Changes in neuromuscular transmission of guinea pig vas deferens produced by decamethrin treatment

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

w,96-102

(1987)

Changes in Neuromuscular Transmission of Guinea Pig Vas Deferens Produced by Decamethrin Treatment’ M. R. MARTINEZ-LARRA~AGA,

A. ANACIN,’

AND M. J. DIAZ

Department of Toxicology, Faculty of Veterinary Medicine, University of Lebn, 24007 Ledn, and Institute of Pharmacology and Toxicology, CSIC/Complutense University, 28040 Madrid, Spain

Received May 16, 1986; accepted March 9, 1987 Changes in Neuromuscular Transmission ofGuinea Pig Vas Deferens by Decamethrin Treatment. ANA&N, A., MARTINEZ-LARRAGAGA, M. R., AND DIAZ, M. J. (1987). Toxicol. Appl. Pharmacol. 90,96-102. Responses of the isolated vas deferens of guinea pig to clonidine (inhibition of contractions to field stimulation at 2.5 Hz), tyramine (inhibition of contractions to field stimulation at 10 Hz), prostaglandin E2 (inhibition of contractions to field stimulation at 10 Hz), and noradrenaline (contraction of longitudinal muscle) were determined after administration of decamethtin (18 mg kg-‘, ip) once a day for 3 consecutive days. Treatment with decamethrin produced a subsensitivity of the prejunctional a*-adrenoceptor system as evidenced by the fact that the az-agonist clonidine was less effective in decreasing nerve-stimulated induced contractions of the vas deferens. In addition, the presynaptic action of tyramine on postganglionic motor transmission was impaired. However, no detectable changes in the inhibition by prostaglandin E2 oftwitch responses were produced by decamethrin. Decamethrin treatment had a significant effect on noradrenaline responsiveness, causing an increase in the maximum contractile response, indicative of an enhanced postreceptor mechanism. The present results suggest that decamethrin treatment reduces peripheral presynaptic adrenoceptor sensitivity. This reduction will lead subsequently to increased noradrenaline release and postsynaptic adrenoceptor upregulation. 0 1987 Academic Press. Inc.

The pyrethroid decamethrin is one of the most potent insecticides used commercially. Pyrethroid insecticides, including the natural pyrethrins, are generally recognized as agents acting specifically on the nervous system. Pyrethroid insecticides cause disoriented movements, convulsions, paralysis, and death when administered to insects (Wouters and Van den Bercken, 1978) and mammals (Barnes and Verchoyle, 1974; Ray and

Cremer, 1979; Ray, 1980, 1982). In nerve preparations of arthropods (Clements and May, 1977) and toads (Van den Bercken et al., 1973; Akkermans et al., 1975), pyrethroids increase spontaneous activity and produce hyperexcitability of motor and sensory fibers and sense organs. These effects are associated with prolonged depolarizing afterpotentials in isolated nerves of squid (Narahashi, 197 1) or toad (Vijverberg and Van den Bercken, 1979), resulting from a decrease in the rate of the sodium channel closing (Narahashi, 197 1; Lund and Narahashi, 198 1; Vijverberg et al., 1982). In addition to the known action on the sodium channel of pyrethroids, Abbassy et al. (1982, 1983) reported that several pyrethroids bind to the channel sites of the nicotonic acetylcholine receptor

’ This work was supported by the Comision Asesora de Investigation Cientiftca y T&mica, Ministry of Fducation and Science, Project No. 582185, and by the University of Leon, Project No. 14/85, Spain. * To whom all correspondence should be addressed at Department of Toxicology, Faculty of Veterinary Medicine, University of Leon, 24007 L.&n, Spain. 0041-008X/87

$3.00

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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with high affinity and thus would have postsynaptic effects at motor endplates. The major action of pyrethroids is to increase the sodium influx in neuronal tissue. However, they may also inhibit ion-dependent transport ATPases. Among the several compounds tested, decamethrin is the most potent inhibitor of Ca2+,Mg2+-dependent ATPase activity in microsomal preparations of squid retinal axons (Clark and Matsumura, 1982). Recently, it has been shown that decamethrin produces a positive inotropit effect in the heart, apparently due to an increase in transmembrane sodium influx. This increased influx causes catecholamine release from the sympathetic nerve terminals and also directly enhances muscle contraction (Berlin et al., 1984). However, limited information is available on the neurotoxic action of decamethrin on the autonomic nervous system of mammals and other vertebrates. The present investigation was undertaken to obtain greater insight into the mechanism of action of decamethrin. A pharmacological analysis of the effects of prolonged treatment with decamethrin of neuromuscular transmission has been carried out. The present work describes a study of the effect of decamethrin treatment on presynaptic and postsynaptic adrenoceptor function in the guinea pig vas deferens. METHODS Animals and injections. Albino guinea pigs weighing approximately 600 g were kept six per cage and maintamed on pellet food and water ad libitum. The animals were treated with decamethrin (18 mg kg-‘, ip) once a day for 3 consecutive days. Decamethrin was dissolved in sesame oil and was injected intraperitoneally at a volume of 1.O ml kg-‘. The animals were killed 24 hr after the last injection of the insecticide. Preparation of vas deferens in vitro. Both vasa deferentia were dissected from control and from decamethrintreated animals and suspended in 2-ml organ baths with built-in vertical electrodes. The method for desheathing and setting up the isolated vas deferens preparations in vitro at 35’C and a full description of essential experimental details, including the composition of the KrebsHenseleit solution, has been previously reported (An-

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adon and Martinez-Larraiiaga, 1985). All recordings were isometric at a resting tension of ca. 0.3 g, achieved by setting up the preparations at an initial tension of 0.5 g. Following an equilibration period of 30 min, electrical field stimulation (trams of five I-msec pulses, 2.5 Hz, at 60-set intervals) was applied. The preparation was stimulated for 30 min to allow for equilibration of contractions, and a dose-response curve for clonidine was then obtained. The inhibitory effect of clonidine upon the height of the twitch responses elicited by field stimulation was examined, the exposure time being 5 min for each dose. The degree of inhibition at each dose level was expressed as the percentage of twitch inhibition. After washing, recovery of the twitch responses from this inhibition was complete within 5-10 min. After the doseresponse curve to clonidine was determined and the contractile responses to field stimulation returned to control tension, the stimulator was turned off, and a dose-response curve for noradrenaline was determined, using an exposure period of 30 set and a between-dose interval of 4.5 min. Preliminary experiments showed that noradrenaline responses were not affected by prior exposure to clonidine using this protocol. Noradrenaline dose-response curves were determined in the absence and presence of 30 pM cocaine to inhibit amine uptake and 3 pM propranolol to block p-adrenergic receptors. The inhibitory presynaptic effects of tyramine and prostaglandin E2 on postganglionic motor transmission were investigated. To avoid undesirable effects attributable to fatigue or depletion after trains of pulses, a fresh vasdeferens preparation was used. Tissues were prepared as above and stimulated at I-min intervals with trains of five pulses of I-msec duration delivered at 10 Hz and supramaximal voltage. The inhibitory effectsof tyramine and prostaglandin Ez upon the height of the twitch responses elicited by postganglionic field stimulation were quantitated, the contact time being 5 min for each dose. The degree of inhibition at each dose level has been expressed as the percentage of twitch inhibition. Drugs. The following drugs were used: bradykinin (triacetate salt) (Sigma); clonidine hydrochloride (Sigma); cocaine hydrochloride (Sigma); (-)-noradrenaline bitartrate (Koch-Light); Dr.-propranolol hydrochloride (Sigma); prostaglandin Ez (Sigma); and tyramine hydrochloride (Sigma). All other chemicals used were obtained from the usual commercial sources and were routinely of the highest grade available. The pyrethroid compound decamethrin, S-a-cyano(lR-cis)-3-phenoxybenzyl-3-(2,2-dibromovinyl)-2-2dimethylcyclopropane-carboxylate, was kindly supplied by Roussel Uclaff. Data analysis. The results are expressed as means f SE. Student’s t test was used to test for significance between mean values. Each dose-response curve to noradrenaline was fitted to the logistic equation

98

ANAtiN, E=

MARTINEZ-LARRANAGA,

100 1

KoaxxC

ED50 + C in which E is the response and C is the concentration of noradrenaline. The unknowns E,,,,, and ED50 are respectively the maximum response and the dose producing half-maximal response. Iterative curve fitting was done using the nonlinear regression program ELSFIT (Sheiner, 198 1) with the aid ofan HP-85B computer. For each preparation the midrange sensitivity was calculated as the negative log ED50 (pD50). Differences in maximum response and pD50 (-log EC50) values were compared using the analysis of variance (ANOVA).

AND DIAZ

-5 z6 ao C .P

60.

a r .c eD

40.

RESULTS Clonidine

Eftects of Prolonged Treatment of Guinea Pigs with Decamethrin on Responses of Isolated Vas Deferens to Clonidine and Noradrenaline There were no significant changes in the strength of the contractile responses of the isolated vas deferens to electrical field stimulation (2.5 Hz) following prolonged treatment with decamethrin (peak tension developed: control, 1068 + 97 mg; decamethrin, 11605 138mg). Twenty-four hours after the last dose of decamethrin there were detectable alterations in responsiveness of isolated vas deferens to the clonidine-induced inhibition of contractile response to field stimulation and also in the contractile response of the organ to noradrenaline. Increased concentrations of clonidine were required for inhibition of contractile response to field stimulation (Fig. 1). Treatment with decamethrin produced a rightward shift in the dose-response curve for clonidine (Fig. 1). Maximal response (mg tension) to noradrenaline was significantly enhanced in the vas deferens removed from guinea pigs treated with decamethrin. The E,,, value was increased from 1042 f 67 to 1966+ 181 mg(n= 12,p
(nl-4)

FIG. 1. Inhibitory effect of clonidine on the twitch response of the isolated guinea pig vas deferens elicited by electrical stimulation (trains of five pulses, I-msec pulse duration, 2.5 Hz, supramaximal voltage, delivered at Imin intervals). Dose-response curves for the inhibition by clonidine of electrically evoked contraction in vasdeferens isolated from control (0) and decamethrin-treated (0) animals. Both vasa deferentia were removed from six guinea pigs 24 hr after the last injection of decamethrin (18 mg kg-‘, ip, once a day for 3 consecutive days) or from six control guinea pigs treated similarly with sesame oil. Each point represents the mean value of 12 experiments with SE shown by vertical lines. ***JJ < 0.001, difference from control value.

tively, for the control and decamethrintreated dose-response curves (Fig. 2a). Dose-response curves to noradrenaline were also determined in the presence of cocaine and propranolol to ascertain whether the observed effects of noradrenaline in guinea pigs treated with decamethrin were due to inhibition of amine uptake by high residual concentrations of the insecticide or to a selective reduction in inhibitory p-adrenoceptor sensitivity. Addition of cocaine (30 PM) and propranolol(3 PM) produced the expected increase in the sensitivity to noradrenaline, without affecting the maximal response of the preparation. In vasa deferentia removed from guinea pigs 24 hr after the last of the three injections of decamethrin (18 mg kg-‘, ip), the Ema, value for not-adrenaline was significantly increased from 1200 -t 86 to

DECAMETHRIN 2000

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respectively (Fig. 2b). Separate experiments were performed with propranolol and cocaine and similar results were obtained (data not shown). In decamethrin-treated guinea pigs, the maximum response to noradrenaline was greater than in controls both in the presence and in the absence of cocaine and propranolol (Fig. 2).

t

I

I

1

3

6

I

12

30

Noradrenaline

60

1

120

( JJM I

Eflect of Prolonged Treatment of Guinea Pigs with Decamethrin on Inhibition of Postganglionic Motor Transmission in Vas Deferens by Tyramine and Prostaglandin EZ

In the vasa from control and from decamethrin-treated guinea pigs, the size of motor responses to transmural stimulation (10 Hz) was almost identical (peak tension developed: control, 1492 -t 29 mg; decamethrin, 1542+31mg). Both tyramine and prostaglandin E2 exerted a dose-related inhibitory effect upon the height of the twitch responses elicited by postganglionic field stimulation. Tyramine inhibited motor transmission in vasa taken from both control and treated guinea pigs, though i 1 the sensitivity of the vasa from guinea pigs 0.1 0.3 0.6 12 3 6 12 24 pretreated with decamethrin to the inhibitory Noradrenaline (p M I action of tyramine was significantly lower FIG. 2. Postsynaptic a-adrenoceptor agonist response than that of the control vasa. This effect of of isolated vas deferens from guinea pig. Dose-response prolonged treatment with decamethrin cancurves to noradrenaline in vas deferens isolated from not be attributed to a nonspecific depression control (0) and decamethrin-treated (0) animals in the of the smooth muscle since the contractions absence (a) and presence (b) of cocaine (30 pM) and proelicited by I-min contacts with bradykinin pranolol(3 PM). Both vasadeferentia were removed from six guinea pigs 24 hr after the last injection of decame(0.2-0.4 PM) were unaltered. Figure 3 illusthrin (18 mg kg-‘, ip, once a day for 3 consecutive days) trates the results of these experiments and or from six control guinea pigs treated similarly with ses- shows that the decamethrin treatment proame oil. Each point represents the mean value of 12 exduced a rightward parallel shift in the doseperiments with SE shown by vertical lines response curve to tyramine. In contrast, pretreatment with decamethrin did not induce 2408 f 152 mg (n = 12,~ < 0.001, ANOVA) any significant effect upon the inhibitory preand there was no significant change in the synaptic effect of prostaglandin EZ (Fig. 4). pD50 value; the pD50 values for the responses to noradrenaline of isolated vas defDISCUSSION erens from control and decamethrin-treated animals in the presence of cocaine and proProlonged treatment of the guinea pig with pranolol were 5.59 * 0.04 and 5.52 + 0.02, decamethrin did not modify significantly

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ANADbN,

I 1

1 1.5

3

6

12

24

Tyramine

MARTINEZ-LARRANAGA,

46

I 96

(MM J

FIG. 3. Inhibitory effect of tyramine on the twitch response of the isolated guinea pig vas deferens elicited by electrical stimulation (trains of five pulses, I-msec pulse duration, 10 Hz, supramaximal voltage, delivered at lmin intervals). Dose-response curves for the inhibition by tyramine of electrically evoked contraction in vasdeferens isolated from control (0) and decamethrin-treated (0) animals. Both vasa deferentia were removed from six guinea pigs 24 hr after the last injection of decamethrin (18 mg kg-‘, ip, once a day for 3 consecutive days) or from six control guinea pigs treated similarly with sesame oil. Each point represents the mean value of 12 experiments with SE shown by vertical lines. *p < 0.05. **p < 0.01, ***p < 0.00 I, difference from control value.

twitch responses elicited by field stimulation of the isolated vas deferens, although the acute administration of decamethrin would probably lead to increase the height of the twitch. This study clearly shows that decamethrin produces changes in presynaptic and postsynaptic adrenoceptor function in the guinea pig vas deferens. Prolonged treatment with decamethrin produced a reduction in a2-presynaptic adrenoceptor sensitivity. Reduction on the inhibitory presynaptic effect of clonidine by decamethrin treatment might be due to a direct receptor blocking effect. Decamethrin, however, has been shown to produce two effects on atrial muscle preparations (Berlin et al., 1984). The main effect of decamethrin is to promote the release of catecholamines from sympathetic nerve terminals;

AND DIAZ

the second effect is independent of norepinephrine release and appears to represent the direct action of decamethrin on atria1 muscle. Both the release of catecholamines and the direct action of decamethrin on cardiac muscle are blocked by tetrodotoxin, which suggests an increased sodium influx into the nerve endings. On the other hand, reduction in qpresynaptic adrenoceptor sensitivity might also be associated with an inhibitory effect on Na,K-ATPase, a mechanism generally accepted to lead to an increase of transmitter release (Vizi, 1978) but the effects of pyrethroid insecticides on the membranebound Na,K-ATPase are controversial. Desaiah et al. (1973, 1975) reported that naturally occurring pyrethrins and synthetic pyrethroids inhibit Na,K-ATPase activity in homogenates of cockroach nerve and fish brain, whereas Clark and Matsumura ( 1982)

OL 91

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0,s

1,2

1 3

6

Prostaglandin

1 12

I 30

E2

FIG. 4. Inhibitory effect of prostaglandin E2 on the twitch response of the isolated guinea pig vas deferens elicited by electrical stimulation (trains of five pulses, lmsec pulse duration, 10 Hz, supramaximal voltage, delivered at I-min intervals). Dose-response curves for the inhibition by prostaglandin Ez of electrically evoked contraction in vasdeferens isolated from control (0) and decamethrin-treated (0) animals. Both vasa deferentia were removed from six guinea pigs 24 hr after the last injection of decamethrin (18 mg kg-‘, ip, once a day for 3 consecutive days) or from six control guinea pigs treated similarly with sesame oil. Each point represents the mean value of I2 experiments with SE shown by vertical lines.

DECAMETHRIN

ON NEUROMUSCULAR

did not find any effect of allethrin, a synthetic pyrethroid, on microsomal Na,K-ATPase enzyme prepared from squid retinal axon. Berlin et al. ( 1984) did not observe any influence of decamethrin on the activity of partially purified guinea pig heart Na,K-ATPase. Therefore, it is not attractive that such mechanism could be associated with reduction in ocz-presynaptic adrenoceptor sensitivity. In the guinea pig vas deferens tyramine and other indirectly acting sympathomimetic drugs exert an inhibitory effect upon postganglionic motor transmission; this inhibitory effect of tyramine is abolished by phentolamine or by pretreatment with reserpine (Ambache et al., 1972). The possibility that decamethrin might act like reserpine was therefore examined; prolonged decamethrin treatment significantly affected the inhibitory action of tyramine on the electrically induced twitches. The specificity of the effect of decamethrin was shown by the fact that bradykinin contractions were not depressed by the decamethrin treatment used in this study. Therefore, it would also suggest the existence of a mechanism of noradrenaline depletion from a sympathetic terminal varicosity by decamethrin, which would also be involved in mediating an effect on postsynaptic receptor sites. Decamethrin modified responsiveness of the vas deferens to noradrenaline after prolonged treatment. The increased postsynaptic effect of noradrenaline following decamethrin treatment (i.e., increased maximal contraction) was still present in cocaine-treated tissues, and, therefore, does not appear to be due to inhibition of neuronal noradrenaline uptake by residual insecticide. Although the use of cocaine has certain limitations (Lew and Angus, 1983) and responsiveness of the vas deferens to noradrenaline shows considerable variability between animals (Ambache and Zar, 1970), the present finding of an increased maximal response to noradrenaline in the cocaine-treated organ following prolonged treatment with decamethrin is consis-

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tent with an enhanced postreceptor mechanism. Finally, it was quite important to know whether the reduction in the presynaptic effect was specific for the a-agonists clonidine and noradrenaline (released by tyramine). This possibility has been put to the test by the use of prostaglandin E2. The high inhibitory potency of some prostaglandins toward postganglionic motor transmission in the guinea pig vas deferens was noted by Euler and Hedqvist (1969) and by Ambache and Zar (1970). It has been suggested by Hedqvist ( 1970) that endogenous prostaglandins may regulate the release of noradrenaline from sympathetic nerves by a negative feed-back mechanism. Since the inhibition induced by prostaglandin E2 has not been reported to involve a-adrenoceptors, the ability of prostaglandin E2 on postganglionic motor transmission was tested both before and after decamethrin treatment. Prolonged decamethrin treatment did not alter the inhibitory effect of prostaglandin E2 on the electrically induced twitches. The unlikely possibility that the loss or reduction of the inhibitory presynaptic effect of clonidine and tyramine by decamethrin treatment was due to a nonspecific antagonism to all drugs producing twitch-inhibition in this preparation was discounted by this finding. In conclusion, we have shown that prolonged decamethrin treatment causes a reduction in the sensitivity of the prejunctional cY*-adrenoceptor system, which would lead subsequently to increased noradrenaline release and postsynaptic adrenoceptor upregulation. The relationship between altered presynaptic receptor sensitivity and noradrenaline release by sympathetic stimulation is suggestive and study of this situation may shed further light on similar processes probably occurring in the CNS. REFERENCES E., ANDELDEFRAWI, A. T. (1982). Allethrin interactions with the nicotinic

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AKKERMANS, L. M. A., VAN DEN BERCKEN, J., AND VERLUIJSHELDER, M. (1975). Comparative effects of DDT, allethrin, dieldrin and aldrin-transdiol in sense organs of Xenopus laevis. Pestic. Biochem. Physiol. 5, 45 l-457.

AMBACHE, N., DUNK, L. P., VERNEY, J., AND ZAR, M. A. (1972). Inhibition of post-ganglionic motor transmission in vas deferens by indirectly acting sympathomimetic drugs. J. Physiol. (London) 227, 433456.

AMBACHE, N., AND ZAR, M. A. (1970). An inhibitory effect of prostaglandin E2 on neuromuscular transmission in the guinea-pig vas deferens. J. Physiol. (London) 208,30-32P. ANAD~N, A., AND MARTINEZ-LARRA~AGA, M. R. ( 1985). Effects of crotoxin on autonomic neuromuscular transmission in the guinea-pig myenteric plexus and vasdeferens. Toxicon 23(6), 963-972. BARNES, J. M., AND VERCHOYLE, R. D. (1974). Toxicity of a new pyrethroid insecticide. Nature (London) 248, 711. BERLIN, J. R., AURA, T., BRODY, T. M., AND MATSUMURA, F. (1984). The inotropic effects of a synthetic pyrethroid decamethrin on isolated guinea-pig atria1 muscle. Eur. J. Pharmacol. 98,3 13-322. CLARK, J. M., AND MATSUMURA, F. (1982). Two different types of inhibitory effects of pyrethroids on nerve Ca- and Ca+/Mg-ATPase activity in the squid, Lo&o paelei. Pestic. Biochem. Physiol. 18, 180-190. CLEMENTS, A. N., AND MAY, T. E. (1977). The actions of pyrethroids upon the peripheral nervous system and associated organs in the locust. Pestic. Sci. 8,66 l-680. DESAIAH, D., CUTKOMP, L. K., AND KOCH, R. B. (1973). The effect of pyrethrins on ATPases of cockroach and bluegill fish. Pyrethrum Post 12,70-76. DESAIAH, D., CUTKOMP, L. K., VEA, E. V., AND KOCH, R. B. (1975). The effect of three pyrethroids on ATPases of insects and fish. Gen. Pharmacol. 6(l), 3 l34.

EULER, U. S. V., AND HEDQVIST, P. (1969). Inhibitory action of prostaglandin El and E2 on the neuromuscu-

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lar transmission in the guinea-pig vas deferens. Acta Physiol. Stand. 77,5 1O-5 12. HEDQVIST, P. (I 970). Studies on the effect of prostaglandins E, and E2 on the sympathetic neuromuscular transmission in some animal tissues. Acta Physiol. &and. Suppl. 345,l l-40. LEW, M. J., AND ANGUS, J. A. (1983). Disadvantages of cocaine as a neuronal uptake blocking agent: Comparison with desipramine in guinea-pig right atrium. J. Auton. Pharmacol. 3,6 l-7 1. LUND, A. E., AND NARAHASHI, T. (1981). Kinetics of sodium channel modification by the insecticide tetramethrin in squid axon membranes. J. Pharmacoi. Exp. Ther. 219,464-473. NARAHASHI, T. ( 197 1). Mode of action of pyrethroids. Bull. World Health Org. 44,337-345. RAY, D. E. (1980). An EEG investigation of decamethrin-induced choreoathetosis in the rat. Exp. Brain Res. 38,22 l-227. RAY, D. E. (1982). Changes in brain flow associated with deltamethrin-induced choreoathetosis in the rat. Exp. Brain Res. 45,269-276. RAY, D. E., AND CREMER, J. E. (1979). The action of decamethrin (a synthetic pyrethroid) on the rat. Pestic. Biochem. Physiol. 10,333-340. SHEINER, L. B. (1981). ELSFIT: A program for the extended least squares fit to individual pharrnacokinetic data. A technical report of the Division of Clinical Pharmacology, University of California, San Francisco, CA. VAN DEN BERCKEN, J., AKKERMANS, L. M. A., AND VAN DER ZALM, J. M. (1973). DDT-like action ofallethrin in the sensory nervous system of Xenopus laevis. Eur. J. Pharmacol. 21,95-106. VIJVERBERG, H. P. M., AND VAN DEN BERCKEN, J. (1979). Frequency-dependent effects of the pyrethroid insecticide decamethrin in frog myelinated nerve fibers. Eur. J. Pharmacol. 58,SO l-504. VIJVERBERG, H. P. M., VAN DER ZALM, J. M., AND VAN DEN BERCKEN, J. (1982). Similar mode of action of pyrethroids and DDT on sodium channel gating in myelinated nerves. Nature (London) 295,601-603. VIZI, E. S. (1978). Na+,K+-activated adenosinetriphosphatase as a trigger in transmitter release. Neuroscience 3,367-384. WOUTERS, W., AND VAN DEN BERCKEN, J. (1978). Action of pyrethroids. Gen. Pharmacol. 9,387-398.