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Neuromuscular blockade of chick biventer cervicis nerve-muscle preparations by a fraction from coral snake venom
Taxiton, 1973 . Vol. li, pp. j05-S0ß, pergamon Press. Printed in Great Britain.
SHORT COMMUNICATION NEUROMUSCULAR BLOCKADE OF CHICK BIVENTER CERVICIS NERVE-MUSCLE PREPARATIONS BY A FRACITON FROM CORAL SNAKE VENOM G. K. Srnvsa,* H. W. RAlHSmr, W. J. TAYLOR and C. Y. C>:nou Division of Cardiology, Department of Medicine, and Department of Pharmacology, University of Florida College of Medicine, Gainesville, Florida 32601, U.S.A . (Acceptedfor publication 14 .luire 1973) ALTHOUGH elapid venoms in general, and coral snake (Micrurus f. fulvius) venom in particular, contain many active principles, their lethal component is generally classified as neurotoxic (L~, 1970 ; M11tANDA et al., 1970). This is based on evidence presented by several authors that elapid venoms induce a curare-like nondepolarizing neuromuscular blockade (Jut~tvl:z-Poiuus, 1968) and lethality studies in which the experimental animals developed flaccid paralysis (RAMSEY et al., 1972). In this report we describe the neuromuscular blocking action of a protein fraction isolated from crude coral snake venom on chick biventer cervicis preparations. The results obtained are in agreement with the thesis that the active fraction of coral snake venom, like that of other elapid and sea snake venoms, may be characterized as neurotoxic. Coral snake venom was fractionated using a carboxymethyl cellulose column and eluting with phosphate buffers (RAMSEY et al., 1972). Fifty mg samples of lyophilized crude venom (Ross Allen Reptile Institute, Silver Springs, Florida) in 0~2 ml starting buffer were eluted through 1 x 20 cm columns of CM 32 (Whatman, 1 "0 mg per g dry), equilibrated with 001 M phosphate buffer, pH 6 at 2-4 ml per hr . The pH and molarity of the starting buffer were increased as a continuous gradient with 0~1 M, pH 8 followed by 0" 5 M, pH 9 phosphate buffers. This contrasts with the stepwise addition of these buffers reported previously. The peak fractions, determined with an inline LKB `uvicord' absorptometer and confirmed with a Zeiss spectrophotometer, were pooled, desalted by passage through a 1 "3 x 85 cm column of Sephadex G-10, lyophilized, and stored at -10°C in sealed vials. The biventer cervicis nerve muscle preparations were isolated according to the methods of GIN3HORG and WAjtlu~tt (1960) as modified by Ciuou (1970) and LorrG and CHlou (1970). Baby chicks weighing 100-300 g were sacrificed with chloroform. The biventer cervicis nerve and muscle were dissected and mounted in a 40 ml organ bath filled with Tyrode solution (NaCI, 8"0; KCI, 0~2 ; CaClB, 02 ; MgClB, 0" 1 ; NaHCO 1 "0 ; NaH,PO 0"05 ; and dextrose, 2~0 g/1.), oxygenated with 95 per cent Og and 5 per cent CO g at 37°C . Dose response curves of the muscle preparation to acetylcholine (ACh), following incubation " Dr. Gregory K. Snyder is presently Assistant Professor, Department of Biology, University of California, Riverside, California 92502. 505
TOXICON 1973 VoJ. 11
506
G. K. SNYDER, H. W. RAMSEY, W. J. TAYLOR and C. Y. CHIOU
with various selected concentrations of the crude venom or venom fraction for 1 hr, were determined using a superfusion technique (Cxtou and Loxc, 1969). The preparation was superfused with Tyrode solution oxygenated with 95 per cent O$ and 5 per cent CO$ at 37°C at 3-4 ml per min maintained by a peristaltic pump (Buchler, Fort Lee, N.J.). The dose response curves were determined as the ratio of the ACh response after incubation and the maximum preincubation response expressed as a percentage . All muscular contraction responses were recorded by attaching the tendon with motor nerve to an isometric force transducer (Myograph, B, E & M Inst. Co., Houston, Texas) . Where indirect stimulations were used, the tendon with nerve was passed through an encasing electrode and interrupted tetanic stimulations of 20-40 V and 5 msec duration applied at a rate of 12 per min. We have previously reported on the fractionation of coral snake venom into seven fractions-one heterogeneous and six homogeneous-{R~MS~r et al., 1972). The separation of the crude venom into separate protein fractions is, with one exception, the same as previously reported . The continuous gradient elution utilized in the present study resolved the previously heterogeneous fraction into three distinct elution peaks (see Fig. 1, RAMSEY et al., 1972). The second elution peak of this group, referred to here as neurotoxic (IVT), produced the flaccid paralysis in mice characteristic of the crude venom, showed the greatest activity on chick biventer cervicis nerve-muscle preparations, and lacked the depolarizing activity characteristic of crude venom and certain venom fractions on isolated heart preparations (RAIrtSEY et al., 1971). Consequently, this NTfraction was selected for use in the present study. ta~
CrOds v~nom
FIO. I.
EFFEC75 Os ]O EIg OF CRUDE OORAL SNAKE VENOM (A) AND I EIg OF CORAL SNAKE NEUROoN NEUROMUSCIlwe zRANS~n.~oN nv c>FncK sn+eNrm cERVxas FREeARA~noNS.
~roxu~ (B)
The action of the crude venom on the chick muscle preparation was typically characterized by an augmented contraction amplitude with progressive failure of relaxation. Secondarily, the elevated contractions diminished until the muscle arrested in partial or complete tetany, usually within 60 min (Fig. lA). Fraction NT (1 lIg/ml) on the other hand, produced neither an augmented contraction nor depolarization response (Fig. 1B). In this case, following a 15-20 min period during which the contractile response appeared unaltered, the amplitude of muscle contractions was progressively depressed and ultimately abolished. The time required for the onset of the diminished contraction response and eventual complete cessation were comparable for the crude venom and the NT fraction. ïn both cases the response to indirect stimulation was not recovered by repeated washing with Tyrode solution ; however, after treatment with NT, the muscle remained fully responsive to electrical stimulation. TOXICON 1973 Yol. II
Neuromuscular Blockade
507
The ACh dose-response curve was progressively shifted to the right with increasing NT concentration (Fig. 2). As with the response to indirect stimulation, the ACh response was not recovered by repeated washing with Tyrode solution . The NT blocking action was prevented when 20 llg per ml of n-tubocurarine were added simultaneously with the NT (1 llg per ml) incubation and followed by several Tyrode washes (Fig . 3). In these preparations, paired right and left muscles were used simultaneously . No systematic difïerence in either the NT or ACh response was observed between right and left preparations ; thus, no attempt has been made to distinguish the pair. Blockade of both the indirect stimulation response and the ACh response were partially prevented by ntubocurarine. The degree of inhibition depended on the duration of incubation and the venom and n-tubocurarine concentrations . Prolonged incubation with n-tubocurarine resulted in partial or complete irreversible blockade to indirect stimulation and ACh. The incubations in these cases were normally much longer than those used in the NT-n-tubocurarine competition experiments. loô
eo
m
i" 0
ao
a 0
20 O 10
30
100
300
ACh,
!cp
1000
3000
FIa. Z. EFFECTS OF CORAL SNAKE NFUROTOXW ON THE DOBE-RESPONSE CURVE OF ACETYLCHOLINE (ACh}INDUCED coNTRACnoNS of CHICK BIVENrER cERwcls MoscLE . (O, Control ; ~, 0~1 lIg per ml ; x , 1~0 iIg per ml ; ,~,, 2~0 ug per ml.) Each point is the mean of 4 values and the vertical lines indicate standard errors.
ta
AChW
d-TCiNT
W W
ACh W
ACh W
ACh W
FIC. 3. EFFECIB of 1 pg OF CORAL SNAKE NEUROTOXIN, OIVEN ALONE (A) AND SiMULrANEOUSLX WITH 2ci /lg OF D-TUHOCURARINE PER ML OF SOLUTION (B), ON NEUROMUSCULAR TRAi ON IN CHICK BiVENTER CERVICIS PREPARATIONS AND ON OONTRACTIONS INDUCED HY ZOO lig OF ACETYLCHOLINE (ACh) PER ML OF SOLUTION. W signifia wash with Tyrode's solution .
7bXICON 1973 Yol. I!
SOS
G. K. SNYDER, H. W. RAMSEY, W. J. TAYLOR and C. Y. CHIOU
In isolated chick biventer cervicis muscle preparations, fraction NT from coral snake venom abolished the muscle response to indirect stimulation, progressively shifted the ACh dose-response curve to the right in lower concentrations and abolished the respose completely in higher concentrations . Neither the ACh nor the twitch response was restored by repeated Tyrode washings ; however, the NT blocking action was inhibited with n-tubocurarine, suggesting that coral snake fraction NT is a neurotoxin producing neuromuscular blockade by irreversibly inactivating the postsynaptic receptor site . These findings are consistent with the mode of neuromuscular blockade reported for elapid and sea snake venoms in general, but they differ from the results of PARNAS and RussE.L (1967) who failed to find any neuromuscular blockade when coral snake venom was applied to crustacean muscle preparations . As previously suggested by LEE (1970), such differences may be attributed to the muscle preparations used. Resistance to the toxic effects of snake venoms in general is species specific . Motor end-plates of the toad gastrocnemius remain sensitive to ACh after treatment with desert black snake (Walterinnesia aegyptia) venom while in nerve-muscle preparations of several homeotherms and the frog rectus abdominus an antidepolarizing blockade was produced (LEE et al., 1971). In the case of the crustacean muscle, a further distinction lies in the fact that elapid neurotoxins are specific for cholinoceptive sites while neuromuscular transmission in the crustacean muscle is possibly mediated by glutamate (LEE, 1970). Acknowledgement-This research is supported in part by Florida Heart Association Grant No . 69-AG-20 and Graduate Cardiovascular Training Grant No . S TOl HEOS784-04 . CHIOU, C. Y.
REFERENCES (1970) Effects of ganglionic blocking agents on the neuromuscularjunction. Eur. !. Pharmac
12, 342. CFnov, C. Y. and LoNa, J. P. (1%9) Acetylcholine releasing effects of some niwtinic agents on chick biventer oervicis nerve muscle preparation . Proc. Soc. exp. Biol. Med. 132, 732. GnvsHORO, B. L. and WwxxnvHe, J. (1960) The isolated chick biventer cervicis nerve-muscle preparation . Br.1. Pharmac.lS, 410. Jl~tvez-Poxxws, J. M. (1968) Pharmacology of peptides and proteins in snake venoms . A. Rev. Pharmac. 8, 299. LEe, C. Y. (1970) Elapid neurotoxins and their mode of action . Clin . Toxicwl. 3, 457. Lam, C. Y., Huwrta, Pa-FFJVO and Tswr, M. C. (1971) Mode of neuromuscular blocking action of the desert black snake. Toxicon 9, 429. Lotva, J. P. and Cmov, C. Y. (1970) Pharmacological testing methods for drugs acting on peripheral nervous system. I. Pharm. Sci. S9, 133. Mnu~w, F., KUPEYAN, C., Rocxwr, H., RocFrwT, C. and Lrssnzxr, S. (1970) Purification of animal neurotoxins . Eur.1. Biochem. 16, 514. PwRNws, I. and RussELL, F. E. (1%7) Effects of venoms on nerve muscle and neuromuscular junction . Animal Toxins, pp . 40115. Oxford : Pergamon Preas. Rw~Y, H. W., StvYnrle, G. K., and TAYLOR, W. J. (1971) The effect of Micrurusf. julviua (coral) venom on myocardial contractility of the isolatod perfused rabbit heart. Clin . Res. 19, 66 . RAMS~r, H . W., TAYLOR, W. J., I{rrcf~v, H. and $NYDER, G. K. (1972) Fractionation of coral snake venom. Toxicon 10, 67 .
TOXICON 1973 Yol. Il
SHORT COMMUNICATION NEUROMUSCULAR BLOCKADE OF CHICK BIVENTER CERVICIS NERVE-MUSCLE PREPARATIONS BY A FRACITON FROM CORAL SNAKE VENOM G. K. Srnvsa,* H. W. RAlHSmr, W. J. TAYLOR and C. Y. C>:nou Division of Cardiology, Department of Medicine, and Department of Pharmacology, University of Florida College of Medicine, Gainesville, Florida 32601, U.S.A . (Acceptedfor publication 14 .luire 1973) ALTHOUGH elapid venoms in general, and coral snake (Micrurus f. fulvius) venom in particular, contain many active principles, their lethal component is generally classified as neurotoxic (L~, 1970 ; M11tANDA et al., 1970). This is based on evidence presented by several authors that elapid venoms induce a curare-like nondepolarizing neuromuscular blockade (Jut~tvl:z-Poiuus, 1968) and lethality studies in which the experimental animals developed flaccid paralysis (RAMSEY et al., 1972). In this report we describe the neuromuscular blocking action of a protein fraction isolated from crude coral snake venom on chick biventer cervicis preparations. The results obtained are in agreement with the thesis that the active fraction of coral snake venom, like that of other elapid and sea snake venoms, may be characterized as neurotoxic. Coral snake venom was fractionated using a carboxymethyl cellulose column and eluting with phosphate buffers (RAMSEY et al., 1972). Fifty mg samples of lyophilized crude venom (Ross Allen Reptile Institute, Silver Springs, Florida) in 0~2 ml starting buffer were eluted through 1 x 20 cm columns of CM 32 (Whatman, 1 "0 mg per g dry), equilibrated with 001 M phosphate buffer, pH 6 at 2-4 ml per hr . The pH and molarity of the starting buffer were increased as a continuous gradient with 0~1 M, pH 8 followed by 0" 5 M, pH 9 phosphate buffers. This contrasts with the stepwise addition of these buffers reported previously. The peak fractions, determined with an inline LKB `uvicord' absorptometer and confirmed with a Zeiss spectrophotometer, were pooled, desalted by passage through a 1 "3 x 85 cm column of Sephadex G-10, lyophilized, and stored at -10°C in sealed vials. The biventer cervicis nerve muscle preparations were isolated according to the methods of GIN3HORG and WAjtlu~tt (1960) as modified by Ciuou (1970) and LorrG and CHlou (1970). Baby chicks weighing 100-300 g were sacrificed with chloroform. The biventer cervicis nerve and muscle were dissected and mounted in a 40 ml organ bath filled with Tyrode solution (NaCI, 8"0; KCI, 0~2 ; CaClB, 02 ; MgClB, 0" 1 ; NaHCO 1 "0 ; NaH,PO 0"05 ; and dextrose, 2~0 g/1.), oxygenated with 95 per cent Og and 5 per cent CO g at 37°C . Dose response curves of the muscle preparation to acetylcholine (ACh), following incubation " Dr. Gregory K. Snyder is presently Assistant Professor, Department of Biology, University of California, Riverside, California 92502. 505
TOXICON 1973 VoJ. 11
506
G. K. SNYDER, H. W. RAMSEY, W. J. TAYLOR and C. Y. CHIOU
with various selected concentrations of the crude venom or venom fraction for 1 hr, were determined using a superfusion technique (Cxtou and Loxc, 1969). The preparation was superfused with Tyrode solution oxygenated with 95 per cent O$ and 5 per cent CO$ at 37°C at 3-4 ml per min maintained by a peristaltic pump (Buchler, Fort Lee, N.J.). The dose response curves were determined as the ratio of the ACh response after incubation and the maximum preincubation response expressed as a percentage . All muscular contraction responses were recorded by attaching the tendon with motor nerve to an isometric force transducer (Myograph, B, E & M Inst. Co., Houston, Texas) . Where indirect stimulations were used, the tendon with nerve was passed through an encasing electrode and interrupted tetanic stimulations of 20-40 V and 5 msec duration applied at a rate of 12 per min. We have previously reported on the fractionation of coral snake venom into seven fractions-one heterogeneous and six homogeneous-{R~MS~r et al., 1972). The separation of the crude venom into separate protein fractions is, with one exception, the same as previously reported . The continuous gradient elution utilized in the present study resolved the previously heterogeneous fraction into three distinct elution peaks (see Fig. 1, RAMSEY et al., 1972). The second elution peak of this group, referred to here as neurotoxic (IVT), produced the flaccid paralysis in mice characteristic of the crude venom, showed the greatest activity on chick biventer cervicis nerve-muscle preparations, and lacked the depolarizing activity characteristic of crude venom and certain venom fractions on isolated heart preparations (RAIrtSEY et al., 1971). Consequently, this NTfraction was selected for use in the present study. ta~
CrOds v~nom
FIO. I.
EFFEC75 Os ]O EIg OF CRUDE OORAL SNAKE VENOM (A) AND I EIg OF CORAL SNAKE NEUROoN NEUROMUSCIlwe zRANS~n.~oN nv c>FncK sn+eNrm cERVxas FREeARA~noNS.
~roxu~ (B)
The action of the crude venom on the chick muscle preparation was typically characterized by an augmented contraction amplitude with progressive failure of relaxation. Secondarily, the elevated contractions diminished until the muscle arrested in partial or complete tetany, usually within 60 min (Fig. lA). Fraction NT (1 lIg/ml) on the other hand, produced neither an augmented contraction nor depolarization response (Fig. 1B). In this case, following a 15-20 min period during which the contractile response appeared unaltered, the amplitude of muscle contractions was progressively depressed and ultimately abolished. The time required for the onset of the diminished contraction response and eventual complete cessation were comparable for the crude venom and the NT fraction. ïn both cases the response to indirect stimulation was not recovered by repeated washing with Tyrode solution ; however, after treatment with NT, the muscle remained fully responsive to electrical stimulation. TOXICON 1973 Yol. II
Neuromuscular Blockade
507
The ACh dose-response curve was progressively shifted to the right with increasing NT concentration (Fig. 2). As with the response to indirect stimulation, the ACh response was not recovered by repeated washing with Tyrode solution . The NT blocking action was prevented when 20 llg per ml of n-tubocurarine were added simultaneously with the NT (1 llg per ml) incubation and followed by several Tyrode washes (Fig . 3). In these preparations, paired right and left muscles were used simultaneously . No systematic difïerence in either the NT or ACh response was observed between right and left preparations ; thus, no attempt has been made to distinguish the pair. Blockade of both the indirect stimulation response and the ACh response were partially prevented by ntubocurarine. The degree of inhibition depended on the duration of incubation and the venom and n-tubocurarine concentrations . Prolonged incubation with n-tubocurarine resulted in partial or complete irreversible blockade to indirect stimulation and ACh. The incubations in these cases were normally much longer than those used in the NT-n-tubocurarine competition experiments. loô
eo
m
i" 0
ao
a 0
20 O 10
30
100
300
ACh,
!cp
1000
3000
FIa. Z. EFFECTS OF CORAL SNAKE NFUROTOXW ON THE DOBE-RESPONSE CURVE OF ACETYLCHOLINE (ACh}INDUCED coNTRACnoNS of CHICK BIVENrER cERwcls MoscLE . (O, Control ; ~, 0~1 lIg per ml ; x , 1~0 iIg per ml ; ,~,, 2~0 ug per ml.) Each point is the mean of 4 values and the vertical lines indicate standard errors.
ta
AChW
d-TCiNT
W W
ACh W
ACh W
ACh W
FIC. 3. EFFECIB of 1 pg OF CORAL SNAKE NEUROTOXIN, OIVEN ALONE (A) AND SiMULrANEOUSLX WITH 2ci /lg OF D-TUHOCURARINE PER ML OF SOLUTION (B), ON NEUROMUSCULAR TRAi ON IN CHICK BiVENTER CERVICIS PREPARATIONS AND ON OONTRACTIONS INDUCED HY ZOO lig OF ACETYLCHOLINE (ACh) PER ML OF SOLUTION. W signifia wash with Tyrode's solution .
7bXICON 1973 Yol. I!
SOS
G. K. SNYDER, H. W. RAMSEY, W. J. TAYLOR and C. Y. CHIOU
In isolated chick biventer cervicis muscle preparations, fraction NT from coral snake venom abolished the muscle response to indirect stimulation, progressively shifted the ACh dose-response curve to the right in lower concentrations and abolished the respose completely in higher concentrations . Neither the ACh nor the twitch response was restored by repeated Tyrode washings ; however, the NT blocking action was inhibited with n-tubocurarine, suggesting that coral snake fraction NT is a neurotoxin producing neuromuscular blockade by irreversibly inactivating the postsynaptic receptor site . These findings are consistent with the mode of neuromuscular blockade reported for elapid and sea snake venoms in general, but they differ from the results of PARNAS and RussE.L (1967) who failed to find any neuromuscular blockade when coral snake venom was applied to crustacean muscle preparations . As previously suggested by LEE (1970), such differences may be attributed to the muscle preparations used. Resistance to the toxic effects of snake venoms in general is species specific . Motor end-plates of the toad gastrocnemius remain sensitive to ACh after treatment with desert black snake (Walterinnesia aegyptia) venom while in nerve-muscle preparations of several homeotherms and the frog rectus abdominus an antidepolarizing blockade was produced (LEE et al., 1971). In the case of the crustacean muscle, a further distinction lies in the fact that elapid neurotoxins are specific for cholinoceptive sites while neuromuscular transmission in the crustacean muscle is possibly mediated by glutamate (LEE, 1970). Acknowledgement-This research is supported in part by Florida Heart Association Grant No . 69-AG-20 and Graduate Cardiovascular Training Grant No . S TOl HEOS784-04 . CHIOU, C. Y.
REFERENCES (1970) Effects of ganglionic blocking agents on the neuromuscularjunction. Eur. !. Pharmac
12, 342. CFnov, C. Y. and LoNa, J. P. (1%9) Acetylcholine releasing effects of some niwtinic agents on chick biventer oervicis nerve muscle preparation . Proc. Soc. exp. Biol. Med. 132, 732. GnvsHORO, B. L. and WwxxnvHe, J. (1960) The isolated chick biventer cervicis nerve-muscle preparation . Br.1. Pharmac.lS, 410. Jl~tvez-Poxxws, J. M. (1968) Pharmacology of peptides and proteins in snake venoms . A. Rev. Pharmac. 8, 299. LEe, C. Y. (1970) Elapid neurotoxins and their mode of action . Clin . Toxicwl. 3, 457. Lam, C. Y., Huwrta, Pa-FFJVO and Tswr, M. C. (1971) Mode of neuromuscular blocking action of the desert black snake. Toxicon 9, 429. Lotva, J. P. and Cmov, C. Y. (1970) Pharmacological testing methods for drugs acting on peripheral nervous system. I. Pharm. Sci. S9, 133. Mnu~w, F., KUPEYAN, C., Rocxwr, H., RocFrwT, C. and Lrssnzxr, S. (1970) Purification of animal neurotoxins . Eur.1. Biochem. 16, 514. PwRNws, I. and RussELL, F. E. (1%7) Effects of venoms on nerve muscle and neuromuscular junction . Animal Toxins, pp . 40115. Oxford : Pergamon Preas. Rw~Y, H. W., StvYnrle, G. K., and TAYLOR, W. J. (1971) The effect of Micrurusf. julviua (coral) venom on myocardial contractility of the isolatod perfused rabbit heart. Clin . Res. 19, 66 . RAMS~r, H . W., TAYLOR, W. J., I{rrcf~v, H. and $NYDER, G. K. (1972) Fractionation of coral snake venom. Toxicon 10, 67 .
TOXICON 1973 Yol. Il