roxkaa, vo1.17,pp 6t-67.

ao41-0fo1~79~oto1-0ostsos .oorom Peraamon Prees Ltd. 1979 . Ikinted in Great Brftain .

REPETITIVE MUSCLE RESPONSES INDUCED BY CROTAMINE

OSWALDO VITAL BRAZIL, JULIA PRADO-FRANCESCHIand *CARLOS JULIO LAURE

Departamento de Farmacologia, Faculdade de Ciências Mbdicas, Univetsidade Estadualde Campings, Süo Paulo, Brasil and *Departamonto de Bioqulmica, Facuhiade de

Medicine de Ribeiräo Preto, Silo Paulo, Brasil

(Acceptedforpublication 3 May 1978)

O . Vrr~r. BRAZIL, J. Pg~no-Ftulvcasc>~ and C . J. L.tuxe. Repetitive muscle responsesinduced by crotamine . Toxicon 17, 61-67, 1979.-The sustained contractions of the doetibialis anterior muscle and the tat ggstrocaemius which may appear after a tetanus or twitchor spontaneously in crotamine injected animals aro associated with the discharge of highfroque~ncy and small amplitude potentials . They are, therefore, asynchronous tetanic con-tractions . These potentials are similar to those observed in myotonia. The potentiatod maximaltwitch observed in crotamine injected dogs is actually a brief tetanus.

INTRODUCTION

CROTAbIINE is a basic polypeptide toxin purified from the South American rattlesnake(Crotalus durissus terr~cus) venom (GoNçALVFS and VIEIRA, 1950 ; GoNçALVES, 1961).Contrary to crotoxin, the main neurotoxin from the venom of this subspecies (VrrALBRAZIL, 1972), crotamine is only present in the venom from specimens collected in certainregions of South America. Mice, rats and dogs, injected with crotamine containing venomor with pure crotamine, show at first involuntary persistence ofvoluntary contractions and,afterwards, continuous or nearly continuous skeletal muscle stiffness. Crotamine containingvenom or pure crotamine causes the following effects in isolated or in situ neuromuscularpreparations (BARRIO and VTfAL BRAZIL, 1951 ; CHEYMOL et al., 1971a, 1971b) : (1) a delayin relaxation and/or an after-contraction following a response evoked by indirect or directmuscle stimulation with single or tetanic pulses ; (2) a sustained contraction (usually afterlarge doses) ; (3) potentiation of the maximal twitch ; (4) depression of the responses toindirect and direct muscle stimulation. The present work was planned to determine if theeffects referred to in (1) and (2) are contractions or contractures . The only satisfactorycriterion for discriminating between them is the existence, in the case of a true contraction,of action potentials in association with the prolonged tension (BROWN and HARVEY, 1939).We also investigated whether the potentiated maximal twitch is actually a brief tetanus .

MATERIALS AND METHODSThe experiments were carried out on 15 dogs and 10 rats anaesthetized with sodium p~tobarbital

(30 m8 per kg, i .v. for dog, SO mg per kg+ i .p . for rats) . In all experiments the electrical and mechanicalactivities of the muscle (tibialia anterior muscle of the dog, ius of the rat) wero simultaneouslyrecorded . The dogi were prepared as previously described (VrrAi BRAZII ., 19C~. The contractions andtension of the tibialis anterior muscle weae recorded, with a flat spring steel myograph (Bnowrt-Scnvsreamyograph stand) . For electrical recording two pin electrodes in connection with a Tektronix Type 122 pro-amplifier were inserted in the tibialis anterior muscle approximately 1 txn apart from each other. Theelectrical activity of the muscle was displayed on a Tektronix Type SODA oscilloscope and photographedwith a Tektronix Type G12 photographk camera . Lv . administration of crotamine and other drugs weremade through a polyeti>,ylene cannula inserted into the femoral vein of the opposite side of the prepared

61

62 OSWALDO VITALBRAZIL, JULIA P1tAD0-FRANCESCHI andCARL03 JULIO LAURE

Ilmb. The trachea was cannulated and artificial respiration applied to dogi igjected with gallamine tri-ethiodide (F7axerül Rhodia). For direct muscle stimulation needle electrodes were inserted into the bellyand near the tendon of the muscle. Peroneal news stimulation was carried out with supramaximal shocksof0 "2meec duration, delivered in two experiments at a rate of0"113z. Ice other eapaimeata stimuli ofhigher(50 Hz) frequency wero used for short (usually 10-20 sec) periods. Direct muscle stimulationwas made withpulses of2 msec,100 Vand 50 Iiz (Grass Model 34stimulator). Icethree earpaiaoents, mechanical atimulatIonwasperformed by strildng tiu skin over the tibialis anterior mordeor its tendon with a percussion hammer.Three mq nb wa+e carried out on dogi with the tibialis anterior mmde ehrn®ically denervated. Elevento twelve days before the experiments the animals woro anaesthetized with sodium pentobarbital (30 mB/kgi.v.) and under aseptic precautions one ofthe peroneal nerves wee cat.

The divided Achilles to~on of one of the legs of anaesthetized rats (240-300 B) was connected to anisotonic lever. Tho hind leg was immobilized by means of a clamp on the limb near the ankle. Electricalactivity of the gastrocaamiua was recorded as described above. In four rats the peripheasl stump of the cutsciatic nerve was placed on platinum electrodes and atimalabed with maximal shocks of0"5 msx durationand 0"1 I3z. In 6 rats, whose sciatic nerve was divided 10 ormore days before the exPaimenta, direct musclestimulation was made with pulses of100 V, 2cosec and0"1 Az.

Crotamine was propared from a pool of Crotalus drrrlssur terr(/tcus venom by one of us (C . J . Laure)using a procedure similar to thatemployed by Goncalvea (Crn:vesoL a a1.,1971a). The i.v. r~,o ofcrotaminein mice was 1"5 (1 "01 to 2"Z3) mg per kg. I~w deieamination was carried out in groups of six mice (17 to21 g) . Four dose levels spaced in geometric pmgrossion (common ratio 1"~were employed . The number ofdeaths was recorded after 24 hr and the ~,o and its 95~ oo~de~ce intervals calculated by the veil pro-cedure (1952) . Crotamine wsa adminiabeaed i.v. to rats in a dose of 0"3 mq/kg (C~YAlOL et al., 1971x) .Preliminary experiments showed that dogi were much less aeasitive to the toxin, so that a dose of 1 mB/kgwas sew for these expalmenta .

RESULTSAttar i.v . injection ofcrotamine (1 mg/kg) in dogs, nerve stimulation with suprar+?A.=imal

shocks of 50 Hz evoked a tetanus which was followed by a long-lasting shortening of thetibialis anterior muscle (Fig . lc) . During this after~ontraction discharges of potentials ofhigh frequency and small amplitude, usually 50-100 ~V were observed in all experiments(Fig. 1-IIn. In dogs which were injected with gallamine (5 mB/kg, i.v.), direct musclestimulation with supramaximal shocks evoked the same phenomena after crotamine ad-ministration, that is, an after-contraction associated with discharges of high frequency andsmall amplitude potentials . On the other hand, stimulation of the chronically denervatedmuscle wasnot followed by an after-contraction . Mechanical stimulation of the innervatedtibialis anterior muscle did not evoke prolonged discharges of potentials .

In nearly all experiments, no spontaneous shortening of the innervated tibialis anteriormuscle occurred following the injection of crotamine, however, fibrillation potentialsalways appeared (Fig. 1-In. In two out of three experiments a few min after crotamineinjection the denervated tibialis anterior muscle developed a slow andprolonged contractionassociated with high frequency potentials of about 50 ~tV.

Crotamine produced a considerable increase in the tension of the responses of the dogtibialis anterior muscle to single maximal shocks (Fig . 2A, B) and also a slight effect onrelaxation (Fig . 2A). Electmmyography revealed more than one action potential duringthese responses (Fig. III, III) . Calcium (6 ml of a 10 ~ solution of CaC12) antagonizedboth the mechanical and electrical effects (Fig. 2B,M.

In rats injected with crotamine, indirect stimulation of the gastrocnemius with singlemaximal shocks elicited twitches ofnearly normal siu followed by a delay in relaxation andan after-contraction . During theperiod in which the muscle remained contracted there wasdischarge ofhigh frequency potentials of small amplitude (25 to 50 ~V). Denervated gastro-cnemü stimulated directly showed similar mechanical and electrical phenomena (Fig . 3),but the amplitude of the potentials was smaller

DISCUSSIONThe nature of the sustained contractions which develop under the influence of pure

Muscla Contractions by Crotamiaa

b3

] SOICVu50msec

Fto. 1 . MYOeueNt ~uro

oB noo~~ ~vraxtoa MvecrE srn~n,~ren wtSO par sac.

Dog (6 .2 >~ was anaathetized with sodium pentobarbital Upper photographs shows myo-gams. The tetani is AsadCwas elicited by nerve stimulation with wpramaximal ahoclm ofSO Hz for 10 sec. Band C, S sad 6 minatecrotamine administration (1 mg par kg, i.v.). Atpoonni >~ II sad 1Tt photographs wastaken oftheawlactivity of the mueda displayed inthe scram of the oedllosoope . Ca]Ibration : 30 sec. Lower photograph : electrical activity

from the tibhlis anterior muscle. Cah'bration : 50 ~V, SO msac .

crotamine or cfotamine~ontaining rattlesnake venom is controversial. BARRIO and Vrrwt,BRAZIL (1951), for instance, considered them veratrinic type responses and therefore,repetitive in nature, while Moussatch~ and co-workers (MovssArca~ and V18ntA, 1953;MovssATCrné et al., 195 suggested that they are induced by aninterference inthe glycolyticcycle of the muscle, that is, by the same mechanism as the contracture called a"Lundsgaardeffort" which is produced by subatanas such as iodoacetateand some vesicants (BAOQ andGOFFART, 1940a, 1940b). In support of the Barrio and Vital Brazil point of view were thefollowing results of their investigation on the muscular and neuromuscular actions of theSouth American rattlesnake (Crotales derlssus terr~cus) venom: (1) Themechanical effectevoked by single shocks to the gastrocnemius of rats injected with crotamine containingvenom was very similar (although not identical) to the classical veratrine mechanicalresponse: of frog muscle . (2) Crotamine containing venom and veratrine produced thecharacteristic response in curarized or denervate:d gastrocnemü muscles stimulated

64 OSWALDO VITAL HRAZIL,~JULIA l'I'ADO-1?RANCESCHI and CARLOS JULIO LAURE

~O.SmVu5msec

CiV. T. MYO(ilAlll AND

OF DOO TIH7AI~8 ANIBATOa MU6CiB ôr~IITLAIFD AT6 per min.

Doer (8"3 ~ was anaeathetmed with sodium pentobarbital . Upper record shows a myograma~f tibialis anterior muscle stimulated with supraafaximal shocks of 0~1 IIz In'~rotsmina"(panel A), the toxin was administered i .v. (1 mQ Per~: in "CaClz" (panel B), 4 ml and 2 mla~fa 10y, solution of caidum chloride were igjected i.v. At points I, II, III (panel A) and IV(panel B), photo~aphs were taken of the muscle action potentials. Calibration : 30 sa.Lower photo~aph : ekctrkal activity of the tibialis anterior muscle indirected stimulated

with single maximal shocks. Calbrations : 0"S mV, 3 msx.

directly. (3) The substances (calcium and magnesium ions, quinidine) which are known toantagonize the effects of veratrine also counteracted the crotamine response . Furthermorequinidine (LAMMBR3 and ltrrcl~, 1955) and quinine (HARVEY, 1939), by increasing themuscle refractory period, may counteract any repetitive response . For instance, in thepresence of quinine (HARVEY, 1939) or quinidine, the ability of a muscle to respond to atetanus is diminished and the anticholinesterase drugs and veratrine are no longer able toevoke a repetitive response to a single stimulus .

Under the action of crotamine both the sustained shortenings and the potendatedresponses to single volleys of impulses are repetitive in nature and, therefore, true con-

Muscle Contractions lry Crotamine

65

50~.Vu50msec

I?oo. 3. MYO(iRAt,! Arm ~Yaat+~ of Tas vsrielevATSU aAarrtocrl8rpt>s or nAT

a~T~D~d~IJLATBD AT Ô per lain .ItYt (300 ~ anaestheti2ed With OWllim pentobarbital. i~a3tOCAemlua d~CiYattd iâ d~cattier. Upper photograph : myo~ram of ~aatrocnemius stimulated with shocks of 100 V, 2cosec, 0"t Hz. In "Gtotamina", the toxin was injected i.v. (300 ~lxr k~. At pointa I and II,ptwtographs were taken of the muscle electrical activity . Calibration : 30 sec. Lower photo"

Graph: electrical activity from the gastmcllemius. Calibration : SO NV, SO cosec.

tractions . The sustained contractions of the dog tibialis anterior muscle and the rat gastro-enemius which appearod after a tetanus, a twitch or spontaneously in crotamine injectedanimals are asynchronous, tetanic contractions . These crotatnine responses are electro-myographically similar to those observed in the myotonias. Cratamine intoxication canindeed be regarded as an experimental myotonia although presenting some peculiar featuressuch as spontaneous discharges of potentials (ßbrillation and myotonia potentials) and lowsensitivity to mechanical stimulation. Nevertheless, the fundamental symptom of themyotonias---failure of the muscle to relax normally due to high frequency discharge ofaction potentials--is observed in crotamine injxted animals. Experiments carried outrecently in this laboratory (PBr r.Eaxixt,1976) support this point of view. They showed thatclinical signs in normal goats injected with crotamine arc very similar to those of animalssuffering from goat congenital myotonia (Kot.s, 1938).

Myotonic symptoms occur in some human and animal hereditary diseases . It can alsobe evoked by drugs such as veratrine, 2,4-dichlorphenoxyacetate (EYZ~aurnB et al., 1948)

66 OSWALDO VITAL BRAZIL, JULIA PRADO-FRANCESCHI and CARLOS JULIOLAURE

and 20,25-diazocholestcrol (Wn~x et aJ., 196 . The abnormality of the muscle fibermembrane which underlies the myotonic phenomenon does not seem to be the same in allof these conditions . In myotonia congertita of human beings and goats (Lrnlc>nt et a1.,1971 ; LIPICIIiI and BRYANT, 1971 ; B1tYANT and Mox~r~-Aau>1~1tA, 1971) as well as in themyotonia evoked by 2,4-dichlorphenoayaoetate and 20,25-diazocholesterol (Run>a. andS$rta>3s, 1972) the repetitive responses are due to a decrease in membrane chloride con-ductance . On the other hand, the myotonic phenomenon is associated with an increase ofpermeability to sodium ions in dystrophia ngyotonica \""OFIIANN et eil., 1966), episodicadynamia (Cx r n et e7L, 1963) and in muscles exposed to veratrine (~Axx, 1958) .Crotamine myotonia also appears to be caused by as increase is sodium conductance(PSLLECiRIIQI, 1976; 1?EC.r.>~Rnvl et a1., 1977) .

Acknowlalgement.~This workwas wpported by s orant (69/334) 4nmthe Fuadaç8o de Amparoè Pesquisado Estado de Sâo Paul (FAPBSP').

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