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Effects of aliphatic dicarboxylic acids on neuromuscular inhibition produced by Naja naja siamensis toxin 3 and D-tubocurarine
Toxkon,1977, Vol . IS, pp . 45960. Peraemon Press. Printed in crest Britain
SHORT COMMUNICATIONS EFFECTS OF ALIPHATIC DICARBOXYLIC ACIDS ON NEUROMUSCULAR INHIBITION PRODUCED BY NAJA NAJA SIAMENSIS TOXIN 3 AND D-TUBOCURARINE K. RATANABANANGKOON and R. ARCHARIYASUCHA Department of Pharmacology, Faculty of Science, IVIahidol University, Rama VI Road, Bangkok, Thailand (Acceptedforpublication 9 January 1977)
W$s>nt and CHANGEUX (1974) and CoLQuxouN and RANG (1976) have provided evidence to suggest that snake postsynaptic toxins and d-tubocurarine (dTc) may interact with similar sites on the acetylcholine receptor (AchR) . Structure-activity studies of these toxins suggested that certain lysine and arginine residues might be functionally essential for the binding of the toxins to the AchR (YANG, 1974). The similarities in pharmacological actions and bindings to AchR of these toxins and dTc and the information on structure activity relationships have led L~ (1972) and YANG (1974) to propose that, probably as in the case of dTc and other curareform agents, two positively charged groups separated by a distance of 12-14 ~ (the inter-nitrogen distant of dTc, decamethonium and succinylcholine) on the toxin molecule may be involved in the binding to AchR. It is thus interesting to see whether compounds having two negatively charged groups separated by a comparable distance could interact with and antagonize the action of postsynaptic toxins. Such interaction, if present, could shed light on the nature of the `active site' of these toxins. In this communication the effects of various straight chain aliphatic dica.rboxylic acids on the neuromuscular inhibitions produced by dTc and siamensis toxin 3 were studied. Isolated phrenic nerve hemidiaphragms were prepared from female albino rats of Fisher strain (150-200 g) and the contractile response was studied according to the method Of BÜLBRING (1946) . The siamensis toxin 3 was purified according to the procedure of KARISSON et al. (1971) . The straight chain aliphatic dicarboxylic acids employed were those having 6-13 carbon atoms and the concentration used was 1~0 mM in each case. The dicarboxylic acids at this concentration did not directly affect the neuromuscular preparation, in the absence of dTc or siamensls toxin 3. It was important first to see whether, and which of, the dicarboxylic acids interact with structure known to have curareform activity and two positively charged groups separated by a distance of 12-14 fir. Only dicarboxylic acids having 9-13 carbon atoms significantly reduced the steady level of neuromuscular inhibition produced by 1~2 x 10 -8 M dTc (P < 005) . The neuromuscular inhibition (expressed as percent of complete inhibition) produced by dTc alone was 95 ~ 3~ (n = 6) ; the corresponding values obtained in the presence of nonanedioic acid, decanedioic acid, undecanedioic acid, dodecanedioic acid and tridecanedioic acid were 87 ~ 1, 74 ~ 2, 80 ~ 3, 74 ~ 7 and 76 ~ 6~, respectively . There was, however, no significant difference between the neuromuscular inhibition produced by dTc in the presence of each of these five dicarboxylic acids. Hexanedioic acid, heptanedioic acid and octanedioic acid did not significantly alter the degree of dTc 459
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inhibition . The most likely explanation for these observations is that dicarboxylic acids having 9-13 carbon atoms interact with dTc chemically resulting in a decrease in the concentration of free dTc in the tissue bath. The observation that dicarboxylic acids with shorter chain lengths failed to antagonize the action of dTc suggests that the distance between the two carboxyl groups of the acids is an important factor for interaction with dTc. Measurments of the distances between the oxygens of the two carboxyl groups (in the most extended configurations of molecular structures constructed on Dreiling stereomodels) of hexanedioic acid, heptanedioic acid and octanedioic acid were 845, 970 and 1095 A, respectively . These values are well below the 12-14 ~distance between dTc nitrogens and this fact could be the reason for the lack of antagonization observed . It is less clear why dodecanedioic acid and tridecanedioic acid which have corresponding values of 1695 and 1720 .~, respectively, could antagonize dTc as well as the dicarboxylic acids having 9-11 carbon atoms whose inter-carboxyl distances fall between 12-14 E1. The $exibility of the polymethylene bridges may permit some `bending' of the molecules so that the distances between the two carboxyls of dodecanedioic acid and tridecanedioic acid approximate the 12-14 ~ distance found between the dTc nitrogens (ICHROMOV-17lJRISOV and MICHEISON, 1966). With the observation that some of the dicarboxylic acids could interact with dTc, the effect of these dicarboxylic acids on neuromuscular inhibition by siamensis toxin 3 was investigated. It was found that the rate of neuromuscular inhibition (as measured by time to 50~ and time to complete inhibition) produced by 7~5 x 10 -8 M siamensis toxin 3 in the presence of each of the dicarboxylic acids were not significantly greater than that in their absence. Thus, none of the acidic compounds tested binds to the sitrmensis toxin 3 molecule. Considering the molar ratio (133 x 10 4) of the dicarboxylic acids and simnensis toxin 3, it is unlikely that any antagonism would be observed at higher dicarboxylic acid concentrations. The absence of antagonism between the dicarboxylic acids and sittmensis toxin 3 suggests that the amino acid side-chains between the proposed two positively charged groups on siamensis toxin 3 may not favor the interaction with the polymethylene bridges ofthe dicarboxylic acids. On the other hand, the negative results obtained from these studies may be explained on the basis that only one negatively charged group on the sitrmerrsis toxin 3 is involved in the interaction with the AchR as is the case with ß-erythroidine . Acknowledgements-The authors thank Dr. 1txvMOtm H. Locxwoon for valuable suggestions. R RFRRRNC~ Büi.siuNO, E. (1946) Observation on the isolated phrenic nerve diaphragm preparation of the rat. Br. J.
Pharmac. 1, 38 . D. and Rwxc, H. P. (1976) Effects of inhibitors on the binding of iodinated a-bungarotoxin to acetylcholine receptors in rat muscle . Molec. Pharrnac. 12, 519. ICnxt..gsox, E., Axxsena, H. and EAREA, D. (1971) Isolation of the principle neurotoxins of two Naja raja subspecies . Eur. J. Btochenr. 21, 1. KFntoMOV-Boxtsov, N. Y. and MiCm~.sox, M. J. (19C~ The mutual disposition of cholinoreceptors of locomotor muscles, and the changes in their disposition in the course of evolution . Pharm. Rev. 18, 1051 . LEe, C. Y. (1972) Chemistry and pharmacology of polypeptide toxins in snake venoms . A . Rev. Pharmac.l2, 265. WEHER, M. and Ct~uxoEVx, C. P. (1974) Binding of Naja nigricollis (3H) a-toxin to membrane fragments of Electrophorus and Torpedo electric organ-II . Effect of cholinergic agonists on the binding of the tritiated a-neurotoxin . Molec. Pharmac. 10, 15. Y~xo, C. C. (1974) Chemistry and evolution of toxins in snake venoms. Toxlcon 12, 1 . COLQUHOUN,
SHORT COMMUNICATIONS EFFECTS OF ALIPHATIC DICARBOXYLIC ACIDS ON NEUROMUSCULAR INHIBITION PRODUCED BY NAJA NAJA SIAMENSIS TOXIN 3 AND D-TUBOCURARINE K. RATANABANANGKOON and R. ARCHARIYASUCHA Department of Pharmacology, Faculty of Science, IVIahidol University, Rama VI Road, Bangkok, Thailand (Acceptedforpublication 9 January 1977)
W$s>nt and CHANGEUX (1974) and CoLQuxouN and RANG (1976) have provided evidence to suggest that snake postsynaptic toxins and d-tubocurarine (dTc) may interact with similar sites on the acetylcholine receptor (AchR) . Structure-activity studies of these toxins suggested that certain lysine and arginine residues might be functionally essential for the binding of the toxins to the AchR (YANG, 1974). The similarities in pharmacological actions and bindings to AchR of these toxins and dTc and the information on structure activity relationships have led L~ (1972) and YANG (1974) to propose that, probably as in the case of dTc and other curareform agents, two positively charged groups separated by a distance of 12-14 ~ (the inter-nitrogen distant of dTc, decamethonium and succinylcholine) on the toxin molecule may be involved in the binding to AchR. It is thus interesting to see whether compounds having two negatively charged groups separated by a comparable distance could interact with and antagonize the action of postsynaptic toxins. Such interaction, if present, could shed light on the nature of the `active site' of these toxins. In this communication the effects of various straight chain aliphatic dica.rboxylic acids on the neuromuscular inhibitions produced by dTc and siamensis toxin 3 were studied. Isolated phrenic nerve hemidiaphragms were prepared from female albino rats of Fisher strain (150-200 g) and the contractile response was studied according to the method Of BÜLBRING (1946) . The siamensis toxin 3 was purified according to the procedure of KARISSON et al. (1971) . The straight chain aliphatic dicarboxylic acids employed were those having 6-13 carbon atoms and the concentration used was 1~0 mM in each case. The dicarboxylic acids at this concentration did not directly affect the neuromuscular preparation, in the absence of dTc or siamensls toxin 3. It was important first to see whether, and which of, the dicarboxylic acids interact with structure known to have curareform activity and two positively charged groups separated by a distance of 12-14 fir. Only dicarboxylic acids having 9-13 carbon atoms significantly reduced the steady level of neuromuscular inhibition produced by 1~2 x 10 -8 M dTc (P < 005) . The neuromuscular inhibition (expressed as percent of complete inhibition) produced by dTc alone was 95 ~ 3~ (n = 6) ; the corresponding values obtained in the presence of nonanedioic acid, decanedioic acid, undecanedioic acid, dodecanedioic acid and tridecanedioic acid were 87 ~ 1, 74 ~ 2, 80 ~ 3, 74 ~ 7 and 76 ~ 6~, respectively . There was, however, no significant difference between the neuromuscular inhibition produced by dTc in the presence of each of these five dicarboxylic acids. Hexanedioic acid, heptanedioic acid and octanedioic acid did not significantly alter the degree of dTc 459
460
Short Communications
inhibition . The most likely explanation for these observations is that dicarboxylic acids having 9-13 carbon atoms interact with dTc chemically resulting in a decrease in the concentration of free dTc in the tissue bath. The observation that dicarboxylic acids with shorter chain lengths failed to antagonize the action of dTc suggests that the distance between the two carboxyl groups of the acids is an important factor for interaction with dTc. Measurments of the distances between the oxygens of the two carboxyl groups (in the most extended configurations of molecular structures constructed on Dreiling stereomodels) of hexanedioic acid, heptanedioic acid and octanedioic acid were 845, 970 and 1095 A, respectively . These values are well below the 12-14 ~distance between dTc nitrogens and this fact could be the reason for the lack of antagonization observed . It is less clear why dodecanedioic acid and tridecanedioic acid which have corresponding values of 1695 and 1720 .~, respectively, could antagonize dTc as well as the dicarboxylic acids having 9-11 carbon atoms whose inter-carboxyl distances fall between 12-14 E1. The $exibility of the polymethylene bridges may permit some `bending' of the molecules so that the distances between the two carboxyls of dodecanedioic acid and tridecanedioic acid approximate the 12-14 ~ distance found between the dTc nitrogens (ICHROMOV-17lJRISOV and MICHEISON, 1966). With the observation that some of the dicarboxylic acids could interact with dTc, the effect of these dicarboxylic acids on neuromuscular inhibition by siamensis toxin 3 was investigated. It was found that the rate of neuromuscular inhibition (as measured by time to 50~ and time to complete inhibition) produced by 7~5 x 10 -8 M siamensis toxin 3 in the presence of each of the dicarboxylic acids were not significantly greater than that in their absence. Thus, none of the acidic compounds tested binds to the sitrmensis toxin 3 molecule. Considering the molar ratio (133 x 10 4) of the dicarboxylic acids and simnensis toxin 3, it is unlikely that any antagonism would be observed at higher dicarboxylic acid concentrations. The absence of antagonism between the dicarboxylic acids and sittmensis toxin 3 suggests that the amino acid side-chains between the proposed two positively charged groups on siamensis toxin 3 may not favor the interaction with the polymethylene bridges ofthe dicarboxylic acids. On the other hand, the negative results obtained from these studies may be explained on the basis that only one negatively charged group on the sitrmerrsis toxin 3 is involved in the interaction with the AchR as is the case with ß-erythroidine . Acknowledgements-The authors thank Dr. 1txvMOtm H. Locxwoon for valuable suggestions. R RFRRRNC~ Büi.siuNO, E. (1946) Observation on the isolated phrenic nerve diaphragm preparation of the rat. Br. J.
Pharmac. 1, 38 . D. and Rwxc, H. P. (1976) Effects of inhibitors on the binding of iodinated a-bungarotoxin to acetylcholine receptors in rat muscle . Molec. Pharrnac. 12, 519. ICnxt..gsox, E., Axxsena, H. and EAREA, D. (1971) Isolation of the principle neurotoxins of two Naja raja subspecies . Eur. J. Btochenr. 21, 1. KFntoMOV-Boxtsov, N. Y. and MiCm~.sox, M. J. (19C~ The mutual disposition of cholinoreceptors of locomotor muscles, and the changes in their disposition in the course of evolution . Pharm. Rev. 18, 1051 . LEe, C. Y. (1972) Chemistry and pharmacology of polypeptide toxins in snake venoms . A . Rev. Pharmac.l2, 265. WEHER, M. and Ct~uxoEVx, C. P. (1974) Binding of Naja nigricollis (3H) a-toxin to membrane fragments of Electrophorus and Torpedo electric organ-II . Effect of cholinergic agonists on the binding of the tritiated a-neurotoxin . Molec. Pharmac. 10, 15. Y~xo, C. C. (1974) Chemistry and evolution of toxins in snake venoms. Toxlcon 12, 1 . COLQUHOUN,