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Reversibility of neuromuscular blockade produced by toxins isolated from the venom of the seasnake Laticauda semifasciata
Tnxkon, 1978, Vol. 16, pp . 219-225. Peraamon Prep . Printed in Great Hrlteln
REVERSIBILITY OF NEUROMUSCULAR BLOCKADE PRODUCED BY TOXINS ISOLATED FROM THE VENOM OF THE SEASNAKE LATICAUDA SEMIFASCIATA A . L. HARVEY and I. W. RODGER Department of Physiology and Pharmacology, University of Strathclyde, George Street, Glasgow Gl 1XW, Scotland (Accepted jor publication l2 July 1977) A. L. HARVEY and I. W. ROD(3ER. Reversibility of neuromuscular blockade produced by toxins
isolated from the venom of the seasnake Laticauda semijasciata . Toxicon 16, 219-225, 1978.The neuromuscular blocking potencies and reversibilities of purified toxins from the venom of the seasnake Latteauda setnTjasciata were assessed on the chick biventer cervicis nerve-muscle preparation . All toxins inhibited the responses to indirect stimulation and to carbachol but not the responses to türect muscle stimulation or to raised KCl concentrations . Fraction 006 M and 009 M were about 100 times less potent than the most potent compound, erabutoxin b but they were similarly partially and slowly reversible. In contrast LsIII toxin was less potent than erabutoxin b but its effort could be readily reversed by washing. Since the toxins differ in chemical structure, it is suggested that separate parts of the toxin molecule may determine aflïnity for the acetylcholine receptor and reversibility . INTRODUCTION
VEIVOM3 of snakes of the Elapidae and Hydrophidae families contain polypeptide toxins that inhibit neuromuscular transmission by binding selectively to nicotinic cholinoceptors at motor endplates of skeletal muscle (for review, see LEE, 1972) . The most active post
junctional toxins, the so-called "neurotoxins", have been divided into two groups according to their chemical structure : short-chain neurotoxins with 61-62 amino acids, and long-chain neurotoxins with 71-74 amino acids (LEE, 1972) . The venom of the seasnake Laticauda semifasciata is unusual since it contains components with neuromuscular blocking activity which do not fall into the above classification .
In addition to the three short-chain neurotoxins (erabutoxins a, b and c) there is a fraction LsIII, which has 66 amino acids (MAEDA et al ., 1974), and fractions 006 M and 009 M, both of which contain about 100 amino acid residues (HARVEY et al., 1977) . The reversibility of the toxins from the venom of Laticauda semifasciata has not been subject to detailed study. The erabutoxins have been reported to be irreversible in the chick biventer cervicis preparation, although partially reversible in the rat hemidiaphragm preparation (LEE et al., 1972). In vivo studies also revealed that erabutoxins were partially reversible (Ct-r~YMOL et al., 1972). In contrast, toxin LsIII is readily reversible in vitro (MAEDA et al., 1974).
Only preliminary results on the reversibility of 006 M and 009 M fractions have been reported (HARVEY et al., 1977). The present report represents an analysis of the reversibilities of the erabutoxins, LsIII toxin, and 006 M and 009 M fractions when tested on the chick biventer cervicis nervemuscle preparation. We have attempted to answer four questions :(1) is reversibility different for toxins of different molecular structure? (2) does reversibility increase with time and amount of washing? (3) is reversibility different for responses to nerve stimulation or to exogenous agonists? and (4) is there greater recovery at lower toxin concentrations? 219
220
A. L. HARVEY and I . W . RODGER
METHODS Biventer cervicis nerve-muscle preparations wero isolated from chicks aged 4-10 days (Gnasaoa0 and WA4RiNPR , 1960) . Preparations were mounted with a resting tension of approximately 0 " 5 g in KrebsHenseleit solution (NaCI, 6" 92 ; KC10" 35 ; MgSO 0 " 29 ; CaQ 0"28 ; KH,PO,, 0 " 16 ; NaHCO,, 2" 1 ; dextrose, 2 "0 gh). The solution was maintained at 334°C and gassed with oxygen containing 5 ~ CO, . For indirect stimulation, contractions were elicited by stimulating the motor nerve in the tendon at a frequency of 0 " 1 Hz by square wave pulses of 0"2 cosec duration and a strength greater than that required for maximal contractions . Some preparations were stimulated directly by Placing tho electrodes is contact with the belly of the muscle and using 1 cosec pulses . Responses were obtained to carbachol and KCl in the absence of nerve stimulation . Carbachol was allowed to remain in contact with tho tissue for 60 sec and KCl for 30 sec. Both were washed out by overflow for 20 sec. Contractions and contractures were recorded isometrically on a Grass 79B Polygraph using Grass FT03C or Statham G10B fot+ee displacement transducers. Fn the experimental procedure, consistent control responses were obtained to carbachol, KQ and indirect stimulation. Toxin was added and the time for 50 and 100 ~ blockade of indirect stimulation was measured. Then responses to carbachol and KCl were tested . Test responses wero repeatod every 100 min after the time of complete twitch blockado . Preparations were washed routinely every 30 min . Recovery is expressed in the figures (Figs . 3-5) in two ways : dose-response curves to carbachol before, and at various times after twitch blockado ; and as histograms illustrating the relative responses to indirect stimulation, to a concentration of carbachol (1 "4 x 10_s M) that produces a response approximately equal in height to the maximal twitch sizo and to a high concentration of KQ (7 x 10' ~ M) . Purified toxins were obtained from the venom of tho sea-snake Laticauda stm~ascfata as described previously (T~mrn and Axe, 1966 ; Te~me and Ate, 1972 ; Maine et al., 1974 ; Haxvasr et al ., 1977) . All toxins behaved as single components on rechromatography on CM-cellulose columns and wore monodispersed on disc gel electrophoresis (N . T~tva, personal communication), Toxin solutions were prepared as 1 mg/ml in distilled water and final dilutions were freshly made in Krebs-Henseleit or 0 "9 ~ sodium chloride solution . Carbamylcholine clilorido (British Drug Houses) and acetylcholine chloride (Koch-Light) were freshly prepared before use by dissolving in 0~9 ~ sodium chloride solution .
Neuromuscular blocking potency
R&SULTS
Erabutoxins a, b and c, toxin LsIII and 0"06 M and 0 " 09 M fractions all inhibited contractions elicited by indirect stimulation . Contractures to carbachol and acetylcholine were abolished when tested immediately after complete twitch blockade. However, responses to raised concentrations of KCl and to direct electrical stimulation were unaffected by neuromuscular blocking concentrations of the toxins . The time to twitch blockade decreased with increasing toxin concentrations (Fig. 1) . For convenience at low toxin concentrations, time to 50~, rather than 100, blockade was plotted in Fig. 1 . The concentration-time curve was very steep : for example, with LsIII toxin, 0 " 5 ltg/ml produced a 50~ reduction in twitches in about 12 min whereas 0" 2 pg/ml produced 50~ reduction in about 200 min . Erabutoxins a, b and c were approximately equipotent ; Fig. 1 illustrates the results obtained with a and b . Fraction LsIII was less potent than the erabutoxins, at concentrations lower than 0 "5 ltg/ml ; at higher concentrations its blocking activity was similar to that of the erabutoxins . As reported earlier HexvEY et al., 1977) fraction 0 " 06 M was similar in blocking potency to fraction, 0 "09 M both being 100-200 times less active than the erabutoxins (Fig. 1) .
Reversibility of eo»rponent hslli
As previously reported (MAEnA et al., 1974), the neuromuscular blockade induced by component LsIII could be reversed by washing . When added at a concentration of 1 lrg/ml twitches were abolished in 15 ~ 1 min (mean f S .E.M .), but the first twitches reappeared about 5 min after the first wash (Fig. 2) and had returned to control height by 50 min after washout of the toxin . Recovery was slower after higher concentrations of component LsIII . At a concentration of 5 ltg/ml twitches did not begin to recover until 30-50 min after removing the toxin from the bath . Two hundred min after twitch blockade, responses to
Reversibility of Seasnake Toxins
FIa. ].
NEUROMUSCULAR HLOCCKINO F0IENCY OP THE TOXIN FRACTIONS FROM LatkaLda faJCIata VENOM ON THB CHICK BIVEN'I8R CERVICIS PREPARATION .
221
semt-
Time taken to reduce indirectly~licited twitches to S0~ of control height (min) is plotted against toxin concentration (lig/ml) for erabutoxin a ("), erabutoxin b ("), LsIII fraction ( "), 0"06 M fraction (~)and 0-09 Mfraction (~). Bach point represents tho mean of at least four determinations . The vertical bars indicate the standard error where greater than the symbol .
FiO. 2.
EFFECT OP FRACTION LsIII (] ~n]I) PROM LatICaI!!Ia BEIilüaSClata VENOM ON THE INDIRECTLY-ELICr1~ T'RRTCF>P~ OF THB CHICK HIVSNTER CERViCI3 MUSCi.E .
LsIII was added at the first arrow and washed out at the second arrow.
carbachol had returned to control levels although responses to indirect stimulation were only about 50~ of control height (Fig. 3). The responses to raised concentrations of KCl were constant throughout the experiments, indicating that the preparations remained capable of sustaining contractures . A similar picture emerges from experiments with 10 ug LsIII per ml although in these experiments twitches returned progressively to 74 ~ 4"0~ (mean ~ S.E.M.) of control height . Reversibility of 0"06 M and 0"09 Mfractions The reversibilities of 0"06 M and 0"09 M fractions were similar, the effects of both being slower to reverse than those of LsIII toxin. In general, responses to indirect stimulation and to similar-sized. contractures induced by carbachol recovered at about the same rate (Fig. 4). The extent of recovery increased with time and washing, although the recovery tended to slow down with increasing time (Fig. 4). There was no evidence that greater recovery occurred in preparations treated with lower concentrations of toxin. For example, 10 Ftg 0"06 M per ml completely blocked responses in 192 ~ 22 min (mean ~ S.E.M.) compared to 38 ~ 4 min for 1001tg 0"06 M per ml but in both cases carbachol responses had recovered to 50-70 of controls after 300 min.
222
A . L. HARVBY
and 1. W.
a 100 r
RODGER
-r
10 60 ~ 40
,a,
20 0
10 1115 4 Carbaehol
T C K T C K T C K 0 100 200 Tfme altar twitch blochada (min)
3M
FIG. 3. REVERSIBiLTTY OF NEUROMUSCULAR BLOCKADE PRODUCED HY LSIII FRACTION (S ug/mI) ON THE CHICK BIVENTER CERVICIS PREPARATION. (a) The percentage of maximal contractuue (produced by 3~4 x 10 -' M carbachol) is plotted
against the molar concentration of carbachol. Open circles (O) represent the control curve to carbachol prior to administration of LsIII. The curves were repeated at time of complete neuromuscular blockade ( ") and at 100 min (Q) and 200 min (") after that limo . (b) The histogram illustrates the relationship between percentage of the control responses fI' _maximal twitch ; C = contracture produced by 1~4 x 10_' M carbachol ; K = contracture produced by 7 x 10 - ' M KCl) at time of complete neuromuscular blockade (0) and at 100 min and 200 min after that time . Each point in (a) and column in (b) represents the mean of at least folu preparations . The vertical bars indicate the standard errors except where they were smaller than the symbols used in (a). b
H
110 r
z
0 16 4 ti5 Carbaehol
z
z
~~TWTW
aL T
3M
xao 0 100 aoo uo Time altes twitch bloduda(rrin)
l'IU . 4. REVERSIBILITY OF NEUROMUSCULAR BLOCKADE PRODUCED BY O~IK M FRACIIUN (IOO ~Ig/ml) ON THE CHICK BIVENTER CERVICIS PREPARATION . (a) The percentage of maximal contractors (produced by 3~4 x 10'' M carbachol) is plotted
against the molar concentration of carbachol. Open circles (O) represent the control curve to carbachol prior to administration of 006 M . The curves were repeated at time of complete neuromuscular blockade (") and at 100 min (p), 200 min ( "), 300 min (p) and 400 min ( ") after that time . (b) The histogram illustrates the relationship between percentage of the control responses îr = maximal twitch ; C = contractors produced by 1~4 x 10 -' M carbachol ; K = contractors produced by 7 x 10 - ' M KCl) at time of complete neuromuscular blockade (0) and at 100, 200, 300 and 400 min after that time . Each point in (a) and column in (b) represents the mean of at least 6 preparations . The vertical bars indicate the standard errors except where they were smaller than the symbols used in (a).
Reversibility of Seasnake Toxins
223
Reversibility oferabutoxins a, b and c The reversibility of erabutoxin c was not studied in detail, although trial experiments indicated that it behaved similarly to erabutoxins a and b. Erabutoxins a and b were essentially identical in reversibility patterns, and only the data for erabutoxin b will be presented here. The neuromuscular blockade caused by erabutozin b was very slowly reversible, and was similar to the effects observed with 006 M and 009 M. The extent of recovery gradually increased with time after washout. There was no consistent difference in rate of recovery of twitch responses or carbachol responses . For example, in preparations treated with 005 lIg erabutoxin b per/ml carbachol responses recovered apparently faster than T
1
_JJ TCK
1QS
1d1
Carbeehd
z z
-L TCK
~J TCK
TYne afbr twiteh
I~M
as f ~ TCK
bloehade(min)
b z
OL~~~~L~ 1ÔS
161
Carbaehol
1dsM
TCK TCK TCK TCK TCK 0 100 200 ~00 100 ~after twitch blochade (rNn)
FIG. S. A. RBVER81HILd1Y OF NBUROMUSCIJLAR BLOCKADE PRODUCED HY IiRABZTPOXIN b (O'OS }l$~ml) ON TSB CHICK HIVENiER CERVICI3 PREPARATION. B. RBVERSIHII.IIY OF NEUROMUSCCTLAR HLOCKADB PRODUCED HY ERAHUTOXiN b (0~8 ltg/ml) ON TSB CSICK HIVENfER CBRVICL4 PREPARATION. (a) The percentage of maximal contracture (produced by 3'4 x 10-' M carbachol) is plotted
against the molar concentration of carbachol. Open circles (Q) represent the control curve to carbachol prior to administration of erabutoxin b. The curves were repeated at time of complete neuromuscular blockade (") and at 100 min (p), 200 min ("). 300 min (~ and 400 min ( ") after that time. (b) The histogram illustrates the relationship between pecentage of tho control responses Cr = maximal twitch ; C = contractors produced by 1~4 x 10'' M carbachol; K = contracturo produced by 7 x 10 -' M KCl) at the limo of complete neuromuscular blockade (0) and at 100, 200, 300, 400 min after that time. Bach point in (a) and column in (b) represents the mean of at least 4 preparations. The vertical bars indicate the standard errors except where smaller than the symbols used in (a).
224
A. L. HARVßY and I. W. RODGBR
twitch responses but the opposite appeared to occur in preparations blocked by 0~8 ug erabutoxin b per ml (Fig. 5). Similarly, there was not a large difference in reversibility in preparations treated with different toxin concentrations . After 005 Fig erabutoxin b per ml (which abolished twitches in about 250 min) the recovery of responses to indirect stimulation was similar after 300 min to the recovery in preparations exposed to 1 !ag erabutoxin b per ml (which caused complete blockade in 17 ~ 1 min. However, there was a greater recovery of responses to high concentrations of carbachol in muscles exposed to the low toxin concentration than in biventers treated with the high concentration (Fig. S). DISCUSSION
The results confirm that component LsIII is an easily reversible neurotoxin (MAEDA 1972). However, the neuromuscular paralysis induced by the erabutoxins in the chick biventer cervicis muscle preparation is, at least partially, reversible. Earlier reports stated that the erabutoxins were irreversible in this preparation (TAnuYA and ABB, 1972 ; L~ et al., 1972; LEe and Ct-err, 197 . The difference between the two sets of results could be explained either by a more rigorous washing procedure coupled with regular exposures to relatively high concentrations of cholinomimetic used in the present study, or by a species difference between the types of chicks used. Toxin LsIII differed from the other components not only in overall extent and rapidity of recovery, but also because the time to recovery depended on the initial toxin concentration. Thus, at low concentrations of Lsiü the recovery was much faster than with high concentrations . Such a relationship was not present with the other toxins : there was little difference in recovery patterns between preparations that took several hr for complete twitch block and others that were blocked in a few min. Presumably a higher concentration means faster spread of toxin into the neuromuscular junctions within the muscle, and hence a more rapid diminution in response to indirect stimulation. However, once the preparation has been blocked, it appears that the "effective" toxin concentration is similar and independent ofthe initial concentration added to the organ bath. Responses to indirect stimulation and to carbachol recovered at approximately the same rate, responses increasing with time and repeated washings. In the biventer cervicis muscle, indirectly elicited contractions are mainly the response of the focally-innervated muscle fibres, whereas the contractures to exogenously added agonist are due to activation of multiply-innervated fibres. The similar recoveries of twitch and carbachol responses may indicate that the toxins are removed from the receptors at focal and multiple endplates at the same rate . Alternatively, the results could be interpreted as showing that the toxins do not possess significant irreversible prejunctional activity (which would be revealed by a failure of responses to nerve stimulation compared to carbachol). Erabutoxins, LsIII toxin and 006 M and 009 M fractions have different amino acid compositions so a comparison of blocking potencies and reversibilities with those toxins is interesting. At concentrations above 0~5 ~g toxin per ml LsIII toxin and the erabutoxins are not very different in ability to block neuromuscular transmission, but the action of LsIII toxin is markedly easier to reverse. In contrast, fractions 006 M and 009 M are over 100 times less potent than the erabutoxins but are similar to the erabutoxins in respect of their reversibility. If neuromuscular blocking potency is a measure of the affinity of the toxin for the cholinoceptor, then ease of reversibility may be determined by binding strengths at sites apart from the receptor . Different parts of the toxin molecule may be responsible for ability to inhibit the receptor and for reversibility. Future chemical studies on the et al.,
Reversibility of Seasnake Toxins
225
structural conformations of toxins may indicate what chemical properties are required for the two biological features of the toxins. Acknowledgements-The authors thank Professor N. Tw~nrw for the generous gift of toxins and Mr. P. TfiERAPONO for assistance with some of the experiments . REFERBNCES C~tisoL, J., Tweavw, N., Bounu rsr, F. and Rocx-AxvBa r ex, M. (1972) Action neuromusculaire du venin de serpent marin "Erabu" (Laticauda semjjasciata) et des erabutoxines a et b. Taxiton 10,125 . GIx3aORO, B. L. and Wwnxnven, J. (1960) The isolated chick biventer cervicis nervo-muscle preparation . Br . J. Pharmac. Chemother.l3, 410. Hwavesr, A. L., ROnaEa, I. W. and Tw~u~rw, N. (1977) Neuromuscular blocking activity of two fractions isolated from the venom of the seasnake, Laticauda semjfasclata. Toxicon. To be published. Lve, C. Y. (1972) Chemistry and pham~acology of polypeptide toxins in snake venoms . A. Rev. Pharmac.l2, 265. Lam, C. Y., Cr3wxa, C. C. and C~x, Y. M. (1972) Reversibility of neuromuscular blockade by neurotoxins from elapid and sea snake venoms. J. Formosan med. Ass. 71, 344. Lee, C. Y. and Ctn;x, Y. M. (1976) Species differences in reversibility of neuromuscular blockade by elapid and sea snake neurotoxins . In : Animal, PlantmrdMicrobial Toxins, Vol . 2, p.196, (Oxswxw, A., HAYASAI, K. and Swwwr, Y. 13ds .). New York : Plenum. Mwanw, N., TAYwßi, K., Tweww, N., GYu:x, Y. M. and Lee, C. Y. (1974) The isolation of an easily reversible post-synaptic toxin from the venom of a sea snake, Laticauda semifasclata . Biochem. J.141, 333 . Twmtrw, N. and Ase, H. (1972) The isolation, properties and amino acid sequence of erabutoxin c, a minor neurotoxic component of thevenom of a sea snake, Laticauda sem#fasclata . Bioclum. .1.130, 547. Twmvw, N. and Axwi, H. (1966) Studies on sea snake venoms : crystallization of erabutoxins a and b from Lattcaada sem(%asciata venom . Blochem. J. 99, 624.
REVERSIBILITY OF NEUROMUSCULAR BLOCKADE PRODUCED BY TOXINS ISOLATED FROM THE VENOM OF THE SEASNAKE LATICAUDA SEMIFASCIATA A . L. HARVEY and I. W. RODGER Department of Physiology and Pharmacology, University of Strathclyde, George Street, Glasgow Gl 1XW, Scotland (Accepted jor publication l2 July 1977) A. L. HARVEY and I. W. ROD(3ER. Reversibility of neuromuscular blockade produced by toxins
isolated from the venom of the seasnake Laticauda semijasciata . Toxicon 16, 219-225, 1978.The neuromuscular blocking potencies and reversibilities of purified toxins from the venom of the seasnake Latteauda setnTjasciata were assessed on the chick biventer cervicis nerve-muscle preparation . All toxins inhibited the responses to indirect stimulation and to carbachol but not the responses to türect muscle stimulation or to raised KCl concentrations . Fraction 006 M and 009 M were about 100 times less potent than the most potent compound, erabutoxin b but they were similarly partially and slowly reversible. In contrast LsIII toxin was less potent than erabutoxin b but its effort could be readily reversed by washing. Since the toxins differ in chemical structure, it is suggested that separate parts of the toxin molecule may determine aflïnity for the acetylcholine receptor and reversibility . INTRODUCTION
VEIVOM3 of snakes of the Elapidae and Hydrophidae families contain polypeptide toxins that inhibit neuromuscular transmission by binding selectively to nicotinic cholinoceptors at motor endplates of skeletal muscle (for review, see LEE, 1972) . The most active post
junctional toxins, the so-called "neurotoxins", have been divided into two groups according to their chemical structure : short-chain neurotoxins with 61-62 amino acids, and long-chain neurotoxins with 71-74 amino acids (LEE, 1972) . The venom of the seasnake Laticauda semifasciata is unusual since it contains components with neuromuscular blocking activity which do not fall into the above classification .
In addition to the three short-chain neurotoxins (erabutoxins a, b and c) there is a fraction LsIII, which has 66 amino acids (MAEDA et al ., 1974), and fractions 006 M and 009 M, both of which contain about 100 amino acid residues (HARVEY et al., 1977) . The reversibility of the toxins from the venom of Laticauda semifasciata has not been subject to detailed study. The erabutoxins have been reported to be irreversible in the chick biventer cervicis preparation, although partially reversible in the rat hemidiaphragm preparation (LEE et al., 1972). In vivo studies also revealed that erabutoxins were partially reversible (Ct-r~YMOL et al., 1972). In contrast, toxin LsIII is readily reversible in vitro (MAEDA et al., 1974).
Only preliminary results on the reversibility of 006 M and 009 M fractions have been reported (HARVEY et al., 1977). The present report represents an analysis of the reversibilities of the erabutoxins, LsIII toxin, and 006 M and 009 M fractions when tested on the chick biventer cervicis nervemuscle preparation. We have attempted to answer four questions :(1) is reversibility different for toxins of different molecular structure? (2) does reversibility increase with time and amount of washing? (3) is reversibility different for responses to nerve stimulation or to exogenous agonists? and (4) is there greater recovery at lower toxin concentrations? 219
220
A. L. HARVEY and I . W . RODGER
METHODS Biventer cervicis nerve-muscle preparations wero isolated from chicks aged 4-10 days (Gnasaoa0 and WA4RiNPR , 1960) . Preparations were mounted with a resting tension of approximately 0 " 5 g in KrebsHenseleit solution (NaCI, 6" 92 ; KC10" 35 ; MgSO 0 " 29 ; CaQ 0"28 ; KH,PO,, 0 " 16 ; NaHCO,, 2" 1 ; dextrose, 2 "0 gh). The solution was maintained at 334°C and gassed with oxygen containing 5 ~ CO, . For indirect stimulation, contractions were elicited by stimulating the motor nerve in the tendon at a frequency of 0 " 1 Hz by square wave pulses of 0"2 cosec duration and a strength greater than that required for maximal contractions . Some preparations were stimulated directly by Placing tho electrodes is contact with the belly of the muscle and using 1 cosec pulses . Responses were obtained to carbachol and KCl in the absence of nerve stimulation . Carbachol was allowed to remain in contact with tho tissue for 60 sec and KCl for 30 sec. Both were washed out by overflow for 20 sec. Contractions and contractures were recorded isometrically on a Grass 79B Polygraph using Grass FT03C or Statham G10B fot+ee displacement transducers. Fn the experimental procedure, consistent control responses were obtained to carbachol, KQ and indirect stimulation. Toxin was added and the time for 50 and 100 ~ blockade of indirect stimulation was measured. Then responses to carbachol and KCl were tested . Test responses wero repeatod every 100 min after the time of complete twitch blockado . Preparations were washed routinely every 30 min . Recovery is expressed in the figures (Figs . 3-5) in two ways : dose-response curves to carbachol before, and at various times after twitch blockado ; and as histograms illustrating the relative responses to indirect stimulation, to a concentration of carbachol (1 "4 x 10_s M) that produces a response approximately equal in height to the maximal twitch sizo and to a high concentration of KQ (7 x 10' ~ M) . Purified toxins were obtained from the venom of tho sea-snake Laticauda stm~ascfata as described previously (T~mrn and Axe, 1966 ; Te~me and Ate, 1972 ; Maine et al., 1974 ; Haxvasr et al ., 1977) . All toxins behaved as single components on rechromatography on CM-cellulose columns and wore monodispersed on disc gel electrophoresis (N . T~tva, personal communication), Toxin solutions were prepared as 1 mg/ml in distilled water and final dilutions were freshly made in Krebs-Henseleit or 0 "9 ~ sodium chloride solution . Carbamylcholine clilorido (British Drug Houses) and acetylcholine chloride (Koch-Light) were freshly prepared before use by dissolving in 0~9 ~ sodium chloride solution .
Neuromuscular blocking potency
R&SULTS
Erabutoxins a, b and c, toxin LsIII and 0"06 M and 0 " 09 M fractions all inhibited contractions elicited by indirect stimulation . Contractures to carbachol and acetylcholine were abolished when tested immediately after complete twitch blockade. However, responses to raised concentrations of KCl and to direct electrical stimulation were unaffected by neuromuscular blocking concentrations of the toxins . The time to twitch blockade decreased with increasing toxin concentrations (Fig. 1) . For convenience at low toxin concentrations, time to 50~, rather than 100, blockade was plotted in Fig. 1 . The concentration-time curve was very steep : for example, with LsIII toxin, 0 " 5 ltg/ml produced a 50~ reduction in twitches in about 12 min whereas 0" 2 pg/ml produced 50~ reduction in about 200 min . Erabutoxins a, b and c were approximately equipotent ; Fig. 1 illustrates the results obtained with a and b . Fraction LsIII was less potent than the erabutoxins, at concentrations lower than 0 "5 ltg/ml ; at higher concentrations its blocking activity was similar to that of the erabutoxins . As reported earlier HexvEY et al., 1977) fraction 0 " 06 M was similar in blocking potency to fraction, 0 "09 M both being 100-200 times less active than the erabutoxins (Fig. 1) .
Reversibility of eo»rponent hslli
As previously reported (MAEnA et al., 1974), the neuromuscular blockade induced by component LsIII could be reversed by washing . When added at a concentration of 1 lrg/ml twitches were abolished in 15 ~ 1 min (mean f S .E.M .), but the first twitches reappeared about 5 min after the first wash (Fig. 2) and had returned to control height by 50 min after washout of the toxin . Recovery was slower after higher concentrations of component LsIII . At a concentration of 5 ltg/ml twitches did not begin to recover until 30-50 min after removing the toxin from the bath . Two hundred min after twitch blockade, responses to
Reversibility of Seasnake Toxins
FIa. ].
NEUROMUSCULAR HLOCCKINO F0IENCY OP THE TOXIN FRACTIONS FROM LatkaLda faJCIata VENOM ON THB CHICK BIVEN'I8R CERVICIS PREPARATION .
221
semt-
Time taken to reduce indirectly~licited twitches to S0~ of control height (min) is plotted against toxin concentration (lig/ml) for erabutoxin a ("), erabutoxin b ("), LsIII fraction ( "), 0"06 M fraction (~)and 0-09 Mfraction (~). Bach point represents tho mean of at least four determinations . The vertical bars indicate the standard error where greater than the symbol .
FiO. 2.
EFFECT OP FRACTION LsIII (] ~n]I) PROM LatICaI!!Ia BEIilüaSClata VENOM ON THE INDIRECTLY-ELICr1~ T'RRTCF>P~ OF THB CHICK HIVSNTER CERViCI3 MUSCi.E .
LsIII was added at the first arrow and washed out at the second arrow.
carbachol had returned to control levels although responses to indirect stimulation were only about 50~ of control height (Fig. 3). The responses to raised concentrations of KCl were constant throughout the experiments, indicating that the preparations remained capable of sustaining contractures . A similar picture emerges from experiments with 10 ug LsIII per ml although in these experiments twitches returned progressively to 74 ~ 4"0~ (mean ~ S.E.M.) of control height . Reversibility of 0"06 M and 0"09 Mfractions The reversibilities of 0"06 M and 0"09 M fractions were similar, the effects of both being slower to reverse than those of LsIII toxin. In general, responses to indirect stimulation and to similar-sized. contractures induced by carbachol recovered at about the same rate (Fig. 4). The extent of recovery increased with time and washing, although the recovery tended to slow down with increasing time (Fig. 4). There was no evidence that greater recovery occurred in preparations treated with lower concentrations of toxin. For example, 10 Ftg 0"06 M per ml completely blocked responses in 192 ~ 22 min (mean ~ S.E.M.) compared to 38 ~ 4 min for 1001tg 0"06 M per ml but in both cases carbachol responses had recovered to 50-70 of controls after 300 min.
222
A . L. HARVBY
and 1. W.
a 100 r
RODGER
-r
10 60 ~ 40
,a,
20 0
10 1115 4 Carbaehol
T C K T C K T C K 0 100 200 Tfme altar twitch blochada (min)
3M
FIG. 3. REVERSIBiLTTY OF NEUROMUSCULAR BLOCKADE PRODUCED HY LSIII FRACTION (S ug/mI) ON THE CHICK BIVENTER CERVICIS PREPARATION. (a) The percentage of maximal contractuue (produced by 3~4 x 10 -' M carbachol) is plotted
against the molar concentration of carbachol. Open circles (O) represent the control curve to carbachol prior to administration of LsIII. The curves were repeated at time of complete neuromuscular blockade ( ") and at 100 min (Q) and 200 min (") after that limo . (b) The histogram illustrates the relationship between percentage of the control responses fI' _maximal twitch ; C = contracture produced by 1~4 x 10_' M carbachol ; K = contracture produced by 7 x 10 - ' M KCl) at time of complete neuromuscular blockade (0) and at 100 min and 200 min after that time . Each point in (a) and column in (b) represents the mean of at least folu preparations . The vertical bars indicate the standard errors except where they were smaller than the symbols used in (a). b
H
110 r
z
0 16 4 ti5 Carbaehol
z
z
~~TWTW
aL T
3M
xao 0 100 aoo uo Time altes twitch bloduda(rrin)
l'IU . 4. REVERSIBILITY OF NEUROMUSCULAR BLOCKADE PRODUCED BY O~IK M FRACIIUN (IOO ~Ig/ml) ON THE CHICK BIVENTER CERVICIS PREPARATION . (a) The percentage of maximal contractors (produced by 3~4 x 10'' M carbachol) is plotted
against the molar concentration of carbachol. Open circles (O) represent the control curve to carbachol prior to administration of 006 M . The curves were repeated at time of complete neuromuscular blockade (") and at 100 min (p), 200 min ( "), 300 min (p) and 400 min ( ") after that time . (b) The histogram illustrates the relationship between percentage of the control responses îr = maximal twitch ; C = contractors produced by 1~4 x 10 -' M carbachol ; K = contractors produced by 7 x 10 - ' M KCl) at time of complete neuromuscular blockade (0) and at 100, 200, 300 and 400 min after that time . Each point in (a) and column in (b) represents the mean of at least 6 preparations . The vertical bars indicate the standard errors except where they were smaller than the symbols used in (a).
Reversibility of Seasnake Toxins
223
Reversibility oferabutoxins a, b and c The reversibility of erabutoxin c was not studied in detail, although trial experiments indicated that it behaved similarly to erabutoxins a and b. Erabutoxins a and b were essentially identical in reversibility patterns, and only the data for erabutoxin b will be presented here. The neuromuscular blockade caused by erabutozin b was very slowly reversible, and was similar to the effects observed with 006 M and 009 M. The extent of recovery gradually increased with time after washout. There was no consistent difference in rate of recovery of twitch responses or carbachol responses . For example, in preparations treated with 005 lIg erabutoxin b per/ml carbachol responses recovered apparently faster than T
1
_JJ TCK
1QS
1d1
Carbeehd
z z
-L TCK
~J TCK
TYne afbr twiteh
I~M
as f ~ TCK
bloehade(min)
b z
OL~~~~L~ 1ÔS
161
Carbaehol
1dsM
TCK TCK TCK TCK TCK 0 100 200 ~00 100 ~after twitch blochade (rNn)
FIG. S. A. RBVER81HILd1Y OF NBUROMUSCIJLAR BLOCKADE PRODUCED HY IiRABZTPOXIN b (O'OS }l$~ml) ON TSB CHICK HIVENiER CERVICI3 PREPARATION. B. RBVERSIHII.IIY OF NEUROMUSCCTLAR HLOCKADB PRODUCED HY ERAHUTOXiN b (0~8 ltg/ml) ON TSB CSICK HIVENfER CBRVICL4 PREPARATION. (a) The percentage of maximal contracture (produced by 3'4 x 10-' M carbachol) is plotted
against the molar concentration of carbachol. Open circles (Q) represent the control curve to carbachol prior to administration of erabutoxin b. The curves were repeated at time of complete neuromuscular blockade (") and at 100 min (p), 200 min ("). 300 min (~ and 400 min ( ") after that time. (b) The histogram illustrates the relationship between pecentage of tho control responses Cr = maximal twitch ; C = contractors produced by 1~4 x 10'' M carbachol; K = contracturo produced by 7 x 10 -' M KCl) at the limo of complete neuromuscular blockade (0) and at 100, 200, 300, 400 min after that time. Bach point in (a) and column in (b) represents the mean of at least 4 preparations. The vertical bars indicate the standard errors except where smaller than the symbols used in (a).
224
A. L. HARVßY and I. W. RODGBR
twitch responses but the opposite appeared to occur in preparations blocked by 0~8 ug erabutoxin b per ml (Fig. 5). Similarly, there was not a large difference in reversibility in preparations treated with different toxin concentrations . After 005 Fig erabutoxin b per ml (which abolished twitches in about 250 min) the recovery of responses to indirect stimulation was similar after 300 min to the recovery in preparations exposed to 1 !ag erabutoxin b per ml (which caused complete blockade in 17 ~ 1 min. However, there was a greater recovery of responses to high concentrations of carbachol in muscles exposed to the low toxin concentration than in biventers treated with the high concentration (Fig. S). DISCUSSION
The results confirm that component LsIII is an easily reversible neurotoxin (MAEDA 1972). However, the neuromuscular paralysis induced by the erabutoxins in the chick biventer cervicis muscle preparation is, at least partially, reversible. Earlier reports stated that the erabutoxins were irreversible in this preparation (TAnuYA and ABB, 1972 ; L~ et al., 1972; LEe and Ct-err, 197 . The difference between the two sets of results could be explained either by a more rigorous washing procedure coupled with regular exposures to relatively high concentrations of cholinomimetic used in the present study, or by a species difference between the types of chicks used. Toxin LsIII differed from the other components not only in overall extent and rapidity of recovery, but also because the time to recovery depended on the initial toxin concentration. Thus, at low concentrations of Lsiü the recovery was much faster than with high concentrations . Such a relationship was not present with the other toxins : there was little difference in recovery patterns between preparations that took several hr for complete twitch block and others that were blocked in a few min. Presumably a higher concentration means faster spread of toxin into the neuromuscular junctions within the muscle, and hence a more rapid diminution in response to indirect stimulation. However, once the preparation has been blocked, it appears that the "effective" toxin concentration is similar and independent ofthe initial concentration added to the organ bath. Responses to indirect stimulation and to carbachol recovered at approximately the same rate, responses increasing with time and repeated washings. In the biventer cervicis muscle, indirectly elicited contractions are mainly the response of the focally-innervated muscle fibres, whereas the contractures to exogenously added agonist are due to activation of multiply-innervated fibres. The similar recoveries of twitch and carbachol responses may indicate that the toxins are removed from the receptors at focal and multiple endplates at the same rate . Alternatively, the results could be interpreted as showing that the toxins do not possess significant irreversible prejunctional activity (which would be revealed by a failure of responses to nerve stimulation compared to carbachol). Erabutoxins, LsIII toxin and 006 M and 009 M fractions have different amino acid compositions so a comparison of blocking potencies and reversibilities with those toxins is interesting. At concentrations above 0~5 ~g toxin per ml LsIII toxin and the erabutoxins are not very different in ability to block neuromuscular transmission, but the action of LsIII toxin is markedly easier to reverse. In contrast, fractions 006 M and 009 M are over 100 times less potent than the erabutoxins but are similar to the erabutoxins in respect of their reversibility. If neuromuscular blocking potency is a measure of the affinity of the toxin for the cholinoceptor, then ease of reversibility may be determined by binding strengths at sites apart from the receptor . Different parts of the toxin molecule may be responsible for ability to inhibit the receptor and for reversibility. Future chemical studies on the et al.,
Reversibility of Seasnake Toxins
225
structural conformations of toxins may indicate what chemical properties are required for the two biological features of the toxins. Acknowledgements-The authors thank Professor N. Tw~nrw for the generous gift of toxins and Mr. P. TfiERAPONO for assistance with some of the experiments . REFERBNCES C~tisoL, J., Tweavw, N., Bounu rsr, F. and Rocx-AxvBa r ex, M. (1972) Action neuromusculaire du venin de serpent marin "Erabu" (Laticauda semjjasciata) et des erabutoxines a et b. Taxiton 10,125 . GIx3aORO, B. L. and Wwnxnven, J. (1960) The isolated chick biventer cervicis nervo-muscle preparation . Br . J. Pharmac. Chemother.l3, 410. Hwavesr, A. L., ROnaEa, I. W. and Tw~u~rw, N. (1977) Neuromuscular blocking activity of two fractions isolated from the venom of the seasnake, Laticauda semjfasclata. Toxicon. To be published. Lve, C. Y. (1972) Chemistry and pham~acology of polypeptide toxins in snake venoms . A. Rev. Pharmac.l2, 265. Lam, C. Y., Cr3wxa, C. C. and C~x, Y. M. (1972) Reversibility of neuromuscular blockade by neurotoxins from elapid and sea snake venoms. J. Formosan med. Ass. 71, 344. Lee, C. Y. and Ctn;x, Y. M. (1976) Species differences in reversibility of neuromuscular blockade by elapid and sea snake neurotoxins . In : Animal, PlantmrdMicrobial Toxins, Vol . 2, p.196, (Oxswxw, A., HAYASAI, K. and Swwwr, Y. 13ds .). New York : Plenum. Mwanw, N., TAYwßi, K., Tweww, N., GYu:x, Y. M. and Lee, C. Y. (1974) The isolation of an easily reversible post-synaptic toxin from the venom of a sea snake, Laticauda semifasclata . Biochem. J.141, 333 . Twmtrw, N. and Ase, H. (1972) The isolation, properties and amino acid sequence of erabutoxin c, a minor neurotoxic component of thevenom of a sea snake, Laticauda sem#fasclata . Bioclum. .1.130, 547. Twmvw, N. and Axwi, H. (1966) Studies on sea snake venoms : crystallization of erabutoxins a and b from Lattcaada sem(%asciata venom . Blochem. J. 99, 624.