Naunyn-Schmiedeberg's Naunyn-Schnriedeberg's Arch. Pharmacol. 381,

Archivesof Pharmacology

179 - 182 (1979)

9 by Springer-Verlag 1979

Short Communication The Action of Lindane in Accelerating the Spontaneous Release of Transmitter at the Frog Neuromuscular Junction S. J. Publicover and C. J. D u n c a n Department of Zoology, University of Liverpool, P.O.Box 14% Liverpool L69 3BX, Great Britain

SUMMARY. Lindane (~ x IO-PM) causes a progressive and marked rise in MEPP frequency at the frog neuromuscular junction. C~ncentrations over a range of 5 x I0-6M to ~ x 10- M were tested. The results suggest that it has two actions in promoting this effect. Its major effect is orobably to cause an increase in Ca 2+permeability and a rise in Ca 2+ entry. Its second, smaller effect, which persists in the absence oi extracellular Ca 2 , ms probably also because of a rise in [Ca2+]i . The ways in which these effects might be produced and the significance of these findings for explaining the known pharmacological actions of lindane are discussed. KEY WORDS: Lindane - neuromuscular junction transmitter release - calcium.

INTRODUCTION The chlorinated hydrocarbons are of importance because of their insecticidal properties, although the details of their toxicological action on vertebrates are frequently not known. The insecticide lindane (hexachloro-cyclohexane, Y isomer) has a number of reported pharmacological actions including that of a rapidly acting potent convulsant in man and other mammals. Primary signs following acute exposures are hyperexcitability~ tremors and convulsions. Lindane also produced changes in the EEG and evoked responses in cats; the responses in the sensory and motor cortex to sensory nerve stimulation were enhanced ~- to 5-fold and the results show that lindsme is directly toxic to the mammalian CNS (Joy, 1976). A small, single oral dose of lind&he also lowers the convulsive threshold for pentylenetetrazol in the mouse (Hulth et al., 1976). Lindane has also been shown to cause the release of neurohormones from the corpus cardiacum of the locust and, concomitantly, the mitochondria were seen to undergo a number of ultrastructural changes. It was concluded from such studies that lindane promotes an excessive entry of Ca 2+ into these cells (Normann and Ssmaranayaka-Ramasamy, 1977). We have found further that treatment of frog muscle with lindane apparently causes 9 marked rise in intracellular calcium ([Ca~+]i) which, in turn, Send offprint requests to C.J. Duncan at the above address

promotes myofilament degradation and, accompanying this phenomenon, the muscle mitochondria were found to be swollen (Publicover, Duncan and Smith, 1979). Such studies suggest that lindane is able to produce alterations in [Ca2+]i in certain tissues. Evidence is now accumulating that the spontaneous release of transmitter at the neuromuscular junction (recorded as the miniature endplate potentials, MEPPs) is primarily determined by [Ca2+] i at the presynaptic terminals (Alnaes and Rah~nimoff, 1975; Statham and Duncan, 1976; Duncan and Statham, 1977; Statham, Duncan and Publicover, 1978). Thus, MEPP frequency at the frog neuromuscular junctiQn can be mod2fied experimentally by releasing Ca L+ frQm 2+ the mitochondria or frgm other intracelluls~ Ca -stores, by altering CaL~-fluxes across the plasma membrane or by reducing the activity of the Ca 2+ pump of the plasma membrane. The purpose of the present experiments was, therefore~ to test the action of lindane on the spontaneous release of transmitter at the frog neuromuscular junction and we report in this short communication that this insecticide is able to increase markedly MEPP frequency and suggest that such findings may throw light on its mechanism of action in the CNS. MATERIAL AND METHODS Electrophysiological recordings of HEPP frequency were made from the isolated cutaneous pectoris nerve-muscle preparation of the frog Rana temporaria. Frogs were maintained in the laboratory at 6~ All salines in which the preparations were bathed contained NaCI 110@~, KCI 1.87mM, NaH2PO 4 O.O}2mM, NaHCO 3 4.76mM, glucose 2g/l at pH 7.1. Calcium concentration was varied; normal saline contained 1.SmM CaCI2, whilst a Ca2+-EGTA buffer was used at N x I0-7M [Ca2+]o . 0.SmM EGTA was added, together with the appropriate volume of AnalaR standard volumetric solution of CaCI2, and free Ca 2+ concentrations were calculated from the method of Portzehl, Caldwell and R~egg (1964). The Na + was replaced by sucrose in salines containing low [Na +] to maintain isosmocity, assuming lmH NaCI was equivalent to 1.824mM sucrose (see Birks: 3urstyn and Firth, 1968). Lindane was dispersed by dissolving in absolute ethanol and adding the solution to the saline. The final concentration of ethanol never exceeded O.N%.

0028-1298/79/0308/0179/$01.00

180 The muscle was excised and equilibrated in the appropriate control saline for 30 min at I0~ It was then pinned out in the experimental bath, the microelectrode inserted in the endplate region and the temperature adjusted to the experimental value. Records of MEPPs began after a further 10 min. Electrophysiological recordings were made by the use of conventional glass microelectrodes filled with 3M KCI; the temperature of the bath was controlled at 22.5~ (+ 0.5~ by a Peltier device. Potentials were fed through a cathode follower to a Tektronix 502A oscilloscope. MEPPs were recorded on moving film and counted. In any one experiment, the MEPPs were monitored in a single fibre at the intervals shown in the figure and at least 100 MEPPs were counted, except at very low frequencies. At least two control readings were taken before application of drugs to give a mean control rate. Statistical analysis was carried out by calculating a regression coefficient, b, and then determining the variance using the difference between successive residuals of the response (y) from the expectation (Y) in the regression line, (ix (y-Y)) (Bliss, 1967). The null hypothesis was b = 0 and the significance level of b was calculated using Student's t test. All inorganic salts and ethanol were Anala~ grade. EGTA and lindane were obtained from Sigma Chemical Co., St. Louis. RESULTS The control MEPP frequency varied between different preparations, although only small oscillations were recorded in any one preparation. Comparisons of the effects of treatments are therefore shown by expressing MEPP frequency as a ratio of the control frequency (expressed as F1/Fo, where F 0 is the control frequency and F I the frequency after treatment). Control experiments (in normal saline plus 0.5% ethanol) showed a small reduction in mean MEPP frequency with time, mean F1/F 0 falling to about 0.9 after 40 min. Similar results were obtained with saline lacking ethanol. Lindane caused a progressive and marked rise in MEPP frequency at a concentration of 5 x 10-5M which was statistically significant (t = I09.36~ 5 d.f.; p < O . O 0 1 ) ~ after 40 min the mean rate had increased 5-fold (Fig. I). The action of k lindane at4the higher concentrations of 2 x IO--M or 5 x 10- M was more dramatic~ MEPP frequency in some preparations rose 15 to 30-fold after 20 min exposure. MEPP amplitude steadily decreased at these higher concentrations of lindane, falling to 20% of control values after 20 min exposure, although there was no change in muscle resting potential. This action of lindane on MEPP amplitude was less marked at 5 x I0-5M. The possible effect of the solvent ethanol was checked again by further experiments in which the preparation was exposed to saline containing 0.5% ethanol for 50 min, during which time only small oscillations in MEPP frequency were detected, and the saline was then changed for

one containing 2 x I0-4M lindane in ethanol (0.5% final concentration). Lindane again caused an identical rapid rise in MEPP frequency (FI/Fo = 5.0 after 10 min). 5 x 10-bM lindane had little effect on MEPP frequency and only very small rises (FI/F 0 = 1.2) were detected after 35 min exposure (Fig. I).

A

2

C

I

0

i 0

I

I 20

1

i 40

Mins Fig. I. Effect of lindane on MEPP frequency at 22.5~ [Ca2+]o = 1.SmM. A: 5 x I0-5M lindsme; mean of 5 experiments; mean F 0 before application of lindane = 8 2 . 1 M E P P s min -I. Calculated regression, y = 0.0876x + 1.004. B: 5 x I0-6M lindane; result of a single experiment. C: Effect of lindane (5 x I0-5M) when [Ca2+] o was reduced to 5 x I0-7M. Mean of 5 experiments. Mean F 0 = 28.9 MEPPs min -I. Calculated regression, y = 0.0178x + 1.211. Ordinate = MEPP frequency (F I) as a ratio of the control frequency (Fo) ; abscissa = time after application of lindane; vertical bars indicate + SEM.

In the light of previous studies (Publicover and Duncan, 1979), such rises in MEPP frequency would suggest that lindane probably causes a rise in [Ca2+] i at the presynaptic terminals. The experiments were therefore repeated with the preparation bathed i~ saline in which [Ca2+]o was buffered at 5 x IO-WM (a value that is approximately equal to [Ca2+] i) and the mean control MEPP frequency was reduced, as previously reported (Duncan and Statham, 1977). Under these conditions the action of lindane (5 x 10-5M) was less marked. Although MEPP frequency rose significantly in all preparations (t = 33.14~ 5 d.f.; p < 0.001), the maximum mean FI/F 0 was only 2.0. After 40 min exposure, m~anF1/F O w a s 1.5 (Fig. I), compared with 5 when [Ca2+]o = 1.SmM. This response at low [Ca2+] o is significantly different from that recorded when [Ca2+] o = 1.SmM (t = 72.54~ 8 d.f.; p ~ 0 . 0 0 1 ) . The experiments were repeated when the preparation was equilibrated in a saline in which [Na ] was reduced to 50% (55mM), normal osmol~rity being maintained by substitution with sucrose. [Ca2+] ~ was normal (1.SmM). A

181 reduction in [Na+]o caused a rise in the basic control ~ P P frequency, as previously reported (Statham and Duncan, 1977). Under such conditions the effect of 5 x I0-5M lindane was much reduced; a significant rise in MEPP frequency was still recorded (t = 46.96; 4 d.f.; p ~ 0.001) but the maximum mean F1/F 0 was now only 1.9. This response at 50% [Na+] o was significantly different from that recorded with normal [Na+] o ( t = 70.98; 7 d.f.; p ~ 0.O01) but not from those with low [Ca2+]o and normal [Na+]o (t = 2.14; 7 d.f.). Further reductions in [Na+] o made detection of MEPPs difficult.

DISCUSSION The results show that lindane clearly accelerates the spontaneous release of transmitter at the frog neuromuscular junction. The threshold for an observable effect on ~EPP frequency is probably close to 5 x lO-UM and the maximum response was recorded at 2 x I0-4M. Since the stimulatory effect of lindane on MEPP frequency is so markedly reduced by reducing [Ca2+]o to a level that is close to [Ca2+]i, we conclude that one of the effects of the insecticide is to increase the Ca2+-permeability of the presynaptic plasma membrane. The stimulatory action can also be reduced to an equally low level by changing [Na+]o from 110mM to 55mM ([Ca2+] o maintained at 1.SmM), and it is therefore possible that lindane acts primarily by causing a change in Na+-permeability and depolarization at the terminals which, in turn, causes an increase in Ca2+-permeability. MEPP frequency at amphibian and mammalian terminals is greatly accelerated by presynaDtie depolarization which causes an increase in Ca2+-permeability (Liley, 1956; Landau, 1969). ENa is reduced by some 17mV when [Na+]o is 55mM so that a smaller depolarization will be produced by lindane and, correspondingly, there will be a smaller increase in Ca2+-permeability. Five-fold changes in MEPP frequency (as shown in Fig. 1) are produced by quite small depolarizations of amphibian (Del Castillo and Katz, 1954) and mammalian terminals (Liley, 1956) and it is also noteworthy that the frequency of miniature excitatory postsynaptic potentials at the frog sympathetic ganglion is sensitive to depolarization by elevated [K+]o only at concentrations above a critical level (Beume and Pott, 1978). Lindane still produces a small~ but consistent, rise in MI~PP frequency, even at very low buffered [Ca2+]o, and we suggest that it may be able to promote the release of Ca 2+ from intracellular storage sites such as the mitochondria. Lindane (1.7 ~mole/mg protein) has been sho~Tn to inhibit electron transport on the substrate side of oytochrome C in isolated beef heart mitochondria (Pardini et al., 1971) and we have found that it inhibits oxidation at 5x10-SM in isolated frog liver mitochondria (Publicover, Duncan and Smith, in prep.). Furthermore, if lindane causes an increase in Na+-permeability at the terminals, [Na+]i may also rise, so

nromoting Ca 2+ release from the mitochondria and an acceleration in MEPP frequency (Statham and Duncan, 1977). There is substantial evidence that a rise in [Na+]~ or [Li+] i causes an increase in MEPP frequency (Carmody and Gage, 1973; Baker and Crawford, 1975; Branisteanu and Volle, 1975; Crawford, 1975) and both Na + and Li + are known to promote the release of Ca 2+ from isolated mitochondria (Crompton, Moser, L~di and Carafoli, 1978). The insecticide dieldrin is also believed to interfere specifically with synaptic transmission; aldrin-transdiol (I0-5M) causes a marked increase in MEPP frequency at the frog neuromuscular junction in Ca• solution (Akkermans, Zalm ~ud Bercken, 1973), again suggesting that this active form of dieldrin probably acts by releasing Ca 2+ from intracellular sites at the terminals. Since the spontaneous release of transmitter at the neuromuscular junction is of little physiological significance in vivo, the actions of lindane may have only a subliminal effect on the control of motor functioning. However, if the insecticide has a similar effect at other synapse~ as in the CNS, accelerating the spontaneous release of transmitter and reducing the amplitude of the excitatory and inhibitory postsynaptic potentials, it is clear that it could have effects on excitability. Lindane (10-4M) is also known to block synaptic transmission at the mammalian sympathetic ganglion (White and Larrabee, 1973). Such a suggestion would explain the actions of lindane on the CNS (as summarized in the Introduction); it produces hyperexeitability, tremors, convulsions and seizures. ACKNOWLEDGEMENTS. We thank Miss S. Scott for assistance in the preparation of the manuscript. S.J. Publicover was in receipt of an S.R.C. studentship. RE~RENCES Akkermans, L.M.A., Van der Zalm, J.M., Van den Bercken, J.: Is aldrin-transdiol the active form of the insecticide dieldrin? Arch. int. Pharmacodyn. 206, 363-364 (1973) Alnaes, E., Rahamimoff, R.: On the role of mitochondria in transmitter release from motor nerve terminals. J. Physiol. (Lond.) 248, 285-306 (1975) Baker, P.F., Crawford, A.C.: A note on the mechanism by which inhibitors of the sodium pump accelerate spontaneous release of transmitter from motor nerve terminals. J. Physio~ (Lond.) 247, 209-226 (1975) Beume, R., Port, L.: Dual effect of external calcium on the frequency of miniature synaptic potentials I! in frog sympathetic ganglion cells. Pflugers Arch. 376, 21-26 (1978) Birks, R.I., Burstyn~ P.G.R., Firth, D.R.: The form of sodium-calcium competition at the frog myoneural junction. J. Gen. Physiol. 52, 887-90? (1968) Bliss, C.I.: Statistics in Biology, Vol. I. New York: McGraw-Hill 1967

182 Branisteanu, D.D., Voile, R.L.: Modification by lithium of transmitter release at the neuromuscular junction of the frog. J. Pharmacol. Exp. Ther. 19~, 362-372 (1975)

Normann, T.C., Samaranayaka-Rm~asamy, M.: Secretory hyperactivity and mitochondrial changes in neurosecretory cells of an insect. Cell Tiss. Res. 183, 61-69 (1977)

Carmody, J.J., Gage, P.W.: Lithium stimulates secretion of acetylcholine in absence of extracellular calcium. Brain Res. 50, 476479 (1973)

Pardini, R.S., Heidker, J.C., Payne, B.: The effect of some cyclodiene pesticides, benzenehexachloride and toxaphene on mitochondrial electron transport. Bull. Environ. Contam. Toxicol. 6, 436-444 (1971) ,, Portzehl, H., Caldwell, P.C., Ruegg, J.C.: The dependence of contraction and relaxation of muscle fibres from the crab Maia squinado on the internal concentration of free calcium ions. Biochim. Biophys. Acta 79, 551-591 (1964)

Crawford, A.C.: Lithium ions and the release of transmitter at the frog neuromuscular junction. J. Physiol. (Lond.) 246, 109-142 (1975) H

Crompton, M., Moser, R., Ludi, H., Canafoli, E.: The interrelations between the transport of sodium and calcium in mitochondria of various mammalian tissues. Eur. J. Biochem. 82, 2551 (1978) Del Castillo, J., Katz, B.: Changes in end-plate activity produced by presynaptic polarization. J. Physiol. (Lond.) 124, 586-604

(1954) Duncan, C.J., Statham, H.E.: Interacting effects of temperature and extracellular calcium on the spontaneous release of transmitter at the frog neuromuscular junction. J. Physiol. (Lond.) 268, 319-333 (1977) 9

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Hulth~ L., Larsson~ M., Carlsson, R., Klhlstrom~ J.E. : Convulsive action of small single oral doses of the insecticide lindane. Bull. Environ. contam. Toxiool. 16, 133-137 (1976) Joy, R.M.: Convulsive properties of chlorinated hydrocarbon insecticides in the cat central nervous system. Toxicol. Appl. Pharmacol. 35, 95-106 (1976) Landau, E.M.: The interaction of presynaptic polarization with calcium and magnesium in modifying spontaneous transmitter release from mammalian motor nerve terminals. J. Physiol. (Lond.) 205, 281-299 (1969) Liley, A.W.: The effects of presynaptic polarization on the spontaneous activity at the mammalian neuromuscular junction. J. Physiol. (Lond.) 134, 427-443 (1956)

Publicover, S.J., Duncan, C.J.: The action of verapamil on the rate of spontaneous release of transmitter at the frog neuromuscular junction. Eur. J. Pharmacol. 54, 119-127 (1979) Publicover, S.J., Duncan, C.J., Smith, J.L.: The action of lindane in causing ultrastructural damage in frog skeletal muscle. Comp. Biochem. Physiol. in press (1979) Statham, H.E., Duncan, C.J.: D~trolene and the neuromuscular junction: evidence for intracellular calcium stores. Eur. J. Pharmacol. 39, 143-152 (1976) Stathsml, H.E., Duncan, C.J.: The effect of sodium ions on MEPP frequency at the frog neuromuscular junction. Life Sci. 20, 18391846 (1977) Statham, H.E., Duncan~ C.J., Publicover~ S.J.: Dual effect of 2,4-Dinitrophenol on the spontaneous release of transmitter at the frog neuromuscular junction. Biochem. Pharmacol. 27~ 2199-2202 (1978) White, G.L., Larrabee, M.G.: Phosphoinositides and other phospholipids in sympathetic ganglia and nerve trunks of rats. J. Neurochem. 20, 783-798 (1973) Received January 23 Accepted July 2,1979