have heen hindered hy this instahilily and hy lhe paucity of

material availahle for study, even from 7,000 frogs.

Batruchotoxin is a weak base oj plt 7.5 as measiired hy parlition coefficients.

lls iiltraviolel spectriim shows only end alisorjilion indicating a lack of conjiigated

doiihle honds. The infrared spectriim indicates hydroxyl groiqis. An intense

haiid al 1690 cm^' coiild he eilher diie lo a carhonyl group or to a vinyl elher,

hiil the usual tests for a carhonyl fimclion are negative in hatrachotoxin and

lhe optical rotary dispersion curve disjilays no Collon effccl in lhe region where

steroidal kelones or aldehydes exhihil such effecls. A slrong ahsorjilion in lhe

infrared sjieclriim at 1250 cni~^ also siiggests iiot a carhonyl group, hut a vinyl

ether.

The nuclear magnetic re.soiiance spcclriim indicates a qualernary melhyl

group, a melhyl group on a Icrtiary carhon altached lo a hctero-alom and 5

/)-

hydrogens al low field which are assigned lo a carhinolamine < ^ Sroii]),

a vinyl ether (f )

í;roup and an olefinic jiroton in jiroximity to a

hetero atoni.

The Itigli resolution niass spectruni of hatrachotoxin gave an empiricul forninla

of C24 H:(:í 1N()4 which indicates the |)resence of 9 rings or douhle honds. The

loss of CHO from lhe jiarenl ioii is evidence for a potential aldehyde group.

The n.m.r. spectrum also indicates a small amount of aldehyde and amine in

equilihrium wilh the carhinol amine fimclion. Fragmenlalion of hatrachotoxin,

with loss of C,H 7 N lo forni lhe ion C.„H„d)4® ,suggests lhal, instead of a carhino¬

lamine, a carhinol amine elher is present in hatrachotoxin. The ion C:;oH 2 o 04 ©

loses in succession 5 molecules of water lo form C 2 (iH 2 (iO®. These Iransformalions

are confirmed hy lhe prcsence of lhe corrcsponding meta-slahle peaks. The ion

C 20 II 20 © íiiay lhen lose an aldehyde group (CHO) lo form CuiHio©- This is

SciELO

LO 11 12 13 14 15 16

Mem. Inst. Bulantan

Simp. Internac.

33(2):425-432, 19(ifí

JOHN W. DALY anti BERNARD WITKOP

429

good evidonce fo

a coiiliniioiis carlioii skeleton of at laasl 19 aloms. The low

niok-cular weiglil fragnienls, CsliiiN() 2 ®, C 7 ll 8 N() 2 ®and C.,H,uN()® lead lo llic

assiirnption thal a inetliyl groiij), lho nitrogeii and Iwo of lhe oxygens are williin

7 carhoiis of each other and liial a inelliyl groiip, lhe nitrogen and one oxygen

are wilh 3 carhons of each other.

The mass spcc^lruni of halracholoxin was also nieasurod afler exchange wilh

DaO. Tvvo exchangeahie hydrogens were fonnd. One was assoeialed wilh llie

CjH.iNOa® fragnient. Tlie nilrogen aloni did not ajcpear to have an exchangc-

ahle hydrogen.

Cheniically halracholoxin gives a positive, immodiate Ehrliclis tesl which iinisl

lie due lo a polential pyrrole groiip in ils slrucUire.

On lhe hasis of other reaclions and lhe speclral data, Icatraclioloxin is a

24 carhon niodified steroid wilh a jcolential 5 niemhered pyrrole ring thal con-

lains a lerliary nilrogen as pari of a carhinol aniine-vinyl ether group and a

donhle hond. Within two carhon aloms of this ring is one of lhe two alcoliol

groiips. The remainder of lhe molecule contains lhe olher Ivvo oxygen aloms,

one in an alcohol group and one in an ether linkage. This part of lhe molecule

prcçhahiy contains a Ictrasuhstitnled donhle hond.

The complete slruclnre of halracholoxin musl now await further studies hy

n.m.r. and ma.ss spectrometry on lhe catalytic and hydride reduetion products

and varioiis other derivatives. Attempts are heing made to jcrejcare a cryslalline

derivative for analysis hy X-ray cryslallography .

Idiaririacologically, halracholoxin is the most toxic known non-protein material

íMLD I jxg/kp,) uàlJi cardioloxic, rnyotoxic and neiirotoxic aclivitie.s.

In rat diaphragm-phrenic nerve preparation, indirect stiimdalion is cjnickly

hlocked and direcl stimulation more slowly. A powerful contraclure of lhe muscle

also develops. These events are irreversihle.

In a scialic-sartorius preparation (Bufo maritius), lhe action jcotential of lhe

nerve is relatively iinaffected al concenlralions which decrease lhe aelion ])olenlial

in lhe muscle. The muscle afler com])lete hlockiug of ils action polential can

still res])ond lo direcl stimulation indicating a hlock in neuro-muscular transmis-

sion. In vivo, in cais and dogs, a dose of halracholoxin (().3-.5 p.g/kg i.v.) which

does not significanlly effecl lhe respottse of muscle lo nerve stimulation did

eaiise inlerference wilh conduction in lhe hearl, exlrasysloles and finally ven¬

tricular fihrillation and dealh. Litlle effect on hlood pressure was noted.

Other dendrohatid frogs have now heen coiamined for toxic alkaioids. These

inchide Dendrohales linctorius (2 color varieties, Playa de Oro, (iolondiia), l‘liyl-

hdiates sal)panclatas (Bogotá, (ioiomhia), l*hyll(d>ates talamancae 11’anama), Phyl-

lohales pratti (Panama), Pliyllohales lagahris (Panama), Dendrohatcs minutas

(Panama), Dendrohates aiiralus (Tahoga, Panama). Of these specics, oídy Den¬

drohales auralus, which is known as lhe poison arrow frog of Panama, the liny

Dendrohates minutas, and some of the eolor varielies of Dendrohates pumilio had

a])|)reciahle toxicity in skin cxtracts. None of these conld, however, he com])ared

lo lhe toxicity of lhe extremely poisonous Kokoi’ of Colomhia. No halracholoxin

could he delecled in any of these other frogs so lhat the Kokoi’ of Colomhia

ap|)ears at present to he quite nnique in this respect.

'riiese studies on Panamanian frogs are lhe result of a slimnlating collahora-

tion wilh (Tiarles W. Myers of lhe Gorgas Memorial Lahoralory, Panama. He

has discovered thal Dendrohates pumilio, a small red and hlack frog in large

SciELO

10 11 12 13 14 15 16

430

THE VENOMS OF AMPHIBIANS

areas of Cenlral America explodes into a great iiumher of varioiisly colored island

pojjulations iti the Bocas regioii of Baiuima. Tlie coloralioii lielweeii |)opulations

is extremely varied l)olh in lhe dorsal and veiitral aspects. Whal factors liave

caused this diversification are at presenl iinkiiowii. Since warning coloration iii

ami)hihians is often assiimed lo he associaled wilh venoinoiis secrelions, a com-

[larisori of the toxicities aj various poputalions wilh hrightness of coloration. was

carried oiil (J. W. Daly and C. W. Myers. In preparalion) .

No correlation heiween color and toxicity was foiind alllioiigli holh faclors

varied widely heiween [) 0 |)nlalions. Cerlain hrighlly colored írogs were almosl

nonloxic while one darkd)lne frog, very proleclively colored, conlained large

araonnls of venoin. The loxicily was measnred hy snhcnlaneous injeclion in

mice and also hy visnalizalion of loxic princi[)les A and R on thindayer chromalo-

])lales. The Iwo loxic princi]des were lhen isolated hy alumina colnmn chroinalo-

graj)hy and silica gel thindayer chromalography. These Iwo loxic principies were

found hy high resolution mass speclromelry lo have empirical formidae of

CioH.isNO^ and ChiHusNO^, respectively. The nltraviolel ahsorption speclra showed

only end ahsorption. The infrared sjrectra showed no carhonyl, donhle hond or

oxazolidine ring. The mass speclra provided evidence for 4 rings, a carhinol-

amine, and one or Iwo hydroxyl groups. Both compound A and B formed 0-methyl

elhers on Irealment wilh melhanolic hydrochloric acid. Compound A forms a

0,N-Diacetyl derivalive. The foregoing data siiggest that eompounds A and B

are related in strnclnre lo the salamander alkaloids. These stndies were carried

oiit on only ahout 1 mg of each comjiound and the final strnctural ehicidalion

wili require additional material.

A greal variety of olher amphihians are known to contain loxic suhslances

iti their skin secrelions, and il apiiears ihal lheir invesligation shonld he qnile

profilahle in terms of discovery of novel chemical slrnclures and com])ounds of

high pharmacological activily.

Uekekences

1. ERSPAMER, V., VITALI, T., ROSEGHIN, M., and CEI, J. M., Experientia, 21,

504, 1965.

2. MÃRKI, F., AXELROD, J., and WITKOP, B., Biochim. Biopinjs. Acta. .58, 367,

1962.

3. ERSPAMER, V., ROSEGHIN, M., and CEI, J. M., Biochem. Pharmacol. 13,

1083, 1964.

4. ERSPAMER, V., ANASTASI, A., BERTACCINI, G., and CEI, J. M., Experien-

tia, 2», 489, 1964.

5. ERSPAMER, V., Abstrncts International Syniposiuni on Hypotensive PoJypep-

tiden, October 25-29, 1965, Florence, Italy.

6. MOSHER, H. S., FUHRMAN, F. A., BUCHWALD, H. D., and FISCHER, H.

G., Science, 144, 1100, 1964.

7. SCHÕPF, C., Experientia, 17, 285, 1961.

8. HABERMEHL, G., Z. Naturforsch., 20, 1129, 1965.

9. MaRKI, F., and WITKOP, B., Experientia, 19, 329, 1963.

10. DALY, J. W., WITKOP, B., BOMMER, P., and BIEMANN, K., J. Amer. Chem.

Soc., 87, 124, 1965.

SciELO

Mem. Inst. Butantan

Simp. Internac.

33(2):425-432, 1966

JOHN W. DALY and BERNARD WITKOP

431

Discussion

F. E. Russell: “Dr. Daly, in the slide showing the effects of the toxin on a

Bulbring nerve-muscle preparation you noted that this demonstrated the neuro-

muscular blocking activity of the toxin. Did not the slide also show a reduction

in the directiy ilicited contractions which would eertainly make it difficult to

evaluate the neuromuscular blocking effect; and secondly, how can you be sure,

in this preparation, that the principal effect is not on the nerve, rather than on

the muscle?”

J. Daly: “I was not clear enough in explaining that in sciatic-nerve-Sartorius

muscle preparation in Bufo rruirinus, that the nerve action potential was unaffected

and the muscle action potential decreased. The muscle at this point stili responded

to direct stimulation thus indicating a block in neuromuscular transmission.”

SciELO

10 11 12 13 14 15 16

KUNO MKYER

Mem. Inst. Butant.an

Simp. Internac.

33(2):433-440, 1966

433

16. CAHDIOTOXIC STEROIDS EKOM TOADS

KUNO MEYER

Pharmazeutisches Institut der Universitãt, Basel, Switzerland

Knowledge of tlie poisonoiisness of llie load goes liack lo antiquity. Fhysiciaiis

of lhe classic ])('riod mentioned in tlieir writings medicines prepared froin loads,

and described their effecl on lhe liearl and respiralion. In lhe sevenleenlh and

eighleenlli cenluries dried loads vvere lemporarily admilled as “hnfones exsiecali”

in lhe official pharniaco|)oeias and were iised as diurelies againsl dro|)sy and

olher diseases, even hefore digilalis was inlroduced l)y Williering. In China and

Jajjan llie dried venomous secrelion of lhe Cliinese load, fonned inlo roíind,

smoolh, dark hrown dises and knovvn as Ch’an Sii or Senso, is slill iised lo-day

againsl various diseases.

The skin glands of elli|)lical form, lhe so-called jraraloids, eonlain lhe hnlk

of lhe venom and are localed in lhe rear of lhe ear. A iniich snialler ainonnl

of venom is also .secreled l)y lhe small verriieose skin glands lhal cover lhe whole

hack of lhe animal. In ihis secrelion tvvo groiqrs of loxic suhslanees are foimd

logelher wilh olher compoiinds.

I . lhe heart-aclive principies, represenling sleroid derivalives, nsnally kiiown

as biijogenins (hufagins), or syslemalically as hiifadienolides, and bujotoxins

(eonjugales of lhe ImfogeTiins wilh suherylarginine), which are |)rimarily res-

|)onsihIe for lhe |)harmacologicaI effecl of lhe poisonous secrelion, and

2. the basic componenls of lhe hufolenine lype as well as lhe hormones

adrenaline and noradrenaline.

Klueidalion of lhe chemieal slruelnre of lhe hnfogenins which are lhe suh-

ject of ihis i)aj)er has heen slarled hy H. Wieland and his associales iti (Jermany,

hy Jensen and Chen in America, and hy several grouj)S in Japan siich as Kolake

and Kuvvada, and Kondo and Olmo. Ilut oídy dnring lhe lasl two decades mi-

eqnivoeal j)roof of lhe sleroid nalure of lhe hnfogenins has heen ])resenled and

lhe delails of lheir slruelnre com])Ielely eincidaled.

On lhe following lahie all load species are lisled whose venomons secrelion

has heen snhjecled lo a closer chemieal invesligalion.

The load ])oisons can he ohlained from lhe following sonrces: froin lhe

dried skins of lhe animais, from sccrelions of lhe skin glaiuls which are slimnialed

hy cleclrical irrilalion of lhe living animais, or hesl, from dried paraloid secrelion.

I In ihis conneclion il shonld he menlioned lhal lhe poisonous suhslanees have

also heen detecled and isolaled in olher paris of lhe hody, e.g. in lhe hlood, and

lhe ovaries.) The eollection of lhe venomons secrelion from lhe living loads can

he effecled as follows: the animais are firmly held wilh one hand and jiressed down

on a hoard that is covered wilh a glass plate. The raised, ohiong gland ac-

SciELO

10 11 12 13 14 15 16

434

CARDIOTOXIC STEROIDS FROM TOADS

('umiilations locatecl Iiehiiid lhe ear are lield near lheir hase, l)etweeii ihuml) and

forefinger and sqneezed firmiy, so lhal lhe milky secrelion squirls onlo lhe uiider-

side of lhe glass jílate. The glass plales cdiarged wilh lhe venom secrelion are

kept in a horizontal position (secrelion on toj)) at room teinperalure wilhoiit

exj)osnre to direct snnlight until lhe secrelion is dry and can easily he scraped

off. This slow drying prohahly permits lhe enzyine contained in lhe raw secre-

tion to split lhe hufotoxins into hnfogenins and snherylarginine. The ahove

])rocednre can l)e repeated after a rest period of 30-40 minutes and yields again

a consideral)le quantity of secrelion. Afterwards lhe venom glands are complelely

empty and regeneration lakes place afler a period of 4 lo 6 weeks. An excellent

soiirce for ohtaining a series of hnfogenins is Ch’an Sn or Senso, menlioned al)ove.

This is still ohlainahle in large quanlities from lhe pharmacies, for instance in

Hong Kong and Tokyo.

SPECIES

SOURCE

Length of

animais

Approx.

amount of

venom

(drled) in mg

per animal

Bufo alvarius GIRARD

Southern Arizona and Southern Cali¬

fórnia to México

80-165

400

Bufo america7tus

HOLBROOK

Eastern part oí North America from

the Hudson Bay southwarti

54-110

Bufo arejiarum HENSEL

(are7iarius LUTZ)

Uruguay, Northern Argentina, South¬

ern Brazii

Bufo asper

Indonésia, Siam, Malayan Peninsuia

260

—

Bufo blombergi MYERS &

FUNKHOUSER

Columbia

200

1200

Bufo bufo bufo LINNAEUS

= Bufo vulparis

LAURENTI

Europe, not including the Mediter-

ranian, temperate zone of Asia

60-80

Bufo bufo ijartjarizans

= Bufo (jarrjarizans

CANTOR

= Bufo asiaticus

China

75-114

Bufo críicifer WIED 1821

Costline of Brazii and Argentina

up to 75

B7ifo for7nos7is BOULEN-

GER

Japan

125

Bufo woodhaiisi fowleri

IimKLEY

New England and New York South to

Geórgia and westward along the

Great Lakes to Michigan. Along the

gulf coast to central Texas

51-82

Bufo granulosus

SPIX subsp. fernandeze

GALLARDO

East-north-eastern part of the South

American contlnent from Panama

to the Southern part of Buenos Ai¬

res in Argentina

50-55

Bufo ictericus SPIX 1824

= Bufo marinus

BOULENGER, 1882,

part.

Brazii

up to 140

190

SciELO

Mem. Inst. Butantan

Simp. Internac.

33(2):433-440, 19eB

KUNO MEYER

435

SPECIES

SOURCE

Length of

animais

Approx.

amount of

venom

(dried) in mg

per animal

Bufo marinus (LINNAEUS

1758) = Bufo marinus

West Inclies, México, Central

and South America

up to 200

580

(L.) SCHNEIDER

Bufo mauritanicus

SCHLEGEL

Morocco, Algeria and Tunisia

122

190

Bufo melanosiictus

Southeast Asía, Indonésia

116

Bufo paracnemis LUTZ

Guiana, Eastern Brazil

130-220

240

Bufo peltocephalus

TSCHUDI

Cuba

130

120

Bufo quercícus

HOLBROOK

North Carolina to Florida vvest from

Louisiana

19-32

Bufo regularis REUSS

África, wide spread

up to 136

180

Bufo fipinulosus

WIEGMANN i=B. chi-

Chile, Peru

up to 100

—

lensifi (TSCHUDI)

Bufo vaUiceps

WIEGMANN

Louisiana, East and South Texas to

New México and Costa Rica

53-125

B ufo virid ifi v irid 1 s

LAURENTI

Europe, not including Iberian penín¬

sula, North África, Near East, east-

ward to MongoIia, Tibet and Hima-

laya area

80-140

For the isolalion of tlic liiirogoniiis from lhe dried j)aratoid sccrelion (e.g.

from Cli’aii Su) lhe íinely j)idverized (povvdered) inalerial is niixed wilh an

e(]iial volume (or more) of sand and exlraeted iu a Soxhiel a|)[)araliis or in a

jjercolalor witii cldoroform. Tlie yellowisli-hrown eoloiired extracts, containing

lhe slerois and lhe hnfogenins, are snlmiilled to an inilial pnrificalion in order

lo se[)arale lhese Iwo classes of com])onnds. 7'he hnfogenins so olilained are

ehromalogra[)hed on alumina or silica Gel. Thus, relatively easily and in a short

time, the major hnfogenins can he ohlained in a crystalline State. However,

tho.se snhstances ])resent in small or miniile cpianlities may reqnire fnrlher ])nri-

fiealion hy ehromalographic melhods, for example parlilion eliromalography, which

may he nsed direclly for se|)aration of lhe crnde venom, instead of ahsorj)tion

eliromalography on alnmina or silica Gel. Mixlures which are difficnll lo separale

may he resolved hy jirejiarative jiaper eliromalography or dispersion on long

colnmns of silica Gel.

Wilh lhe aid of Pajier Chroniatography or Thin Layer Chromalogra])hy it

is possihle in most cases to ohtain an nneqnivocal identificalion of the hnfogenins,

thus enahling the chemisl to analyse ininnle amonnts of the venom. Fnrther-

more, the.se melhods are most reliahle and easy for delermining the hornogeneity

of the crystalline materiais. The chromalograms are dried and the migrated

snhstances can he detected hy sjiraying wilh a .solnlion of ShCh, in cldoroform

(20 gr/lOO ml) and heating lo ahont 80‘’-120'’ for several minutes. The dif-

436

CARDIOTOXIC STEROIDS FROM TOADS

ferenl colours ihus appcariiig in day- or iillraviolel liglit lielj) furthcr lo charac-

lerize tlieso suljstances. Since tlie l)iifogenins have a sirong ultraviolet al)sorpüoii

at al)oiil 290-300 iim lhey can lic located direclly oii lho j)aper hy a pliolocopy

with filtred ultraviolel light, or eveti })etter wilh a nionochromator. By this way

0.005 mg of a hufogeniii can hc detected.

The different coloralions or lhe change of lhe colours which occur when lhe

Inifogeniiis are trealed wilh strong acids are very usefui in idenlificalion. ll

should he emjdiasized here lhat reliahie residis are only ohlained if pure cryslals

are iised and at lhe same time aullientic sulistances are lested as well. Witli

concentrated sulphuric acid or 84% sniphnrie acid it is liest lo use white spot

plates, Inil colour reaclions wilh a solulion of SliCls iu chloroform (20 gr/100 ml)

are jierformed on filler paper. Only minute amounls of suhstances are needed

(0.05 mg). The coloured sjiots should also he examined uuder ultraviolel light.

The fãehermann colour reacliou and its rnodifications have hecome ohsolele.

The chemistry of the rufogem.xs

In contrast lo lhe C 23 -sleroids of the digilaiis and strophanlhus tyjie, the

hufogeuins are C 24 -steroids. Their sleroid nalure was first deducted from de-

hydrogenalion experiments: chrysene was ohlained from hufotalin, and -melhyl-

cyclojienteno-phenanthrene from cinohufagin, cinohufolalin and marinolmfagin.

Direcl transformalion of a hufogenin inlo a sleroid of kuown struclure has not

heen achieved iinlil 1939.

1) The Laclone groitp

An inlegrant jiart of all hufogenins is lhe laclone group. Wieland and Wey-

land first formulalcd ihis laclone grouji as five-memhered on lhe hasis of experi¬

ments with ozone fformation of formic acid). Later a slructure with only one

douhle hond in a six-rnemhered laclone was jirojiosed. In 1936 Wieland and

associales correctly inter|)reled lhe ultraviolel spectrum [X 290-300 um

(log e = 3.75) I of the hufogenins as derived from an «-jiyrone structure. The

same s|)eclrum is found wilh coumalic acid rnelhyl ester as well as wilh the scylla

glycosides and their aglycones. The struclure of lhe laclone ring is unamhigous-

ly jiroved hy the givcii .spectral datas, hy degradation ex|)erimenls and also hy

synthesis.

2) 77m Stcroid ring skelefon

Slereochciniatry. The stereochemislry of the ring junctures at C-5, C-8, C-9,

C-10, C-13, is the same for all naturally occurring hufogenins and corresponds

lo lhe ring junctures of the hile acids series, wilh lhe only exception of the C/D

ring juncture, which is cis instead of trans.

3) The Degradutwn

Can he effecled hy different ways. By hydrogenolysis the hufogenins can

he transforrned into a hile acid. As relalively large amounls of suhstauce are

needed, this method has heen giveu iip. It has heen shown lhat the hest method

2 3

5 6

11 12 13 14 15 16

438

CARDIOTOXIC STEROIDS FROM TOADS

of degrailation is lhe permangaiiatc oxidation of Üu- peracelylalcd liufogeniiis.

I?y this melhod llic corrcsponding clianic acids caii hc ol)laitu‘d iii a relalively

high yield (iij) lo 50%).

'p-Hydroxycholanic acid

COOH

5P-Hydroxy-5p-etlanic acid

Fig. 3

4) Thr Oxy^eti fiinclions

Tlie htifogenins diffor primarily iti lhe mindicr and posilion of llie hydroxyl

groiij>s, wliicli an‘ scatlercd all ovor lhe .skelelon. Eacli hufogenin has al leasl

a secondary hydroxyl groiip al C-8 (reeenlly 8-kelol)iifogenins have heen isolaled

in miniile cpianlilies) and niosl hufogenins have as an inlegranl paii a leiiiary

liydroxyl grou|) al C-14 (similar lo lhe eardio-aclive aglyeones of llie digilalis-

slrophanlhiis group). Those hufogenins carrying an oxido ^roítp al C-14/C-15

helong lo a separale gronp. Addilional hydroxyl gronps are fonnd al C-5, C-11,

C-12, C-10 (moslly in aeelylaled forni) and al C-19. A keto group was deleeled

al C-12 and an rddehyde, group al C-10.

R„ R,, R„ R,

= Bufalin

R„ R„ R, =

R..

= IIO =

(lamabufotalin

R„ R., R, =

R.,

= OCOCH., = Bufotalln

R,, R,„ R, =

= HO =

Telocinobufagin

R„ R, = 11;

= HO =

Hellebrigenol

SciELO

LO 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(2):433-440, 1966

KUNO MEYER

439

R,, Rj, Rj = H = Reslbufogenln

Rj, Rj = H; R, = HO = Marinobufagln

R,, Rj = H; Rj = OCOCHj = Cinobufagin

R, = H: R, = OCOCH.,: R, = HO = Cinobufaginol

R, = HO; R, = OCOCH^; R, = H = Cinobufotalin

The bufotoxins

Toad venoms coiitain hesido the l)ufogeidns a fiiillier lype of cardio-active

suhslaiices, lhe so-called hiifotoxiiis. These represciil conjugales ot hufogenins

witli suherylarginitie and wcrc isolalc-d and investigaled in a numlier of lahora-

tories dnring lhe I930’s. With lhe exceplion of Wielainrs hufotoxin, all are

rather poorly cliaracterized dne lo lhe fact lhal lhese snhslances are exlremely

difficnil lo ohlain in honiogeneous iryslals. This is even lhe case when modern

chromalographic melhods are applied. No doul)l llie hiifoloxins so far descrihed

are niixlures, and il seems very prol)ahle lhal WielaiKhs Inifoloxin also was ini-

pure. Oiir assum])lion lhal lhe snherylarginine resl (in Wieland’s Biifoloxin)

is linked to lhe hytiroxy groiip at C-14, seems, as Fieser aiready suggested, very

iiidikely. New investigations made in onr lahoratory tenlatively confirm lhal lhe

linkage poinl is al C-3.

Enzyme ,NH 2

Bufotoxin->■ Bufogenin +

COOU

SUBERYEARGININE

The BlOGEiNESlS OF THE TOAI) POISONS

Very liltle is knowfn ahonl lhe hiosynlhelic pathway of lhe formation of lhe

toad j)oisons. Tfie close relalionship in lhe slrueliire of lhe hnfadienolides and

lhe hile acids indicate that these tvvo lypes of sleroids are formed froin tlie saine

SciELO

10 11 12 13 14 15 16

440

CARDIOTOXIC STEROIDS FROM TÜADS

hasic suhslancf. rscliesclic and Korte assiinied llial llic laclonn ling nf tlu’

liiifadicnolidcs is liuill iip frorn oxalaceloacclic acid. Tlu- invesligaüons so far

lindcitaken wilh lahedled siibslancc-s, vvliitli coidd serve as l)uilding slones for

lhe hiosvnlliesis of lhe hiifadieiiolides, have only shovvn, that radioaclive ear-

l)onale, or sodiuni aeetate-l-(i''*, and sodiurn acelale-2-C'‘‘ are nol incorporated.

()n Üie olher hatid, l)y feeding fi. marinus wilh C “-eonlaitdng algae or hy pa-

renteral adniinistralion of eholeslerol-d-C'** it was jiossihle lo ohtain radioaclive

marinolnifagin and rnarinohufoloxin, res|)eelively. These residis show lhal

eholesterol is a precursor in lhe synlhesis of Üie liufogenins and hiifoloxins.

This ohservalion wonld siiggesl lhal eholeslerol or a elosely relaled eonipound

is lhe major soiirce of lhe eardiotonie slerols in lhe load.

Tlli: SYMHKSIS OK lUlFADIEXOiaDES

While lhe synlhesis of a eardenolide, i.e. Digiloxigenin was |)repared oídy

reeenlly, lhe synlhesis of a Co, sleroid of lhe hufogenin lype has lieen aehieved

in 1961 l)y llerlin and eo-workers. Ihil lliis laelone possessed neilher lhe 14/3-

configiiralion nor llie hydroxy groiip al C-14, whieli means lhal no “irue” Inifa-

dienolide eould lie synlhesized.

IIkferences

1. MICHL, H., and KAISER, E., Toxicon, I, 175, 1963.

DiscnssioN

E. A. Zteller: “In order lo find out lhe hydroxyl grouj) to which lhe suberyl-

arginine is attached, enzymic studies wilh simple model substances and the enzymo

liresent in crude venom prejiaration eould be carried out. Did anybody try this

way?”

K. Meyer: "The isolation of the enzyme was not yet tried.”

SciELO,

LO 11 12 13 14 15 16

JOHN J. PISANO

Mem. Inst, Butantan

Simp. Internar.

3;i(2):441-446, 19(16

441

47. WASP KININ

JOHN ,1. PISANO

Ldborcttory of Cliniatl Biochemistry, Natiomil Heart Institute, Natioiuil Institutex

of Hedlth. Bethesâa, Mítrylwnd, U.S.A.

)

Diiritig aii ('xaminalion for liistaiiiiiic-releasÍM<r aclivily in walor oxiracts of

(Iricd vfiiom a])|)aralii.‘i(“s (.sacs atui f!;latuls) of Vespa valp,aris, Jaqiies atui Scluuli-

ler in 19,54 (1) foiitul hi.stainine. serolonin. and a polenl sniootli nnistle stininlanl

vvliieh produeed a (liaraeteristic delayed. slow conlraetion of lhe <iiiinea-|)ijí ileiini

in lhe presenee of alropine and inepyrantine and also afler lhe mnsele had heen

desensilized lo serolonin. Aclivily was slahle lo lualiiift; al neulral pll hui vvas

ra])idly destroyed in hot dilule NaOH or eoncentraled flCl. The similarily of

lhe lasl snioolh imisele stimiilant in Vespa viilgaris lo hradykinin was noted and

ils ])oly[)e|)lide naliire was slrongly indiealed hy Sehaehler and Thain (2), when

il was shovvn lhat eryslalline lry[)sin or ehymolrypsin readily deslroyed SO-TOÇf

of lhe aclivily. The faelor was nol |)reeipilaled hy 60^/c ammoninin siilfale,

Irichloroaeelic aeid or 95% phenol hui was insoluhle in 95% elhanol. 95%'

acelone and anhydrons elher. In addilion lo prodncing lhe delayed slow eon-

traelion of lhe isolaled gninea-pig ilenin, ernde venom also eonlraeled lhe isolaled

rahhil jtqunnm and ilenni. niarkediy lowered arlerial hlood pressnre of lhe rahhil

and lo a lesser degree lhal of lhe cal. Differenees helween lhe aelite |irineiple

in wasp venom and known peplides led lo ils lenlalive designation as was|) kinin

or simply kinin. Kinins are now rcgarded (5) as a gronp of peplides whi<h

have a variely of polenl |)harmaeologieal properlies in eoinnion. They are hypo-

lensive, inerease eapillary |)ernieahilily, eonlrael mosl isolaled snioolh ninsele

prejiaralions hui relax lhe ral dnodennni, jirodnee |)ain and canse Itroinho-

eonslrielion in lhe gninea-[)ig.

Wasp kinin has heen jnirified froni glacial acelic acid exlracls of venom

sacs (4 ). Addilion of 15 volumes of anhydrotis elher lo lhe exlracl qnanlilalivtdy

pretipilaled lhe kinins whidi were washed wilh 95% elhanol. dissolved in waler

and lyophilized. The dry powder was slahle al room lem|)eralnre when slored

in a desiccalor. Using pa])er chromalography (hntanol. acelie acid. waler solvenl

System), pnrified wasp kinin conld readily he distingnished from lhe known

kinins as lhe former remained at lhe origin and lhe known kinins had an Kt

of 0.55. Trypsin, which does nol inactivate hradykinin and kallidin, destroyed

70-100% of wasp kinin aclivily. A |)harmacological distinction was also notiul

in lhal venom kinin prodnced a secondary depression in lhe arterial hlood pres-

sure of lhe rahhil whith was nol consistenlly seen wilh hradykinin or kallidin.

In olher lests wasp kinin was 10-100 times more effeclivt' than histamine in

increasing eapillary jiermcahilily afler inlradermal injeclion and 10 limes more

effeetive than acetyh holine in evoking pain on a culaneons hlister hase.

SciELO

10 11 12 13 14 15 16

442

WASP KININ

(]liromalogra|)hy of wasj) kiniii on coliiinns of carljoxylic acid exchaiige resin,

amhcrlilc XE-ól (= C(’»-5()| aiso revealcd dil fcrences Í5). Of significance was

llu‘ clear sej)aration of tlu* knowii kinins from the major wasp kitiin which eluled

laler. This siil)slance accounted for ahonl 90% of the eliiled activity. Two

niiiior eompoiients (ahoiil ]()% of lhe ehited aetivily) vvhich eluted earlier lhaii

hradykiniii aod kallidiii were aIso ofiperved. Thiis d kinins are presenl in Vespa

vulgaris venom. Kecovery of aetivily from the ion-exchange cohimn was dO-50%

for lhe wasp kinins and 15-80%' for lhe known kinins. All ihree was]) kinin

j)eaks had the same relative activity on the guinea-pig ileiim and ral uleriis and

all were completely destroyed when incuhaled wilh ehymolrypsÍTi and nnieh rediieed

inactivity l>y Irypsin.

Ilornet venom {Vespa crabro) like thal of the wasp conlains large amoimts

of histamine and serolonin Init has in addition very high leveis of acetyleholine

and a single kinin wilh the charaeterislie kinin activity hiit whieh conld he

clearly distingnished from wasj) kinin hy chromalographic and enzymalic tests(6).

Hornel kinin, like hradykinin, was fonnd to he re.sislani lo inaclivation hy Irypsin.

However, relative lo hradykinin it was only al)onl one lenlh as aclive in con-

tracling lhe gninea-pig ilenm as in causing relaxation of lhe ral dnodenum or

lowering rahhil arterial hlood [)ressiire.

Uniike was]) or hornet venom, hee venom {Apis tnellijica) contains lillle or

no serolonin or kinin (2). It does, however, produce a delayed, slow conlraction

of lhe mepyramine-treated gninea-|)ig ileuni. This activity is characterized hy a

rapid desensilization and may he due lo an enzyme.

The inosl ihorough sludy of the pharmacologically aclive suhslances in hee

venom, s[)anning 16 years, has come from lhe Ihiiversity of Wurzhnrg (7).

Histamine, hyaluronidase, phos|(holi[)ase A and an active hasic fraclion were re-

ported in earlier sludies. Recent work on lhe fiirlher characterization of hasic

fraction has cniminated in lhe isolation of two poly|)e))tides, meliltin and apamin (8).

Meliltin forms rnicells in solnlion and this ])ro])erty may conlrihiite lo an

imderslanding of ils polency in lowering the snrface tension of acjueous Solutions

and ils powerfnl hemolylic action (9). The amino acid sequence of mcdiltin, a

2 3 4 .5 6 7 b !) 10

major comiionenl of hee venom is (X)-(lly-lleii-(íly-Ala-Val-Leii-Lys-Val-Leu-Thr-

Another minor meliltin has aiso heen characterized and differs from lhe major

peplide hy an unknown amino acid at the N-lermituis hefore glycine and after

2'i 21 22 2.0 24 25 20

the idenlical secpience of lhe firsl 19 residues, Ileu-Ser-Arg-Lys-Lys-Arg-dhi (NHj)-

-CÍnlNH,)., (10).

Apamin is [iresenl in miich lower leveis lhan meliltin and is jiliarmacological-

ly characterized hy a long-lasling excitation of lhe central nervous syslem of

mice. It has lhe following amino acid composition: Ala,), Argj, Asp,, l/^Cys 4 ,

Glu:), HiS], LeUa, Lysj, PrO], Thr, (8).

Uniike ihose of the hee, wasp and hornet ])ej)tides have jiharmacological

jiroperlies like the known kinins (Tahle 1). These imjiortant actions as well

as their jiotency make it of inleresl to determine lheir structures. The present

reporl conlains hoth a snnimary of earlier work (11,12,18) and lhe mosl recent

findings which permil a proposal for lhe slrnclnre of lhe major kinin in Pa-

I i s I e. s venom.

2 3

5 6

11 12 13 14 15 16

JOHN J. PISANO

Mcm. Inst. Butantan

Simp. Internac.

33(2):441-44H, 1966

443

STÜDIES ON POLISTES

MaTKIíIALS AM) MKTHODS

Ifasjjs

riirec s|)i‘cies of wasp Polistes annularis LiiiTiaeiis, P. juscatus Lepcletier,

and P. exclanutns Vicreck were delivi-red alive to lhe lalioratory. These were

niixed aiid wlien nol iininediately used they were slored al 3-5" for several days

or uj) to Iwo weeks al room lem|)eraliire wlien fed eanned or fresh frnit. Other

foods siicli as cooked, strained ineat or live silk works were rejecled. Most of

lhe was])s, nj) to 16,(K)0 were immediately frozen at —10". Their alidomens

were disseeled and the terminal 3 segments which eontained lhe venom glands,

saes, dnels, and lanceis were slored iip lo 6 nionlhs at —10".

Exiraction of venom and isolation of kinins

Several exiraction jirocednres were coin|)ared in order lo find lhe most

convenient procedure for ohtaining snitahle qiiantities of material. An electric

shock techniqne (14) was nsed to ohtain piire venom which served as a standard.

Extraction of venom ajiparatuses with glacial acetic acid as jirevioiisly descrihed (4)

and homogenization of lhe disseeled ahdomen in 6% trichloroacetic acid were

lhe other procedures employed. The essential ste[)s in the latler procedure are;

laj hornogenizalion of lhe disseeled ahdomens in 6% trichloroacetic acid, (h)

removal of trichloroacelic acid frorn the extract with ether, (c) adsorption of

the kinins on a jirejiarative cohimn of CM-Sephadex and elution with ammoniiim

formate, (d) fractionation on Sejihadex G-10. (e) fractionation on an analytical

cohimn of CM-Sephadex hy gradient elntion with ammoninm formate, (f) repeat

of stej) 5 nsing a flalter gradient.

Hioassays

Kinins were measiired on lhe eslriis rat nlerns siispended in de Jalon’s sohi-

tion (12). Whenever scrolonin may have heen presenl, 1 íig/ml lysergic acid

diethylamide was added lo the halhing fluid. Anlihislaminics were not added

hecanse lhe nterus was insensitive lo the leveis of histamine enconntered. Rio-

a.ssays nsing the gninea-])ig ileum and rat duodeiuim were performed in the

nsual manner(15). Vahies were hased on the responses to single and douhle

doses of lhe nnknown sample and slandards. Hat hlood pressiire was recorded

with a Stathain Strain gange and Sandhorn Hecorder.

Results

Essentiallv the same results, i.e., kinin activity equiactive to 1.2-1.5 /rg

hradykinin in the rat nteriis assay, were ohtained when venom was collected hy

electric shock, or extracted from ajiparatuses hy glacial acetic acid, or extracted

from ahdomens with trichloroacetic acid. Hecanse the latler procedure was more

convenient. it was nsed lo extract kinins from 6000 wasps. After the jireparative

CM-Sephadex slep. kinin activity was ohtained ecpiiactive to 6.4 mg hradykinin.

Chromalography on tlu' analytieal CM-Sephadex cohimn resnlled in the isolation

SciELO

10 11 12 13 14 15 16

444

WASP KININ

of 3 ])eaks of kiiiiti activity. The sanie resiills were olilainccl wheii glacial acelic

aeicl exlracls of venoni apparaluses were ehromalographed on CM-eelliilose (12).

The firsl Iwo ])eaks of activity. Polistes kiiiitis 1 and 2, accoiinted for less

than 10% of the total kinin activity and Polistes kinin 3 approxiniately

90%. Keeovery of kinins from the cohitnns vvas essentially (|iiantilative.

In preliminary tests einploying the estnis ral uterus, duodemim and hlood

pressure tests and the guinea-i)ig ilenrn, all three kinins shovved activities similar

to hradykinin and kallidin iTahle I); however, they eould he distinguished from

the known kinins in j)reliminary stiidies. For example when hradykinin was

iised as the standard Polistes kinin 3 was found to he ahoiit 7 times more

active on the guinea-pig ilenrn than rat dnodennm, 1 times more active in lower-

ing ral hlood pressnre than on the ilenrn, 3 times more active on the ilenrn than

on lhe nlerus and 2 limes more active on the nlerns than dnodemim. Thns

relalive to hradykinin Polistes kitiin 3 was most aclive in lowering hlood

|)ressure, the order of activity hcing hlood pressnre > ilenm > ntenis > dno¬

dennm.

The structnre of Polistes kinin 3 is cnrrently nnder invesligalion. In-

cnhation of the kiidn with chymolry|)sin deslroyed hiological activity hnt lryi>sin,

[)epsin or collagena.se did not. The action of tryjjsin was particniarly inlerest-

ing becanse it cleaved lhe pejitide into a fragment which was slighlly more aclive

on lhe nlerus and which was readily isolated on a coinmn of CM-Se])hadex.

In a typical experimenl, 1.0 mg of Polistes kinin 3 (recovered from

CM-Sephadex and conlaining some sall and moislnre) was incnhated in 0.5 ml

of 0.05 M ammonium hicarhonate pH 7.0 and 0.02 ml of 0.1% Irypsin for 3

hours al 37°. This digestion cansed an increase in hradykinin-like activity from

300 lo 400 jjLg when lested on lhe ral nlerus. When analyzed on a CM-Sephadex

colnmn no activity was recovered where Polistes kinin 3 is normally foimd.

Inslead activity equivalent to 390 pg hradykinin was fonnd as a single ])eak

mnch earlier than lhe position of Polistes kinin 3. Annno acid analysis of

the new aclive jicplide revealed lhat it had lhe same composition as hradykitiiti

hnt, in addilion, also contained glycine. Comi)ari.son hy TLC of lhe dansyl

derivative of the active lry])lic pe|)lide and synlhelic glycylhradykiiun showcd

they were indistingnishahie (13). Dansyl glycine was identified in an acid hydro-

lysale of the pe[)tide derivative showing lhal glycine was al lhe N-lerminns.

Additional evitience for the idenlity of lhe active Iryplic fragment of Polistes

kinin 3 as glycylhradykinin was the identical potencies on the rat ntenis of the

nalnral and synthetic pejilides.

Acid hydrolysales of lhe wasp kinin showed thal in addilion to lhe amino

acids in glycylhradykinin lhere are 3 residnes of lysine and 1 each of glntamie

acid, threonine, aspartic acid, lencine and arginine. Fxperiments performed in

our lahoralory hy Dr. T. Nakajima have indicated lhat lhe N-lerminus is hlocked,

as the jieptide did nol reacl with lencinaminopeptidase nor in lhe kàlman jiro-

cedure. Furlhermore only r-dansyl-lysine was ohserved in hydrolysales of the

dansylated peptide. Upon Ireatment with lry|)sin lhe following were ohserved:

free lysine, lencylarginine, glycylhradykinin and an N-terrninal hlocked telrapejitide

which afler acid hydrolysis was fonnd lo contain glntamie acid, threonine, as-

parlic acid and lysine.

Heaction of the wasp kinin with carhoxy|)ejilidase B yielded only free arginine

which was also ohserved afler Ireatment with chymolrypsin. Horse nrinary kal-

likrein, like tryjrsin, also s])lit glycylhradykinin from the wasp kinin hnt only

2 3

5 6

11 12 13 14 15 16

446

WASP KININ

oiie addilioiial |)ej)li(]e was foimd which u])on acid hydrolysis yielded 3 residiies

of lysitie and onc nach of gliilainic acid, llirconinc, asi)artic acid, leiicine and

arginine. Thesc residts arc in agrecnicnl vvilli prcscnl knowlcdge of the spccificily

of liorse nrinary kallikrcin which is known lo hc an endope])lidase which splils

oídy arginyl liondsilG). If lhe vvasp peplide is firsl dansylalcd, in which caso

all lhe f-amino groups of lysinc rcaci, lhen Irypsin and horse nrinary kallikrcin

gave lhe satne jirodiicts, i.e., glycylliradykinin and a second peplide conlaining

all lhe reinaining aniino acids, jiliis f-dansyl lysine. The data iip lo ihis |)oint

indicate lhe following slrnclnre: ((dn, Thr, As|), Lysj) Lys-Leu-Arg-ídy-hrady-

kinin. Enzymatic digeslion of P o I i s I e x kinin 3 wilh a coinhinalion of l^ro-

nase, carhoxype|)lidases A and 15, Irypsin, chyrnotry|)sin (5 and prolidase yiehUal

as])aragine, |)yroglutanne acid atui pyroglnlarnylthrconine. Asjtarlie or glntaniic

acid was nol ohserved and ihreonine was lowered hy an ainonnl cxpecled froni

the |)yroglutamylthreonine foimd. Other data ohtained wilh Iryjisin and chymo-

trypsin C indieale lhal lhe 3 lysine residnes are .se(|iienlial. Ilence lhe follow¬

ing tenlative slrnclnre for /* o I i s I e s kinin 3 is: l’yr-Thr-Asn-I,ys-Lys-Lys-Len-

-Arg-Gly-Arg-Pro-Pro-(51y-Phe-Ser-Pro-l’he-Arg.

Idenlificalion of lhe hradykinin slrnctnre in was]) venom, logelher wilh an

earlier re[)orl on its presenee in amphihian skin (17) indieale lhe widespread

ocenrrenee of lhe kinin in nalnre and .snjiport accninnlating evidence for lhe

significance of kinins in rnan. Certainly, lheir presenee in snch a highly s])ecial-

ized finid as venom which aiso contains well-known pharmacologically active

snhstances, inclnding hislamine, serolonin, acetylcholine, phospholypase A. hyaln-

ronidase, snggesls lhal kinins and jiarlienlarly lhe hradykinin moleenle are nniipiely

active snhslances of physiologieal imjiorlance.

Kekekencks

1. JAQUES, R., and SCHACHTER, M., Brit. J. Phttrmacoh, 3, 53, 1954.

2. SCHACHTER, M., and THAIN, E. M., Brit. J. Pharmacol., 9, 352, 1954.

3. SCHACHTER, M., Anu. Rev. Phimnacol., 4, 281, 1964.

4. HOLDSTOCK, D. J., MATHIAS, A. P., and SCHACHTER, M., Brit. J. Phar-

mdcol., 12, 149, 1957.

5. MATHIAS, A. P., and SCHACHTER, M., Brit. .]. Pharvuicol., 13, 326, 1958.

6. BHOOLA, K. D., CALLE, J., and SCHACHTER, M., .7. Physiol.. 1.59, 167, 1961.

7. BEARD, R. L., Ann. Rev. Entomoí... 8, 1, 1963.

8. HABERMANN, E., and REIZ, K. G., Biochem. Z.. 341, 451, 1965.

9. HABERMANN, E., and REIZ, K. G., Biochem. Z., 343, 192, 1965.

10. HABERMANN, E., and .lENTSCH, ,J., Arch. exp. Path. Piumnakol., 253, 40,

1966.

11. PRADO, J. L., TAMURA, Z., FURANO, E., PISANO, J. J., and UDENFRIEND,

S., in E. G. ERDOS, N. BACK, and F. SICUTERI (Editor.s), Hypotenifive

Peptulex, Springer Verlag, N.Y., 1966, p. 93.

12. PISANO, J. J., PRADO, J. L., FURANO, E., and UDENFRIEND, S., Bio¬

chem. Pharmacol., in press.

13. TAMURA, Z., PISANO, J. .1., and UDENFRIEND, S., Biochem, Pharmacol.,

in press.

14. 0’C0NN0R, R., ROSENBROOK, W., JR., and ERICKSON, R., Science, 139.

420, 1963.

15. PRADO, E. S., PRADO, .1. L., and BRANDI, C. M. W., Arch. int. Pharmaco-

dyn.. 137, 3.58, 1962.

16. PRADO, E. S., Personal communication.

17. ANASTASI, A., ERSPAMER, V., and BERTACCINI, G., Comp. Biochem.

Phy.siol.. 14, 43. 1965.

2 3

5 6

11 12 13 14 15 16

Mcm. Inst. Butantan

Slmp. Internac.

33(2):447-432, 19(i6

CATIIERINE ROCHAT, HERVÉ ROCHAT, FRANCOIS

MIRANDA et SERGE LISSITZKY

447

KS. PUKIFÍCATION DES NEEHOTOXINES DU SCOHPION

ANDROCTONUS AUSTRALIS

CATHERINE ROCHAT, HERVÉ ROCHAT, FRANÇOIS MIRANDA

et SERGE LISSITZKY

Luhomtoire de Biocliimie Médicale, Fuculté de Médecine, Bd. d’Alès,

MarseiUe 5e, Frunce

Des locherc lies aiilérieiiros (l-.S) oii moiilré qiu> les veiiiiis de deiix espèees

de scorjtions Nord-Afrieaiiis coiileiiaieiil chaeuii deiix iieiiiotoxiiies dont la jtiirifi-

calioii a élé réaiisée par réteiilioii réversilile stir Sephadex G-25 et ])ar ehronia-

logra])liie irécliaiige (Pioiis siir Anil)erlite IIÍC-50. Les jtrotéiiies Itasitpies ohte-

mies élaient honiogènes en idlraeentrifiigation, eit éleclrophorèse de zone sur gel

(ramidon et eii ehromatograpliie d’éqiiililire sur Amherlite IHC-50. Un poids

moléculaire de 11.000 à 18.000 avait été ohtenti |)ar idtracentrifugation. Des

Iravaux idtérieiirs oiit niontré que le traitement des toxiues à des ])H éloigiiés

de la neutralité eonduisait à leiir dissoeiatiou eu luoiionières.

late nouvellc métliode de |)urifieation a été niise au |)oiut, jtermettaut d’ol)te-

nir les toxiues moiionières avee uii reiidemeut élevé. Le uiatériel de dé|)art était

le venin lirut proveiiaiit <rauiniaux eollectés à Tozeur (Tunisie), recueilli par

stimulatioii électrique et desséché sous vide. La purifieation a eomporté esseii-

tiellenieut, une extraction par Teaii, une filtratiou sur Sejthadex G-5(). suivie de

deux elirouiatograpliies sueeessives sur Amherlite lllC-50 à pH 6,70 et sur DEAli-

Sephadex A-.50 à jtH 8.50. Une dernière cliromatograpliie d’équilil)re sur Ani-

lierlite IHG-50 à pH 6,'M) pour la toxine I et à pH 6,70 jtour la toxine II a

eouduit à rolitentioii des toxiues pures. Goiume dans la inéthode précédente, les

dirférentes séparalions eliroiiiatograjthiques out été réalisées eu utilisant des laiu-

])ons aeétate d’aiumoiiiuni, dont le sei peut être eomplètement élimiué par une

doitblc lyophilisatioti. II a été aiusi jtossiltle d’éviter des o|)ératious de dialyse-

eoneentration, source de jtertes ajtpréeiahies de toxiues. Un exposé détaillé ile

la proeédure de purifieation pourra être trouvé dans un artiele à jtaraítre pro-

eliainenient (4). Le lalileau I résuine les étajtes de la [uirifieation. 52 nig de

toxine I soil l,6*/r et 88 tng de toxine II .soit 2.5% du venin Itrul de déparl

out été oliteuus. La loxieité retrouvée dans les toxiues pures corresjtond à 65Ç(i

de eelle du venin hrut. Des résnitats parfaitement reproduetihle.s ont été olitenus

au eours (ropérations réjtétées de purifieation jtortant sur 20 g de venin.

La DL,,„ des toxiues |tures déterruinée sur la .souris de 20 g en présence

(rallniinine et par voie intraveineuse est de 19 /rg/kg (toxine 1) et de 10 /rg/kg

(toxine II). Les toxities pures .«ont done 10 et 19 fois plus neurotoxitpies que

le venin hrut. La eoniposition en aeides aminés des toxiues pures est rajiportée

dans le tahleau II. ()n notera Talisenee de inéthionine dans les deux toxiues et

eelle d’aeide glutauiiqne dans la toxine 1. L’ahsence de eystéine a été eoiistatée

])ar titrage par le |)-eldoromer('urihenzoate (9). Ge résultat a été eonfiriné par

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Slmp. Internar.

3;t(2):'M7-452, líHKi

CATIIERINK KOCHAT, IIERV£ ROCilAT, FRANCOIS

MIRANDA et SERGE LISSITZKY

449

riiydroly.se (U(4 CiN à 11(1" pfiidanl 20 ol 70 li I des toxiiies alkylées par Pacide

moiioiodoacélitpie (|iii onl foiinii 7,10 (toxiiie l| et 7,.52 résidiis (loxiiie II) de

denii-cystiiie el aiieiin résidii de S-earl>oxyinéllivlcysléiiie.

Le poids niolécidaire délerniiné par éqiiilil)re de sédimentatioii selou Sved-

mouoiodoaeétique qui oiit fourni 7,10 (toxiiie I) H 7,52 résidus (loxiiie II) de

minations oiit été réalisées dans une ullraeenlrifugeuse Spinco E à 15.220 el

15.410 rev./iiiin (loxines 1 et 11) pendant 88,5 el 116 li à 20". Les loxines

étaienl dissoiites dans Tacélate d’animoninni 0.20 ,1/ [iH 6,90 à une eoncentration

de 0,897. Ees valeurs olitenues soul três proelies du jxiids luoléeulaire mininuim

ealeulé (faprès la eoniposition en aeides aininés (6.822 el 7.249 pour 1 et II).

Dans Taeide aeétique 0,5 N, les loxines I et II pré.sentenl des niaxinuini

(ral)Sor|)tion 275 el à 276 mp res|)eetivemenl. Pour ees longueurs (Ponde les

coeffieients tPextinetion moléeulaire soul 10,71X10" (loxine 1) et 18,08X10'

(toxitie 11).

TABLEAU II

COMPOSITION EN ACIDES AMINÉS DES NEUROTOXINES

D'ANDROCTONUS AUSTRALIS

1 à 2 mg (le toxine pure ont cté hydroly.sés pendant 20 et 70 h dans IICI (íN ã IIC» .selon

MOORE et STEIN (7) et analysê.s par chromalographie sur colonne selon PIEZ et MOR¬

RIS (8) avec un Autoanalyzer Technicon. Chaque valeur repré.sente la moyenne de deux

analyses pour chacun des temps d’hydrolyse. Les valeurs de la sérine, de la thrííonine

et de la tyrosine ont été calculées par extrapolation au temps d’hydroIyse 0.

Acide aminc

Toxine I

(rapport molalrc ♦)

Toxine II

(rappoiT molaire *)

A. asiiartique

9,04

(9)

8,13

(8)

Thréonine

2,00

(2)

3,07

(3)

Scrine

5,70

(0)

2,12

(2)

A. glutamlque

0,0

(01

4,13

(4)

Prolinc

5,90

(0)

2,80

(3)

Glyclne

0,04

((>)

7,02

(7)

Alanine

1,00

(1)

3,12

(3)

Cystlne (1/2)

7,55

(«)

7,92

(8)

Valinc

4,29**

(4)

4,08

(4)

Méthlonlne

0,0

(0)

0,0

(0)

Isolcucino

2,40**

(3)

0,98

(1)

Leurlne

4,01**

(4)

1,75

(21

Tyrosine

2,S3

(3)

7,04

(7)

Phcnylalaninc

1,01

(1)

0,99

(1)

Lysine

5,S7

(0)

5,00

(5)

Ilislidine

0,99

(1)

1,90

(2)

Arginine

2,03

(2)

2,99

(3)

NIIj amklé

(0)

(9)

Tryptophane

(1)

Total

(i3

Poids moléeulaire minimum

0.822

7.249

En prenant phénylalanlne = 1,0. Entre parenthèses 1'entler le plus proehe.

Valeur obtenue après 200 h d'hy(lroly.se.

Détermination spectrophotométrlque selon BEAVEN et IIOLIDAY (6).

SciELO

10 11 12 13 14 15

450

PURIFICATION DES NEURÜTOXINES DU SCORPION ANDROCTONUS

AUSTRALIS

Lcs acides amiiiés N-tcrminaux oni clé détcrminés |)ar <litiilro|)héiiylali()Ti

sclon I’raeiikcl-(^onral c/, «/.(IO). Pour I(‘.s dcux loxiiics, une seulc laclie a élé

oli.scrvéc sur Ics chromalograinmes. Kllc corrcspondail à la (li-l)]NI’-lysÍTie dans

plii.sieurs solvanls chroniatograj)hi(|ues et, ajjrès éliition du paj)ier, ellc eu jirésen-

tail le speetre earaetérisliqiie. La reeherehe des acides aniinés C-lerminaux a

été réalisée par hydrazinolyse selou Akaliori e/ «/. (lll. Après analyse ehroma-

togra])hir(iie sur eolonue, on a trouvé la thréoniue pour la loxine I et le glyeo-

colle |)our la loxine 11. Aitcim aiilre aeid aminé n’a élé déeelé.

Les [)reuves de I homogéiiéilé des loxines soul les suivanles: 1 ) par éleelro-

phorèse en gel de ])olyacrylamide à pH d,6 cl eu préseuee (riirée <SM (12), iha-

eune des toxines migre .sous forme (rime haude uni(]ue. 11 en esl de niême en

gel (Pamidon à pH 8,6 ou les handes des toxines se déplaeent du e(*)té eathodique,

indiquant leur caractere fortemenl électropositif. 2) le fail que chacune des lo-

xines sédimente dans rultracentrifugeuse analytique en donnant nne fronlière

unique et symélrique ne ])eut être eonsidéré que eomme une présomplion d lio-

mogénéité en raison de rexistenee dans le venin hrui (Pautres jiroléines nou lo-

xiques de failde poids moléculairi'. 8) a|)rès reehromalographie (Péquililire sur

Amlierlile IKC-50 les fraelions eonsliluanl le pie loxi(]ue symélriipie olitenu ont

une loxieité sjiéeifique eonslanie el maximum. 4) Pahsenee de mélliionine dans

les deux loxines el (Pacide glulamique dans la loxine 1 eonstitue un critòrc Irès

sensilde de la pureté des fraetions ohlennes. 5) un senl acide amine

terminal a été mis en évidcnce pour ehacimc des loxines.

N-

C-

Un certain nomlire cPargiimenls milite en outre en faveur de ee que chaipie

loxine esl eonstituée par une ehaíne ])olyj)ej)lidi(]ue í/«íV/«e:

1) A])rès réduetion eom|)lète des jionls disulfures el alkylalion (acide mono-

iodoaeéliijue ou iodaeélamide), on devrail s’allendre, si les loxines élaienl eons-

tiluées par deux ou |dusienrs ehalnes |)(dypeptidi([ues, à ee qiPelles jiénèlreul dans

le Biogel Pn (qui exelut les moléeules (Pim poids égal ou sujiérieur à 4.600) et

quVlles soient jilus relardées que les toxines natives sur Sephadex (1-50. Tel

tPesl pas le eas. Les loxines réduiles et earhoxyméthylées sonl exeliu‘s dn Iliogel

P,i el rnoins relardées que les loxines nalives sur Se])hadex G-50. De plus elles

dialyseni heaueoup moins vile que les loxines natives à Iravers la rnême mernlira-

ne semi-perméalde.

2) Un seul acide aminé N-lerminal a élé Irouvé. llien ipie la jirésenee

éventuelle (Paeides aminés lerminaux i\-aeylés rPait pas élé reeliereliée, leur exis-

tence est rendue |)eu vraisemlilalde du fait qiPun seul acide aminé C-lermiual a

été tronvé dans (liaeune des deux loxines dans des |)roportions iPexeédant pas

im résidu [lar rnoléeide.

IPensemlde des résullals ohlenus permel (Passigner aux nenroloxines iVAn-

droclonus aiistrulls les formules linéaires suivanles:

Toxine I: H-Lys-(As])», Tlirj, Sern, Pro,,, dy,,, Ala,, CyS-SCy,, Vai,, llcua,

Liui.,, Tyr,,, Plie,, Lys.-„ His,, Arg^, Try,)-Tlir-OII.

Toxine II: 11-Lys-(As]),s, Tlir,,, Ser.., Ulu,, Pro;,, Uly,,, Ala,,, UyS-SCy,, Vai,,

lleu,. Leu,;, 'Pyrj, 1’lie,, Lys,, His-j, Arg,,, 'Piy,)-Uly-OII.

Ces formules, (jui iPimpliquenl pas de séquenee enlre les acides aminés in-

elus dans la parenthèse, montrent eerlaines analogies enlre les deux loxines: 1)

SciELO

0 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(2):447-452, 1960

CATIIERINE ROCHAT, IIERVÉ ROCHAT, FRANCOIS

MIRANDA et SEROE LISSITZKY

451

lt“ Momhri' total d acides aminés (63 et 61). 2) l’al)st“Tice do iiiétliiouine ol la

préseiuo de quatre poiits disulfiires. 3) la ricliesse en acides aminés aromatiqnes

(5 et 9 résidus poiir I et II). 1) le même résidii de lysine i\-terniinal.

Des caractéristiqiies voisines ont été mises en évidence dans la toxine I de

Hulhiis occilaiuis (2), compte tenn de ce qne Ton sait maitUenanl qne les ana-

lyses jirécédeminent puldiées jiortaient siir le diinère de la proléine.

La svmptoniatologie de renveiunemenl prodnit par rinjection des toxines esl

identiqiie ponr chacime d’elles et ponr le venin linit desséclié on non. Etanl

donnc rahsence jiratiqnement totale (ractivités enzymatiqiies dans le venin d’/l/;-

droc/onus uuxtralis, on peut conclure qne la symptomatologie de renvenineinent

esl directement et senlement liée à la jirésence des nenrotoxines dans le venin.

Malgré lenr différence de coni|)OSÍtion en acides aminés, les analogies de

slructnre existant entre les denx nenrotoxines étndiées el Tidentité de lenr action

])liarmacologiqne permettent (renvisager qn’un motil stnictnral nnique dans cha-

cnne des protéines ponrrait être responsahie de lenr activité nenrotoxiqne.

Hini.ioGKAfitii-;

1. MIRANDA, F., et LISSITZKY, S., Nature (Loncl.), 190, 443, 1961.

2. MIRANDA, F., ROCHAT, H., et LISSITZKY, S., Toxicon, 3, 51, 1964.

3. MIRANDA, F., ROCHAT, H., et LISSITZKY, S., Toxicon, 2, 123, 1964.

4. ROCHAT. C., ROCHAT, H., MIRANDA, F., et LISSITZKY, S„ soumis à Bio-

chemistry.

5. BEHRENS, B., et KARBER, C., Arch. cxij. Pulh. Phurmak.. 177. 379, 1935.

6. BEAVEN, G. H., et HOLIDAY, E. R. Adv. Protein Chem., 7, 319, 1952.

7. MOORE, S., et STEIN, W., Methods in Enzymology, Vol. 6, Academic Pres.s,

Inc., New York, 1963, p. 819.

8. PIEZ, K. A., et MORRIS, L., Analyt. Biocliem., 1, 187, 1960.

9. BOYER, P., ■]. Amer. Chem. Soc., 76, 4331, 1964.

10. FRAENKEL-CONRAT, H., HARRIS, J. I., et LEVY, A. L., Methods of Bio-

chetnicul Anulysis, Vol. 2, Interscience Publ., Inc., New York and London, 1955,

p. 359.

11. AKABORI, S., OHNO, K., IKENAKA, T., OKADA, Y., HANAFUSA, I., TSU-

GITA, A.. SUGAL, K., et MATSUSHIMA, T., Biül. Chem. Soc. Japnn., 29,

507, 1956.

12. DAVIS, B. J., An7i. N.Y. Acud. Sei., J31. 404, 1964.

Discussion

A. do A7nunil: “Est-ce que le pouvoir anlitoxinogénique de ces denx toxines

a déjà été essayé comimrativement?”

S. Lissitzky: “Non”.

D. Mebs: “Have you found a direct relation between the toxicity and lhe

reduction of the SS-bonds of your neurotoxin preparation?”

2 3

5 6

11 12 13 14 15

452

PUHIFICATION DES NEURÜTOXINES DU SCOKPION ANÜROCTONUÍi

AU ST RA LIS

S. Lissitzky: “The fully reduced and carboxymethylated toxins have lost their

toxicity.”

E. G. Mendes: “Les deux toxines ont-elles le pouvoir de modifier Tactivité

cholinestérasique ?”

S. Lissitzky: “Cet effet éventuel n’a pas élé essayé.”

A. Shulov: "Our group has an impression that Ihere is a difference in cons-

tituents whether the venom is lyophilised, fresh or from direct bites. We shall

be glad to help your group in the preparation of anti-serum against Androctonus

australis if you would like to try to elucidate this difference, according to the

method which led to excellent results your group achieved.”

S. Lissitzky: “I thank you for your suggestion, Dr. Shuiov, and I should be

glad to get in touch with you.”

N. Sarkar: “It is unusual that a polypeptide of such molecular weight as

the scorpion venom is so easily denatured. Do you know which are the structural

properties responsible for this behaviour of the polypeptide?”

S. Lissitzki: “I think we should wait until more Information on the structure

of the toxins is available.”

C. Y. Lee: "Is there any study on the action of your purified neurotoxins on

the neuromuscular transmission?”

S. Lissitzki: “We have not done such studies.”

J. M. Gonçalves: “Etant donné que vous avez, par hydrazinolise, identifié la

glycine comme C-terminal de la toxine II, avez-vous trouvé une résistence de la

toxine à Taction de la carboxipeptidase du pancreas?”

S. Lissitzki: “Ces essais sont en cours de réalisation.”

.0 11 12 13 14 15 16

Mcm. Insl. Butantan

Simp. Internac.

CARLOS R. DINIZ, MARCUS V. GOMEZ, ABÍLIO

ANTÔNIO, and ALEXANDRE P. CORRADO

453

33(2):453-45(i,

49. CHP:M1CAL PHOPKHTIES AM) BiOLOGlCAL ACTIVITY OF

Tirrus ví;n()m

CARLOS R. DINIZ, MARCUS V. GOMEZ. ABÍLIO ANTÓNIO,

and ALEXANDRE P. CORRADO

DeiKtrtamento de Bioquímica, Faculdade de Medicina, U.F.M.G., Belo Horizonte

and Departamento de Farmacoloqia, Faculdade de Medicina U.S.P., Ribeirão Prêto,

São Paulo, Brasil

I hi‘ tvvo inosl pievaleni spcc ies of sior])ioiis in I5razil are tlie yellow seorpioii

7. scrrulalus and lhe l)lack seorpion T. I>a/iiciisi.s. Their venoms produce on

inanimal.s similar symplonialoloiíies Inil lhe degree of loxieily is tiol lhe sanie (1,

2). Allempls lo fraelionale lhese venoms, ol)lained l)y eleelrieal slimulalion, led

Diniz and (/Onçalves (4, 1) lo demonslrale lhal lhey are mixlures of proleins,

moslly of l)asic naliire; lhe main melliod used was ])a|)er and slarefi zone eleelro-

jjhoresis. The following eoraponenls were fonnd: a) toxic h) smoolh miisele

slimidaling e) inereasing eaiiillary permeahilily atid d) hvíihironidase; a and h

were inaelivated hy ])roleolylic enzymes. From several enzymes assayed. only d

showcd measiirahie aelivily.

Kecenlly Gomez and Diniz suhmilled /’. scrrulalus venorn lo a fraelionalion

proeednre nsing a eomhination of |)reeipilalion and colmnn chromalography on

Se])luidex (T25 and CjVT-eelhdose; lhey have eonfirmed llie prolein com|)lexity of

lhe venorn and, in addilion, isolaled a higtdy |)iirified loxic eomponenl which

migraled as a single eom|)onenl on eellnlose aeelale pa|)er eleelroplioresis ( Fig. 1).

Titpus serrulaliis toxin

CelluIo.se acetate paper electrophoresl.s

Acctate buffer pll 5.5, 0.05, 7 hours

Fig. 1 — Cellulose acetate paper eiectrophore.sis ot the toxic component obtalned

after chromatography of the component in a CM cellulo.se column. Acetate buffer

pll 5.5 lí = 0.05. A single component was obtalned.

.Mkcha.msim or .vctid.x of T i t v u s vknom

As was |)oinled onl in previoiis paragraplis, lhe venomons secrelions are

complex mixlures. When llie wliole venorn or exlraels of lhe venorn ap|)aralus

aro used lo sludy llie venorn meehanism of aelion, diffieullies lo inlerprel resulls

2 3

5 6

11 12 13 14 15

454 CHEMICAL PROPERTIES AND BIOLOGICAL ACTIVITY OF T I T YU S VENOM

arisc. In lhe case of scorpion venoms, use has l)een niade in general eilher of

lelsons exlracts or of whole venom ohtained l)y electrical or manual sliinulalion.

In most cases, following injeelions into whole animais, lhe effects ohserved on

hlood pressure, liearl rate, respiration, glandular secrelions, etc., are used lo at-

lcmj)t an underslanding of lhe mechanism of action. In spite of lhe difficulties

involved several authors as Houssay (5), Magalhães (1), Carvalho (6), Ramos

and Corrado(7), Freire-Maia and Ferreira (8) have reached tlie conchision that

lhe T ityus venom contains neurotoxic sul)slanees.

The sludy of lhe effeets of T it y u s venom on isolated organs disclosed

some effects that point to lhe parasympalhetic nerves as lhe mediators of the

loxic action (3,9). T ityus venom contracts the smooth musele of the guinea-

pig ileum. This effect is iidiihited hy atropine and jiotentiated hy eserine;

ganglion l)locking agents such as hexamethonium have no effect and rnorphine

jiartially antagonizes the aclivity of the venom on this preparation. These re-

sults (Fig. 2) led lo the supposition that T ityus venom contained at least

HAT

0 3 6

atf ooif'?

Flg. 2 — Gulnea-plg ileum preparation suspended In 10 ml Tyrode solutlon. 0.4 Ag

of atropine sulphate left 2 minutes in contact wlth the preparation Inhlblts the

action of subsequent additlons of 5 tig of toxin preparation (T) or 0.08 /íU oí acetyl-

choline chloride (A); 0.04 iig of histamlne (H) is not affected. The recovery time is

about the same for the toxin and acetylcholine. (Aceording to Diniz and Valerl, 1959).

One component acting on jiarasympathetic jiostganglionic fil)ers(9). This effect

migfit he either a direct one or mediated ihrough an autojiharmacological stili-

slance jiresenl in lhe nervous endings of guinea-pig ileum plexuses. Recently,

in collahoration with Torres (10), we incuhaled fragments of guinea-])ig ileum

wilh T i ty u s venom and lesled the release of acetylcholine aceording lo Pa-

lon(ll); in the flasks containing venom, a smooth musele conlracling suhslance,

with |)harmacological and some chemical projierties similar lo acetylcholine, ac-

cumulaled. We concluded that the conlracting effect of the venom on the guinea-

pig ileum is indireet and mediated through acetylcliolinc. Since lhe venom does

not inhihit cholineslerase (4) lhe release of acetylcholine mnst he due to an effect

of lhe venom in some otlier place of lhe melaholic pathway of lliis suhslance.

Many symptoms of the scor|)ion venom intoxicalion could fie interpreted hy ad-

milting lhe release of acetylcholine in the nervous slrucTures.

These resiilts were, however, in a|)parenl discordance with lhe findings of

Ramos and Corrado(7); Freire-Maia and Ferreira (8) wlio found that in some

condilions lhe venom produced hyperlension, lachycardia, hyjjerglycemia, relaxed

lhe airojiinized rahhit duodenum and jirodnced olher symptoms siiggeslive of

sympalhelic medialion. We decided ihus lo investigale the action of lhe venom

iising the perfused guinea-jiig heari which offered lhe possihilily to analyse in

lhe same slruclure effects mediated hy acetylcholine or adrenaline, wilhoul the

inlerferencc of lhe eomjilex slructures of lhe whole animal.

SciELO

0 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(2):453-456, 1966

455

CARLOS R. DINIZ, MARCUS V. GOMEZ, ABÍLIO

ANTÔNIO, and ALEXANDRE P. CORRADO

456 CHEMICAL PROPERTIES AND BIOLOGICAL ACTIVITY OF T I T Y U S VENOM

We were ahle to sliovv thal 20 /xg of veiiom pcrfiised throiigh tlie heart

jirodiiced first liradycardia simullaneousiy willi a reduclion of slrenghl of hearl

beals. These exjieriments suggested a participation of cdiolinergic, simultaiieously

or followed liy adrenergic mechaiiisnis in lhe actiou of llie venom (Fig. 3).

Tliis hypothesis was confirmed liy use of driigs whicli interfere vvitli holli me-

chanisms. The negative chronolrojiic and inotropic effects were hloeked hv

atropine and hemicholinium anc! |)otentiated hy jirosligniine. Hexamethoiuuin

had no effecl; heta sympatholitics, as Inderal, ir.hihiled lhe ])ositive effects.

Previous treatment of lhe giiinea-pig with reser|)ine, a sid)Stance knovvn to deplete

catecholamines, prevented the positive inotropic and chronotropic effects, allovv-

ing however lhe negative effects lo sliow.

The imiiortanl poinl now is to decide if these effects are prodiiced liy dif-

ferent components or l)y the same suhstance. We cannot decide at lhe moment.

hiil the toxin ohtained in the various steps of the piirificalion procedure, jiresenled

hoth effects on the heart. Based on these resnlts, we can specniate ahoiil llie

site of action of lhe venom. A liypothesis which sàlisfies lhe resnlts ohtained

in onr experiments is lhat lhe venom is a postganglionic fiher nenrotoxin. If

we admit with l]nrn and Rand tliat even noradrenaline is liherated hy a previous

release of acetylcholine in lhe adrenergic nerves, only one toxic snhstance, that

may acl always liy releasing acetylcholine from the anlonomous nervons syslem,

can ex|)lain all lhe effects hitherto ohserved in lhe isolated organs.

We may conclnde these remarks hy pointing ont the interesting jiossihilities

offered hy the stndy of interaction of T i t y ii s toxin with the strnctures of the

nervons syslem.

Acknowledgement — Part of this work became possible through a grant re-

ceived from the Fundação de Amparo à Pesquisa do Estado de São Paulo. One

of us (C. R. D.) wish to thank the Faculdade de Medicina de Ribeirão Prêto, the

opportunity to visit the University of Minas Gerais during the realization of

this work.

HkKEHEiNCES

1. MAGALHÃES, O., Mem. Inst. Osw. Cruz, 31, 5, 1928.

2. BÜCHERL, W., Mem. InM. BuUintan, 25, 53, 1953.

3. DINIZ. C. R., and GONÇALVES, J. M., in E. E. BUCKLEY, and N. PORGES

(Editors), Venonis, Amer. Ass. Advanc. Sei., Washington, 1956, p. 131.

4. DINIZ, C. R., and GONÇALVES, J. M., Biochim. Biophys. AcUi, 41, 470, 1960.

5. HOUSSAY, B., J. Physioh Patliol. Gen., 18, 305, 1919.

6. CARVALHO, P., Arch. Inst. Benj. Bnpt., 3, 101, 1937.

7. RAMOS, A., and ÇORRADO, A. P., An. Fac. Med. Univ. São Pauto, 28, 81,

1954.

8. FREIRE-MAIA, L., and FERREIRA, M. C., Mem. Inst. Osw. Cruz, 59, 11,

1961.

9. DINIZ, C. R., and VALERI, U., Arch. Int. Pt\urmacodyn., 121, 1, 1959.

10. TORRES, J. M., and DINIZ, C. R., Ciência e Cultura, 16, 197, 1964.

11. PATON, W. D. M., Brit. J. Pharmacol., 12, 119, 1957.

2 3

5 6

11 12 13 14 15 16

FARMACOLOGIA

P HARMACOLOGY

Mem. Inst. Butantan

Simp. Internac.

33(2):457-460, 1966

M. ROCHA E SILVA

457

50. PHAKMACOLOGY OF VENOMS — INTKODUCTOHY REMAHKS

M. ROCHA E SILVA

Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto,

Universidade de São Paulo, Ribeirão Preto, São Paulo, Brasil

The present Iiitroduclory Hemarks on lhe Pliaimacology of Venoms should

cover a large area treated in this Symposiiim, since most of lhe suhjects which

are going to lie discussed here have a heariíig on lhe Pharmacology of Venoms.

Especially all papers on lhe hiochemical aspeets of lhe action of venoms are of

lhe litmost importanee to iinderstand their ])harmacological aclions.

Olivionsly, I have to limit my talk to a fevv personal recollections al)ont

onr past and present work involving animal venomo.

In the heginning, 1 was mainly interested in the mechanism of anaphylaxis

and relatcd jdienomena, from the [)oinl of view of a release of histamine. Con-

sequenlly, 1 hecame ehiefly interested in the work hy Feldherg and Kellaway

(1937-38), in Australia, ahonl lhe release of histamine hy the Australian and

Indian cohra, the Denisouiu sitpcrha and the Naja naja. Both venoms, when

perfused throngh the gninea-pig hmg, |)rodnced a sharp release of histamine,

leading almost to exhanstion of the stock of this amine in that organ of the

gninea-pig. However, cther experiments hy the Australian workers, on the cir-

cnlalory effects of the venoms, as well as on lhe effects npon lhe isolated smooth

mnsele of the gninea-pig ilenm, vvere eonsistent wilh lhe idea that hesides his¬

tamine, other endogenonsly released ])rinci|)les might jiarticipate in sneh enveno-

mations. An im|)ortant onteome of these experiments was lhe eonclnsion that,

nnder certain conditions, a slow-reacling sid)stance (SHS) was released and eould

exj)lain some of the featnres of lhe shoeks hy animal venoms.

These experiments showing the release of histamine and of a slow-reacting

snhstance, hy snake venoms, estahlished an ohvions ])arallelism helween lhe symp-

toms of envenoniation and ihose of anaphylaxis, as had heen descrihed in the

düg, hy Kichet and Porlier (1902), in lhe guinea-|)ig, hy Theohald Smilh (1906)

and in lhe rahhil, hy Arthns and Breton (1903-5).

This analogy was the most striking having in view the way hy which ana-

])hylaxis was discovered hy Kichet and Portier. in 1902. It is well known that

the Erench physiologists were stndying the venom of lhe sea anémona, of the

genns P h y .s a I i a- and A c I i ti i a, using glycerinaled extracts of their tentacles.

When injecled wilh a lelhal dose of these extracts the animais showed a [nctnre

similar to that lhey conld have iindcr the ana])hylaclic crisis: diarrhea, abdominal

cramps, fali in hlood jiressnre, coma and dealh.

By section, lhe animais showed profnse hemorrhages and slagnalion of hlood

in lhe portal region.

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458

PHARMACOI.OGY OP' VENOMS

The new |)lieiiomenon, ohserved hy líichel and Porlier and lo vvhieh lltey

"ave lhe name of Anaphylaxis, vvas lhat a similar ])ictiire eonld he ohlained

vvilh a small fraclion of lhe lelhal dose, if such a small amounl of lhe exlraels

was given 15 lo 20 days afler lhe animal had heen suhmilled lo a previoiis

injeclion of a non-lelhal dose of lhe extraels. Therefore, lhey have coneluded

lhal inslead of heeoming |)roleeled (imnumized) hy a previoiis admitiislralioa

of lhe loxin, lhe animais ])resenled an increased sensilivily lo il. fo denole ihis

ahsenee of ])roleclion or pliylaxis, Hichel and Forlier eoined lhe name which

hecame famoiis of Anuj)liylaxis.

This slory is well known lo all ihose who have worked in ana|ihylaxis, hui

lhe ])oint I wish lo raisc, is lhe im[)licalion eonlained in lhal ?iame of Anaphy¬

laxis, lhal lhe |)rimary agenl should he lhe loxic malerial and lhal lhe symploms

arising al lhe second injeclion were oídy exacerhalion of lhe primary loxic ef-

fecls of lhe iililized malerial. Whal Hichel and Forlier tried lo im|)ly wilh lhe

name of ana])hylaxis was lhe rediiclion of protcction lo lhe loxic effects of lhe

glycerinaled exlracls of P h y s a I i a or Actinia lenlacles. Laler on. lhe

name was widely ulilized lo indicale lhe developmenl of loxic effecls hy malerials.

such as seruni, ovalhumiu and so forlh, which are prirnarily non-loxic and he-

come so afler lhe repelilion of lhe Irealmenl.

In 1904, Arihus discovered sero-anaphylaxis, i.e., lhe developmenl of loxic

effecls in lhe rahhil hy rejrealed injeclions of horse serum, and in 1906, lhe

immiinologisls wilh Theohald Smilh, descrihed in guinea-|)igs lhe ana|ihylaclic

reaclion lo lhe reinjeclion of sera lo which lhe animal is normally insensilive.

We know whal greal success had ihis name of Ana])hylaxis lo indicale a greal

(leal of palhological |)henomena in animais and in humans, where von Firquel

and Schick descrihed serum disease and allergy.

liul, why should Hichel he fooled in his ex])erimenls lo lhe poinl lo ihink

lhal whal he was ohserving al lhe .second injeclion was in realily lhe ahsenee

aj prol.ecíion lowards lhe |)rimary loxic effecls of his malerial exlracled from

lhe lenlacles of lhe Acliniae? Il is easy now lo answer such a rpieslion. In

facl. lhe sym|)lomalology [)roduced hy many venorns from animal origin, re-

semliles or ruimies lhe effecls lhal we know lo he lypical of Anaphylaxis. In

olher words. whal he look as ati inlensifiealion of lhe primary effecls of his

loxin. lhal he used lo call aelinoeongeslin. were aclually lhe similar .symploms

j)roduced hy Anaphylaxis, which oceur as a conscípience of an endogenous in-

loxicalion. hy release of aclive suhslances, among which hislamine is cerlaiidy

one of lhe mosi imporlanl. al leasl in dogs and guinea-|)i"s. Hui, of course.

lhe aelinoeongeslin when acling as a loxic malerial releases hislamine and/or

olher medialors direcllv from lheir endogenous slores, ihough when aclÍTig in lhe

sensilized animal lo produce anaphylaxis. lhe symplomalology develo])s ihroiigh

an enlirelv differeni mechatiism, namely hy comhinalion wilh anlihodies formed

afler lhe firsl itijeclion. Il is ihis profound analogy helween envenomalion hv

animal poisons and lhe symplomalology of Ana[)hylaxis lhal was so nicely explored

hv Arlhus in his hook “De TAnaplivlaxie à rimmunilé”. ])uhlished in 1921. in

Faris.

Hui, in lhe meanlime. lhe imporlanl conlrihulion hy Sir Henry Dale. in

Kngland. sludying lhe effecls of hislamine and poslulaling ils parlici|)alion in

lhe mechanism of Anaj)hylaxis, should he considered.

Iti 1919, Dale pro|)osed lhe name of Aniopliarniaeology lo denole ihis class

of |)henomena ilevelo|)ing in lhe aniiTial hody hy lhe release of endogenous aclive

malerials, especially hislamine.

SciELO

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Mem. Inst. Butantan

M. ROCHA E SILVA

459

Simp. Internac.

83(2):457-460, 1966

Aiifl then, lhe excelleiil work which followeck hy Feldherg atui Kelhnvay

(1937-38) atui others to shovv lhe importaiice of lhe release of histamiiie atui

slow-reacting siihstances (SRS) iii lhe mechatiism of productioii of shocks hy

suake veiionis.

Anolher kiiid of shock in wliieh histamiiie ajtpears lo paitici|)ale lo a very

imporlaiit exletil is lhe shock jtrodiiced hy A s c a r i s, iii guinea-pigs and dogs.

4’liis kiiid of shock was stiidied hy myself, with Grafia, Porto atui Andrade (in

1915-16), froni lhe point of view of a release of histaniine. It is enoiigh lo

take a portion of a single worni, lo niacerate it in saline and inject inio a 10 kg

dog lo prodnce an extreniely severe shock, reseinhiing in all details anaphylaxis

in ihis sjiecies. For lhal reason vve have called this shock an Anaphylaxis-Uke

reaction, instcad of nsing lhe coniinon expression of Anaphylactoid. Ry lhal

time. I thonght lhal lhe phenonienon conld he explained as Irur anajthvlaxis.

assnmitig lhal lhe dogs were [trohahly sensitized to lhe Ascuris material.

In agreement with this point of view, Heraltlo and his colleagnes, in 1961, have

presented evidences thal lhe effecl of A s c a r i s in the gninea-pig a]i]jears lo

depend npon a real State of sensitizalion, prohahiy as a conseqnence of infesla-

lion hy NEMATODA parasites, which are common in snch animais.

I cannol dwell any longer npon this interesling asjtect of the jthenomenon.

that once more estahiished so dose connections helween Anaphylaxis and the

envenomation hy animal venoms.

In lhe ]ueceding discussion we have mentioned mainly histamine as the

])rinciple released hy snake venoms and Ascuris extracts.

It was. indeed. with this idea in mind lhal, in 1918, we slarled doing ex-

perimenls to decide whelher the venom of liollirops jararaca prodnces its sym|)-

lomatülogy in dogs, hy releasing histamine from lhe liver. \X’e were doing. with

Rerahio. liver perfnsions and have assayed lhe venom, hronght lo ns hy Hosen-

fehl. to .^ee wlu-ther it releases histamine from dog’s liver. Since we had show n,

in onr previons experiments with Ascuris extracts, that the hlood was im-

jtorlanl for lhe relea.se of histamine, we perfnseti lhe liver with defihrinated hlood

to which lhe venom was added al lhe momeni of lhe |)erfnsion. The ontpnt

was nol histamine, hnl hradykinin and it hecame clear that the venom released

this material from lhe glohnlin fraction and that hesides lhe venom, aiso Irvpsin

wonld release hradykinin. Hui. whal was mosl imporlant, lhe liver itself had

nothing lo do with lhe release of lhe new snhstance, and if we added lhe venom

direclly lo the defihrinated hlood or to the ])seudo-glohulin prepared from it.

lhe same activily or even more, was released and conld he demonstraled npon

lhe isolaled gninea-pig ileuni, made insensitive to lhe venom hy repealed |)revious

addilions of the same.

Hnl here, again, we had a sirong analogy hetween lhe action of lryi)sin.

which was fonnd lo [)roduce very sirong anaphylactoid symptoms when given to

dogs and rahhils, and lhe envenomation hy snake venoms. Those who are

presenl to this section of Pharmacology know very well, and some hetter lhan

I, lhe conditions in which hradykinin is released hy venoms and toxins. I wili

nse lhe few minnles stili Icft lo discnss some of lhe ])harmacological actions of

hradykinin: as a vaso-dilalor ageni not only npon lhe systemic hnt aIso npon

lhe coronary circnlation, as a powerfnl slimulating sid)stance npon the smooth

mnscle of the intestinal Iract in certain animal s|)ecies and lhe isolaled nlerns

of many sjiecies lesled. as lhe mosl jiowfrfnl agenl lo jirodnce increased cajiil-

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33 ( 2 ): 461 - 466 , 1966

E. KAISER anci W. RAAB

461

51. LIBERATION OF PHARMACOLOGICALLY ACTIVE SUBSTANCES

FROM MAST CELLS BY ANIMAL VENOMS

E. KAISER and W. RAAB

Department of Medicai Chemistry, University of Vienna, Vienna, Áustria

In many inslances, reactions following contacl or ])arenteral adminislration

of animal venoms are indislinguishahle from anaphylactoid reactions. For the

first lime lhe analogy helween antigen adminislration in a sensitized animal and

lhe action of a snake venom (Crotalus adamanleus) was clearly demonstrated

hy Arthiis (1).

It is now generally accepted that diiring anaphylaxis several Itiologically

active suhstances are liherated and activated from precursors (histamine, 5-hydro-

xytryptamine, hejjarine, slow reacting suhstances, kinins) (2,8,4).

On the hasis of the similarity helween the effects of snake venoms and

histamine (5, 6, 7), lhe liheration of histamine from perfused lissues hy animal

venoms was investigated hy Feldherg and Kellaway (8). The release of histamine

was first attrihuted to the jihospholipase activity of the venom and to the lyso-

])hosphatides formed (9). Rocha e Silva and coworkers (10, 11) demonstrated

lhe liheration of histamine from isolated organs following perfnsion with ])roteo-

lylic enzymes and snake venoms. Resides histamine, slow reacting suhstance

a|)pears in lhe perfnsion finid of isolated Inng n?Kler the infhience of snake

and Itee venoms ( 12). Sdiydroxylryptamine is liherated hy animal venoms from

platelets (18). Bradykinin can he formed from lhe glohnlin fraction of normal

plasma (hradykininogen) nnder lhe infhience of snake venoms or trypsin (14, 15).

In anajihylactic and anaphylactoid reactions (administralion of animal ve¬

noms) identical mediators are formed and liherated (16). In hoth types of

reactions, tissne mast cells play an important role. Certain chemical snhslances

as well as enzymes prodnce anaphylactoid reactions hy degrannlating mast cells

and hy liheraling hiologically active snhslances; concomitanlly kinins are activated

from precursors.

In Tahle I, a snmmary is given of direcl and indirect mediators of ana-

phylaclic shock, which are found in animal venoms.

Histamine has heen identified in the venoms of HYMENOPTEBA (17. 18),

DIPTEIÍA ( 19 ), LEPIDOPTEUA (20), HEMIPTEUA (21 ) , S c o / O p e ;i á r o. (22)

and OCTOPODA (28).

S-Hydroxytryptumine is present in lhe venoms of ACTINAKIA (24, 25),

GASTIIOPODA (26), OEPHALOPODA (27,28) HVMENOPTERA (29),AIIACH-

NIDA (30,81) and in skin secretions of A5IPHIBIA (32,88,84).

1 SciELO

462

LIBERATION OF PIIARMACOLOGICALLY ACTIVE SUBSTANCES

FROM MAST CELLS BY ANIMAL VENOMS

TABLE I — ANAPHYLACTOID SUBSTANCES IN ANIMAL VENOMS

DIRECT EFFECTORS:

Illstamine

5-hy(lroxytryptamine (5-HT, serotonin)

klnins

INDIRECT EFFECTORS:

(Ilistamlne, 5-IIT, kinins)

Enzymes (proteases, phospholipase, esterases, hyaluronkiase)

Kinin-proiiucing fraction

Anaphylatoxin-producing fraction

Iligh molecular mast cell dcpletors of unknovvn structure

I,ovv molecular mast cell clepletors of knovvn structure

Surface active agents

A characteristic kinin was foiiiid iii wasp vt-noni (26,35,36,37). In lhe

venom of Bolhrops jararaca a l)ra(lykiniii potcntiatiiig factor lias heen foiind (38).

Il sliould he mentioiied lhal liistaniine (39, dO), 5-hydroxytryplaniine (41. 39,

10) and kinins (42) exert a mast-cell depleling effect; for this reason. these

siihslances are eonsidered lo lie indireet effectors of anaphylaetoid shoek, too.

Several animal venorns eonlain mast cell depleling (13) enzymes (44)

(proleases, phospholipase, esterases. hyahironidase).

The kinin-prodiicing fraclions are mainiy related to lhe enzymes of'lhe

veiionis.

An anaphylaloxin-proãucing fraction was found in Cohra venom (45); il is

knovvn lhal anaphylatoxin degrannlales mast etdls (46,47).

Iligh inoiccalar mast cell dcpletors have heen exlracled from lhe jeily fish 148,

19. 50, 51), from lhe eelworm of swine (52) and from lhe skin seerelions of

A >l 1* 111 B IA (53,31, 54, 55, 56).

Spcrininc whieh is knovvn to liherale histamine (57) is fonnd in spider

venom (.58). Ilolotiiarin, a sa|)onin4ike snhslanee, was isolaled from Uolothuria

vagabunda (59). It has heen shown lhat surface active agenls are potent mast

eell deplelors (60).

In Kig. 1, a simplified siimmary is given on llie tneelianisni wliicli |)rovokes

anaphviaetoid shoek, with s|)eeial referenee to lhe aetion of animal venorns.

Morphologieally, masl eell degramilalion hy animal venorns ean ea.sily he

ohserved. liiopsies taken at various iniervals afler injeelions of animal venorns

(Agkistrodon piscivorns, skin secrelion of Honibina variegata) demonslrate lhe

differenl stages of mast eell degraniilalion. 3 hoiirs afler injeelion, praelicallv

all mast eells are found (l(>granulale(l. The severe vascular ehanges at sites of

injeelions seem to result from liherated masl eell suhstauees as vvell as from direct

loxie effeets of lhe vmioms (55. 61 ).

Mast eell degranulation hy animal venorns ean also he demonstraled in

isolated eells hy mieroseopie exarninalion or hy hioehemieal analysis of lhe

siis|)etision fluid ( 50 ).

SciELO

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Mem. Inst. Butantan

Simp. Internac.

33(2):461-466, 1966

E. KAISER and W. RAAB

463

Fig. 1 — Anaphylactoid shock provoked by animal venoms.

Fig. 2 — Inhibitory effect of antihistamine-, anliserotonin- and antikinin-

substances on mortality íollovving snake venom.

Aiia[)hylactoi(l reactioiis caii In- [ireveiitod by admiiiistralion of aiilaniiiie

sulistances: antihislamiiies, aiitiserotoiiins and antikiiiins. Aiitiliistainines did iiot

exerl any sigiiificaiil influence oii loxicity of siiake venoms (62, 6ií), experi-

SciELO

10 11 12 13 14 15

4fi4 LIBERATION OF PHARMACOLOGICALLY ACTIVE SUBSTANCES

FROM MAST CELLS BY ANIMAL VENOMS

ments with aiitiserolonins and antikinins have iiol yel l)cen rcporled. In a

series of preliminary experimenls, we invesligated lhe prolective action oí dif-

ferent antamines on lhe loxicity of the venom of Agkistrodon piscivorus in mice.

Pretreatment with antihistamines or antiserotonins did not show any signifieant

influence on survival time following injection of a lethal dose of Agkistrodon

piscivorus venom (20 mg/kg

However after administralion of a comhined

antihistamine-antiserotonin suhstance (cyprohej)tadine), a longer survival time

was foimd. In several animais rapid death due to anaphylactoid shock was

prevented. Optimal resnits were ohtained with a eomhined antihistamine-anti-

serotonin-antikinin suhstance (WA-335 Dr. Karl Thomae, Germany). A pre¬

treatment with this suhstance (15 minutes hefore injection of the venom) did

not only ehange survival lime, hui the lethal dose of the venom was tolerated

liy a numher of mice. The high antikinin aclivity might exert a direct antitoxic

effect hy neulralizing toxic fractions of lhe venom (Fig. 2). The prevention

of shock l)y hroad spectrum anli-anaphylactoid suhstances might he of therapeutic

value in man.

References

1. ARTHUS, M., De Vanaphylaxie à Vimmunité, Masson, Paris, 1921.

2. MONGAR, J. L., and SCHILD, H. O., Physiol. Rev., 42, 226, 1962.

3. RAAB, W., and KAISER, E., Klin. Wschr., 43, 345, 1965.

4. UNGAR, G., and HAYASHI, H., Ann. Allergy, 16, 542, 1958.

5. ESSEX, H. E., and MARKOWITZ, J., Amer. J. Physiol., 92, 698, 705, 1930.

6. KELLAWAY, C. H., Brit. J. exp. Pathol., 10, 281, 1929.

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13. MARKWARDT, F., BARTHEL, W., GLUSA, E., and HOFFMANN, A., Arch.

exp. Pathol. Pharmacol., 252, 297, 1966.

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222, 1957.

15. ROCHA E SILVA, M., BERALDO, W. T., and ROSENFELD, G., Amer. J.

Physiol., 156, 261, 1949.

16. KAISER, E., and RAAB, W., Z. angew. Zool., 52, 1, 1965.

17. HABERMANN, E., in Recent Advances in the Pharmacology of Toxins, Per-

gamon Press, Oxford, 1965.

18. SLOTTA, K., and BORCHERT, P., Mem. Inst. Butantan, 26, 279, 1954.

19. ECKERT, D., PAASONEN, N., and VARITIANEN, A., Acta Pharmacol., 7,

16, 1951.

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SciELO

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Mem. Inst. Butantan

Slmp. Internac.

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YAMASAKI, M., J. Orient. MecL, 34, 401, 1941; Chem. Abstr., 35, 8124, 1941.

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KONISI, M., Okayama Igakkai Zasshi, 48, 1309, 1936; Ber. ges. Physiol., 95,

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BETAZZI, P., Arch. int. Physiol., 18, 313, 1921.

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MATHIAS, A. P., ROSS, D. M., and SCHACHTER, M., J. Physiol. (London.) 151,

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FROM MAST CELLS BY ANIMAL VENOMS

50. UVNAS, B., Ann. N.Y. Acad. Sei., SO. 751, 1960.

51. UVNÂS, B., J. med. pluirrn. Chem., 4, 511, 1961.

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scand., 42, Suppl. 145, 1957.

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58. FISCHER, F. G., and BOHN, H., Ann. Chemie, 603, 232, 1957.

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Discüssion

E. A. Zeller: “If serotonin, histamine, and other biogenic amines play an es-

sential role in the action of certain venoms, then it should be possible to change

the response to venom by changing the metabolism of those biogenic amines. Since

amine oxidases, e.g. diamine oxidases (histaminase) and monoamine oxidases can

be blocked in vivo, I wonder whether anybody has pretreated laboratory animais

with inhibitors of the enzym.es before the venoms were administered?”

E. Kaiser: “As far as I know substances blocking monoamine oxidase have

not been used until now. Preliminary experiments in our laboratory have shown

that pretreatment with compound 48/80 (synthetic mast cell depletor) significantly

diminishes anaphylactoid reactions following snake venoms.”

II. Edery: “Would you please tell us more details about this anti-kinin substance

you mentioned. It is specific, it acts in other organ system reactive to kinins?”

E. Kaiser: "I am sorry to say that only very limited Information is available

on this substance (WA-335, Dr. Thomae, Western Germany). At the present mo-

ment I can only tell you that the substance has a high antihistamine, antisero-

tonin and antikinin activity. I am not informed about pharmacological tests i)er-

formed by Dr. Thomae.”

E. R. Trethewie: "Do you think adenosine and related enzymes released are

significant in that cardiac effects? One can select a venom that is simpler in

effect, e.g., Pseudechis porphyrincus (Australian Black Snake), antihistamine with

lieparine does reduce its mortality. Polyvinylpyrrolidone will i)rolong life with

lhe hemotoxic Tiger Snake venom.”

E. Kaiser: “Our experiments were restricted to the venom of A. jji.scivorus

until now. We have no personal experience on the cardioto.xic effects of the

venom.”

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ADOLFO MAX ROTHSCIllLD

467

52. MECI1AN4SM OF HISTAMINE HELEASE HY ANIMAL VENOMS

ADOLFO MAX ROTHSCHILD

Depdrtumento de Farmacologia, Faculdade de Medicina de Ribeirão Preto,

São Paulo, Brasil

Tlif aldlity lo rploase histaniine from lissues is a pliainiacodynaniic projjerty

of many animal venoms. Allliougli not gtmerally considered as a major cause

o[ lhe lethal actions of siich venoms, lhe hypolensioii and increased peripheral

vascular j)ermeahilily which are j)roduced hy hislamine, can eidiance lhe loxic

aelioiis of olher venom componenls. as vvell as of endogenous faclors released

hy lhe venom in lhe hillen suhjeel. In ihis presetilalion I shall limit myself lo

a discussiou of lhe hiochemical aspecls of lhe release of hislamine indueed hy

Iwo venoms; lhat of lhe Hrazilian ralllesnake Crotalu.s diiri.s.sii.s terriliciís and

lhal of lhe honey hee Api.'! nieilijcra. The resulls lo he presenled are moslly

ihose of our own sludies, performed in eollahoralion wilh Dr. Mercedes P. de

Oliveira, A. Caslania, Vera Porlieri and Suzana Hiheiro da Cosia.

ft has heen known siuee 1956 lhal crotamine, a hasic jnotein foimd in lhe

venom of CROTALIDAE of soulhern Brazil (1), is capahle of releasing hislamine

from ral lissues. This resull was firsl ohlained hy Moura Gonçalves and Rocha

e Silva (2) using lhe perfused hind leg pre|)aralion of lhe ral; lhe aulhors

concluded lhal crolamine is lhe cause of lhe well-known hislamine releasing

ahilily of ihis crotalic venom. There are hovvever, cerlain varielies of Hrazilian

ralllesnakes, more ahimdanl in lhe norlhern and cenlral regions of lhe country,

which do nol conlain crolamine in lheir venoms. In a sludy of lhe jdiarmaco-

dvnamic properlies of such venoms, vve noled lhal lhey were highly aclive

hislamine releasing agents, having a ])olency e(|ual or higher lhan lhal of

crolamine ilself. Eig. 1 shovvs lhe release of hislamine from isolaled ral masl

cells hy crolamine and crolamine-free whole ralllesnake venom. In ihis, as vvell

as in most suhsequenl sludies, hislamine release was assayed on lhe washed masl

cells isolaled from lhe periloneal cavily of lhe lal. Such cells are good re-

pre.senlalives of lhe lissuediound forms of lhese cells which, as il is well-known,

are hislamine slorage siles in many s|)ecies. Il is hy lhe slimulalion of lheir

granular secrelion or hy unspecific cylolylic damage, lhal hislamine release is

hroughl ahoul in vivo.

Il is known lhal lhe so-called liemolylic snake venoms are oflen ])owerful

hislamine releasing agenls in vivo or in perfused lissues. Trelhewie in Auslralia,

has aclually suggesled (3) lhal lhese Iwo aclivilies e.g. hemolysis and amine

releasing aclion are due lo lhe same chemical enlily. We have Iried lo verify

whelher in crolamine-free ralllesnake venom, hislamine-releasing aclivily was as-

socialed wilh liemolylic or ralher, lo use a more precise lerm, ])hos|)holipase A

Supportcd, in part, by grant No. DA-ARO-49-()92-66-Gini U.S. Army Re.search Office.

2 3

5 6

11 12 13 14 15

468

MECHANISM OF IIISTAMINE RELEASE BY ANIMAR VENOMS

aclivity. To ihis ciid, wc made a chromalograiihic analysis of crotamino-frec

ralllesnake veiiom oii a And)erlite lrc-5() ion-excliaTige columii accordiiig to lhe

techiii(]iie descrihed l)y Hahermaiiti (4). Fig. 2 shows lhe residis. Il ean he

Flg. 1 — Histamine release from

Isolated rat mast cells by crotamine

and crotamine-free vvhole rattle-

snake venom.

ml effluent

Flg. 2 — Chromatographlc analysis oI crotamine-

free crotalie venom on Amberlite XE-64, CG-50

lonic resin.

seeii that hemolytic aetivity, measiired as phospholipase A liy lhe egg-yolk eoagu-

lation lest, coidd he clearly differenliated from hislamine releasing aclivily as

measiired hy lhe isolated mast cell teclmique. This aclivity was eluted sliortiy

after, hut definilely iiot together willi crolacliii I tall peak), lhe major protein

of lliis ralllesnake venom, which aiso seems lo he lhe major factor res])on.sihle

for lhe lelhality of snch venom (4). It thiis hecomes clear lhal lhe histamine re¬

leasing factor of erolalic venom, which vve have lentalively called enzyme fraclion

I, is neither crolaclin nor phospholipase A. By nsing lhe comhined fractions

showing highest histamine releasing aclivity, we jiroceeded wilh an analysis of

ils projierties. These are shown in Tahie I, which aIso comjiares them wilh

TABI.E I — HISTAMINE RELEASING ACTIVITY OF CROTAMINE AND OF ENZYME

FACTOR I FROM RATTLESNAKE VENOM

Treatment

Histamine releasing

aetivity ♦

Enzyme factor I

Crotamine

Ileat (5’, lOCPC)

Lost

Retained

Dialysis

Retained

I-ost

Digestion by trypsin

Lost

Electrophoretlc moblllty (pH 7.7)

Slow, anionlc

Fast, cationic

Assayed on rat isolated peritoneal fluid mast oell.s.

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Mem. Inst. Butantan ADOLFO MAX ROTHSCHILD 46Í)

Simp. Internac.

33(2):467-476, 1966

ihose of crolamine. Il can he seeii lhat \ve are most prolialily tlealiiig with an

enzyme as the lhermal instaliilily, |)rotein natiire and high molecular vveight data

indicale. \Ve suspect lhal enzyme factor I is a proteolytic enzyme having chymo-

trypsindike properties and base this assumption on three facts: a) chymotryptic

activity has lieen demonslrated in crotalic venom by Deutsch & Diniz (5) in

1955; 1») cbymolrypsin is a hislamine-releasing agenl (6,7) in contrast to trypsin

which will not act on the isolaled mast cell; c) our product was able to destroy

])radykinin, a pro])erty of cbymolrypsin, and which is an extremely sensitive,

although not specific test, for this enzyme.

The absence of a direct histamine releasing effect of crotalic phospholipase A

was a somevvbat unexpected finding, even more so because of reports in the

literature (Uvniis and collaborators (8)) showing that snake or bee venom phospho¬

lipase A was indeed a potent histamine releasing agent on isolated mast cells

of the rat. It seemed possible to us that the preparations used by Uvnas could

have been contaminated with other venom factors capable of releasing histamine.

In order to investigate this problem, we decided to study the histamine releasing

properties of a highly piirified and very active jíreparalion of phospholipase A

írom bee venom, obtained through the courtesy of Prof. Habermann (9) of

Germany. It turned out, that this enzyme was only slightly active on the isolated

mast cell, even when assayed in relatively high doses as shown in Table II. Not

TABLE II — EFFECT OF PHOSPHOLIPASE A TREATED EGG YOLK ON THE RELEASE

OF FIISTAMINE FROM ISOLATED RAT MAST CELLS

Releasing agent *

Percent histamine released

Phospholipase A, 20 íig/ml

Egg yolk (4í7) pretreated with phospholipase A

7.3

(37°, 40')

91.0

Egg yolk (4%)

0.0

* Allovveci to act for 20 min, 37°C on the mast cell .suspension.

imexpectedly, |)hospholi[)ase A was also inactive as a hemolytic agent, an obvious

concliision from the known fact that lhe red ceirs membrane phospholipids are

shielded from attack by this enzyme (10). They are however highly sensitive

to the action of the product of this enzyme’s action on a susceptible substrate

like egg yolk’s fresb lecithin. The data on Table 11 indicate that rat mast cells

readily loose lheir histamine when treated with lhe producls of the action of

jihospholijiase A on egg yolk, which we know to conlain lysolecithin. Thus,

in vitro release of histamine bv phospbolipase A can only take place in an in-

direct way.

The question which now arose in our minds was: would the enzyme bchave

in the same manner in an in vivo test for hislamine-releasing activity? Would

lherefore the numerous results about lhe histamine releasing activity of hemolytic

vcnoms in vivo or in perfused tissues demonslrated by other authors, have to

bc ascribed enlirely lo lhe presence of non-phos|)holipase A components? In

order lo answer lhese questions, we jierformed a very simjile test for the deteclion

of histamine releasing activity in vivo. This is lhe well-known Trypan blue skin

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470

MECHANISM OF HISTAMINE RELEASE BY ANIMAL VENOMS

capillary [lermealnlily k>sl, which consists of lhe followiiig: a ral or a giiinea-pig

is iiitravenousiy iiijected wilh a dye like Trypaii or Evans hhie, which nonnally

does nol leave lhe circtdalioti lo enler lissiie fluid spaces l)ecause of ils iiialiilily

lo cross capillary wall ])crmeahilily harriers. If sucli harriers are liovvever allered

hy aii experimenlal procedure siich as lhe local release of hislamine. a suhslance

highly aclive on ca|)illary permeahilily, diffusion of lhe dye across lhe vascular

Wall can lake |)lace and a hhie si)Ot will ap|)ear al lhe sile of hislamine release.

l'ig. 3 shovvs lhe resulls of inlradernial injeclion of hee venoni j)hospholipase A

B'ig. 3 — Effect of bee venom phosphollpase A on capillary per-

meabllity In rat skln. Dark areas correspond to the injection of

respectively 0.01, 0.1, 1,0 and 5.0 jig of enzyme. Contra-lateral

injectlons of saline failed to produce comparable respon.ses.

inlo ral skin

doses ranging frorn 10 /rg to ,5 /<,g. ll can he seen lhal the

enzyrne was highly effeclive in hnvering ca|)illary diffusion harriers and lhal ihis

effect hore relation to lhe dose iised. This effecl was nol a direcl one; it is

prohahly medialed hy the release of skin hislamine and serolonin since it coiild

he shovvn (13) lhal the [lermeahility effects of phospholi|)ase A coiild he completely

siippressed in animais |)revionsly Irealed wilh a mixlure of an inhihitor of the

vascular effects of hislamine I diphentiydramine) and an inhihilor of the vascular

effects of serolouiu |H()L-M8, hromiysergic acid dielhylamide). This laller had

Io he iised hecatise as it is well-kiiovvu, wheuever mast cell damage occurs iu the

ral. uol only hislamine luit serolouiu as well, is released. Serolouiu is even more

jioteul on rat capillary |)ermeahility than hislamine. The ohviotis couchision from

lhese resulls was lheu, lhal jihosjiholijiase A was ahle to release mast cell amines

in vivo, eveu ihough it proved iucapahie of doiug the same on the isolated mast

cell in vitro.

Talile 11 had shown lhat phosjiholipase A can iuduce copioiis liistamiue

release from isolated mast cells in vitro hui only iu au iudirect way, namely,

lliroiigh lhe formaliou of lysolecilhiu. As it is well-knowu, this suhslance, a highly

cytolytic. agent, is lhe resnit of the spliltiug of an acid radical from lecithin Ijy

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10 11 12 13 14 15 16

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MECHANISM OF HISTAMINE RELEASE BY ANIMAL VENOMS

472

Fig. 4b

^..11 I

Fig. 4c

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Mem. Inst. Butantan

Simp. Internac.

33(2):467-476, 1966

ADOLFO MAX ROTHSCHILD

473

Fig. 4d

histamiiu' releasiiig stini

llial histamiiie liroaks 1

pliarinacological actioiis

lhe effect of conipound

ig stiniiili and il is likely, lliat il is oiily iii llie slied granules

reaks loose froni ils intracellular lies, hecoinitig free lo exerl

utions iii lhe hody. Fig. Id shows, for lhe sake of comiiarisoit,

npoiind 48/80, lhe inost potent chemical histamine releaser iii

of another sani])le of

shows

lhe

kiiown, on

lhe

niasl

mesenlerv.

rat

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474

MECHANISM OF IIISTAMINE RELEASE BY ANIMAL VENOMS

llie effects ot ineliltin; ihis is a polype])li(le siil)statKc, isolated fiom l)ee veiiom

liy Hahermann (14), which has an iiitense and a[)part‘iitly unspecific cytolytic

effect. It induces 100 per cent release of histamine froni isolated mast cells (13).

As it can be seen, its effects are not so imich a degranulation hiit rather a dis-

solution of the celFs houiidaries. This as[)ect is lypical of unspecific cell damage,

and is, in its mechanisni of aclion quite different from granule secretion as evoked

by compound 48/80 or the antigen-antibody reaction. The action of melittin is

however similar to tbat of lysolecithin. Table III sbows that tbere is a ineans

TABLE III — MODES OF ACTION OF IIISTAMINE RELEASING AGENTS

Group I: Stimulants of mast cell secretory activlty (effects inhlblted by metabolic in-

hibltors (anoxla, DNP, NaCN)

Antigen-antibody reactions

Epinephrine

Compound 48/80

Curares

Chymotrypsin

Enzyme factor I from crotalic venom

Crotamine

Group II: Non-specifie cytolytic agents (effects not Inhlblted by metabolic Inhlbitors)

Surfactants (octylamlne, Tween 20, etc.)

Phosphollpase A

Lysolecithin (egg yolk)

Melittin

Crotamine

of distinguisbing belween lhe niecbanisin of action of histamine releasing agents.

1 consider those agents wiiose action is inhihited by metabolic inhibitors like

oxygen lack, dinitrophenol or cyanide, lo be Irue stimulants of the secretory

macbinery of the mast cell. In contrast, the group of histamine releasers wbose

action is not blocked by metabolic inhibitors, are to be considered unspecific

cytolytic agents, capable of riiiiluring lhe mast cell in lhe same way as lhey

would injure most any cell in lhe mammaliaii organism. It can be seen that

among histamine releasing agents conlained in animal venoms, phospholipase A,

melittin and, to a partial extent, crotamine, are included in this group.

Kefeuences

1. MOURA GONÇALVES, J., in E. E, BUCKLEY, and N. PORGES (Editors),

Venoms, Amer. Ass. Advanc. Sei., Washington, D.C., 1965, p. 261.

2. MOURA GONÇALVES, J., and ROÇHA E SILVA, M., Ciência e Cultura,

Brasil, 1(), 163, 1958.

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ADOLFO MAX ROTHSCUILD

475

Simp. Internac.

33(2):467-47fi, 1966

3. TRETHEWIE, E. R., Aust. J. exp. biol. med. Sei., 17, 145, 1939.

4. HABERMANN, E., Biochern. Z., 329, 405, 1957.

5. DEUTSCH, H. F., and DINIZ, C. R., J. biol. Chem., 216, 17, 1955.

6. UVNÂS, B., Ann. N.Y. Acud. Sei., 166, 880, 1963.

7. SAEKI, K., Jap. J. Pharmacol, 14, 375, 1964.

8. UVNÃS, B., Cliemotherupiu, 3, 137, 1961.

9. HABERMANN, E., Biochern. Pharmacol., Suppl., 12, 187, 1963.

10. CONDREA, E., MAMMON, Z., ALOOF, S., and DE VRIES, A., Biochhn. Bio-

phys. Acta, 84, 365, 1964.

11. KELLER, R., Helv. Physiol. Pharmacol. Acta, 22, C76, 1964.

12. FELDBERG, W., and KELLAWAY, C. H., Aust. J. exp. biol. med. Sei., 15,

461, 1938.

13. ROTHSCHILD, A. M., Brit. J. Pharmacol., 25, 59, 1965.

14. NEUMANN, W., and HABERMANN, E., Arch. exp. Pathol. Pharmacol., 22,

367, 1954.

Discussion

F. Kornalik: “How do you explain the fact, that ELAPIDAE venoms, especial-

ly the venom of Naja naja which are known to have big amounts o£ phospholipase

A are not able to liberate any histamine from the tissue? Have you te.sted the

phospholipase A from Prof. Habermann in another way than the skin test for the

presence of spreading factor?”

A. M. Rothschild: “I have no personal experience with such venoms. How-

ever the statement that the venom of Naja naja does not release histamine from

tissues contradicts observations reported by a considerable number of authors wbo

have found this venom to be quite powerful as a histamine releasing agent. We

have not done a characterization of this enzyme because Prof. Habermann (9) has

presented convincing proof that it is essentially free of enzymatic contaminants

including hyaluronidase. Furthermore, our skin lests definitely implicate histamine

and serotonin as mediators; hyaluronidase is known not to release these amines.”

J. L. Prado: “The bradykinin destroying effect of enzyme I fraction of snake

venom seemed too slow to me; would it really be a chymotryptic enzyme?”

A. M. Rothschild: “There is, I believe, fair evidence that the histamine releas¬

ing activity of Fraction I is of an enzymic nature. Aithough our results do not

conclusively characterize this activity as being chymotrypsin-like, they point in

this direction. Slugishness of action does not necessarily rule out this possibility

since even whole crotalic venom is slow in attacking a typical chymotryptic sub-

strate like ATEE.”

E. A. Zeller: “Have you tested the substrate pattern of your enzyme factor

which you suggested appears to be a chymotrypsin-like proteinase?”

A. M. Rothschild: “No.”

2 3

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Mem. Inst. Butantan

Simp. Internac.

33(2):477-50K, 1968

PHILIP ROSENBERG

477

53. l’SE OF VENOMS IN STUDIES ON NEHVE EXClTATIOiX

PHILIP ROSENBERG

Departinent of Neurology, College of Physicians and Surgeons

Columbia University, New York, N.Y.

Introduction

Various techniques have Iteeii utilized over the past several decades in at-

tcmpliiig to understand hioeleclrogenesis, lhe jirimary eveiil in neiiral aclivdty.

Ulilizing eleetrophysiological techniques it was ]) 0 ssil)le lo analyze inany aspecls

of electrical activity of conducting lissues (1,21. However, for elucidaling the

Chemical and molecular hasis of nerve activity it was necessary that techniques

of modem hiochemislry and pharmacology he used. Over lhe pasl 25 years the

sludies of Nachmansohn and coworkers have [irovided us with lhe essential steps

involved in lhe generation of hioelectricity allhough many details require further

investigation (3-5).

The impetus which led to our studying of the effecls which slruclure and

organizalion of hiological memhranes and their environment have on the function-

ing of lhe ACh syslem, and on lhe action of compounds apjilied externally lo

l)iological lissue, will he hetter understood if we hriefly review the role allrihuted

to acetylcholine (ACh) in the excitahie memhrane during nerve conduction (Fig.

1). ACh in resting condition is jiresent in a hound form. Any stimulus reach-

ing lhe memhrane leads to release of the ester which comhines with a receptor.

In the reaction of ACh with the rece])lor a change in conformalion of lhe receptor

is thought to occur which leads lo a shifl of charge initiating lhe permeahility

changes of lhe mend)rane associated with the passage of a nerve impulse, i.e.

sodium ions enter lhe axon and potassium ions leave. The rapid inactivation

of the ester hy acelylcholinesterase (AChE) permits the recejilor to return to its

resting condition. A ])rerequisite for this lheory was the demonslralion of lhe

presence in all conducting tissue of choline acelylasc (ChAc) lhe enzyme which

synthesizes ACh and AChE the enzyme which hydrolyzes ACh. Of crucial im-

|)ortance to lhe development of the theory has heen, however, the evidence that

hlock of eilher AChE or ChAc hy jiotent or comjictitive inhihitors leads inevitahly

to hlock of electrical activity.

Thls Work was supported by the Divlsion of Research Grants and Fellowships, U.S.

Public Health Service, Grants NB-03304, NB-04367 and B-400 and training grant 2B-5216;

by the National Science Foundation Grants 12901 and 1913 and by the Muscular Dys-

trophy Associatlons of America Inc.

Recipient of a Public Health Service Research Career Development Award, 5-K3-

NB-21, 862.

2 3

5 6

11 12 13 14 15

478

USE OF VENOMS IN STUDIES ON NERVE EXCITATION

ELEMENTARY PROCESS

O-r "

2 A T P

1 s!

2 PYRUVIC ♦ 02

VIA CITRIC ACID CYCLE

30 ATP

fCHOLINE ACETATE j

. _)

PHOSPHOCREATINE

Co A t ACETATE

:holIN E \

cetylaseJ '

-ACETVL-Coâ

a M p + PP

Fig. 1

Some invesligators liave, hovvever, not accepted lhe lheory lhat ACh lias

an essential role in axonal conduction, and liave inainlained that ACh is essential

for transmission of impulses only al certain jimclional regions. One of their

major ohjections to Nachmansolm’s theory was the failiire of ACh, and other

quaternary nilrogen derivatives lo affect conduction in nerve axons in coutrasl

to their povverfui aclion on junctions. The excilahie and conducting axonal

memliranes, in coutrasl to those al synaptic junctions, are surrounded hy slructures

rich in liiiid material, the Schvvann cells. which form a iiermeahilily harrier

jirevenling li[)id insoluhle compounds from reaching the memhrane. Exjierimenlal

evidence for the existence of liarriers was ohtaincd hy the demonstration that

lioth ACh and lhe quaternary cholinesterase inhihitor neosligmine, failed lo jie-

netrate into the axo])lasm of lhe squid giant axon afler their a])|)licaliou to lhe

fiher in the externai solution, whereas lipid solulile lertiary nilrogen derivalivE's,

•sucli as physosligmine, that lilocks electrical aclivity, were fouud in the axo-

plasm (6,7). lii pre|)aratious such as the ralihit vagus and axons from lhe lohsler

walking leg in which the permeahility liarriers are apparenlly incomplele, direct

effecls on conducliou have heen ohtained with ACh, and related comjiouuds, such

as d-tuliocuraririe (curare) knowii lo compele with ACh for recejilor sites at

junctions (8-11).

If A(ih and curare are inactive on lhe squid giant axon and other axonal

prejiarations hecause of permeahility liarriers it appeared possihle that hy (hemical

Ireatment one could decrea.se lhese liarriers and iheii demonslrate actions of lijiid

insoluhle com])ouuds which iuleract with lhe ACh syslem. After exjiosure of the

frog scialic nerve to a detergent. axonal conduction was reversihly hlocked hy

ACh, curare, neostigmine and other quaternary nitrogen derivatives (12). In

our sludies we elected to use the squid giaut axou (Fig. 2) hecause it is a large

single fiher and is non-myelinated, i.e. is surrounded hy a relatively ihin Schwann

cell. In such a prejiaration chemical treatment migliL he ex|)ected to he more

effective and hetter conlrollahle lhau in a mulliíihred [ireparation. This preparation

also has lhe uniipie advaulage lhat its axoplasm lan he readily extruded in suf-

íicient amounts lo allow penetralion sludies to he carried oul. In addition it

was usefui lo sludy a preparation for which a considerahle amounl of data are

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Mem. Inst. Butantan

Simp. Internac.

33{2):477-508, 1966

PHILIP ROSENBERG

479

already availahie, and willi which oiir residis coidd lie correlaled. Tliis review

shall presenl and snmmarize some of llie findings we have made over lhe past 5

years (14-25).

Fig. 2

Effects of tertiary and quaternary mtrogen dekivates on conduction

While a few phannacologieal agenis have heen tesled on eonduclion of lhe

sqnid giant axon, no systenialic eornjiarison had heen made hetween terliary and

qualernary nilrogen conlaining eomponnds. If a permeahility harrier ]irevenls

]i])id insolnhle qualernary nilrogen comjjounds from reaehing lhe conducting

niemlirane. lhen we shoidd find lhese compoimds lo he inactive on lhe squid

axon even if lhey interacl wilh lhe ACh syslem at junclions where lhe jiermealiility

harriers are mueh w'eaker. \\ e should also find lhal lijnd solnhle compoimds

which affecl jnnctional Iransmission shonld also affecl axonal conduction. This

is whal we did find on conirol squid axons in Tahle 1 where we have also

cornpared lhe |)otency of various compoimds on lhe squid axon wilh lhal ohserved

at lhe synaplic junclions of lhe isolaled single eleclroplax where harriers are

either miuimal or ahsent, and where approximale affinily lo rece[)tors jireseut

1 SciELO

480

USE OF VENOMS IN STUDIES ON NERVE EXCITATION

inay Ix' delermineíl (28,29). The terliary iiitrogeii derivatives are effective in

verv similar eoncentrations oii lliese two [jreparations. One couid not, however,

expect exaet agreement, even if lhe |)ermealiility harriers were tom])letely ahsent,

l)ecause of species differences, sornewhat differciil experimental eondilions and

most likely other faelors involved. In eonlrast all cjuaternary nilrogen eontaining

componnds except noracelylcholine and jiyridine aldoxime dodecyliodide (PAD)

are ineffeetive on lhe sqnid axon in eonlrast to their high ])olency al lhe synapse.

Noracetylelioline and PAD are li])id solulile derivatives of acetyleholine and

jjyridine aldoxime methiodide (PAM) having a dodecyl group vvhieh replaces

a methyl group on the nitrogen. We shall disciiss lhe resulls ohlained on the

venom Ireated sqiiid axon in seetion 8. ACh and curare were equally inert ou

control sqnid axous which were very carefully dissected free of all adhering sniall

nerve filiers as well as axons iu which not all of the small nerve fihers were

removed(28). Physosligmine was also found to he almost equally potent in

hoth pre|)aralions. These resulls indicate lhal lhe adhering small nerve fihers

and assoeiated connective tissue do not constitule a strong ])ermeaf)ility harrier.

The major harrier is a|)parently the Schwann cell surrounding the giant axon.

For convenience we have lherefore usually used j)reparations in which no S])ecial

effort was made to remove all adjacent fihers, although as will he diseussed in

seclions 8, 4 and 8 venom effects and penetration of ACh were comjiared wilh

hoth ly])es of prejiarations.

These results show lhat lipid soluhle com|)ounds expected to interact wilh

lhe ACh syslem affecl conduction along lhe axon as well as at the junction,

whereas lipid insoluhle componnds hlock junctional Iransmission hul not axonal

conduction.

Venom pretreatment of squid giant axon

In attempling lo reduce the {)ermeahility harriers surrounding the squid giant

axon, various enzymes, detergenis and other componnds in the highesl eoncentra¬

tions which had no effect on conduction were a|)|)lied to the axon for 80 minutes.

After removal of lhe pretreatmenl ageiil and rinsing in normal sea waler for 15

to 80 minutes, 1.4X10~’‘ M enrare was applied for 80 minutes. Curare was

used as a lesi compound for iudiealing disruplioti of permeahilily harriers siuce

il is highly ])oteut al synapses and is not readily melaholized hy enzymes of

hiologieal tissue sueh as for examjtle ACh is hy AChK. Curare had no effect

on conduction following ex|)osure of lhe axon lo digitonin, hyaluronidase, Irypsin,

chymotry|)sin, hydrolase mixture, lij)ase, lysozyme, pa])ain, alkaline ])hosphatase,

sodium desoxycholale, Span 20, Tween 20, neuraminidase, saponin, arsenite, dim-

clhyl formarnide, dimelhyl sulfoxide or hislamine (14,21). Following prelreat-

ment with Naja naja I hooded cohra) snake venom, however, curare was found

lo reversihly hlock conduction (14). An axon rendered sensilive to lhe action

of curare remained so over a long ])eriod of lime. For example curare was

equally effective whelher [)relrealment was followed hy |)lacing in sea waler for

10 min. or 95 min. (Fig. 8). This figure shows effects ohlained wilh a com-

hination of hooded cohra venom plus a delergent, however, similar resulls were

ohtaiued with venom alone. Since snake venoms apj)eared lo render the axons

sensitive to curare a .syslematic sludy of lhe effects of curare following venom

jjrelrealmenl of lhe axon was underlaken. Of a series of 21 venoms lesled Agkis-

trodon p. piscivoriis (Collonmoulh moccasiu) venom was most effective in render-

ing the axon sensitive to curare (Tahle II). Fxamples of the effects of curare

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Inlernac.

33(2):477-508, 1966

PHILIP ROSENBERG

481

Flg. 3

TABLE I — MINIMAL CONCENTRATIONS OF SEVERAL COMPOUNDS REQUIRED TO

BLOCK CONDUCTION OF CONTROL AND VENOM TREATED SQUID GIANT AXONS

AND SYNAPTIC TRANSMISSION IN THE ISOLATED SINGLE ELECTROPLAX

Compounds were appliecl for 30 minutes. Venom treated axons vvere pretreated for 30

minutes vvith 15 ng/ml A()kif:trodon p. piscivonis venom. > indicates that at concentra-

tion shown no effect on conduction was observed. Noracetyicholine = ;S-acetoxyethyl-

dimethyidodecyl ammonium lodide; PAD = Pyridine aidoxime dodecyiiodide; PAM = 2-

Pyridine aidoxime methiodide.

COMPOUND

[M]

Squid

TO BLOCK CONDUCTION

Axon *

Control

Venom Treated

Synapse *•

Tertiary Niirogen Derwativefi

Atroi)ine

2 X lO--'

3X10-'

3 X10-'

Methantheiine

2 X 10-^

—

5X10-1

Physostigmine

7X10-3

1 X 10-3

7 X 10-'

Procaine

3 X 10--’

—

1X10-=

Dibucaine

3 X 10-'>

—

3X10-=

Diphenhydramine

4X10--*

—

2 X 10-'

Chiorpromazine

1 X 10-«

—

1 X 10-1

Qnaternary Nitrogen DerivaCives

Acetylcholine

> 10-'

2 X 10-1

3X10-0

Curare

> 10-=

3X10-3

3X10-»

Decamethonium

> 10-'

2 X 10-'

3X10-0

Benzoylcholine

> 10-'

2 X 10-=

1X10-=

Chlorisondamine

> 10-'

1X10-=

2 X 10-1

PAM

> 10-1

1X10-=

—

PAD

5 X 10-'

—

5 X 10-3

Noracetyicholine

2X10--Í

—

2 X 10-'

Choline

> 10-1

> 10-'

Data from Ro.senberg et al. (14-16).

Data from Ro.senberg et al. (26, 27).

SciELO

10 11 12 13 14 15

482

USE OF VENOMS IN STUDIES ON NERVE EXCITATION

íollovviiig coltonmoulh moccasiii veiiom are sliown in Figs. 4 and 5. lii lhe

conceiitralions used for prelreatmenl tlie venoms had iio effect on the aclion

polenlial. Hooded cobra venom depolarizes the lobster axoii (30), biit il is not

apparent whether depolarizalion is the cause of the conduction hlock or occiirs

suhsequenl lo the hlock. On the squid axon lhe resting and action potentials

SCA

WATIR

I-

3.8v

COTTONMOUTH

VENOM

8.Sv

SE A

WATER

-1

50m V

1.4x10-3 M

CURARE

Fig. 5

S E A

WATER

1 SciELO

Mem. Inst. Butantan

Simp. Internac.

33(2):477-508, 1966

PHILIP ROSENBERG

483

vvere foiind to decline simidtaneously (Fig. 6), whereas after coltoninoiith moc-

casiii venom, depolarization occurred only after the action potential was niarkedlv

reduced (Fig. 1). There was no difficulty of deterinining wliether hlock of

conduction was diie to curare or venom since the effects of the venoms on the

action potential were always irreversihle whereas conduction hlock hy curare was

usually readily reversihle. Curare had little or no effect on the resting potential

after venom pretreatment (Fig. 5) which agrees with its effects at junctions where

Venom

SciELO

10 11 12 13 14 15

484

USE OF VENOMS IN STUDIES ON NERVE EXCITATION

íL also Iilocks comliictioii luit does iiol depolarize. It ai)|)arenlly ialeraels wilh

the receptor lo lilock the effecls of ACli. Iiut does not cause llie chaiige in con-

formatioii of lhe receptor responsil)le for altered conduclance. IL may be called

a receptor inhihitor iii contrast to comj)ounds siich as ACh whicli depolarize and

may lie classified as receptor activators.

The ahility of the snake venoms to render curare active ahnost exactiy cor¬

relates with their direct potencies on electrical activity of the squid giant axon

(Tahle II) indicating that the same venom component may he resjjonsihie for

TABLE II — CONCENTRATIONS OF VENOM PRETREATMENT WHICH RENDERED

THE SQUID GIANT AXON SENSITIVE TO CURARE, AND CONCENTRATIONS REQUIRED

TO BLOCK DIRECTLY THE CONDUCTED ACTION POTENTIAL (AP). VENOMS WERE

APPLIED FOR ABOUT 30 MINUTES

> incllcates that at concentration shown no effect on AP was observeU. -I-+-h, -h+,

-h, and - indica te respectively a 80 - 100 %, 50-80%, 20-50% and 0-20% decrease in the

AP produced in 30 minutes or iess by 1.4 X 10-^ M curare alter venom pretreatment.

The eflect of curare was readiiy reversible. Results taken from Refs 14-16, 24.

VENOM

íig/mi

Direct block

of AP

Pretreat.

Curare effect

Afjkistrodon p. piscivoriis

+ + -h

Notechis scíitatus

100

+ + 4-

Acanthophis antarcticns

100

+ +

Naja naja

100

-f -1-

Heloderma horridum

100

+ -1-

HeJoderma suftpectuni

100

+ -1-

Enhydrlna schistosa

100

—

Dendroaspis poJi/Iepin

100

-1-

Bunffarus coeruleus

250

-I--H

Ophloph agu .s han n ah

400

100

-1- -l

Crotalufi atrox

500

150

-t-

Affkistrodon c. mokeson

1000

200

fíothrops atrox

1000

200

Bitis arietans

2000

500

+ +

Vipera russeUii

> 1000

500

Centruroides scuipturatufi

> 100

100

—

Vespula arenaria

> 100

100

—

Latrodectus f/eomeiricus

> 100

100

—

La trodeet ua varidn s

> 250

250

—

CrotaluH h. horridus

> 500

500

—

Cr o t a 1 UM a da m a n te us

> 2000

2000

—

holh effects. Stiidies hy others of venom effects at lhe neuromuscular junction (31,

.12) have also shown collonmouth venom to he more ])Otenl lhan Crolaliis ada-

niaiitcus (Lastern diamondhack ralllesnake) venom, which in our sltidies was

one of the leasl effeclive of the snake venoms. Likevvise lhe relalive effeclive-

ness of hooded cobra and ralllesnake venoms in our studies and in cansing

demyelinaling changes in the central nervons system (33, 34) are similar. In

contrast ralllesnake venom is more loxic lhan cottonmouth moccasin venom (35,

1 SciELO

Mem. Inst. Butantan

PHILIP ROSENBERG

485

Simp. Internai'.

33 ( 2 ):- 477 - 508 , 1966

riG). It is t>s])i‘ciallv inlfiesting lhal lhe heitiolylie and heniagghilinin aclivities

of .«everal siiake veiiorns as foiiiid liy Miiilon (351 is iii excelleiit agreemenl witli

their ahilities lo render eiirare aclive, hor example lie reporls lhal Kastern

diamondhack ralllesnake venoni has lillle or no hemolytie aelivily whereas Cro-

Niliis atro.x (Vieslern dianiondhaek ralllesnake) venom is more |iotenl whieli also

agrees wilh onr findings. He also fonnd Agkisirodon c. mukcson (eojiperhead

moecasin) lo he Icss jrolenl llian eollonmoulli moceasin. The marked similarity

lietvveen his and oiir residis may indieale lhal llie same venom eom])onent is

resjronsihie for holh effeels measnred.

Sinee eollonmonlli moceasiti venom apjteared mosl effeclive in rendering llie

axon sensilive lo eiirare it was selected for additional slndies. The venom Ireated

axon not only lieeame sensilive lo curare Inil as shown in Tahie I. .4Ch. deca-

melhoniiim and several olher qnalernary nilrogen derivalives l)Ioeked condnclion.

Examjiles of lhe ACh effeels on collonmoiith venom Ireated axons are showii

in Figs. 8 and 9. As can l)e seen in Fig. 9 llie effeels of ACh were nol aiways

reversihle. In a large series ahoiil 1 onl of !■ experiments wilh ACh were

reversihle. In experiments wilh intraeellnlar eleelrodes, 1 X 10“" M .4Ch follow-

ing venom jirelreatmenl hloeked eondnelion and deereased lhe resting irolential

ahonl in 30 min. while in eonlrol axons ACh had no effeel on lhe resting

or aetion potenlial. However. 4X10“^ 41 ACh following venom prelrealment

had a marked effeel on lhe aelion jiolenlial hiil no ap|)arenl effeel on lhe resling

])olenlial. This is in conlrasl lo olher ])re|)arations where lhe effeels of ACh

on lhe aelion potenlial are paralleled hy eonciirrenl effeels on lhe resling po-

lenlial 19-11).

The eoneentralions of qnalernary nilrogen derivalives shown as heing effeclive

in venom Irealed axons iTahle I) are llie minimal eoneenlralions whieh in

al leasl a few exjierimenls hloeked eondnelion. For fairly eonsistenl effeels it

was neeessarv to nse eoneentralions ,5 lo 10 fold higher. The eoncentration

of t(nalernary nilrogen derivalives lisled in Tahie I as heing effeclive on lhe

venom Ireated axon are 10 to 1000 fold grealer than ihose reqnired al lhe

svnajise. However, al leasl pari of this differenee is ipiile ohvionsly dne to

lhe ineomplete rednction of permeahilily harriers snrronnding lhe axon, .so

lhal ordv a relatively small fraelion of lhe exlernally ajiplied ACh. enrare ele.

is ahie to |)enelrale Isee .seetion 4). The inaetivity of eholine in venom Ireated

axons, despite lhe fael lhal il penetrales nnder lhese eonditions tsee seetion 4)

show lhal lhe effeels of ACh are sjieeifie. At those synapses where ACh is highly

))olent. sneh as for example lhe eleelroplax synapse. il has also heen fonnd lhal

eholine is inert (Tahie I). In conlrasl to lhe (jnalernary eomponnds shown in

Tahie I whieh were effeclive on venom Irealed axons earhamvleholine and neo-

sligmine even in 5 X 10~- 41 eoneentralions were inaetive. These eomponnds

are apparenlly even more lipid insoinhie than ACh. enrare, or eholine sinee neo-

stigmine does not jienelrale inlo lhe sqnid axon nnder eonditions where lhe venom

markedly inereased lhe ])enetration of lhe olher eomponnds (seetion 4). In these

exjieriments we ohserved only hloeking effeels of ACh and nol anv eleetrogenic

aelion. Wilh lhe melhod of appiieation nsed, one eannot expeel to mimie lhe

snggested physiological aelion of ACh. Physiologieally ACh wonld he liheraled

wilhin a memhrane of ahonl 80 A" thiekness. Il wonld lhen ael, in mieroseeonds,

on a iirotein reeeplor in elose proximily, prohahiy only a few A" away. W hereas

ihis aelion may he very effieient and rajiid. ACh when ap|)lied exlernally miisl

pcnelrate throngh a relatively large amonnt of lissne hefore reaehing lhe reie[)lor

of lhe active axonal memhrane. Thns. lhe eondilions are nol eomparahle.

2 3

5 6

11 12 13 14 15

SciELOiio

Mem. Inst. Butantan

Simp. Internac.

33 ( 2 ): 477 - 508 , 1986

PHILIP ROSENBERG

487

It was foiind (Talile [) thal lhe potency of atropine and physostigmine,

tertiary nitrogen derivatives, was also increased hy venom pretrealmenl so that

lhe concentrations effective on lhe axon hecome very dose to those effective at

the electro])lax synapse. These findings are in agreement with the idea that

venoms are disriipting permeahility harriers, since even relatively lipid soluhie

materiais prohahly cannot freely diffuse through the memhrane surroiinding lhe

giant axon. The findings with atropine, physostigmine and the other compounds

shown in Tahle I appears to estahlish that a fiinctional ACh receptor and AChK

are present in the sqiiid axon.

Effect of ve.xoms on perme.ability of squid giant axon

We interpreted the demonstration that ACh and curare affect electrical activity

after exposiire of squid giant axons to coltonmoiilli venom as heing due to a

rediiction of the permeahility harrier which prevents lipid insoluhle compounds

from reaching the conducting memhrane. In view of lhe crucial question in-

volved, viz. whether ACh and curare act on the receptor in the axonal memhrane,

this iuterpretation was suhmilted to a direcl tesl; i.e., whether the venom which

has heen shown to allow ACh and curare to affect electrical activity also allows

them to penetrate.

Axons were exposed to Solutions of venom or in the case of control axons

to normal sea water for 30 min., followed hy a 10 min. washiug and hy a 60 min.

exjjosure to the C'^ lahelled compound heing studied. The axoplasm was then

extruded and C’^ assayed. A summary of resulls ohtained are shown in Fig. 10.

These data clearly estahlished that ACh and curare do penetrate after trealment

with lhe low concentrations of cotlonmouth moccasin venom which renders these

compounds active. In contrast after exposiire to even high concentrations of

Eastern diamondhack ratllesnake venom, curare neither penetrates nor does it

affect electrical activity. In control axons lhe penelration of ACh, choline and

dimelhylcurare is less lhan 1 per cent of that exjiected if no harrier were present.

Even these low values however may not rejiresent aclnal penelration hut may he

dne to conlamination during lhe process of extrusion, or the presence of trace

amounts of C’‘-tertiary nitrogen containing compounds or trace im|)nrities in the

radioactive samples of the qnaternary compounds. This suggestion is snpjiorted

hy the finding that the penelration of ACh in venom trealed axons appears to

increase with increasing lime of ACh incuhation, whereas in axons not pretreated

with venom the apparent ACh penelration after 5 min. incuhation is as great

as after 60 min. The amonnt of contaminalion occurring during extrusion would

he expected lo remain approximalely constant regardiess of whether the ACh

incuhation time was 5 or 60 min.

We fonnd lhe penetration of ACh to he equally low hoth in axons dissected

free of all adhering small nerve fihers as well as in axons containing mnch con-

nective tissue and surrounding small nerve fihers. This agrees with our findings

that ACh had no effect on conduction in hoth lypes of prejiaralions (23), hut is

in disagreement with a report that ACh rajndly enlers lhe axoplasm of finely dis¬

sected axons (37). In that study, moreover, the electrical activity of the axons

was not checked, which makes the meaning of the ex[)erimenls questionahie. In

contrast to the qnaternary compounds lhe li[)id soluhie tertiary nitrogen derivativo

Irimethylamine readily penelrated (Fig. 10) even without venom treatment which

confirms an earlier report (6).

,1 1 SciELO

488

USE OF VENOMS IN STUDIES ON NERVE EXCITATION

ACh Choline Dimethyl Trimelhy!

Curare omine

Fig. 10

[n lhe studies (lescril)ed radioactivity has l)eeti iiieasiired iii lhe axoplasm,

althoiigh our iiiterest is iii knovviiig lhe cortcenlraliou of lhe compounds reaching

lhe aclive siles of lhe mendirane which are surroimdÍTig lhe axoplasin. Allhoiigli

only aliout 80 A'’ ihick (38,89), lhe ineinhraiie is eomplex and lhe exact localioii

of lhe ACli syslein is nol kiiown. ll is impossible lo kiiow whal lhe concenlralion

of ACh or curare may he iti lhe meml)rane compared lo llial foimd iu lhe axo-

plasm. Even llie meaning of concenlralion in such a memhrane is nncertain.

Considering lhe wide variely of compoimds lo which nerve axons diiring lheir

enlire lenglh may he exposed in lhe liody. one woidd ex])ecled llial lhe receplor

areas of lhe excilahie memhrane may he especially well prolecled againsi lhe aclion

of externai comjioimds. Measuring lhe concenlralion of lhe compoimds lesled in

lhe whole axon would he of qiieslionahie significance, since lhere is non specific

hinding of charged molecnies such as ACh and curare wilh mauy macromolecules

in lhe Schvvann cell, i.e., exlernal lo lhe aclive neuronal memhrane )23 ).

1 SciELO

Mem. Inst. Butantan

Slmp. Internac.

33(2):‘477-50S, 1966

PHILIP ROSENBERG

489

Since \ve tiad foiiiul tlial lhe ]) 0 \veiful CliE inhihilor iieostigniiiie does not

affeel eoiiductioii eveii after venom pretieatnieiit. \ve nieasiired its per ceiit ])e-

iielralioii iiito lhe axoplasin iising a magnetic diver deviee(21l. Oii coiitrol

axons or afler trealmeiit of lhe axon wilh 25 p-g/nil cotloiinioiilh moccasiii venom

only 0.1 per cenl penelration of neo.sligniine vvas ohserved. vvhile afler 100 ,u.g/rnl

of venom, a concenlralion whicli hloeked eonduclion. 1 |)er cenl petielralion vvas

ohserved. The poor penelralion of neosligmine lherefore appears lo l)e in good

agreemeiit vvith ils ineffecliveness in Idoeking eleclrical aclivily. Neosligmine

thus is apparenlly even more lijvid insoluhle lhan ACh or curare.

The increased jvermealiilily prodiiced hy llie venoms agrees wilh snggeslions

made many years ago. even hefore any experimenlal evidence vvas availalde, lhat

niany effecls of venoms are dne lo increased permealiilily of hiological memlvranes.

More recently il was foimd lhal moecasin venom increases llie passage of per-

fnsion fliiid ihrough frog alria(40) and lhe penelralion of jvrocaine into õog

seiatie nerve(41). Cohra venom releases ACh from hinding particles of lhe

eenlral nervous system ( 421 vvhile collonmoulh moecasin venom causes lhe release

of gliilamine oxalacelic transarninase from cerlain rat lissue(4). Easlern dia-

mondhack ralllesnake venom vvhich is ineffeclive in our sludíes vvas akso imahle

lo disrnpl rahhil liver microsomes in conlrasl lo anolher venom vvhich vvas foimd

effective (44). The ahilily of many venoms lo hemolyze red hiood cells is vvell

known (45-47). Ralllesnake venom hovvever has lillle or no aclivily as a hemo-

lysin or hemagglulinin vvhereas coltonmoulh moecasin is very aclive (35). Il is

significanl lhal in many of lhe sludies moecasin or cohra venom is more [lolent

lhan ralllesnake venom in disrnpling memhranes vvhich agrees wilh our residis.

TABLE III — PENETRATION OF VARIOUS C* LABELLED CO.MPOUNDS INTO THE

AXOPLASM OF CONTROL AND AGKISTRODON P. PISCIVORUS VENOM TREATED

SQUID AXONS

AU compouncls vvere appiied for one hour in a concenlralion of 4.5 X 10-“ M excepl

for 1 X 10-“ M indoleacelic acid, 1.9 X lo-'> M dieldrin and 2.5 X 10-‘ M diphenyl-

hydanloin, corlisol and dehydroepiandroslerone sulfale. AgkÍKtrodon p. piscivoriis venom

applied for 30 minules in a concenlralion of 100 ,/ig/ml blocked eonduclion. None of

lhe olher compounds had any effecl on eonduclion during lhe 1 hour appiicaliun.

GABA = 7 Amino bulyiic acid, DOPA = 3,4 dihydroxyphenyialamine, DOPA mine = 3,4

dihydroxyphenylelhylamine. Dala from Iloskln and Ro.senberg (22).

COMPOUND

Per

Conlrol

cenl penelralion

Venom prelrealed

Glucose

Mannitol

Sucrose

Glulamale

Glutamine

Aspartate

GABA

DOPA

DOPA mine

Serotonin

Indoleacetic acid

Acelylsalicylic acid

Diphenylhydanloin

Corli.sol

100

Dieldrin

100

Dehyd roepland roslerone .sui fale

—

SciELO

10 11 12 13 14 15

490

USE OF VENOMS IN STUDIES ON NERVE EXCITATION

The stiulies of the |)erineahility jjroperties of the squid

exlentlcd hy ineasiiring Üie penetration iiito the axojjlasm of a wide variety of

compoiinds (Tahle III). Excepl for glucose, ])enetration of the siil)stances studied

ap])ears to depend largely on the exteiit of the non-polar, lipo[)hilic charaeter of

the eompoimd. This conclusion is hased on experiments with squid axou, how-

ever it appears possihle thal cell memhranes from widely different sources may

have sufficient properties iu eommon to give validity to such generalization. The

glucose penetration appears to he metaholically mediated, indeed studies with

specifieally laheled glucose indicated the participation of the jientose phos])hate

pathway as a metaholic route in axonal memhrane and associated cell wall material

and partial or complete ahsence of the oxidative system in the axoplasm (22).

Clutamate, glutamine, GABA, aspatate, DOPA, DOPA mine and serotonin, all of

which penetrated poorly have two or more iotnzahle groups each, selected from

carhoxyl, amine and j)henolic hydroxyl groups, and exist almost always in a

charged form. In contrast aspirin, indolacetic acid and diphenylhydantoin which

have only one ionizahle group, and which would he in equilihrium with a

significant amount of the dissociated lipophilic form, penetrated the untreated

squid axon much more rapidly. Dieldrin is an extremely water-insoluhle in-

secticide, and cortisol is a lipid-soluhle steroid. In contrast dehydroe])iandro-

Fig. 11

SciELO

Mem. Inst. Butantan

Slmp. Internac.

33(2):477-508, 1966

PHILIP ROSENBERG

491

slerone sulfate is a water-solulile com]jletely ioiiized derivative of an olhervvise

water-insoluhle steroid. The steroid and steroid sulfate thus constitute a striking

exaniple of the effect on iiermealúlity of introducing a highly polar coni])letely

dissociated substituent into a lipophilic moleeule. Moccasin venom treatment seems

to iucrease the |)enetration of many poorly penetrating compouiids in a not too

specific way.

The permeahility harrier of the squid axon may he the axolemma, Schwann

cell or connective tissue surroimding the axon. According to as yet unpid)lished

ohservations of Dr. David Rohertson with electron microscojiy of venom pretreated

axons, the venom produced marked alteration and disintegration of the Schwann

cell; the axonal plasma memhrane appeared to he not affected (Fig. 11). Ville-

gas and Villegas (38) have shown that Schwann cells are traversed by relatively

large channels, however their tortuous nature may still make penetration difficult.

Higher concentrations of venom may hlock electrical activity hy a gross disru|)tion

of the axonal memhrane or its receptor areas. The venoms might also increase

the concentration of curare and ACh which reaches the active sites hy disrupting

nonspecific hinding sites with which charged molecules such as curare and ACh

might he ex])ected to interact (e.g. nucleic acid, chondroitin sulfate etc.).

Ai.tekatiox oe acetylcholi.ne penetration into, and effects on

VENOM-TREATED SQUID AXONS

An ap])arently surprising result in our studies with venom pretreated squid

axons was the finding that low concentrations of physostigmine applied in com-

hination with ACh did not enhauce its action as would have heen expected with

a |)otent ChE inhihitor, hut markedly antagonized its action. Curare in contrast

did not antagonize the action of ACh (Tahie IV). Similar unexpected ohserva¬

tions had previously heen made on the vagiis and lohster nerves (10,48). Floth

of these findings are opposite to that observed at junctional sites where low con¬

centrations of physostigmine enhauce the action of ACh and curare antagonizes

TABLE IV — PENETRATION INTO AXOPLASM AND EFFECTS ON ACTION POTENTIAL

(AP) OF VENOM TREATED SQUID GIANT AXONS BY C“ ACETYLCHOLINE (ACh)

ALONE AND COMBINED WITH OTHER COMPOUNDS

Axons were exposed to 4.5 X 10-' M ACh for one hour. AH axons were pretreated with

25 /ig/ml Apkistroilon p. piscivorvs which had no effect on the AP. Data taken from

Hoskin and Rosenberg (19), and Rosenberg and Podieskl (16).

COMPOUNDS

Decrease

Per

cent

Penetration

ACh

ACh-FchoIine 2.7X10-" M

ACh + oholine 5.4X10-3 M

—

ACh-l-choline 2.7X10-'' M

ACh-|-neostigmine 1.7X10-2 M

ACh + physostigmine 5X10-* M

ACh + physostigmine 5X10-“ M

ACh + curare 1X10-“ M

—

ACh-F curare 7X10-' M

100

SciELO

10 11 12 13 14 15

492

USE OF VENOMS IN STUDIES ON NERVE EXCITATION

ils acüon. Botii clioliiie aiul iifosligmine. vvhicli cvcii iii liigli coiifentralions havc

110 cffect on coiuliiction of lhe venoni trealed squid axon aiso inarkediy antagonize

lhe effeel of ACh on lhe action potential (Tahle IV).

Nachmansohn had suggesled lhal there may he a competition helvveen lhese

(:oin|)onnds for tlie palliways of penetration in tlie structnres surrounding lhe

eondueting memhrane; i.e., externai to the reee|)tor areas of lhe nieinhrane. We

llierefore undertook slndies wilh radioactive eoinpoiinds to see hovv well the effects

on electrieal activity eorrelated with the iienetration of ACh. Choline. neoptigmine

and physostigmine decreased the [lenetration of ACh in venom treated axons froin

ahoiil 4 per cent to ahoiit 1 per cent whieh is lhe levei of penetration ohserved

in eontrol axons not treated wilh venom (Fig. 10 ). In conlrasl curare did nol

decrease hui actnally increased the jienetration of ACh (Tahle IV).

The resuits indieate that physostigmine. neostigmine and choline decrease the

effects of ACh on conduction hy eompeting wilh ACh for ])enetration jiathways.

The competition ajipears lo he concentralion dependent for choline, and choline

seems to compete with ACh for penetration on a mole for mole hasis since con-

centrations of choline ahont eipial to that of ACh are needed to dernonslrate

anlagonism. In contrast the antagonistic action of physostigmine is mueh more

potent than that of choline sinee 5X 10“' M physostigmine decreased lhe pe-

netralion of ACh as mnch as 5 X lü“" M agreeing wilh lhe ohservations on

electrieal activity. Since neostigmine, choline and lhe low concentralion of jihyso-

stigmine have no direct effects on conduction il would he difficull to assume

lhal lhey are com|)eling with ACh for a speeific site in lhe memhrane essenlial

for electrieal activity. This conelusion is further su|)[)orted hy the ohservalion

that it is no longer [jossihie to demonstrate anlagonism of ACh |)enelration hy

choline when the jiermeahility harriers lo ACh are no longer the limiting faclor

when the axons are jiretreated with 100 /xg/ml eotlonmoulh moccasin venom, a

concenlration whieh hlocks electrieal activity and inarkedly increases the |)enelra-

lion of ACh (Kig. 10).

The.se resnits eonfirm and exlend the previous ohservations on the importance

of structural harriers snrroimding lhe axonal eondueting memhranes in determin-

ing effects of Chemical compounds on electrieal activity. ACdi. choline, neostigmine

and curare are all qiiaternary nitrogen derivatives while physostigmine al the pH

used is ])artly in lhe eharged fonn. They may all reacl with negatively charged

gronps in the rednced hui slill persisting harrier and may modify lhe effeels

ohserved when two of lhem are ap|died simultaneousiy. A|)parenlly, howev(>r.

lhey do not react with the same gronps in lhe sqnid axon preparation since

curare facilitates lhe passage of ACh while choline and neostigmine decrease ils

passage. In conlrasl curare markediy decreases lhe penetration of a (piaternary

nitrogen compound in skeletal miiscle (49) emphasizing lhe variahility of inler-

action helween compounds whieh may he ohserved in differeni |)reparations.

Il was of inieresi to invesligate whether there was a degree of siiecificity

ahont this comjielilion phenomenon. The |)enetralion of ACh was lherefore lesled

in the presence of sucro.“e. a non charged jiharmacologically indifferent com|)ound.

Sucrose had no significanl effeel on ACh jienelralion (28). indicating lhal charged

molecules may more effeclively compete wilh A(.'h for jienetration sites than un-

charged molecules. Our findings on lhe axon are due to the enlirely different

environrnent from that al the jimclion where for exam[)le neostigmine and phy.so-

sligmine at low eoncenlralions increase the action of ACh hy lhe inhihition of

AChK and neostigmine in addilion hy activating the reee[)tor. 'lhe exacl sites at

SciELO

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PHILIP ROSENBERG

493

Simp. Internac.

33(2):477-5Ü8, 19H6

vvliifh tliese toin|joiin(ls aiitapoiiizt' lhe |K‘iielialion uf ACh is iiol known. hiit it

must he kepl iii mind, lhat llie Schwatiii eell siirroumhng lhe squid gianl axon

is alioul 2000 A thick eompared to lhe 80 A thickness of lhe excilahle niemhiaiie

itself.

Externally added ACh decreases lhe action |)oteiilial and de|)olarizes lhe

rahl»it vagus after renioval of llie sheath (48). Hovvever. lhe likelihood of lliere

l)eing a physiological significaiice to this effect was eoiisidered remote hecause

neostigmine and olher conij)ounds wliicli had no effecl on conduction antagonized

lhe action of ACh (50). As in llie sqnid axon lhe condneting niemhrane in lhe

vagus pre])aration is surrounded l)y a large layer of otlier structures. and thus

il appears lo us likely lhal an effect on penetralion similar to lhat descrilied

here is lhe exjdanation for their findings.

InCREASEI) CnOl.INESTERASE ACTIVITY GE IXTACT CEI.LS CAESEI) BY VE.XOMS

To determine vvhether snake venom increa.sed permeahilily to ACh in jjie-

paralions olher than lhe squid giant axon it was necessary to use techniques nol

requiring lhe extrusion of lhe cells contents. It lias heen shown thal lhe ratio

of Chf] activity in an intact and homogenized preparation, to vvhich we shall

refer to as the jiermeahility constant (PC), serves as an index of lhe iiermeahility

harrier (51). If the CliE of the tissue were completely accessihle to ACh, which

is used as the suhstrate for measiiring the ChE activity, then the PC wonld he

1, whereas complete inaccessihilily would give a PC ratio of 0.

The PC ratios in control |)reparalions of squid stellar nerve, eel eleclrojilax,

walking leg nerve of lohster, frog sartorius muscle and rahhit cerehral córtex

indicated lhat the eel eleclrojilax has the strongest harrier to ACh whereas the

squid stellar nerve has lhe weakesl (Tahle V). The squid stellar nerve is composeil

of lhe gianl axon plus small nerve fihers. Since gianl axons contain only

of lhe total ChE activity of the squid stellar nerve (52) these figures would no)

indicate lhe harrier iiresenl in lhe giant axon. Hecause of lhe relalively low

euzyme activity of the giant axon it is nol possihie to measure small changes in

its activity. The grealest fraction of ChE of lhe gianl axon is located in the

envelojie. The surface arca of lhe giant axon is niuch le.ss than lhe total surface

area of lhe small nerve fihers. Therefore, it is nol surprising lhat lhe majorily

of lhe (diE is in lhe small fihers.

Agkistrodo/i p. piscivorus and lo a lesser exlent Crotalus adainanletis venom

caiised an increase in the ChE activity oí intact axons of lhe sqnid stellar nerve.

lhe fihers of the lohster walking leg nerve and lhe isolated eel electroplax (Tahle

V). The grealer effectiveness of cottonmouth lhan ratllesnake venom in increas-

ing |)ermeahilily agrees wilh ])revious findings in olher systems lhal ratllesnake

venom is less effective in its ahilily lo disrupl memhranes. Since lhe venoms

used have no ChE activity themselves (20,53,54), their effects must he altrihuted

lo a greater accessihilily of ChE in the intact preparations as previousiy disciissed.

In contrasl to the complete ineffecliveness of Eastern diamondhack ratllesnake

venom on lhe squid gianl axon (Tahle 11 and Eig. 10), il increased the permeahil-

ity of the sqnid stellar nerve ailhongh less lhan the collonmonlh moceasin venom.

The increased permeahility cau.sed hy lhe venoms in lohster nerves agrees wilh

lhe findings lhat collonmonlh venom decreases the concenIralioTi of ACli required

to affect electrical activity 111 ).

2 3

5 6

11 12 13 14 15

494

USE OF VENOMS IN STUDIES ON NERVE EXCITATION

TABLE V — EFFECT OF VENOMS ON PERMEABILITY OF VARIOUS PREPARATIONS

AS JUDGED BY THE ABILITY OF 5 X 10-» M ACETYLCHOLINE (ACh) TO PENETRATE

AND ASSAY ALL OF THE AVAILABLE CHOLINESTERASE (ChE) IN INTACT BIO-

LOGICAL PREPARATIONS

The permeabillty constant (PC) is the ratlo oI ChE activity of intact and homogenized

tissue. AP = Agkistrodon piscivorns venom; CA = Crotalus adamanteus venom. AIl

ChE activities are expressed as ACh hyd/g/hr except for the electroplax results

whlch are presented as iiM ACh hyd/cell/hr (Average cell vveight is 40 mg). Data taken

from Rosenberg and Dettbarn (20, 25).

TISSUE

Venom

/ig/ml

ChE

Homog.

Intact

Squid Stellar Nerve

0.74

0.77

0.96

0.79

200

0.91

Eel Electroplax

0.17

0.25

400

0.34

1000

0.63

0.20

1000

0.28

Walking Leg Nerve of

Lobster

—

925

337

0.36

100

900

442

0.49

1000

886

698

0.79

1000

900

475

0.53

Frog Sartorius Muscle

0.41

1000

0.84

Rabbit Cerebral Córtex

260

138

0.53

1000

285

152

0.53

1000

256

156

0.61

Permealiility l>ariiers are j)re.çent iii the intact ral)l)it cereliral córtex pliees

as is sliown hy the a])|)roximately doiihling of ChE activity oh.served ii])on homo-

genization, hut neither venom vvas ahle to significantly increase the accessihility

of the enzyme to ACh in tliese slices. This is in agreement with earlier stndies

showing that Naja naja venom cannot hydrolyze ])hos|)holipids in hrain slices (55).

As we shall discnss in Section 8. s])litting of phospholipids might he essential for

increasing permeahility. An interesting and imexpected finding was the demons-

tration in homogenized electrojilax of strong permeahility harriers. This was the

only |)reparation in which the venoms increased the ChE activity even of homo¬

genized tissne. The harriers in the homogenized tissiie conld he ])artially eliminated

hy longer jieriods of homogenization or hy freezing and thawing of cells; how-

ever, the use of venoms appeared to yield the highesl enzyme activity. Jn the

homogenized electroplax hoth venoms were ahout eqnally effective in contrast to

SciELO

10 11 12 13 14 15 16

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PHILIP ROSENBERG

495

Slmp. Internac.

33(2):477-50í<, 1966

llie weaker effects of Crotulus venoin iii iiilact jireparations. This niay

iiulicate an inahility of rattlesnake veiiom comjionents to ])enelrate lo siilistrate

sites in the inemhraiie of intact preparatioiis (see section 8).

Ueraiise, as disciisscd ahove, suhstrates siich as ACh do nol jienelrate to all

of the ChE it is diffieiilt to determine the total enzyme aclivitv in intact nerve

fihers. In studies aimed at measuring ChE activity of a tissiie following exposnre

lo an “irreversil)le” organophosphale ChE inhihitor, it is essential lo measnre

activity in intact preparations, since excess inhihition diie to tlie jiresence of

lincomhined inhihitor inay occiir at the time of homogenization. Several ])ro-

cediires have heen nsed in attempts to measiire the total activity in the intact

preparation. Hovvever none have heen conipletely satisfactory (56, 57). Our re¬

sidis indicate that venoms may he useful in future studies where it will he essential

to ohtain some indication of lhe total ChE activity in intact jireparatious.

IJSE OF VENOMS IN TESTlNG FOK ESSENTIALITY OF CHOLlNESTERASE IN CONDLCTION

The ahility of venoms to decrease permeahility harriers in the sqiud axon

and ex])ose ChE to ACh (Fig. 10, Tahle V) offered lhe jiossihility of designing

experinients to test for the essentiality in nerve conduction of ChE, the enzyme

vvhich hydrolyzes ACh. Organophos])hates, such as diisopropyl jihosphorofluoridate

(DEP) or diethyl-p-nitro]ihenyl phosphate (Paraoxon) jihosphorylale and therehy

irreversihly inhihil ChE and hlock electrical activity (3-51. It is possihle to re-

activale the phos|)horylated enzyme wilh certain nucleophilic compounds among

which pyridine-2-aldoxime methiodide IPAM) is extremely potent and has a high

degree of specificily for ])hos[)horylated ChE (58). ff hlock of conduction hy

organophosphates is specificallv due lo hlock of ChE and if ChE is essential for

conduction as postulated hy Nachmansohn (3-5) it might he jiossihle hy using

PAM lo specifically restore conduction. Experinients of this lyjie are extremely

difficult to carry ont however, hecause PAM, like ACh and curare heing a lijiid

insoluhle quaternary nilrogen compound cannot penetrale lhe conducting mem-

hranes of nerve and nurscle. In the lohster axon where lhe permeahility harrier

is less as evidenced hy the ahility of ACh to directly hlock conduction (10,11).

it was possihle to overcome conduction hlock hy Paraoxon with PAM (59). It

was lherefore worthvvhilc' lo lesl on lhe venom Ireated squid axon, where the

permeahility harriers are retluced and the aclive sites of ChE exposed, whelher

PAM could restore electrical activity which had heen liloeked hy DEP or

Paraoxon.

It was found that in axons not venom pretreated lhe hlock of conduction

hy Paraoxon, iu the concenlrations used and lhe lime of exjiosure appiied, was

reversihle whereas in axons ireated wilh 25 jiig/ml coltoumoulh moccasin venom

lhe hlock was irreversihie (25). At lhe lime of reversihle hlock of conduction

ChE activity is stili presenl and in no case was conduction found in the ahsence

of enzyme activity (3-5,60,61 ). This indicates that Paraoxon may he reacting

with some component of lhe memhraue other than ChE, possihly with the active

site of lhe receptor, to cause reversihle hlock. The irreversihie conduction hlock

hy Paraoxon after venom may he either hecause of the greater exposnre of ChE

to Paraoxon or hecause venom prelreatrnent inactivales the enzyme which hydro¬

lyzes organophos|)hates. It was found that lhe squid axon has eonsiderahle

amounts of this enzymatie activity, holh in the envelope and in the axoplasm (62).

Conduction iu 11 out of 16 venom Ireated axons was irreversilily hlocked hy

2 3

5 6

11 12 13 14 15

496

USE OF VENOMS IN STUDIES ON NERVE EXCITATION

Paraoxon, whereas iii axons exposed lo PAM followiTig Paraoxoti llu> lilock of

conduction l)v l^araoxoii was irreversilile iii onlv 6 of 17 axons (25). An aclnal

ex|)erjment wilh Paraoxon and PAM is sliown in Fig. 12. I’AM aiso restored

electrical activity which had I)een lilocked hy DFP|25). These exjicrinienls

confirmed lhe inseparahle associalion helween eleclrical and CliF aclivily.

B _

Fig. 12

MeCHAMSM of VENOM ACTIO.N ON THE SQUII) GIANT AXON

In addilion lo non-enzyniic íoni|)onenls venonis conlain inany enzymes in-

clnding plios])holi|)ase A, hyaliironidase, jiroleolylic enzymes, 1-ainino acid oxidase,

phosjdiodieslerase, ele. (15, 16,61-66). Venoms oí lhe ELAPIDAE such as Naja

naja venom liave FhF aclivily (55,51). ll is ohviously of greal inlerest lo de-

lermine lhe comiMinent (or coinjionenls) of lhe venonis responsihie for increasing

llie jierrnealiilily of lhe sqnid giani axon.

The effecls of varions venom fraclions, enzymes and jihospholipids are shovvn

in Talile VI. Thosí' componnds which hlocked lhe aclion |)olenliaI did so irre-

versihly. The concenlralions of lhese agenls nsed as |)relrealmenl had no effecls

on lhe aclion polenlial. The effeci of curare following phospholipase D was ir-

reversihle whereas all olher curare effecls were reversihle.

A non dialyzahie fraclion of Naja. naja venom rich in |)hos])holipase A (17,

67) was more polenl lhan Naja naja venom (Tahie II) holh in ils direcl effecls

on lhe aclion polenlial and as a prelrealmenl ageni (Tahie VI). ACh aIso

affecled condnclion afler lhe |)hos|)holi|)ase rich fraclion. In conirasl a dia¬

lyzahie fraclion of cohra venom poor in ])hospholi])ase A (17,67), recpiired

relalively high concenlralions lo hlock condnclion, and following jirelrealmenl of

lhe axon wilh il curare was com|)lelely inaclive (17). The comjiaralive phos|)ho-

li|)ase A aclivilies of lhe rich and poor fraclions shown in Fig. 15, indicale lhal

lhe rich fraclion is al leasl 200 limes as aclive as lhe poor fraclion.

SciELO

10 11 12 13 14 15 16

Simp. Internac.

Mem. Inst. Butantan

33(2):477-508, 1966

PHILIP ROSENBERG

497

TABLE VI — CONCENTRATIONS OF SEV^ERAL VENOM FRACTIONS, ENZYMES AND

PHOSPIIOLIPIDS USED AS PRETREATMENTS TO RENDER THE SQUID AXON SEN-

SITIVE TO CURARE, AND CONCENTRATIONS REQUIRED TO BLOCK THE CONDUCTED

ACTION POTENTIAL (AP)

Compouncis were applled for about .30 minutes. > indicates that at the eoncentration

shown no effect on AP was observed. + + +, ++, and — indicate respectlvely a 80-

100%, 50-80% and 0-20% decrease in the AP produced in 30 minutes or less by 1.4 X 10-» M

curare after pretreatment ot the axon. OH— and H-h indicate.s that venom solutlon was

bolled for 15 minutes at pH 8.5 or 5.5 after which the solutlon was cooled, pH readjusted

and then tested. Data taken from Rosenberg and Podleski (15) and Rosenberg and Ng (17).

COMPOUND

tig/m\

Curare effecl

AP block

Pretreat

Phospholipase A rlch fractlon

+ -H-

Phospholipase A poor fractlon

200

100

- -

Agkistrodon j). piscivorus

OH-

> 1000

200

—

Agkistroãon p. piscivorus

H4-

200

-n- -t-

Naja naja

OII-

> 400

200

—

Naja 7iaja

H-t-

100

+ +

Phospholipase C

> 500

500

—

Phospholipase D

> 100

-I- +

L-Amino acid o.xidase

> 100

100

—

Hyaluronldase

> 100

100

—

Cobroxin

500

200

—

Cobra venom Neurotoxin

> 250

250

—

Lysoleclthln

500

100

—

Egg lecithin

> 1000

Beef heart lecithin

> 1000

Pliospliolijiase A in venoins is rtisislanl lo lioiling al acid pH wherras it is

destroyed liv hoiling at aii alkaiine ])H (68-70). The otlier enzynies known lo

lie present in venom are destroyed l)y lioiling al acid or alkaiine ])H. Some

SciELO

10 11 12 13 14 15

498

USE OF VENOMS IN STUDIES ON NERVE EXCITATION

investigators tlierefore use acid heated veiiom Solutions as their source of plios|)ho-

li])ase A. As seen iti Tahle VI alcaliuc heated Solutions of A. piscivorus and Naja

naja did not render curare active while acid heated Solutions vvere effective. An

acid heated solution of cobra venom has also been found to de])olarize and hlock

conduction in lohsler axons(30). The comparative phospholipase activities of

lhe acid and alkaline heated Solutions are shown in Fig. 14. These results also

indicated that ])hospholipase A is res])onsihle for decreasing the ])ermeahility bar-

rier in the squid giant axon.

Neither ]-amino acid oxidase nor hyaluronidase, both of which are ])resent

in snake venoms, had any effecl on the squid axon (Tahle VI). A preparation

of phos[)hodiesterase frotn venom ajijieared to be highly |)otent, however \ve found

it to contain eonsiderable phospholipase A activity (17). Two different ])repara-

lions of nenrotoxin from N. naja venom (Cobroxin and cobra venom neurotoxin),

although higbly toxic to animais were also inactive on the squid axon. No

relalionshij) has been found between venom toxicity and any of the enzymatic

fractions in venom (64, 71-74). As noted in Tahle VI three |)hospholi])ids were

inactive as also were ])ho.s[)hatidylelhanolamine, [)hos|)balidylserine, L-a-cephalin and

L-«-lecithin. Phospholipases C and D are not presenl in snake venoms and we

shall discuss their actions laler.

The results shown in Tahle VI indicate that phospholijiase A of venoms is

responsible for their ability lo iticrease lhe jiermeability of the squid axon to

normally impenetrahle lipid-insoluble com|)ounds. Also in suj)j)ort of this su])-

posilion is the finding that Nolrchis scutata.s a venom highly potent in our sludies

(Tahle 11) also contains high eoncentralions of ])hospholipase A (75). In contrasl

Centruroide.s scalpturatas (scorpion) venom which contains no ])hos])holipase

A (76) was inactive in onr test syslem (Tahle II), although it produced repetitive

firing of the sqidd axon (24). As shown in Fig. 15 ])hospholi])ase A hydrolyzes

a íally acid ester from the (i ])osition of eertain glycerophosjíhatides yielding the

SciELO

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Mem. Inst. Butantan

Slmp. Internac.

3S(ü):477-508, 1966

PHILIP ROSENBERG

499

CH,—o—X,

R—C—O—CH o

i t

CHj—O—P—O—X,.—N(+)—Xj

/ I

O(-)

Phosphatidylcholine (lecithin)

C—R

(CH.,).,

(CH,);

Phosphatidylcthanolamine

C—R

(CH.,),

H 3

Phosphatidylserine

C—R

CH.^—CH

Phosphatidalchoiine

CH=CH—R -

COÜ(->

(CH,),

(CH,);

(choline plasmalogen)

Fig. 15

corres])oiiding lysopliosphatide. 1’revious studies hy many workers have showii

lhat phospholipase A is caj)alile of disrupling memhranes (33, 34,69, 77,78).

The soiirce of ])hosj)liolipase A in mosl of these sliidies was acid heated solulions

of venom. Since we ol>served such a great variahilily in venom j)olency (Tal)le

II) we decided lo compare the |)liospholipase A activities of cerlain venoms. In

most of the nieasiireinents of ])hos]diolipase A activities we used egg or heef heart

lecithin rather than ])urified sul)slrates l)ecause hydrolysis was slower and in-

complete wdth piirified snhstrates (17). Snake venom phospliolipase A has l)cen

reported to hydrolyze egg yolk and seriim ])hos])holipids at a rale 10 to 20 fold

greater than that olilained with juirified ovolecitliin ])reparations, and it was sug-

gested lhat this may he diie to the specific natnre of the comhination of phospho-

lipid and protein in lhe crude snhslrate ])rcparation (79). The sample of heef

heart lecithin iised contained ahoul 60% phospliatidalcholine (choline j)lasma-

logen) and 40% phosphatidylcholine (lecithin). It was re])orled hy Gottfried

and Kapport (80) lhat cohra venom hydrolyzes egg lecithin at only a slighlly

faster rate than heef lecithin whereas AVeslern diamondhack ratllesnake venom

hydrolyzes egg lecithin considerahly helter than heef lecithin. In addition when

thcy ohtained purified plasmalogen from heef lecithin the difference in hydrolysis

of lecithin and jilasmalogen hy ratllesnake venom was even more marked. Since

Kastern diamondhack ratllesnake venom did not render curare active whereas

cohra and coltonmoiith moccasin venom did it appeared lhat an action of the

venom iijion j)lasmalogen might he responsihle for their ahility lo render curare

active. As seen in Fig. 16 we found that collonmouth venom hydrolyzed the

two snhstrates at similar rates, whereas rattlesnake venom is somewhat more ef-

fective in hydrolyzing egg than heef lecithin. Quantitatively however this dif¬

ference in reaction rale does not a|)pear snfficient to aceonnt for the marketl

differences in potency of these two venoms on lhe sqnid axon. When the hydro¬

lysis of heef lecithin hy various venoms was com])arcd it was fonnd that two of the

venoms most cffeclive in rendering curare active also hydrolyzed heef lecithin at

lhe highest rates, hnt hvdrolysis hy lhe other three venoms did not correlate with

their ahility lo render curare active (Fig. 17). It will he necessary to actnally

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USE OF VENOMS TN STUDIES ON NERVE EXCITATION

mcasure the phosjjholipids in tlie squid axoii as wcdl as lhe changes ])roduced hy

cotlonniouth and rattlesnake venoms to see if there is a correlaüoii hetween s])lit-

tiiig of any j)articular phospholipid and potency of the venoms. The nervous

syslem of course eontains many ])hospholi])ids in addition to lecithin and choline

j)lasinalogeti ([)hosphatidalcholine) ; for example, phos[)hatidylethanolainine, phospha-

tidylserine, |)hosj)hatidalethanolamine, phospfialidalserine, inositol pliosphatides and

sphingomyelins (81-87). The concenlralion of phosphalidaleholine is actnally nnich

less than thal of phosphatidalelhanolainine (87, 88). Lecithin is nol even a coin-

])arcnt of the myelin lipids whereas j)lasmalogens, inositol pliosphatides and s])hingo-

inyelins are (81,89,901. Quantitalively plasinalogens forni one of the largest

jiart of lhe jihosjihalides fonnd in while niatler 183, 91).

In addition lo lhe possihilily thal lhe phospholipase A in coltoiimoulh and

rattlesnake venoms have different siieeificities tow'ard phos|)holi|)id suhstrates it is

also possihle thal there are isozymes of phospholipase A vvhieh have differenees

in lheir molecular structnre, whieh may explain the marked differenees in lheir

jiotencies. ll has heen reported that there are differenees in lhe electro|)horetic

migration of lhe ])hos|)holipase A fraetions from different venoms (92, 93). Il

has also heen jioinled onl thal it can no longer he assnmed that a single enzyme

in each venom is responsihie for the hydrolysis of all phosiiholipid snhslrates (80).

In cerlain venoms there liavi^ heen foimd more than one |diospholi|)ase A, each

wilh different |)hysical properties (75,91), and different sjiecifie activities when

tesled on varions phospholi|)ids. It is of s|)ecial pertinence to our data thal the

j)hospholi|)ase A from llinghals and Naja naja venom are ahle lo split phospho-

lipids in certain memhranes whereas lhe phos])holi[)ase A from V. palf.slinac is

imahle to do so (55,95). Similar differenees conld he res|)onsihle for the marked

difference in |)olency hetween rattlesnake and colloninoiilh venom.

In addition to the possihilily that the phospholi|)ases from rattlesnake and

cottonmonlh venoms have different snhstrale specificities or different molecnle

strnclnre there is also lhe possihilily that there is anolher faclor in lhe active

venoms whieh |)repares the memhrane for attack hy venom pliospliolipai^c. Such

a factor, the ilirect lytie faclor (DLF) has heen isolated from cohra and hee

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PHILIP ROSENBERG

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venoms. liiil is nol ])iest>iil iii lerlaiii vijier venonis ( 7H. 93. 96-98). For i'xaiii|)le

waslied erytlirocytes are resistani lo lhe aelion of pliospholiiiase A ohlaioed from

varioiis veiioms. whereas lhe addilioii of DLF, whieh ilself has no |)hospholi])ase

A aclivily rendeis lhe red cell phospliolipids snsceplihle lo atlack liy jiliospliolipase

A of lhese venoms (96). Df.F mij 2 ;hl allow venoni jihospholipase A lo slijil llie

phospholijiids in lhe inlael sqnid axon. If this faclor were ahsenl in raltlesnake

hiit ])resenl in eollonnioiilh venoms this conld exjdain our results.

Assnming thal phosjiholipase A is lhe aclive eomjionent of tlie venoms it is

of inleresl to knovv whether lhe venom effects are due to a direct aelion of

phospholipase A on phos]dioli])ids or dne to lhe liheration, hy lhe aelion of

phospholipase A on pliosjdialides, of lysophosphalides in lhe memhrane. For

example lysolecilhin a eompoimd wilh delergenl projierlies is formed hy lhe hydro-

lysis of lhe fatly aeid esler al lhe 13 posilion of leeithin hy phospholipase A (Fig.

15). It is ihoiighl lhal indirect hemolysis of red hlood cells hy venoms is due

lo lhe aelion of lhe lysophosphalides formed from lhe aelion of jdiosjiholipase A

on lhe mernhranal phos])holipids (99). Bolh j)hos|)holi])ase A and lysoleeilhin

cause demyelinaliug ehanges iu lhe ceulral nervous syslem (83), while lysolecilhiu

and venom eause a release of ginlamie-oxaloaeelie Irausamiuase from vvhole eell

preparalions (13). Il was aiso suggesled lhal hloek of eondueliou in lohsler

nerves hy healed venom solulious may he due lo lhe formalion of lyso|)hos])ha-

lides(3()l. Olher invesligalors (77, 78, 100, 101), for various reasons, ihiuk lhal

lhe effects they have ohserved are due to a direct aelion of phosjiholipase A and

nol due to a liheration of lysojihosphatides. For example, in two of lhese sludies

(78,100), ])hospholipase C had a similar effecl as healed venom Solutions which

was takeii as evidence lhal a lyso|)hosphatide is nol imporlant in lhe aelion of

phosidiolipase A. The aelion of ])hos|)holipase C on ])hos|)hatides does nol give

rise to lysophosphalides (Fig. 15). Iu our sludies however phospholi|)ase C was

inactive (Tahie VI).

Iu altemptiug to decide whether phos[iholipase A is actiug direclly or whether

lyso[)hos|)hatides are responsihie for increasiug permealiility an ohservalion made

on squid giant axons after removal of olher fihers is of relevante. Afler having

found thal ACh was inactive on conduction and unahie lo penelrate hoth on

cleaned and less earefully dissecled axons (see section 2 and 3) we have foi-

lhe sake of eonvenience used gianl axons wilh only partial removal of surrouud-

ing fihers. The apparentiy sur|)rising ohservalion was later made thal collon-

nioiith venom is |)ractically inerl on axons earefully dissecled, lhal is, wilh all

sinall iierve fihers aud as much conuective tissue as possihie removed(15). Fol-

lowing ap|)licatiou of even high coucenlrations of cottonmouth venom on finely

dissecled axons, curare and ACh were inactive and peuetraled no hetter lhan iu

axons nol prelrealed wilh venom (23). These results are iu marked conlrasl to

effects of lhe delergenl celyltrimelhviammonium hromide whith is known lo direcl¬

ly disru|)t mcmhraues, and which was even more polent ou lhe fiuely dissecled

axon lhan on lhe preparation wilhout removal of small fihers (23). A likely

explanation for lhe ohservalion wilh venom on cleaned axons is thal lhe venom

is actiug through lhe formalion in or near lhe memhrane of a secoudary |)roduct

which iu luru affects electrical aclivily hy disru|)ling liarriers eilher withiu or

externai lo lhe memhrane. Iu lhe closely dissecled axon there would he less

suhslrate on which phospholipase A conld act aud therefore less lysojihosjihatides

formed. The lyso comjiouuds may aIso increase lhe aetivity of |)hos|)holipase A

(102). Attempts were made to demonstrale lhe produetiou hy coltoumouth venom

of a eompound wilh polenl effeels on electrical aetivity. The venom was ineuhated

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USE OF VENOMS IN STUDIES ON NERVE EXCITATION

vvith sqiiicl axons and with t'gg and lieef lecilhin, hiit no coni|)onnd was ohlained

froni the incuhation mixture with potent effects on electrical activity. Tliese

negative results ohviously do nol exclnde lhe possiliility that such cornpoimds may

he íormed since adding a lyso[)hosphatide extcrnally may not give results eqnivalent

to its formation within lhe preparation.

Although phospholi])ase C and D are not present in venoms, the effects of

lhese two comj)oiinds on the squid axon are interesting. While neither of them

had any direct effects on the action ])otential, ])hospholipase D even in relatively

low amounts rendered curare active (Tahie VI). The hlock of conduction hy

curare after phospholipase D could however not lie reversed. Hydrolysis of

phosphatides hy {)hospholipase D removes a cationic nitrogen 11'ig. 15). One

may specidate that the cationic nitrogens of curare may he attracted to the

negatively charged phosphates. The curare molecules may pass from one ])hos])hate

lo another and finally penetrate to the active sites of tlie neuronal memhrane and

affect electrical activity. The concentration gradient of curare and its hinding

might he such that with washing it would he difficult to remove enough of lhe

curare to reverse its effects on electrical activity. Hydrolysis of phosphatides hy

phospholij)ase C removes the j)hosphate grou[)ing as well as the cationic nitrogen

leaving an uncharged diglyceride which would not he expected to hind curare.

SUMMARY

It has been possible with the aid of venoms to analyze some of the effects

which the structure and organization of biological membranes and their environ-

ment have on the physiological functioning of the acetylcholine (ACh) system and

on the action of compounds when appiied externally to biological tissues.

1) Lipid soluble tertiary nitrogen containing compounds such as atropine and

physostigmine which are known to interact with the ACh system at junctions also

block conduction in lhe squid giant axon, indicating the presence of the ACh

system in this preparation. In contrast lipid in.soluble quaternary nitrogen con¬

taining compounds such as ACh and curare do not affect conduction of the a.xon.

2) ACh and curare reversibly block conduction after pretreatment of the

squid axon with concentrations of certain snake venoms which by themselves have

no affect on conduction. Of 21 venoms tested Agkistrodon p. piscivoriís (cotton-

mouth moccasln) venom was the most effective in rendering the axon sensitive

to curare whereas Crotulus acUivwmteus (Eastern diamondback rattlesnake) venom

was completely inactive.

3) ACh and curare do not penetrate through the structural barrier surround-

ing the excitable membrane of the giant axon in contrast to lipid soluble tertiary

nitrogen derivatives which can penetrate. Cottonmouth moccasin venom markedly

increased the permeability of this structural barrier as evidenced by increased

penetration of radioactive ACh, curare and choline. In contrast Eastern diamond¬

back rattlesnake venom did not increase permeability. Cottonmouth venom also

increased the permeability of the barrier to various sugars, amines and amino acids.

The barrier is formed presumably to a large extent by the Schwann cell. Pre-

liminary electron microscopic studies indicate that fragmentation of the Schwann

cell surrounding the giant axon by venom may be responsible for the increased

permeability.

4) Choline, neostigmine and physostigmine when appiied in combination with

ACh to venom treated axons markedly decreased the effect of ACh. This action

was shown to be due to a competilion for sites of penetration, thereby allowing

less ACh to reach the excitable membrane!.

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PHILIP ROSENBERG

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5) In addition to increasing penetration of ACh in the squid giant axon,

cottonmouth venom aiso increased its penetration in the squid stellar nerve,

isolated single eei electroplax, walking leg nerve of lobster and frog sartorius

muscle.

6) It was possible to demonstrate the essentiality of cholinesterase for con-

duction on the venom treated squid giant axon. The organophosphate choline¬

sterase inhibitor Paraoxon caused a block of axonal conduction which could not

be reversed by washing with sea water but which was readily reversed by pyridine

aldoxime methiodide, a specific reactivator of organophosphate inhibited choline¬

sterase.

7) It appears that phospholipase A is at least one of the components res-

ponsible for the action of venoms on the squid giant axon.

Acknowledyements — For encouragement and advice as well as assistance in

preparation of this manuscript, I am grateful to Professor David Nachmansohn.

I am pleased to acknowledge the collaboration in many of these studies of Drs.

W-D. Dettbarn, F.C.G. Hoskin and T. R. Podleski, all of whom I would like to

thank. The Marine Biological Lab., Woods Hole, Mass. where many of the studies

were carried out deserves special thanks for the facilities which they made available

to us. I also would like to express my appreciation to all those who provided

samples of several of the compounds used in these studies, as acknowledged in

the original publications.

Legends

Fig. 1 — Schematlc presentation of the elementary process controlling membrane

permeabllity during electrlcal activity and integration of ACh into the metabolic path-

ways. The role of ACh In the permeabllity cycle may be pietured as follows: 1) In rest-

Ing condltion ACh is bound to a storage proteln (S). The membrane is polarized.

2) ACh is released by current flow; the free ester combines with the receptor protein (R).

A conformational change of the receptor (symbolized by dotted llne) leads to a shift of

charge; this process increases permeabllity; it is the trlgger action by which the ionic

concentration gradient becomes effective. 3) The ester receptor complex is in dynamic

equilibrium with free ester and receptor. The free ester is attacked by ACh-esterase (E).

4) The hydrolysls of the ester permits the receptor to return to its original condltion.

The permeability barrier is reestabllshed and the membrane is repolarlzed (From Nach¬

mansohn, 3).

F\g. 2 — Cross section of the giant axon of squid (above) compared to that of rabbit

sclatlc nerve (below) at the same magnlficatlon (From Young, 13).

Fig. S — Effect of 1.4X10-'’ M curare on squid axon rinsed in normal sea water CW)

for 10 min. (left) and for 95 min. (rlght) alter the application, per ml, of 10 fig of cobra

venom (CV) plus 20 jig of cetyltrimethylammonium chloride (CTA) (From Rosenberg

and Ehrenpreis, 14).

Fig. 1) — Effect of d-tubocurarine chloride (curare) on the giant axon of squid fol-

lowlng pretreatmcnt with 15 /jg/ml cottonmouth moccasin venom. S. W. indicates return

to sea water (From Rosenberg and Podleski, 15).

Fig. 5 — Effect of curare on the restlng and action potential of the squid giant

axon following exposure to cottonmouth venom. A, control; B, after exposure to 15 /rg/ml

venom for 15 min.; C, 15 min. after return to sea water (stlmulus voltage remained

constant for this period); D, E, 4 and 8 min. after exposure to 1.4 mM curare; F, 22 min.

after return to sea water (From Rosenberg and Podleski, 16).

Fig. 6 — Effect of 67 íig/ml of hoodcd cobra venom on the resting potential (. —.)

and action potential (o — o) of the giant axon of squid (From Rosenberg and Podleski, 15).

Fig. 7 — Effect of 50 /íg/ml of cottonmouth moccasin venom on the restlng potential

(. —.) and action potential (o—o) of the giant axon of squid (From Rosenberg and

Podleski, 15).

1 SciELO

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USE OF VENOMS IN STUDIES ON NERVE EXCITATION

Fi(i. S — Effect of acetylcholine (ACh) on the electrical activity of the giant axon

üf squkl following exposure to 15 /Jg/ml o£ cottonmouth moccasln venom. S. W. indicates

return to sea vvater (From Rosenberg and Podieski, 15).

Fi;i. !> — Effect of acethylcholine on the re.sting and action potential of the squid

giant axon following e.xposure to cottonmouth venom. A, controi; B, after exposure to

15 íig/mi venom for 30 min.; C, 15 min. after return to sea vvater; D, E, 15 and 25 min.

after exposure to 4.4X10-= M ACh; F, 30 min. after return to sea vvatei (From Rosen¬

berg and Podieski, 16).

Fif/. 10 — Penetration of radioactivcly (C") labeied ACh, choline, dimethylcurare and

trimethylamlne into the axoplasm of squid giant axon with and vvithout exposure to

cottonmouth moccasin venom. The percentage indicates the radioactivity of the axoplasm

compared to that In the outside fluld. The figures belovv the columns Indlcate the /tg

of venom/ml. In contrast to the moccasin venom, that of the Eastern dlamondback

rattlesnake (R. S.), even In 1000 /ig/ml had no signlflcant effect on the penetration of

dimethylcurare. Trimethylamlne readily penetrates with and vvithout exposure to venom

(accnrding to data of Rosenberg and Iloskln, IS).

Fi;/. 11 — Electron micrographs of squid giant axons A, controi and B, after treat-

ment with 100 /ig/ml Nuju naja venom. KMnO,-Pb stalns used. Magnificatlon of A =

17,000, B = 25,000. AX = a.xoplasm; SC = Schwann cell; CT = connective tlssue. The

a.xolemma of the squid giant axon is between Schwann cell and axoplasm. Vesiculatlon

and fragmentatlon of venom treated Schwann cell is scen. (By courte.sy of Dr. David

Robertson unpubllshed observations).

Fíd. 12 — Restoratlon by pyridine-2-aldoxime methiodide (PAM) of electrical activity

blocked by Paraoxon in venom treated squid giant axon. A, controi; B, after exposure

to 25 vug/ml cottonmouth moccasin venom for 30 min.; C, D after exposure to 0.01 M

Paraoxon for 2 and 5 min.; E, 30 min. after return to sea vvater; F, G, after e.xposure

to 0.05 M PAM for 5 and 20 min. Time slgnal is 750 cycles per second (From Rosen¬

berg and Dettbarn, 25).

Fiíi. 13 — Phospholipase A activities of 5 /ig of phospholipase A rich (Rich) and

100 /ig of phospholipase A poor (Poor) fraetlons of Naja naja venom and activity of

5 /ig of Naja naja venom (Venom). 1 mg of egg (-) or beef (— — —) lecithin

vvas the substrate. Results are shovvn as means ± S. E. of the mean (From Rosenberg

and Ng, 17).

Fiil. Ht — Phospholipase A activity of cottonmouth moccasin venom heated at pll 5.5

(11+) and pll S.5 (OH — ). Results are shovvn as means ± S. E. of the mean. Egg leci-

thln (1 mg) vvas the substrate (From Ro.senberg and Ng, 17).

Faj. 15 — Structure of severai glycerophosphatides. A, C and D indicate the points

of hydrolysis by pho.spholipa.se A, C and D, respectively. R = hydrocarbon Chain (From

Rosenberg and Ng, 17).

Fii/. 16 — Phospholipase A activity of cottonmouth moccasin (C.M.) and Eastern

dlamondback rattlesnake (E.R.) venom. 1 mg of egg (-) or beef (— — —) lecithin

vvas the substrate. Results are shovvn as means ± S. E. of the mean (From Rosenberg

and Ng, 17).

Fiii. n — Phospholipase A activities of 5() /ig of cottonmouth moccasin (C.M.), hooded

cobra (II.C.), Russells vlper (R.V.), Eastern diamondback rattlesnake (E.R.) and king

cobra (K.C.) venoms. 1 mg of beef lecithin was used as substrate. Results are shovvn

as means ± S. E. of the mean (From Ro.senberg and Ng, 17).

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Internac.

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PHILIP ROSENBERG

505

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RITCHIE, J. M., and ARMETT, C. J., J. Pharmacol. exp. Ther., 139, 201,

1963.

51. ROSENBERG, P., and DETTBARN, W-D., Biochim. biophys. Actn (Amst.),

69, 103, 1963.

52. BRZIN, M., DETTBARN, W-D., ROSENBERG, P., and NACHMANSOHN, D.,

J. Cell Biol., 26, 353, 1965.

53. ZELLER, E. A., Experientia, 3, 375, 1947.

54. ZELLER, E. A., Helv. chim. Acta, 32, 94, 1949.

55. KLIBANSKY, C., SHILOAH, J., and DE VRIES, A., Biochem. Pharmacol.,

13, 1107, 1964.

56. DETTBARN, W-D., and HOSKIN, F. C. G., Biochim. biophys. Acta (Amst.),

62, 566, 1962.

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1962.

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Slmp. Internac.

;<3(2):477-508, 1966

PHILIP ROSENBERtJ

507

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WILSON, I. B., and GINSBURG, S., Biochim. biophijs. Acta (Amst.), 18, 168,

1955.

DETTBARN, W-D., ROSENBERG, P., and NACHMANSOHN, D., Life Sciences,

3, 55, 1964.

BULLOCK, T. H., GRUNDFEST, H., NACHMANSOHN, D., and ROTHEN-

BERG, M. J., J. Neurophysiol, 10, 63, 1947.

GRUNDFEST, H., NACHMANSOHN, D., and ROTHENBERG, M. A., J. Neu¬

rophysiol., 10, 155, 1947.

HOSKIN, F. C. G., ROSENBERG, P., and BRZIN, M., Proc. nat. Acad. Sei.

(Wash.), 55, 1231, 1966.

ZELLER, E. A., Adviinc. Enzymol., 8, 458, 194S.

YANG, C. C., CHEN, C. J., and SU, C. C., J. Biochem., 46, 1201, 1959.

BRAGANÇA, B. M., and ARAVINDAKSHAN, 1., in K. A. C. ELLIOTT, I. H.

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GHOSH, B. N., DUTT, P. K., and CHOWDHURY. D. K.,

Soc., 16, 75, 1939.

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HUGHES, A., Biochem. J., 29, 437, 1935.

BRAGANÇA, B. M., and QUASTEL, J. H., Biochem. J., 53, 88, 1953.

MAGEE, W. L., and THOMPSON, R. H. S., Biochem. J., 77, 526, 1960.

YANG, C. C., CHIU, W. C., and KAO, K. C., .7. Biochem,, 48, 706, 1960.

YANG, C. C., KAO, K. C., and CHIU, W. C., J. Biochem., 48, 714, 1960.

NEUMANN, W., and HABERMANN, E., in E. E. BUCKLEY, and N. PORGES

(Editors), Venams, Amer. Ass. Advanc. Sei., Washington, 1956, p. 171.

MASTER, R. W. P., and RAO, S. S., J. biol, Chem., 236, 1986, 1961.

DOERY, H. M., and PEARSON, .1. E., Biochem. J., 78, 820, 1961.

MASTER, R. W. P., RAO, S. S., and SOMAN, P. D., Biochim. biophys. Acta

(Am.st.), 71, 422, 1963.

NYGAARD, A. P., DIANZANI, M. U., and BAHR, G. F., Exp. Cell. Res., 6,

453, 1954.

PETRUSHKA, E., QUASTEL, J. H., and SCHOLEFIELD, P. G., Catiad. J.

Biochem., 37, 975, 1959.

CONDREA, E., DE VRIES, A., and MAGER, J., Biochim. biophys. Acta

(Amst.), .58, 389, 1962.

GOTTFRIED, E. L., and RAPPORT, M. M., J. biol. Chem., 237. 329, 1962.

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(Editors), Neurochemistry, Vol. Hl, C. C. Thomas, Springfield, 1962, p. 10.

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BRANTE, G., Acta physiol. scand., 18 (suppl. 63), 1, 1949.

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USE OF VENOMS IN STUDIES ON NERVE EXCITATION

86. McCAMEN, R. E., and ROBINS, E., J. Neiirochem., 5, 18, 1959.

87. HACK, M. H., GUSSIN, A. E., and LOWE, M. E., Comp. Biocliem. Physiol.,

r., 217, 1962.

88. RAPPORT, M. M., and ALONZO, N. F., J. biol Chem., 235, 1953, 1960.

89. JOHNSON, A. C., McNABB, A. R., and ROSSITER, R. J., Arch. Neuroh

Psychkit. (Chie.), 64, 105, 1950.

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(Editors), Neurocliemistry, Vol. III, Thomas, C. C., Springfield, 1962, p. 870.

91. RAPPORT, M. M., and LERNER, B., Biochhn. biopltys. Actn (Amst.), 33,

319, 1959.

92. WAKIU, K., and KAWACHI, S., J. PJutrmacol. Soc. (Japan), 79, 1177, 1959.

93. CONDREA, E., DE VRIES, A., and MAGER, J., Biochim. biophys. Acta

(Amst.), 84, 60, 1964.

94. SAITO, K., and HANAHAN, D. J., Biochemistry. 1. 521, 1962.

95. KIRSCHMANN, C., CONDREA, E., MOAV, N., ALOOF, S., and DE VRIES,

A.. Arch. int. PlKtrmacoclyn., 150, 372, 1964.

-V

96. CONDREA, E., MAMMON, Z., ALOOF, S., and DE VRIES, A., Biochim. bio¬

phys. Acta (Amst.), 84, 365, 1964.

97. GENNARO, J. F., and RAMSEY, H. W., Amer. J. trop. Mecl.. 8, 546, 1959.

98. CONDREA, E., and DE VRIES, A., Toxicon, 2, 261, 1965.

99. ROY, A. C., Nature, 1.55, 696, 1945.

100. MELDRUM, B. S., and THOMPSON, R. H. S., Ginfs Hosp. Rep., 111, 87, 1962.

101. ARAVINDAKSHAN, I., and BRAGANÇA, B. M., Biochem. •/., 79, 84, 1961.

102. ROHOLT, O. A., and SCHLAMOWITZ, M.. A) f/í. Biochem.. 94, 364, 1961.

Disclssion

C. Y. Lee: “I would like to ask your comment on the paper by Feng & Hsieh

who demonstrated that the nerve action potential is unaffected after complete in-

activation of the acetylcholinesterase by TEPP.”

P. Rosenberg: “The difficulty in studies atlempting to correlate cholinesterase

activity and electrical activity is the method used to measure enzyme activity. The

tissue cannot be homogenized since excess inhibition may oceur al the time of

homogenization due to presence of e.xcess uncombined inhibitor. If one uses intact

tissue with acetylcholine as substrate then one is only measuring the readily

available or “externai” cholinesterase since acetylcholine cannot penetrate to all

of the enzyme. We have discussed all of these problems in a publication a few

years ago in “Biochemical Pbarmacology”. Feng and Hsieh did not take into

consideration all of the sources of error in this type of study.”

F. Kornalik: “Have you got all the snake venoms from one source only?”

P. Rosenberg: “Most of lhe venoms were obtained from the Ross Allen Reptile

Institute, Silver Springs, Florida, except for some of the Australian venoms which

were obtained from Light & Co., England, and a few which were kind gifts of

investigators from various parts of the U.S.A. and which are acknowledged in

my publications.”

SciELO

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509

54. SEHOTO.MN AND KELATED THYPTAMINE DEHIVATIVES LN

SXAKE VENOMS

J. H. WELSH

BioloçjicaJ Laboratories, Hurvurd Vniversity, Cambrklge, Mass., U.S.A.

IXTRODLXTION

Cerlain liiogenic amÍTies siich as acptylcholiiie, serotoiiin aiul histamine often

are foiind iii veiioms in verv large aniounts compared with other natural sources

(Talile ll. Since tliese suhstances are known lo produce ])ain wlien a])|)lied to

Elister areas it has Eeen siiggested tlial lliey ])lay a defensive role rather thaii

conlriliute iii any importaiit degree to lhe toxieity of the venom (1,2).

TABLE I — SOME EXCEPTIONALLY HIGH LEVELS FOR CERTAIN OF THE I.OVV

-MOLECULAR WEIGIIT COMPONENTS OF VENOMS

Species

Common

name

Value Acetylcholine

Reference

Vespa crahro

(hornet)

10-100 mg/g dry venom

(11)

10-50 mg/g dry venom

(12)

Zyoaena spp.

(moths)

1.6-60 mg/g (Accessory se.x

glands and ducts. Organs of

defense?)

(13)

Deiulrodfipis 13 species)

(mambas)

7-30 mg/g dry venom

(3)

5-Hydroxytryptamine

(Serotonin)

Leiurus quinquestriatus

(scorpion)

up to 4 mg/g dry venom

(14)

Phoneutria fera

1 Brazllian

spider)

1.5-2.7 mg/g dry venom

(15)

Vespa, crahro

(hornet)

up to 19/mg/g dry venom sacs

(12)

Synoeca sur\y\ama

(wasp)

13 mg/g dry sting apparatus

(15)

Rana plpiens

(írog)

0.3-1.0 mg/g wet skin

(2)

Other AMPHII3IA ímany species)

up to 4.5 mg/g dry skin

(16)

Histamine

Ap\s melUfera

(honey

bee)

10 mg/g dry venom

(17)

Vespa- vulqaris

(wasp)

10 mg/g dry venom sacs

(18)

Vespa, crahro

(hornet)

14-30 mg/g dry venom sacs

(12)

Supported hy Grant NB-00623 from the Instltute of Neurological Diseases anU Blind-

ness, USPHS.

SciELO

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510

SEROTONIN AND líELATED TRYPTAMINE DERIVATIVES IN SNAKE VENOMS

Until recenlly, liüle allenlion lias lieen paid lo lhe possihie occurrence of

such siilistances in snake vonoms. Acelylclioline has now lieen fourid in large

amounts in the venoms of three speeies of D e n d r o a s p i s {'ò). However, the

occurrence of serotonin and catecholamines in snake venoms is in disjinte. Zara-

fonetis and Kaias (4) report lheir presence in venoms of Crotalus atrox, C. ada-

matUcus and Agkistrodon pLmvoriis, luit Anlon and Gennaro (5) failed to find

serotonin or norejiinejihrine in the venoms of the two latter s|)ecies.

In a further stndy of this ])rohlem the venoms of 20 speeies of elapid, vijierid

and crotalid snakes have heen examined hy hioassay, sjieetroflnorometry, and

chromalography for the possihie presence of serotonin and related indolealkyl-

amines. The results wili he presented in this jiaper.

Materiai.s and methods

The speeies of snakes whose venoms have heen studied are listed in 'Pahle

II. Venoms from seven speeies of Ilrazilian Bothrops were ohtained from

the lintantan Institnle throngh the kiiulness of Dr. Hoge. Venom samjiles of

the remaining speeies were ])urchased from the Miami (Florida) Serpentarinm.

In most instances only one pooled sam[)le of venom from a given s|)ecies was

studied.

Three methods were employed to iletect the presence of serotonin or related

substances. However, some venoms were examined hy only one or two of the

following methods:

1) Hioassay. The isolated heart of the ciam. Mercenária [Venus) merce¬

nária. which detects small amonnts of serotonin and related componnds (6), was

used aceording to the method descrihed hy Welsh and Twarog{7).

2) Spectrojluorometry. Venoms were extracted

of Hogdanski ct al. (8) and finoresiamce and excitation

with a Farrand speetroflnorometer in the [iresence of ?>N

aceording

to the method

sjiectra were recorded

HCI.

8) Chrornatopraphy. Venoms were extracted with 50 (ler cent acetone or

70 per cent ethanol and the eoncentrate suhjeeted to paper and silica gel chromato-

gra[)hy. Several developing systems suitahle for separation indolc componnds were

used, as well as several standard methods for visualizing snch snhstances.

lIlCStILTS

The

cedures,

follow:

results are snmmarized in Tahie II. Certain details eoneerning jiro-

and the significance of the results ohtained with eaeh method of assay

BIOASSAY — No hioassays were done with venoms of lhe Brazilian speeies

of Bothrops. Whole venoms of all other .speeies were tested on one or more

isolated Mercenaria hearts. After dissolving in distilled water, from 0.01

mg to 0.25 mg of venom per ml of ])erfusion finid was added to the hath. Tests

were made hefore and after treatment of the hearts with henzoquinoninm iMyto-

lon) in order to hloek the jiossihle inhihilor action of acetylcholine which is

presenl in some venoms (e.g. Dend roas pis in very large amounts (.8)).

SciELO

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J. H. WELSH

511

TABLE II — SUMMARY

OF RESULTS OF THE

SEROTONIN

THREE METHODS

FOR DETECTING

5-HT

Indole-reacting

SPECIES

(Spectrofluoro-

spots

(Bioassay)

metry)

(Chromatography)

ELAPIDAE

Elapinae

Bungarus fasciatns

+ ?

None detected

None

Hemachatus haemachatus

+ ?

None detected

Naja naja

—

None cietected

Pseudechis mortoneiisis

+ ?

None detected

None

Dendroaspinae

De^idroaspis polylepls

—

None detected

Dendroaspis ancjusticeps

—

Not run

VIPERIDAE

VIPERINAE

Vipera russeUii

+ ?

1.9 íig/g

Bitis gabonica

30 /ig/g

ClíOTALINAE

Crotalus horrulus

—

None detected

2 or 3

Sistrurus m. harbouri

6.3 /ig/g

Agkistrodo7i contortrix

Not run

Agkistrodon piscivorus

1.7 íig/g

Boihrops utrox

None detected

B 0 th r o p s

(7 Brazilian .specles)

Not run

None detected

Not run

Serotoniii and certain rtdaled conipoimds cause an increasc in ampliUide

and freqnency of l)eat of tlie Mercenária heart, wilh little increase in toniis

lintil relatively liigh concentralions are reached. The catecholamines and histamine

also have an excilor action hut the M e r c e n a. r i a heart is far less sensitive

to lhese llian to serotonin. Also, lhey cause an increase in tonus resulting in a

niarked rise in baseline. Methysergide (UML 491) is an cffective hlocking agent

for serotonin on tlie M e r c e n a r i a lieart hut not for catecholamines and hista-

inine (9). When a venom was scen to excite the heart it was tested again after

the addition of methysergide to the hath.

A sample recording of the actions of liitis gabonica and Sistrurus m. hur-

boiiri venoms hefore and after methysergide is seen in Fig. 1. The exciter action

of liitis venom is completely aholished hy methysergide and that of Sistru¬

rus venom is much reduced. This suggcsts that serotonin or a dose relativo

is j)resent in these venoms. The failure of methysergide to hlock completely the

exciter action of S i s t r ii r u s venom, at the given dose levei, might he due to

the presence of a catecholamine or histamine in this venom.

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513

Simp. Internac.

33(2):5()9-51S, 1966

Heferring lo Tahle II. it iiiay I)i' .seeii lliat. of lhe six elapid venoms tested.

aii exeiter aetion on lhe Mercenária heai l was found in B u n u r ii s.

II ew achatas and Bseudechis venoms hnl lhese were nol ly|)ically sero-

lonindike. Of lhe seven viperid and crolalid venoms lested on lhe Merce¬

nária heari, all hnl lhal from Crotalas hórridas had some serotonindike exciler

aetion.

In some venoms. notahiy ihose of Bseudechis, S i s I r u r n s, A. pis-

civorus and Bolhrops alrox, a calecholatnine or histamine may he ])resent hnl \ve

have made no aUem])l lo look for lhese compoimds.

The residis from the hioassays are only indicalive of lhe presente of sero-

tonin or relaled snhstanees and ihey hecome significant only when vievved in lhe

light of the residis from speelrofiiiorometric and chromatogra])hic examinalion

of lhe same venoms.

SPECTROFLUOROMETRY — In niosi instances. only one samjile of venom

of a given species was exlraeted and ils fliiorescence and excitalion s[)eelra reeorded

wilh a speelrofluoromeler. Wilh the iiistriiment lhal was nsed, the imcorreeted

íliioreseence jieak for serolonin in 8 N HCI ajipears al 510 mp and the excitalion

])eak at 805 mp. (Fig. 2). Venoms of 14 species so examined showed no peaks

corresponding wilh ihose for serolonin. Sample records of reeorded specira of

ihree of lhese species are reprodiiced in Fig. 2.

Extracts of venoms of foiir of the VIPERIDAE showed excitalion peaks or

shoulders at 305 m/x hiil a strong fluorescence al 450 m/x ohsciired lhe characterislic

540 m/x peak for serolonin (see, for example, records for Bilis gabonica, Fig. 2).

In addilion to the excitalion jieak or shoulder al 805 m/i. Bilis and Sisiru-

r u s venoms gave a second excitalion peak near 280 m/x. This might he dne

to try|)tamine or one of ils melaholites lhal carne throngh the extraction procedure

(see residis from chromalography).

Only 10-25 mg of each ernde venom were exlraeted for the sjiectrofhioro-

melric analysis. If some venoms contained low' lewels of serolonin lhe characteiistic

excitalion jieak might nol have heen detected. Cerlain viperid and crolalid ve¬

noms. however, do give evidente for lhe presente of serolonin hy ihis melhotl.

CHROMATOGRAPHY — The residis of oídv one cliromalograjihic jnocednre

which was nsetl for all of the venoms (excejtl B o t h r o p s venoms from Bn-

tanlan) will he re|)ortetI. This involvetl the nse of ascentiing paper chromato-

graphy wilh 10-honrs developmenl in hntanol acelic acid:H20 (60:15:25). Sero-

tonin anti olher inilole-reacting spots were matie visihie hy sjnaying wilh |)-di-

melhylaminocinnanialdehytle (10). This DM(iA reagent is more sensitive hiil less

seleclive lhan EhrlichV reagenl. The nimihers of inilole-reacting s|)ots, made

visihie hy ihis procetinre. are given in Tahle II. Of lhe six ela|)id venoms.

three gave one s])ot each; two gave none; one was ilonhtfid. Chromalograms

of seven viperid anil crotaliil venoms showeti from two to five sjiots made visihie

hy the DMCA reagent.

In an allempi to iilentify lhe components of the venoms responsihie for

lhese several spots, serolonin anil cerlain relaled conijionnds were spotted wilh

lhe venoms. The following known snhslances were nsetl: tryjitophan. 5-hydroxy-

Iryjitophan. Iryptamine. 5-hydroxytry])tamine (serolonin). hnfolenine. N-methyl-

tryplaniine. !\.N’-dimelhyllryptamine. indole. indolyI-8-acetic aciti. and melatonin.

2 3

5 6

11 12 13 14 15

514 SEROTONIN AND RELATED TRYPTAMINE DERIVATIVES IN SNAKE VENOMS

FLUORESCENCE EXCITATION

I. Pse u de c hl s

i 0 0

50 0

6 0 0

30 0

4 0 0 MU

Flg. 2 — Examples of fluorescence and excitation spectra oí a serotonin (5-KT) standard

of three venoms shovving no detectable 5-IIT; and of Bitis gaboiüca venom with a

characteristic excitation peak at 305 mii. See text for further detail.

An examjile of a cliromatograin of S i s t r u r u s venoin rim with six of

the standards is reproduct'd in Fig. d. Tlie venom liad heen exiracted with 50

per cent acetone and volumes eqnivalent to 10 and 20 mg of venom sjiotted.

Tlie venom may lie seen to have vielded two jnominent and two faint s])ots made

visihie hy lhe DMCA reagenl.

SciELO

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Mem. Inst. Butantan

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J. n. WELSII

515

Melat.

Venom T-phan Venom T-mine

20 mg 10 mg

5-HT

Fro n t

Venom Bufo.

20 mg

Venom

10 ma

N-CH^-T

Fig. 3 — Chrnmatogram of S i s t r u r u s venom (10 and 20 mg) run in butanol; acetlc

acid: vvater with 5 /ig amounts of each of the followlng standards; melatonln (Melat);

tryplophan (T-phan); tryptamine (T-mine); .serotonin (5-HT); bufotenlne (Bufo) and

N-melhyllryptamine (N-CII^-T). Spraycd with DMCA rtagent. This ehromatogram w:)s

dcvelopcd for 12 hrs and the Hf value.s are higher than those for a run of optimal

duration (8-10 hrs).

riie lowt'sl venom s|)()l lias an lif corrcsjionding closely with thal for sero-

lonin (5-HT). The iiexl higher, and niost jirominent, venom s])ot is at a levei

near lhal of hiifotenine. There is a faint venom sjiol at the same Hf as N-

methyltry|)tamine. Ilowever, this solvent system fails to sejiarate this uinitie from

N,I\'’-dimethyltry|)tamine. A fourth faint venom spot is seen near the front.

Melatonin rims to this jiosition hiil so do indole and indolyl-5-aeetie aeid.

Chromatograms of the venom of Agkistrodon jHscirorus (water moeeasin)

gave fonr indole-reaeting sjiots that eorrespond with those from Sistruriis

SciELO

11 12 13 14 15 16 17

516

SEROTONIN AND HELATKD TRYPTAMINE DERIVATIVES IN SNAKE VENOMS

vciioin aiul, iii addilion, a fiflli, latc-apijcariíi" s|)()l willi aii Hf .10 and

40. Tlii.s is dose lo wlicre S-liydroxylryploplian runs in luilanol laeetie aeid:vvaler.

Wilh A. piscivorus venotn (20 mp) tlie fast-ninninjí sjiol al Hf 95 was mneli

more olivioiis lhan vvilii S i s ! r ii r u x venom (20 rng) : il gave a purple eolor

vvith tlie DM(fA reagenl wlueli is aiso Irne for indolyl-,l-aeetie acid. Agkixtro-

dofi conlortrix (eopperficad) venom (20 mg) gave no spol al Hf 95. The ollier

fonr s|)ols corresponded eiosely willi lliose from A. piscirorns venom.

An insnffieient variely of developing syslems liave l)een Iried, tliiis far, lo

permil tlie eerlain idenlifiealion of any of lhe indole-reaeling suhslanees in snake

venoms invesligaled in ihis sliidy. Il wili he neeessary lo use addilional solvenl

syslems and a wider variely of slandard iudole derivalives hefore lhe idenlily

of serolonin and relaled snhslauees in any given snake venom is more cerlainly

delermined.

Discussion

Keele and Armslrong (I ). in lheir hook Siihslimcrs Producinf’ Pain and

Itch, diseiiss lhe eonslilnenls of snake venoms lhal eonlrihule lo jiroduelion of

pain. From lheir eilalious, pain jiroduelion al lhe sile of lhe hile would ajijiear

to he more eommou aniong lhe vijierids and erolalids lhan among lhe elajiids.

Rradykinin releasing faetors eonslilute oue ela.ss of agenls resjionsihle for jiaiii

Jiroduelion in snake venoms. They slale (ji. 217) “Snake venoms do nol eonlain

ACh, 5-HT or hislamine, ihongh many of them release hislamine from lissues.”

We novv know lhal venoms of lliree sjieeies of l) e n d r o a s p i s eonlain large

arnoiinls of aeetyleholine (1). and from lhe residis of lhe jireseul sliidv il is aji-

jiarenl lhal some snake venoms eonlain a variely of indole-reaeling eomjionnds,

one of whieh is jiroliahly serolonin. This is a highly jiolenl jiain-jirodncer.

However, Armslrong has found lhal Iryjilamine and eerlain of ils derivativos

other lhan serolonin are aIso very effeelive jirodueers of jiain vvhen ajijilied

to enlaneous nerve endings. Aniong lhese are l-hydroxylryjilamine, I\'-melhyl-

Iryjilamine, N.N’-dimethyl-5-hydroxylryiitannne ( hnfolenine), and N-melhyl-5-hydro-

xylryjilamine. The last is even more effeelive lhan .serolonin (5-hydroxylryjitamine).

From lhe ehromalograjihie residis of lhe jireseni sltidy il ajijiears lhal some snake

venoms may eonlain al least ihree of lhe.se aetive Iryjilamine derivalives, namely

serolonin, liiifolenine, and N-melhyllryjilamine. If serolonin and hufolenine are

jiresent in some venoms, then lhe inlermediale. N-melhyl-5-hydroxylryjilamine,

may aiso he jiresenl. Il vvill he of mueh inleresl if fiirlher sindy reveals lhal

some snake venoms do, in faet, eonlain an assemhly of Iryjilamine derivalives

knovvn to he aniong lhe mosi aelive in jirodueing entaneoiis jiain in man.

AckHoivledgements — Mytolon was supplied by lhe Sterling-Winthrop Research

Institute and methysergide by Sandoz Pharmaceuticals. Carolyn S. Batty. Joyce

B. Zipf and Lois D. Williams assisted in Ihis sludy.

Hkeehknces

1. KEELE, C. A., and ARMSTRONG, D., Subutances Pro(tucin<j Pain and Itch,

E. Arnold, (London), 1964.

WELSH, ,T. H., Ajm. Rev. Pharmucol., 1, 293-304, 1964.

SciELO

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33Í2):509-518. 19G6

J. H. WELSll

517

3. WELSH, J. H., in F. E. RUSSELL (Edit.), Animal Toxins, Pergamon, in press.

4. ZARAFONETIS, C. J. D., and KALAS, J. P., Amer. J. Med. Sei., 340, 764-

768, 1960.

5. ANTON, A. H., and GENNARO, J. F., Nuture. 208, 1174-1175, 1965.

6. GREENBERG, M. J., Brit. J. Pharmacol., 15, 375-388, 1960.

7. WELSH, J. H., and TWAROG, B., in H. D. BRUNER (Edit.), Methods in

Medicai Research, Year Book Publi.shers, Chicago, 1960.

8. BOGDANSKI, D. F., PLETSCHER, A., BRODIE, B. B., and UDENFRIEND,

S., J. Pharmacol. exp. Ther., 117, 82-88, 1956.

9. WRIGHT, A. M., MOORHEAD, M., and WELSH, J. H., Brit. J. Pharmacol., 18,

440-450, 1962.

10. HARLEY-MASON, J., and ARCHER, A. A. P. G., Biochem. J., 69, 60, 1958.

11. NEUMANN, W., and HABERMANN, E., BUCKLEY, E. E., and PORGES, N.

(Editors). Venums, Amer. Ass. Advanc. Sei., Washington, 1956, pp. 171-174.

12. BHOOLA, K. D., CALLE, J. D., and SCHACHTER, M., J. Physiol. (Lond.),

1,59, 167-182, 1961.

13. MORLEY, J., and SCHACHTER, M., J. Physiol. (Lond.), 168, 706-715, 1963.

14. ADAM, K. R., and WEISS, C., Nature, 178, 421, 1956.

15. WELSH, J. H., and BATTY, C. S., Toxicon, 1, 165-173, 1963.

16. ERSPAMER, V., Handbook of Exp. Pharmacol, Vol. XIX, Springer-Veriag,

1966.

17. REINERT, M., Quoted by KEELE, and ARMSTRONG. 1964.

18. JAQUES, R., and SCHACHTER, M., Brit. J. Pharmacol., 9, 53-58, 1954.

Discussion'

P. Sawaya: “1. When you mention wasps, do you mean the European wasps

or the Brazilian wasps from which you have isolated the venoms? 2. The dif-

forent venoms that you have investigated, how many have a curare-like action?”

J. H. Welsh: “1. The results pertaining lo histamine, acetylcholine and kinin

referred to the European wasp and hornet venoms. I e.xamined Brazilian wasp

venoms only for the presence of 5-hydro.xytryptamine. 2. Most of the invertebrate

venoms that we have studled have a i)aralysing action on suitable test animais.

However, with the e.xception of those containing tetramine this paralysing action

is probably due to proteins and not to the low molecular weight components.”

F. E. Russell: "Would you care to speculate on the mode of action by which

serotonin produces pain, perhaps with special consideration of sodium exchange and

the possibilily of vasoconstriction about sensory endings? Secondly, how might you

explain the observation that the serotonin content of two of the snake venoms, you

noted Bitis tjabonica and Vipera russellii, while having a 15 plus fold difference

in serotonin content have no such marked difference in their pain producing effect

in clinicai cases?”

J. //. Welsh: “1. The precise mechanism by which serotonin produces pain

is not known to me but, at least in some invertebrates, it is known to produce

repetitive firing of certain sensory neurons (e.g. the crayfi.sh stretch-receptor sensory

SciELO

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33(2):519-522, 19G6

TOMOJI SUZUKI

519

55. I^MAHMACOLOGICALLY AM) RIOCHKMICALLY ACTIVE

COMPONEATS OF JAPANESF ()I»HII)IAN VFNOMS

TOMOJI SUZUKI

iHutitute for Protein Research, Osaka University, Kita-ku, Japan

\Ve liavi' liceii niakiiig a systematic sliuly on llu' se])aration of eiizynies of

venoins froni Japanese and Fonnosan snakes, and nuitiv enzymes wi-re ohtaiiied

in a piirn slati'.

l’lie most cliaracterislic syinploms |)rodiiced hy injeclion of lho venom of

Agki.sirodoii halys blomhojjii, callcd “Mainushi” in Japanese, a representatiye of

lhe Japanese j)oisonoiis CROTALIDAE snake. are: lieniorrliage, necrosis. museular

degeneration and lovvering of hlood pressure.

Ainong tlie enzymes from llie venom of Af^kislrodon halys blomhojjii, one

of ils ihree proleinases had hemorrhagie aetivity, and three were arginine ester

liydrolases vvith "hradykinin releasing". “elolting” and "capiliary |)ermeahility

increasing” aclivities, they were considered to he coimecled wilh lhe ])harmaco-

logical aclions of lhe venom. Re.sides lhe |)harmacologically active enzyme.s, a

siriking hemorrhagie protein de[)rived of anv enzymalic aetivity was also present

in llie venom. This paper deals with the pharmacologieally active comjíonenls

of lhe venom of Agkislrodon halys blomhojjii.

Mosl of tlie ca.seinolytic aclivities was distrihnted in three fraclions which

were designaled, in lhe order of llieir elulion from lhe cohmm of DFAF-celliiIose,

as proleinase A. li, and C. Hemorrhagie aetivity was foimd maitdy in lidtes

100 to 170 and 170 lo 260. There was liltie ])roleolylie aclivily in lhe firsl

fraclion, designaled as lllM. wliile in lhe second fraetion, IlH-lI, there was

proteinase 15 aetivity. Proteina.ses A and C were not hemorrhagie laetors and

no hemorrhage was ohserved even when they were injeeted at 500 fold the

mitiirmim hemorrhagie dose of proteinase 15. When HI5-I was injeeted into a

mouse iniravenoiisly. marked intestinal hemorrhage was ohserved, while on injeclion

of 1115-11, peteehial hemorrhage was ol).served in lhe suhentaneoiis lissiies. Nexl.

we allempled lo j)urify 1115-11 lo see vvhelher lhe hemorrhagie aetivity was due

lo the proleinase aetivity. After M15-11 fraetion, namely the j)roteinase 15 fraetion.

was desalted hy a Sephadex G-25 eohimn, the sample of ])roleinase 15 was re-

ehromatographed on DFAF-eellnlose iising gradient elulion. The curve of ah-

sorhaucy at 280 m/x did tiol coincide wilh lhe curve of hemorrhagie or [)roleiuase

aetivity, lhese lalters heiug found iii a single peak. After lyophilization, the

sample was ai)plied lo a hydroxyIai)atite eohimn, and lhe hemorrhagie aetivity

was eluted logelher wilh lhe ca.seinolytic aetivity. To luirify the resulting proteinase

15 jireparation, DFAF-Sephadex A-25 eohimn ehromalogra|)hy was used. 15y this

proeediire some im|)urilies were renioved. The hemorrhagie and easeinolytie

aetivilies of proteinase 15 were not separaled hy lhese |)urifieation proeedures and

SciELO

LO 11 12 13 14 15 16

520 PHAUMACOLOCICAI.LY AND BIOCHEMICALI.Y ACTIVE COMPONENTS

OF JAPANESE OPIIIDIAN VENOMS

llie increase iii |)otenc'y oí llia liomorrliaf^ic activily al each slcp \vas psscnlially

iti j)aralli-l wilh that of lhe caseiiiolytic aclivity oí |)ioleinase R. The avera<ie

yields of lhe purified jiroleinase 15, froni erurie venoni was al)out 2.5 per renl.

The ]nirified pre|)aratioii was {•ltroiiialogra|)liieally, eleclro])fioi(Tieally and aiso

iiltracenlrifugally hoinogeneoiis.

Proteinase R showed to he a inelai-protein, and lhe heinorrhagie and caseino-

lytic aclivitie.s fully reinained even afler lhe renioval of sialic aeid hy sialidase.

Rnt its aclivilies were iidiihiled hy EDTA and ey.steine. In EDTA-iidiil)ilion

experiment.s, lhe extenl of the decrease in heinorrhagie activily was |)arallel wilh

lhe decrease in caseinolytie aclivity. Also, in cysleinc-inhihition cxpcrimenls, lhe

hemorrhagic activily decreased parallel wilh lhe caseinolylic aclivity, in jiropor-

lion lo lhe amount of cy.steine added.

There was another heinorrhagie fraclion, iiamely lhe fraction of HR-I con-

taining arginine esterases, froni wliicli the arginine cslcrascs were easily reinovcd

hy hydroxylapalite chromatogra|)hy. The HH-I ])iV|)aralion ihiis ohiained conlain

no eiizyme aclivity. AlÜiongii the |)rolcin conlent of lliis HR-I pre])aralion was

low, lhe loxic iirinciplc nnisl lie a prolein and whcn this was ln‘aled wilh varioiis

[iroleinases, lhe hemorrhagic activily decreased.

As the hemorrliagic activily of HK-I appcared to play a Icading role in lhe

lelhality of lhe venom of Agkistrodoii hulys lãomhofjii, a coin])arison of lhe he¬

morrhagic and jiroteinase aclivilies in varions venoins were examined. The ernde

venom of A. conlortrix contartrix had no hemorrhagic activily even ihoiigh it

had high |)rolcinase activily. ()n lhe olher hand, lhe venom of Crolatiis aihi-

riianteiis, which showed sirong hemorrhagic aclivity had low proteinase aclivity.

Moreover, whcn lhe latter venom was fractioncd on a DfiAE-celhdose colimm. 9R

per cent of lhe total hemorrhagic activily was recovered in a fraclion which had

no |)roteinase aclivity. This hemorrhagic faclor was pharmacologically differenl

from HH-I isolalcd from Agkistrodoti Indys blarnliojjii venom. So, it seems that

even in venoins of lhe same family, there may hc different hemorrhagic faclors

and differenl siihstances wilh lethal loxicily, and for lhe characterization of lhese

loxic [irinciplcs, it is essenlial lo |)nrify each faclor from each snake venom.

In the same way, we inirified Iwo kinds of arginine esterases, namely lhe “clol-

ling” and lhe “capillary permeahility increasing enzymes" in physico-chemically

jnire States. We also ohiained lhe hradykinin releasing enzyme, free from olher

jihysiologically active componenls.

As a considerahie amonnl of lhe '‘cloiling enzyme” and lhe “capillary ])er-

meahilily increasing enzyme” were |)resenl in lhe venom, we altempled lo pnrify

the firsl lo a physico-chemically homogeneons slatc. Piirificalion procedures con-

sisled of foiir steps and hy lhese |)rocediires lhe clotiing enzyme was |)mificd lo

a pliysico-chemically homogeneous State.

Among the arginine ester hydrolases in lhe eliiate from the DhiAE-celhdose

column, an enzyme which has a hypotensive action and increases capillary per¬

meahility was foimd. The arginine ester hydrolylic aclivity of this enzyme was

.'ÍO lo 40 per cent of the total arginine ester hydrolylic aclivity of lhe venom,

and whcn it was injccied into the skiii of an alhino rahhil, the permeahility of

lhe cainllaries were distinclly increased as shown hy lhe Evans Rhie Tesl.

When this iirejiaralion was incuhated wilh piirified hradykininogen, no release

of hradykinin was delected hy assay on gninea-pig ilciim. So, it is noi clear hy

whal nieidianism lhe permeahility of lhe capiliaries is increased hy this enzyme.

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TOMOJT SUZUKI

521

lii ollier ex|)i'iimt‘iils, \ve foiiiid lhal a considerahlo amoiint of tliis capiliary

])i-i'in('al)ility incroasing; cnzynie was aiso |)iesiMil iii Crolahis aJanianIcus vonoiti

and in Triinervsuriis jlavovirhUs vtniom. Tliis enzyme of lhe veiiom of Agkislro-

don hulys blomhojjü was ])urified hy similar procediires as lliose vvliicli were used

for lhe elülling: enzyme. The speeifie aelivily of ihis enzyme was more llian 70-fold

thal of lhe enide veiiom, and lhe j)nrified ])re])aralion was honiogeneons on ullra-

eenlrifngation and eyanogum eleelrophoresis al varioiis pH valnes.

Tlie nllracenlrifngation f)aUerns of llie “cloiling” and “capiliary ])ermeahilily

increasing” enzymes show lhe homogeneilies of lhe enzymes.

When llie pnrified “elolling enzyme” was inenhaled wilh 98 |)er cenl pnre

fihrinogen, whieh had l)een j)re])ared in onr lahoralory, aceording lo lhe melhod

of liiomhaek and Laki, ihree kinds of fihrinopeplides were lil)eraled. Two of

lhese eorresponded lo fil)rinope[)lides A and H whieh were liheraled írom fihrinogen

hy lhe aelion of ihromliin or hy llie venom of Bollirops jararaca. Hnl, oiie of

lhe ihree peplides was a new fihrinopeplide. Hy amino aeid analysis and end

gronp analysis of ihis new fihrinopeplide, il was eoneluded lo he a |)e|)lide whieh

resniled from lhe loss of one mole of arginine from lhe C-lermimis of fihrino-

pejilide H. The reason why lhe physieo-ehemieallv |)nre “elolling enzyme” of lhe

venom. whieh showed only arginine esler hydrolylie aelivily, liheraled ihis new

pe|)lide is nol yel known. When lhe enzyme was inenhaled wilh fihrinope|)lide B,

no reaelion was ohserved.Therefore, lhe new fihrinopeplide seemed lo he released

direelly from lhe hovine fihrinogen moleenie hy lhe aelion of lhe elolling enzyme.

The elolling aelivily of lhe enzyme of Agkistrodon halys hlotahojjii was

lower lhan lhal of lhe elolling enzyme of Bollirops jararaca. The aelivily of lhe

elolling enzyme of .ígkisirodon haly.s hloniliojjii was nol inhihiled hy jilasma

anli-lhromhin. In ihis il is similar lo lhe elolling enzyme of ih evenom of Bo-

throps jararaca.

Hradykinin releasing enzyme fraelion whieh was ohlained from lhe ehiale

on lhe firsl DEAE-eellnlose eohmm ehromalograjihy of Agkistrodon halys hlom-

hojjii venom, eonlained elolling enzyme. And il was fnriher appiied lo a CM-

eelhilose eohimn. Thns lhe hradykirdn releasing enzyme, free from elolling en¬

zyme, was ohlained, hnl only 5 ])er eeni of lhe lolal imils of arginine esler

hydndylie aelivily of lhe venom were reeovered in ihis parlially pnrified enzyme

])re|)aralion. 'Iherefore, no fnriher pnrifiealion was allempled. AIlhongh lhe

snhslrale .sjieeifieilies of lhe elolling. hradykinin releasing, and ea|)illary per-

meahilily increasing enzymes were (pialilalively llu' same, lheir physiologieal

aclivilies were eomplelely differenl. The hradykinin releasing was only inhihiled

hy Irasylol. whieh is a poleni inhihilor of nrinary and panerealie kailiereins, and

lhe elolling and capiliary |)ermeahilily increasing enzvmes were nol inhihiled hy

Irasylol al all. Erom lhe.se resnils and lhe s|)eeifieilies fonnd hy lesls on several

synlhelie snhslrales, lhe hradykinin releasing enzyme in lhe venom seems lo he

a .salivary kailikrein of lhe snake.

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H. I. BICHER

523

56. SFltCIFIC SITES OF ACTION OF SNAKF VFNOMS [N THE

CFNTHAF NEKVOUS SYSTEM

H. I. BICHER

Department of Pharmacotogy, Tel-Aviv University Medicai School, Rogoff Medicai

Research Institute, Beilinson Hospital, Petah Tikva, Israel

Intiíoduction

Ntniroloxins from snake venoms are lheir aclive comitoiients responsihle for

(lislurliances iii lhe ceniral and |)eri|)lieral aetivities of llie nervoiis syslem. Tlie.se

(lislurhances are produced hy llie venom of all species of the fatnily ELAPIDAE,

sucli as lhe Indian Cohra [Naja naja), all Aiistralian venoinoiis snakes(T) and

Walterinnesia. acgyptiu, the only ELAPIDAE nalivc lo Israel (2). Neuroloxins

are also eomjtonenls of lhe venotn of lhe American pilvipers, CROTALIDAE (3)

and have heen isolaled from lhe venoms of many VIPERIDAE, although hemor-

rhagic and cylolytic aclivities jtredominale in lhe aclion of the latler venoms (4).

In onr previous puhlications we have descrihed the eleclrophoretic (5) and

ehromatographic (6) separalion of neuroloxic fraelions from Vipera palcslinae

venom. The adminislralion of these fractions to mice produces nenrotoxic signs

and causes dealh of lhe animais vvithoul hemorrhages laking place. The hemor-

rhagic fractioti jtrodiiees widespread hleeding in mice, hnl is devoid of any

netirotoxic aclion.

Dislinct eleclrophorelically sejiarated neuroloxic fraclion.s from lhe venom of

lhe Indian Cohra (Naja naja) have also heen reporled (7), hnl lhe relalions

helween lhe loxic and enzymalic aclivilies in ihis and olher venoms have noi

heen finally eincidaled. IMiosjtholipase A, prolease, L-amino acid oxidase, choline-

slerase, nucleases, phosphodiesterase, monoeslerase, hyahironidase have heen fonnd

in most venoms and some of lhem, nucleases (8), cholinesterase (9), j)hosj)holipase

A (10) have heen suspecled lo cause various envenomalion syndromes. Ahove

all, lhe phosj)holi|)ase A of lhe Indian cohra (Naja naja) venom has heen con-

sidered lo possess neuroloxic aclivily (10) ailhough in rectml jtuhlicalions this

asserlion is heing dissented (7,11).

The jtreseul series of ex])erimenl.s was underlaken as an allempt lo ehicidale

lhe specific aclion of these nenrotoxins on hrain inechanisrns, aniouomic funclions

and lheir loxicity to different species.

Matehiai.s

Whole venom from adull Vipera palestinae specimens was ohiained from lhe

fnslitute of Naiural Science of lhe Tel Aviv llnivcrsily (Frof. M. Mendelssohn

and Dr. E. Kochwa, to whom lhanks are due). Nenrotoxic and hemorrhagic

1 SciELO

524

SPECIFIC SITES OF ACTION OF SNAKE VENOMS IN THE CENTUAE

NERVOUS SYSTEM

fraclions wcrc separalcd as prcvioiisly descrilicd I 12,6). Maxiimiiri snlilollial

dosis, cqidvaleiil lo dO mouse LD^o unils of vciion; or veiiom fraclion, jx-r kg

hody weighl, wen‘ admiiiistcred intravenously to cats and raliliils.

Iiidiati Col)ra [Naja naja) freezi; dricd veiiom from L. Liglil and (lo. I.ld.,

(lolidirook. Knglaml was used.

Plios[)lioIipase A from Naja mija venom was ohtained hy |)a[)cr ('l('('tropli()rcti('

separalion al pM 6.0, using plios|)hale Imffer M 15. Afler elcclrojilioresis a nar-

row scgmenl (of akoiit 1 cm widlli) was ciil off from llic middlc of cach sirip

(al)oul 7 cm widih) and staincd willi tiaplillialencdilack. Kig 1 sliows 6 hands

3 4

START

Fig. 1 — Paper electropherogrnm oI N'nja vaja venom.

“Mktiiods”.

Condition.s as described in

in tfie cle(dro|)licrograni. The ajjprojnialc prolein hands wcrc ent off from lhe

imstained strips and elnted over night with dislilled water al 1". Sodium chloridc

was added until isolonicity and j)rolein concentration was cstimaled hy Lowry

reaction (18). Only lhe fraclion remaining al lhe ap|)licalion line (fraclion 6)

conlaincd lhe jihospholipasc A activily. 1'he adjacent fraclion 5 proved in niosl

separalions lo hc devoid of |)hos|)holi|)asc A aclivily hnt in a fcw inslanccs was

contaminatcd wilh lhe cnzyme. The ])ho.spholipasc A coiilaining fraclion 6 was

tested for lhe following activities: j)rotease (1), jirocoagulant (18), L-amino acid

oxidase (14) and hyaliironidase (15) and was foimd devoid of all of them.

Analytical ullracenlrifngation of fraclion 6, dialyzcd againsl salinc, was carricd

out wilh a modcl K, S[)inco nitracentrifnge al 16..5", using lhe standard 12-mm

ccll, and a single |)eak was ohtained corres])onding lo a sedimenlalion conslani

S 20 = 4.65 X 1()“^'‘. Fraclions 1 and 2 conlained lhe direct lytic faclor, a

hasic prolein, ca])alile lo lyse washed red hlood cells (12),

For some experiments lhe venom was hoiled and Irealed with he])arin. For

ihis ])nr])os(í a saline-venotn solnlion, 1 mg/l ml, was healed for 15 min in a

hoiling waterhath al pH 5.,5 and lhen cenirifnged. The eleclro])horelic pallcrn

of lhe snpernalanl was similar lo lhal of lhe ind)oiled venom i.e. com])rising 6

hands. 1 mg heparin (Naiional Iliochemicals (lorporalions, lleparin-sodinm 100

U/mg) in 0.1 itd saline was added lo 1 ml of lhe sn|)ernalanl and lhe mixinre

was ke|)l for 20 min al room lem])eralnre. A sedimeni ap|)eared which is known

lo contain lhe direci lytic faclor (12). Il was se|)arated hy cenirifngalion and

{•lecirophoresis of lhe clear snpernalanl showed lhal fraclions 1 atui 2 went

ah.sent.

l‘h()si>/i(>lii)asc A aclivily

One ml of lenfold saline dihiled egg yolk was incnhaled al 87" for incnxis-

ing [teriods of lime wilh 0.05 ml venom or phospholipase A fraclion conlaining

0.5 p.g prolein. lhe mieslerified fally acids (UFA) were del('t'mined hy lilralion

using lhe melhod of Dole (16). 'Plie ticlivily was calcnialed as m-ctpiiv. of free

SciELO

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Mem. Inst. Hutanlan

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33{2):523-5^0, 196(5

II. I. BICHEIl

525

ari(] n-leased por enzynio fraolioti por niiiiulo. For ox|)orimonls only llioso plios])lio-

lij)ase A proparalioiis wore usod wliioh lihoralod al loasl 1.6 ni-ocpiiv. lIFA/rnin

por 1 /ig proloiii.

Metiious

Expcrimrnls

on mico

Inlraporiloiioal mouso-LD.-,ii of wliole voiioni and ils separate fraclions wero

ilolorniinod on looally hrod Swiss all)ino inioo, using 5 animal-s por doso. Fal-

rulation of LD.-,„ was mado aooording lo Ilood and Miioncli (17).

Exiicrimcnls on cats

Tlio oxporiinonts vvoro oarriod oul on anao.slholizod inlaot oals and s[)inal

cais, woiglnng 2-d.,5 kg. Anaoslliosia was induood liy ollior and rnainlainod

ihrongliont lho ex])oriment hy ropoatod injoolion inlo a fenioral voin of 0.5-1 nd

of a mixtiiro oonlaining 10 ing lliioponlono sodiiim and dO /xg alropino sulfalo

por nd. Illood clolling was provonlod hy inlravonous adndidstralion of hoparin

(5,000 iiidts/nd/kg hody woighl).

All aclivilios stiidied wero rooordod on a Grass modol 5 |)olygra])h. Blood

pressiiro was rogistered from lhe eannidated feinoral arlery using a Stalham

Iransdueer. Hespiralion was moasurod l)y nioans of a iherniooouplo inlroduced

inlo llie canniilaled Iraeliea. The oleelrooardiograni was ohlained using needie

eloelrodes insorlod inlo lho oxlrondtios. lVri])heral eiroidalion was sliidied l)y

[)holo])lelhysniography, using a Hoffman 55-e silicone ])holocell wilh high sen-

silivily in lhe infrared range, |daced around llie oaFs |)aw. Variations in inlensily

of lho liglil reaohing llio scnsilivo area of lhe ])holooell on Iransillutninalion of

lhe oxposod oxlrendly are a measure of varialions in lhe ainounl of hlood in

llio liglil |)alh and ihus indioalo va.sooonslriclion (deoroasing volunio) or vaso-

dilalion (iticreasing volunio) (1<S). The direol ciirronl olilainod from lhe jiholo-

coll was ainplifiod hy a Gra.ss D.C. amplifior. In ordor lo faoililalo lhe rooording

of |)rolonged changes in hlood volume, amplifiealion was reducod lo a ])oint where

|)idse wavos almost disa[)pearod.

F.lcclrooorlieograms (EeoG) wero recorded hi|)olarly using insidaled slainless

sloel eloelrodes wilh hare lips placed in eontact wilh lhe hrain surface ihrough holes

drillod in lho .skull on holh sidos in lhe fronlal, lem|)oral and occipilai arcas.

Changes in aulonomio nervous syslem aclivily wero deleoled hy reeording

lhe aclion |)olenlials of a fow fihrcs of lhe lefl cervical sympalhelie chain. The

fihres wero dis.secled from lhe main nervo Irimk and mounled on plaliniim wire

eleclrodes which were isolaled exce|)l for lhe immediale area in conlael wilh lhe

nervc fihres. The preparalion was immer.sed in physiological saline solulion al

37" eovered wilh liquid paraffin. The eleclrodes were eonnecled lo a Teklronix

lype 132 low levei pream|)lifier and lhe nerve polenlials conlinuously viowod on

a Teklronix 515 A oseillo.seope. A record of lhe polenlials was aiso slored on

a Grundig TK '15 la])e recorder for delayed Iranscriplion.

Spinal cais were pre|)ared acoording lo lhe melhod of Dale (19). Arlificial

reajiiralion was slarled using a Slarling pump and lhe hrain was deslroyed wilh

a [irohe inlroduced ihrough an orifice made in lhe second cervical verlehra;

SciELO

LO 11 12 13 14 15 16

526

SPECIFrC STTES OF ACTION OF SNAKE VENOMS IN THE CENTIIAI.

NEHVOUS SYSTEM

hiecíling

was conlrolled l)y

spacc. Etlier aiiaeslliosia

tissiie was accoinplislied.

inserling a laperiiig jiack of gaiize iiilo lhe luain

was niaintaiiied iiiilil llic dcsiniclioii of tlie condiral

d’lie

contiaction of tiic nictitaliiig mpnd)rano joodiiced hy jooganglionic

sliiiiidalioii of llie ipsilalcral corvical sympallielic cliaiii was rocorded siimdlanooiis-

ly witli Idood ()n‘ssiir(' iti cais Ixdorc and aflcr adtninisiralioti of lhe ncuroloxiii.

Hcxamelhoniimi was uscd as a coiilrol. Dcpressioti of llie response of lhe nielilal-

ing niemi)iane occiirring sininltaiieously willi a fali in hlood pressure was regarded

as indicalive of ganglion hloeking aclivily in lhe lesled com[)onnds.

Experiments on ruhhils

(ionseions rahhils, linder local anaeslhesia (novocain) only. and weighing

2-Í-5 kg, were filicd wilh a slercolaxic a|)paralus aceording lo lhe melhod of

Monider and (langloff 120). Reeording elecirodes were inserted inio holes drilled

over lhe left and righl fronlal atid parielal lohes of lhe cerehral corlex, and

slirnidaling elecirodes insnialed excepl for lheir lips were placed in lhe niidhrain

reliciilar formalion.

Elecirocorlicograms were recorded on a (írass eleciroencephalograph, and

slimuli in lhe reticular formalion were j)rodiiced wilh a (frass slinudalor model

Sf wilh an atlached isolation nnil, and monitored wilh a Tektronix model 502

oscilloscoj)e,

The cortical arousal response was induced hy slimulalion of lhe relicniar

formalion; lhe vollage of llie stimnius varied hetween 0.2-1.5 volts. lhe diiralion

was 0.5 msec, and frequency was 2'!() p[)s. Varialion in lhe ihreshold and diiralion

of lhe evokcd corlical resjionse, as well as changes in lhe elecirocorlicogram

(KCofi) itself after injection of lhe venoms were noted.

['orly rahhils were used in ihis study, cach experimenlal groiip consisled

of a minimnm of 5 animais. lhe venoms and lheir fraclions were injecled intra-

venonsly al a dose of 10-50 mouse Id)r,o per kg, cansing dealh wilhin 1-2 honrs

aflcr injcciion.

Exprriiiirnls on lho isolaírd Jr</g Scinliciis nerve

.Sindics on lhe infinence of snakc venoms and lheir se|)araled nenroloxic

fraclions on lhe aclion polenlial of isolaled nerve jireparations were cariied onl

in vilro. \ lenglh of 7-8 cm of frog seialiens nerve was cnl onl and di.ssecled

frorn thc spinal cord and lhe rnain Irnnks were furlhei- separaled from lhe smallcr

hranches. Hy threads connected lo holh ends, lhe nerves were snjiporlcd in a

nerve chamher, consisling of ihree separale com])arlmenls wilh a small gniding

groove for lhe nerve passing ihrongli all lhe com|)arlments. The nerve was

[daced npon Iwo platinnm elecirodes in each of lhe ihree compartments. Trans-

mission was ohserved hy slimnialing one end of lhe nerve in lhe firsl compart-

menl and reeording lhe provoked aclion polenlial al lhe olher end in llie ihird

cornjiarlmenl. (.hanges in ihreshold dnring lhe experimeni were fonnd hy slimnial¬

ing the rniddle [larl of lhe nerve which was in direci conlaci wilh lhe loxins.

A (/rass 4 slimnialor and a IVklronix 502 oscilloscope were nsed for sli-

mnlalion and reeording. In some cases lhe aclion polcniials were pholograjihed

nsing a polaroid camera.

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Inteinac.

38(2):523-54(), 1966

ri. r. BICIIEH

527

l{i:si:i.Ts

I p e r a v e ii o ni

1. Exprrinteiiln on cdis

Foiir cais wore injaclecl cvilh wliole Viprra palcslinae vcnoni and sixtffii

with lhe iieiirolüxic fraclioii. In order to ohtain a siirvival period of at least

foiir liüiirs afler lhe first injeelion, tnaxinial suhiellial doses were used. The

following phenomena were ohserved hoth afler whole venom and nenrotoxie

fraction administration. A sliarp fali in hlood pressnre took place within 20 s.

afler injeelion. This rediiclion in hlood pressnre conlinned ihroughout lhe dnralion

of lhe experiinenl in most of lhe animais, althongh in some cases lhe hlood

pressnre retnained eonstanl al lhe iinlially estahiished lovver levei. The jndsc-

pressnre diminished |)rogressively lo a very low amplitude. The fali in hlood

[cressnre was assoeialed wilh eontinnonsly jcrogressive jceriiiheral va.sodilatalion,

as shown in Fig. 2, althongh a deerease in lhe peripheral hlood volume may

he ohserved for a shoii period vvhile lhe acnte hlood jcressnre dro[) is taking

[)laee, dne lo aentely imjcaired hlood snjcply.

135

Mssâss

AAAAAA-^

Fig. 2 - Pharmaeodynamic effect of Vipera polextinae neurotoxin. Order of tracing.s

frorn top to bottom: ECCt, hlood prcssure, photoplethy.smography, rcsplration. For

e.xplanutlon, see text.

lfes])iration shovved decreased frecpieney and inereased vohime, wilh hrief

])eriods of a|)nea (Fig. 21; later, lhe respiralory pallern is stijierficial and slow.

The F(i(; reeord shovved litile ehange; lhe vollage decreasing conlinnotisly dnring

lhe experimenl.

'Phe eleelrieal aetivily of lhe eeniral and antonomie nervons sysiem was

sirongly affeeled.

ly aholished for

neurotoxin (Fig. H).

K(io(! vollage

shorl periods.

was diminished in all leads and .somelimes enlire-

ihree minnles afler lhe injeelion of venom or

eases lhe aelion polentials of lhe cervical syinpalhelie

SciELO

LO 11 12 13 14 15 16

528 SPECIFIC SITES OF ACTION OF SNAKE VENOMS IN THE CENTHAL

NEIIVOUS SYSTEM

chaili (lisapijcared complelely williin 50 s. aflcr iiijcclioti (Fig. 5 li). Il aj)-

paars, therefore, llial syrnjiatticlic firing vvas crascd miicli earlicr llian E(Jo(;

waves; aiso lhe laüer were siippressed incornjiletely or only for a limited jieriod

of time (Fig. 5 C).

« vAv^-

,. t V

- - - —

— —HtHiir*'

30 sec

3 min.

Fig. 3 — Effect oI V i pera neurotoxin on lhe ECoG and .spontaneous activlty üf

lhe cervical .sympathetic Chain. Order of tracings from top to bottom: ECoG

(frontai, parietal and occipital ieads) and s'ympathetic nervo action potential.s. A:

hefore, B: 30" and C: 3' alter neuroto.xin injection. D: HO min. alter injection.

The ncrve action potentlals dlsappear much earller than the ECoG.

Si.\ cais recovered from the firsl injection of Fí/tcrd palestimit’ neuroto.xin

or whole venom, shovving decreased hnl steady patteins of lilood pressnre, res|)ira-

lion and clectrocorticogram. A second injection of lhe same dose of nenroloxin

or of whole venom was given lo tliese animais np lo 50 min following

dose. This second adrninislralion of venom did not provoke a fnrther

in hlood jiressiire. Tachy|)hylaxis was not ohserved when lhe firsl

eonsisted of lhe nenrotoxic fraction and the second was whole venom.

case, hlood pressnre again dro|)ped sharply and imrnedialely.

Doses of 20 mouse Lü.r,,, nnils of lhe nenroloxin, eqnivalent lo half the

lhe firsl

decrease

injeclion

In this

maximnm snhlelhal dose for intact cais, were adminislered intravenonsly to sjiinal

cais. ünring 2-5 min. after lhe injeclion a slow increase in hlood pressnre

amonnling lo 10 to 20 mm JIg was ohserved.

The increased levei was mainlained

for ahonl len minnles, afler whieh a retnrn lo lhe previons levei took place.

FC(i, recorded simnilaneonsly with lhe hlood pressnre, was not affecled and lhe

cardiac rhylhm was nnchanged. In six cais, lhe adrninislralion of 20 monse

FDoo nnils of lhe nenroloxin did noi affeci lhe response of lhe niclilaling

rnemhrane to pre-ganglionic sym])alhelic slimnlalion. even al the heighl of lhe

acnte hlood pressnre decrease deserihed previonsly. In eonirol ex|)erinients, hexa-

melhoniiim injecled prior to lhe nenroloxin in doses of 1 mg/kg, prodnced a

IransienI lowering of lhe hlood pressnre with a simnitaneons reversihie depiession

of niclilaling rnemhrane coniraclioir.

Irrjectiorr of maxirrrnrrr sidrlethal doses of lhe herrrorrhagic fracliorr of Vipara

pdlcslinar venorrr inio niire cais cansed no alleralion in hlood |)ressnre in seven

of lhe animais. A slighl trairsieirt fali iir hlood jrressnre was rrolieed iirrirredialely

SciELO

10 11 12 13 14 15 16

Mem. Inst. Bulantan

Slmp. Intcrnac.

3S(2):523-54Ü, 19(i6

H. I. BICHEIl

529

afler injection in lhe Iwo romaining animais only. The sympathelic ncrve

potenlials vvere iinaffeeted or only slightly deereased and no ehanges vvere oliserved

in the ECod and peripheral hlood volume. Mice injccled inlraperiloneally with

2 LI)|i)o nnils of lhe same fraclion died wilhin 2 honrs, showing wide-spread

hemorrhages in aeeordance with j)revions ohservations (12).

2. Experimcnts on ruhhits

The injection of lO-SO rnonse LD^n per kilogram of whole Vi pera venom

cvoked a gradual decrease in the amplitude and freqnency of the corlical potentials,

in several cases finallv resnlting in complete disa[)pearance of detectahle cortical

aclivily. Iti general no effecl was noled on lhe threshold or dnration of the

arousal response elicited hy stiinulalion of the reticnlar formation, althongh oc-

casionally ils dnration was prolonged.

3. Isolated ncrve

Vipera ixileslinae nenroloxin was aiso fonnd to depress the provoked action

[)otenlial in lhe isolated frog sciatic nerve preparation. Transmission throngh

lhe intoxicaled nerve was completely hloeked afler incidialiori with the nenro-

toxin for I-IV 2 1'onrs at a concenlration of nol less lhan 150 monse LDr,» imits

|)er ml. This hlock was only partially reversihie on washing with frog Kinger

(see Fig. 6). A response was ohlained on direct stimnlation of the loxin-incnhated

])orlion of lhe nerve, des|)ile lhe transmission hlock. Ilowever, in some |)repara-

tions lhe excitahility threshold was that of the initial threshold.

11 — C o I) r « V e n o rn

1 . Experiiuenls on tu ice

Aclion of whole Cobra venoni

'lhe intra|ieriloneal LDr,» for mice was O.FS mg/kg. Mice injected with

2 X LD.^d showed convnlsions. excitemenl and impaired movements, and died in

respiratory arrest wilhin SÓ minutes.

Cobra venom jradions

Of lhe 6 ])aper electrophorelic (iohra venom fractions, three-comhined fraclions

1 and 2, fraclion 3 and fraclion 6, had nenroloxic activity, althongh having

different s|)ecies specificity and mechanism of action.

The Ll).-,i, for the condiined elnates of fraclions 1 and 2 was ahonl 1.5 mg

Iprolein) ])er kg hody weighl. Mice injccled inlrai)eriloneally with 2 LI).-,,, of

this comhined einale showed nenroloxic symploms — apalhy, motor depression.

and dose lo dealh 3 honrs after venom injection, gas])ing respiration, jnm))ing

and clonic convnlsions.

Fraclion 3 posse.ssed sironger nenroloxic activity. the inlraperitoneal FD.-.o

heing 0.5 mg/kg. Mice injected with this fraclion showed excitemenl, jnmping,

SciELO

LO 11 12 13 14 15 16

530 SPECIFIC SITES OF ACTION OF SNAKE VENOMS IN THE CENTRAL

NERVOUS SYSTEM

coiiviilsions, fasl rospiralion willi use of aiixiliary respiralion imisclas, atui ciirliiig

of lha lail. Daalli occiirrcd carly. nol latar lhaii 30 miii afiar lha injaatiori.

Animais raaaiving fraation 4 axhiliilatl in some cases similar symptoms hiit

lha LDjo was miiah liighar, ahova 2.5 mg/kg. Fradion 5 was not toxia at lhe

“r'/

dosas injaclad, iip lo 2.5 mg/kg. Fraalion 6, lha only ona whiah aonlainad

phosj)holi[)asa A aativily, was nol loxic, avan al dosas of 5 mg/kg. This con-

forms lo lha findings of Maslar and Hao (7) wilh Itidian aolira phospholipase A

pnrifiad hy slarah gel alaclrophorasis.

2. Experimenls un cuts

Cobra venorii

'riia intravanoiis LOj,,,] in aneslhelizad cais was 1 mg/kg, lha animais dying

wilhin 2 honrs frorn lha momanl of injaalion. In experimenls on 12 aals, lha

injeclion of one í..l)i(i(, caiisad a dij)hasic aircidalory shoak. Immadialaly afiar

lhe injaalion a Iransianl dro[) in hlood j)rassnra ocanrrad, as.socialad wilh a[)naa

and hradyaardia, high P wavas and ST daprassion (Fig. 4' /l). Laiar, afiar

lha maan hlood |)ra.ssura had ralnrnad lo lha prainjealion lavai, lhera ocanrrad

a lemporary daprassion in lha FCoG. In lha pariod from ahonl 10 min afiar

injeclion lill shorlly hafora dealh, raspiralion was fasl and pnlse [iressura da-

araasad j)rograssivaly, KCoG and KCG ramaining normal (Fig. 4 C). Naar lo

dealh, 80-120 min. afiar lha injaalion (Fig. 4 D), lha EGoG slowly wanad, lha

res|)iralion hecame prograssively hrady[maia imlil respiralory arrasl oacnrrad.

Tharaaflar lhe hlood pressnra drojjped lo zero and lha animal diad.

Síüíüií “

1 t

1..

««•

--S/ ■ ■ -~v~'

/I B ^

Fig. 4 — Ellec-t of Cobra vanom (1 ED„„) in an ane.slhetizeci cal. Order of tracing.s (from top to bottom)

ECG, biood pressurc, re.spiration and ECoC — frontai, parietal (two Icads), occipital areas. Record A:

normal tracing bclore venom injection; B: immediately after venom injection; C: 30 min alter injection;

D: 80 min after injection at time of death.

Kecording of lha aalion polanlials from lha aarviaal synipalhalic ahain showad

incraasad firing slarlifig shorlly afiar venom injaalion, gradnally inaraasing in

fraqiianay and ampliinda, np lo lha lima of dealh. Incraasad sympalhatia firing

logalhar wilh maximally tlaprassad KCoG 10 min. hafora dealh wara nolicad. Al

ihis lima hradypnaia raspiralion parsislad, lha synipalhalic firing inaraasing al

inspiralion.

SciELO

11 12 13 14 15 16 17

Mem. Insl. Butantan

Slmp. Internac.

33(2):523-540, 1966

H. I. BICIIEH

531

Cobra venom jruclions

Conil)ine(l fractioiis 1 and 2 were injecled inlo 2 cais inlravenously in lhe

arnount of 1 ing (protein)/kg. One of thc cats shovved Iransienl clianges in lhe

ECG-lovvering of QHS voltage and ST elevalion, lasting for ahoiit 10 min. after

injcction. No otlier toxic efíects were recorded.

Fraction 8 was given to 3 cats inlravenously at doses of 0.25 mg/kg (1

LDjii(i). No changes were seen in hlood pressnre, ECG, EGoG and respiratioii

at lhe niornenl of venom injeclion (Fig. 4 .4), Init 120 min. later EGoG slowly

waned (Fig. 4 B), respiration l)ecame sporadic and llien slo])])ed completely.

Pulse pressnre diminislied progressively during the Iwo hours following injeclion

and fell to zero. During lhe i)eriod of aj)nea lhe ECG showed increasing hrady-

cardia and anoxie changes (Fig. 4 B ).

Fraction 4 was as a rule non-toxic, hut sometimes symptoms similar lo

those produced hy fraction 3 could he elieiled al higher doses (0.8 mg/kg),

prohahly due lo deficicnt separation from fractio]) 3.

Fraction 5 ])rej)arations were found non-loxic, even in high doses up lo 1.5

mg/kg, when devoid of |)hospholii)ase A. In those cases in which fraction 5

possessed |)hospholipase A activity hy contamination from fraction 6, the |)repara-

lion did show neuroloxic activity.

Fraction 0, which had strong |)hospholipase A activity, was neuroloxic. lhe

FD,i„i heing approximately 1 mg/kg. The pharmacologieal effecl was identical

to thal of lhe whole Gohra venom with lhe two typieal shock phases descrihed

ahove.

The activity of phos])holipase containing hoileddieparinized Gohra venom was

indistinguishahie from thal of fraction 6 and of whole venom. The Ff), nu was

similar, ahout 1 mg/kg.

3. Expcriniciils oti rabbils

Cobra venom

The injeclion of whole venom caused an early quickcned respiration which

was later replaced hy res])iralory difficully and gasping. Death occurred due

lo resi)iralory arresl which was preceded hy clonicolonic eonvulsions. Earlier,

])artial paralysis of the limhs, particularly lhe hind legs, was noted.

Immediately after injeclion, lhere was a mueh increased corlical aclivily

and awarcness, as shown hy a strong hcta rhylhm in lhe eleilrocorlicograrn.

This was accompanied hy quickcned respiration and fiypermotility. Within the

following 10-15 minutes, respiration considerahly slowed, limh paralysis hegan lo

appear, and the córtex was characterized hy a gradually increasing de[)ression

of activity, as evidenced hy a steadily increasing deita rhythm in the FGoG.

(Fig. 5-3a). This stalc of cortical depression and lahored respiration was

mainlained for iq) to 2 hours. Ahout 10 minutes hefore death, the animais under-

went great respiratory stress, characterized hy slow gasping respiration. Al this

lime, strong eonvulsions hegan to a|)|)ear, hut these convulsious were noi ae-

com])anied hy characleristic seizure patlertis in lhe eleclroenee|)halogram. Cortical

polcntials then waned and completely disap|)eared (Fig. 5, 5a); only after com|)lele

disappearance of the EGoG record did death occur due to respiratory arresl.

SciELO

LO 11 12 13 14 15 16

■i.-

la ''■ -r"-'::;-' ■

3^ U V -; V — 3t

Hfe

y^''V'‘\/^'^'

"-^vV-

'' '.' •

Sfe

Fig. 5 — KCoG Record. Left sUle — PXoG before and aftcr venom injcction. Ulght sido

— Cortical arousal response elicited by direct stimulation of the reticular Tormation. 1 a,

b — before injection; 2 a, b — immedlately after injection; 3 a, b — 20 minutes aftei

injectlon; 4 a, b — 60 minutes after injection; 5 a, b — 70 minutes after injection.

SciELO

0 11 12 13 14 15 16

Mem. Inst. BuUintan

Simp. Intcrnac.

33(2):523-540, 1966

H. I. BICIIER

533

The aroiisal response threshohl aiso ehaiiged cliaracterislically iipon lhe in-

jeelioii of lhe venom. Diiring llie inilial perio<] of excitation follo\vÍTig lhe

injeclion, lhe ihresliohl was elevated. It lhen gradually decreased lo a ijoinl

helovv lhal delennined liefore lhe injeclion, and remained al ihis low levei for

lhe niajority of lhe remaining lime, paralleling leniporally lhe a|)pearance of

corlical depression. Shortly hefore dealh (Fig. 5, 4h| and hefore lhe lolal flal-

tening of lhe ECoC waves, lhe arousal res])oiise ccidd no longer he eliciled even

wilh relalively high voltage slimnlation.

Cobra venom jruetions

Three of lhe electro])horelically-separated prolein fraclions showed nenroloxie

aclivity, naniely fraclions 1-2, 3 and 6.

The effects cansed hy fraction 1-2 were similar lo ihose of whole venom,

except lhal lhe corlical depression was much less marked and lhal lhe lerminal

flallening cf lhe ECoG |)Olenlials was jirononnced and occnrred much earlier in

ihis fraclion lhan in olhers. The arousal resjionse disap|)eared only after a

complete ECoG flallening; unlil lhe poinl of its disap[)earance, lhe ihreshold

remained lower lhan lhe conlrol levei and coincident wilh corlical depression.

Fraclion 3 was lhe most loxic of lhe fractioTis. lls ncurotoxic action differed

from lhal of whole venom in lhal lhe corlical depression was very marked and

])ronounced, and persisled unlil almosl immedialely hefore res])iralory arresl when

lhe corlical polenlials finally disappeared. The arousal response disap|)eared much

earlier lhan wilh any olher fraclion, in lhe midsl of lhe period of corlical de¬

pression (delta rhythm).

Fraclion 4 and 5 possessed no nenroloxie aclivity. No change was ohserved

in lhe ECoG or lhe arousal resi)onse after injeclions, atui lhe artimals in this

group remained alive al leasl four hours afler injeclion.

Fraclion 6, ou lhe olher hand, cansed almosl identical effecls as lhe whole

veuom.

1. Isolaírd nerve

All three nenroloxie fraclions hlocked conducliou ihrough lhe isolated froa

Sciaticus nerve, in a |)attern and concenlralions similar to those descrihed for

lhe V i p V r a neuroloxin (see Fig. 6).

Non nenroloxie fraclion 4-.5 were inaclive, even al a eoncentration of

2 mg/ce.

Discnssiox

The eleclrical aclivity of lhe cervical sympalhelie chain reflecls lhe slale of

lhe medullary cardiovascular centres which regidale hlood pressure hy controlling

|)eri[)heral arteriolar s|)hincters. Consetpienlly, sympathetic firing is decreased

when these centres hecoine dejtressed (21). During hlood pressure fali due to

causes olher lhan de|)ression of lhe medullary centres, sym])athetic firing is in-

creased (22,23), jtrohahiy due lo comjtensalory foedhack mechanisins.

SciELO

LO 11 12 13 14 15 16

534 SPECIF'IC SITES OF ACTION OF SNAKE VENOMS IN THE CENTRAL

NEKVOUS SYSTEM

Flg. 6 — Effcct of neurotoxins on the evokcd Irog

soiatic nerve actlon potentlal. The electric response

(upper left) Is progressively depressed (upper right

alter 30’, mlddle ieft alter 60', mlddle right after 80’),

and dlsappears eompleteiy alter 90’ incubation. Bottom

traelng; alter 2 h washing in frog Ringer, partlal

recuperatlon is seen.

Vipera pairstinw wliole vetiom as vvell a.s its se[)arate(l tieiirotoxic fractiotis

|)ro(liice imtnediale stipiiressioii of lhe cervical sympallictic actioii jiolcntials, af-

fccliiig thc clcclrical activity of the córtex otily later. Iii additioti, the iiiarkcd

atid prolonged rise of the pholo|)lethysmogra|)hic record iiidicales increased hlood

volume in the dilatcd perijihcral vascular hed.

The ])rcsent resuits have aiso shovvn lhal Vipera puleslinue neurotoxiu has

no cardiotoxic or ganglion-hiocking activity. It seems prohahie that lhe slighl

transient hlood |)rcssure rise ohserved in lhe s|)iual cat is ])roduccd hy a moderale

vasoconslriction induced hy lhe toxin.

The instaiitaiieous de|)ression of hlood pressure associatcd vvith simultancous

inhihition of sym|)athelic actiou |)olcnlials and jicripheral vasodilatation indicale

that a primary action on medidlary vasojtressor centres rnay he responsihie for

lhe neurovegelative effects of Vipera pedestinae whole venom or neurotoxin.

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H. I. BICHEIl

535

ELAPIDAE (Naja naja) and CROTALIDAE (Crolahis atrox) venonis are

also knovvii lo cause aii immediate aud severe fali iit systemic Idood pressure.

Aecording to earlier ohservalioiis (21,25), colua venoni, vvhen adminislered iii

high doses, produce Iwo phases of circulatory shoek, aii initial íall in Idood

pressure, altriluiled to histamine release iii lung tissue with eoncomitani ohsiruction

of pulmouary eircidalioii, aud late respiratory failure. Lower doses of Naja naja

venoiti ])roduce neurotoxic effeets wilhoul appreeialdy affeeting cardiovascular

activity. Experimeuls carried out in our lahoratory showed that lhe iuitial hlood

])ressure deereasc as a result of iuiravenous adniiuistration of cobra veuoui lo

cats aud associated with ininiediate disap|)earaucc of ECoC waves is not associated

with dejiressiou of cervical syni|)alhetie actioii ou poleutials (26).

The preeipitous fali iu arterial hlood pressure eaused hy lhe veuom of

Crotalus atrox was shown hy Hussell et al. (27,3) uot lo he due to de])ression

withiu lhe central nervous system, hui lo a déficit in left heart output seeoudary

to chauges in resistance in lhe |)uhnonary circulalion.

Cardiovascular effeets and shoek have also heen descrihed in relation to

veuoins of other animais. L a t r o d e c t u s veuom (28) was fouud lo have a

dircct action on lhe heart aud coronary circulalion. Stonefish veuom imjtairs

the coutractile force of the heart aud jnoduces vasodilatation uot of central

origin (29).

It therefore seems warranted to assume that medullary cardiovascular centres

are specific rece])lor sites for Vipera palestinae neurotoxin hut not for the venoms

of some other |)oisonous snakes, s|)iders or fish. Consequently, the mechanism

of the jnimary cardiovascular shoek produced hy cach of thcse venoms is dif-

ferent. The hlocking of peripheral nerve transmission and lowering of ils excit-

ahility indicate a direct loxic action of Vipera palestinae neurotoxin on the nerve

memhrane.

The hemorrhagic eomponeni of Vipera palestinae veuom does not ])roduce

primary shoek and has no influence on lhe sympathetic nerve ])otenlials. This

fraction produees hleeding in experimental animais, which evenlually leads to

dealh.

Earlier studies hy Houssay (30) suggested a correlalion hetween lhe neuro¬

toxic activity of CROTALIDAE venoms and lheir hemolytic action. Fehlherg

aud Kellaway (21) attrihuled the slee]) fali in hlood pressure after intravenous

iujectiou of Cohra veuom to lecilhinase which, hy |)roduciug lysolecithin, liherates

histamine frorn the tissues. Bragança and Quastel(lO) reaffirmed the idenlily

of neurotoxin aud eohra venom ])hospholipase A. Cohra veuom heated for 15

min. al lOO" relained hoth its neurotoxic aud phosjjholipase A activilies while

other cnzymes were inactivalcd. On lhe other hand, ])hospholipase A-conlaining

fractions, se[)araled from various snake venoms hy electroj)horesis or chromato-

graphy, were reecnily reporled lo he devoid of neurotoxic activity, the latter

heing recovered in other fractions (6).

fn the ])resent study an animal s])ecific action of different jiaper electro-

j)horetically separalcd Naja naja venom neurotoxins. oídy one of lhem — fraction

6 — having ])hospholipase A activity, was demonstraled. This was a|)parent

not only from lhe ohserved resistance of mice to fraction 6 and of cats lo fractions

1-2, hut al.so from the diffcreut syndromes ap])earing iu cats aud miee. Eractioii

6 produced iu cats di|)hasic circulatory shoek, de])ressiou of respiratiou, evenlually

leadiug to respiratory dealh, aud early oceurring ECoG de])ression aud chauges

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536

SPECIFIC SITES OF ACTION OF SNAKE VENOMS IN THE CENTRAL

NERVOUS SYSTEM

iti syin])alh('li('. aclivity. l'’ra(tion 3 |)rovokf(l carly dcalli and coiivulsions iii

inicc, whercas in cais delaycd rcspiralory arrcst and dcalli vvilli late occurrence

of similar eleclropliysiolofíical response.

'I’he di])hasic circulalory shock ensning in cais follovving intravenous ad-

niinislration of wliole Naja naja venom, as well as of fraclion 6 and of liealcd

liepariidzed venoin I])hos))lioli|)ase eontaining. devoid of direct lyüe faetor( 12 )

and of lhe mice-nenroloxie aclivities relaled to fraclions 1-2 and 3) reseinhies

tliat descrihed l)y l'\ddl)erg and Kellaway (24) for vvliole Naja naja venom. Afler

reeovery from lhe immediate sleep drop in hlood ])ressiire, there was a late oc-

eiirrence of a seeondary gradual descent lintil res])iralory dealh sniiervened.

Fraction 3 did nol |)rodiice diphasic shock hut only a Iate lethal effect a])pearing

synchronousiy wilh lhe second pari of lhe diphasic shock caused hy whole venom

or fraclion 6 . Thns, whcrcas in whole venom-induced inioxication the phos|)ho-

li])ase A-containing fraction 6 ])lays a role in lhe cansation of holh shock ])hases.

fraclion 3, which is devoid of phospholipase A, conirihules lo the delayed shock

phase only.

Although the juimary shock has hcen altrihiiled hy Fcidlierg and Kellaway

lo lhe hislamine liheraling aclion of lhe venom. a direci aclion of lhe venom

])liospholi])ase A on the central nervous sysiem, manifested in onr ex|)crimenls

hy transient ECofl depression and changes of res()iralory rhylhrn, cannol he

exchided.

Il was estahiislied iti the |)resenl sludy thal Naja naja venom |)rovokes in-

creased cervical sym|)alhetic aclion |)otetitials wilh primary depre.ssion of £6067

waves. In contradislinclion, F/pem palcstinac venom neiiroloxin has hecn shown

lo rcduce cervical sympathetic firing immediately npoti injection wilhoul simultane-

ously affecling lhe corlical activily (31). Il ajepears lherefore thal neurotoxins

from differetU snakes act oti differeni sites íti the central nervous sysiem.

Phospholi|)ase A from differenl snake venoms have heen shown to have dif-

ferenl suhsirale specificity. The |)hospholipa.se A of holh cohra and Vipera

painslinae venoms hydroly.se phospholipids in soluhle slate, such as in egg yolk

and plasma. However, whereas lhe cohra phos])holi|jase A readily altacks lhe

])hospholii)ids in human osmolic red hlood ccll ghosls( 12 ) and hlood ])late-

lets (32), cat hrain homogenates and cal hrain milochondria (33), lhe Viprra

palcstinar phos|)holi|)ase A has no such activily. This may reflecl a |)ossihle

hiochcmical correlalion wilh lhe diffcrence in neiiroloxicily of lhe separaled

phospholipase-conlaiidug fractions of lhe Naja naja and Vipera palcstinac venoms,

lhe former heing toxic to cats, lhe laller non-toxic lo holh mice and cais ( 6 ).

A spccific aclioti of cohra venom |)h()S|)holipase A on nervous memhranes

was demonslraled hy Poliias (34), who used lhe isolated lohsler giani axon. The

inaclivalion of lhe aclion potenlials was nol accom|)anied hy clcclron micro-

scopically dcmonsirahie mcndirane (hanges. The assumjclion of a direci central

aclion of Naja naja phos])liolipase A. however, as yel lacks an in vitro corrolary,

sítkc the cnzyme, which is ahle lo split phos|)holi|iids in hrain homogenates, does

nol act on iniact hrain slices(33).

Although several invesligators have dcmoTislraled a ])eripheral curare like

aclion of cohra venom neuroloxin. a central neurotoxic activily has aiso heen

sludied. Wcslerman ct al. {'^5) demonslraled aholilion of a central vagai reflex

and (iiiyol (36) ohscrved ncuroloxic' sym|)loms whcn lhe venom was injected

intraventricniarly, and Ciuchla(37) and olhers have demonslraled changes in

lhe FCoG of inioxicatcd arnmals. Whelher lhese cffecls are primary resuits of

ueuroloxic aclivity or are s(‘condary lo othcr physiological changes is slill imclear.

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H. I. BICHEU

537

Since il was demonstralod llial lhe animais coiiliniied lo res])ire even afler

coilieal ])oletilials could no longer he deleeled, il may he assiimed that lhe central

aclion of cohra venoni nenroloxin is a |)riniary aclion of the venom itself; lhal

lhe ol)served nenroloxic (dfeels are nol secondary lo respiratory difficnlly and

cansed simply l)y anoxia lo the hrain tissue. This is not to say that the respiratory

arrest is necessarily cansed hy the same central-acting components, hnt it seenis

clear that a strong contrihntory canse, if not the main nnderlying factor, in the

death dne to cohra venom intoxication is tlie deleterioiis effect of tlie toxin on

the hrain and tlie disapiiearance of cortical activity.

It was noted ahove that fraction 1-2 cansed early disa[)]iearance of cortical

potenlials as evidenced hy complete flattening of the ECoG record. Fnrther-

more the aronsal res])onse threshold was not significantly elevated diiring the

period of cortical de|)ression, and disapjieared onl} after the disappearance of

cortical activity. These Ivvo facls lend lo indicale thal ihis neiirotoxic fraction

has a |)articnlar affinity to lhe ccrlex itself, and lower centers are affected only

laler and more weakly.

According lo lhe same line of reasoning, il was conclnded that fraction 3

acts jiarticniarly on lhe relicniar formalion and ])erha]js olher lower cenlers. As

lhe aronsal resjionse disa])peared long hefore flallening of lhe ECo(i hecame

ajiparent.

Fraction 6. whose action paralleled that of whole venom, seems to act on

hoth cortical and snhcortical slrnctnres, allhongh the action is firsl on the córtex

and later on lhe reticular formalion. This seems lo elahorale onr previons oh-

servalion thal this phospholipase A-conlaining fraction is toxic for cats, althongh

it is non-toxic in mice as demonstrated hy Master and Hao (1961) (7).

The facl thal the terminal conviiisions ohserved were nol accomiianied hy

the characteristic s|)ike. and some palterns usnally noted in seiznres as well as

the ohservalion that often the convnlsions appeared in the midst of the period

of ECoG delta rhylhm (paralleling cortical depression ) scem to indicale that lhe

convnlsions ohserved are of snhcortical origin.

The negalive findings thal lhe venom of Viprra palcslinac has no effect on

the aronsal resjionse seems to lend sn[)|)ort to a sjiecificily in central action of

ihis nenroloxin, selectively dejiressing central anlonomic vasoregnlalory mcchanism.

Il may he conclnded from the ahove w'ork that these neuroloxins lend to

act on sjiecific centers in the central nervons syslem, and lhal nenroloxic aclion

is generally nol random and diffnse. Nevertheless, il is clear that all nervous

lissiie is to some exlenl vnlnerahle lo nenroloxic action. allhongh specific affinities

too clearly seem lo exisl.

The lücns of aclion of Vip cru nenroloxin seems lo he more reslriclive

and definilive lhan lhal of Naja naja nenroloxin. This jierhajis is dne lo ils

jiredominanl aclion on more jirimilive cenlers. as ojijiosed to lhe jiredilection

of cohra venom for lhe higher cenlers.

Heeeüences

1. COCHRAN, D. M., Poiftonom lieptiles of lhe World Smithsonian Institute.

Washington, 1943.

2. GITTER, S., MOROZ, C., LIVNI, E., and DE VRIES, A., Proa. Beilinson Med.

Cen., 10, 160, 1961.

1 SciELO

538 SPECIFIC SITES OF ACTION OF SNAKE VENOMS IN THE CENTRAL

NEHVOUS SYSTEM

3. RUSSELL, F. E., MICHAELIS, B. A., and BUESS, F. W., Proc. west. Phur-

mucol. Soc., 4, 27, 1961.

4. DE VRIES, A., RECHNIC, J., and GITTER, S., Proc. Beilinson Med. Cen., 10,

168-178, 1961.

5. GITTER, S., KOCHWA, S., DE VRIES, A., and LEFFKOWITZ, M., Amer. J.

trop. Med. Hytj., 6 , 180, 1957.

6. KOCHWA, S., PERLMUTTER, CH., GITTER, S., RECHNIC, J., and DE VRIES,

A., Amer. J. trop. Med. Hyg., í), 374, 1960.

7. MASTER, R. W. P., and RAO, S. S., J. biol. Chcm., 2.36, 1986, 1961.

8. TABORDA, A. R., TABORDA, L. C., WILLIAMS, J. N., and ELVEHJEM, C.

A., J. biol. Chem., 194, 227, 1952.

9. ZELLER, E. A., Advances in Enzymology, Vol. 8, Inter.science, N.Y., 1948,

p. 459.

10. BRAGANÇA, B. M., and QUASTEL, J. H., Nature (Lond.), 169, 695, 1952.

Biochem., J., 53, 88, 1953.

11. YANG, C., SU, C., and CHEN, C.. J. Biochem. (Tokyo), 46, 1209, 1959.

12. CONDREA, E., DE VRIES, A., and MAGER, J., Biochim. biophys. Acta, 84,

60, 1964.

13. LOWRY, O. H., ROSENBERG, N. J., FARR, A. L., and RANDALL, R. J.,

J. biol. Chem., 193. 265, 1951.

14. BOMAN, H. G., and KALETTA, U., Biochim. biophys. Acta, 24, 619, 1957.

GLICK, D., Methods of Biochemical Analysis, Vol. 1, Interscience, N.Y., 1954,

p. 439.

DOLE, V. P., J. clin. Invest., 35, 150, 1956.

17. REED, L. J., and MUENCH, H., Amer. .]. Hyg., 27, 493, 1938.

18. WEINMAN, ,1., BICHER, H. I., and LEVY, D., J. appl. Physiol., 15, 317, 1960.

19. BURN, ,1. H., Practicul Pharmacology, Black well Scientific Publ., Oxford,

1952, p. 35.

20. MONNIER, M., and GANGLOFF, H., Rabbit Brain Research, Vol. 1, ELsevier,

Amsterdam, 1960.

21. KREPPEL, E., Med. Exp., 7, 223, 1962.

22. ADRIAN, E. D., The Mechanism of Nervoiis Action, University of Penn.syl-

vania Press, Philadelphia, 1959, p. 74.

23. McCUBBIN, J. W., and PAGE, I. W., Circulat. Res., 6, 816, 1958.

24. FELDBERG, W., and KELLAWAY, C. H., Aust. J. exp. Biol. med. Sei., 15,

159-172, 1937.

25. FELDBERG, W., and KELLAWAY, C. H., J. Physiol. (Lond.), 90, 257, 1937.

26. BICHER, H. I., GITTER, S., and ROSEN, S., Bibl. Anat. (Basel), 7, 429, 1965.

27. RUSSELL, F. E., MICHAELIS, B. A., and BUESS, F. W., Proc. west. Pharma-

col. Soc., 4, 27, 1961.

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H. I. BICHER

539

28. MARETIC, Z., Toxicon, 1, 127, 1963.

29. SAUNDERS, P. R., ROTMAN, S., MECHANO, V. A., and CHIN, H. P., Avier.

J. Plnjs., 203, 429, 1962.

30. HOUSSAY, B. A., MAZACIO, P., and NEGRETE, J., Rev. Ass. méd. argent.,

35, 699, 1922.

31. BICHER, H. I., ROTH, M., and GITTER, S., Med. Pharmacol., 14, 349, 1966.

32. KIRSCHMANN, CH., CONDREA, E., MOAV, N., ALOOF, S., and DE VRIES,

A., zirch. int. Pharmacodyn., 150, 372, 1964.

33. KLIBANSKY, CH., SHILOAH, J., and DE VRIES, A., Biochem. Pharmacol.,

13, 1107, 1964.

34. TOBIAS, J. M., J. Physiol. (Lond.), 43, 57, 1960.

35. WESTERMAN, E., and KLAPPER, W., Arch. exp. Pathol. Pharmacol., 239,

68, 1960.

36. GUYOT, P., and BOQUET, P., C. R. Acnd. Sei. (Paris), 251, 1822, 1960.

37. CIUCHTA, H. P., and POLLEY, E. H., Red. Proc., 23, 1964.

38. VICK, J. A., and CIUCHTA, H. P., Nature, 203, 1387, 1964.

Discussion

C. Y. Lee: “Is lhere any evidence lhat cobra neurotoxin, which is a basic poly-

peptide, can pass through the blood-brain barrier in sufficient quantity to produce

central effects?”

H. I. Bicher: “There are some publications using radioactively labelled venom,

that indicate that smail amounts of cobra venom reach indeed the central nervous

System. The specificity of the observed electrophysiological effects makes us sup-

pose that this smail amount may be enough for a delimitated effect. Similar

experiments by Gotter and his co-workers demonstrated that Vipera palestinae

venom also can penetrate the blood brain barrier.”

J. C. Vidal: “With the fraction (neurotoxin of 6th band) have you found

hydiolysis produets due to phospholipase A activity?”

H. I. Bicher: “We did not test ourselves, but people of our Institute, and

specially Dr. Klivansky and Dr. Condrea demonstrated phospholipid splitting in vivo.

However, we think that the action of the neurotoxic fraction is a direct one upon

the CNS cells, and not rnediated through the liberation of Chemical produets in

the blood stream. May be so at the cell membrane levei.”

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JEAN CHEYMOL, FRANCOIS BOURILLET,

MONIQUE ROCII

541

57. ACTION NEUHOMUSClíLAlHE DES VENINS DE QUELQUES

CROTAUDAE, ELARIDAE ET IIYDROEIIIIDAE

JEAN CHEYMOL, FRANÇOIS BOURILLET, MONIQUE ROCH

Institut de PharmacoUxjie, Faculté de Médecine, Paris, France

Dans lc's trois grands groii|)('s des serpeiits veninieiix, OPISTOGLYFHES,

PHOTEIiOíiLYPHES, SOLENOGEYPHES, les deux derniers soul i)iati(|ueniciit

les seiils dangereiix poiir rHomnie.

Parnii les sigiies de l’iiitoxi(alioM on iiole ])our heaiK'oii|) une aclion neiiro-

niuscidaire. Nous nous soiniiKxs aüaeliés à I étiide de eel effet produil ])ar les

veiiins de:

PHOTEUOGLYPHES

ELAPIDAE

FIYDROPHIIDAE

s()een()(;lyphes

CROTALIDAE

N a j a

E a p e m i s

II y d r o p h i s

E n h y d r i n c

C r o I (I I II s

Naja naja

Naja liaje

Naja nigricollis

Lapeniis hardwickii

llydrophis cyaiiocincliis

Enhydrine schistosa

Cro/ala.s durissas Icrrificas

C. d. ter ri ficas

C. d. terrificus var. ciolarni-

niciis (erotamiiié)

Poiir res|)ecler Tordie eliroiiologiípie de iios reclierclies, nous exaniineroiis

d’al)ord les effets des venins de Grolale et la erolamine, ])uis les vetiins des

Naja et des HYDROPHIIDAE, raelioii inhihilrice iieuromusculaire de ees deux

derniers grou|)es élant voisine.

Les cpiaiitités réduites de venins mises à notre disposition nous onl oliligé

à elierclier des préj)arations les éeonornisant le |)lus ])ossil)le.

Les seliémas de ees essais couiprennenl:

la loxieité,

l('s essais siir le muscle sirié, stimulation iudireele ])ar le nerf, exeitation

direele du muscle.

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542

ACTION NEUROMUSCULAIRE DES VENINS DE QUELQUES

CROTALWAE, ELAPIDAE ET HYniiOPHIIDAE

pré|)aralion lil)ial aniéricur de Fiat in situ, innervé ou dénervé,

pré|)aration iierf scialiquo, muscle lil)ial aiilérieur, niuscle soléairc iti silii

de (dial,

])ré|)aration dia|)hraguie isolé de Hat innervé on dénervé,

jiréjiaralion dn recliis ahãominis isolé de Grenonille,

parfois nne conric inenrsion snr le ninscle lisse,

pré|)aration (riléon isolé de Coliaye.

VENIN DE CROTALUS DUKISSUS TERRIFICUS var. CROTAMINICUS

ET CHOTAMINE

(Venins provenanl de ITnstilnt linlantan (l)r. Schenlierg), erotamine fonrnie

|)ar le Pr. J. Monra (íoiiçalves).

íls onl des aelions (pialilalivemenl semlilaliles, ils seront done trailés en-

seinlile.

TOXICITÉ (250 nicg/kg crolamine inject. i/v qnene de la sonrie Idanclie),

la crise a nu asjiect lyjiique d’nne dnrée de 2 à 8 rninnles, on constale nne

coiilraclnre dn dos avec rejet des palies poslérienres en arrière, les doigls des

[lalles anlérienres recroqnevillés snr enx-mêmes. L'aninial se déplace en ram-

])anl sur les moignons de ses palies anlérienres ainsi qne snr sa qnene. Les crises

se reprodnisenl |)lnsienrs fois. Enlre leni|)s il y a nne aeeéléralion rcs|nraloire

el nne démarelie anorniale “en canard” sni la jioinle des doigls des palies pos¬

lérienres.

PRÉPARATION NEUROMUSCULAIRE IN SITU DE RAT

Action pkorhe difkiókkntk ski.on i.ks i)osi:s:

25-50 nieg/kg i/v, jias de modifiealion de r.implilnde des eoniraelions mais

diniinnlion de rapliinde à manlenir nn lélanos.

100-200 ineg/kg, le nuisele réjiond anx exeilalions mais il y a gêne à la

déeonlra<'lion jns<pi’an nivean de liase. On noie nne laeliypliylaxie jiar répéli-

lion des do.ses, fail dejà signalé par les anienrs hrésiliens.

400-500 meg/kg, donne phénomène eomplexe:

a -- une forle eonlraelnre avee forle élévalion dn nivean de ha.se, gêne de la

déeoniraelion el iliminnlion de ranqiliinde des eoniraelions lani par slimn-

lalion indireele (nerf) que jiar slimidalion direele (musele).

Il — relonr à la ligne de liase iniliale el diminnlion de rampliinde.

e — relonr à rampliinde dn dépari el même angmenlalion de eelles-ei jiar rap-

])orl à la normale.

d — dnrée phénomène: 80 minnles environ, ])nis après lemps varialile (80-15

min.) ajiparaissenl des eonlraelnres sjionlanées moins forles mais lyjiiqnes.

e — nne nonvelle injeelion esl sans effel.

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:í;í(2);541-,'554, 19Ü6

JEAN CHEYMOL, F'RANCOIS BOURILLET,

MONIQUE ROCII

543

Lc fail ((ue li‘s iCFulUUs soiit ideiiliquos siir los moiivenioiils cnlraíiiés ])ar

oxcilalioti indirecto ou diroole est cn juveur d'une origine niusciilaire du phé-

notnène.

PrÉrakatiom déneiívÉe (lil)ial antérioiir déuorvéo). Sur un lol imisolo ox-

cilé diroolomonl une injeolion do orolamino ])rodiiil uno conlraolure mais elle

osl nioins forto qiio cello onlrainée par stiniulatiou dii norf do Fautre patto. Do

plus la laohypliylaxie apparaíl dòs la 2.*' iiijoction sur lo musole déuervé. Ceei

esl, uiissi en juveur d'une aclioii directe sur le inuscle.

I.\TEHACTK)N AVEC LES PAHAEYSANTS NEIIUOMUSCUI.AIKES — NouS avOUS Uti-

lisé uu aoétylolioliuooompétitif (d-luliocurariiio), un acétyloliolinomiméli(|ue (Cm).

d-lul)ocururine — La oonlracturo par la crotaniino so [jroduil comitie si la

|>ré|iaration iFélail pas curariséo, mais dans un 2.*' tonips los 2 substancos agis-

sont |)our donnor une itdiibilion complèto avant rotour des mouvomonts (Fig. 1).

Fig. 1 -- RAT; Contractions du muscle tibial antérieur in sitii. Stimulation

alternee du nerf (Indlrecte) et du muscle (directe) toutes les 10 secondes

(nerf sclatique: 0,U msec, 3 V — muscle 0,1 msec, KO V). En dT: d-tubo-

curarlne — 40 meg/kg l/v: seule la contraction indlrecte est déprlmée. En

C: Crotamine — 500 meg/kg i/v: contraclure typique avec reprise fugace

des contractions indirectes puis Inhibition complete des deux contractions.

Ce muinlien de la contraclure après curarisation est un argumenl de plus

pour une uetion niusculaire.

La orotamino diminuo la sousibilité à tiiio 2.'' injeolion tillérioure do d-lubo-

oiirariuo.

décarnéthoniuni — Après Cm, la oonlracturo est maiuteuue mais est moins

forte que sur une pré|)aration non parliollemont inhibéo. La crotamine aiigmenle

la sousibilité à uno 2.'^ injoction do Cm.

AnTACONISME des lONS

Antagonisme des ions L'«++ — Comino poiii la véralrino Finjoctiou de

CaCb. aiuiule la coutracluro orotaminiquo ot la jtréviont si CaCL est injocté préala-

blomont.

SciELO

LO 11 12 13 14 15 16

544

ACTION NEUROMUSCULAIRE DES VENINS DE QUELQUES

CROTALinAE, ELAPIDAE ET HYDROPHIIDAE

Aniagonismc des ions + — L’aclion i-st dii mêmo lype (]u’avec' (Ài"''

ils soiil nioins toxiques mais leiir effet est ])lus fugarc.

Tacuyimiylaxie ckoisÉE — Nous avons moiilré une lacliypliyiaxie eroisée.

entre crotamiue et venin à crotamine el vice-versa. Par coiitre le veniu saus

crotaminc et la crotamiue administrés run a|)rès Tautre et vice-versa n’onl en-

traíné aucuue altéuuation de la coiitracture crotaminique. La tachyphyla.xie se-

rait donc duc. à ce.ltc dernière siibstance.

PRÉPARATION NERF PHRÊNIQUE — DIAPHRAGME ISOLÉE DE RAT

Action |'kopI!E — Crotamine (7 mcg/ml) cntraine une aclion contractu-

rante avec élévation du niveau de hase et décontraction difficile, la contracture

est généralement immédiate ou peu différée. Klle décroil saus lavage, progres-

sivement mais reparail par lavage. Puis des conlractures ai)])araissent s|)onta-

ucment à intervalles lougs imjjrévisililes.

Actiom des ions — Ca++ [)révient la contracture (CaCL 2()() mcg/ml)

celle-ci ap])araít aii lavage. Ca++ sup])rime la contracture cu cours. Mg+^-d"-

mais aholit plus difficilcmeni une contracture en cours.

iNTElíACTION AVEC EES 1’AHAI.YSANTS NEUKOMESCULAllíES

d-tuhocurarine (dose enlraínant une faihle paralysie). La crotamine entraí-

iie une faihle contracture qui ap[)aralt nettemcut au lavage.

décaméllwriiiun, après Irès faihle contracture par crotamiue et lavage u'eu-

traiiie pas de contracture importante.

Taciiyphyi.axie CHOiSÉE ohicnue sur ce lest jiar venin :i crotamiue ct cro-

tamine ou vice-versa.

RECTUS ABDOMINIS DE GRENOUILLE

Action pkopke — Aucun effet contracturanl sur ce tesl.

2 à 5 mcg/ml de crotaminc seusihilise considérahiemeni le rectas au K +

douhlanl ou triplant les effets du K"*" (30 à '15 miii. a|)rès), cette sensihilisation

persiste après lavage et se prolonge 12 hcures et plus. Les ious Ca"'"“’“ (250

mcg/ml) supprimeiit les effets de la crotamine vis à vis du 1n + .

10 mcg/ml de crotamine douhlent les effets de racétylcholine, mais cette

action disparait après lavage.

ILÉON DE COBAYE

I.a crotamine (2,5 mcg/ml) provoque la contracture des fihres lisses avec

apparilion des rnouvements spontaués de la fihre lisse, disjiaraissant juir lavages

répétés. Peu ou ])as d’action sensilulisante à racétylcholine.

SciELO

LO 11 12 13 14 15 16

Mem. Inst. líutantan

Simp. Internac-,

JEAN CIIEYMOL, FRANCOIS BOURILEET,

MONIQUE HOCII

545

33(2):541-554, 1966

VENIN DE CROTALUS DURISSUS TERRIFICUS SANS CHOTAMINE

TOXICITÉ — 250 nicg/kg i/v chez la Souris jirovoqueiit une gênc respi-

raloire de loutes les souris après 5 minutes. SOÇf meurent en 24 heures. Ce

venin est donc plus toxique que celui à crotainine, coticliision donnée égaleinent

|)ar Vital-Brazil qui [)ense que Ia toxieité du venin dii C. d. terrijicus est due

à la crotoxine.

PRÊPARATION NEUROMUSCULAIRE IN SITU DE RAT — Après une lé-

gère augmenlalion de l’aniplitude des eontraclions au nioment de Einjeetion, ou

ohserve une j)aralysie de la préparation três lente à s’étal)lir et irréversiblc, les

myogramnies sont identiques sur le tibial aiitérieur excite directement comine

sur le tiliial excite indirectement par son nerf. II scmhle donc que le venin

agisse direciement sur le niuscle.

Très léger et fugace antagonisme par Ca++ et clilorhydrat de Clioliue.

Après venin sans crotamiue le venin à crotainine exerce son effet contractiirant

liahitiiel si la paralysie n’est pas trop avancée, mais Eeffet est atténué. Le venin

sans crotainine sensihilise aiix inhihiteurs neuromusculaires tant d-tuhocurarine

que siiccinyldicholine.

Si sur une j)ré|)aration neuromusculaire de Chat, on injecte dans Eartère

tihiale de racétylclioline, on a une contraction ])lus grande ou égale à celle don¬

née jiar Eexcitation indirecte [lar le nerf, ajirès le venin Eeffet acétylcliolinique

est diminué. Donc les récepleurs de la plaque nioirice paraissent également

toiicliés. Sur le diapliragme isolé de Hat on note une jiaralysie |)rogressive lente

et irréversihie.

RECTUS ABDOMINIS DE GRENOUILLE — Ajirès un certain teinjis (do¬

ses > à 10 mcg/ml) on note des contractions de grande amplitude mais de

courte diirée atténiiahles ou su])|)rinialiles jiar les ions Ca"'" + . Pas des niodifica-

tions des seusihilités au K+ ou à EA/C.

L’action contraclurante de la crotainine est-elle vératrinique? Ce rapproclie-

luent a été présenté par Moussatché et Conçalves en 1956. Quatre points sont

comimuis entre les effets de ces deiix substances:

1 — Mêiiies symptònies d'intoxication chez la Souris.

2 — Sensibiliseut le reclas abdoniinis de Grenouille aiix K+ (c’est Eeffet

veratriniqiie de llACri).

o — Cênent la décontraction du muscle.

4 — Ont leurs effets antagonisés par les ions Ca++ et Mg++.

Par contre lEaiitres effets sont nettement différents. Entre autres:

1 — Sur une préparation neuromusculaire non stimulé, Ia crotainine jirovo-

(|ue une contracture, la vératrine ne le fait pas.

2 — Sur une préparation neuromusculaire stimiilée directement ou indirecte-

ment, isolée ou in sita, la vératrine provoque une augmentation de Eamplitude

des contractions sans élévation de la ligue de base alors que la crotamiue ])ro-

voipie une élévation de la ligue de base et diminution jiendant la contracture de

Eamplitude des contractions.

2 3

5 6

11 12 13 14 15

546

ACTION NEUROMUSCULAIRE DES VENINS DE QUELQUES

CROTALIDAE, ELAPIUAE ET HYDROPIIIIDAE

3 — La crotamiiH’ presente iine paralysie seeoiulaire jamais ol)servée avec

la vcralrine.

1 — La crolainine inésente le ])liénomèiie cie lacliyphylaxie et non la veratrine.

5 — Lc's effels cie la vératrine varient en fonclions cie la frécpience cie sli-

nnilation, alors cpie ceux cie la erotamine sonl iclenlic^ues, quelle cpie soil la fre-

ejuenee cie stimulalion.

6 — A|)rès cl-liil)ociirarine (dose entrainanl une paralysie parlielle), la cro-

tamine ])rocliiil ime coiitraeture ]mis la paralysie soiivcmt lolale. La vératrine

entraine la rejerise cies contraetions et rangmentation crani[)litncle aii-delà clii

nivean crorigine.

VENINS DE NAJA {N. naja, N. haje, N. nigricullis)

(Venins provenant de Elnstitut Pastenr (annexe de Garches — Dr. Bocpiet, Paris)

Les actions (jiialilalivcs sont cin mêine type pour les trois, noiis déerirons |)liis

particniièrenient eelles cin N. naja indiqnani à la fin les différenees snrtout dor-

clre qnantitalif entre les trois.

TOXICITÉ — L’aspect qualitatif est tonjonrs seinhlalile mais la valenr quan¬

titativo assez varialile cTiin échantillon à raiilre liien (|ne eenx-ci soient eonservés

à Fctat see en tuhes liien lioncliés, |)arfois scellés et en glacic're. Cliiffres varia-

Ides. Souris hlanche (DL.-.n i/v allant de 390 meg/kg à 850 meg/kg). I.es

Solutions sont tonjonrs préjcarées extem])oranément. Paelivité diminiiant en 21 à

48 lienres de façon sensihle.

La mori [)résente tonjonrs le mêrne aspect. Apirs nn tem|)s de latenee ])lns

on moins long, gêne respiratoire qiii s’aeeentne jiiscpFà Taspliyxie, le coenr eon-

timiant à liattre après Tarrêt respiratoire.

Même tahlean, ehez le Poussin de 8 jonrs, hien qne ceini-ci soit [)lns sensi-

lile que la Souris, la DL.-,» i/v se situant vers 100 meg/kg.

PREPARATION NEUROMUSCULAIRE IN SITU DE RAT

Action PiíonHK — A|)rès injection par voie i/v ou i/artérielle, la jiaralysie

s’installe a|)rès un ternps de latenec' jilus ou moiiis loug, seloti la dose adminis-

trée, elle est lent (|)lusieurs lieures), progre.ssive et irréversilile clans les 7 à 8

lienres des expcriencos.

Si Ton enregistre rexcitation clirecte et indirecte du musele, on constate qne

le musele reste eontractile jusqiFà Ia fin, même (|uancl Texcitation indirecte ])ar

le nerf est inefficaee.

Cet aspect est comparahle à une curarisation classiíjue, la Iransniission ner-

veuse est annulée alors que la contraetion directe par excilaíion du musele persiste.

Ess.ai i)'.\>'TACt)NTSTES — La néostigmine, récirophcmium sur une paralysie

jiar venin de Naja encore à moitié des contraetions uormales, assure une re¬

prise nelte mais fugace.

SciELO

LO 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(2):541-554, 1966

JKAN CHEYMOL, FRANCOIS BOURILLET,

MONIQUE ROCH

547

Sur une même |)réparation eu cours de paralysie par veiiin de N a j a, le ve-

niu de Crotaliis durissus lerrijicus crotaminicus, donne sa contracture hahituelle,

puis une reprise des mouvements, mais la j)aralysie progressive et déjinitive ré-

apparait après.

IntERACTION AVEC INHIBITEUKS NEEKOMUSCULAIRES, D-TUBOCURARINE - Alors

que Ton ])eul injecter j)ar voie i/v jusqidà 6 fois 20 mcg/kg de d-tul)ocurarine

sans curarisation, les mêmes iiijections aj)rès une seide injection de 100 mcg/kg

de venin de N. naja soul toujours progressivement efficaces.

Avec la succinylcholiue les résultats sont conlradictoires. Chose peu êlon-

nante Ic Rat nc réagissant pas de jaçon piire aux acétylcholinomimétiques, la pre-

mière injection agit selon ce type, mais par répétition des doses 1' évolution se

fait vers le type compétitij.

CHUTE DE LA TÊTE DU LAPIN

Sur des la])ins étalomiés par rapporl à la d-tidiocurarine ou à la succinyldi-

choline, nous avons rnontré qu’une injection de venin de N. naja (80 mcg/kg)

diminuait de 50% les doses de d-tuhocurarine nécessaires pour ohtenir la chute

de la tête et ceci, sensihle 9 heures après Finjection du venin se maintient en

s’atténuant jusqiFau 9.® joiir.

Rour les raisons indiquées ci-avanl, les résultats avec la succinyldicholine

sont contradictoires.

DIAPHRAGME ISOLÉ DE RAT — Le venin de N. naja entraíne après un

certain temps de latence une contracture (élevation du niveau de hase) dispa-

raissant par lavage. En même temps s’installe la paralysie progressive, lente, ir-

réversihle. Néostigmine, édroplionium antagonisent nettement mais passagèrement

la ])aralysle. Les ions Ca++ et Mg++ agissent de même. Le venin de C. d.

terrijicus crotaminicus donne sa contracture haliituelle, mais la paralysie réappa-

rait inexorahle.

Commc pour la pré])aration in sita, le venin de N. naja sensihilise le dia-

phragme à la d-tuhocurarine. Avec la succinyldicholine ])as de sensihilisation

mais parfois antagonisme. Ceci augmente la parenté de Taction du venin de

N. naja avec les acétylcholinocompétitifs.

DIAPHRAGME DÉNERVÊ ISOLÉ DE RAT — Sur une telle pré|)aration les

mouvements provoqués })ar excitation directe du muscle étani enregistrés si on ar-

rête les mouvements et ajoute de TA/C durant Farrêl, on a une contraction d’am-

plitude normale. Après addilion de venin on voit Faction de FA/C disparaítre

(Fig. 2). On retrouve ces effets sur une préparation in situ de Chat par in¬

jection d’A/C dans Fartère tihiale avant et après venin de N. naja.

On peut donc admettre (jiie le venin de Naja diminue la sensihilité des

plaques motrices à Facétylcholine.

RECTUS ABDOMINIS DE GRENOUILLE — A doses faihles le venin de

N. naja n’a aucun effet. A doses fortes on ohtieiit des contractures, ne dispa-

raissant ])as par lavage. A la dose de 0,5 mcg/kg, on a siqjpression des effets

de Facétylcholine, mais aucune action sur les effets du K + .

1 SciELO

548

ACTION NEUROMUSCULAIRE DES VENINS DE QUELQUES

CROTALIDAE, ELAPIDAE ET HYDROPHIIDAE

Fig. 2 — RAT; Hémidiaphragme chroniquement dénervé et isolé: Stimulatlon

du muscle (0,1 c/s — 0,1 à 0,2 msec — (50-80 V). La stimulatlon électrique

est interiompue pour permettre 1'additlon au bain de 1’acétylcholine (30 sec)

et le lavage eontlnu de la préparation (9,5 min). La stimulatlon électrique

est reprise 5 min avant une nouvelle additlon d’acétylcholine. En Ac, Tacé-

tylchollne (0,75 mcg/ml) est ajoutée avant et 30-45 et 60 min après Taddition

<en VNh) de 1 mcg/ml de venln de Naja haje. Solutlon de Tyrode — Baln

de 50 ml à 36-37°C — Oxygénatlon par 95% O, + 5% CO,.

Qiiciqiies difjérences entre les Irois renins de l\ n j a: l{é!-erve faite des dif-

íérences d’activilé iriin écliantillon à iin aiilre, il iious a |)ani que le venin de

N. haje étail qiianlilalivenienl le plus jiaralysanl (dose iiiiniina aeüve 100 nieg/kg

i/v). L’élude iieiiromuseiilaire du veniu de A', nigricoilis esl difficile à nioulrer

sur rauimal eiilier à eause de sou aelioii eardiovasculaire linilale elio(|uanl lieau-

eoup les sujeis. L’injeeli(>n répéléi" cl lente de faililes doses (50 à 100 nicg/k")

nous a [lerinis (rexpérirtieuler.

Noloiis égalenieut que sur le diajiliragme isolé de Hal, raiilagouisme par

la néostigmine esl plus niarqué sur le veniu de N. nigricoilis.

VEMNS D’lf]I)ROPHIII)AE: I.upeinis hurdwickii, Uydrophis cyanocinctus

Enliydrinu .schisíosa

(Venins [irovenant de rinstitut Pasteur de Saigon, Dr. Bariue)

Les Irois venins expériinenlés ont des aetions qualilatives eomnnuies, nous

pouvons donc traiter leur aetion neuronniseulaire en niêine leinjis.

TOXICITÊ — V. Eapemis harduickii: DL.,,, i/v Souris 110 nieg/kg voisine

de celle du Naja. haje, le jiliis loxitpie des 5 Naja que nous avons éludiés.

On eonslate une (*xcitalion légère après rinjeclion, après un temps de latenee de

SciELO

LO 11 12 13 14 15 16

Simp. Internac.

33(l):45-54. 1966

JEAN CIIEYMOL, FRANCOIS BOURILLET,

MONIQUE ROCII

549

33(2):541-554, 1966

10 à 15 mimiles, une gêne res|)iratoire s’installe, suivie d’une paralysie progres¬

sivo généralement morlelle. Après une dose non mortelle, il faut au nioins 5

heures pour qne les trouhles res|)iratoires disparaissent.

PRÉPARATION NEUROMUSCULAIRE DE RAT — L’excitation électrique iu-

direcle par le nerf devient inefficace, 1’excitalion électrique directe par le muscle

reste efficace (dose paralysante senil 75-100 nieg/kg). II y a une grande res-

semhlance avec Teífel du venin de Naja, mais ici pas d'effet hypotenseur con-

joinl gênanl.

Donc typc curarisalion classique, mais la paralysie est irréversible. Néostig-

niine et édro[)honium ont une actiou antagoniste fugace. 11 y a sensiOilisation

uette aux doses non ])aralysantes de d-tnhocurariue.

SuR LA PRÉPARATION' isolÉe diaphragme DE Rat, OU note une paralysie len-

tement irréversiltle sMnstallant après nn temps de latence plus ou moius long

selou la dose (2 mcg/ml — la paralysie a[)parait après 20 à 80 min., devient

totale, malgré les lavages, 80 à 40 minutes après). Là encore, Texcitatiou électri-

qne indirecto ])ar nerf devient inefficace, rexcitalion électrique directe du muscle

reste efficace. Mêines antagonismes fugaces jiar néostigmine et édropliouium.

Même sensiliilisatiou uette aux doses non paralysantes de d-tuhocurariue.

Actiox de l’acétylcholine exogène

a) Sur un diajihragme clironique dénervé, isolé 10 jours ajuès. rexcitalion

directe dn muscle étanl inlerrompne, Taddition d’A/C nVntraine aucnne con-

traction après venin d’ HYDROPHIIDAE.

li) Sur une ])réparation in silit de Chat. Texcitation indirecte étanl inter-

rompue, rinjection dans Tartère tihiale d’A/C. nVntraine aucnne contraction

après venin de L a p e m i s (l’ig. 8).

RECTUS ABDOMINIS ISOLÉ DE GRENOUILLE

Sur ce lest venius tl’ HYDROPHIIDAE: pas (radiou pro|)re, diminulion con-

sidérahle des effets de rA/C, ]ias de sensihilisalion aux ions K'*'.

Kn conclnsion adiou nenromusculaire du même type que celle des Naja,

mais peut-êtrc plus dépouillée. car il n’y a pas de fadenr cardiovasculaire donc

radiou hy[)olensive surajoulée rend ])arfois pour ceux-ci rexpérimenlalion diffi-

cile sur les ])ré])aralions in siln.

Mécanisme daclion péripliériquc nenromusculaire des venins de Naja et

d’ HYDROPHIIDAE étudiés.

Le trois venins de Naja (N. naja, N. haje, N. nigricollis) d les trois ve¬

nins d’ HYDROPHIIDAE {Lupemis hurdwickii, Jíydrop/iis cyanocinclus. Enhydri-

na. schistosa) éludies ont une adiou neuromnscidaire du même Ivjie. Celle adiou

ressemhle ndtement à une curarisation typiqne. On conslale après lenr admi-

nislralion;

Argnmcnls ])our lype curarisalion classique (ly|)e Claiide Bernardi

1 — IVxcilalion éledri(|ne indirecte ])ar le nerf devient inefficace, rexcitalion

éledritjne dn muscle reste efficace.

2 3

5 6

11 12 13 14 15

550

ACTION NEUROMUSCULAIRE DES VENINS DE QUELQUES

CROTALIDAE, ELAPIDAE ET HYDROPHIIDAE

mín

Fig. 3 — CHAT: Contractlons du muscle tlbial antérieur in situ. Stimulatlon

du nerf sciatique (0,3 msec — 3 V — 0,1 c/sec) interrompue pendant 2 mín

pour permettre ITnjectlon rapide d’acétylcholine en Ac (2,5 mcg) dans l’ar-

tère tibiale (portlon dlstale à contre courant). A: contracture lémoin en V

Lap, injection i/v de 50 mcg/kg de venln de Lapemis (doíe paralysante

seull). La contracture e.st presque totalement inhibée au bout de 12 min:

clle récupére ensuite progressivement au bout de 30 min (B), 45 min (C) et

1 h (D), mais sans jamais pouvoir atteindre Tamplitude initiale.

2 — iiéosligmhie, édroplionium, antagonisme, nc’t mais fugace.

3 — synergic avec les acétylclioliiiocompíílitifs, antagonisme (?) avec acétylclio-

rmcmiméti(|ues.

4 — injection directe dans Tartère (Prep. nerf-muscle in silu) inefficace addi-

tion d’A/C diapliragme isolé innervé ou énervé (dejniis 15 joiirs) inefficace.

Ceei montre que TA/C exogène n’agit |)lus sur les récepteurs s])écifiques et

laisse supposer (juMI en est de même pour PA/C eudogène. S’il y a diminutiou

de la syntlièse de TA/C (?l, cela ne tient pas à uu déficit en clioline, Tadini-

nistration de celle-ci ne modifiant pas la paralysie.

Aux doses supraliminaires de venin, raugmentation de fréquence des excita-

tions (augmentant la consommatiou d’A/C endogène) parait accélérer la paralysie.

5 — diminutiou ou sujipression des effets de TA/C sur le rccíus ahdominis de

Grenouille.

Arj^umenta contre une ciirarisaíion. clu.saique:

1 — ternps de lateuce plus ou moins jírolougé et jiaralysie irréversihle,

2 — pas (Tantagonismes vrais, jniisque fugaces.

SciELO

LO 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

JEAN CHEYMOL, FRANCOIS BOURILLET,

MONIQUE ROCH

551

33(2):541-554, 1966

Bieii qu’il senihle y avoir avec ces veniiis une actioii siir les récepteiirs spé-

cifiqiies à racétylcholine de Ia ])laqiie niotrice, on ne peul doiic Fassimiler à la

paralysie réversil)le par les acétylcliolinoconipétitifs (curares historiqiies ou subs-

taiices de synlhèse). Le blocage des réceplcurs |)arail ici irréversible.

CONCLUSIONS

D’inie façon uii peu sinqdisle, ou peul opposer:

L’actiou musciilaire des veuins de CROTALIDAE: contracturaule dans le cas

des veuins à crotamine, paralysanle pour les veniiis sans crolaniine.

L’a(lioii curarisante de lype compétitif des veuins de Naja et tl’ HYDRO-

PHIIDAE.

SuMMARY

I. The nenronuiscidar aclions of several cobra (common cobra. Naja naja,

Egyplian cobra, N. haje, and spitling cobra. N. nigricollis) and sea-snake (Lapc-

mis hardivickii, Jlydrophis cyariocinctas and En/iydrina schistosa) venonis were

studied as well as ihose of both varieties of ibe South American ratllesnake

iCroUdiis durissus lerrijicas) venoni. lhe crolamin conlaining onc and lhe variely

devoid of lhal substance. The aclions of crolamin itself, a basic ])rolein isolated

l)y Moura Gonçalves, was also invesligaled.

II. All tbree cobra venoms studied are neuromuscidar blocking agenls: tbey

exert a perij)beral, slovvly indiiced and irreversible action. The jiaralysis of

neuromuscular transmission a])[)ears concurrenlly wilh a cardiovascular depression

on iti .‘iilii |)reparations or wilb a conlraclure on isolated ])reparations. N. haje.

venom is a more potcnl ueuromuscniar blocking agenl tban a cardiovascular de-

pressing or conlraclure inducing substance vvbile N. nigricoilis venom is more

aclive in producing cardiovascular depression or skelelal muscle conlraclure tban

in cliciting neuromuscular blockade. The venom of N. naja occu])ies, from tbis

poinl of view, an inlermediate position.

1. The skelelal muscle paralysis is not due lo a direct action of lhe cobra

venoms on ibe muscle fibre; lhe conlraclure, on lhe conlrary, is ])roduced at

tbis levei.

2. l'bc inlerruption of nenromuscular transmission produced by tbe Naja

venoms is, at least in some measnre, exj)lainable by a slow and irreversilile

inhibition of end-plale recej)tors: therefore ibeir neuromuscular blocking action

resemble a curarization in Cl. Bernard sense. It is wilh tbe venom of N. naja

ibat tbis itdiibilion of recejilors cau be beller evidencc<l.

.1. Il is assumed, wilboul direcl evidence. tbal tbcse venoms also exert

a presyna])lic action.

III. All ibree HYDROPHIIDAE venoms studied sbow qualitalively identical

neuromuscular blocking properlies. Tbcir mecbanism of action is very dose lo

tbal of tbe Naja venoms: |)aralysis of neuromuscular transmission by a slow

2 3

5 6

11 12 13 14 15

552

ACTION NEUROMUSCULAIRE DES VENINS DE QUELQUES

CROTALIDAE, ELAPIDAE ET HYDROPUIIDAE

and irreversilile inhihition of cnd-platn receptors. On lhe other liand, lhe

HYDROPHIIDAE venoms, cotUrary to llie cobra venomp, do nol prodiice cardio¬

vascular depression or contracturcs.

IV. The crotamin conlaining variety of ralllesnake venom and crolamin

ilself show contracture indncing ])ropertics on skeletal mnscles which can l>c oh-

served on inlact and conscious animais as well as on in sitii or isolated prc|)aralions.

This effect is jjroduced hy a direcl action of crotamin on mnscle fihres. A

secondary paralysis is inconstantiy prodnced.

fn s])ite of some similarilies with veratrin, lhe action of crotamin and cro-

taminic venom can not he compared with a veratrinie one.

V. The non crotaminic ralllesnake venom shows a weak and slowly indueed

paralytic effect whicli can nol he easily evidenced on accoimt of its slrong cardio¬

vascular depressing properlies.

This |)aralytic effect is dne lo a ilirecl action on lhe mnscle fihre without

inliihition of lhe end-plate recei)tors.

Rekereaces

VENIN DE CROTALE ^ CROTAMINE

1. BARRIO, A., et VITAL BRAZIL, O., Rev. Inst. Malbnin, 16, 22-40, 1954,

2. ESSEX, H. E., et MARKOWITZ, J., Arner. J. Physiol., 92, 317, 348, 695-706,

1930.

3. FRAENKEL-CONRAT, H., et SINGER, B., Arch. Biochem., 60, 64-73, 1956.

4. GRALEN, N., et SVEDBERG, T., Biochem. J., .32, 1375-77, 1938.

5. HABERMANN, E., et NEUMANN, W. P., Biochem. Z., 327, 170-85, 1955.

6. HABERMANN, E., et NEUMANN, W. P., Biochem. Z., 329, 405-415, 1957.

7. HOUSSAY, B. A., et coll., C. R. Soc. Biol., 87, 821-824, 1922.

8. HOUSSAY, B. A., et coll., C. R. Soc. Biol., 88, 367-68, 1923.

9. KLAUBER, Rattlesnakes, Berkeley University Press, 1956.

10. MOURA GONÇALVES, J., Venoms, 261-274, 1956.

11. MOUSSATCHÉ, H., et VIEIRA, G. D., An. Acud. brasil. Cienc., 25, 249-58,

1953.

12. MOUSSATCHÉ, H., GONÇALVES, J. M., VIEIRA, G. D., et HASSON, A„

Venoms, 275-79, 1956.

13. SCHENBERG, S., Science. 129, 1361-63, 1959.

14. SLOTTA, K. H., et FRAENKEL-CONRAT, H. L., Nuture, 142, 213, 1938.

15. VELLARD, J., C. R. Soc. Biol., 130, 463-64, 1939.

SciELO

LO 11 12 13 14 15 16

Mem. Inst. Butantan

-Slmp. Intcrnac.

33(2):541-554, 1966

JEAN CHEYMOL, FRANCOIS BOURILLET,

MONIQUE ROCII

553

VENINS DE NAJA

16. ARTHUS, M., Arch. int. Physiol., 10, 161-191, 1910.

17. BOQUET, P., Toxicon, 3, 243-79, 1986.

18. BRUNTON, T. L., et FAYRER, J., Proc. roy. Soc., 31, 358-374, 1872.

19. BRUNTON, T. L., et FAYRER, J., Proc. roy. Soc., 32, 68-133, 1873 .

20. CICARDO, V. H., C. R. Soc. Biol., 120, 732-733, 1935.

21. SU, C., J. Formosan med. Ass., 59, 1083-1091, 1960.

22. DETRAIT, J., IZARD, Y., et BOQUET, P., C. R. Soc. BioL, 153, 1722-24, 1959.

23. ELLIOT, R. H., PhiU. trans. roy. Soc., 197, 361-406, 1955.

24. GAUTRELET, J., HALPERN, N., et CORTEGGIANI, E., Arch. int., 38, 293-

352, 1934.

25. KELLAWAY, C. H., BuH. John’s Hopk. Hosp., (50, 18-39, 1937.

26. KELLAWAY, C. H., CHERRY, R. O., et WILLIAMS, F. E., Aiist. J. exp. Biol.

med. Sei., 10, 181-194, 1932.

27. LEE, C. Y., et PENG, M. T., Arch. int. PhurmacoL, 133, 180-192, 1961.

28. MELDRUM, B. S., Brit. J. Phurmacol., 25, 197-205, 1965.

29. MELDRUM, B. S., Physiol. Rev., 17, 393-445, 1965.

30. RAGOTZI, V., Arch. Pathol. Aniitom. Physiol., 123, 201-234, 1890.

31. SARKAR, N. K., et MAITRE, S. R., Amer. J. Physiol., 163, 209-211, 1950.

32. VICK, J. A. et coll., Arch. int. Phurmacol., 153, 424-429, 1965.

33. VITAL BRAZIL, O., Thèse fac. med. São Paulo. 1983.

34. VITAL BRAZIL, O., et BARRIO, A., Rev. Inst. Mulbrán. 16, 1-18, 1954.

35. WALL, A. J., Proc. roy. Soc., 32, 333-362, 1881.

VENINS D'HYDROPHIJDAE

36. BARME, M., Ven. and pois. animais and noxious plants of the Pacific region,

Pergamon Press, 1963, pp. 373-378.

37. BARME, M., et DETRAIT, J., C. R. Acad. Sc:., 218, 312-315, 1959.

38. CARREY, J. E., et WRIGHT, E. A., Trans. roy. Soc. trop. Med. Hyg., 54, 50-

67, 1960.

39. CARREY, J. E., et WRIGHT, E. A., Trans. roy. Soc. trop. Med. Hyg., 55, 153,

1961.

40. FRASER, T. R., et ELLIOT, R. H., Proc. roy. Soc., 74, 104-109, 1904.

41. FRASER, T. R., et ELLIOT, R. H., Pliilul. Trans. roy. Soc., 107, 249-279, 1905.

42. HALSTEAD, B. W., Dangerous marine animais, 1959, pp. 93-99.

SciELO

10 11 12 13 14 15

554

ACTION NEUROMUSCULAIRE DES VENINS DE QUELQUES

CROTALIDAE, ELAPIDAE ET HYDROPHIIDAE

43. MARSDEN, A. T. H., et REID, H. A., Brit. med. J., 1290-93, 1961.

44. REID, H. A., Brit. med. J., 2, 73-78, 1956.

45. REID, H. A., Brit. med. J., 2, 1284-89, 1961.

46. REID, H. A., Symyosium sur an. et yl. ven. du Pacif., Pergamon Press, 1963,

pp. 355-362.

47. ROGERS, L., Proc. roy. Soc., 71, 481-496, 1903.

48. ROGERS, L., Proc. roy. Soc., 72, 305-319, 1903.

49. TAUB, A. M., et ELLIOTT, W. B., Toxicon, 2, 87-92, 1964.

50. WERLER, J. E., et KEEGAN, H. L., SymyosUim sur an. et yl. ven. du Pacif.,

Pergamon Press, 1963, pp. 219-325.

Discussion

A. Barrio comments: “Nos cupo a Oswaldo Vital Brazil y a mi seiialar por

primera vez la existência de dos tipos de veneno de Crotalus durissus terrificus

en cuanto a su acción neuromuscular: uno (tipo I) que producia paralisis y otro

(tipo II) que en forma muy llamativa prov'ocaba espasmos. Posteriormente pude

demonstrar que este cuadro era producido por la crotamina, substancia aislada por

Moura Gonçalves, dei veneno de C. díirissus terrificus procedente de la Argentina.

Comparamos la acción dei veneno tipo II con la veratrina. Aclaro que denomina¬

mos a esta ponzofia “v'eratrine-like”, nunca quisimos senalar identidad absoluta y

total entre estas dos substancias.”

SciELO

LO 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(2);555-572, 1966

C. Y. LEE anti C. C. CHANG

555

58. MODES OF ACTJONS OF P15FIF1ED TOXINS FROM ELAPID VEXOMS

ON NEUROMISCULAR TKAXSMISSIOX

C. Y. LEE and C. C. CHANG

PJiarmacological Institute, Colíege of Medicine, National Taiwan University,

Taipei. Taiwan, China

The actions of venoms from snakes lielonging lo tlie faniily of ELAPIDAE

011 neuroiTuisciilar traiisniission liave recenlly lieen reviewed liy Meldnini ( 1 ). The

view lhat peripheral jiaralysis of res])iratory imiscles is lhe principal cause of

dealh from elapid venoms aj)])ears lo he well eslahlislied (2, 8, T)- The ciirare-

like aclion of elapid venoms has iiecn exlensively sliidied and several aulhors

havo presenled evidence lhat neiiromiiscular lilocking venoms have a ])ostsyna])tic

site of aclion (for references see Meldrum (1 ) I. Altliough a “non-depolarizing’'

lilock of neuromusciilar transmission. like lhal of curare, has heen |)OStulated as

lhe mode of aclion for Formosan cohra {Naja naja atra) venom (5) and Iianded

krait (Bungarus inalticinctus) veuorn (6). some differences lielvveen actions of

lhese venoms and ihose of curare were also noled. Thus, lhe neuromuscular

hlock was not effectively relieved hy anticholin-eslerases or reversed hy washiiig-

the muscle paralysed liy lhese venoms at higher concentrations; and acetvlcholiiie

release from llie presynaptic terminais was more or less imjiaired. Resides, lhe

aclion of cohra venom is complicaled wilh a direcl musculolropic effecl (5). and

a depolarizing aclivity on skelelal muscle has heen found in ihis venom (7).

Since snake venom could he regarded as a mixiure of proleins or jioly-

pcjilides, il was considered lhal lhe complexily of lhe venom ailions mighl he

due lo comhined effecls of differeni componenis contained in lhe same venom.

Using zone electrojihoresis on slarch at pH 5.0, lhe venom of B. multicinctus

was separated inlo four fractions (8). One lacks neuromuscular hlocking jiro-

perties Init contains cholinesterase. One called “n-Rungaroloxin'’ jiroduces a

neuromuscular hlock of relalively ra])id ousei in vitro and in vivo. Il does

nol aller lhe acelylcholine outpul from lhe ral phrenic nerve endiugs hui il

aholishes lhe respon.se of lhe chick hivenler cervicis muscle lo acelylcholine. The

Iwo most eleclroposillve fraclions, called ‘‘yS- and y-Bungaroloxin” respeclively.

juoduce neuromuscular hlock and a severe reduction in acelylcholine ouljnil in

lhe ral dia|ihragm afler a laleni jieriod of ahoul one hour and this jieriod is

not shorlened hy increasing lhe dose. Xeuromuscular hlock produced hy lhese

two fraclions in lhe chick hivenler cervicis muscle is nol associaled wilh anv

diminulion of sensilivily lo acelylcholine. Mice given large doses of lhese fractions

show hyperirrilahilily al firsl and, afler a lalenl period of ahoul one hour. die

suddenly wilh dysjmea and convulsions.

Aidetl by thc U.S. Army Meti, Re.s. Develop. Command Research Grant DA-MD-19-

193-6d-G10S and by the National Councll on Science Development, Republic of China.

2 3

5 6

11 12 13 14 15

MODES OF ACTIONS OF PURIFIED TOXINS FROM ELAPID VENOMS

ON NEUROMUSCULAR TRANSMISSION

Frotn Naja naja a/ra vciiom two loxic fractions were sejiaralod uiuler the

sainc cleclrophoretic ('011(11110118(9,10). One callcd Najaloxin or cobra nciiro-

toxin produces non-depolarizing Idock of neiiromii.sciilar transniission liiil, iinlike

lhe vvliole veiiom, doe.s iiol diminisli lhe acetylclioliiic oul]nil froni lhe rat phrenic

iierve, nor has aiiy direcl actioii oii lhe miisele fihres. This neiiroloxiii is also

free frotii phospliolipase A, histaniiiie releasing and local irritanl aclivilies conlaiiied

in lhe vvhole veiiom. Anolher fractioii, which is lhe mosl eleclroposilivc and

called carclioloxin, produces a neuroniusciilar Idock vvilli coiitracinre, cardiac arrest

in syslolic .«tale and niany ollier |)harmacological effects(ll).

From lhe ahove-mentioiied studies, il is ohvious lliat al lea.®! Iwo tyfies of

effect.s are oh.servah]e al the neuromiiscular junclion. A curare-Iike iion-depolariz-

ing lilock is produced liy colira neurotoxin and «-Bungaroloxin. Tlie olher type

of effeci is impairment of acelylcholiiie release from the iiresynaptic terminais,

which is produced hy /?- and y-Hungaroloxiiis.

The [iresent pajier descrihes lhe resuits of our recent eleclrophysiological

study of lhese purified loxins on the iieuromuscular Iransmission. A preliniinary

accounl of some of lhe residis has hcen givcii in a coinmunicalion al lhe XXIII

Internalional Congress of Physiological Sciences (12).

Materials and methods

Parijicalion of toxin.i: — «- and y8-l?ungaroloxins were isolaled from lhe

venom of Hiinganis nuillicinclus accordirig lo lhe mcthod descrilied hy Chang &

Lee (8). Cohra neurotoxin and cardioloxiu were juirified from the venom of

Naja naja atra hy CM-Scphadex column chromalograjihy as descrilied hy Lo,

Chen & Lee (13). Potencies of lhese purified loxins were checked hy their

toxicities in mice and effecis on the chick hivenler cervicis muscle.

I niraccilular inicrocleclrodc rccordtng: — The conventional microelectrode

rccording techniipie (11) was adopicd using glass rnicroelectrodi^s filled wilh 3M

KLl and having 6-10 Mí2 resislance. No ca|iacily compensation for lhe micro¬

electrode was incorporaled. (Irass model P6 DL jireamplifier wilh ils calhode-

follower prohe and leklronix 5()2A oscilloscojie were used. For the ral jdirenic

iierve-diajiliragm preparaliou, Tyrode solution oxygenaled wilh ÇSÇF Oo + 5%

C ()2 was em|)loye(l. The temperalnre was kept al 32-35 ± ().5'’C. For the

frog nerve-saiTorius mnscle lhe jireparalion was susjiended in Kinger solution,

conlaining NaCl 117 mM. KCI 2.0 mM. CaCl. 1.8 niM and NallCO., 6 niM,

at rooni temperalnre (20-21'’C).

'I’he end-plale foeus was localized wilh lhe aid of lhe lime-coiirsc of lhe

niinialure end-plale ]iolenlial (FPP) or evoked FPP. Indirecl sliniulalion was

ajiplied ihrough a pair of electrodes on lhe nerve wilh snpramaximal reclangular

])ulses of 0.2 msec duralion, and direcl sliniulalion ihrough electrodes. one on

the niuscle-tendon junclion and lhe olher in lhe halh fluid, also wilh 0.2 msec

rectangnlar ]ndses.

'I crniinal nerve .spikc: — Lxiracellnlar reeording of lhe terminal nerve sjiike

wilh a microelectrode having resislance of ahoul 5 MU was ])erformed on lhe

frog sarlorius muscle, aceording lo lhe techniijue descrilied hy Kaiz & Milcdi (15).

The niusde was imniohilized hy adding 11 niM MgLL lo the Hingir solution.

Lhidor such condilion, lhe lerminal nerve sjiike potenlial could he rccorded to-

gcther wilh an EPP.

SciELO

LO 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(2):555-572, 1966

C. Y. LKE and C. C. CIIANG

557

Antidronüc activily: — The nicthofl clescriliod liy Katulic & Straugliaii 1161

for the recording of anlidromic aclivity of the isolated ral j)hrenic iierve was

followed. To llie niodified Tyrode solution, conlaining 3.6 mM CaCl 2 and 0.12

niM MgCU, iieostigmiiie melhylsid|)liate was addcd to give a final concenlralion

of 0.3 /ig/nil. Tlie lemperaliiro was ke])l al 22 dz O.S^C. linder ihis condition

it was |)ossilile to rocord the antidromic repetitivo discharges of lhe nerve follow-

ing single nerve volleys for more lhen 2 honrs if the stimulns frequency was

kej)t at 0.05/see or less.

Bivcnter cervicis iierve-muscle preparalion oj lhe chick: — The isolated hi-

venter cervicis nerve-muscle j)re[)aration (17) was siispended in 20 ml of Krehs

soliilion which was maintained at 37 ± 0.5"C and l)id)hled with 9S^/c IT and

5% CO 2 . The preparalion was stimnlated indirectly with snpramaximal reet-

angular pulses of 0.5 msec duralion at a rale of 6 per min.

Hesui.ts and discussion

Efjecl on resting membranc poleritlals: — As shown in Tahle I, all of lhe

three purified neiirctoxins, cohra neurotoxin, a- and /3-Bimgarotoxins, did not

cause any changes in the resling memhrane |)olentials of mustle fihres at eithcr

end-plate or non-end-plate zone of the rat diaphragm at a concentration as high

as 10 /ig/rn], In eontrast, cardiotoxin as well as erude cohra venom caused a

])rogressive reduction of the resting potentials. Our resuits are al variance with

ihose of Meldrum (7) who reported that lhe neurotoxic fraction isolated from

Indian cohra {Naja naja) venom depolarized the frog sartorius muscle. It is

likely that ^Ieldrum’s neurotoxic fraction mighl he co}Uaminaled hy lhe cardio-

loxic comjronent.

TABI.E I — EFFECT ON RESTING MEMBRANE POTENTIALS

Membrane potential.s were recordcd from both the end-plate and non end-plate zone.s

of muscle fibers of the rat diaphragm at the Indicated periods after addition of 10 /ig/ml

of each agent. n = Number of observations.

TOXIN

Membrane

potentials (mV ± S.D.)

Control

0-5 min

5-10 min

10-15 min

15-20 min

(»-Bungarotoxln

75.3

± 3.7

77.3 ± 6.5

79.3 ± 3.2

81.6 ± G.ü

74.7

± 4.1

= 33)

(n = 9)

(n = 11)

= 6)

^-Bungarotoxin

76.5

± 5.0

—

72.9

± 8.2

= 27)

= 23)

Cobra neurotoxin

78.1

± 4.4

81.4 ± 4.4

77.0 ± 3.9

78.4 ± 4.2

76.0

± 4.8

= 28)

(n = 11)

(n = 5)

(n = 10)

= 7)

Crucie cobra venom

83.0

± 3.7

49.3 ± 8.3

.34.0 ± 17.2

29.0 ± 6.2

23.0

± 6.8

= 30)

(n = 9)

(n = 11)

(n = 9)

= 8)

Cardiotoxin

81.4

± 4.1

73.7 ± 8.5

54.4 ± 9.7

45.3 ± 14.2

28.9

± 12.8

= 30)

(n = 11)

(n = 10)

(n = 9)

= 12)

SciELO

10 11 12 13 14 15

558

MODES OF ACTIONS OF FURIFIED TOXINS FROM ELAPID VENOMS

ON NEUROMUSCULAR TRANSMISSION

Kflccl ou aclion potentiuls: — As sliown in Fig. 1, no a|)i)rec'ial)le clianges

liolh in lhe am|)Iitiule and lime course were fonnd in lhe aclion polcnlials elicited

hy direct stimnialion of llie mnscle fihres paralysed l)y a high concenlration (10

/xg/ml) of eilher a-Bungarotoxin or colira neuroloxin. Tliis is in agreement

willi our previous findings lhal lliese nenroloxins do not affect lhe ninscle fihre

itself.

«-BUNGAROTOXIN

(I X lO'5)'

COBRA NEUROTOXIN

(I X 10-5)

2 msec

50 mV

Fig. 1 — Effect on action potentials. Action potentials recorded from rat diai)hragm

preparation.s are shown. The flrst traoing of each pair (a & c) shows control action

potential evoked by indirect stimulatlon. The second tracings are action potentials evoked

by direct .stlmulatlon after the neuromuscular transmls.slon was blocked by 10 íig/ml of

(v-Bungarotoxin (b) and 10 /tg/ml of cobra neuroto.xln (d), iespectlvcly.

Ay/ccf on rnd-platc polcnlials (EPI\s): — EPFs were easily recorded from

lhe mnscle fil)res of ifie ral diaphragm, immedialely afler llieir mechanical res-

jionses on nerve slimnialion had heen aliolished l>y /Idhmgaroloxin al a con-

cenlralion of 1 /ig/ml. üy conlrasl, in lhe cases of «dhmgaroloxin and cohra

neuroloxin, sn|)erficial mnscle fihres were innch more rapidiv paralysed lhan lhe

deeper ones and no KPPs conld he recorded from lhe superficial mn.scle fihres

when lhe mnscle was paralysed. In order lo record KI’Ps from lhe diaphragm

[laralysed hy cohra neuroloxin, lhe prejiaralions was firsl immersed in a con-

eentralion of 1 /xg/ml for ahonl one honr lo hlock lhe ncnromnscniar Iransmission,

lhen washed for one honi', and finally swilclied lo a lowcr conceniralion, snch

as ().0.'l-0.0,5 ;ng/ml, which was jnsl enongh lo ])revenl reappearance of mechanical

response on nerve slimnialion. Uniike cohra neuroloxin. lhe effecl of «-Piiingaro-

loxin was progressive and irreversihie and hence no sleatly slale conld he al-

lained in lhe pre.«ence of lhe loxin. In lliis case, lower concenlralions, snch

as 0.4-0.,S /j,g/ml, were a|)plied for ahonl 2-8 honrs anil then, lhe preparalion

was washed wilh fresh Tyrode solnlion for recording of KPPs.

The lirne-conrses of llie KI’Ps ihns ohiained were compared wilh ihose

ohlained in llie preparalions jiaralysed hy olher nenromnsctdar hlocking agenls,

snch as dimelhyllnhocnrarine (DMTfi), decamelhoninm iCinl and MgCK. As

shown in Fig. 2 and Tahie II, lhe KPPs ohlained in lhe diaphragm Irealed wilh

each neuroloxin, whellicr acling |)resynaplically or poslsyna|)lically. invariahiy

showed a shorler lime-conrse lhan ihosc ohlained in lhe mnscle Irealed wilh

SciELO

LO 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(2):555-572. 1966

C. Y. LEE and C. C. CHANG

559

a-BUNGAROTOXIN

^-BUNGAROTOXIN

DECAMETHONIUM

COBRA NEUROTOXIN

MAGNE51UM

-ju-Í

2 msec

2 mV

Fig. 2 — EPPs as aftected by toxlns and ofher neuromuscular blocking agents.

EPPs reeorded from the rat phreníc nerve-diaphragm preparation vvhich was treated

wlth the respective neuromuscflar blocking agent as descrlbed in Table II.

TABLE II — TIME-COURSES OF THE EPPs AS AFFECTED BY NEUROTOXINS AND

OTHER NEUROMUSCULAR BLOCKING AGENTS

The concentration of each agent shDvvn in the table is the final one except that of

a-Bungarotoxin and cobra neurotoxin; foi details see the text. * For spontaneous miniature

EPPs only those vvhich had amplitudes comparable vvith the EPPs vvere selected. n =

Number of observations.

AGENT

Concentration

Amplitude

(mV±S.D.)

Time from

onset to peak

(msec+S.D.)

Time from

peak to

14 decay

(msec±S.D.)

a-Bungarotoxln

0.4 /ig/ml for 3 hrs.,

then wash-out

2.35 ± 1.28

0.71 ± 0.14

0.62 ± 0.18

/3-Bungarotoxln

1.0 Hg/ml

3.04 4 1.12

0.57 ± 0.11

0.97 ± 0.21

Cobra neurotoxin

1.0 /ig/ml for 1 hr.,

then reduced to 0.05

/ig/ml

3.14 i 1.29

0.59 ± 0.13

1.02 ± 0.29

Dimethyltubocurarine

0.8 íig/ml

3.43 ± 0.94

0.82 ± 0.20

1.54 ± 0.12

Decamethonium

40.0 ^g/ml

3.41 ± 1.16

0.95 ± 0.28

2.38 ± 0.78

Mg + +

12 mM

3.04 ± 0.60

0.41 ± 0.03

0.71 ± 0.09

Miniature EPP*

—

2.27 ± 0.46

—

0.78 ± 0.16

1 SciELO

560 MODES OF ACTIONS OF PURIFIED TOXINS FROM ELAPID VENOMS

ON NEUROMUSCULAR TRANSMISSION

eitlier DMTC or Cn, and vvere ralher dose to ihose of lhe magnesiiim-paralysed

muscle. These results suggest that lhe more prolonged EPP ohtained in lhe

muscle treated either vvith DMTC or Cm as compared vvith that ohtained in the

magnesium- or neurotoxin-paralysed muscle may he due to some jjharmacological

effecl exerted hy DMTC or C,,, on the end-i)late.

Ejjecl on spontaneous miniature EEPs: — Brooks (18) has ohserved a decline

in the frequency of miniature EPPs in a jjreparation treated with holulinum-

loxin. Neuromuscular hlock occurred either hefore or after the complete dis-

appearance of miniature EPPs. This result led him to conclude that hotulinnm-

toxin blocks the neuromuscular transmission at the nerve terminais rather than

at the arhorization as postulated hefore hy himself (19). The effect of /?-Bun-

garotoxin was sludied in the conjunction since it also hlocks neuromuscular

transmission at presyna|)tic site (8). The frequency of a|)pearance of miniature

EPPs was first increased 2-8 times hy /8-Bungarotoxin (0.3-3 p.g/ml) during

1-2 hours after the application, then gradually decreased and finally no miniature

EPPs conld he fonnd (Figs. 3 & 4). Stimulation of the j)hrenic nerve revealed

that EPPs were al)oli.shed hefore lhe com|)lete disappearance of miniature EPPs.

It was occasionally found that a hurst of miniature EPPs ap|)eared for several

minutes hefore their complete disappearance (Eig. 5 ).

In contrast. hoth a-Bungaroloxin and cohra nenrotoxin reduced the amplitude

of miniature EPPs progressively without affecting the rate of discharge, and final¬

ly the miniature EPPs had disappeared hefore neuromuscular hlock took place.

200 mse^

I mV

Flg. .3 — Effect of fi-Rungarotoxin on tlie spontaneou.s miniature EPPs. Continuou.? record-

ing on moving film, 2 .sec per svveep. (a) Control recordeii 45 min after .setling up of

the preparation; (I)) 80 min after addition of 0,3 /i-ttungarotoxin. Note the

increase in lhe frequency of miniature EPPs.

SciELO

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Mem. Inst. Butantan

Simp. Internac.

33(2):555-572, 1966

C. Y. LEE and C. C. CHANG

561

Flg. 4 — Effect of /3-Bungarotoxin on the trequency of miniature EPPs. A dif-

ferent preparation from that shown in Fig. 3. Control was taken 30-60 min atter

setting up ot the preparation and shown as the filled cirele in the leít side. /?-Bun-

garotoxin (3 Mg/ml) was added at 0 time. At 240 min, none ot muscle fibres

contracted on nerve stimulation. Each elrcle represents the mean trequency ot

miniature EPPs obtained trom 20 sec observatlon of one end-plate.

Fig. 5 — Burst ot miniature EPPs induced tay ^-Bungarotoxin. The same preparation as

described in Fig. 3. A burst ot miniature EPPs from the same end-plate is shown. It

was recorded at about 200 min atter addition of 0.3 (Ug/ml /t-Bungarotoxin. There was

about 1 min interval between (a) and (b). This end-plate did not respond to nerve

stimulation when the record was taken.

Antuí^onism ivitli neostig,mim’: — As shown in Mg. 6 (a & ii), in the ral

(liaphragni, treated wilh eillier «-lUingarotoxin or eohra neuroloxin. tlie EPF was

increased in ils size and jnolongpd in its linie-conrse l)y neosliginine (1 /j.g/nd).

Some of lhe Kl’l’s hecame large enongh to generale action potentials. Neo-

stigmine aiso exerted a similar effect on llie miniature KPPs recorded in lhe

diaphragm afler the evoked EPPs liad lieen aholished hy /3-I]imgarotoxin (Eig.

6, c & (1). It mnsl l)e staled here. however, lhal <v-Himgarotoxin hlocks lhe

neiiromuscular Iransmission more rapidiy in the presence of anlicholinesterase

agents (8).

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562

MODES OF ACTIONS OF PURIFIED TOXINS FROM ELAPID VENOMS

ON NEUROMUSCULAR TRANSMISSION

o<-BUNGAROTOXiN

[51

COBRA NEUROTOXIN

y?-BUNGAROTOXIN

2 msec

I mV

Flg. 6 — Efíect of neostigmlne on EPPs. Rat phrenic nerve-dlaphragm preparations

were respectively treated vvUR the three neurotoxlns as described in Table II. In

a & b, lower tracing shovvs EPP before, and upper tracing 20 min after addition

of 1 /Jg/ml of neostigmlne in diaphragm paralysed by o-Bungarotoxin (a) and

cobra neurotoxin (b), respectively. In c & d, it is shown superimposed miniature

EPPs before (c) and after addition of neostigmlne (d) in the fi-Bungarotoxin-treated

diapraghm.

(lia[)hragm jireparatioii (20). It

the siiccossiví' EI’Ps in res|)onsc

lo ffel a sustainonl coiilraction.

Ejject on the traiu oj EPPs: — ll lias lioon slicwn llial llie ral diajiliragm

|)rt’i)aration treated vvitii a-Hiingarotoxin responds wilh a sustained eonlraetion to

repetitive nerve slitnidalion, vvhereas cohra neurotoxin ( 10) and lhe jiresynaplieal-

ly-aeting /3-Bnngarotoxin (8) heliave jtist like d-tnhocurarine, eausing Wedensky

inhihition. On lhe other hand, liotniinuni-loxin, anolher jiresynajilie jioison, does

nol shovv Wedensky inhihition in lhe gtiinea-pig

is generally eonsidered llial the rapid decline of

lo repetitive stimtilation is tlie cause of failtire

Inahility of the mtiscle to inaintain tlie height of KPPs on repetitive stimnlation

has lieen interjireled to he dne lo a |)resynaptie phenonienon (21) or allernatively

to desensitizalion of the posisynaptic meinhrane to acetyleholine (22). In order

to gain more insighl into the niode of action of these nenroloxins, Irains of EI’Ps

were elicited wilh successive jitil.ses at various iniervals in lhe ral dia])hragm

hloeked hy each neurotoxin and corn])ared willi (liose ohlained in tlie inuscle

paralysed hy other hloeking agents. The EPP seqnenees ohtained with pulse

ig. 7. ll is apparent lhat EPPs deelined

anolher. Eor lhe sake of eomparison, lhe

])er

interval of 10 msec are shown in I

at different rales frorn one agenl to

amplitude of successive EPPs was calenlaled as jier cenl of lhe amplitude of lhe

SciELO

LO 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(2):555-572, 1966

C. Y. LEE and C. C. CHANG

563

TABLE III — THE THAIN OF EPPs AS AFFECTED BY POSTSYNAPTIC BLOCKING

AGENTS

Phrenlc nerve was stimulated vvlth a traln of supramaximal pulses íor 100 msec. at

various pulse Intervals. Amplitude of the second, fifth and tenth EPP was expressed as

per cent of that of the first EPP, respectlvely. n = number of observations.

Agent

Pulse

intervals

(msec)

E.P.P.

Ist 2nd 5th lOth

(mV±S.D.) (ri ± S.D.) (% ± S.D.) (% ± S.D.)

4.95

2.04

73.9 ±

16.4

B ^

1 s

4.50

2.08

79.2 ±

19.7

45.6

15.5

4,38

1.56

83.2 ±

19.7

48.1

19.6

28.5

10.3

x: ao

22.9

3.72

1.22

92.4 ±

50.8

22.2

30.0

13.1

5 S

2.01

H-

0.96

50.8 +

0.84

2.01

0.84

47.9 ±

7,2

28.7

6.6

sz ^

aj £

5 w)

s =>•

2.09

0.85

46.1 ±

7.6

25.1

6.4

24.7

5.8

2.10

0.97

47.6 ±

6.3

22.4

7.4

16.4

4.5

£2 in

-C o

2.36

1.22

74.1

15.7

O o

fcí

2.28

1.10

84.2

21.2

50.6

18.7

E a

2.34

1.38

89.2

26.8

56.9

25.1

39,1

± 22.5

M C

^ 0)

2.22

0.97

100.9

24.9

61.6

25.6

41.8

± 18.7

H *->

3.86

1.72

84.4

12.9

3.17

1.32

93.7

16.0

71.4

19.3

3.76

1.19

106.0

23.9

70.7

±:

24.8

47.6

16.2

3.56

0.70

98.4

16.9

77.5

±_

11.6

57.6

17.3

pulse intervals in llie J3MTC-treate(! miiscle were similar to lliose ohlaincd hy

Huhliard (23). To our siirprise, however, Cm elicited a mucli more raiiid decline

of EPPs in tlie rat diapliragm pre|)aration and eansed a marked \Wdensky

inhiliition, eonlrary to lhe finding lliat C,,, eansed a snslained contraction in

the eal lihialis mnsele(24).

The FiPPs in cohra nenroloxin-lreated ])reparation deelined almost as rajiidly

as ihose in I)l\1TC-lrealed one. On the other hand, the EPPs in lhe jirejiaralion

Irealed wilh n-l>ungaroloxin shovved mneh slower decline lhan did lhe EPPs in

preparations Ireated with other hloeking agents descrihed ahove. Actnally in

some jnnetions lhe EPPs maintained a conslant levei I Eig. 7. a).

SciELO

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564

MODES OF ACTIONS OF PURIFIED TOXINS FROM ELAPID VENOMS

ON NEUROMUSCULAR TRANSMISSION

I I I I I

Flg. 7 — Patterns of trains of EPPs. The phrenic nerve was stimulateci vvith a

train of 10 pulses at the frequency of 100/sec. The experimental condltlons vvere

the same as clescribeci In Tahle II. (a) n-Bungarotoxin; (b) fi-Bungarotoxin; (c)

cobra neurotoxin; (d) Dlmethyltubocurarlnc; (e) Decamethonlum; (f) 12 mM MgCl,^.

In fontrast to lliese agenls, all of wliicli are helieved to act on the post-

synajitic memlirane, ^-Hiingarotoxin eauseti the amplitude of KPPs to fliietuate

irregiilarly (Kig. 7, h), and there was oeeasional conduction hlock so thal some

EPPs were left ont. Analysis of the EPP sequence revealed thal no decline in

its amjiliinde oecurred dtiring the train of 10 pnises (Tahle IV). As shown in

Tahle IV and Fig. 7 f, trealmeni with high magnesiiim ion also eansed a fhicluation

of EPPs and facilitalion as rejiorled hy dei Castillo & Katz (25) and Hiihhard 125)

hiiL no eondnclion hlock was ohserved. Il is inleresling in this regard that hoth

/8-Bnngaroloxin and magnesinm ion hlock lhe nenromiiscnlar transmission hy

inhihition of acetylcholine release.

TAHLE IV — THE TRAIN OF EPPs AS AFFECTED BY PRESYNAPTIC BLOCKING AGENTS

Phrenic nerve was stimulated with a traih of suprama.ximal pulses for 100 msec at

10 msec pulse interval. Amplitude of the EPP was shown as the mean (mV + S.D.)

since the EPP fluctuated irrcgularly (Fig. 7-b) and had no definite correlation with the

first one.

E.P.P. (mV ± S.D.)

AGENT

(mV

Ist

± S.D.)

(mV

2nd

± S.D.)

(mV

5th

± S.D.)

(mV

lOth

± S.D.)

^-Bungarotoxin

1X 10-“ for 3 hours,

lhen wash-out

1.47

± 0.74

1.(52

± 0.80

1.48

± 0.71

1.35

± 0.72

Mg, 12 mM

2.44

± l.lü

3.14

± 0.83

3.38

± 0.97

3.70

± 1.16

SciELO

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Mem. Inst. Butantan

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C. Y. LEE and C. C. CHANG

565

However, condiiction hlock, occiirred in lhc> /3-Hiingarotoxiii-treaU-d nuiscle

duriíig repetitiva stiimdatioii, lieeame more marked and the average amplitude

of EPPs vvas reduced as the slimidation was prolonged. As sliown in Fig. 8

and Tahie V, the conduetion faihire inereased to more than twiee during 500

msec and the average amplitude of EPP decreased hy ahout 66%. Jn eoiUrast.

neither conduetion hlock nor decrease in the amplitude of E!PP was ohserved

iu the magnesium-paralysed musele during ])rolonged stimulation. These results

may ex])lain why the musele treated with /S-Rungarotoxin tends to shovv Wedeiisky

itdiihition.

...iiiiiiÉwiiiiuiinüilillliivi

Fig. 8 — EfCect of /i-Bungarotoxln and Mg ion on the train of EPPs. The phrenic

nerve vvas stimulated with a train of 50 puises at the frequency of 100/sec. (a) Treated

with 1 /íg/ml /3-Bungaroto.xin for 185 min and then washed. Note the faiiure in re.sponse

to the stimuiation and decrease in the ampiitude of EPP. (b) 12 mM MgCL.

TABLE V — COMPARISON OF THE RATE OF FIRING AND THE AMPLITUDE OF FIRED

EPP ON REPETITIVE STIMULATION BETWEEN PREPARATIONS TREATED WITH

/5-BUNGAROTOXIN AND THOSE WITH Mg ION

The diaphragm vvas treated either with 1 /vg/ml /i-Bungarotoxin or with 12 mM Mg-

Tyrode’s soiution. A train of 50 puises at the frequency of lüO/.sec vvas given. The

number of responses to the first 10 pulses and that to the last 10 pulses of the train

vvere counted and the average amplitude of EPPs vvas compared.

AGENT

No. of junctions fired

(mean ± S.D.)

First

10 stimuli

Last

10 stimuli

Amplitude of EPP

(mV ± S.D.)

First

10 stimuli

Last

10 stimuli

+ +

/S-Bungarotoxin

3.66 ± 1.06

1.10 ± 0.76

3.35 ± 1.08

0.37 ± 0.32

Klfect on aniidroniic aclivitirs: — li has heen shown lhat eurare aholishes

repelilive anlidromie discharges of molor nerve fihres, evoked hy orthodromic

nerv(> impul.se under the influenee of anticholineslerase agenls, at a dose levei

mueh lovver than that required for hloeking neuromuseular transmission (26,27).

As shown in l'ig. 9, the repetitive discharges of the rat phrenic nerve evoked

SciELO

10 11 12 13 14 15

Calibration 40;jV

MODES OF ACTIONS OF PURIFIED TOXINS FROM ELAPID VENOMS

ON NEUROMUSCULAR THANSMISSION

hy a single volley in the presence of neosligmine (0.3 p,g/nil) were abolished

hy all of lhe ihree neuroloxins hefore lhe oceiirrence of complele neiironuiscular

hloek.

oi-BUNGAROTOXIN ^-BUNGAROTOXIN GOBRA NEUROTOXIN

(IXIO'^) (lxlO~^)

lOO mm

130 min

60 min

20 msec

Fig. 9 — Eífect on antUiromic activities. Lovver traclng In each figure shovvs the

antiíiromlc activities of the rat phrenic nerve prepaiation in the presence of neo-

stigmine (0.3 /;g/mi) as (iescribeci in the Method. Upper tracing shovvs the muscle

action potential monitoreci with an extraceiiuiar microeiectrocie. Upper figures (a, b

& c) Show the control activities and lovver figures, (ci) 100 min after «-Bungarotoxin

(1 /ig/ml), (e) 130 min after /?-Bungarotoxin (1 íig/ml) and (f) 60 min after cobra

neurotoxin (1 /jg/mi), respectiveiy.

Ejfcci on Icnninal nerve sjnke: — While the lerminal nerve s|)ike recorded

with extraceiiuiar mieroelectrode in lhe frog sartoriu.s was aholished hy eardio-

loxin (Fig. 10), il remained unaffeeted after lhe F.IM’. simullaneonsly recorded

with the satne eleelrode, had heen aholished hy eaeh of lhe ihree neuroloxins

(Fig. 11). These findings indieate lhat none of the.se neurotoxins interferes with

the eouduetion in nerve axons up lo the nerve terminais, whereas eardioloxin

hloeks nerve eouduetion juoliahly hy ils dejiolariziug effeet. This effcel of eardio¬

loxin inay explain lhe inhihilion of aeetyleholine out|)ut froni the nerve endings

hy the ernde eohra venoinllO). On the other hand, sinee lhe eouduetion in

nerve axons is unaffeeted hy j8-Bungarotoxin up lo lhe nerve terminais, lhe

inhihition of aeetyleholine release hy this loxin is jirohahly due to its action ou

the exeilation-seeretion eou|)liug systeni.

Kjject oj stimnliis jreqnency on the lime-conrse oj neurotniiscnlnr hloek hy

P-Bun^urotoxin: — Hughes and Whaler (2Í)) have reeentiy shown lhat nerve

stimnialion with higher frecpieneies canses a marked enhaneemeni of neuro-

mtisenlar hloek hy holnlinum-toxin. This lihenomenon was inlerpreled as jirodueed

hy an inerease of the permeahility lo lhe hotidinnm toxin in consetjuenee of

a sustained depolarizalion of lhe nerve endings eaiised hy nerve stimnialion al

SciELO

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568

MODES OF ACTIONS OF PURIFIED TOXINS FROM ELAPID VENOMS

ON NEUROMUSCULAR TRANSMISSION

scarcely affccted liy increasing the rate of stimiilation. Siiice’ lhe iiplake of

/3-Bungarotoxin appears to Ite a rather ra])i(l ])rocess(8), it is iinlikely that the

enhancement of neiiromiiscular hlock is due to an increase in penneahility to

/8-Bungarotoxin, caused hy stimuli of liigh freqiiencics as postulated for hotulinum-

toxin hy Hughes and Whaler (28). This resiilt rather indicates that the action

of /5-Biingarotoxin oii the nerve endings is infhienced hy the activity of the nerve

endings.

Pulse intervals

Flg. 12 — Etfect oT stlmulus frequency on the time-course of neuromuscular block.

The phrenic nerve was stimulated with single pulses at frequencles varylng from

0.75/mln to 48/mln, Each polnt represents the mean of 4-15 experlments.

Ejject of calcium and magnrsiiim ions on the time-course of neuromuscular

hlock hy /3-Ihingaroloxin: — One heinidiaphragm prejiaration was immersed in

modified Tyrode sohition containing either low calcium (0.45 tnM) or high

magncsiiim (12 niM). The pre|)aralion usually failed to respond to nerve stimula-

tion williin 20 min. and j^-Bungaroloxin (1 /tg/ml) was llien added. Inleresling

enough, the resjionse to nerve stimulalion was [lartially re.slored on addition of

jS-Biingaroloxin and llien decreased jirogrcssively (Big. 15). The conlralalcral

hernidiaphragm was immersed in normal Tyrode sohition and similarly treated

with /3-Bungarotoxin to serve as the eontrol. When j8-Bungarotoxin induced a

com[)lete neuromuscular hlock of the eontrol jireparation, liolh of lhe jireparalions

were washed oul wilh normal Tyrode sohition. While no recovery of respon.se

occiirred in lhe eonirol preparalion, lhe response of lhe test pre|)aratioii was

restored after rejieated washing, and it took anolher 100-150 min. to eaiise iieiiro-

mirscular liloek again. Thiis, low ealeiiim as well as high rnagnesium can mark-

edly [irolong the time for neuromuscular hlock hy /3-Bungarotoxiii (Tahle VI).

Sueli antagonism helweeii j8-Bungaroloxin and ealeitim or magnesiiim ion may

suggesl that lhey share a eommoii site of action at the nerve endings.

SciELO

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Mem. Inst. Butantan

Simp. Internac.

33(2):555-572, 1966

C. Y. LEE and C. C. CHANG

569

Effect of low calcium on the N-M blocking action of ^-Bunga^otoxin

CaCf20.45mM yS-Bungarotoxin I x IO'

140 min

t 240min

CaCl 2 I 8mM

Fig. 13 — Effect of calcium and ^-Bungarotoxln on neuromuscular transmisslon. Rat diaphragm prepara-

tion; for detail see the text. Note the restoration of neuromuscular transm.ission on addltlon of ;8-Bungaro-

toxin 1 jug/ml to the paralysed preparatlon.

TABLE VI — EFFECT OF CALCIUM AND MAGNESIUM ON THE NEUROMUSCULAR

BLOCKING ACTION OF /1-BUNGAROTOXIN

For the experimental detail see the text. The time taken for the test preparatlon

to cause neuromuscular block was that from the addition of ^-Bungarotoxln (1 /jg/ml) to

complete arrest after washlng. n = no. of experiments.

Pretreatment

Concentration

Time for N-M block by

j3-Bungarotoxin

(Min. ± S.D.)

None

—

153 ± 12.8 (n =

15)

MgCl,

12.00 mM

266 ± 63.3 (n =

CaCL

0.45 mM

313 ± 45.0 (n =

Ejfect of d-liibocururine pretreulnient on the neuromuscular blocking action

of a-Bungarotoxin: — It has recently heeii demonstrated that pretreatment with

fl-tul)OCiirarine protecls the chick hiveiiter cervicis miiscle from the neuromuscular

hiccking action of cohra neurotoxin (10). In order to see whether d-tulio-

curarine also can ])rotect lhe muscle from the paralytic effect of /3-llungarotoxin,

one of lhe Ivvo hivenler cervicis muscles from lhe same chick was pretreated

with d-tuhocurarine (10 /rg/ml) for 10 min. and the other was immersed in

Krelis solution to serve as tlie control. ív-Hungarotoxin (0.3 p,g/ml) was added

to lioth prejjarations simiiltaneously. When the resjionse of the control prejiara-

ticn to nerve stimulation was aholished, hoth pre])arations were washed with fresh

Krehs solution once every 5 min. As shown in Fig. 14, while no recovery was

SciELO

570

MODES OF ACTIONS OF PURIFIED TOXINS FROM ELAPID VENOMS

ON NEUROMUSCULAR TRANSMISSION

éOmin

CHICK BIVENTER CERVICIS

Fig. 14 — Effect of ti-tubocurarlne on the neuromuscular blocking actlon oí «-Bungarotoxin. Chick biventer

cervicis muscles. a: control treateci only vvith «-Bungarotoxln, 0.3 ;ig/ml. b: contralateral preparatlon pre-

treated vvith 10 /jg/ml of d-tubocurarine. Note the remarkable restoratlon of response after repetitlve

vvashlng in the d-tubocurarlne-pretreated preparatlon In eontrast to the control.

found in the cotUrol |)rc|)aralioii despile re|)(‘ated vvashiiigs for d lioiirs, lhe

response of llie ])reparalion ])retrealed wilh d-tuliociirarine recovered sleadily lo

ahout 70% of the original height after d hoiirs’ washing. In eontrast. no pro-

tection ])y d-luhociirarine was found against the neuromuscular hlocking aetioii

of ^-Hungaroloxin. These findings, logether with our ])revious ohservatiou vvith

eohra neuroloxin, strongly suggest lhat «-Ifuiigaroloxin as vvell as eohra neuro-

toxin l)locks neuromusenlar transrnission hy eoml)ining vvith the S])ecific reee[)lor

of acethylcholine al the end-plate, just like d-tuhocurarine does.

SuMM.VHY AND CONCl.USIÜNS

Cobra neuroloxin: — Cohra neuroloxin de|)resses EPF vvilhout affecling rest-

ing nienihrane potential, niusele aelion ])otential. and terminal nerve s|)ike. The

amiilitude of lhe EPP is increased and ils time-eourse prolonged liy neostigmine.

Antidromic aetivities of the phrenic nerve in the |)resenee of neostigmine are

aholished. The amjililude of successive EPPs on repetitive stimulation deelines

markediy as in lhe curarized miisele. Erom these findings it is eoncluded lhat

SciELO

11 12 13 14 15 16 17

Mem. Inst. Butantan

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33(2):555-572, 1966

C. Y. LEE and C. C. CHANG

571

lhe inode of iieiiiomuscular l)lockiiig action of cohra iieuroloxiii is essciitially

similar to tliat of curare, although lhe former acts mucli more slowly and less

reversihlv than lhe latler.

a-llimguroloxin: — The effects of a-l]imgaroloxin on lhe nenromiiseular

Iransmission are similar lo ihose of cohra neurotoxin in lhe following aspects;

(1) depression of EPP wilhout affecting resliiig memhrane ])olenlial, muscle action

potenlial and terminal nerve spike; (2) enlargement and |)rolongation of EPP

hy neostigmine; (3) iidiihilion of anlidromic discharges of lhe motor nerve;

and 14) protection from ils ])aralytic effect hy d-luhocurarine. Hovvever. «-

Bungaroloxin differs from cohra neurotoxin in lhat its jiaralylic. effect is irre-

versihle and not restored hy neostigmine and thal the decline of successive EPPs

is much less marked, so that sustained contraction is ohserved on repetitive

stimulalion.

ji-Hungaroloxin: — ;S-Bimgaroloxin increases lhe frequency of sponlaneons

miniature EPPs in the early stage anil reslorcs ueuronmscnlar transmission im-

jiaired hy eilher low calcinm or high magnesium, hnl in lhe later stage, lhe

numher of miniature EPPs is diminished without change in size. Neuronmscidar

hlock as well as aholilion of EPP takes place hefore the comjilete disapjiearance

of miniature EPPs. All lhese findings are in good accordance wilh onr previous

conclusion thal j8-Bimgarotoxin acts jifesynaptically, reducing the acethylcholine

output from the nerve endings and leaving the sensitivity of the end-j)late to

acetylcholine unaffected. Since the terminal nerve sjtike remains unaffecled, it

ajipears that /3-Bungaroloxin acts on lhe motor nerve endings, prohahly affecting

the excilalion-secretion conpling System.

Cardiotoxin: — Cardiotoxin causes conlraclure of the muscle as well as

neuromuscular hlock hy deiiolarizing hoth the muscle and nerve fihres; the

terminal nerve s])ike is aholished and direct slimulation of lhe muscle íihre íails

lo evoke an action [lotenlial. This effect of cardiotoxin on nerve tissue is prohahly

resjionsihie for lhe rednction of acetylcholine output from the nerve endings caused

J)y crude cohra venom.

Hekehk.xcks

1. MELDRUM, B. S., Pharmacol. Rev., 17, 393-445, 1965a.

2. KELLAWAY, C. H., CHERRY, R. O., and WILLIAMS, F. E., Aiist. J. exp.

Biol. med. Sei., 10, 181-194, 1932.

3. LEE, C. Y., and PENG, M. T., Arch. int. Pharmacodyn., 83, 180-192, 1961.

4. VICK, J. A., CIUCHTA, H. P., and POLLEY, E. H. — Arch. int. Pharmacodyn.,

153, 424-429, 1965.

5. SU, C., J. Formosan med. Ass., 59, 1083-1091, 1960.

6. CHANG. C. C., J. Formosan med. Ass., 59, 315-322; 416-426, 1960.

7. MELDRUM, B. S., Brit. J. Pharmacot., 25, 197-205, 1965b.

8. CHANG, C. C., and LEE, C. Y., Arch. int. Pharmacodyn., 144, 241-257, 1963.

9. LEE, C. Y., J. Showa med. A.s.s., 23, 221-229, 1963.

10. SU, C., CHANG, C. C., and LEE, C. Y., Toxicon, in press.

11. LEE, C. Y., CHANG, C. C., CHIU, T. H., TSENG, T. C., and LEE, S. Y.,

Int. Symp. Animal Venoins, São Paulo, Pharmacological properties of cardio¬

toxin isolated from the venom of Naja naja atra, 1966.

12. LEE, C. Y., and CHANG, C. C., Abstracts of the XXIII Int. Congress Physiol.

ScL, Tokyo, Modes of neuromuscular hlocking action of neurotoxins isolated

from elapine venoms, 1965.

SciELO

10 11 12 13 14 15 16

572

MODES OF ACTIONS OF PURIFIED TOXINS FROM ELAPID VENOMS

ON NEUROMUSCULAR TRANSMISSION

13. LO, T. B., CHEN, Y. H., and LEE, C. Y., J. Chin. chem. Soc., II, 13, 25,

1966.

14. FATT, P., and KATZ, B., J. Phyftiol. (Lond.), 115, 320-370, 1951.

15. KATZ, B., and MILEDI, R., Proc. roy. Soc., B, 161, 453-482, 1935.

16. RANDIC, M., and STRAUGHAN, D. M., J. Physiol. (Lond.), 173, 130-148, 1964.

17. GINSBORG, B. L., and WARRINER, J., Brit. J. Pharmacoh, 15, 410-411, 1960.

18. BROOKS, V. B., J. Physiol. (London), 134, 264-277, 1956.

19. BROOKS, V. B., J. Physiol. (Lond.), 123, 501-515, 1954.

20. BURGEN, A. S. V., DICKENS, F., and ZATMAN, L. J., J. Physiol. (Lond.),

109, 10-24, 1949.

21. OTSUKA, M., ENDO, M., and NONOMURA, Y., Jap. J. Physiol., 12, 573-583,

1962.

22. THESLEFF, S., J. Physiol. (Lond.), 148, 659-664, 1959.

23. HUBBARD, J. I., J. Physiol. (Lond.), 169, 641-662, 1963.

24. PATON, W. D. M., and ZAIMIS, E. J., J. Physiol. (Lond.), 112, 311-331, 1951.

25. DEL CASTILLO, J., and KATZ, B., J. Physiol. (Lond.), 124, 560-573, 1954.

26. FENG, T. P., and LI, T. H., Chin. J. Physiol., 16, 37-50, 1941.

27. RIKER, W. F., WERNER, G., ROBERTS, J., and KUPERMAN, A., Ann. N.Y.

Acad. Sei., 81, 328-344, 1959.

28. HUGHES, R., and WHALER, B. C., J. Physiol. (Lond.), 160, 221-233, 1962.

Discussion

P. E. Russell: “First, may I compliment you on a very interesting and definitive

study. It is perhaps infortunate that curare was studied in depth before venoms,

for we have a tendency to drift into the habit of calling things “curare-like”. In

reality, many substances have a “curare-like” activily, and these substances are

in no way related chemically, and indeed the difficulties may not be as closely

associated with chemistry as they are with the physiological abuse of “curare-like”.

Your Work indicates that the common acceptance of certain venoms and their

fractions as post-synaptic agents .should be reexamined. I agree with you, and I

would like to ask whether or not you care to discuss the specific site of action

involved or if you have condueted studies in this direction?”

C. Y. Lee: “Although cobra neurotoxin as well as alpha-Bungarotoxin has some

presynaptic action (e.g. such of antidromic discharges of motor nerve), we believe,

N-M block caused by these two neurotoxins is post-synaptic and lhe specific site of

action is acetylcholine receptor on the end-plate, since d-tubocurarine pretreatment

can protect the paralysing action of these neurotoxins. On the other hand, the

action of beta-Bungarotoxin is exclusively presynaptic, leaving the sensitivity of

end-plate to acetylcholine unaffected. The site of action is on motor nerve end-

ings, probably on the excitation-secretion coupling system, since the terminal nerve

spike remains unaffected.”

E. Garcia Mendes: “How about the molecular weight of lhe alpha- and beta-

Bungarotoxin?”

C. Y. Lee: “The molecular weight of alpha-Bungarotoxin has been estimated

to be about 8,000. Since beta-Bungarotoxin has not been obtained in pure State,

the exact molecular weight is unknown, but we have evidence that it is higher

than that of aipha-Bungaroto.xin.”

D. Mebs: “Is your AT (t j (í-neurotoxin-preparation the same of Mr. Yang? Is

it íree of enzymes?”

C. Y. Lee: “Judging from the LD50 in mice, our cobra neuroto.xin is almost

identical with Dr, Yang’s cobrato.xin and it is free from various enzymic activities,

such as protease, cholinesterase, phospholipase A and hyaluronidase (unpublished

observations).”

2 3

5 6

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Mem. Inst. Butantan

Slmp. Internac.

33(2):573-582, 1966

ANIMA DEVI and N. K. SARKAR

573

59. CAHDIOTOXIC AND CAKDIOSTIMULATING FACTORS IN

COBRA VENOM

ANIMA DEVI and N. K. SARKAR

Ueyartment of Fhysiology, University of Calcutta, índia and Canada Department

of Aíjriculture, Animal Research Institute, Central Experimental Farm,

Ottawa, Canada

The suhjecl of snakes and lheir venoms is of special interest lo people of

all tropical coiR.tries, as lhe numlier of deaths lhat occiir every year all over lhe

world runs, according to lhe World Health Organizalion is as high as 40,000. In

Índia alone. some 12,000 lo 15,000 people die every year and in South America

3,000 to 4,000 deaths occur each year. The numher of deaths from snake hites

is likewise considerahly high compared lo their total population; in Burma, Indo-

China, Australia, África, West Indies, and the troj)ical helt of North and South

America. The temperate parts of the glohc are far less severely affected.

The venoms of snakes helonging lo ELAPIDAE are generally more toxic

than the venoms of VIPERIDAE as is evident from Tahle I, where the toxicities

of venoms of some snakes helonging to ELAPIDAE, VIPERIDAE and CROTALIDAE

which are generally responsihle for causing most of lhe deaths, are recorded (1).

The toxicity of a venom depcuds u[)on many faclors, snch as seasonal chauges.

lenght (size) of the snake, amount of venom collecled, frequency of milking,

whether or not fonud in the field (foresl) or takeu from snakes in captivily, etc.

The toxicity of a venom also varies with the nature of the animal emjiloyed for

lhe cxperiment (2).

Venom is a mixlure of several |)roteins. Relatively fevv of them have lieeu

separated until recenlly. 1’artial or complete sejiaraliou of them has bcen achieved

liy emj)loying such procedures as Am;,S ()4 fractionalion, starch-gel eleclro|)horesis,

jjaper electrophoresis and anion and cation cxchauge chromatography. Nearly

all venoms depending npon lhe species of origin contain at leasl eight to ten

(somclimes more) well characlerized com])onenls. Most of them if not all, are

enzymes; each one is differeni from the other in their liehaviour lovvards suhslrales.

Their activities vary from s])ecies lo s])ecies and even from venom lo venom,

collected from lhe same snake, at different seasons of the year (3).

The enzymes that are commoidy found in lhe venom of all s})ecies helong-

iug lo ELAPIDAE, CROTALIDAE and VIPERIDAE, are phosidiolipase A, L-amino

acid oxidase, deoxyrihonuclease, rihonuclease, phos|)hodiesterase, non-specific |)hos-

jihomonoesterase, 5'-nuclcolidase, adenosine-5’-triphosphatase and are recorded in

'Pahle II. Proteases and amino acid eslerases are getierally found in lhe venoms of

VIPERIDAE and CROTALIDAE. Colira venom exhiliits oídy feehie proteolylic

activity, liut coulains several pei)tidases. Venoms of ELAPIDAE in contrasl lo

lliose of VIPERIDAE contain enzymes capahie of hydrolyzing acelylcholine and i

nou-cholinesters (4 ). The venom of Formosan cobra contains besides acelylcholin-

eslerase, an irdiibilor of ihis enzyme too, which can be reversibly blocked by

2 3

5 6

11 12 13 14 15

TABLE I — RELATIVE TOXICITIES OF VENOMS OF SOME COMMON SNAKES OF

ELAPIDAE, VIPERIDAE AND CROTALIDAE

SNAKES

Estlmated MLD for Man

(150 Ibs. body welght)

In mgs.

ELAPIDAE

Bungarus candidus (blue krait)

1.5

Nntechis scutatttn (tlger Gnake)

2.0

Naja naja (Intiian cobra)

20.0

Dendroaspis angusticeps (mamba)

20.0

CROTALIDAE

Bothrops atrox (fer-de-]ance)

70.0

Crotalus atrox (Western dlamondback rattlesnake)

140.0

Crotalus adamanteus (Eastern dlamondback rattlesnake)

2S0.0

VIPERIDAE

Vipera russellii (Indlan daboia)

Bitis arietans (African puff adder)

120

TABLE II

Enzymes

Cobra venom Vipera runsellü

Venom

C. adamanteus

Venom

C. atrox

Venom

5'-Nucleotldase (AMPase)

Dlphosphopyrldlne nucleo-

tldase (DPNa.se)

not known

4 -

Adenoslne trlphosphatase

(ATPase)

Deokyrlbonuclease

(DNa.se)

Rlbonuclease (RNase)

Phosphodlesterase

Phosphomonoesterases

(nonspeclflc)

Phosphollpase A (In some

venoms In addltlon to

thls Phosphollpase B or

C Is found)

Protease

L-amlno acld oxídase

Acetylcholinesterase (and

cholinesterase)

—

ESTEROLYTIC

Proteases

ENZYMES IN SNAKE

VENOMS

Proteolytlc enzyme (pro-

teln degradlng enzyme)

Synthetic amíno acid es-

ters

Clotting enzymes

—

Bradykinin — releasing

enzyme

not known

Bradykinin — destroying

enzyme

not known

Clotting Inhlbltor (Inhlblts

clotting due to Its actlon

on phophollplds)

—

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

ANIMA DEVI and N. K. SARKAR

575

SImp. Internac.

33(2):573-582, 1966

Mg ioiis (5l. Colira venoni exhil)its adenosiiie (li|)lio?|)halase and pyrophospliatase

aitivities in addilion lo it8 well known ATPase activity altlioiigli lhe activities

of the?e tvvo enzyines are low (6). KiisselTs viper venom can iniliale coagulalion

of lilood, whcreas colira venom acts as an iíduliilor. The presence of ])hospho-

lipase I? in venonis of l’seinlcchis porphyriacm (Ansiralian snake), C. aduinan-

teus and A. piscivorits (North Anieriean snakes) has heen recenlly reporled(7).

Sorne venorns also exhil)it phos|)holipase (i activily.

Althongh many of lhe enzymes fonnd in lhe venorns of ELAPIDAE, VIPERIDAE

anil CROTALIDAE are eornnion hnl lhe eanse of dealh appears to he different

in eaeh case, e.g. it is generally helieved lhal the priniary cause of doath in

colira |)oisoning is the faihire of respiratory system lirouglit alioiit either liy the.

paralysis of the central nervous system or hy the paralysis of diajdiragm mnscle

(8-11). The heart is affected at a miich later stage. Tlie changes in hlood

pressure following the intravenous injection of cohra venom into anesthetized

animais, have heen recordei! hy many vvorkers; lhey all invariahly fonnd failnres

of rcs|)iration and cirenlation 18-131. Many of these investigators helieved that

the heart vvas primarily and direetly affected 18 , 9-13) . Feldherg and Kcll-

awaylTl) pointed ont tliat the circnlatory faihire vvas secondary to the effects

of the venom on other organs. Marked electrographic changes in the rhylhm

and eondnction of the heart resnlting from an intravenous administration of C.

d. terrijicus venom led Soaje-Eehaqüe to consider myocardial inefficiency as the

jirimary canse of the faihire of circidation (13).

The action of cohra venom on isolated frog heart has also heen extensively

stndied hy many vvorkers (11, 15-19). They all foimd an initial increase in the

amplitude of contraction follovved hy complete stoppage of all the movements of

an isolated frog heart, when it vvas ])erfnsed vvith venom solntion. The effeet

as noted hv most of the vvorkers dependei! largely on the concentration of the

venom solntion nsed.

These stndies, carried ont in silu, in vivo and in vilro, indicate that the

ohserved effects of the venom may he ex|)lained on the hasis of its action on

myocardinm. Whether or not it vvas actnally so, vvas investigated many years

ago in onr lahoratory 119). The vvork to he jiresented here vvas hased on three

different series of exjierirnents and vvas initiated to determine the seipiences and

relationshi]) hetvveen the changes that follovv after the intravenous injection of

venom into animais.

MkTHOOS A.M) MATIClilAI.S

as follovvs: For the stndy of the effeet of the venom on circnlation and resjiira-

tion, ailiilt eats vveighing hetvveen 2.0 to 2.5 kg. vvere chosen. Urethane I 1.8

g/kg. of hodv vveight) vvas introdnced intramnsenlarly into the animal tvvo lionrs

prior to dissection. Femoral vein vvas cannnlated for the administration of the

venom Inenrotoxin or cardiotoxin) dissolved in 2 ml. of saline. Carotid artery

vvas cannnlated for measnrement of the changes of hlood pressnre, and the

changes in respiration vvhere recorded hy rneans of a pnenmograph.

For the stndy of the effects of cohra venom on excised heart, toads of

average vveight of 100 to 120 gms. vvere chosen. The heart vvas perfnsed vvith

Solutions of cohra venom Inenrotoxin or cardiotoxin I of desired concentration.

The eontractions of the heart vvhere recorded on a kymograph. The heart vvas

2 3

5 6

11 12 13 14 15

576 CARDIOTOXIC AND CARDIOSTIMULATING FACTORS IN COBRA VENOM

first perfuscd witli lhe Hiiiger soliilion iiiilil it heeame |)liysiologic'ally normal;

al lliis poini, lhe eaniuila was filled wiüi 2 ml. of llie l{inger-venom soliition

of desired eoncenlration and lhe drip slarted and lhe eoncenlrations reeorded.

Wlien all lhe venom soliUion was iised np, il was replaced liy Hinger solulion.

The sludy of lhe effeets of lhe venom (neuroloxin or cardioloxin) on lhe

transmission of nerve impulse were carried out wilh frog nervc-musele preiiarations

kej)t immersed in venom solulion for differeni lenghls of time. The eonlraction

of lhe muscle was elicited hy indireet (ihroiigh lhe nerve) and direci electrical

stimulalion.

The venoms were collected írom snakes of lhe same species {Naja naja)

and lhe same size, as far as possihle, during lhe monlhs of Ajiril and Jime, freeze-

dried, and kepl in an ice hox (2-l'’C) in a darkd)rown slojipered glass hollle

imlil use.

Hesui.ts

The residis ohiained can hc summarized as follows: Kollowing lhe injeclion

of a large do.se of cohra venom (2 mg/kg of hody weighl), a sharp fali in

syslemic arterial jiressure is noted. The cnsiiing respiralion hecomes more and

more shallow and rajiid, and tends lo he ahdominal in tyjie imlil finally ccases;

lhe IkP. drops lo zero mm ílg and lhe animal dies, aiiparenlly hecause of lhe

faihires of res])iration and circulalion. The a[)|)licalion of artificial respiralion

can not jirevent lhe dealh of lhe animal. Even if a smaller dose of lhe venom

(0..5 mg/kg of hody weighl) is administered, lhe changes in re.sjiiralion and

hlood [iressnre follow lhe same pallerns. If artificial re.spiration is ajijilied, lhe

hearl gradually improves, 15.P. slowly and |)rogressively rises imlil it is reslored

lo normal levei. If lhe artificial respiralion is discontinned at ihis poinl, natural

resjiiralion revives slowly and lhe animal survives for many hours. If now a

larger quaniity of venom (1.5 mg/kg) is administered, hlood pre.ssure drops

almosl inslantaneously lo zero anil respiralion ceases imrnedialely. The lelhal

effecls of ihis second dose can nol he counieracled hy lhe ap|)licalion of aiiificial

res|)iralion. If on lhe olher hand, a smaller do.se (0.2 mg/kg) of venom is

inilially iniroduced, a very slight fali in arlerial hlood pressurc oflen accomjuinied

wilh certain irregnlarilies in res])iralion, is ohserved. If no furlher venom is

adminislered, lhe ll.P. slowly rises iinlil il reaches lhe normal levei and remains

steady thereafter. Adminisiralion of ihree lo four doses increasing each time

lhe arnonnl of venom iniroduced, (tolalling 2.25 mg/kg) al regular inlervals,

does not affecl eilher 15.P. or res])iralion. Al lhe end of líie fotirlh adminisiralion.

a large quaniily of venom can he iniroduced even in a single dose, wilhoiil af-

fecling eilher lhe 15.P. or respiralion. Afler 1 lo 6 hours, 15.P. drops lo zero,

res|iiralion ceases and lhe animal dies. If lhe hearl is examined jusl imrnedialely

afler lhe dealh of lhe animal, il is found in syslolie coniraclure.

If neuroloxin, [lurified from lhe same venom, is injecled inio aneslhelized

cais, faihires of resjuralicn and circulalion are ohserved almosl al lhe same lime.

The laller can he reslored lo normal levei if ariificial respiralion is ajiplied.

wliich remains sleady lhereafler for many hours (6 lo 8 hours) uniil lhe animal

dies hecause of olher secondary effecls. If lhe chest is opened and lhe hearl

is examined, no syslolie arrest of lhe hearl like lhe one lhal has heen noied

hefore wilh crude venom can he delecled (19).

If lhe venom is healed ihrough various lempeialures lo ilestroy one or lhe

olher aclive eonsliluenls of lhe venom and lesled lhereafler for ils aclion on

2 3

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11 12 13 14 15

Mem. Inst. Butantan

ANIMA DEVI and N. K. SARKAR

577

Simp. Internac.

33{2):573-5S2, 1966

resi)iratioii and circiilation, il heconies cvideni lhal il rclaiiis this |)iO]K‘rly iinlil

il is heated lo 85‘’C for 30 minutes. At this temperature mosl of the enzynies

are destroyed. Heinolysin wliieh is not destroyed at this temperature is inef-

feelive in eausing res])iratory and cireulatory failures.

Hecenlly Viek and his colleagues isolaled froni eol)ra venom three major

[)hysiologically identifiahle eompouents (actually 12 coinpouents were separated).

The first eomponent, whicli vvas a mixture of 3 to 4 sei)arate fraclions ])roduced

a loss of cortical eleetrical activity when injecled intravenously into dogs. Tlie

second compouenl (fraetions 5 to 8) eauses res])iratory ])aralysis and lhe ihiril

component I fraction 12) affected cardiovascular system. On lhe hasis of tlie

effect of fraction 12 on arterial hlood jiressure and heart rate, these workers

coucluded thal colua venom has a definite action on cardiovascular system of

the dog.

Action of cohra venom on excised fhog heart

If au isolaled frog heart is perfused vvith an exlremely diluled solulion of

venom (1 in 100.000) no changes in lhe amjililude of conlraction could he

noticed; no slimulating or de])ressing action of venom could either he detecTed

if the heart is perfused with a less diluled venom solulion (1 in 10,000). An

increase in the amjilitude of conlraction for a hrief jieriod is noticed when a

venom sohition of 1 in 5,000 is used, which disa])pears on washings and lhe

heart hecomes normal. If a more concentraled solulion is used, say 1 in 1,000.

an increase iu am[)lilude is first ohserved and lhen certain irregularities and slow-

ing of heart heats are noticed. The heart finallv slops heating. These effects

of the venom can also he jiaiTially removed hy jirolonged washings with lhe

Hiuger. If a more concentraled venom solulion (1 in 200) is used, no slimulal-

ing action can he detected, instead, a ])rogressive decrease in lhe am|)litude of

conlraction is ohserved. The heart finally sto])s iu syslolic coulracTure and can

not he revived in this case eveu afler conlimious washings with Kiuger. The

effects of venom on excised heart could uol he couuteracTed hy alropine and

aulihistamine drugs.

If the heart is perfused with a venom solulion )] in 200) previously heated

ihrough varioiis temperatiires, il is found lhal venom loses its action at 85‘’C (20).

Phospholipase A or neurotoxin has no such effect on excised heart.

It is inleresting lo note that similar findings have heen recorded with dif-

ferenl concenlralions of digilalis drugs (digilalis, slrophanlheine, etc.). In all

cases where high concentralioiis ol these drugs have heen used. lhe heart slops

in systolic contracture hut contrary lo lhe action of lhe venom, their effects

could he easily washed oul with llinger. RecenI histo|)alhological sludies indicate

lhal venom produces sjieclfic changes in lhe isolaled heaiTs of frogs and of guiuea-

pigs which are quite differenl from those produced hy histamine, slro]ihan-

iheine. etc.

When an isolaled frog-nerve jireparaliou is ex]iosed lo a venom solulion, it

loses its excitahilily in 4 lo 6 minutes depeudiiig upon lhe concenlralion of lhe

venom solulion used (21,22). Neither direcl nor indirecl slimulalioa can produce

conlraction. The ahility of lhe eleetrical stimulus lo eliciled conlraction when

appiied through the uerve is lost earlier than if appiied direclly lo lhe muscle.

The muscle contracts lo one third of its original lenghl. hecomes jiale and opaque.

Nerves or mnscles ex])osed to oídy Hinger’s solulion maintain eleetrical |)ropeiTy

2 3

5 6

11 12 13 14 15

CARDIOTOXIC AND CARDIOSTIMULATING FACTORS IN COBRA VENOM

for 4 lo 6 lioiirs. lí lhe ticrve-niuscle projiaralion is exjiosfd to tifuroloxiii, lhe

iniiscle, retaiiis its excitahilily; iinder identical condilioiis oídy niuscle loses ils

exritahilily if exposed lo cardiotoxin sohitioii. The ahilily of veiiom lo lilock

contraetion of lhe imiscle is losl if il is healed at 55'’C for 10 miii. or 85"(i for

30 min., de|ieiiding iipon hovv llie eotilraclion is eliciled. i.e. whelher stimniated

throiigh lhe nerve or ihrough lhe niuscle (21). At lhese lemperaliires acetyl-

cholinesterase aclivily of tlie venom and ils cardiotoxic property are destroyed.

The action of cardiotoxin can nol he prevenled or coimteracted hy K ions, anli-

histainine driigs or liy EDTA, known lo inhiliil llie |)rotease action of tlie venom.

Col)ra venom or cardiotoxin ihtis appears to have direct |)aralysing aclion on

cardiac and skelelal niuscle hui hovv il acts is nol known. Neilher colira venom

nor cardiotoxin can jiroduce any coniraction of aclomyosin filiers either extracled

direclly from rahhil skelelal muscle or pre|iared synthelically hy mixing cryslailin

myosin and adiu. Il has neilher any action either on myosin or aclin(23).

Il ihns ajipears thal venom has no aclion on individual comiionenls lhal eonslilnle

aclomyosin, lhe contraetile elemeni of lhe muscle. Ils aclion is visihie when only

inlacl muscle cells are involved.

The results olilained indicale lhal lhe ohserved changes in hlood pressure

thal occurred following lhe injection of cobra venom, might he due lo its dirccl

action on myocardium ralher than on central nervous system since nenrotoxin

has no such effect. Il is possihlc lhal cohra venom conlains anolher factor

resjionsihie for all lhe ohserved changes in lhe circnialion when lhe venom is

injected into au aneslhetizeil cal and lhe changes in lhe movements of an isolated

frog heart when il is jierfnsed wilh lhe venom sohition. This factor was isolated

from lhe venom using lhe convcntional methods vvhich involved hcal denatnralion,

fractional precijiilalion wilh AmoSO,, adsorplion and elnlion from Ca-()hos])hale-

gel, iso-electric ])recipitation, elc. (21). The purified material was 20 times more

aclive than lhe crnde venom, and lhe rnclecnlar weight as determined hy diffnsion

melhod was fonnd lo he 30,()()() (25). Hecenlly il has heen purified using snch

methods involving heal denalnralion, AnioSOj fraclionation, chromatography on

DKAE-cohinm and starch-gel electro|)horesis. The molecular weight of ihis purified

material as determined from sedimenlalion cisnslanl, is approxirnalely 13,()()() to

11,000. Il is 30-35 times more aclive than crnde venom.

(inrionsly enongh, when a single dose of cardiotoxin (0.1 mg/kg) is ad-

ministered intravenonsly into an anesthetized cat. an immediale sharj) fali in

arterial hlood pressure to zero nnn Hg is ohserved; respiralorv failnre also occnrs

at lhe same time. Artificial res|nralion if applied fails lo reslore thc hlood

pressure lo normal levei. When lhe heart is exarnined, it is fonnd in sysiolic

contraclnre. If an isolated toad heart is |)erfnsed wilh a cardiotoxin soinlion,

there is at firsl some angmenlalion in lhe amplitude of lhe contraetion for a

hrief period followed hy a gradual decrease in am])lilnde nnlil finally all lhe

movements are slopped. The heart goes lo sysiolic contraclnre. The effect of

cardiotoxin can nol he washed ont hy prolonged washing with Hinger. Il has

no aclion oti nenromnscniar jimction nor on nerves hnt skelelal muscle kepl im-

mersed in cardiotoxin solntion loses ils excilahilily.

'l he resnils so far ohtained. indicale lhal all lhe effecis recorded eilher wilh

crnde cohra venom or cardiotoxin snggesi lhat lhese effects are nol possihly dne

to lhe aclion of hislamine or histamine like componnds, released from tissnes hy

the aclion of cohra venom or cardioloxin, as lhe laltcr has neilher any proleo-

lylic activity nor any |)hospholipase A aclivily and can nol he implicaled in

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(2):573-582, 1960

ANIMA DEVI anel N. K. SARKAR

579

releasing liislamiiic or histaniiiie like compoiinds froni mast cells. There aro many

rejioris in lhe literatiire siipporling lhe hy|)olhesis lhal llie effects of venom are

(lue lo llie actioii of liistaniine liheraled. Tliey are niaiiily Iiased on reports of

successfiil Irealmenl of snake I)iles wilh untihiiítamiiie drugs, ACTH (adreno-

eorticolropic hormone) and corlisone (26-28). Tliese reports are no don!)l of

some interesl to ns hnt are not as significant as imagined since a criticai analysis

of the resnits reveals many short comings wliicli do not allow to draw any sla-

tistically jiistified conclusions as lo eillier llierapenlic values. This is due to

the lack of reliahle standard l)y vvhich the severity of snake hile ])oisoning can

he judged and hecanse of lhe limiled niimher of cases Irealed snccessfnlly. The

merits of lhese drngs have Iteen ifierefore reassayed in onr lahoratory hy more

accuralely conlrolled animal experimenls.

The antihistamine dnig used was 10-12()-dimelliyIaminoisopro]iyl I-])heno-

ihiazine. The corlisone was a commercial preparation of 1 l-dehydro-17-hydroxy-

corticoslerone-21-acelale olilained in lhe forni of a snsjicnsion in salinc. The

doses of ACTH or corlisone adminislered were 25 lU per kg of hody weighl.

The drngs were adminislered in all cases along with the venom.

The resnits ohlained wilh lhese drugs clearly indicate their ineffectiveness

to counteract the action of cohra venom on circnlalion and respiration; in other

words, they can not |)revent the fali of arterial hlood jircssiire to zero mm Hg

and counteract lhe resjiiralory faihire that oceur following the atiministralion of

the mixture. None of the drugs can also prevent the slojipage of the movements

of tlie excised frog heari when il is perfnsed with lhe venom or cardiotoxin

solntion, they can not eilher counteract the action of cohra venom on nenro-

muscular junction or miiscle fihers. Scholtler (29) did not also find any heneficial

effect of antihistamine drngs in snake jioisoning in contrast to its a[)[)reciahle

ciiralive effect againsl fatal dose of histamine. He did not also find any effeclive-

ness of AOTH, corlisone, etc., againsl venom [loisoning.

(ÍARI)IOSTIMUI.ATING PKINCI IM.i; IN COBRA VENOM

If an isolaled (detached from lhe hody) frog heart is kept immersed in a

venom solntion, il loses its ahilily lo prodiice rhythmic movements in 6 to 10

minutes depending upon the concentralion of the venom solntion used. If il is

exposed to a very dilnted solntion say 1 in 5,000, it takes 2 to 3 honrs to stop

heart heat. If on lhe other hand the heart is exposed to a venom solntion previons-

ly heated at 98'’C for one honr, lhe heart continues to heat for as long as 12

to 14 honrs. The rhythmic' movements hecome more and more feehie wilh lime.

Under identical conditions, the heart continues to heat for onlv 4 lo 6 honrs if

kept immersed in Ringer solntion.

Since at 98‘’C all the known components of the venom with exception of

nenrotoxin are destroyed and the pnrified nenrotoxin has no snch effect on heart

or skelelal mnscle, it may he possihle that the factor directly responsihle for

maintaining the rhythmic movements of the heart for 12 to 14 honrs, has heen

masked liy the presence of other factors, particnlarly hy cardiotoxin, which has

just lhe op])osile effect on heart. This factor may he associated with cardio-

loxin; and withonl the destrnetion of the latter, lhe stimnlaling effect pf venom

conld not he delected. If instead of the heated venom solntion lhe isolaled heart

is exj)osed to a mixtnre of trijihosphales of all the fonr nncleosides, ATI’, (ITP.

CPT and UTP or TTP in eqnivalent proporlions, the heart continues to heat

SciELO

10 11 12 13 14 15

580 CARDIOTOXIC AND CARDIOSTIMUDATING FACTORS IN COBRA VENOM

for oiily Ct to í! liours. When siicli a inixime is rcplaced hy aiiy individual

nucicolide say AMP, (dVlP, CMP, l^Ml^ or TMP otily a sliglil slimidaliiip; effect

is ohserved wilh AMP.

Since llic isolaled lioari íontinues lo l)cal for 12 lo 14 hours iu heated vcnoni

solulioii and tlu> fact llial none of lhe foiir micleolides eillier individually or

('olicctively caii mainlain lhe rhylhniic niovemenls of llie lieart longer lhan 6

lo u liours, it is helieved llial lhe faclor responsihle for lhe sliniulating effecl

of lhe venom is a proteiu, quite resistanl lo heal and has lovv molecular weight.

Venom loses ihis properly if il is heated al 100‘’C for oue hour. This faclor is

a eardioslimulaling faclor, as opposed to eardiotoxie faclor. Ils separalion and

characlerizalion are novv under aclive iuvesligalion in our lahoratory.

(iohra venom conlains hesides neurotoxin and enzymes, Iwo more faclors.

'lhe one, cardioloxin, is resjionsilile for lhe failure of circulalion and sloppage

of all lhe niovemenls of an exci.scd heart. 'Plicse effecls can noi he aholished

or reversed hy any prolonged washing or liy any antihistamine driigs, alro|)ine

or corlicosteroids. Since lhe effecl of venom or cardioloxin is primarily dne

lo either aclion ou cardiac or skeletal mnscle rallier lhan on central nervous

sysiem, it is helieved cardioloxin should he lhe mosl ajipropriate name llial can

he a.ssigned lo ihis faclor. The olhei faclor stimniales lhe hearl, ils effecl is

only visihie when lhe olher faclor is destroyed.

'Plie presenl commiinicalion emphasizes lhe irnporlance of lhe isolalion of

various components in relalively purcr form. and lhe sludy of lheir nature, hio-

chemical, and pharmacological actions separalely as well as colleclively. Such

comprehensive sindies are helieved lo he more usefnl and significanl in lhe under-

slanding of lhe cause of dealh from snake hiles.

l{i;i'EHE.\ci;s

1. MINTON, S. A., Sei. Amer., Illfi, 114, 1957.

2. KLAUBER, L. M., RutÚesnukes, tlieiv hdhitN, Vife historiex, (uul influenco on

vuinkind, Press and Zoological Sociely of San Diego, Universily of Califórnia,

1956.

3. DEVI, A., in W. BÜCHERL, V. DELOFEU, and E. E. BUCKLEY (Editors),

Venoms and Venomous Anrnials, Vol. II, Academic Press, Inc., New York, in

press.

4. DEVI, A., and SARKAR, N. K., in W. BÜCHERL, V. DELOFEU, and E. E.

BUCKLEY (Editors), Vetioms and Venomoua Animais, Vol. I. Acad. Press,

Inc., New York, in press.

5. LEE. C. L., CHANG, C. C., and KAZUYA, K., in E. E. BUCKLEY, and

N. PORGES (Editors), Venoms, Amer. Ass. Avanc. Sei., Washington, 1956

p. 197. B , . ,

6. JOHNSON, M., KAYE, M. A. G., HEMS, R., and KREBS, H. A., Biochem. J.,

ãt, 625, 1953.

7. DOERY, H., and PEARSON, ,1. E.. Biochem. J., i)2, 599, 1964.

8. ELLIOT, R. H., Philad. Trans. roy. 8oc., B, 197, 361, 1905.

9. MACHT, D. I., Med. Rec., 1.53, 369, 1941.

2 3

5 6

11 12 13 14 15

Mem. Inst. Hutantan

Simp. Intcrnac.

33(2):573-582, 1966

ANIMA DKVI and N. K. SARKAR

581

10 .

11 .

12 .

13.

14.

15.

16.

17.

18.

19 .

20 .

21 .

22 .

23.

24.

25.

26.

27.

28.

29.

HOUSSAY, B. A., NEGRETE, .1., and MAZZOCCO, P., Rev. As. ynéd. argent.,

Sec. Soc. Biol., 35, 185, 1922.

CUSHNY, A. R., and YAGI, S., Philad. Trans. roy. Soc., B, 308, 1, 1916.

SARKAR, B. B., MAITRA, S. R., and GHOSH, B. N., Indian J. med. Res., 30.

453, 1942.

SOAJE-ECHAQUÊ, E.. Rev. Soc. argent. Biol., Ifi. 475. 1940.

FELDBERG, W., and KELLAWAY, C. H., Ausl. J. exp. Biol. med. Sei., 15.

441, 1937.

KUSNETSOR, A. I., Byiãl. éksp. Biol. Med., 3, 295, 1936.

GOTTDENKER, F., and WACHSTEIN, M., J. Pharmacol. exp. Tlier., 69, 117,

1940.

GUNN, J. W. C.. and EPSTEIN, D., Quart. J. Pharmacol., 6, 182, 1933.

BROWN, R. V., Amer. J. Physiol.. 134, 202, 1941.

SARKAR, N. K., Ann. Biocheyn., 8, 11, 1948.

SARKAR, N. K., MAITRA, S. R., and ROY, P. K., Toxicon, Ann. Biochem.

Med., 6, 81, 1946.

SARKAR, N. K., and MAITRA, S. R., Amer. J. Physiol., 163, 209, 1950.

DEVI. A., MAITRA. S. R.. and SARKAR. N. K., Ann. Biochem. Med., 13, 23,

1954.

SARKAR, N. K., Proc. Soc. exp. Biol. (N.Y.), 78, 469, 1951.

SARKAR, N. K., J. Indian Chem. Soc., 34, 227, 1947.

SARKAR, N. K., J. Indian Chem. Soc., 34, 61, 1947.

MAIER, H. K., Vet. Med., 46, 463, 1951.

CLUXTON, H. E., Proc. 2nd. Clin. Acth Conf., 3, 445, 1951.

HOBACK, W. W., and GREEN, T. W., J. Amer. Med. Ass., 153, 236, 1953.

SCHÕTTLER, W. H. A.. Amer. J. Trop. Med. Ilyg., 3, 1083, 1954.

10 .

11 .

12 .

13.

14.

15.

16.

17.

18.

19 .

20 .

21 .

22 .

23.

24.

25.

26.

27.

28.

29.

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(2):583-588, 1966

FINDLAY E. RUSSELL

583

60. CARDIOVASCULAR RESPONSES TO SNAKE VENOMS AND THEIR

FRACTIONS

FINDLAY E. RUSSELL

University of Southern Califórnia, School of Medicine, Los Angeles,

County General Hospital, Los Angeles, Califórnia, U.S.A.

It has long heen kiiown lhal cerlain sitake venoms exert a deleterious effect

ori the cardiovascular syslem. During more recenl years, altempts liave heen

made to determine which fraction or fractions of snake venoms are res])onsil)le

for these deleterious effects. Some attempt has also l)een made to define the

relationships hetween the direct effects of the venom on the cardiovascular system

and tliose which may he precipitated hy auto|)harmacological changes initially

provoked l)y lhe venom. In the course of these various investigations, many

imporlant data, which indicaled the complexity of the vascular response, have

heen ohtained. The purpose of lhe j)resent paper is to present a shorl review

of lhe compendium of knowledge on lhe cardiovascular effects produccd hy snake

venoms, and to reflect on some experiences which, I feei, are imporlant as guide

lines for future research on the pharmacology of these fascinaling toxins.

First, I should like to discuss, in a very general vvay, some relalionshi|)s

l)elween lhe j)hysio|)harmacological aclivities of snake venoms and lheir chemislry.

We are wíell aware, as has heen pointcd oul hy numerous workers, thal snake

venoms are complex mixlures, chiefly proteins, many of which have enzymalic

aclivities. There was a time in our ihinking when all of lhe deleterious effects

of venoms were linked wilh lhe enzymatie components of the loxin. We now

know, of course, lhal ihis is nol tnie, and indeed, lhe more lelhal aclivities of

most venoms — whelher lhey he snake, arthrojmds or fish — are nol generally

associaled wilh one enzyme or evcn with several, although it does appear lhal

certain enzymes may he closely hound with cerlain non-enzymalic |)roteins in a

venom, and thal these condiinalions can he rpiite lelhal. On llie olher hand,

lhe enzymes of snake venoms are cerlaitdv responsihie for some of lhe deleterious

chauges provoked hy the crude toxin, and perhaps in particular with those

changes which ajjjjear lo occur al lhe cell memhrane, whelher lhe cell hc one

in the intima of a vessel or in suhcutaneous tissues, or even if it he a hlood cell.

These unusual comhinations of mullijde enzymatie and non-enzymalic ])roleins,

along with certain non-prolein suhstances, not only indicale very com[)lex or-

gauization and |)hysio])harmacological activily, hui mighl apjiear to reflect upon

a series of adaptive mechanisms during lhe evolulion of lhe venom. On lhe

olher hand, snake venom enzymes may he involved in far more com|)Iex hiological

functions, as has long heen pro])osed hy Professor Zeller in his many fine papers.

I ani sure we are all hecoming impressed wilh lhe fact lhat. in general, there

is a great deal of conformity in lhe organization of lhe chemislry of snake

venoms, and while cerlain fractions are indeed quite differenl, chemically as

1 SciELO

584

CARDIOVASCULAR RESPONSES TO SNAKE VENOMS AND TIIEIR FRACTIONS

vvell as physio[)harmac'ologic:ally, ])arlitiilai'!y al lhe Family levei, lhere appear

to he more similarities lhan (liffereiiees tlum vvhal \ve luul llioughl oiie or Ivvo

(leeades afio. Tliis same general im[)re.ssion is also ohtaimnl when one eonsiders

tlie marine venoms and poisons, and indeed lo some cxteni even willi aiihropod

toxins.

There is a lendeney, ])erlia[)s forUinalely so, in lhe vvell-oiled seienliFie mind,

to gather data inlo orderly groups For [)ur|)oscs oF elassiFieation. This is an

admirahle Irait hnt it ean he Fraught wilh some dangers, and perhajis nowhere

in hiology is this more evideni lhan in our ovvn Field. In lhe ahsenee oF reliahie

dala there has heen a lendeney to arrange snake venoms into loose indeFinahIe

elassiFicalions. These elas.siFiealions have, in many instanees, heen more eonFus-

ing than hel[)Fnl. Even sueh widely iised lerms as luniroloxiii and cardiotoxin

are inadeíjuale and misleading. For lhey are oFten ap[)lied lo lhe whole venom.

and most venoms are eomplex rnixliires having several or many hiologieal pro-

perties. Il has also heen shown thal neuroloxiiis ean. and oFten do, have cardio-

loxic or hcmotoxic aetivity, or hoth; vurdiotoxins may have ncitroloxic or hemo-

toxic aetivity, or hoth, and hcriioloxins may have the other aetivities. The laheling

oF a venom as a neuroloxin is not oniy conFusing hnl dangerous. For it may lead

a physieian to make unwise elinical decisions (1 ).

No one has hrought this |)oint eloser to Foens lhan 11. Alistair Heid, vvho

has reeenlly demonstraled that the prinei|)al clinieal Featnre oF eohra poi.soning

in Malaya is local necrosis, and that ncuroloxic eFFeets in hiiman vietims are

indeed rare(2). Furthermore, as Dr. Heid poinis oiit, and as has heen noted

hy [jrevioiis workers, the cardiovaseular eFFeets oF lhese venoms are oFten times

marked, and in many ea.ses are the eaiise oF death. It would seem, that imtil

the Fraetions responsihie For the deleterioiis eFFeets oF a snake venom have heen

isolated and studied individually and in eomhination, we need to exereise extreme

eare in systematizing data vvhieh are hased parlly on hiologieal assay methods,

])artly on hioehemieal stndies, parlly on elinical ohservalions and |)arlly on in-

Inilive himehes.

There is anolher vveakness in our over-all approaeh lo lhe sliidy oF lhe

physio|)harmaeologieal jiroperlies oF snake venoms. There is a lendeney to link

.s])eeiFic ehemieal struetures wilh speeiFie hiologieal aetivities; i.e., thal suhstanee

A |)roduees eFFeet A, and lhal suhstanee 15 produees eFFeet 15, ele. Il would

appear lo me that it would he highly uniikely and unheeoming For nalure lo

have developed venoms íti this manner. Partieularly, sinee perha|)S one oF lhe

most irnporlant Faelors in the evolution oF a venom is lhe role |)layed hy lhe

ada|)tion oF the prey or the oFFending animal. Bul whelher or not lliis eoneern

is Founded, lhe very |)rinei|)le oF looking For a s|)eeiFie Fraetion lo exeri a speeiFie

eFFeet is a dangerous one, For among olher things, il may oFten limit our eoneept

and our experimental ap|)roaeh to determining the meehanism oF aelion oF a

venom hy ihinking thal il neeessarily Follows thal one s])eeiFic Fraetion has one

speeiFie Fimelion in or on one organ syslem, ailhough in some cases this may

prove lo he true.

Some oF lhe so ealled neuroloxins thal have heen seni lo us For Furlher

evaluation in cardiovaseular preparalions during iluí ])asl Five years have had a

more marked eFFeet on lhe cardiovaseular syslem than on lhe nervous system,

and in some cases, in Fael, this eFFeet has Far oiil-shadoweil lhal on the laller

syslem. In most oF lhese ea.ses lhe error in judgemeni has not weighed wilh

lhe venom hut ralher wilh the experimenter, who, For example, having secn a

mou.se in eonvulsions Following lhe injeelion oF lhe venom has presuined that iIkí

SciELO

10 11 12 13 14 15

Mcm. Inst. Butantan

FINDLAY E. RUSSKLL

585

Simp. Internac.

33(2):5,S3-58S,

venoiii was a neuroloxin. wlicii iii rcalily llu- nioiisc is coiivulsing liecaiise' oí

fcrcliral aiioxia caiiscd liy a niarkcdly raducad hlood supply lo ils hrain secoTidary

lo syslcniic Iiy|)ol(‘nsion.

'Flieri' is onc olhar araa of cardiovascidar researcii oti wliicli I slioidd likc

lo coninu-iil. '1’his involvas lhe ahoiaa of llia propar ax])arinianlal animal. Ona

iniisl ha axaaadinply aarafid in applying dala darivad frorn sludies in ona gron|i

of animais lo aonalnsions ahonl lha hiologiaal affaals of a vanom in anolhar

gronp of animais or lo dala on lha dasign, usa and adaplion of a vanom. \Va

ara all familiar vvith lha markad diffaranaas in lhe lelhality of vanonis for dif-

faranl animais and hovv in soma aasas lha.sa ara ])ailieidarly ralatad lo s])aeifie

aeologiaal prohlams. 1'nforlunalely, mueh of oiir informalioti on lha zooloxiao-

logiaal ])roparlias of vanoms is hasad on sliidias vvilh niammals. vvhiah, of eoursa,

liniils lhair a|)pliealion as far as oiir nndarslanding lha design of lhe loxin in

lha animars armamanl. On lha olhar hand, il Is acpially dangarous lo appiy

dala ohiainad on a fish narva-musala pra])aralion or lha frog haari, or llia aoak-

roaah haarl, lo aonalnsions ahonl lha aalion of lha vanom on man, or avan on a

rnammal. CerlaÍTdy lha mora divarsifiad onr sltidias llia mora imjiorlanl dala

lhal vva vvill ohlain, hnl lha a|)pliealion of lhase dala nnisl ha gnardad zaalonsly.

Kven in ap])lying dala ohiainad in mammals one mnsl ha exaaadingly aarafnl.

For inslance, dala ohiainad from aardiovascniar slndies in dogs aannol he liherally

ap|)liad lo hnmans. Il has haaome increasingly a])|)arpnl, jiarlicniarlv from slndies

on shoek, lhal lhe dog may res|)ond quila diffarenlly lhan lhe human nnder

similar experimenlal aondilions. The imporlanee of lhe porlal eiranlalion in lhe

dog dnring varions hy|)olensive arises is far mora markad lhan in lhe hnman.

In eeiiain cardiovascular condilions lhe dog mnsl he considered a porlal animal

while man cerlainly is nol. Anolher difference is in lhe renal eiranlalion. In

lhe rahhil and eal, as well as in lhe hnman, lhe renal vein is known lo dilala

(piile raadily in responsa lo machanical and earlain chamiaal slimnii. It is far

less rasponsiva in lhe dog. whila on lhe olher hand lhe s|)lenic vein in lha lallar

animal dilalas (jnila easily vvilh s|)acifia drngs and slimnii. This jiiohalily raflaels

lha high dagrae of sjilaen raservoir fnnalion in ihis s|)aaias. In lha eal. rahhil

and hnman lhe S|)lenia reservoir is mnah less marked. In cerlain mammals,

paiiienlarly lhe dog, aerlain venons valnes |)lay a more im[)orlanl role in lha

vascular res|)onsa lhan lhay do in lha hnman. The cal, on lhe olher hand. ap-

jiears lo res|)ond lo snake vanoms in a mannar mnch more lika lhe hnman lhan

does lhe dog. In holh lhe cal and in lhe hnman lhe hypotensive crisis evoked

hy C r o t a I tt s venom, for inslance. is associaled vvilh changes in lhe |mlmonary

eiranlalion and perhaps in lhe larger ves.sels of lhe chesi (1,8, 1).

These faw examples poini onl lhe greal care one mnsl lake in ap])lying dala

from one gron|) of animais lo anolher, or lo hnmans. One niighl aonclnde lhal

jierlnqis manv' of lhe differences noied in lhe literalnre helvveen resiion.ses lo

varions venoms ara mora direclly ralalad lo lha choice of animal n.sed lhan lhe

venom nsed or aven lo lhe leehniqne applied.

Halher lhan deal vvilh speaific' dala on lhe eardiovasaniar effeels of snake

venoms and lheir fraclion (and of lhase affaals mosi of ns are acqnainled), I

shonld like lo revievv some of lha hasic eonea|)ls lhal one mnsl eonsidar if he

ehooses lo measnre and inierprel lhe changes provokad hy vanoms in lhe de-

pendenl variahies of lhe varions paramelers of lhe cardiovascular syslern.

I■’ig. 1 illnslrales lhe ])rinciple of parallel cirenils and resislances in lhe

aardiovascniar sysiam. Sinae lhe cardiovascular syslam aceomplishes ils hiological

2 3

5 6

11 12 13 14 15

586

CARDIOVASCULAR RESRONSES TO SNAKE VENOMS AND TIIEIR FRACTIONS

fiincTions in a mechaiiical way, il can he seen lhat any chaiige in oiie of the

paraineters will affect changes in tho resistance of oiu-, several or all other jiara-

melcrs of lhe syslein. These changes will he reflected in lhe dependenl variahles,

lhal is in the heart output, and the prcssures on lhe arterial and venous sides

of the systemic and puhnonary circulations. The relationships can he piolled as

HEAD

NECK

ARMS

.UNGS

TRUNK

LIVER

^^1_ABDOMINAL ORGANS

1 .. .1

SPLEEN

KIDNEY

PELVIC ORGANS

LEGS

Fig. 1 — Diagram of tho circulation showlng parameters, Inclufl-

ing reslstances (moUitied from Wezler and Bõger, 1939).

a matheniatical formulation, and this has heen done hy several workers. In

1954, hefore lhe days of eleclromagnetic flowmelers and other flow-jilessure

nionitoring devices, Prof. Van Harreveld and I jiroposed a inodel of lhe eirculalion

(Fig. 2), froni whieh eerlain equalions eould he developed and iised in defining

more earefully llie changes jirovoked in lhe vascular parameters hy various

venoms (5 ).

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(2):583-58S, 1966

FINDLAY E. RUSSELE

587

The relevant eqiialioiis descril)iiig lhe relations hetvveeii lhe parameters and

lhe dependent variahies of lhe cireidation may lie sunimarized as follows:

P.si =

Pl:

Csi (Rs fs) Csz Ts -j- Cin (Rl “1“ i‘l ) Ci,2 1‘l

_V ( Rs + r.s )_

Csi (Rs + r,) + Cs2 rs + Cu (Rl + vl ) + Ci,2 r,,

_ V n,

Csi (Rs “h rs) “l” Cs2 rs - 1 - Cu ( Rl rn) -|- Cl 2 rL

In these equalions, I is lhe hearl oiilpiil. P s, rejjresenls lhe syslemie arlerial

[jressure. P l, indicales lhe lefl alrial i)ressure. V is relaled, ihough nol sy-

tiüiiymous, wilh lhe lolal volume of lhe cireidaling l)lood. Kg and Kl are llie

resislanees in lhe arlerioles and ea|)illaries of lhe syslemie and |)ulmonary eireula-

lions respeelively (periplieral resislanee). The lolal resislanee in lhe venoiis

syslem and all lhe faelors limiling lhe hlood flovv inlo lhe venlrieles are re])resenled

hy lhe resislanees rg for lhe righl hearl and rj, for lhe lefl hearl. The largest

faclor for lhe vahie r is lhe elaslieal resislanee of lhe venlrieular wall againsl

filling. Cg^, C , ele...., are lhe eonslanls of eapacilanee (volume ehange per

nnil ehange of pressiire) for lhe varioiis com|)onenls of lhe eireulalory ap|)aralns.

These eornpoiuMils are an index of lhe lone of lhe larger vessels of lhe syslemic

and |)ulmonary arlerial and venons syslems.

íl ean lie seen lhal hy varying a parameler one can delerrnitie, ihrough

meusuremenls in lhe dependenl variahies, lhe pressures and flows in lhe various

eomponenls of lhe cardiovascular sy.slem. Today, ihis has heen made more simple

hy lhe advenl of lhe eleclromagnelic flowmeter. Il is now ])ossihle, willi a lillle

eare, lo measure hlood jjressnre and flow eoncomilanlly in a single vessel, and

lo earry onl ihis ])roeednre in fonr or five vessels dnring a single experiment.

The informalion ohlained from snch measnremenls nol only gives ns valnahle dala

on lhe changes lhemselves and lheir relalionsliips lo olher vascular ])henomena,

hui il gives us a ready insighl inlo lhe mechanism of aclion of lhe venom.

This mighl he demonslraled hy several receul experimenls wilh C r o t a I ii s

venoms. Using Iransducers lo measure cerlaiu arlerial and venons jjressures on

ho Ih sides of lhe syslemic and jndmonary circnialions (some measnremenls heing

SciELO

10 11 12 13 14 15

588 CARDIOVASCULAR RESPONSES TO SNAKE VENOMS AND TllEIR FRACTIONS

takcii llirougli callieti-rs llireadcfl lliroiigh llio licari tindcr fliioroscopy), and hy

taking simullaiieous Idood flow rccords willi ('l('clroinagiuTic ílowniotnrs fiom llic

same and different vesscis, and liy concomilanl recording of llic eleclrocardio-

grani and (‘Icctroenceiilialograni, and 1)V mcasnring ccrfhrosiiinal finid prcssnre

and lhe rate and de|)th of respirations il [ias lieen ))Ossihli‘ lo delermine lhe

seípience of evenis of lhe imniediale and |)reei|)ilons hypolensive eriíiiíi lhal oc-

curs following lhe iniravenons injeetion of C r o ! a ! u s venoinld, I ). Il lias

also lieen possihie, nsing lhese teehniqnes and cerlain isolaled organ teeliniqnes (d,

4), lo ohiain some insighl inio lhe meehanisni of aetion of ihis venoni.

Frorn lhese sludies il has heen eoneinded lhal lhe hyqiolensive crisis is dne

lo ehanges in lhe resislance vvilhin lhe puhnonary eirenil. Tliese elianges lead

lo a decreased left heari oiil|nil ])ressnre and flow, wliieh in lurn jirovoke allera-

lions in lhe flow and ])ressnre wilhin varions vesseis, ehanges in pei'i])heral

resislance, ehanges in respiralion, eerehrospinal finid jiressnre, and in lhe eleelro-

cardiogram and eleclroencephalogratn. Il was snggesled lhal lhe (hange in

|iiilmonary resislance may he allrihuled lo lhe jiooling of hlood in lhe Inngs and

larger vesseis of lhe chest, dne lo posl-cajiillary resislance froni eilher vascular

conslriclion and/or lhe formalion of mnlliple ihromhi. It apjiears lhal each of

lhese phenomena may |)lay a pari in lhe crisis. These ehanges are evideni in

lhe hiiman, monkey and cal. They are much less conspicnoiis in lhe dog, where

lhe mechanism for lhe hy[)olensive crisis may he quile differenl.

The sludies previoiisly noted mighl .serve as a giiide for fulure work ou lhe

cardiovascular effecls of venoms and venom fraclions, for lhe leihniques ])rovide

a cotisideralile amounl of dala not olherwise ohtainahie, while also permilling a

carcful l•heck and evalualion of inler|)retations. Bul even loday new lechniques

are heing developed, and these may give us considerahiy more insighl inIo lhe

mode of aclion of loxins lhan we aniicipale. Parlicnlarly encouraging is lhe

possihilily of measnring definilive vascular ehanges vvilh lagged venom and hlood,

ailhough even lhese lools have ohvious limils of usefniness. In all of lhese

sludies, however, lhe crilical conlrihiilion will slill resi wilh lhe ex|)erimenler.

As one sludies lhe progress in onr Science, as in all Sciences, il is ohvions lhal

lhe mosi significani conirihulions are fnlly dejiendenl iqion lhe invesligalor and

nol u|)on his eipiipmenl. The cardiovascular effecls of venoms and lheir fraclions

are slowly heing unraveled hy scholars and not hy gadgeis, and we have seen

in ihis meeling a demonsiralion of lhe kind of iniellecinal slimnialion and en-

coiiragemenl lhal will greally enhance onr underslanding of lhe pro|)erlies of

lhese mosl remarkahie loxins.

Beitciíence.s

1. RUSSELL, F. E., in F. A. DAVIS (Editor), Cyclopedia of Medicine, Surgery

and. tlw Specialtics, Vol. II, Philadolphia, 1962, p. 197.

2. REID, H. A., Brit. med. J., 2, 540, 1964.

3. RUSSELL, F. E., BUESS, F. W., and STRASSBERG, J., Toxicon, I, 5, 1962.

4. HALMAGYI, D. F. J., STARZECKI, B.. and HORNER, G. J., J. appl. Pliysiol.

(U.S.A.), 20, 709, 1965.

5. RUSSELL, F. E., and VAN HARREVKLD, A. Árch. int. PJmiol.. 62, 322, 1954.

2 3

5 6

11 12 13 14 15

Mem. Inst. Bulíintan

Simp. Internac.

33(2) :5S9-6n2, 1966

MASAIIIRO OKADA

589

61. PHAKMACOLOGY OF THE COMPONENTS OF TOAD VflNOM AND

ALLIED SUBSTANCES

MASAHIRO OKADA

Department of Pharmacology, Faculty of Medicine, Tokyo, Japan

The dried venom of lhe Chinese load has beeii used externally íroin aiitiquily

as a home remedy called Senso iClFaii Su) for canker sores, looth-ache, and many

local inflammatory conditions. Biil most pharmacologists have concenlrated them-

selves on the sludy of ils cardiolonic action, and little is known on ils local

actions.

V7e have investigated lhe local aneslhetic aclion of each purified suhstance

exlractcd from Senso (Tahie I). As descrihed in previoiis papers (1,2,3) lhe

strong local aneslhelic properly was foiind generally in the steroid fraclions while

the water-soluhie fraclions have local irritating aclion with very weak or almost

no local aneslhetic jnoperlies. For example hufotenidine, a water-.solnhle derivalive

of Iryptamine, has no aneslhetic action.

For [jhartnacological lests, lhe snhstances insoinhie in waler were dis.solved

in a mixtnre of waler and pro])ylene glycol. In ihose cases conlrol lests were

made with lhe same concentralion of pro])ylene glycol.

Snrface aiií^slhetic action on córnea was delennined with adnll inale rahhits.

The conjunctival sac was filled with 0.2 ml of lhe solnlion of various concenlra-

tions, and the lids were released after one minute. The wink reflex was elicited

hy a horse-tail hair (0.17 mm in diameler, 0.7-0.75 g in jiressnre). The lesl

of 6 jiricks was ajiplied on córnea every 5 minutes for 30 minnle‘S. The nnmhcr

of limes lhe prick failed lo elicil a hlink reflex during the 30-minnle jieriod was

added nj) and the sum gives an indicalion of lhe degree of aneslhesia. Therefore,

6 X 6 = 36 is the value thal indicales complete anesthesia for lhe 30-minnle

period. Tahies If and III show .some resnlts.

When lhese residis for each snhstance were jilotled on a log dose-response

coordinale, the apiiroxiinalely slraighl lines so ohlained were not qiiile jiarallel

to each otlier, which rnakes it difficnlt to slale exaclly the relative strenglh of

anesthetics to cocaine (Fig. 1), hnt in roimd figures, when aneslhetic ])olency

of cocaine hydrochloride is taken as 1, thal of jirocaine hydrochloride is 0.1,

while that of Fraclion No. 200 is 30-60 I mean 40) as Tahie IV shows. This

facl is remarkahle and what is more, No. 200 has almost no irritating aclion.

SciELO

10 11 12 13 14 15

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(2):589-602, 196{i

MASAHIRO OKADA

591

TABLE II — NUMBER OF TIMES THE BLINK REFLEX IS ABSENT DURING A

30-MINUTE PERIOD (No. 200)

Time (min)

CONCENTRATION

0.011%

0.017%

4 2 3

2 2

5 5 6

4 5

4 2 4

1 3

2 0 2

0 0

0 0 0

0 0

COO

0 0

Totai

15 9 15

7 10

Time (min

)

0.025%

Tolai

TABLE III

— NUMBER OF

TIMES '

THE

BLINK

REFLEX

ABSENT

DURING A

30-MINUTE PERIOD. COCAINE-IICl

Time (min)

CONCENTRATION

0 . 2 %

0.4%

Total

Time (min)

CONCENTRATION

0 . 8 %

1 . 6 %

Totai

3ÍÍ

SciELO

10 11 12 13 14 15

CSQ9 PIIARMACOLOGY OF THE COMPONENTS OF TOAD VENOM AND

ALLIED SUBSTANCES

Fig. la — Log Dose-Response curves oI

Bufogenins and allied Compounds.

TABLE IV — POTENCY

IN TERMS OF COCAINE-IICl (=1.0).

IN RABBIT CÓRNEA

SURFACE ANESTHESIA

Substance

Number of

animais

Potency of surface anesthesia

R.^

Mean

Cocalne-IICl

Procalne-IICl

0.13

0.11

0.09

0.11

193

12.G

14.8

17.4

14.9

196

13.8

16.2

19.1

16.4

194

()

9.6

11.2

13.5

11.4

197

1.6

2.2

3.2

2.4

198

()

19.1

24.6

28.8

24.2

199

200

28.8

39.8

57.5

42.1

201

(í

5.0

4.7

Antllog (X,.-XO X

!■; log dose

of cocalne-IICl

X»: log dose of substance

R,:

Response

lO-levcl

R,:

Response

20-ievel

R,:

Response

30-level

SciELO

10 11 12 13 14 15

Mem. Inst. Hutantan

Simp. Internac.

33(2):589-6(>2, 1966

MASAIIIRO ÜKADA

593

The onset of local aiieslhcsia by No. 200 is soiiiewhat slovvcr lhan cocaiiie, l)iil

its duralioii is mucli lotigcr. In infiltration ancslhesia test (wheal mclliod) on

mati, lliis sulislance (No. 200) ])iodiices total local anesthesia in 5 minutes, whicli

continues dnring 30 minutes to 1 liour in 0.0003% solntion and for 3 bonrs

in 0.03% solntion, and no local tissne damage vvas leít.

The fraction No. 200 corresponds to the snbstance of fraction niimber Fr. 52

in the repori hy Olmo and Komatsn (4). As was therein descrihed, this snbstance

gave four spots with different Kf values by ])aper partition chromatography, biit

repeated ])iirification finally afforded a snbstance giving only a single sj)Ot at

Hf 0.61.

Komatsn |3| examined the ehemieal and physical i)ro|)erties (melting point, ele-

mentary analysis, Hf valiie, colour reaetion, ultraviolet and infrared S|)eelra) of

this fraction and fonnd il to be identical with bnfalin isolated from toad venom

by Knno Meyer. Local anesthetic activities of Meyer’s sample of bnfalin (No.

201!) and the sample (No. 209) isolated by Komatsn were eompared and we

fonnd no difference between the tvvo, both qnalitatively and cpiantitatively.

The assay method for local anesthetic activity was the same as previonsly

descrihed, and the resnlts are shown in Tables V and VI. Samples No. 208 and

No. 209 in 0.0025% solntion showed the maximnm hyjresthesia after abont 10

minutes and the drng action disap|)eared after abont 20 minutes. With 0.005%

solntion, hypesthesia continned for abont 30 minutes, with 0.01% solntion for

over 30 minutes, and an ahnost perfect anesthesia was exhibited. With 0.02%)

solntion. complete anesthesia continned for over 30 minutes and there .seemed

to be no irritation on the córnea.

TABLK V — NUMBER OF TIMES THE BLINK REFI.EX IS ABSENT DURING A

30-MINUTE PERIOD — No. 208

Time

(min)

CONCENTRATION

0.0025 %

0.005 %

Total

Time

(min)

CONCENTRATION

0.01 %

0.02 %

()

(í

Total

SciELO

10 11 12 13 14 15

594

PlIARMACOLOGY OF THE COMPONENTS OF TOAD VENOM AND

ALLIED SUBSTANCES

TABDE VI — NUMBER OF TIMES THE BLINK REFLEX IS ABSENT DURING A

30-MINUTE PERIOD — No. ;209

Time

(min)

CONCENTRATION

0.0025 %

0.005%

í)

Total

CONCENTRATION

Time

(min)

0.01%

0.02%

Total

Do.«(‘-r(>S])oiisc curve is giveii in Fig. 2. Conijiarisoii of lhe values from

this gra|)li wilh tho.se of eoeaine hyclroehloride aiul procaiue liydrochloride is

giveii iii Talile VII.

Fig. 2 — Log Dose-Re.sponse Curve.s.

The local aiiesthelic adiou of samples No. 208 and 209 is over Iwice that

of No. 200, and ahoul 90 times that of cocaine hydrochloride, and duration of

lheir action is markedly lotiger, allhough it takes somewhat longer lime for the

action lo ajipear.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(2):589-602, 1966

MASAHIRO OKADA

595

TABLE VII — POTENCY IN TERMS OF COCAINE HYDROCIILORIDE ( = 1.0) SURFACE

ANESTIIESIA IN RABBIT CÓRNEA

Compouncl

No. of

animais

Poteney of surface aneslhesia

Mean

Cocalne-HCl

1.0

l.ü

1.0

Procalne-HCl

0.12

0.11

0.C9

0.11

No. 209

78.7

87.9

98.0

88.2

Antllog (X,-Xs) Xi-: log dose of cocalne-IiCI

Xs: log-dose oí sub.stance

R, Response 10-level

R, Response 20-Ievel

R.i Response 30-level

riiis fact was also confirmed iii iiilradernial vvheal lesl on hiinian skin. lii

lliis lest 0.1% soliition (iii 50% propylene glycol) of No. 209 was diliiled with

physiological saline to niake 0.01% and 0.001% Solutions, 0.1 nil of eacli solution

w'as injected intracutaneousiy in tlie forearm, a local |)atcli of anestliesia was

jirodiiced and its degree and duration was tested l)y pin-pricks.

With 0.001% solution, anestliesia lasted for ahout 30 minutes, while with

0.01% solution complete anestliesia lasted for several (3-7) hours, without leav-

ing any tissue damage. These results are in marked contrast with the effect

of cocaine hydrochloride and jirocaine hydrochioride, whose intracutaneous in-

jection of 0.1 ml of 0.125% solution causes immediate apjiearauce of the action

hut its durations are respectively 15 aud 6 miuutes.

The local tissue-irritating jirojicrties of these fractions were estimated hy

iutradcrmal injection of 0.3 ml of the following local anestheties into the clipped

ahdominal skin of a rahhit hy Trypan Hlue test descrihed hy Hop|)e, Alexander,

and Miller (5) and the magnitude and intensity of local hlue spots on the

ahdominal skin caused hy the intravencus injection of trypan hlue (1%, 1 ml/kg)

were measured after 30 miuutes, 1 hour, and 3 hours. With 2% or 5% jiro-

caine-hydrochloride (5% propylene glycol solution) and 2% xylocaine-hydro-

chloride (5% j)ro|)ylene glycol solution), large intensive hlue sjiots soou ap])eared,

which could not he ohserved with 0.03% of hufalin (5% |no|)ylene glycol

solution) and 0.01% of gamahufotalin (5% |)ropylene glycol solution).

As has aiready heen descrihed (1) some local anesthetic activity has heen

found in cinohufagin, cinohufatalin, gamahufotalin (Fig. la), etc., so that the

local analgesic action of the Chinese crude drug, Ch’an Sn, the toad venom,

cannot he accredited entirely lo hufalin. However, hufalin is the suhstance found

until lhe present date that has the strongest local anesthetic action among hiifa-

dienolides, and a way will he opened for the use of sleroid as a local analgesic

hy com|)arative examination of such suhstances to lie j)re|)ared hy changing their

side chain.

Cinohufagin aiul citiohufalalin are ahout one-sixlh of hufalin in local anesthetic

poteney, while resihufogenin has no such adiou. Digitoxigenin, a cardenolide

jiossessing the same Chemical slructure as hufalin excejil for its 5-memhered lactone

ring, has a very weak local anesthetic actioti, ahout one-fortieth that of hufaliu

in surface anesthetic poteney.

SciELO

10 11 12 13 14 15

596

PHARMACOI.OGY OF THE COMPONENTS OF TOAD VENOM AND

ALLIED SUBSTANCES

Ciliohufaginic acid, fonncd Iiy cleavage of the laclone ring through Iiydro-

lysis, was found lo l)t' entircly dt-void of aiiestlietic adiou iu lhe eye. The

sigiiifieaiue of lhe |)ref;etiee of uiiíaliiraled ladoiie ring, hesides lhal of hydroxyl

iii Ca-posilion of sleroid ring, seenis lo l»; essenlial for lhe local aneslhdic adion.

Hufaliii has, however, a very vveak anei-lhdic adion on llie nerve filicr as

compared vvilh procaine (Tal)les VIII and IX).

TABLE VIII

Threshold anesthetic

concentration

Duration of anesthesia

Toxicity

(mouse)

cu p

.ií

Compound

nerve

)

s g

§ B

C 3

ctí x:

córnea

(min.;

o ,5

c .

Cd —

3 Td

O Cd _

c + °

« -S 2-

bjo

Sciatic

(trog)

c ^

o i"

U w

O ^

Cd Cd

U Ot

C > 1^-

M r*

Rabbit

1% sol.

Human

intracut

0.5 % SC

Human

intracut

0.5 9c SC

lin l:õC

S.C. LD

I.V. LD.

Procaine

0.250

4.0

0.04

0-2

171

750

Butacaine

0.125

0.31

0.02

200

50*

12*

Dibucaine

0.004

0.003

0.0025

51 (0.1%)

100

221

10*

2.5-'

Cocalne

0.125

0.32

0.02

200

110

Butalin

0.0025

0.001

30 (0.02%)

■ 30

—

4.0

0.35

(0.001% )

150-480

(0.01 % )

• Rabbit

TABLE IX

Compound

.Surface anes-

thetic potency

(rabbit córnea)

ED,„

(rabbit córnea)

LD,^ (mg/kg)

(mouse)

LD.„/ED,,„

I.V. s.c.

I.V.

S.C.

Cocalne

1.0

0.85

0.437

11 110

12.9

129

Procaine

0.11

8.50

4.37

45 750

5.3

Xylocaine

0.27

3.10

1.74

31.5 172

9.!)

Bufalln

88.2

0.01

0.ü(í

0.35 4.0

29.2

400

The effed of hiifalin and procaine on lhe eledrical excilahilily of nerve

fiher wa.s examined, iising lhe scialic nerves of loads or hullfrogs. As descrihed

hefore (6), hiifalin, in spile of ils very strong |)Olency in siirface aneslhesia,

has otdy a vveak effed on lhe nerve fiher. Il was lherefore sujvposed lhal lhe

sile of loeal aneslhdic adion of hufalin niight he lhe recejftor of sensory nerve

ending. However, lhe sile of adion in queslion is now under delailed cxaminalion.

SciELO

10 11 12 13 14 15

Mcm. Inst. Butantan

Simp. Internac.

33(2):589-602, 1960

MASAHIRO OKADA

597

Hufadienolides as vvell as cardeiiolides induce a loiig-lastiiig contraclure iii

llic isolated frog rectas ahdonünis inuscle, wliile cocaine, and otlier local anesthetics

inliil)it llie acetylcholinc conlractiire of tlie imiscle. Tlie muscular contraclure

curve consists of two stages: the stage of gradually increasing lonus, and tlie one

inducing faster contracture follovving tlie foriner.

The second stage is inhihited hy pretreatnient with d-tid)ocurarine, as well

as hy letrodoloxin, while the firsl stage is not affected hy its juetrealment

(presenled in Fig. 3).

This contracture-inducing activity of hufadienolides and cardenolides runs

parallel to the cardiotonic activity of these coinpounds.

Arh Bufalin

1 : 2 000 001) 1 1 000 000

Arh Bafilin

1 : 100 000

.iher cl Tr. I : 10 000 0

Fig. 3 — Rufalin on Rectns Abdominis

Muscie of the Frog.

Method

(i) Sciatic nerve trunk was arranged in a separating box, as inclicated in

Fig. 4, slimulus was given to it at po.sitions a and b (S), action-current was led

off from positions d and e (R) and recorded. Drug was applied to desheathed

part c. (ii) Direct stimulus was given to a single nerve fibre. As shown in Fig. 5,

single medultated nerve fibre was excised, internode of Ni-Nj-Nj was dried in the

air, and Nj was stimulated directly after application of drug to the node.

U. N

Fig. 4 — Experimental nrrangement for

nerve trunk. N: Nerve trunk. D.N: De¬

sheathed part. S: Stimulus. R: Reeord-

Ing apparatus. a, h, c, d; Normal Ringer.

C: Test solutlon.

Fig. 5 — Experimental arrangement. N ij 3

Nodes exposed. N 13 : in 0.3% Coealne-Ringèr.

N^: In test .solutlon. N,-Nj; Slimulatlng

Circuit. Nj-Np Recordlng apparatus.

SciELO

10 11 12 13 14 15

598

PHARMACOLOGY OF THE COMPONENTS OF TOAD VENOM AND

ALLIED SUBSTANCES

Action-current was conducted from space between Nj and N, by means of a

low input resistance (IM ij ) D.C. amplifier combined with a cathode ray oscillo-

scope, and recorded. Moreover, node Ni and Nj were previously anesthetized with

0.3% cocaine-Ringer solution. The stimulus was a square pulse of duration of

0.5 m sec. provided by a stimulator in (i) and (ü).

Thus, the aspect of decreasing action-current at applied region, together with

the elapse of time, was observed.

Concentrations of the examined Solutions were 2x10~ 1x10 and 1 x 10

bufalin-Ringer’s solution and 0.05-1.0% procaine-Ringer’s solution, propylene glycol

alone was examined at the same time.With the applied concentration of procaine-

Ringer solution, action-current was found to decrease or disappear in nerve trunk

and single nerve fibre. After the materiais were brought to Ringer solution,

and thus fully recovered, they were applied with bufalin.

With Ix 10 ~ 5 concentration there was noted almost no effect but with

IXlO""* to 2x10“'* concentration there occurred reduction in action-current or

inhibition on the conduction of excitation.

However, almost the same effect appears in propylene glycol itself with the

same concentration as of the solution used as solvent, so that the action of bufalin

on nerve trunk and single medullated nerve fibre may be far weaker than its

surface anesthetic action on córnea, and strong local anesthetic action may be

presumed to be due to its specific effect on the receptor of sensory nerve ending.

Its mechanism is now under investigation.

As to the cardiotonic activity it has long heen l)elieved that the presence of

OH groups in Cs- and Cj 4 -positions was a requisite in cardenolides. In resi-

bufogenin there is no OH group in C^-position and there is a j 8 -epoxide («-

epoxide is inactive) ring between C 14 - and Cis-positions. In spite of this Chemical

structure, resihufogenin was found to have cardiotonic action nearly as strong

as that of ouabain (Table X) hesides marked activities in respiratory excitation

TABLE X — CONTRACTURE-INDUCING ACTIVITY OF THE VARIOUS CARDIO-

ACTIVE STEROIDS AND THEIR DERIVATIVES

(A)

Digitoxigenin

4 - 4 +

Bufalin

4 4 4

Oleandrigenin

4 4

Bufotalln

4 4 4

Digitoxin

4 4

Reslbufogenln

4 4

Dihydrodigitoxin

Cinobufagin

4 4 4

Ouabain

Digitalinum verum mono¬

4 4

Gamabufotalln

4 4

acetate

Strospeside

4 4

Lanato.slde C

4 4

17 ii-Dlgitoxlgentn

/I-Anhyrirodlgitoxlgenin

14fv, 1.5ti-Epoxy- ( p ) -anhydrodlgitoxlgenln

17 (v-Digitalinum verum monoacetate

/I-Anhydrogamabufotalin

Deacetylclnobufaglnic acld

(C)

+ 4 +

+ 4-

Tincture of dlgitalis purpurea leal (plgeon LD,'50 74,0 mg/kg) eftcctlve

in 10-»

cffective in doses of 1-3X10-*

“ 1-3 X 10-“

“ IX10-5

“ IX10-“

1 SciELO

Mem. Inst. Butantan

Slmp. Internac.

33(2):5S9-602, 1966

MASAHIRO OKADA

599

and elevation of Llood pressure tiotwilhstanding its very low toxicily. This facl

is considered to play an importanl role in furlher sliidy of the cardiolonic action

of these suhslances (Fig. 6, Tahles XI-XII).

Cinobufagin

Fig. 6

TABLE XI — LETHAL DO.SE OF BUFOGENINS AND AELIED COMPOUNDS IN THE CAT

(HATCHER-MAGNUS METHOD)

COMPOUND

Bociy vveight (g)

and sex- of

animai

Time of

infusinn ímin.)

M.L.D.

(mg/kg)

M.L.D.

reported by

ot.her workers

(mg/kg)

Bufalln

26S0

0.157

0.137

Gamabufotalin

2000

0.134

0.101

2400

0.137

Cinobufagin

2700

0.296

0.200

Resibufogenin

23.Õ0

110

3.2

Digitoxigenin

1900

0.411

0.450

Digitoxin

3450

130

0.365

0.325

Strophanthin

2400

0.118

0.116

SciELO

10 11 12 13 14 15

600

PIIARMACOLOGY OF THE COMPONENTS OF TOAD VENOM AND

ALLIED SUBSTANCES

TABEE XII — FROG SYSTOLIC STANDSTILL METHOD (hr.)

COMPOUND

ED„ (/ig/g)

95%

Confidence limit

(pígie)

Bufalln

1.01

0.95-1.08

Gamabufotalin

1..15

1.26-1.44

Resibufogenln

7.11

5.29-8.93

Strophanthin

0.28

0.24-0.33

Digitoxin

1.75

1.62-1.89

Digitoxigenin

fi.io

4.80-7.75

í)ii ihis mechanisni of respiralory excitatioii of resiliufogeniii lhe following

expcriinent was made oii urethaiiized ral)l)il. Even afu-r intravenous itijeclion

o[ 10 nig/kg of procaiiK-, wliich iidiihits llu' carolid sinus reflex, and causes

com|)lete disa|)pearance of res])iratory-stiinulating action l)y 0.05 tng/kg nicotine

tarlrale and O.d mg/kg loheline hvdrochioride, tlie resjiiratory stimiilaling action

of resihiifogenin could lie ohserved, and it was foiind lo I)e alinost eqiial lo

that induced hy single a<lniinistration of lhe satne suhstance. Moreover, respiralory

excitation liy nicotine disappeared coni[)letely due to removal of carolid sinus

nerve and nodular ganglion, luit resihufogenin revealed a reniarkahle resjiiralory

slinndaling effecl even after lhe saine oj)eralion. The res|)iralory excitation hy

resiliufogenin, heing different frotn lhe refleclive one hy Tiicotine and loheline

tlirough lhe carolid sinus, is supposed to residi from its direct action on lhe

res[)iralory center.

As for lhe niechanisin on lilood pressure, intravenous injection of 5-10 mg/kg

of hexamellionitini in urethanized raliliil inhihited lhe action of picrotoxin on

the elevation of lilood pressure, luit had ahnosl no effect on lhe saine action of

resiliufogenin. In sjiinal cat, llie action of |)icrotoxin disapjieared coinpletely,

luit resiliufogenin revealed a dislincl effect. F.ven after adininistralion of 10

ing/kg of Priscol and 5 ing/kg of chiorprornazine lhe aetion of resiliufogenin

on lhe elevation of lilood |iressure was noled lo he ahnost similar lo that hy

single administralion of the saine suhslancí

pover, resiliufogenin

causeil

consideralile elevation of lilood pressure even when it was previousiy lowered

liy intravenous injection of 5 nig/kg of NaNO^. This faci suggesls that resiliufo-

genin affects lilood pressure peripherally differing from jiicroloxin which aets

cenlrally only.

On lhe olher hand hufotenidine, lhe helain of hufolenine, of lhe water-solulile

fraction of Senso, was found to have an action which closely resemhles that of

nicotine thoiigh far more potenl lhan the latter.

Heferences

1. OKADA, M., and ISHIHARA, T.. A. R. ITSUU L<tb., 8, 66-70, 1957.

2. OKADA, M., and ISHIHARA, T., A. R. ITSUU Lah., 0, 91-95, 1958.

3. KOMATSU, M., A. R. ITSUU Lah., í), 95-99, 1958.

4. OHNO, S., and KOMATSU, M., A. R. ITSUU Lah., 8, 71, 1957.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

MASAHIRO OKADA

601

Simp. Internac.

33(2):589-602, 1966

5. HOPPE, J. O., ALEXANDER, E. B., and MILLER, L. C., J. Amer. pliarm. Ass.,

39, 147, 1950.

6. OKADA, M., SUGA, T., and MATSUMOTO, S., Asuin med. J., 5, 405-414, 1962.

A. R. ITSUU Lab., 11, 75-82, 1960.

Discussion

R. Kornalík: “Have you observed any olher local reactions such as vaso-

constriction or any other effects upon the cardiovascular system in locally anesthetic

doses of bufalin?”

M. ükada: “Bufalin and others have weak vasoconstrictory actions, bul they have

almost no other actions in the concentration as they are used as local anesthetics.”

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(2):603-614. 1966

K, R. ADAM and CU. WEISS

603

62. SOME ASPECTS OF THE PHAKMACOLOGY OF THE VENOMS

OF AEHICAN SCORPIONS

K. R. ADAM and CH. WEISS

Department of Biochemistry, Unwersity of Khartoum, Sudan and

Department of Plnisiologi/, University of Hamburg, Germany

Intiíoduction

The scope of this revievv is ronfinerl to work carried oiit, mainly in recent

years. on the venoms of African scor])ions, and iiiay thus include such species

as Androctonus uusíralis, Biithus occitaniis, Leiiirits quinqueslriatus, and Butiiolus

rninax, hut in lhe ])resenl eontext, iiidess olherwise stated, lhe terni “venom”

will refer to the venom of Leiuriis quinqueslriatus. The j)harmacological stiidies

nientioned have all heen carried on vertelirate lissues.

Most commonly, the venoms have heen ohiained hy eleclrical stimulation

of the lelson and have lieen promptly dried. It is generally agreed lhat, in the

dry State, lhe venoms of these species jtreserve lheir toxicities for months or

years (1, 2, 3). Most re|)orts have stated lliat the toxic factors are non- or poor-

ly-dialysahle, hnl recent sludies l)y Sliulov and his colleagues (4,5) claim tliat

lhere is a dialysahie faclor in fresli venom vvhich is toxic to mice. ft is, there-

fore, ])ossil)le that lhe common use of dried or freeze-drieci venom has ohscured

the exislence of another toxic suhstance and furtiier work sliould lie devoted lo

this |)OÍnt.

Il is proliahly triie lhat llie fraction of lyophilized venoms of the ahove

species, which is toxic to vertehrate lissues, consisls of one or two hasic pro-

teins. The evidence for this was originally hased on electro[)horelic. studies, dia-

lysis, and inaclivation l)y proleolytic enzymes (2, .3) and has heen siihslantialed

the work of Miraiula and liis colleagues al Marseilles on the natnre of lhe toxic

components of A. auslralis and B. occilanits venoms (6, 7, 8). In our experience,

it would appear that the toxic constilnents in L. quinqueslriatus venom hear a

strong resemlilance to tliose sludied hy Miranda et al., hut imfortunately we

have noi lieen so successfiil in ohtaining clear separations of lhe two hasic

jiroteins in L. quinqueslriatus venom thus far. The use of eleclrophorelic ehiales

indicated that nenromiiscidar was associated mainly wilh lhe slower-moving of

lhe two protein hands, which is interesting as Miranda and Lissitzky (Q) foimd

that the two hasic jiroteins in A. australis venom shared almost equally lhe

toxicity to mice. In our case, insiifficient material was availahle in the eluates

for toxicity testing; il is clearly desirahle that pure jirotein fractions should

hecome availahle and enahie us lo he more precise in allocaling pharmacological

effects to individual comjionents.

2 3

5 6

11 12 13 14 15

«04 SOMK ASPECTS OF THE PIIARMACOLOGY OF THE VENOMS OF

AFRICAN SCOHPIONS

Til lhe aelions lo he discussed nexi, lhe fraelion of lyophilized venoni jire-

eipilaled hy 80% (v/v) aeelone al —15"C vvas iised, and conlained holh of lhe

hasic proleins seen on eleelrophoresis.

Neuiíomuscui.ah actions: Considerahie inlerepl oenires on llie neuroloxie,

aclivily of venoms. One of lhe niost ohvious effccls of pcorjiion venoni is lo

provoke Iwilcliing and filirillalion of skelelal nuisele, and il is naliiral lo iiupiire

as lo lhe sile or siles al whieh ihis effeei lakes jilace. Hoiissay (10) shovved

lhal lhe ap|)liealion of venoni lo a inolor nerve eould cause Iwilcliing of llie

innervaled niiiscle, hiil he eonsidered lhal nerve was less sensitive llian rnuscle

heeaiise he had lo appiy nineii higfier eoneeniralions of venom lo nerve in order

lo ohlain an effecl. However, if one de-shealhs jiari of llie nerve, lhen lhal pari

heeonies al leasl as sensilive lo venom as is lhe imiscle, indiealing lhal lhe

e|)inenriiiin fornis a eonsideralile harrier lo lhe venom moleeiiles (11 ).

To analyse llie effeei on nerve more elosely. il is advanlageons lo use

isolaled nerve fihres, jierfusing a Hanvier ncile vvilh venom solulion and noling

lhe effect on lhe eleclrical aclivily. This has been done recenlly (12). With low

Imsec

^msec 4mseq

pig. 1 ^ Actioíi potenlials of a sensory fibre in normal Ringer solution then 1, 2.5, and 3 minutes

after addition of venom (5X10-'^). Node stimulated once per second.

11 12 13 14 15 16 17

Mem. Inst. Butantan

Simp. Internac.

33(2) :603-614, 1966

K. R. ADAM and CH. WEISS

605

coiiceiilrations of veiioin (5X10~' — 5X10“®) íhe most striking effect is tlic

prolongation of action jjotentials (Fig. 1). Tliese iiicreased iii duration froin

the tiormal one msec. or so ii[) to a second or more, and occasionally lasted as

long as ten seconds. Since, inilially, the rising phase of the action potential

was unaltered, it scemed prohahie that the fast increase in Na permeahility was

nnaffected. The falling phase of the normal action potential is dne to a comhined

change of Na and K jjermeahilities, hut as no effect on delayed rectification

was found it seemed uidikely that the venom affected K permeahility. Therefore

it was considered that the ohserved |)rolongation of the action potential was due

mainly to delay in the inactivation of Na permeahility. This view was su[)ported

hy voltage clamp ex])eriments.

With higher concentrations of venom I > 5 X 10“') and longer exposure,

depolarization and spontaneous action potentials occiirred (Fig. 2). Perfusion of

O-*

Q2 sec

Fig. 2 — Spontaneous activity of a motor fibre supertused with

Rlnger containing venom (5X10-’). Photograph taken after 18

minutes exposure to venom.

the node with low-soditim Solutions aholished the effect of venom, and increasing

the sodiiim to normal Hinger leveis restored the effect again. (lenerally, the action

of venom on the resting potential was strongly dependent on the concentrations

of sodiuin, the depolarization amplitude rising with increasing sodinni concentra-

tion at a rate snggesting that the resting memhrane was rendered mnch more

jicrmeahle to Na hy the venom (Fig. 3).

The venom-indnced dejiolarizations and the spontaneous firing of impulses

in the nerve fihres of a nerve-muscle jireparation wili, of cour.se, initiate Iwilch-

ing of the muscle. Is there evidence for a direct action on miiscle as well? A

contracture of isolated skeletal muscle can he ohtained with venom in the pre-

sence of sufficient tuhocurarine to antagonize quite large qnantities of acetyl-

choline (2). In Fig. 4, a rat diaphragm is heing stimulated directiy (suh-

maximally) in the |)resencc of tuhocurarine. At lhe arrow 5 pg venom/ml was

added, resulting in a contracture and increased amplitude of lhe twitches.

Gradually. the effect diminishes as time goes on. and over the nexi 15-30 minutes

SciELO

10 11 12 13 14 15

G06

SOME ASPECTS OF THE PHARMACOEOGY OF THE VENOMS OF

AFRICAN SCORPIONS

Flg. 3 — Relatlonship tietvveen Na concentration of the médium and membrane potential

before and after exposure to venom (l()-“). Ordlnate, change of membrane potential In

mV ídepolarizatlon positive). V = 0 is the normal resting potential before applicatlon

of venom. Mcan values and standard errors of the means of 14 flbres.

the twitch amplitiule decreases until lhe muscle hecoines almost iiiexcitahle. With

higher concentrations of venom, violent spontaneoiis twitrlies are .«uperimposed

initially.

Il seems likely, therefore, lhal there is a direet effert on skelelal miiscle

and, inkeeiiing witli lliis, n-eords resting memlirane jiotentials from Sartorius fihres

show a slovv depolarization imder the inflnenee of venom tinlil an action ])Otential

is elicited (Adam and Weiss, un])ul)lished oliservations). I’ossihly ihis, too, may

l)e due to an effect on Na eonductanee throngh lhe memltrane hiit, as yet, there

is no experimental evidence. Thiis lhe effeei olitained witii venom on an isolated

or m situ nerve-musele prejtaration is [trohahiy dne to an aetion on lioth muscle

and nerve, though it seems likely lhat lhe latler is affected more readily.

CoMPAitisoN wiTH VEiiATHiNE: Since lhe early vvork of Hoiissay (10), the

effects of several venoms on nerve and skeletal muscle have heen noted to re-

semhle those of veralrine, or one of ils constitiienl alkaloids, veratridine. Fig. 5

shows the effect of dh /xg veralrine/ml on lhe direclly, std)maximally, slimulated

rat diaphragm, inducing a coniracture and incn'ased Ivvitch am|)littide very

similar to that shown in Fig. 4. Various differences, e.g. lhe smaller effect of

SciELO

10 11 12 13 14 15

2 3 4 5 6 SciELO ;lo 11 12 13 14 15

fiflQ SOME ASPECTS OF THE PHARMACOLOGY OF THE VENOMS OF

AFRICAN SCORPIONS

veratrine on muscle relaxalioii afler a twitcli, l)elwcen vcnom and veratriíie can

1)0 ficmonstrated (2,12) hiit oii tlio whole thoro is a strikiii" similarity. Vora-

Irine has keen showii to liavo direot depolariziiiji effects on skoletal muscle and

on nerve fihres, and tliese effoots have heen interpreted as duo lo increases in

mcmhrane permeahility to Na (Id, 14, 15, 16). Tluis llie observed similarity in the

actions of venom and of voralrine may havo its hasis in a oonirnon meclianism.

Again, the effects of holh venom and veratrine resemhle tliose induced hy low-

calcium Solutions and hy Ca-chelating agents, and can he al least partially an-

lagonized l)y increasing lhe calcinm concentration of the médium (2, 16, 12).

Such effects have heen demonstralcd with various venoms for many years(17).

Displacement of (ia from memhranes was suggested as lhe meclianism of vera¬

trine aclion hy (lordon and Welsh (18) and it seems possihle that venoms could

displace Ca in a similar way. One might sjieculate that the jiositively-charged

venom molecules disfilay an affinity for the memhrane acidic jihospholipids in

])reference to (ia or lo Na, ihus interferinf

Na-c

System, hut it is

prohahly not profilahle lo pursiie further any disciission of (ia-venom antagonism

al the memhrane at presenl. However, it should he rememhered that various

memhrane “slahilizers”, such as adrenaline or local anaesthetics, can he shown

to hlock or reverse phases of venom action, and it has heen poslulated that their

stahilizing effects may he mcdiated via lhe memhrane Ca (19, 20, 21).

Pain production by VENOM: This is anolher nolahle effect of scorpion

venom, and may well he related lo lhe actions on nerve fihres discussed previ-

ously. In some cases it is possihle that lhe presence of large qnantilies of

serotonin may conlrihute to the pain of a sting(22, 23) hut [irohahly the major

jiain-producing suhslance is protein. Using lhe Armslrong-Keele cantharidin

hlister lechnicpie (24), \ve found that venom could still cause jiain when its

serotonin contenl was helovv ihreshold; that jiain conld he provoked hy lhe

ajiplication of eluates from a protein hand after eleclrophoresis; and that it

was usually |)ossihle to detect a cpialitative difference hetween lhe pain produced

hy pure serotonin and that due lo venom (Adam, Smith & Weiss, unpuhlished

ohservations). It .seems possihle that lhe same suhstanees which provoke neuro-

muscular activity are resiionsihle for most of lhe jiain of a sting, and that this

may he due to a direct stimulalion of sensory nerve fihres hy the meclianism

already discus.sed. It is interesting to note that holh veratrine and citrale can

induce pain al lhe hlister hase(25).

PiiosPHOi.iPASE A ACTtviTY: Althougli /.. quinquestriaíiis venom has heen

shown to have some haemolytic activity (26), it does not appear to contain jilios-

pholipase A. Incuhation of venom with pnrified |ihospholi|)id suhsirales, human

plasma, rat-hrain or rat-muscle homogenates resulls in a negligihie increa.se in

free fatty acids, and a lack of degradation of di-acylphospholijiids ean he con-

firmed chromatographieally (27). One wonders if lhere is a direct lylic faclor

and whelher this might he the neuroloxic protein interfering with calion lrans|)oiT

across lhe erylhrocyle memhrane. However. lhere is some evidence that the

toxicities of various venoms do not parallel their haemolytic aclivities (28).

To SUMMAHIZE: It seems likely that the main aclion of L. quínquestriatus

venom is to interfere with the meclianism switching on and off lhe Na ])ermeahilily

of cell memhranes. While this is a suilahle working hypolhesis for cnrrenl

jiharmacological studic‘s, it is evident that lhe immediate recpiirement for further

advances is lhe provision of pure venom fraclions in adequate amounts.

SciELO

10 11 12 13 14 15

SciELO ^0

610

SOME ASPECTS OF THE PHARMACOLOGY OF THE VENOMS OF

AFRICAN SCORPIONS

Discussion

O. Vital Brazil (Department of Pharmacology, University of Campinas, Campi¬

nas, São Paulo: “Although it was known since the investigations of Maurano (1915),

Vital Brazil (1918) and Houssay (1919) that the venom from the South American

scorpions belonging to the genus Tityus was immunologically different from that

of the African scorpions, their pharmacological actions were believed to be almost

identical. However, by comparing the results obtained by Prof. Adam employing

the venom of Leiuriis quinquestriatus and that of other Buthinae, with our own

results using the venom of Tityus serrulatus, we can now appreciate that there are

also important pharmacological differences between these venoms. In fact, the

venom of T. serrulatus never produced, in our experiments, the contracture of

the isolated rat diaphragm as described by Prof. Adam for the venoms of the

African Buthinae.

FIg. 1 — Isolated phrenlc nerve-dlaphragm preparatlon of the rat. The venom

(10 mcg/ml) of Tityus serrulatus evoked contractlon.s of the diaphragm whlch

were abollshed by d-tubocurarlne (10 mcg/ml).

Instead, it produced intense twitchings of the muscle which were promptly

abolished by d-tubocurarine (Fig. 1). Therefore, the venom of Tityus serrulatus

seems to be devoid of the direct muscular action exhibited by the venoms of the

African Buthinae.

The twitchings as well as the increase caused by T. serrulatus venom in the

amplitude of the response evoked by isolated supramaximal shocks applied to the

nerve (Fig. 2), can be explained by acetylcholine release from the motor nerve

terminais. Such a release of acetylcholine caused by the venom has recently been

demonstrated in my laboratory at the University of Campinas. The isolated

inervated, and sometimes the isolated chronically denervated hemi-diaphragm, were

SciELO

K. R. ADAM anti CH. WEISS

Mem. Inst. Butantan

Simp. Internac.

33(2):6n3-614, 1966

611

Fig. 2 — Isolated phrenic nerve-diaphragm preparation ot the rat. The venom

of T. ser7'vlatus caused a great increase in the responses produced by nerve

stimulation wlth supramaximal shocks delivered at a rate of 6 per minute.

used in these experiments. They were suspended in Tyrode solution containing 0.2

per cent of glucose and 5 x oí neostigmine methylsulphate. The bath volume

was 5 ml and its temperature, 37°C. The preparations were oxygenated by bub-

bling a m.ixture of 95 per cent O; and 5 per cent COj. The fluids after bathing

the diaphragm for 20 minutes were removed and immediately assayed for acetyl-

choline by its depressor effect on the arterial blood pressure of anaesthetized small

cats injected with hexamethonium and ephedrine. The results can be summarized

as follows:

1. A very small spontaneous release of acetylcholine sometimes occurred, lhe

acetylcholine content of the fluid being always less than 0.4 ng per 0.2 ml. There-

fore, the spontaneous release of acetylcholine by the hemi-diaphragm was always

less than 10 ng.

2. The venom promoted the release of acetylcholine from the inervated hemi-

diaphragm. The acetylcholine content of the venom containing fluids which bathed

the hemi-diaphragm for 20 minutes was seldom less than 2 ng per 0.2 ml; in most

instances it varied from 2 to 4 ng per 0.2 ml (Fig. 3). Therefore, the acetylcholine

chlorine released by the venom from the hemi-diaphragms could be estimated to

be from 37.5 to 100 ng.

3. The v'nom did not release acetylcholine from chronically denervated hemi-

diaphragms. *

4. Curarization ot the hemi-diaphragms with d-tubocurarine did not seem to

reduce the release of acetylcholine by the venom.

5. The venom did not released acetylcholine when the fluid bathing the hemi-

diaphragm contained procaine.

G. The acetylcholine released by venom seemed to be calcium dependent. When

Ihis ion was suppressed from the Tyrode solution no acetylcholine release could be

demonstrated. When it was increased above the usual concentration in the Tyrode

solution, an increase in the acetylcholine output was verified.

The mechanism of acetylcholine release by lhe venom is unknown. Depolar-

ization of the nerve fibres by the venom as Prof. Adam has verified, would explain it.”'

1 SciELO

612

SOME ASPECTS OF THE PHARMACOLOGY OF THE VENOMS OF

AFRICAN SCORPIONS

Flg. 3 — Arterial blooti pressure oí the cat. 1. and 2. — 0.2 ml of Tyrode solution

vvlth neostigmine which bathed the diaphragm for 20 minutes (spontaneous release

of acetylchollne); 3. — 0.2 ml of Tyrode solution vvlth neostigmine contalning

20 mcg/ml of T. serrulatus venom; 4. and 5. — 0.2 ml of Tyrode solution vvlth

neostigmine contalning 20 mcg/ml of T. serrulatus venom after bathing the

diaphragm for 20 minutes (acetylchollne released by the venom); 6. — 4 ng

of acetylchollne chlorlne; 7. — the same as 4. and 5.; 8. — spontaneous release

of acetylchollne after Tyrode solution vvlth neostigmine contalning the venom was

removed and the diaphragm vvashed; 9. — the same 4. and 5; 10. and 11. —

acetylchollne (4 ng) and acetylchollne relea.sed by the venom (0.2 ml) after the

injection of 2 mg/kg of sulphate of atropine. Cat anaesthetlzed by pentobarbital

(30 mg/kg, l.v.) and Injected vvlth hexamethonlum bromide and ephedrine sulphate.

Heferences

ÍIOUSSAY, B. A. — Action physiologique du venin des scorpions {Buthus quinquestrtatus

et Tityus hahiensis). J. Physiol. Pathol. Generale, 18, 305-317, 1919.

MAURANO. II. — Envenenamento escorpiônico e seu tratamento. Tese, 1915.

RRAZIL, V. — Sôro anti-escorpiônico. Mem. Inst. Putantan, 1, 47-52, 1918.

Disclission

A. Slmlov: “1. Whether are there any differences in results obtained through

use of fresh and lyophilised venom? 2. Whether any experiments were carried

out with scorpion venom such as from Scorpio nmurus occurring in Sudan?”

K. R. Adanis: “We have no evidence regarding the first question, but have

this point very much in mind. We have not had the opportunity of investigating

the venom of Scorpio maurus."

P. Efruti: “I was deeply interesled in the observations presented by Prof.

Adam. My experience concerns, unfortunately, human beings stung by Leiurus

quinquestriatus. Besides of pains, observed predominantly in adults, we have observed

SciELO

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Mem. Inst. Butantan

E. C. DEE POZO

615

Slmp. Internac.

33(2):615-R26, 1966

68. PHAKMACOLOGY OF THE VENOMS OF MEXICAN CENTRUROIDES

E. C. DEL POZO

Instituto de Salubridad y Enfermedades Tropicales, México

Hecent reports from electro])hysio]ogical aiui hiorhemical work seeni to

show qualilative (liffereiices iii scor])ion venoins. Old descriptions of l)Oth diiiical

and experimental poisoning are similar and only qnantitative discrepaneies are

found in ol)servations eorresj)onding to a wide variety of speeies from distant

parts of the world. Differenees in symptoms or signs of intoxieation seemed to

be diie to variable amounts of venom or different content of aetive principies (1).

The general actions; muscular Iwitchings, ptyalism, progressive respiratory

irregularities reaeliing respiratory paralysis in some cases, pilo-ereetion, mydriasis,

hlood pressure rise, signs of local pain, signs of laringeal constrielion, are to l)e

found in aecounts of seorpion poisoning from any part of the world. Howevcr,

the mechanism invoked to exj)lain such actions varied very much.

At ])resent, fundamental work is heing done wilh seorpion venoms in many

lahoratories and agreement seems to he reached ahout the effects on nervous

System and neuromuscular junctions. Neverlheless new discrepaneies could be

found in re[)orts from electrophysiological studies. Different teehniques are be-

ing nsed and one shonld kee|) in mind lhal discre|)ancies are early and stimulat-

ing aspecls of original research.

In s])ite of the faet thal new |)aths of research in j)harmaeology of seorpion

venoms may lead to the discovery of a common mechanism of action, it is es-

sential to mention always the seorpion speeies nsed in each work.

The descriplion that follows is limited to |)harmacological j)ro|)erties of venoni

from Mexiean C c n I r ii r o i d c s, and it is based on works from my' laboratory.

Onr comi)arative studies of the actions of venoms of C. siijjiisus siijjusus Pocock,

C. noxiiis Hoffmann, C. Umpidus tecomanus Hoffmann and C. Umpidiis llmpidiis

Karseh, grant a common consideralion. Only qnantitative differenees have heen

found. l.arger doses of lhe less aclive venoms re])rodnce the effects of smaller

doses of the more active ones.

Muscui.ah KKKECTS — One of the niosi immediate effects of inlravenoiis

injections of seorpion venom in cais, dogs, mice and rats is lhe aj)pearance of

generalized muscular twitchings and fascicniar coniractions. This activity ori-

ginates in lhe spinal cord. It persisls afler section of lhe brain slem, deaf-

ferenlation or Iransection of the spinal cord. The fascicniar conlraclions helow

lhe levei of lhe section are more marked than above, but they disa|)pear complctely

when lhe s|)inal cord is destroyed or lhe motor nerves are ciit (Fig. 1).

2 3

5 6

11 12 13 14 15

616

PHARMACOLOGY OF THE VENOMS OF MEXICAN CENTRUR0IDE8

WBHi

Fifí. 1 — A: Muscular efrects of an intravenous Injection of scorplon venom. B; No

effecl is obtained when an equal amount of venom is injected after the muscular nerve

was cut. (This and Figures 3 to 7 taken from dei Pozo and Anguiano (2)).

Muscular twilchings and filirilialions are aiso ohlained if lhe venom is aj)-

plied directly lo one mnscle or injeeted intrarlerially even with lhe corres])onding

nerve cut. Tliis muscular aclivily does not oecur when lime has heen allowed

for Wallerian degeneralion after denervation. Local ajipliealion of venom lo a

muscular nerve trimk does noi |iroduee any effects on lhe museles 12).

The local action of the venom lakes jilace on the muscidar end-plate region.

This inference was eonfirmed when eleclrical records were taken from muscle

and nerve. Kepelilive potentials were foimd following single shoeks a[)plied to

lhe nerve cenlrally cut. This re[)etitive activity is conducled antidromically in

lhe nerve and disappears when this nerve is disconnecled from lhe muscle. When

records were taken from anterior and dorsal rools. the repelitive aclivily was seen

in the former and not in the lalter (4) (Fig. 2).

Big and longdasting conlradions are ohlained when single shoeks are aj)-

plied lo museles under lhe adiou of scorpion venom. When suceessive resjionses

are provoked al short intervals, the conlradions are |)rogressively smaller in

amplitude and dnralion. Eledrical recordings show at the same lime a gradual

ílirninution of the repetitive activity I, Figs. d and 4).

SciELO

10 11 12 13 14 15

E. C. DEL POZO

lOmseg.

Flg. 2 — Eléctrica! recordlng from gastrocnemius muscle of the

cat. Five superlmposecl responses to shocks applied to the nerve

in each segment. A, betore; B to D, after successlve dosls of

scorpion venom. (This and flg. 8 taken from dei Pozo, Salas

and Pacheco, in press).

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Mem. Inst. Butantan

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According lo this evidence th(' grcat and long niiisctilar resjtonses to single

shocks correspond to short letanic conlratlions consecnlive lo lhe repetitive aclivily

jirovoked at lhe end-])Iate regions of jioisoned mnscles hy lhe arrival of single

impulses.

These effects on time and duralion of muscular contraction and the influencc

of rejietilive stimulalion have heen extensively analyzed at differenl frequencies

of stimulalion. The grajths of tension development of the tetanic contractions

shows mechanograms resemhling those ohtained when higher frequencies of

stimulalion are applied to normal mnscles 12) (Fig. 5).

Fig. 3 — Effect of scorpion venom on the amplitude and duration of muscular res¬

ponses. Maxlmal contractlons ot gastrocnemius of the cat, before (A) and atter the

injeetlon of the venom (B). In successive contractions, the amplitude and duration

are progressiveiy reduced (C). (D), 20 minutes later.

Fig. 4 .— The length of the interval betvveen stimulations change the type ot responses

In a muscle under the action ot scorpion venom. A, 3 minutes interval betvveen

shocks. B, one second intervals.

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10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(2):615-626, 1966

E. C. DEL POZO

619

Fig. 5 — Ettect of scorpion venom on muscular responses to high frequency

stimulation. Pairs of responses before and after injectlon of the venom to

stimulation to the following frequencies per second: A and D, 5; B and E,

13; C and F, 17; G and L, 25; H and M, 30; I and N, 60; J and O, 120;

and K and P, 200. Gastrocnemius of the cat.

The letaniziiig niinimal frequeiicy of lhe stiinuliis for one particular muscle

is snialler tvheti poisotied than for a normal one, hiil after the initial complete

letanus the individual responses lo each shock ajipear on the graph. This pecidiar

develo])ment of lhe contraction is the oiijiosile of the normal graph ohtained at

the minimal telanizing frequency, i.e., initial individual response lhal jirogressively

i SciELO

620

PIIARMACOLOGY OF THE VENOMS OF MEXICAN CENTRUROWES

fuse iii a complete lelaiuis. The long lasting initial coiitractions of the jioisoned

miiscle and llie |)rogressive shorleniiig of lhe siiccessive res|)onses aceoimt for ihis

phenomenoii (Fig. 6).

Flg. 6 — Inversion of the stages of the responses to stimulation at a minimal tetaniz-

ing frequcncy In a musrle under scorpion venom. A, 3 per second; B, 5 per seeond.

When neiiromiiscular transmission has lieen liloeked l)y curare, scor|)ion venom

given hy intravenons injecTion decnrarizes (2). Ilowever, large doses of lhe

venom prodiice a complete hlock of neiiromnscular transmission. This hlock is

not snjjpressed hy either |)rostigmÍ7i or curare. The miiscidar res|)onses to direct

electrical or acetylcholine stimidalion are preserved (F igs. 7 and 8).

Scorpion venom displays anli-cholinesterase aclivily as shown hy onr experi-

mcnls tesling the fali of hlood pressure [)roduced l)y fixed arnonnts of aeelylcholine

after hydrolysis wilh hlood senim with and withoul venom.

This |)ropeiTy was also assayed com|)aratively with eserine on frog ahdominal

mnscles (5). The anticholinesterase activity of differenl venoms was fonnd lo he

plighl and withoul correlation wilh loxicity or lhe mnscle adivating jirojterlies.

A stndy of lhe effects of venom (C. naxiiis Hoffmann ) on cholineslerases

was examined on lhe isolated inteslines of guinea-i)igs and rahhils. Il was

fonnd that the venom oídy inhiliit cholineslerases holh from hnman sernm (pseudo)

and from the caudale nucleus of rahhits (specific) when present in very high

concentralions (6).

ín hrief, lhe muscular effects of scorpion |)OÍsoning are prodnced hy two

actions of the venom: one, central, located on lhe s()inal cord, and lhe other,

peripheral, on lhe neuromuseidar jnnclions. Both actions take jtlace at regions

SciELO

Fig. 7 — Decurarization by scorpion venom (B) anci by prostigmine (A). The first

signal in both segments correspond to the injection o£ curare, The second In B to

a second injection of curare (in A to atropine). The third signal in B maiks the

time ot the Intravenous Injection of .scorpion venom. (In A, prostigmine).

2mvT

10 mse g

Fig. 8 — Slmultaneous recording from sciatlc nerve (above) and gastrocne-

mius muscle (below) to stimulations applied to the nerve. A, before, B, 1

minute after injection of scorpion venom, C, 5 minutes later. D, after curare.

i SciELO

PHARMACOLOGY OF THE VENOMS OF MEXICAN CENTRUROIDES

of excilaMe ineinhranes, and il is rfasonaMe lo assume lhal lhe venom affects

lhe [)ermeal)ilily of lliese niemhranes antl chaiige lhe ionic eqiiilihrium lielween

holh sicles of lhe same.

Macroeleetrode reeordings from lhe end-jilale regions of lhe sarloriíis musele

of a cal showed a progressivo iiiversion of lhe loeal poleiilial imder lhe aelion

of seorpion veiiorn (7). This is a direet evidenee of ehaiiges iti ioiiie dislrihiilion

produced hy lhe seorpion loxiiis.

Hesimkatory effects — The accidenlal or experimenlal |)oisoniiig wiüi

seori)ion venom ])rodnee marked irregnlarily in differenl animais holh in frequency

and amplilnde of lhe respiralory movemenls which in eases of slrong inloxiealion

reaches a Cheyne-Slokes lype rhylhm and finally paralysis. The eenlral origin

of lliis paralysis was altesled hy keeping lhe animais (eals) alive afler lhe para¬

lysis hy means of arlificial respiralion hroiight ahonl hy rhylhmie slimnlalion

of holh |)hrenic nerves. In lhese exj)eriments, lo onr knowledge, was for lhe

firsl lime appiied what aflerwards was inlrodneed under lhe name of e/ec/ro-

phreiiic respiralion. The paper ])nhlished in 1945 refnled lhe hy])olhesis of

enrarizalion as the origin of lhe respiralory j)aralysis (8).

Bronehiolar ohstrnclions hy ahnndanl secrelions, laryngeal and hronchiolar

mnseles eonlractions conlrihnte to lhe respiralory distress hut are not the canse

of the asphixia. Same may he said of the fascicnlar eonlractions lhal appear

in respiralory as well as in all skeletal mnseles. Periodic rhylhms of respiralion

from central origin can he perceived among lhe hackground of irregular jerks

uj) to the time of a{)pearance of Cheyne-Slokes ty|)e rhylhms.

An addilional proof of lhe central aelion is lhal a minute amonnt of the

venom injected into the cisterna magna prodnces immediale respiralory paralysis.

Cardiovascui.ar effects — The inlravenous injection of seorpion venom

prodnce in cais and other mammalians an initial increase of hlood pressnre and

simnilaneons hradicardia. The j)ressnre then comes down slowly to normal values.

During lhe periods of cyclic, respiralion, increases in hlood jiressiire aceompany

the periods of apnea and hlood pressnre falis during hyj)ervenlilation. Finally.

if the dose of venom was high the ])re.ssnre comes down gradually lo zero (9).

The initial increase in hlood pressnre long time ascrihed lo a j)eripheral

aclioti of the venom is dne lo effets on lhe S|)inal vaso-conslrictor |)resynaplic

nenrons and lo the liheration of ej)inephrine aiso ihrongh slimnlalion of lhe

adrenal activating |)re-gangliotdc nenrons of lhe spinal cord.

Spinal animais afler total deslruction of the hrain, give equal or higher

hlood pressnre increases wilh lhe venom, hnt lhe effecl does not aj)pear when

the spinal cord is deslroyed even when lhe hlood ])ressnre levei is [)revionsly

raised lo normal valnes. Denervated ])innae of white rahhits and cais showed

vaso-constriction dne to liheraled epinephrine; it did not ocenr when lhe sym-

))athetic fihers lo lhe glands were cnl or the s|)inal cord was deslroyed (Figs.

9 and 10).

Hradicardia corresponded also lo a central aelion. It disa|)peared when lhe

medula was deslroyed or when the vagi were cnl. In lhese cases lhe heart was

slightly acceleraled under lhe infinence of scorjiion venom. This effecl was also

of Central origin hecanse il did not ocenr when the heart was denervated.

The vascular reflexes produced hy lhe slimnlalion of the central end of lhe

sciatic nerves peripherally cnl were not modifieil hy scor|)ion venom on enrarized

2 3

5 6

10 11 12 13 14 15

E. C. DEL POZO

623

Mem. Inst. Butantan

Slmp. Internac.

33(2); 615-626, 1966

160-

—

140-

120-

_ h

80-

60-

40-

20-

■■ \)>

'■t-J-.-A

Fig. 9 — Increascs of blooci pressure produced by a first (A) and a second (B) intravenous Injection oí

scorplon venom in a spinal cat by destructlon of the brain. (This and the following figure are taken from

dei Pozo, Anguiano and Gonzãiez (9)).

Fig. 10 — Scorplon vrnoin dce.s not produce increa.se of blood pressure when brain and spinal chord are

destroyed. A, first in;ection of venom; B, injection of an equal volume of saiine; C, injection of 10 mcg

of epincphrine; D, 5 mcg of epinephrine.

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11 12 13 14 15 16 17

624

PHARMACOLOGY OF THE VENOMS OF MEXICAN CENTRUROIDES

animais. This dissociation hetween lhe tonic vasopressor dircct effects and lhe

reflex responses is an old physiological finding reporled hy Sherrington in

1906 (10), Porler in 1910(11) and Langley in 1924(12).

Otiier effects — Other effecls of lhe scorj)ion venom siich as plyalism,

mydriasis, j)iloereclion are also lo he cxplained l)y cenlral aciion on lhe syinpathelie

])re-ganglionic neurons heeaiise ihose effecls are greally rednced or disappear

complelely hy denervation.

Wc have seen thal lhe rich pharmacological aclions of scorpion venoms seem

lo derive from a fundamental common mechanism present at different analomical

regions. We have discovered already lhat ihis venom acls on lhe ly])ical })laces

so-called centcrs and at neuromnscular junctions. We know lhat it works hy

increasing lhe permeahilily of excilahle memhranes as shown hy lhe inversion of

end-plale potential (7). Others investigators have reached similar results with

finer tcchniqiies (13).

Iti addition, valuahle hlochemical work is heing done in several lahoratories

for lhe isolation of scorpion toxiris; we may he hopeful lhat now we are on lhe

road to a hasic underslanding of lhe mechanism of aciion of these peculiar

venoms.

This first International Symposium has brought together the people working

on the same problems in all parts of the earth. It will be an historical event.

The importance of this meeting is increased because we meet at this famous Bu-

tantan Institute, the leader of the venomological studies. We pay tribute to

the illustrious Vital Brazil, inspired founder of the Institute and to all the group

of workers that have followed his steps in this fascinating path of research.

Kefekences

1. DEL POZO, E. C., in E. E. BUCKLEY, and N. PORGES (Editors), Venoms,

Amer. Ass. Advanc. Sei., Washington, 1956.

2. DEL POZO, E. C., and ANGUIANO, G., Rev. Inst. Saliibr. Enferm. trop. (Mex.),

8, 231-263, 1947.

3. DEL POZO, E. C., Gac. méd. (Mex.), 78, 387-397, 1948.

4. DEL POZO, E. C., SALAS, M., and PACHECO, P., Extr. Comunic. VI Congr.

Nac. Cienc. Fisiol., Mexlco, 1963, pp. 109-110.

5. DEL POZO, E. C., and DERBEZ, J., Rev. Inst. Salubr. Enferm. trop. (Mex.),

10 , 203-213, 1949.

6. DEL POZO, E. C., Brit. J. Pharmacol., 3, 219-222, 1948.

7. DEL POZO, E. C., SALAS, M., and PACHECO, P., Extr. Comunic. IX Congr.

Nac. Cien. Fisiol., México, 1966, p. 141.

8. DEL POZO, E. C., GONZÁLEZ, Q. J., and MÊNDEZ, T., Rev. Inst. Salubr.

Enferm. trop. (Mex.), e, 77-84, 1945.

9. DEL POZO. E. C., ANGUIANO, G., and GONZÁLEZ, Q. J., Rev. Inst. Salubr.

Enferm. trop. (Mex.), 5, 227-240, 1944.

10. SHERRINGTON, C. S., The Integrative Action of the Nervous System, London,

1906, p. 242.

SciELO

Mem. Inst. Butantan

E. C. DEL POZO

625

Simp. Internac.

S3(2):615-626, 1966

11. PORTER, W. T., Amer. J. PInjsiol., 36, 418-422, 1910.

12. LANGLEY, J. N., J. Physiol., 59, 231-258, 1924.

13. ADAM, K. R., SCHMIDT, H., STÂMPFLI, R., and WEISS, C., Brit. J. Phar-

macoL, 26, 666-677, 1966.

Discussiox

H. Edery: “AU these features you described on the neuromuscular transmission

such as twitchings, and fasciculations are also produced by anticholinesterase organo-

phosphorous compounds. I learnt from your paper that you tested the venom

for anticholinesterase activity and it has a high concentration. I would like to

ask if you would accept the idea that at least part of the effect on neuromuscular

transmission were due to inhibition of cholinesterase at the end-plate levei. When

you inject a substance intraarterially, you do get high levei concentrations. And

thinking in this way of inhibition of cholinesterase, I would be keen to know the

effects of the so-called reactivations of cholinesterase on the block produced by

the venom.”

E. dei Poso: “We did find anticholinesterase activity and we thought that this

could be the explanation for the muscular effects of scorpion venom. However, when

tested that possibility we found that the anticholinesterase effect was slight and did

not keep relation or correlation with the muscle activating properties. For this

last testing we compared venoms from different scorpion species with regards to

anticholinesterase activity, to.xicity and muscle activating properties. One particular

venom could have less anticholinesterase and more muscle activating properties

than other.”

P. Efrati: “Also my experience concerns mainly stings by Leiurus quinques-

irxatus, I think, at least two clinicai signs, observed in general envenomation, could

support the observation of E. C. dei Pozo about the influence of scorpion venom

on the spinal cord: urinary retention and priapism, observed very often indeed.”

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

3S(2):627-638, 1966

S. SCUENBERG and F. A. PEREIRA LIMA

627

61. I’HAHMAC()L()(;Y OF THE FOLYFEFTIDES FI{()M THE VENOM

OF THE SPIDEK PHONELirRIA FERA

S. SCHENBERG and F. A. PEREIRA LIMA

Seção de Fisiologia, Instituto Butantan, São Paulo, Brasil

Tliis jjaper resumes pharniaeo-hioehemical invesligations made witli lhe venom

of tlie siiider Rhoneutria fera Perly, 183H iCteini.s jcrii,s, and C/em/.s nigrivenicr

Keyserling, 18911, a speeies of lhe AKANEIDA order, LABIDOGNATA sidiorder,

CTENIDAE Family and Cteninae suhfamily.

Very dangerous hy its aggressivity and venom toxieity, this speeies is

responsihie for lhe higher lethalily percenlage amongst olher venomous speeies

from lhe Stale of São Paulo. The individual extraetions of the.se spiders may

furnish a maximum of 1.8 mg dried venom in winter and 2.5 mg in sumnier (1,

2). After dried lhe venom has a grayish-white eoloiir. Notvvithslanding its

toxieity heing eom|)arahle to that of C rol a I ii ,s and B o 1 It r o p s ophidian ve-

noms, the small availahle venom arnoimts of individual spiders are generally only

eai)ahle of attaiuing lelhal coucentrations in children.

Venom efi ects on dogs

In dogs lhe P. fera venom was preferentially admiuislered hy suheutaneous

roule due to its high toxieity and lhe iniense hlood pressiire drop it induees

wheu injeeled intravenously.

Suheutaneous low lelhal doses (180-200 /ig/kg hody weight) provoke in

dogs successively: intense loeal pain, violent sneezes, lacrimation, ahundant sialor-

rhea, vomitiug, jiriapism, ])roslralion, semen elimination and in some eases death.

Suhlethal doses generally are insuffieieul lo induee ])ria|)ism hy suheutaneous

route. VVilh lhe exeeplion of priapism(8), all lhe aforementioned venom effeets

were anteriorly reporled hy Vital Hrazil and VellardiT).

Local pain — The venom suheutaneous injeetiou is excrueiatingly |)ainful,

it makes dogs yelp for nearly an hour, foreiug lhem to maintain contraeted lhe

injeeted hind leg for longer jieriods. The pain faelor is neiitralized hy the

S])eeifie antivenin, whieh exeludes the possihility of this effeet heing eaused hy

venom eontained hislamine or serotoidu. On the olher hand, the jtain faelor

heing dialyzahle. its moleeule must he relatively small lo he eonsidered as a

hradykiiun releasiug euzyme. Priapism onset may take plaee dnring the pain

[)eriod.

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10 11 12 13 14 15

628

PIIARMACOLOGY OF THE POLYPEPTIDES FROM THE VENOM

(JF THE SPIDEU PHONKUTRtA FERA

Sncezes — lii dogs siioezcs constilule one of llie firsl eiivonomatioii sigiis.

'I'licy are iiitermillent l)eing ohserved for more lhaii 24 h. The firsl attacks

are very stroiig, lhe animal ihrows its head violently and nncontrollal)ly towards

lhe floor, very often, liurting ils nose and lips.

Lacrimalion and mydriasis — Dnring envenomalion lears drop conslanlly

from lhe dog eyes, even in anesthetized animais.

Mydriasis is indiiced early after venom injecTion, it ])ersists for many hours.

The dogs ])resenl visual dislurhanees as a consequence of mydriasis.

Siulorrhra — The venom ])rovoked sialorrhea is ahimdanl, pilocar|nne-like.

IJnswallowed salive drops from lhe animal moulh continuousiy for hours. This

effecl is hlocked hy alrojjine and doses of eserine and hexamelhoniurn. whieh

inerease lo a small exlenl lhe poisoning signs, seem nol lo inlerfere in ils ma-

nifeslation (3). Suheulaneous effeclive doses are ineffeetive in dogs under ehlo-

ralosc, chioroform and harhiluries aneslhesia, hovvever, lhe same doses induee

sialorrhea when appiied inlravenously.

I‘riapism — The venom indueed priapism is aiso an intermillent and long-

standing effecl I Hg. 1). It manifests re])ealedly for hours, very often exceed-

iiig 24 h, in whieh cases edema is generally formed at lhe i)enis distai extremily

correspondenl lo lhe glans.

1 — Priapi.sm inducctl by P. fcrct venom. The do" presents: adynamia, locomotion

dirflculties and drow.siness.

Priapism generally lakes ])lace at a more advanced envenomalion phase when

lhe dog has alrinidy presented intoxicalion signs. Differiug from sialorrhea, venom

j)riapism coidd nol he indueed in anesthetized dogs eveii when lhe venom was

admiuislered inlravenously in larger doses than those whieh are effeclive hy sidi-

culaneous route. Priapism is indueed in dogs having lhe medulla eul at DXII.

SciELO

Mem. Inst. Butantan

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S. SCHENBERG and F. A. PEREIRA LIMA

629

indicating that it is independent froni the excitalioii of higher centers (5). Hiilike

caiitharidin priapism, this of the arachnidan venom does not seem lo residt from

reflexes caused l)y irritation of the iirinary tracts. In dogs, the venom provokes

priapism hefore any micturition, as well as in animais with hoth urelers implanted

into the skin; it is also not evoked hy perfusing venom Solutions ihrough the

nrethra into the hladder.

Senien eliminalion — In dogs lhe P. jera venom provokes eliminalion of

semen during or after ])riapism onset. This effect seems to result from the

excitation of other structiires than the seminal vesicles since dogs have not gol

them (5 ).

Toxicity — The venom intoxicalion pattern of dogs is characterized hy:

adynamia, locomotion difficulties, prostration, drowsiness, vomiting, dyspnea, san-

guinolent feces and dealh. By iising 200 jug/kg suhcutaneoiisly, lhe dog siirvives

for several honrs hefore dealh occurs. Dogs also present sneezes, sialorrhea and

priapism during lhe severe intoxicalion phase.

Giiinea-pig ileum coiitractiori — C. Diniz separated from this venom Iwo

polypeptides which contract the ileum of guinea-pigs; according lo his findings

the fraction containing one of these polypeptides was also responsihle for the

venom toxicity (6).

Blood pressure jall — Endovenously, small venom doses ])rovoke a sharp

hlood pressure fali. The loo small histamine ccntent of these venom doses have

no comjiarahle effects on hlood pressure.

Tachyphylaxis — Bafael H. L. Sampayo (7) reported that components of

lhe hlack widow spider (Lalrodectiis rnaclana) venom provoke tachyphylaxis in

dogs. Tachyphylaxis ])henomena vvere not ohserved for any one of the effeels

eliciled hy the /h fera venom. The intermillenl actions would not have this

characleristic in the case that the agenls res[)onsihles for these effects would have

lach.yijhylaxis properties. Tachyphylaxis also shows that hoth these venoms dif-

fer hy lhe molecular slructure of their components.

Venom ekkects o\ mice

C. Diniz was ahle to reproduce on alhino mice the jera venom ])riapism

first ohserved in dogs (8). Lxcluding lacrimalion, mydriasis, sneezes, semen

eliminalion and vomiting, which are difficull to he followed in mice, all the

other P. jera venom effects can easily he tesled on these small rodents (5).

Mice slrain and weighl are essential for reproduetive quantitative assays.

The hest re.si)onses, for mice of lhe Instituto Butantan strain, are ohlained hy

using animais of 22-25 gm. With less than 20 gm they are more sensihle lo

loxic effects than to priapism. The method of Beed and Muench (9) shown

lo he very suitahle lo eslimale statistically this venom actions on mice.

Loca! pain — Not so noticiahle as in dogs, it is, however, exteriorized in

mice hy contraction of the injected hind leg. The animais also hite lhe injectiou

area in a scratching form.

i SciELO

630

PHARMACOLOGY OF THE POLYPEPTIDES FROM THE VENOM

OF THE SPIDER PHONICUTRIA FERA

Sialorrhca — As Fig. 2 shows, ihis effecl can easilv l)e followed in inice.

Al the Feginriitig small hiilihies of saliva acciimulale at lhe moulh. Later on,

dcpending on the venom dosage, lhe anirnals inay have a large part or nearly

all of its fur wel vvith saliva. Small doses of venom ])rovoke sialorrhea wilhoul

any sign of toxicity. Wilh larger doses, sialorrhea a])|)ears hefore loxic mani-

feslalions and eonlinues on its ap|)earance. The venom ED50 for sialorrhea is

0.43 mg per kg hody weight.

Fig. 2 — Beginning of P. fera venom provoked sialorrhea in mire. The mouse is

apparently normal, no sign of intoxication is evident.

Priapism — Fig 3 shows clear manifeslalion of priapism in mice. The

pria[)ism dose-effect relationshij) in mice is maintained until a maximum of

respoirses in a gronp of animais are altained, thereafler, inereasing doses of

venom jirovoke a deerease in lhe niimher of animais whieh presenl priajiism,

and death oeenrs in some of ihern hefore ihis effeet a|)pears. In a similar way

to sialorrhea, small doses of venom can induce ])riapism in mice wilhout any

loxic manifestalions; in ihis point mice reacl differentiy from dogs. The jiriapism

Fl)5() for mice is 0.25 mg per kg hody weight.

1 oxicity — The venom toxicity manifestalions in mice are: dyspnea, pros-

tralion, distensive paralysis and death. The toxicity Ll)50 is 0.76 mg per kg

hody weight.

Distensive ixtnilysis — As can he seen in Fig. 4, in mice erude venom

provokes a distensive jiaralysis of the hind legs and tail; this latter, diiring a

certain period, remains hent in a distensive form npon the animal hack. The

SciELO

Fig. 3 — Priapism Induced In mouse by 10 iig ot P. fera venom. This dose is effective

for priapism and sialorrhea but does not provoke intoxication signs.

FIg. 4 — P. fera venom distensive paralysis. Characteristic position of hind legs and

tail; the mouse died In a precocious rigor mortis.

632

PHARMACOLOGY OF THE POI.YPEPTIDES FROM THE VENOM

OF THE SPIDER PHONEUTRIA FERA

liiiul Ifgs paralysis lakcs place afler lhe induclion of sialorrhea and priapisni

and remains iinlil dealh, which occurs with lhe animais aiready presenüng a

precocious sorl of rigor mortis.

VeNüM EFFECTS O.N OTHflR KODENTS

(;uinea-])igs re|)rodiice ahnost all lhe venom actions provoked on dogs and

miee, however. lhey have lieen less invesligaled lhan miee due to llieir larger

venom eonsnm[)lion (3,5).

Kals and rahhits are very resislant lo this venom actions, 500 /xg on rats

of 150 gm only provoke secretion of lhe glands of Harder, and 1,000 /ig on

rahbils of 2,000 gm only indncc small sialorrhea and lighl inloxicalion signs (3).

HlIMAN BFING ACCIDENTS

A ])aUern which resemhles lhat of dog envenomalion is also noticed in

human heings siiffering from P. fera stings: local unhearahle ])ain, sialorrhea, visual

<li.slurhances, sw'eating, jiroslralion, ])ria|)ism and dealh. Priapism is predominanl-

ly ohserved in hoys nnder 10 years presenling signs of severe inloxicalion.

(lenerally |)atients do nol complain ahout pria[)ism, and it remains nnjterceived

hy lhe clinicians. In lhe last years, avvare of ihis venom effecl, clinicians have

given more atlenlion to ihis sym])lom of P. fera stings, [)rincipally in lhe cases

hroiighl lo lhe Inslitulo llutanlan Hos|)ilal.

lllOCHEMISTRY OF THE PHONEUTRIA FERA VENOM

Some [)nhlications have heen made regarding lhe constiluenls of ihis venom.

A. llarrio reporled nenromuscular and muscular actions of ils elecirophorelic

íraclions ( 10). (k Diniz demonslrated hy cleclro[)horesis and chromalography

lhal il conlains histamine, serolonin and two poly[)eptides which coniraci lhe

ileiirn of guinea-pigs (6). F. (1. Fischer se|)araled from it histamine and sero-

tonin hy electro])horesis, demonstraling that this venom also contains, in free

form. glulamic acid (23.69f), as|)artic acid (l.OÇh) and lysine (0.2%) (11).

J. H. Welsh studied comparatively its serolonin (12). An hyaluronidase and a

proleolylic enzyme of ihis venom were rejiorled hy F,. Kaiser (13). The histamine

conlenl of lhe fera venom varies, according lo different reporis. from 0.06%

lo 1.0% and this of serolonin from 0.03 to 0.25%.

Anli^eiiicity — Kxcluding lhe venom small molecules (hislamine, serolonin),

all lhe olhers aclive constiluenls are neutralized hy lhe specific antivenin, a fact

which allrihules lo lhem characTeristics of large molecules of a |)rohahle proleic

natiire (M).

The venom antigenic composition, determined hy agar douhle-diffusion im-

munoprecipilalion (Ouchlerlony melhod) and also hy agar imnumoeleclrophoresis,

showed lhal it musl contain approximalely 14 antigens (15). These findings

])OÍnled out lhe [lossihility lhal lhe venom mighl have several distinci componenls,

each one corres[)()nding lo one or more of its effects. They also permilted fore-

.seeing the .separalion possihililies of lhe loxic from lhe non toxic faclors, and

SciELO

10 11 12 13 14 15

S. SCIIENBERG anci F. A. PEREIRA LIMA

Mem. Tn=t. Butantan

Slmp. Intcrnac.

SSCii) ;fi27-63.S, 1966

thesc from olliers jjrohalily not yel idcMilified active venoni j)riiici|)les that, otice

altained. woidd ])rovide helter condilions for [)rocee(fing wilh lhe investigalioti

of their pharmacological |)roperlies.

Fhiccid ixiralysis ejjcct — In the course of lhese imnuinological determina-

lioiis, a venom fraction vvas separated l)y electrophoresis in agar plates, pH 5.0,

whicli induced flarcid ])aralysis in mice when injected suhcutaneciisly (Pig. 5).

Fig. .5 — Flacciíl paralysi.s Induced in mlce by Sephadex G-5() fraction of P. fera

venom heatcd at lOQoC for 6 min. in pho.sphatc buffer, pll 8,0, 0.05 M. The hind

legs are paralysed in a flaccld form; one of the mice ia trying locomotion by using

the fore leg.s; both preaented flaocid bodies after death.

The sanie coin])onenl was also separaled laler on in celhilose acelale strijis, jill

5.0(16). This paralysis differs froni lhe distensive one, tlie hind legs are ])ara-

lysed in a flaecid hnt not distensive form. as lhal which is provoked hy cnide

venom. lhe tail aiso remains flaecid, and lhe animal motionless; for its loco-

molion lhe fore legs are nsed to pnll the hody. This fraction is very loxic, and

death occurs wilh lhe mice presenling a flaecid liody, aIso differing, in this

])articnlar. from lhe cnide venom lelhality in which lhe animais tlie in rigor

mortis. PTaclions conlaining the principie resjionsihle for this effecl were aiso

sejiarated hy olher methods and will he descrihed laler on. In ernde venom

assays lhe distensive paralysis dominant effecTs acl as fimctional antagonizers

of the flaecid jiaralysis actions, which conld only he revealed after se|)aration

of holh their components.

//('«/ inuclivalion — .'\ll the venom pharmacologicaliy active components are

resislanl lo heating. Venom Solutions in saline of mammals can he heatcd at

100"(] for 20 min wilhoul losing aelivily. However, inaclivalion occurs when

SciELO

634

PIlARMACOr.OGY OF THE POLYPEPTIDES FROM THE VENOM

OF THE SPIDER PHONEUTRIA FERA

heated al tlie sanie leinpcralurc for 6 miii al ])H 8.0; llie recovcred aclivity

of some of lhe corniiottenls lieing of: ileum eonlraeting polypejjtides — lOO^r.

[jriapisin — 39.2%, sialorrhea — 34.2% ami toxicity — 19.4% (17).

Chemical and cnzymic inaclivalioa — The venoin effeels are iiiactivated liy

eoncentrated acetic acid. The |)riapisin, sialorrliea, loxieily and ileum contractitig

effeels are more resislant lo aeids (HCl 0.1 N) lhan lo alkaliiie solulions (NaOH

0.1 N). The eomponenls eorres|)onding lo lhese four effeels are inaelivaled hy

NaOH 1.0 N.

Sulpluirie elher, acelone, ehioroform and Imlanol do nol inaetivale any of

lhese effeels, lhe first ihree exlrael from lhe venom a signifiealive amount of an

inaelive malerial logelher wilh some of ils hislamine and serolonin 115).

Excejjting lhe venom hislamine and seroloniti effeels, all lhe olhers are in-

aclivated l)y Irypsin, chymolrypsin and |)epsin, indiealing lhe |)roleie nalure of

llieir eorresponding eom[)onenls (15,17).

The ])roleolylie enzyme of P. fera venom inaelivales all ils own large moleeule

eonsliluenl.s, hovvever, miieh more rapidly lhe guinea-])ig ileum eonlraeling poly-

peplides lhan lhe eomponenls res])onsihle for lhe remaining effeels. This enzyme

aiso degrades easein mueh more rapidly lhan alhnmin.

Dialysis — The Visking luhe 18/32 (im[)ermeahle lo insulin) is more per-

meahle lo lhe priapisrn lhan lo lhe sialorrhea and loxie prineiples. All venom

aclive conslilnenls dialyse ra])idly ihroiigh luhe 8/32 as aIso does insulin (14.

15,17). As ihése venom eomponenls dialyse ihrough luhe 18/32 (im|)ermeahle

lo insulin), il musl he admilled lhal lheir molecular weighl is smaller lhan 5,733

(insulin). So, lhey musl he eonsidered as poly[)eplides since lhey are aiso tle-

graded hy lhe ])roleolylic enzymes, hesides heing aiso immiinogenie.

Venom fhactionation

Arumonium siilplialc and clcclropltorc.sis — lhe flaectid ])aralysis and lhe

ileum eonlraeling eomponenls were separaled hy 65-75% ammoiunm sul[)hate

saluralion. Eleelrophoreelieally, all aclive eomponenls are ])osilively eharged and

migrale lo lhe ealhode. Tlu^ flaeeid ])aralysis eotnj)onenl was lhe only one

separaled hy eleclrophoresis (agar |)lales or eellulose aeelale slrips) in acelic

acid ammoninm aeelale huffer, jiH 5.0 (16,17).

Gel filtralion eolnnm.s — All lhe large moleeule eomponenls are exeluded

in Sephadex (1-25, exeepling lhe ileum eonlraeling polyjieplides whieh have nol

yel heen assayed, indiealing lhal lheir molecular weighls are grealer lhan 5,()0(),

hnl smaller lhan 10,000 since lhey diffuse in Sephadex (1-50, dala whieh eon-

firm olhers anleriorly ohlained hy dialysis 114, 15, 17). Holh seem lo indieale

lhal lhe larger pharmaeologieally aclive moleeules of lhe P. fera venom have

molecular weighl larger lhan 5,000 and smaller lhan 5,733 linsidin). These

comjionenls heing immunogenie and hydrolyzed hy ])roleolylie enzymes, and hav-

ing molecular weighls helween 5,000 and 6,000, mnsl really he eonsidered as

])olypeplides.

Fig. 6 shows a ehromalogram of 20 mg P. fera venom run iu a Sephadex

(1-50 eolurtm (760 X 22 mm), eluled wilh jdiosphale huffer, pH 7.5, 0.05 M

and NaEl 0.1 M (15). The venom was |)reviously healed al 1()0"(1 for 6 min

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Slmp. Internac.

33(2):627-638, 1966

S. SCIIENBERG and F. A. PEREIRA LIMA

635

al ])hosi)hal(! hiiffer, pH 8.0, 0.05 M. Tfiis trealmeiil denatures the proteolylic

enzyine and a large part of lhe lomponenl responsihle for lhe dislensive ])aralysis,

perniilling lo sejjarale lhe flaccid |)aralysis comjionenl.

StPHADtX 6-50

PHONCLITRIA FERA HEATED VENOM

Sialorrhea

Toxicity (lethality) — —

Distensive paralysis

riaAcid paralysis

IlBuin contraction - -

Histamipe —

Serotonin —

Blood pressure fali -

Fig. 6 — Chromatogram of a 20 tig P. fera venom run on a Sephadex G-50 (fine) column

(760X22 mm). The venom vva.s previously heated at 100“C for 6 min In phosphate buffer,

pH 8.0, 0.05 M. 13 pharmacologlcally active component.s, dl.strlbuted In tvvo zones, are

reincsenled; flaccid paraly.sls was revealed after venom heatlng at pll 8.0.

All lhe identified effects are foniid amoiigsl lhe effluent fraclions. The

aelivilies are dislrihiited in Iwo zones; lhey have no relalionship wilh lhe 280 nifi

ahsorjdion jieaks, which seeins lo indieate lhal lhe venom aclive polypeplides are

poor or dejirived of tvrosine, Iryjilophan and plienylalanine. Some of lhe same

effecls are fonnd in lhe Iwo aclivily zones, whose comjionenls are well differenliated

hy llieir molecular size, shovving lhal lhe venom conlains componenls of different

molecular weighls having, hovvever, lhe .same jiharmacological aclivily.

'riie ilenm polypeplides, hislamine and serolonin were ohlained individually

in some Inhes free from olher componenls, evidencing lhal jirohahly loxicily and

ilenm conlraclion are exerled hy differenl venom moleciiles. Priapism, sialor¬

rhea and loxicily polyp('plides are not sejiaraled hy Sejihadex G-50; Inhes which

conlain lhe Iwo former jirohahly also conlain lhal of loxicily.

'riie flaccid jiaralysis com])onenl. hy heing also loxic, ils fraclion overlaiis

a small pari of lhe loxicily line. The venom st“ems lo conlain a ihird loxic,

componeni deprived of any paralysing effecl.

lonic cülumns — Fig. 7 shows lhe fraclionalion chromalogram of 20 mg

venom in a CM-Sephadex (i-50 coinnm (270 X 10 nim), in gradient and slep-

SciELO

10 11 12 13 14 15

636

PIIARMACOLOGY OF THE POLYPEPTIDES FROM THE VENOM

OF THE SPIDER PHONEUTKIA FERA

wise elutions. Similarly to ollicr coliimiis, ovt'rlap|)iiig of lhe efflueni aclivities

wilh 280 mp, Ifcluro poaks does iiot t-xisl. Four acTivily zones were formed liy

lliis venom fraclionatioii, lhe saine mimher of zones were also ol)lained with DEAE-

celliilose eolumns. Histamine and serotonin were tiol assayed in lhese fraelions,

two of llie fonr fraelions which eonlrael the ilenm miisl eorres|)ond to lliese

ainines. Elaecid paralysis, which is se|)araled from distensive jiaralysis in DEAE-

cellnlose colnmns, was not separaled hy ihis cohitnn, and was nol revealed.

Toxicity is not representetl in the graph under ])riapisni and sialorrhea aclivities

of lhe firsl and third activity zones, which would indicate lhal toxicity, |)riapism

and sialorrhea are not provoked hy a single coniponent. However, lhese findings

were nol considered as definitive j)roofs, since large volumes were necessary to

indnce j)riapisni and sialorrhea, holh reípiiring lower minimal effective doses

than toxicitv.

Priapism

Stalorrh^a

Toxicity flethalityj

lleum contraction

Blood pressure rise

Blood pressuré -ralt

Fig. 7 — Chromatogram of a 20 mg P. fera venom run on a CM-Sephatiex C-SO column

(270X10 mm), in gradient and stepvvise elutions. Some of the same effects are di.stributed

in four zones. The priapism effert is absent in the fourth zone, indicating the possibility

that its moleeules might ditfer from those resi)onsibles for siaiorrhea and toxicity.

Very significanl, however, is the ahsence of priajiism in lhe fourth zone.

since its ininirnal ('ffeclive dose is sinaller than lhal of toxicity, which was induced

withoul any priapism manifestation. demonsiraling the ])ossihilily that jniajnsm

and toxicity might he jirovoked hy two dislincl polypeptides.

SciELO

10 11 12 13 14 15

Metn. Inst. Butantan

Simp. Internac.

33(2) :627-638. 196(i

S. SCHENBERG and F. A. PEREIRA LIMA

637

Two other comjioneiils werc sojiarated liy this and alío hy Sophadex 0-50

coliimns. Adniiiiislered hy iiitravciious route, one provoked Idood pressure rise

and lhe other hlood pressnre fali. Bolh have nol heen well stndied. neverthelcss,

some of lheir fraclions are not contaminaled wilh histainine or serolonin. In

crnde venoin lhe hy|)otensive effects dominate and niask lhe hyperlensive ones (17).

These colnmn e.xperimenlal findings fnrnish t\idences which seem to de-

inonstrate that lhe molecules of each activily zone differ froni ihose of other

zones in whal regards lheir size and eleetrical charges. Those of each zonc

aiso differ one frorn each olher hy lheir pharniacological ])roperties. According

to these dala, lhe venom would contain 16 pharmacologically aclive components,

incinding hislamine and serolonin.

The prohleni increases in complexity when it is admitted lhat each one of

some of lhe venorn effects can he provoked hy fonr different poly|)e[)tide molecnies.

It wonld he difficnil to admil that lhe veneniferoiis glands wonld secrete snch

a ninnher of components provoking, some of lhem, lhe same effects, that is lo

say, having lhe same finalilies.

A heller investigation of the venom proleolytic enzyrne might ihrow some

lighl on these apparentiy paradoxical facts. This enzyrne differs from trypsin,

chymotrypsin and jiepsin hy its sid)Strale linkage sjiecificity. It seems verv

prohahie that lhe veneniferons gland wonld secrete lhe jnoteolytic enzyrne lo-

gether wilh a few large molecnies, having one or more of these particular linkages.

l)e|)pnding on lhe stage of these large rnolecnle Cascade degradalion hy lhe

venom proleolytic enzyrne, the venom accnmidated in lhe veneidferons gland

might [nesent molecnies having lhe same ])harmacological aclive centers honnd

lo larger or smaller chains, and these would condilion lheir differences in size

and eleclrical charge. The degradalion process of these large hy])othetical mole¬

cnies should he inlerrnpted at a cerlain eqiiilihriiim ])oinl, olherwise all the

venom wonld he Iransformcd irilo amino acids.

'riie large amount of free amino acids of lhe venom seems lo represent

residnes of this degradalion, it also conslilntes an argnment in favonr of lhe

ahove mentioned hy|)olhesis. The different amoinUs of each one of the three

amino acids fnrnish a light indicalion of the molecular slrnclnre of these hypo-

thelical large molecnies secreled hy lhe veneniferons gland. The venom conlenl

in gintamic acid (23.6%) seems lo show lhe enzyrne preference for a linkage

containing this amino acid, presnmahly it wonld constitnte the terminal amino

acid of one or more of lhe venom molecnies.

Ht:i'EnE.\ci;s

1. BüCHERL, W., Mem. Inst. Butantan, 2.5(2), 1. 1953.

2. BÜCHERL, W., in E. E. BUCKLEY, and N. PORGES (Editors), Venoms,

Amer. As.s. Advanc. Sei., Washington, D.C., 1S56, pp. 95-97.

3. SCHENBERG, S., and PEREIRA LIMA, F. A., Cirncái e Cultura, II, 237, 1962.

4. VITAL BRAZIL, O., and VELLARD, J., Mem. Inst. Butantan, 20, 569, 1925.

5. SCHENBERG, S., and PEREIRA LIMA, F. A., Ciência e Cultura, 15, 267,

1963.

6. DINIZ, C. R., An. Acad. Bras. Ci.. 35, 283, 1963.

SciELO

10 11 12 13 14 15

638

PHARMACOLOGY OF THE POLYPEPTIDES FROM THE VENOM

OF THE SPIDER PHONEUTRIA PERA

7. SAMPAYO, R. L., Latrodectus muctans y latrodectismo, “El Ateneo”, Buenos

Aires, 1942, pp. 108-111.

8. DINIZ, C. R., Personal communication.

9. REED, L. J., and MUENCH, H., Amer. J. Hyg., 27, 493, 1938.

10. BARRIO, A., Acta Physiol. Latinam., 5, 132, 1955.

11. FISCHER, F. G., and BOHN, H., Z. yhysioL Chem. Hoype Seyler’s, 306. 265,

1957.

12. WELSH, J. H., and BALT, C. S., Toxicon, 1, 165, 1963.

13. KAISER, E., in E. E. BUCKLEY and N. PORGES (Editors), Venoms, Amer.

Ass. Advanc. Sei., Washington, D. C., pp. 91-93, 1956.

14. SCHENBERG, S., and PEREIRA LIMA, F. A., Cicncia e Cultura, U, 237, 1962.

15. PEREIRA LIMA, F. A., and SCHENBERG, S., Cicncia e Cultura, 16, 187, 1964.

16. PEREIRA LIMA, F. A., and SCHENBERG, S. Cicncia e Cultura, 15, 268, 1963.

17. SCHENBERG, S., and PEREIRA LIMA, F. A., Abstracts III Internac. Pharma-

col. Congr., 1966, São Paulo, p. 189.

Discussion

C. Y. Lee: “Is the flaccid paralysis caused by the fraction of the spider venom

central or peripheral in origin? Have you studied its effect on the neuromuscular

transmission?”

S. Schenberg: “Studies were not yet made to determine the local of action

of the flaccid paralysis component. Work is being done to separate and purify

this polypeptidh as to permit a better investigation of its pharmacology.”

SciELO

10 11 12 13 14 15

Mcm. Inst. Butantan

Simp. Tnternae.

33(2):639-fí44. 1966

PETER N. WITT

639

65. SPIDEH GLANDS AND PSYCHOTHOPICS

PETER N. WITT

Hiate University of New York, U]>stute Medicai Center at Syracuse, N.Y., U.S.A.

There exists evideiice llial lhe silk glaiuls of lho spiifor Aranciis diademuliis

Cl. Work iii olose ooiniection witii lhe central nervoiis system (CNSl. Tlie Ci\S

a|)|)ears lo send signals to lhe glands, "teiling’’ ihtni to produce more or less

silk; it peenis also “informed ” of lhe amoiiiit of silk availal)le in lhe ampullate

glands at a given lime. Silk heing so importani in lhe s])ider’s life — for Ira])-

huilding, moving aroimd, and eommimication — siich Iwo-way mechanisms a])-

pears of greal survival value: enough material is made availahle, hut no energy

is wasted on excess. The close interrelationship helween CNS and glands exjjlains

lhat driigs which affect CNS fimction, the [)sychotro|)ics, influence silk gland

activity and lhal wíehs hiiill afler these dnigs reflect lheir effects in weh-weighl

and -])altern. The evidence vvill he reviewed.

To estahlish the amoimt of silk which a s|)ider uses for one weh, the whole

slruclure is ciil off from its siipports, rolled up on a j)iece of relalively nilrogen-

free filter pa])er, and digested in selenium-siilfiiric acid. The amoimt of nitrogen

determined Wíith an optical micromethod in lhe weh digesl is a measure of lhe

lolal amoimt of polypejitide in the weh. Fig. 1 shows the residts of an experi-

ment with 23 sjiiders which were treated wilh 1 mg/kg physostigmine hy mouth

in sugar water and 23 control animais. The wehs were digested daily and on

Tuesday hoth grou[)s showed lhe same mean of 39 microgram N per weh. 36

hoiirs afler lhe drug had heen given, on Wednesday morning. lhe ]diysostigmine-

trealed animais had huilt wehs containing a mean of 48.9 ± 1-.4 microgram N

per weh, while Controls huilt significanlly lighler wehs with 20-30 micrograms N.

Tlie following day, h^riday, all animais, Controls and drugged sjiiders, huilt wehs

with the same meau of 34.5 microgram N per weh. It was concluded that wehs

afler physostigmine are heavier or contain more polypejilide ihread.

In order lo lesi the hypolhesis tliat the cholinergic drug stimulates silk jiro-

diiclion rather than promotes a more thorough scjueezing out of the ampullate

silk glands, Peakall performed ex|)eriments using 3 kinds of methods (1, 2) :

A: Glands were pulled emply of ihread at regular iniervals. f.e. every 6

hours, and lhe (jiiantity thread (udled was determined in lhe selenium-siilfiiric

acid digesl. Such jirocedure lakes advaniage of lhe experience lhal a spider

which sils on a rough surface leis oul thread onlo lhe rotaling axle of a motor

imlil the glands are em|)ty. PeakalFs results wilh jihysostigminc and carhachol.

2 cholinergic drugs, and atro|)ine, an anticholinergic drug, show lhal with in-

creasing dose of the drug, silk (juantily increased after lhe cholinergic and

decreased after lhe anticholinergic drug.

2 3

5 6

11 12 13 14 15

640

SPIDER GLANDS AND PSYCHOTROPICS

Fig. 1 — Nitrogen values in the digest of webs of 23 spiders

treated with 1 mg/kg physostlgmlne on Tuesday nlght (12 hours

before the Wednesday vveb-buUdlng time) and 23 Controls. Each

polnt represents the mean ot all measurements, and lhe vertical

Une the standard error ot the mean. The tvvo Thursday values

are signiticantly ditterent belovv the 1% probability levei. Note

the increase ot web nitrogen 36 hours atter the drug.

li: Anollier procediirc usos laliclliiig of a silk preciiisor, alailinc, willl CM,

iiijdcling il inlo the siiiders’ ahdomen, and measiiring lhe sjieed vvilh vvliich the

lal)el liiriis iip in lhe silk. G hours afler alaiiine was given, signifieanily more

lahel ajipeared in lhe silk afler |ihysosligmine, carhachol and jiaraoxon lhan in

iinlrealed spiders (Tahie I). ll was aiso foiind lhal jiisl eni|)tying lhe glaiids

proinoted incorporation of lhe lahel, and llial eniplying phis driig Irealnienl was

iiol additive, hui ralher made the glands hehave like afler one Ireatmenl alone.

We ean lherefore assume lhal tvvo mechanisms regulale sjieed of silk production:

1 ) a feed-hack from lhe lumen of lhe gland via lhe inner ejiilhelial memhrane

and 2| a possihly neurohumoral meehanism via lhe outer epithelial memhrane.

C: A ihird way of meastiring changes in silk production is the use of radio-

aiilography: the lahelled silk precursor, again CM alanine, leaves hiack dois on

a pholographic fihn which is sjiread ihinly over lhe sliced lissiie. By iising

tliis metliod al different limes afler alanine injection, its progress from lhe hody

fluids inlo lhe gland epitlielium and from there inlo lhe gland lumen can he

followed. Tahie II shows lhal 4 hours afler injection of CM alanine, mosi of

the lahel had left lhe intestine and hlood of lhe sjiiders and ajipeared in lhe

gland lumen in jiulled or physostigmine Ireated animais, wliile atrojiine treated

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(2) :639-644, 1966

PETEU N. WITT

641

TABLE I — AMOUNT OF INCORPORATION OF C-14-ALANINE INTO WEB PROTEIN

DURING 6 hr.

The labelled alanine was given at the start of the experlment. Threaci was pulled

for determination 6 hr aftervvards. (From D. B. PEAKALL, Comp. Biochem. Physiol., 13,

467, 1964).

TREATMENT

No.

of spiders

Actlvity

counts/min

Standard Micrograms Standard

error of nltrogen/ error of

the mean 100 mg b.wt. the mean

Unpulled,

no drug

123

11.1

—

Unpulled,

physostigmine

290

27.4

—

Unpulled,

carbachol

301

25.4

—

Unpulled,

paraoxon

329

60.7

—

Pulled, no drug

314

13.5

15.0

1.4

Pulled, phy.sostlgmlne

346

17.9

16.2

1.6

TABLE II — COUNTS FROM 7-DAY AUTORADIOGRAPIIS OF INTESTINE, BLOOD AND

SILK GLAND EPITHELIUM AND LUMEN IN THE INTACT SPIDER

Counts are per mm=

with standard deviatlon,

ground count (Bkd) were

Sll, 1965).

at magnlflcatlon

from 50 count.s i

1-4/mm-. (From

of 60. Each figure represents the average,

[10 counts from each of five spiders). Back-

D. B. PEAKALL, Coinp. Bioche^n. Physiol., 15,

Time

in hr

Treatment

Hind intestine

Blood

Epithelium

of silk gland

Lumen of silk

gland

None

61.0 ± 8.0

29.7

4.3

4.4 ± 1.9

Bkd

Re-pulled

55.1 ± 6.9

26.3

5.1

8.8 ± 3.7

Bkd

Physostigmine

54.9 ± 7.9

29.0

2.9

6.1 ± 1.9

Bkd

Atropine

64.1 ± 9.1

27.1

-±

5.0

Bkd

None

15.1 ± 2.5

17.8

3.0

25.5 ± 4.1

4.9 ± 0.8

Re-pulled

10.3 ± 1.8

13.0

5.2

54.3 ± 8.3

18.0 ± 4.2

Physostigmine

8.4 ± 2.1

8.9

3.1

48.0 ± 7.5

12.5 ± 4.3

Atropine

16.7 ± 3.0

22.1

4.6

25.5 ± 4.8

6.1 ± 2.1

None

Bkd

31.8

1.9

33.8 ± 4.8

38.4 ± 4.0

Re-pulled

Bkd

4.7

1.9

15.3 ± 2.8

55.6 ± 4.2

Physostigmine

Bkd

5.6

2.7

16.2 ± 3.5

62.8 ± 9.8

Atropine

Bkd

14.0

7.4

44.4 ± 9.6

22.4 ± 7.5

SciELO

10 11 12 13 14 15

642

SPIDER GLANDS AND PSYCIIOTHOPICS

or restiiig glands had iiol yot laken u[) lhe hulk of tlie lahel. Iii liislological

slides lhe size and shapes of lhe amj)ullale glands and lhe position of lhe lahel

ean he idenlified.

If we assume thal aeelyleholine is lhe neurolransmiller snhslanee whieh is

responsihle for carrying lhe signal for silk iirodnelion from lhe nerve lo lhe

gland lissue, we mnsl look for places on lhe gland whieh eoidd hind aeelyleholine.

Peakairs work shows auloradiographie ])roof thal lahelled aeelyleholine is ae-

cnmnlaled on lhe gland e|)ithelium. This is possihly lhe area for reeeption ol

lhe nenrohnmoral signal.

Lei us now take a look al lhe whole animal and see lhe geographieal loeation

and possihie interrelalionships of silk glands and Ci\S in Amneua diadcmutus Cl.

F. Meier I personal eommnniealion) has idenlified nerves leading from lhe

hig snheso|)hageal ganglion in lhe eej)halothorax lo lhe silk glands. Sneh nerves

eould earry signals in hoth direetions, eoordinating leg movemenls of lhe weh-

hnilding spider with silk suppiy in lhe gland.

Behavioral experimenls wilh weh-huilding s])iders have shown thal there are

3 |)ossihle ways whieh ean lead lo wehs Iniill wilh a shorter ihread:

1. Atro[)ine sulfate, 1, 2 or 4 mg/kg, was given hy mouth to 19, 19 or

39 s|)iders 12 hours hefore weh-huilding time. The two lower doses eansed wehs

whieh showed no change in size or regniarity hnl were huilt with wider meshes,

eovering lhe same area with less ihread. The highesl dose eansed signifieanlly

smaller and less regular wehs hiiill wilh less silk. This latler ehange lasled

through lhe seeond day after drug a])])liealion. The inlerpretation assumes thal

alro|)ine shows its effeet on polypeptide synlhesis in lhe spiderV glands as well

as interferes with eentrally regulaled exaelness of movemenls. It is inleresting

to note lhal not lhe size of lhe eatehing area in lhe weh was deereased wlien

less ihread was availahle, hnl lhe shorter ihread was wider spaeed .so thal lhe

Irap was full size and otdy losl lhe smallesl inseels.

2. S|)iders aiso hnilt wehs wilh shorter ihread afler a weighl had heen

allaehed lo their haeks. These experimenls (3) were underlaken lo lesl lhe hy)) 0 -

ihesis lhal ])siloeyhin, lhe hailueinatory mushroom poison, eansed in s|)iders similar

effeets as an increase in hody weighl. This snhslanee is known to ehange in

man pereeplion of one’s hody. Does a spider afler psiloeyhin “feel” heavier

and lherefore huilds a weighl-weh? It eonld he shown thal 150 mg/kg |)silo-

eyhin given to 9 and 23 s|)iders in Iwo independenl experimenls 12 hours he¬

fore weh-huilding lime, as well as a 30% increase in hody weighl of 15 s|)iders,

deereased average thread lenglh signifieanlly hy ahonl 30%. However, when

lhe wehs were digesled and N delerrnined, a signifieant differenee in lhe amounl

of silk was found hetween lhe psiloeyhin and weight-wehs: lhe wehs after psilo¬

eyhin were huill wilh less silk, lhe shorter thread was as ihin as hefore; lhe

heavier spiders, in eontrasl, huilt wehs wilh ihicker thread, using e(pial amoimts

or even more prolein lhan hefore. The interprelalion for lhe results of lhe ex-

periment wilh heavier s[)iders assumes thal lhey huilt a thieker thread lo hold

lhemselves uj); as lhey had no more material lhan nsual in lheir glands, lhe

thieker thread had to he shorler. The psiloeyhin wehs mnsl he inlerpreled in

a differeni way, as will he seen laler. If lhe inlerpretation is eorreel, lhe s])iders’

CNS musl integrate information on ihread lenglh as well as silk cpianlily slored

in lhe glands during weh eonstruclion.

2 3

5 6

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(2)1639-644, 1966

PETER N. WITT

643

3. Experinieiits ])erfoi'me(l iiiider lhe iiifluence of llie Iraiiquilizer Valium

(diazepani) may Help to inlerpret psilocybin effects (4). Wheii 100 nig/kg

Valium were giveti lo 40 spiders 36 liours hefore web-building lime, all animais

hiiilt smaller wehs wilh a shorler ihread and less material. This conld lie lhe

result of a deerease in silk production in lhe glands ihrough lhe tranquilizei', or

lhe glands were nol eompletely em|)tied at lhe end of weh construction. Experi-

ments wilh ihread pulling afler Valium auswered lhe question in favour of lhe

second inlerpretalion: when silk was |)ulled from 13 spiders one and Iwo days

after 100 mg/kg Valium and from 9 eontrol spiders, no difference in quanlity

could he found. The effeet of Valium is lherefore' interjireted as possihly af-

fecting lhe spiders’ “drive” so lhat lhey huild smaller wehs usiug only part of

lhe material. The glands stay jiarlly filled at lhe end of lhe eonstruetion jieriod.

This, in lime, would slow down new silk synthesis hy lhe feed-hack mechanism

which was shown hy Peakall.

Thus, lhe three ways in which wehs wilh shorter thread were |)roduced

ihrough drugs and weight changes, provide some evidenee for the close inter-

eoimeclion helween silk glands and CNS. If the Iranquilized CNS “inslructs”

lhe legs to huild a smaller weh, less silk is pulled from the glands. Eig. 2.

Flg. 2 — Araneiis diaúematux CJ. sitting on a rough surface

anel facing away from the camera. Note the posterior spin-

nerets from which the 8th leg pulls two threads, and the

anterior spinnerets from which a thread runs to the ground.

illuslrates how the s]nder pulls silk from lhe spinnerets hy means of its hind

leg. It can also lower itself on lhe ihread ihrough ils weighl and regulales S])eed

and ])ossihly ihiekness wilh lhe eighlh leg. The funeition of WilsoiTs conirol

valve (5,6) in this proeess and its interrelalionship wilh CNS and glands is a

matter for ftilure investigalions which will we ho|)e furlher elarify |)syehotroj)ic

drug effects on weh-huilding.

1 SciELO

644

SPIDER (3LANDS. AND PSYCIIOTROPICS

Heferknces

1. PEAKALL, D. B., Comp. Biochem. Pliysiol., 12, 465, 1964.

2. PEAKALL, D, B., Comp. Biochem. Physiol., 15, 509, 1965.

3. CHRISTIANSEN, et al., J. PharmacoL, 1,S6, 31, 1962,

4. WITT, P. N., and REED, C. F., in press.

5. WILSON, Qiuirt. J. microhiol. Sei., 103, 549, 1962.

6. WILSON, Quart. J. microbiol. Sei., 104, 557, 1962.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(2):645-650, 1966

CHARLES F. REED

645

66. SPIDEH WEBS AND PSYCHOTROPICS

CHARLES F. REED

Temple University, PhíUideJphut, Pu., U.S.A.

Perliaps, from the v'ie\v|)oint of lhe stiuleiit of venoms, llie spider wch is nothiiig

more thaii an elahorate deviee for delivery of venoni lo lhe prey. (Jranling lhal

inlerprelation, ihose of iis wlio are interesled iii iinderslaiiding animal hehavior

are gralefid for lhe inlimacy required for tlie injeetion of venom: in lhe orh-

weh we have a concrele segmeiit of hehavior es|)eeially susceplihle lo (jiianlilalive

sludy. The weh is also a leslimony to the sensory and motor integrity of the

•spider. While it may lie fnrther agreed lhal it is the result of innale hehavior

patterns, we are not lherel)y relieved of the recpiirement to explain tlie s|)ider’s

response to eontingencies of the moment (e.g. vvind and hroken strands), nor

nltimately to explain the elahorate sensory, nervons and motor Systems whieh

mediate ihose innate patterns.

Eor it is the case that the lechnology of weh-hnihling is rather imperfectiy

known. There have heen excellent naturalistic observations of lhe proeess and

several attempts at motion piclures. There have also heen experimental inler-

ferences in huihling: hy selectively injuring the animal (for instance hy removal

of legs and hy the jirodnclion of lesions in the central nervons System), hy dis-

rupting the ordinary luiilding eireumstances (as hy destroying threads or rotaling

lhe frame dnring constrnetion ), and hy feeding lhe spiders drngs whieh affect

lhe hehavior of rnany animais, (he psychotro|)ic drugs, and whieh in lhe sjrider

have re|)roducihle and particular consequences for cerlain featnres of the weh.

Drng effects on wehs have heen investigated exlensively for Iwo species.

Zygiella x-nolula and Arancus diadematus. In these stndies, the ohject of measiire-

ment has heen the compleled weh rather than the motions of the animal in the

course or acl of huihling. There are many good rtasons for ihis choice, among

olhers the reiuoducihilily of events whieh oceur at greal speed under eonditions

of limited visihility, hut a prime consideration has heen the variahilily in the

dimensions of the weh and lhe eonsequent necessity lo eniploy slatistieal com-

j)arisons of many wehs. eilher of many animais under experimental and conlrol

eonditions or of the saine aidmal treated afler a suitahiy lengthy weh-hnihling

history had heen accnmnlated.

Fig. 1. whieh shows lhe eom|)leted weh of .druncus diadematus, makes it

ai)parenl lhal several different measnres or aspeets of the weh may he ehosen

for evidenees of influenee of treatme^its. The eom|)nler, allowing as it does. many

and rapid eonqnitations to he inade, diminished somewhat the limitations of lhe

choice of parameters. It also |)ermitted ns lo pick nj) rather snhlle changes.

apjjarenl in a series of wehs hnl perhajis not in lhe comparison of Iwo single

represenlalives of treatment and control samples. (See for example Fig. 2, show-

ing a represenlative drng effeel.)

SciELO

10 11 12 13 14 15

Fig. 1 — Completed vveb of Araneus diudematus Cl. Structure

in upper right comer is weight supported at 20 mm intervals

by three thread.s.

Fig. 2 — Radial and frame structure of Araneus vveb. The

spider has been removed before beginning provisional spiral from

hub tovvard periphery. (From BREED, A. L., LEVINE, V. D.,

PEAKALL, D. B. and WITT, P. N-, Ilehavtonr, 23. 43, 1964).

Mem. Inst. Butantan

Simp. Internac.

33 f2):645-650, 1966

CHARLES F. REED

647

The inaiiy jiaramelers of lhe wel) niay he reduced to ihree classes: the size.

lhe shape, and llic regidarily of ihread placenieol in lhe wehs. Psycholropic

drugs may he descrihed iii siniply emjcirieal leriiis of lhe effeets lhey wreak oii

lhese classes. D-amphetamiiie, for example, in a dose of 600 mg/kg slimidates

llie hnilding of smaller wehs than normal wilh irregular placeinent of certain

ihreads (lhe spiral and radial ihreadsl. LSD 25, on lhe olher hand, can he

adminislered in doses 10.3-0.5 nig/kg) which rednce lhe freqnency of vveh-hiiild-

ing, luit have no effecl on the regularity of angles and spirals; in faci lhe

regidarily of placement may he grealer than usual. Higher doses do have an

adverse effect on angle regularity.

These eni])irieal ohservalions, however, can only he lhe starling point for

explanalion. ll is important of course lo note lhal lhe wehs change in ways

sjiecific to lhe drug, hnl il is necessary to identify hasic elcments in lhe execution

of lhe wel) struclure.

There are four sources of alleralion which are now' apparcni.

Dr. Witl has discussed the possihie role of thrcad supply in delermining

weh dimensions. The inhihilion or stiniulation of protein-synthesis requires ad-

juslmenls in the weh. This is nol an ohvious relalionship: it could he enteiiained

as a possihilily lhat lhe s])ider comjiletes its weh wilh thread material slill

availahle or that il runs short in lhe process. Ordinarily, however. the availahle

material is rather com]iletely utilized, the sjiider coming to a slop which nol only

completes lhe geometry of the weh hui empties lhe ampullate and aggregale glands.

Diazepam, as Dr. Witl has shown. is an exce|)tion to ihis rule. An important

point is lhal dearlh or ahiindance of material need nol imply loss of precision

of placement of ihreads.

However, if cholinergic action enlers inlo ihread produclion, il niiisl also

he conlemiilated as one |)ossihle conlrihnlor lo the second source of weh change.

se.nsory-molor disliirlxincc. Thal is. neural Iransmission may he affecled. Phvso-

stigmine. however, does nol affecl jjrecision of jilacemenl ailhough it does slimnlate

ihread-synthesis. Two olher suhstances potenlially involved in neural Iransmis¬

sion, on lhe olher hand. nor-epine])hrine and 5-hydroxylriplamine, affecl holh size

and legularily of thread-|)lacing. Whatever lhe source of lhe dislurhance, il is

clear lhal sensory-motor integralion is a second factor underlying weh alteralions.

A relaled hnl conceplually inde])endenl source of disturhance may he called

jailiire or clian^r in infonnalion-processins'. The ca])acily of lhe s])ider lo inle-

grale sensory informalion can he affecled. lor inslance, hy central nervous system

lesions. A simpler forni of interference is produced hy depriving lhe s[)ider of

visual or laclual informaliou hv hlinding or hy removing legs.

Pinally. il may he necessary lo invoke a higher-order adaptation on lhe part

of lhe animal, reUridion of enprp;y output. If a weh is produced al all, it is

smaller in area and in thread lenglh; il may or may not show thread irregularities.

Slrychnine (30()-82() mg/kg) for inslance, diminishes frecpiency of hnilding and

results in wehs of smaller spiral area and less oval shape. Caffeine (ahove 1

g/kg) also produces rounder wehs, disni|)lion in threads and long weh-huilding

limes (several hours inslead of lhe usual 20 lo 30 minnles.)

While each or several of lhese faclors may euler inlo lhe |)aiiicnlar dislurhances

of weh conslrnclion, we have recently heen concenlraling on lhe malter of in¬

formalion. chiefly on lhe sensory informalion emjiloyed hy lhe s|)ider in ihread

i SciELO

SPIDER

8/64 t

4. p m

600 ^/g

d-Amphetamine |

fti'

CONTROL DAY

AREA 430.0 cm“

radii 35

AN6LES 1.29

DAY AFTER DRUG

149.6 cm*

5.96

Fig. 3 — Effect of d-amphetamine on web construction. The webs are separaled by

one (lay, but the spider has been given 600 mg/kg 12 hours before beginning the second

web. Reduction in area and in number of radii are observable, but it can also be

demonstrated lhat central angles and spiral separation are more irregular than usual.

This figure and the following illustrate rather than establish drug eífects; variation

of webs requires statistical analysis of experimental treatment. íFrom WITT, P. N.

BRETTSCIINEIDER, L., and BORIS, A. P., J. Pharmaeol. exp. Ther., iS2, 183, 1961).

A B C

Fig. 1 — Control, treatment and recovery webs, psllocybln. Drug vvas given 12 hours

before web shown in B, dosage 150 mg/kg. Web A is web of previous day, C that

of day following Web B. Reduction in area and number of radii in the treatment

web are apparenl. (From Cl IRISTIANSEN, A., BAUM, RICARDA, and WITT, P. N.,

■/. Phannarol. exp. Ther., 13H, 31, 1962).

Area (cm'-')

293.6

227.3

31'1.5

Number of

spirai turns 35

Numl)er of

radii 36

150 mg/kg psilocybin was given 12 hours before web B was built

SciELO

10 11 12 13 14 15

Mem. Inst, Butantan

Simp. Internac.

33(2); 645-650. 1966

CHARLES F. REED

649

jdacement. Bolh analysis of the coni|)lele(l webs and of ongoiiig huilding suggesl

lhat lhere are stages in cüiistruclioii iii vvhich testing fimclions alteriiate witli

actions taken iipoii tliose tests. A few examples will illustrate lhe ohservations.

The firsl ihreads oí the weh are dependcnl U|)on lhe eonlingencies of localion :

u])on the availahility of fastening points. F.ven in the lahoralory cages, the moor-

ing ihreads differ froni day to day, perhaps heginning from lhe apparently random

residues of lhe previoiis day. One means or anolher will he employed to set

the firsl threads; if an air stream from a fan is directed across the cage, the

s|)ider will let the thread he carried along it rather than lahorioiisly carrying the

dragline from ])lace to place. Biit as constriiction proceeds, it is possihle to

s])eak of typical structnres, where actions are giiided hy a narrower range of

circiimstances than in the initial stages and where it is possihle to ohserve rather

more invariahie events. It is interesting lhat while the spider may he easily

distnrhcd dnring the selling of the initial threads, it is considerahly more difficnit

to do so once the later stages of constrnction have hegnn.

Assume lhat the sjiider has achieved a Y-structure of three threads witli a

sparse perimeter of additional framing threads. Taking a station at the cenler

of ihis plexus or huh, the animal appears to lest existing threads hy circling,

lonching each of the lines with several legs; lhe necessity for an additional radial

thread and/or siipporting stnictures is apparently estahiished during lhese move-

ments, hui it is not clear what sensory data are heing utilized hy lhe animal.

One jjossihility is lhat feedhack occurs from movement of a leg over a cerlain

arc wilhoul contacting thread; anolher possihility is lhat lhe spider is utilizing

some sensory data related to the degree of tension in lhe existing threads. Al

any rate, if lhe first legs on one side of Araneus diadematus are removed, there

are clear and irreversihle disturhances of lhe regularity of thread ]ilacement —

ailhough such an experiment does not allow us lo dislinguish helween lhe Iwo

foregoiug hy|)olhesis.

Depending n|)on lhe results of lhe "tesl”, lhe spider exils from the huh

along an existing thread, pulling a dragline vvhich is thereu])on atlached to the

frame. The spider relurns to lhe huh along the new radial thread. If lhe new

|)lacement is lo occur in the lower ((uadrauls of lhe weh. lhe animal may drop

hy means of lhe dragline instead of crawling: lhere does seem to he an “ojrlion”

and not a fixed aclion-])attern followiug lhe onlcome of lhe [nohing funclion.

There is anolher o]vtion, a kind of suhslage which results not only in an

additional radial line, hnl in an acce.ssory sup])oiiing or framing memher. In¬

stead of relurniug direclly lo lhe huh along lhe new radial line the s|)ider makes

an allachmenl along lhe way. The altachmenl serves lo secure the radial line

a second lime to the frame, and deviales it from its original direction. Bolh

threads may receive additional reinforcement as lhe spider |)asses over them.

The assemhling of lhe radial and frame elements jnoceeds hy this alternation

of testing and placement until lhe sjjider succeeds in making a complete circuit

of lhe hnh wilhoul receiving whatever signal it is lhat reí^uires additional su[)-

ports. The inauguration of the nexl stage is so irnhedded in lhe preceding move-

inenls thal it is dispntahle not only when it hegins hut whelher it is a separale

stage at all. The se(|uence of movements thal have accompanied the radial-lest-

ing continue, hut it is a|)parent lhat lhe s|)ider has also heen altaching thread

to lhe radii as it circied, hridging the central angles. The resull of a com|)lele

Circuit of the weh wilhout need for a radial strncture is lhe heginning of a

light s])iral. Gradually the spider moves faiiher cul from the center; the pro-

1 SciELO

650

SPIDEU WEBS AND FSYCHOTROPICS

visionai spiral lias hegim. This teniporary spiral ajipears only as a Iracp in the

conijilelfd weli: it is (lovoiircd hy llu- s|)id(‘r as il losos ils iisefidncss as a giiide

and su[)porl for the laying of the |)('rmanenl viscid s|)iral.

The permanenl, tacky spiral is constructed in a elearly sejiarahle slage. First

of all. the sjiider jiauses at the coinjiletion of the provisional spiral. The aggregate

glands now ht‘gin to siipply material, and there seems to he a reipiireinent of

time for this glandular shift-over. (It is possihie that the clear pause iu the center

of the vveh, which oeeurs iu the radial hiulding is aiso a period in vvhieh the

gland duct is filling vvith material.)

The laying of the permanent spiral takes up hy far the greater |)roportion

of wehdniilding time: the hasic structures are huild within the first ,5 minutes,

the [lermaneut spiral oecujiies the remaining 15 to 20 minutes. The jirovisional

sjiiral had proeeeded from within outwards and has hrought the spider to the

periphery of the weh; now the work goes from out inwards. Thread-plaeing is

halted short of the huh, leaving an o])en space or free zone hetvveen the inner-

most turn and the huh.

The S[)ider takes u|j its |)osition of vigilanee on the huh. It may remove

some of the threads of the huh hy hiting thein out.

Ks[)ecially elear movie se(]uences of these and other aetivities of A r a n c u s

have reeentiy heen ohtaiued hy Bariium and VVitt. These films have revived the

.speeulation that the ])ositioning of a |)artieular thread is determined hy the array of

forces then e.xtant in the weh, that the spider tugs the threads and resjionds hy lay¬

ing a new thread if the resistanee to the tug falis helow a hy|K)thetieal hut unknown

vahie. In order to test this eonjeetiire, we have heen attenijiting to make films

of the seipienee of structures iu a single weh, with the ohjeetive of testing where

the spider appears to test and iiredieting the jiosition of the next thread. A

siiccessful emulation of the single event of new plaemnent should (lermit a state-

ment of the range of tension to which the spider is sensitive in huilding, if indeed

this is the teehnologieal hasis of its eonstrnetion at all.

Discession

yl. SJiulov: “Is there any correlation between the influence of drugs on the

structure of the web and the changes in web’.s conslruction in time as a result of

mutilation as cutting every one or two tarsi (Dr. Sehieps experiments) ?”

P. N. Witt and C. F. ReecI: “The same structural change may be produced

by different treatment.s. For instance, irregularities of central angles can be the con-

sequenee of losing one first leg or 600 mg/kg d-amphetamine.”

W. Bücherí: “1. In what kind of spiders you made your studies? 2. Have

you used also other species of spiders or only orb-web-spiders?”

P. N. Witt and C. F. Reed: “1. Araneus dUidematun and Zygielía-x-notata. 2. The

orb-weavers have the distinct advantage of iiroducing webs with geometric fornis

susceptible to mathematicat anatysis. Departures from ordinary variation can there-

fore be specified in a way which is not possible with other webs.”

D. Valente: “1. Há alguma correlação entre a feitura das teias e o sistema

neuro-secretor? 2. O ciclo sexual e a idade têm influências na feitura das teias?”

P. N. Witt and C. F. Reed: “1. Endocrine structures have been histologically

identified in spiders, but their function is still unknown. 2. All of our drug experi¬

ments were done with adult female Araneus diadematus, so that the drug would

be the main variabte. Males are irregular builders in later life.”

2 3

5 6

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(2);651-658, 1966

WERNER RATHMAYER

651

67. THK EFFECT OF THE POISON OE SPIDEIi Ax\D DIGCEHWASPS

ON THEIH PREY {HYMENOPrERA: POMPÍLÍDAE. SPIIECIDAE)

WERNER RATHMAYER

Zoologisches Institui. Frankfurt u.M., Germuny

Ainong lhe HYJIENOPTKKA \ve find lhe most so])liistieate(l venonioiis aniinaP.

With regard lo [jotencv I do iiot kiiovv of a slrongei poi.soii thaii thal oí l/ahro-

bracoii jiiglundis, a iH acoiiid wasp, whieh ])aralyzes its ])rey, lhe G a 11 e r i a

larva, cveii at a dilulioii of 1 pari veiiom in 200,000,000 ])arts of hemolymph (ll.

AEo willi regard to duralioii of ])aralysis lhe ])redaceoiis vvas])s come first: a

sj)ider sOmg hy lhe pom])ilid Cryptochilus uj/ini.s lived for foiir monlhs iii dee])

paralysis (2). Otie woiiders vvhy ])hysiologists and j)hannacologi?ts liave so far

takeii liltie interest iii tlie solilary wasps.

I wiil confine myself in lhe follovving lo lhe POMPILIDAE and SPHECIDAE.

Wilh llieir sting ap])araliis llie females of lhese families inject venom into llieir

])rey whieh residis in paralysis of varying dnralion. The iiomjdlids hunl exclnsivelv

for siiiders. On lhe lisl of prey of lhe SPHECIDAE we find all orders of inseels

ano sjiiders loo. Imnioliilizalion of lhe prey is ilways related to eare of lhe off-

S])ring. An egg is laid on lhe viclim and lhe haldiing larva feeds on lhe liv¬

ing ])rey.

The niain snhjecl of ihis pajier is lhe ])hysiologieal effecl of lhe poisou.

The dnralion of jiaralysis jirovides a firsl elneld). When we look al lhe pom-

jdlids we find ihree calegories of paralysis: 1. In lhe mosl ]irimilive case re-

])resenled hy some memhers of lhe genns II o lu o n o I u s lhe females [laralyze

s])iders, lay an egg on lhe viclim hnl hury lhem nol in lhe groimd. According

to (4,51 all lhe sjiiders reeover a few miniiles aflei lhe sting and resume lheir

regular aclivity. The larva lhal halehes a few days later wili feed on lhe sjiider

and kill il. 2. females of lhe genus Anopliiis also suhdue spiders hy sliug-

ing. 1 hey hury lhem aflerwards in chamhers in lhe ground and altaeh an egg.

If one digs lhem oul one finds thal jiaralysis lasls much louger lhan wilh Hu-

tu (> ll o t u .s, neveiiheless lhey usually reeover after a few hours. In lhe chamhers.

however, lhey are. even after reeovering. imahle lo move hecause lhey are

jiaeked so tighlly. Temporary paralysis is also re|)orled for N o t o c y p h ii s (6).

3. AIosl of lhe |)ompilids paralyse lhe s|)iders dee|ilv and permanently. The

])rey remains fresh, i.e. il is nol desieeated or eonlaminated for weeks. (ierlainly

lhe vietims live dnring this lime.

The striking facl is thal lhe prey slung hy cerlain jiompilids is aiways ahie

lo reeover, lhal of others is pafalysed deejily and irreversihly. Since lhe stinging

The recelpt of a travei grant by the Deutsche Forschungsgemeinschaft i.s gratefully

acknouleilged.

SciELO

10 11 12 13 14 15

652

THE EFFECT OF THE POISON OF SPIDER AND DIGGERWASPS ON

TllEIR PREY (HYMENOPTERA: POMPILIDAE, SPHECIDAE)

heliavior is very similar iii tlie whole faniily (7) oiie may assiinip tliat we liave

to (leal willi (jiialitative or qiiaiililalive differetiees iii the eom|)osilioTi of the

poisou or tlial differeiit pliysiological systems are affeeled. Kxjrerimeiits oii tliis

poiiil are niiieh iieeded. 1’aralysis lasling only a fevv minutes eould lie caiised

liy an instaliility of the active compound of the venom. The ohserved differeiices

iii diiration of paralysis among memhers of the group that normally paralyse ir-

reversihly ean he attrihuted to the amoimt of poisou iujeeted as ex])eriments vvilh

a sphecid wasp, re|)orted later, show (8).

How aud where the poisou of pompilids acts iu the orgainsm of the ])rey

is eulirely uuknovvu. There is oue paper reportiug that the veuom gets iuto the

hemolvmph of the prey(9). Teuetratiou of the stiug iuto the central uervous

System therefore seems not to he a prere((uisite for jiaralysis aud prohahiy does

iiot happeu iri lhe field.

To sum up, the veuom of lhe POMPILIDAE has to he hroiighl iuto the

hemolymph of a s])ider aud it paralyses the motor-system. There are iiilerestiiig

geiius-sj)ecifie differeuces iu regard lo duraliou and completeuess of ])aralysis.

Whelher lhey are due lo rpialitative or fuuclioual differeuces of lhe iujeeted

veuom stili has to he eslahlished.

Novv let us eousider lhe SPHECIDAE. In ihis family we also find differeuces

iu duratiou aud com[)leteuess of paralysis. Molecrickets stimg hy L u r r u re-

cover aiways .some minutes after stiiiging f 10, 11 I. Crickets slored hy Sphcx

lobulus also recover 10 to 15 minutes after heiug stuug as well as the viclims

of some species of Li ris and No togou ia. These examples, hovvever, and

the a|)pareul killiug of the |uey hy the venom of uiany IloMHlCINl (12,15),

.4 s í a I a sj)ecies ( 14, 15) and some Ckabkonim are exce|>lional. Delay hy mauy

hours of onsel of ])aralysis is reported for crickets stung hy Liris nigra{\6)

and for auts hunied hy Aphilanlhops jrigidus (17). The greal hulk of lhe sjihecoid

wasps hovvever paralyse lheir prey dee|)ly and irreversihlv.

I |)erformed ex])eriments with l'liilaulhiis Iriangiili/ui Wi) vvhich provided

a first chie as lo the effeci of lhe poisou of ihis species ou its prey, lhe hon('y-

hee. In mauy cases I eould walch lhe stinging direclly. The hee is alvvays

slimg oídy onee iuto lhe ciitaneoiis memhrane hehiud lhe first jiair of legs I Fig.

1). Histological seclions demonsiraled that lhe stiug iu most of lhe cases in-

vpstigaled did iiot pierce lhe veniral nerve, cord. Oue caii have hees stuug hy

L li i I a u I li II s exjierimeulally al auy desired poiul hy cultiug a small vvindow

iuto lhe cuticle aud placiug the stiug there. Il vvas shovvu that it is siifficieul

for paralysis that lhe veuom luerely gets iuto lhe hemolymph. The furlher lhe

poiul of [)unclnre is from lhe locomolor-sysiem lhe longer it takes uiilil lhe legs

cea.se lo move (Fig. 2). The natural |)oiul of jmnclure hehiud lhe first pair

of legs guaraulees the (piickest ousei of paralysis. Fvideuce vvas ohtaiued as to

lhe mechauisrn of immohilizaliou. There are Ivvo íacts iudicatiug that [varalysis

is not due lo a central adiou ou the uervous system hut ralher lo a peripheral

hlocking. The immohilizatiou does noi stari inslaustaueously hy hlockiiig lhe

movemeuts of all parts of the legs hui spreads progressively lovvard lhe periphery.

The distai ])arls of lhe legs cau slill move al a lime vvhen the proximal oues

are aiready immohilized. Siuce all muscles of a giveu leg are innervaled from

lhe same maiu nerve truuk leaving lhe correspouding thoracic gangliou aud siuce

lhe cell hodies of the moloneuroues lie closely logether al lhe periphery of lhe

gatigliou, a central adiou of the veuom should had to a uearly simullaneous

hlock or iuterruplion of the effereni iuuervatiou of lhe various leg muscles. This

SciELO

10 11 12 13 14 15

WERNER RATHMAYER

653

Mem. Inst. Butantan

Simp. Internac.

33(2):651-658, 1966

marked lime delay can lie ex|)lained hy lhe anatomy of lhe insect leg. The

miiseles for movenieiit of lhe coxa and feimir lie in lhe hody cavity of lhe hee

and lherefore wili he reached l)y lhe venom firsl. The muscles for lhe tihia are

allaehed in lhe leg itself, lhe ones for lhe metatarsiis lie still further dislally.

Jl will lake lime imlil via lhe hlood eirctdalion enoiigh poison has enlered lhe

narrow leg and lherefore \ve encoimier lhe delay in onset of paralysis.

Fig. 1 — Positlon of the dlggervvasp Phtliinthus triangulum

whon attacking and stlnging, behind lhe firsl palr of legs of

ils victim, the honeybee.

A second ohservalion |)oinls in lhe same direclion. Wilh local adminislralion

of small doses of venom il is possihle lo ])aralyse muscles vvhieh are innervaled

hy lhe verv same ganglion as for example lhe muscr.lalure of the forevvings with-

oiil affecling lhe muscles of the middlelegs. Again one comes to lhe conchision

that ihis shonld he im|)ossihle hy an effecl of the venom on lhe CNS of the prey.

It was shown further for lhe venom of I'h i I a n I h ii s thal il acis nol

S])ecifically on the honeyhee hut ralher paralyses all tested insecls and s])iders

loo (Tahle 1). The completeness of jiaralysis varies wilh lhe amoimt of poison

injected. Wilh little doses lhe liees can comjilelely recover after initial im-

SciELO

fic;4 THE EFFECT OF THE EDISON OF SPIDER AND DIGGERWASPS ON

THEIR PREY (IIYMENOPTERA: POMPILIDAE, SPHECIDAE)

Flg. 2 — The variatlon of onset of paralysls of a honeybee’s legs

when venom of Philanihus was injected at points 1, 2 or 3.

mobilizalion. For a differetil spccii-s (19), demonstralcd lhat duralion and coni-

plcteriess of paralysis varied willi llin niimlaT of .dings a])plied. I lliink in Uns

case it is aiso an effecl of tlie amoiitil of venoin injected.

Wliellier lhe resnlt llial lhe sling of F li i I u n I li ti s iisnally does noi reach

lhe CNS may l)e generalized lo olher si)ecies of spliecids reqnires hislologic'al

sludies of llie |(aralysed |)rey. The |)i'eeision willi which Liri.s iiig,ru ap|)lies four

stirigs to lhe cricket (19) and lhe fact lhal lliey are placcd in dose topograi)hical

rclalion lo lhe snhoesophagcal and lhe ihree thoracic ganglia leads lo lhe as-

sumption lhal in this case lhe venom has to l>e hronght vcry dose lo lhe nervons

centers responsilile for locornolion. A varialion of lhe mechanism of innnohilizalion

and aIso in lhe effecl of llie poisou vvonld not hc snrprisitig when one ihinks of

lhe greal nnmher of different insect families nsed as prcy.

SciELO

10 11 12 13 14 15

Simp. Internac.

Mem. Inst. Butantan

33(2);651-658, 1966

WERNER RATHMAYER

655

TABLE I

EFFECT OF INJECTED VENOM OF PHILANTHUS TRIANGULVM ON

VARIOUS ARTllROPODS

ORDER

Xo. of families

tesled

No. of genera

tested

No. of species

tested

Effect

INSFXTA:

HYMENOPTERA

LEPIDOPTERA

COLEOPTERA

ORTHOPTERA

HEMIPTERA

ODONATA

p,<l

DIPTERA

P, (1

AHACHXIDA

p = paraiysis, (i = after initial paralysis apparentiy cleaci

As \ve iiave seeii. lecovery after initial j)ai'alysis is also ])ossil)le in the

SPHECIDAE. One cannot inulerstand that in tliese cases either inechanical injnry

of the ventral nerve eord hy the intruding sting or a heavy degeneration of the

CXS as (20) elaiined for cater|)illars paralysed liy A rn m o p li í I a can ha[)j)en.

Most prohahiy. hlocking of the loeoniotor-system is also peri|)herally hy S])reading

of the ])oison throiigh the lieniolyniph to tlie effeetors. This leads to a ])oinl of

great ])hysiologieaI significanee: is it the exeitaliility of the mnsele, the nenro-

mnsenlar transniission at the jnnetions or the inipnlse propagation at the ])eripheral

axons tliat are affeeted? In onr lahoratory ex|)eriinents are in progress to stndy

these cpiestions in detail. Inlracellnlar reeording with microeapiliaries from ventral

hody mnscles of Eph estia larvae ])aralysed hy Bracon serinope show that lhe

nienihrane polential after 2 or H days is slill hetween 43-65 mV eompared with

12-71 mV of normal mnsele fihers. The exeitahility of lhe mnselefiher memhrane

therefore stili exists. Meehanically stimniated. the imiselefihers of |)aralysed larvae

can contract. It is, however lo early at the moment, to reporl more details on

these experiments. Hecentiy l’iek(21) shovved that the extracelhdarly recorded

action ])otential of museles of moth larvae declined and was finally aholished

when venorn of lhe hraconid Microbracon hcbetor was injected.

A few fnrther ohservations shonld he mentioned which confirm lhe highly

S])ecific effeci of the s])hecid veiiom on lhe locomolor syslem. The heart mnscles

of the hee are nnaffected hy the venorn of B li i I a n t li u s. The heartheats of

a completely paralysed hee continues np to 37 honrs. It is inleresting to note

that lhe frecpiency of heating is no longer regular hui the hearl heats in irregular

hiirsls. Since it is gcnerally assumed that the heart of adnll insects is regnlatcd

hy a neurogeiuc pacemaker it is lempting lo assume that the venorn affccts this

pacemaker and inijiairs neuromuscniar transniission, nol however the myogenie

aniomatism.

SciELO

10 11 12 13 14 15

fiCTfi THE EFFECT OF THE POISON OF SPIDER AND DIGGERWASPS ON

THEIR PREY (IIYMENOPTERA: POMPILWAE, SPHECIDAE)

The cligestion of food iti the giit is iiol stojjped iii ])aralysed caterpiliars as

the discharge of small fecal ])ellets, eveii several weeks afler heing slung hy

A m rn o [) li i I a, shows(22, Hathniayer iin|)uhlished ). The weh-spiiiiiiiig ahility

of j)aralysed spiders still is |)resent (21) demotistrated lhal the metaholism measiired

as the oxygen consumption of paralysed insects does not differ niuch froni thal

of a normal starving insect of the saine s])ecies (Fig. 3).

Fig, 3 — Oxygen consumption of caterpiliars paralysed by AmmophlJa campestris

compared vvith that of normal starving caterpiliars (after NIELSEN, 1935).

With the assiim|)tion that vve may draw a coinmon coiiclusioii from the

experiments vvith h i I a n t h u s and the ohservations on the very few other

species stndied till now, the following jioint is reached. The venoin of the s|)hecid

wasps acts not on the CNS of the jirey hut peripherally hy a selective inhihitioii

of the locomotor-system. By the current electrophysiological ex|)eriments 1 hope

to locate this perijilieral site of aetion in the near future.

BeFEREiXCES

1. BEARD, R. I., A(jr. exp. Sta. Buli., 562, 27. 1952.

2. MINKIEWICZ, R., Polsk. Pismo Entomol., 13, 43-60, 1934.

3. EVANS. H. E., and YOSHIMOTO, C. M., Misc. Piibl, Entomol. Soc. Amer., 3,

65-119, 1962.

4. IWATA, K., Anvot. Zool. Jap., 13, 305-317, 1932. *

5. RICHARDS, O. W., and HAMM, A. H., Trcnis. Soc. Brit. Entomol., 6, 51-114.

1939.

SciELO

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Mem. Inst. Butantan

Simp. Internac.

.33(2):651-658, 1966

WERNER RATHMAYER

657

6. CLAUSEN, C. P., Entomophagous Insects, McGraw Hill Book Co., New York-

London, 1940.

7. EVANS, H. E., System. Zoo/., 2, 155-172, 1953.

8. RATHMAYER, W., Z. vergl. Physiol., 45, 413-462, 1962.

9. RABAUD, E., C. R. hebd. Seanc. Acad. Sei. Paris, 165, 680-683, 1917.

10. WILLIAMS, F. X., Hawaii. Sugar Planters Ass. exp. Sta. Buli. Entorno}., Ser.

19, 1928.

11. IWATA, K., and TANIHATA, M., Jap. Trans. Sitikoliu Entomol. Soc., 7, 101-

105, 1963.

12. EVANS, H. E., Studies on the comparative ethology of digger wasps of the

genus Bômbix, Comstock Publ. Assoe., Ithaca, N.Y., 1957.

13. EVANS, H. E., and GILLASPY, J. E., Amer. Midland Naturalist, 72, 257-280,

1964.

14. PECKHAM, G. W., and PECKHAM, E. G., Wisc. Geol. Nat. Hist. Survey

Buli., 2, 1-245, 1898.

15. EVANS, H. E., J. N.Y. entomol. Soc., 65, 159-185, 1957a.

16. STEINER, A., C. R. hebd. Seanc. Acad. Sei. Paris, 247, 150-152, 1958.

17. BEARD, R. L., Ann. Rev. Entomol., 8, 1-18, 1963.

18. RATHMAYER, W., Nature (Lond.), 196. 1148-1151, 1962a.

19. STEINER, A., Ann. Sei. Nat. Paris, Ser. 12, 4, 1-126, 1962.

20 NIELSEN, E. T., Videns. Medd. Dansk. naturhist. Foren. Kopenhagen, 99, 149-

231, 1935.

21. PIEK, T., J. ins. Physiol., 12, 561-568, 1966.

22. EVANS, H. E., Amer. Midland Naturalist. 62, 449-473, 1959.

SciELO

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