Toxtcon, 1977, Vol. 13, pp . 371-376. Pergamon Prean. Printed in Groat Britain
SHORT COMMUNICATIONS THE PRESYNAPTIC NEUROMUSCULAR BLOCKING ACTION OF TAIPOXIN. A COMPARISON WITH ß-BUNGAROTOXIN AND CROTOXIN C . CHIUNG CHANG and J. DONG LEE Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China
and
DAVID EAKER arid JAN FOHLMAN Institute of Biochemistry, Biomedical Center, University of Uppsala, Uppsala, Sweden (Accepted for publlcatian 1 February 197
arid THESLEFF (1974) and CULL-CANDY et al. (1976) reported that the neuromuscular blocking action of taipoxin has many features in common with that of ß-bungarotoxin (CHANG et al., 1973 ; CHEN and LEE, 1971) and crotoxin (CHANG and LEE, 1977), i.e. the sensitivity of the endplate to acetylcholine was not significantly decreased, the release of neurotransmitter was severely depressed, the frequency of miniature endplate potentials (m.e.p.p.s) was depressed, block occurred after a latency but continued to progress after washout of the toxins, block was accelerated by increasing the rate of nerve stimulation, and there were pronounced morphological changes in the nerve terminals some time after neuromuscular blockade. However, there appeared to be important differences among the reported actions of these presynaptic toxins . A significant initial facilitation of neuromuscular transmission was observed with ß-bungarotoxin (CHANG et al., 1973 ; KELLY and BROWN, 1974) and crotoxin (VTIAL BRAZIL and EXCELL, 1971 ; CHANG and Lam, 1977) . This effect is manifested as an increase in- the quantal contents of endplate potentials, increases in the frequency of m.e.p.p .s and increased contractile response of the muscle partially paralysed by low Cas+ . On the other hand, no such facilitation was reported for taipoxin with the mouse muscle (KAMENSKAYA and THESL .EFF, 1974 ; CIn.IrCANDY et al., 1976) . It is essential to know whether these differences are due only to the use of different experimental conditions or whether the reported differences are real and re$ect different modes of action of these toxins . Moreover, all of these presynaptic toxins of snake venom origin are known to have phospholipase A activity (WERNICKE et al., 1975 ; STRONG et al., 1976 ; RüBASAMEIV et al., 1971 ; HENDON and FRAENKEL-CONRAT, 1971), and the question remains whether the presynaptic effect involves the enzyme activity. The presynaptic effect of taipoxin was therefore compared with that of ß-bungarotoxin and crotoxin . The results indicate that no qualitative difference exists among the effecta of these toxins, but that their relative potencies are different in different species of test animals. ß-Bungarotoxin and crotoxin were purified from the venoms of Bungarus multicinctus and Crotalus durissus terrificus (South Brazilian origin, Miami Serpentarium, Florida), respectively, by repeated chromatography on CM-Sephadex C-25 and Sephadex G-75 as described previously (CHANG and LEE, 1977) . Taipoxin was isolated from the venom of Oxyuramis scutellatus scutellatus by gel filtration on Sephadex G-75 followed by column KAMENSKAYA
571
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572
zone electrophoresis on cellulose powder as described by FOIiLMAIV et al. (1976). The Lnbn in mice were 0002 ltg/g (i.v.) for taipoxin, 002 lIg (i.p.) for ß-bungarotoxin and 005 ltg/g (i.p.) for crotoxin . The recordings of physiological parameters were as previously reported (CHANG and LEE, 1977). The times needed to block neuromuscular transmission in the isolated rat and mouse phrenic nerve~iaphragms and in the chick biventer cervicis muscle are compared in Fig. 1 . ~_
300
4520' v
E
v
vy 1 v 1
1
. 0 E
H O
S
-_
i~ -~ 0.01
11
0.03
.._ J- i-_-L-0 .1
0.3
Concentration,
I
3
._
10
',cg/ml
FIG. I . COMPARISON OF INE NEUROMUSCULAR BLOCKING ACTIONS OF TAIPOXIN, ß-BUNGAROTOXIN AND CROTOXIN AT VARIOUS CONCEN'IRAÜON3 . THE TIME TO CAUSE COMPLETE PARALYSIS WAS COMPARED EXCEP7IN THE CASE OF CROTOXIN ON THE CHICK MUSCLE, WHERE 9O~ BLOCKING TIME IS SHOWN .
~, Taipoxin ; p, B-bungarotoxin ; p, crotoxin ; , rat diaphragm ; - -, mouse diaphragm ; ----, chick biventer cervicis muscle . The points arc means of 4-7 experiments except in the case of taipoxin where only 2-3 experiments wcre performed.
Taipoxin was three times more potent than ß-bungarotoxin and five times more potent than crotoxin in blocking the mouse diaphragm preparation, but was 30 and 100 times less potent, respectively, than crotoxin and ß-bungarotoxin in the chick muscle . The baby chick muscle is extremely sensitive to ß-bungarotoxin (LEE and TSENG, 1969; CHANG and I-IUANG, 1974) and to crotoxin (ViTaL BRAZIL et al., 1966 ; CHANG and LEE, 1977). Compared with the mouse diaphragm, the rat diaphragm was about 10 times more resistant to crotoxin and taipoxin but was more sensitive to ß-bungarotoxin . These results indicate that the three presynaptic toxins differ with regard to species specificity. Upon application of taipoxin (1 ug/ml) to the rat diaphragm, the frequency of m.e.p.p .s increased from 103 ~ 012 (n = 7) to 280 ~ 030 (n = 4) per sec during 20-60 min, and thereafter declined to less than 0~3 by about 300 min, by which time the response to nerve stimulation was completely blocked. When 20 mM KCl was introduced to the blocked muscle the m.e.p.p. frequency increased dramatically to 20-30 per sec in 5-20 min. With untreated muscle the m.e.p.p. frequenc y usually increases to about 100 per sec upon raising KCl to 20 mM. In the mouse diaphragm treated with taipoxin üt vivo or in vitro, KAMENSKAYA and TIiFSLEFF (1974) found no `striking' increase in m.e.p.p. frequenc y during the course of intoxication . In the low Cas+-medium, all three toxins caused an immediate transient depression of contraction in the mouse diaphragm, followed in about 10 min by augmentation and then paralysis of the muscle (Fig. 2). No such change was observed in the muscle upon direct stimulation. In the rat diaphragm, the immediate depression was not marked with either taipoxin or ß-bungarotoxin, whereas the facilitation was still quite prominent (Fig. 3). The
9 10 mi n
t
Taipoxin 5 ug/ml
~ g-Bungarotoxi n 3 u g/rt~l
5 9
10 a~in
FIG. T. EFFECTS OF THE PRESYNAPTIC TOXINS ON THE MOUSE PHRENIC NERVE-DIAPHRAGM BATHED IN LOW Ca' + -MEDIUM. THE Câs + CONCENTRATION WAS REDUCED TO I/C>-1/% SO THAT THE TWTICH AMPLTTUDE WAS ABOUT l/3 OF THAT IN NORMAL TYRODE . (a) Taipoxin (0~3 lIg/ml) ; (b) ß-Bungarotoxin (3 lIg/ml); (c) Crotoxin (1~21Ig/ml). FIG.
3.
EFFECTS ON THE RAT PHRENIC NERVE-DIAPHRAGM BATHED IN LOW EXPERIMENTAL CONDTTIONS WERE THE SAME A3 IN FIG. i.
Note the diff'etence in calibration.
Ca°+ -MEDIUM.
Short Communications
57 5
augmentation of transmitter release thus seems to be the same for all three toxins . In both the rat and mouse muscles partially blocked with high MgE+, the initial facilitation by the toxins following the depression was much less marked than in the low CaE+ medium . In normal Tyrode solution, the time required by taipoxin (1 ltg/ml) to cause complete paralysis was 103 ~ 15 min (mean ~ S.D. ; n = 3) in the mouse diaphragm . When the CaE+ concentration was raised to 9~0 mM, the paralysis time was 186 ~ 23 min (n = 3). When the MgE+ concentration was raised to l2 mM during the l20 min incubation with the toxin, the time to paralysis was prolonged to 290 min . Low (045 mM) CaE+ during the 150 min incubation with taipoxin (0'3 ltg/ml) also prolonged the paralysis time from 274 min to 380 min. Due to lack of taipoxin the experiment was not repeated, however, the results are in accord with those obtained with ß-bungarotoxin and crotoxin . The experiments demonstrate that taipoxin has initial facilitatory effects on transmission. The frequency of m.e.p.p.s. in the isolated rat diaphragm was increased about 2-fold and the release of transmitter evoked by nerve activity was enhanced, especially in the medium with reduced CaE+, just as was observed with ß-bungarotoxin and crotoxin . There was also an immediate depression of transmission by all of the presynaptic toxins tested, although this can be seen only when the safety factor of transmission is lowered, as in the media with low CaE+ or high MgE+ . These snake presynaptic toxins are therefore characterized by a triphasic action . Since the first two responses, depression and facilitation, are evident without latency, it is possible that these immediate effects are not due to enzymatic phospholipase A action but are direct consequences of the binding of the toxins to the terminal membrane of motor nerves . Indeed these immediate effects of ß-bungarotoxin were still evident when CaE+ was replaced with SrE+, which markedly reduced the enzyme activity of the toxin (Su, Lee and Chang, unpublished). Another question to be settled regarding the effects of taipoxin and ß-bungarotoxin is whether high K+ can induce an increase ofm.e.p.p.s `immediately' after blockade with the toxins . It has been shown that the mouse diaphragm treated with ß-bungarotoxin or crotoxin was completely paralysed at the stage when respiratory depression become apparent (TSAi et al., 1976 ; CxnxG and Lam, 197?) . Blectronmicroscopic examination at this stage of intoxication revealed that the structure of the nerve terminal of the diaphragm was still unchanged (Tsni et al., 1976). The ultrastructural changes, such as the decrease in synaptic vesicles and the appearance of ~-shaped indentations in the neurolemma, were seen only after complete paralysis of the diaphragm. These observations indicate that the depletion of the transmitter is not the immediate cause of the toxin-induced blockade but is rather an advanced effect of the intoxication . Apparently the excitation-secretion coupling system is affected first. The present experiment showed that when the K+ was raised immediately following the failure ofnerve impulse-evoked release oftransmitter, there was still a vigorous increase in m.e.p.p.s, although slightly less than that observed in the normal muscle . This result resembles those obtained with ß-bungarotoxin (Cxa rra et al., 1973) and crotoxin (Cxnxc and Lam, 1977) and suggests that the nerve impulse-evoked release is preferentially affected before the inhibition of release induced by K+-depolarization . The failure to increase m.e .p.p.s in the previous experiments with taipoxin (KAMENSKAYA and THESLEFF, 1974) would be understandable if the test was not performed immediately after the establishment ofneuromuscular blockade . Protection against the action of taipoxin, upon increasing the concentration of CaE+ and MgE+ or upon decreasing CaE+, reflects the situation observed with ß-bungarotoxin and crotoxin . It is thus apparent that the presynaptic effects of taipoxin, ß-bungarotoxin and crotoxin are very similar, although their potencies toward different animals differ
576
Short Communications
markedly . We therefore suggest that these snake venom toxins act by the same mechanism. Whether the effect is a consequence of their phospholipase A activity remains to be solved . The immediate depressant and facilitatory effects of these toxins seem to suggest that the nerve terminal is changed upon binding the toxins. The influences of changing concentrations of Mgs+ and Ca$+ on the rate of blockade by these toxins cannot be simply explained on the basis of effects on enzyme activity . Since Mgs+ is not an inhibitor of the enzyme action (unpublished) and Cas+ is an activator, the antagonistic effects of these ions might involve either the binding of the toxin to the target site, the nerve activity, the stabilization of the nerve terminal, or the intraaxonal concentration of Ca'+ . REFERENCH.S
Cxwxo, C. C., CFtsrr, T. F. and Lee, C. Y. (1973) Studies of the presynaptic effect of ß-bungarotoxin on neuromuscular transmission. J. Pharmac. exp. Ther . 184, 339. Cr;wxa, C. C. and Huwxo, M. C. (1974) Comparison of the presynaptic actions of botulinum toxin and B-bungarotoxin on neuromuscular transmission . Naunyn-Schmiedebergs Arch. exp. Path . Pharmak. 282, 129. CI3wxo, C. C. and Lss, J. D. (1977) Crotoxin, the neurotoxin of South American rattlesnake venom, is a presynaptic toxin acting like B-bungarotoxin . Naueyn-Schmiedebergs Arch . exp. Path. Pharmak . To be published. CYisx, I:L. and LHB, C. Y. (1970) Ultrastructural changes in the motor nerve terminals caused by li-bungarotoxin. Yirchow. Arch . Abt. B. 6, 318. Ctn.UCwxnY, S. G., Fofuatwx, J., Gusrwvssox, D., LtlrJaswx-Rwuce, R. and Tfissrp~, S. (1976) The effect of taipoxin and notexin on the function and fine structure of the marine neuromuscular junction . J. Neurosci. 1, 175. Fo~usiwx, J., Ewxsx, D., I{ARr ccnN, E. and TaESUSeF, S. (1976) Taipoxin, an extremely potent presynaptic neurotoxin from the venom of the Australian snake taipan (Oxyuranus s. scutellatus) . Eur. J. Biochem. 68, 457. Hstvnox, R. A. and FkwsxxsUCorrttwr, H. (1971) Biological roles of the two components of crotoxin . Proc. natn . Acad. Sci., U.S.A . 68, 1560 . Kwa~t~tsxwYw, M. A. and ~~~+, S. (1974) Neuromuscular blocking action of an isolated toxin from the elapid (Oxyuranus scutellatus) . Acta physiol. stand. 90, 716. KELLY, R. B. and Bxowx, F. R. (1974) Biochemical and physiological properties of a purified snake venom neurotoxin which acts presynaptically. J. Neurobiol. S, 135. LEE, C. Y. and TsErra, L. F. (1969) Species differences in susceptibility to elapid venoms . Toxicon 7, 89 . Rtlawswa~x, K., BxErrxwurr, H. and HwsstuKwxx, E. (1971) Biochemistry and pharmacology of the crotoxin complex-I. Subfractionation and recombination of the crotoxin complex. Naunyn-Schmiedebergs Arch . exp. Path . Pharmak. 270, 274. Srxorra, P. N., GoExxE, J., OHERG, S. G. and KELLY, R. B. (1976) ß-Bungarotoxin, a presynaptic toxin with enzymatic activity . Proc. natn . Acad. Sci., US.A . 73, 178. Tsar, M. C., Cxwxa, C. C. end LEE, C. Y. (1976) The relation betwcen the blockage of transmitter release and the ultrastructural changes of the motor nerve terminals caused by i~bungarotoxin . In : Memorial Volume to President Chiar~g Kai-Shek, p. 289, (S . L. C~x, Ed.) . Taipei : Academia Sinica . VrrwL Bxwzu,, O. and ExcELL, B. J. (1971) Action of crotoxin and crotactin from the venom of Crotalus durissus terrißcus (South American rattlesnake) on the frog neuromuscular junction . J. Physiol. (Load.) 212, 34 . VrrwL Bxwzn., O., FRANCI3ESCFII, J. P. and Wwtssicx, E. (1966) Pharmacology of crystalline crotoxin-1 . Toxicity . Meets. Inst. Butantan 33, 973. WExxicxE, J. F., VANRER, A. D. and Howwxn, B. D. (197 The mechanism of action of Li-bungarotoxin . J. Neurochem. 25, 483.
SHORT COMMUNICATIONS THE PRESYNAPTIC NEUROMUSCULAR BLOCKING ACTION OF TAIPOXIN. A COMPARISON WITH ß-BUNGAROTOXIN AND CROTOXIN C . CHIUNG CHANG and J. DONG LEE Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China
and
DAVID EAKER arid JAN FOHLMAN Institute of Biochemistry, Biomedical Center, University of Uppsala, Uppsala, Sweden (Accepted for publlcatian 1 February 197
arid THESLEFF (1974) and CULL-CANDY et al. (1976) reported that the neuromuscular blocking action of taipoxin has many features in common with that of ß-bungarotoxin (CHANG et al., 1973 ; CHEN and LEE, 1971) and crotoxin (CHANG and LEE, 1977), i.e. the sensitivity of the endplate to acetylcholine was not significantly decreased, the release of neurotransmitter was severely depressed, the frequency of miniature endplate potentials (m.e.p.p.s) was depressed, block occurred after a latency but continued to progress after washout of the toxins, block was accelerated by increasing the rate of nerve stimulation, and there were pronounced morphological changes in the nerve terminals some time after neuromuscular blockade. However, there appeared to be important differences among the reported actions of these presynaptic toxins . A significant initial facilitation of neuromuscular transmission was observed with ß-bungarotoxin (CHANG et al., 1973 ; KELLY and BROWN, 1974) and crotoxin (VTIAL BRAZIL and EXCELL, 1971 ; CHANG and Lam, 1977) . This effect is manifested as an increase in- the quantal contents of endplate potentials, increases in the frequency of m.e.p.p .s and increased contractile response of the muscle partially paralysed by low Cas+ . On the other hand, no such facilitation was reported for taipoxin with the mouse muscle (KAMENSKAYA and THESL .EFF, 1974 ; CIn.IrCANDY et al., 1976) . It is essential to know whether these differences are due only to the use of different experimental conditions or whether the reported differences are real and re$ect different modes of action of these toxins . Moreover, all of these presynaptic toxins of snake venom origin are known to have phospholipase A activity (WERNICKE et al., 1975 ; STRONG et al., 1976 ; RüBASAMEIV et al., 1971 ; HENDON and FRAENKEL-CONRAT, 1971), and the question remains whether the presynaptic effect involves the enzyme activity. The presynaptic effect of taipoxin was therefore compared with that of ß-bungarotoxin and crotoxin . The results indicate that no qualitative difference exists among the effecta of these toxins, but that their relative potencies are different in different species of test animals. ß-Bungarotoxin and crotoxin were purified from the venoms of Bungarus multicinctus and Crotalus durissus terrificus (South Brazilian origin, Miami Serpentarium, Florida), respectively, by repeated chromatography on CM-Sephadex C-25 and Sephadex G-75 as described previously (CHANG and LEE, 1977) . Taipoxin was isolated from the venom of Oxyuramis scutellatus scutellatus by gel filtration on Sephadex G-75 followed by column KAMENSKAYA
571
Short Communications
572
zone electrophoresis on cellulose powder as described by FOIiLMAIV et al. (1976). The Lnbn in mice were 0002 ltg/g (i.v.) for taipoxin, 002 lIg (i.p.) for ß-bungarotoxin and 005 ltg/g (i.p.) for crotoxin . The recordings of physiological parameters were as previously reported (CHANG and LEE, 1977). The times needed to block neuromuscular transmission in the isolated rat and mouse phrenic nerve~iaphragms and in the chick biventer cervicis muscle are compared in Fig. 1 . ~_
300
4520' v
E
v
vy 1 v 1
1
. 0 E
H O
S
-_
i~ -~ 0.01
11
0.03
.._ J- i-_-L-0 .1
0.3
Concentration,
I
3
._
10
',cg/ml
FIG. I . COMPARISON OF INE NEUROMUSCULAR BLOCKING ACTIONS OF TAIPOXIN, ß-BUNGAROTOXIN AND CROTOXIN AT VARIOUS CONCEN'IRAÜON3 . THE TIME TO CAUSE COMPLETE PARALYSIS WAS COMPARED EXCEP7IN THE CASE OF CROTOXIN ON THE CHICK MUSCLE, WHERE 9O~ BLOCKING TIME IS SHOWN .
~, Taipoxin ; p, B-bungarotoxin ; p, crotoxin ; , rat diaphragm ; - -, mouse diaphragm ; ----, chick biventer cervicis muscle . The points arc means of 4-7 experiments except in the case of taipoxin where only 2-3 experiments wcre performed.
Taipoxin was three times more potent than ß-bungarotoxin and five times more potent than crotoxin in blocking the mouse diaphragm preparation, but was 30 and 100 times less potent, respectively, than crotoxin and ß-bungarotoxin in the chick muscle . The baby chick muscle is extremely sensitive to ß-bungarotoxin (LEE and TSENG, 1969; CHANG and I-IUANG, 1974) and to crotoxin (ViTaL BRAZIL et al., 1966 ; CHANG and LEE, 1977). Compared with the mouse diaphragm, the rat diaphragm was about 10 times more resistant to crotoxin and taipoxin but was more sensitive to ß-bungarotoxin . These results indicate that the three presynaptic toxins differ with regard to species specificity. Upon application of taipoxin (1 ug/ml) to the rat diaphragm, the frequency of m.e.p.p .s increased from 103 ~ 012 (n = 7) to 280 ~ 030 (n = 4) per sec during 20-60 min, and thereafter declined to less than 0~3 by about 300 min, by which time the response to nerve stimulation was completely blocked. When 20 mM KCl was introduced to the blocked muscle the m.e.p.p. frequency increased dramatically to 20-30 per sec in 5-20 min. With untreated muscle the m.e.p.p. frequenc y usually increases to about 100 per sec upon raising KCl to 20 mM. In the mouse diaphragm treated with taipoxin üt vivo or in vitro, KAMENSKAYA and TIiFSLEFF (1974) found no `striking' increase in m.e.p.p. frequenc y during the course of intoxication . In the low Cas+-medium, all three toxins caused an immediate transient depression of contraction in the mouse diaphragm, followed in about 10 min by augmentation and then paralysis of the muscle (Fig. 2). No such change was observed in the muscle upon direct stimulation. In the rat diaphragm, the immediate depression was not marked with either taipoxin or ß-bungarotoxin, whereas the facilitation was still quite prominent (Fig. 3). The
9 10 mi n
t
Taipoxin 5 ug/ml
~ g-Bungarotoxi n 3 u g/rt~l
5 9
10 a~in
FIG. T. EFFECTS OF THE PRESYNAPTIC TOXINS ON THE MOUSE PHRENIC NERVE-DIAPHRAGM BATHED IN LOW Ca' + -MEDIUM. THE Câs + CONCENTRATION WAS REDUCED TO I/C>-1/% SO THAT THE TWTICH AMPLTTUDE WAS ABOUT l/3 OF THAT IN NORMAL TYRODE . (a) Taipoxin (0~3 lIg/ml) ; (b) ß-Bungarotoxin (3 lIg/ml); (c) Crotoxin (1~21Ig/ml). FIG.
3.
EFFECTS ON THE RAT PHRENIC NERVE-DIAPHRAGM BATHED IN LOW EXPERIMENTAL CONDTTIONS WERE THE SAME A3 IN FIG. i.
Note the diff'etence in calibration.
Ca°+ -MEDIUM.
Short Communications
57 5
augmentation of transmitter release thus seems to be the same for all three toxins . In both the rat and mouse muscles partially blocked with high MgE+, the initial facilitation by the toxins following the depression was much less marked than in the low CaE+ medium . In normal Tyrode solution, the time required by taipoxin (1 ltg/ml) to cause complete paralysis was 103 ~ 15 min (mean ~ S.D. ; n = 3) in the mouse diaphragm . When the CaE+ concentration was raised to 9~0 mM, the paralysis time was 186 ~ 23 min (n = 3). When the MgE+ concentration was raised to l2 mM during the l20 min incubation with the toxin, the time to paralysis was prolonged to 290 min . Low (045 mM) CaE+ during the 150 min incubation with taipoxin (0'3 ltg/ml) also prolonged the paralysis time from 274 min to 380 min. Due to lack of taipoxin the experiment was not repeated, however, the results are in accord with those obtained with ß-bungarotoxin and crotoxin . The experiments demonstrate that taipoxin has initial facilitatory effects on transmission. The frequency of m.e.p.p.s. in the isolated rat diaphragm was increased about 2-fold and the release of transmitter evoked by nerve activity was enhanced, especially in the medium with reduced CaE+, just as was observed with ß-bungarotoxin and crotoxin . There was also an immediate depression of transmission by all of the presynaptic toxins tested, although this can be seen only when the safety factor of transmission is lowered, as in the media with low CaE+ or high MgE+ . These snake presynaptic toxins are therefore characterized by a triphasic action . Since the first two responses, depression and facilitation, are evident without latency, it is possible that these immediate effects are not due to enzymatic phospholipase A action but are direct consequences of the binding of the toxins to the terminal membrane of motor nerves . Indeed these immediate effects of ß-bungarotoxin were still evident when CaE+ was replaced with SrE+, which markedly reduced the enzyme activity of the toxin (Su, Lee and Chang, unpublished). Another question to be settled regarding the effects of taipoxin and ß-bungarotoxin is whether high K+ can induce an increase ofm.e.p.p.s `immediately' after blockade with the toxins . It has been shown that the mouse diaphragm treated with ß-bungarotoxin or crotoxin was completely paralysed at the stage when respiratory depression become apparent (TSAi et al., 1976 ; CxnxG and Lam, 197?) . Blectronmicroscopic examination at this stage of intoxication revealed that the structure of the nerve terminal of the diaphragm was still unchanged (Tsni et al., 1976). The ultrastructural changes, such as the decrease in synaptic vesicles and the appearance of ~-shaped indentations in the neurolemma, were seen only after complete paralysis of the diaphragm. These observations indicate that the depletion of the transmitter is not the immediate cause of the toxin-induced blockade but is rather an advanced effect of the intoxication . Apparently the excitation-secretion coupling system is affected first. The present experiment showed that when the K+ was raised immediately following the failure ofnerve impulse-evoked release oftransmitter, there was still a vigorous increase in m.e.p.p.s, although slightly less than that observed in the normal muscle . This result resembles those obtained with ß-bungarotoxin (Cxa rra et al., 1973) and crotoxin (Cxnxc and Lam, 1977) and suggests that the nerve impulse-evoked release is preferentially affected before the inhibition of release induced by K+-depolarization . The failure to increase m.e .p.p.s in the previous experiments with taipoxin (KAMENSKAYA and THESLEFF, 1974) would be understandable if the test was not performed immediately after the establishment ofneuromuscular blockade . Protection against the action of taipoxin, upon increasing the concentration of CaE+ and MgE+ or upon decreasing CaE+, reflects the situation observed with ß-bungarotoxin and crotoxin . It is thus apparent that the presynaptic effects of taipoxin, ß-bungarotoxin and crotoxin are very similar, although their potencies toward different animals differ
576
Short Communications
markedly . We therefore suggest that these snake venom toxins act by the same mechanism. Whether the effect is a consequence of their phospholipase A activity remains to be solved . The immediate depressant and facilitatory effects of these toxins seem to suggest that the nerve terminal is changed upon binding the toxins. The influences of changing concentrations of Mgs+ and Ca$+ on the rate of blockade by these toxins cannot be simply explained on the basis of effects on enzyme activity . Since Mgs+ is not an inhibitor of the enzyme action (unpublished) and Cas+ is an activator, the antagonistic effects of these ions might involve either the binding of the toxin to the target site, the nerve activity, the stabilization of the nerve terminal, or the intraaxonal concentration of Ca'+ . REFERENCH.S
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