Inhibition by decamethrin and resmethrin of hormone release from the isolated rat neurohypophysis—A model mammalian neurosecretory system

PESTICIDE

BIOCHEMISTRY

AND

PHYSIOLOGY

17, 42-47 (1982)

Inhibition by Decamethrin and Resmethrin of Hormone Release from the Isolated Rat Neurohypophysis-A Model Mammalian Neurosecretory System R.E.J. Department

of Anatomy,

University

of London

DYBALL King’s

College,

Strand,

London

WC2R

2LS,

England

Received June 15, 1981; accepted November 4, 1981 The isolated rat neurophypophysis, which shows a calcium-dependent hormone release when depolarized in vitro was used as a model system to investigate the effects of the pyrethroids decamethrin and resmethrin on mammalian nervous tissue. Both compounds inhibited neurohypophysial hormone release in response to electrical stimulation, decamethrin being more potent than resmethrin. Decamethrin reduced the hormone content of the neurohypophysis. Resmethrin did not reduce stored hormone significantly and its effects on release were dose dependent. They could be mimicked by raising the [Na+] of the medium but not by lowering the [Ca*+]. Resmethrin had no effect on the release of hormone following depolarization of the tissue with a raised [K+]. The results are consistent with the suggestion that the compounds do not act on the potential-dependent secretion process but rather on the mechanism linking depolarization of the secretory terminals with the arrival of action notentials possibly by interfering with sodium-channel activation and inactivation.

sequently to a failure of action potential generation and paralysis (5 - 7). It is probable that similar effects also occur in amphibia (2,8) and mammals (3,4) but the evidence is very far from complete. The difficulty arises because it is not easy to perform critical electrophysiological experiments involving intracellular recording on mammalian nerve cells so that indirect methods are frequently sought. The present investigation uses a preparation of mammalian excitable tissue, the isolated rat neurohypophysis in vitro, which has been extremely useful in elucidating the mechanism involved in neurosecretion, particularly stimulus- secretion coupling (9). It consists of the isolated terminals of the neurosecretory neurons of the neurophypophysis. The tissue releases the neurohypophysial hormones (oxytocin and vasopressin) when stimulated in vitro by depolarizing stimuli (9- 11). Secretion can be blocked by verapamil derivatives which block Ca2+ ion transport and is promoted by calcium ionophores (12). It thus provides a useful model for studying release of stored transmitter substances by exocytosis. Hormone

INTRODUCTION

Pyrethroids are very widely used insecticides and although they are much more toxic to insects than mammals (1) their toxic effects on mammals, including man, have a compelling interest. The signs of pyrethroid intoxication in insects include hyperexcitability, tremors, and convulsions which ultimately lead to death. After administration of large doses of pyrethroids to vertebrates similar signs develop quickly (2-4). There is a considerable and growing body of evidence on the mode of action of pyrethroids on the invertebrate nervous system (5-7) but less evidence is available on their effects on mammals. In invertebrates the mode of action of pyrethroids on the electrical excitability of nerve cells is probably to inhibit the increase in sodium-channel permeability which occurs on the initiation of the nerve impulse, to inhibit sodiumchannel inactivation, and to inhibit potassium-channel inactivation. These changes initially lead to a repetitive discharge when a nerve cell is stimulated in the presence of the pyrethroid and sub42 0048-3575/82/010042-06$02.00/O Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

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release by electrical stimulation with appropriate parameters can also be blocked by the Na-channel-blocker tetrodotoxin (13). It thus provides a very useful model in which materials active in the mammalian nervous system can be studied and can be used to investigate both axonal excitability and synaptic function. The present investigation shows that two pyrethroids, decamethrin (NRDC 161) and resmethrin (NRDC 104), both inhibit neurohypophysial hormone release from the isolated neural lobe in vitro. The inhibition does not appear to affect the exocytosis mechanism itself but rather the excitability of the nerve cell membrane and is consistent with interference with sodium-channel activation. METHODS

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43

Composition of the incubation media. The composition of normal Locke’s solution was NaCl, 15OmM; CaCl,, 2.2 mM: MgC12, 1.0 mM; KHC03, 5.6 rm?4; glucose, 10 mM. When [Na+] was reduced the change in osmolality was compensated for by the addition of choline. A medium containing 100 mM+(pH 6.9) was used for most of the incubations since such a medium had been used extensively for in vitro studies of the neurohypophysis (9). Further, the increased hormone released into such a medium makes assay of inhibited release much more precise since the assay used is not at the limit of its sensitivity. Hormone assay. Both neurohypophysial hormones are released from the isolated neural lobe in vitro. In the experiments reported here, the procedure adopted by Nordmann (11) was followed and milkejection activity was estimated by the method of Bisset et al. (14). The quantity of hormone released was then expressed in terms of milliunits of synthetic oxytocin.

Animals. The experiments were performed on isolated neural lobes dissected from male rats (Porton Wistar strain) weighing between 200 and 250 g, maintained in a constant environment and allowed water and food (Diet 41B, Grain harRESULTS vestors) ad fihitum. Before the incubations The influence of resmethrin and dethe rats were given, by stomach tube, either camethrin on neurohypophysial hormone decamethrin (NRDC 161) or resmethrin release following electrical stimulation. (NRDC 104) dissolved in glycerol formal Within 4 hr of the administration of de(Fluorochem Ltd) at the stated dose. Incubation procedures. After adminiscamethrin (either at 50 or 25 mg/kg) the rats tration of the pyrethroid (4 hr before re- became unconscious and showed marked moval of the tissue for decamethrin; 5 hr for tremors. Tremors were not seen with resmethrin at any dose used but 5 hr after its resmethrin) the rats were stunned by a blow on the head and decapitated. The neuroadministration at the higher doses (25 and hypophyses were dissected out, impaled in 50 mg/kg) the animals appeared to be in the groups of three on one of a pair of platinum early stages of anesthesia. Both resmethrin stimulating electrodes and immersed for (given 5 hr before) and decamethrin (given 4 two periods of 10 min in normal Locke’s hr before) inhibited neurohypophysial horsolution (13). They were then transferred to mone release from isolated rat neural lobes a modified Lock’s solution containing 100 incubated in vitro. No hormone release could mM Na for five successive periods of I5 be detected before stimulation started but, min and stimulated electrically for the last during stimulation, release was inhibited by four periods. Other media and stimulation both drugs. At 25 mg/kg decamethrin deregimes were used when specified and all pressed hormone release significantly more the incubations were carried out at 37°C than resmethrin (P = 0.0332, Mann-Whitand bubbled constantly with O2 (95%) and ney U test). Both compounds prevented the significant reduction in hormone release co, (5%).

44

R. E. J. DYBALL

during stimulation which occurred during stimulation of control glands (inactivation of release; see Nordmann (11) and Figs. la and 2a). Both drugs also tended to reduce the neurohypophysial content of stored hormone (see Figs. lb and 2b). To calculate the values for gland content in Figs. lb and 2b the average quantity of hormone released during the incubation was added to the quantity of hormone remaining in the glands at the end of the incubations. Only in

A E

a)

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h c

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0

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= A

b)

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2-

b)

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1’5 $0 4’5 6’6 timetmin)

E’ Y c ii

4 cio c 2 :: 260 c Q 5' Iii 0-

4-

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T In 040‘0

ui 01 0 cd resmethrin(mg/Kg)

b

1 k 3’ 4’5 6’0 time Pmin)

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0-

FIG. 2. (a) Release of hormone into the incubation medium during four successive periods of electrical stimulation of control glands (0) and glands from animals given 6.25 (0) , 25 (0) and 50 (m) mglkg resmethrin. Each point represents (except in the case of 50 mglkg where n = 5) the mean (LSEM) of at least six different incubations. The initial point represents the release before stimulation started and was below the satisfactory limit of the assay in each case. (b) The mean (+SEM) gland content of hormone at the different dose levels (n = at least 10 each case). Asterisks indicate significant difference from control values: *P < 0.02, **P < 0.01.

decamethrin(mg/Kg) Release of hormone into the incubation medium during four successive periods of electrical stimulation of control glands (0) and glands from animals given 25 (0) or 50 (W) mglkg decamethrin; each point represents the mean (%SEM) of four incubations. (b) The mean (%SEM) gland content of hormone at the different dose levels. Asterisks indicate significant difference from control values: **P < 0.01. FIG.

1. (a)

the case of decamethrin at 50 mg/kg was it significantly below that in control glands.

A comparison of other procedures which reduce initial hormone release. A number of other procedures reduced neurohypophysial hormone release in vitro. Depletion of hormone stores by 15% by applying a dehydration stimulus to the

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AND

animals (drinking 2% NaCl solution instead of drinking water for 1 day) reduced initial hormone release but did not prevent inactivation of release with time. Similarly reducing the [Ca”‘] in the medium from 2.2 to 1.5 mA4 reduced the initial release of hormone but did not prevent the inactivation (Fig. 3). Raising the sodium concentration of the medium from 100 to 150 mA4 also reduced initial release but in this case no significiant reduction of hormone release was seen during the course of the stimulation. Raising both [Na+] and [Ca2+] had no detectable effect on unstimulated release and increased the initial release rate during stimulation back toward control but inactivation of release during stimulation was not seen in this experiment either. The influence

of resmethrin

on neuro-

HORMONE

45

SECRETION

hypophysial hormone release during potassium-induced depolarization. If the nerve

terminals in the isolated neural lobe were depolarized by raising the [K+] in the medium from 5.6 to 56 mA4 a substantial hormone release occurred ((11) and see Fig. 4). This release rapidly inactivated and neither initial release rate nor inactivation was influenced by resmethrin at 50 mgkg (Fig. 4). Potassium-induced release, like that due to electrical stimulation, was reduced by raising the [Na+] of the medium from 100 to 150 mA4 but, in the case of potassium-induced depolarization, inactivation of release was seen despite the raised [Na+]. DISCUSSION

Release of neurohypophysial hormone from the isolated rat neural lobe was clearly reduced by the prior administration of pyrethroids to the animals from which the tissue was taken. The drugs were given in advance by stomach tube because of the diffi-

a

-.------------

L

0

=

O1

b

1’5 3’0 4’5 6’0 time(min)

FIG. 3. A comparison of difirent incubation procedures which inhibit hormone release during electrical stimulation of isolated neural lobes for I hr: l , control glands in IO0 mM; A, control glands in 150 mM Na; A, control glands in 150 mM Na and raised (4.95 mM) Ca; 0, control glands with reduced (1.5 mM) Ca; and q glands with the hormone store reduced from 423 ? 17 to 358 h 27 ma by drinking 2% NaCl solution for I day.

FIG. 4. The effects of resmethrin on hormone release stimulated by raising [K+]from 5.6 to 56 mM; 0, control stimulation (100 mM Na); W. stimulation aftet resmethrin; and A, incubation with 150 mM Na.

46

R. E. J. DYBALL

culty of maintaining them in aqueous solution and also because any drug or its vehicle added to the incubation medium might have affected the assay animal used to determine the quantity of hormone released. The method of administration described here was clearly effective since it reduced hormone release but any interferences with the assay seems unlikely. Any pyrethroid washed from the tissue into the incubation medium would probably have been present in the media from both electrically and potassium-stimulated tissue but the compounds had no detectable effect on potassium-induced release. An alternative method of overcoming this interference with the assay would have been to use an in vitro assay system. Reduced release of neural lobe hormones in vitro after pretreatment of the rats in vivo with decamethrin or resmethrin could have been due to depletion of stored hormone available for release, interference with hormone release by calcium-induced exocytosis, interference with depolarization-induced calcium influx, or interference with the general excitability of the nerve cell membranes. It is unlikely that depletion alone played an important role in reducing release, except with the higher dose of decamethrin. Neither the lower dose of decamethrin, nor resmethrin at any dose level used, significantly reduced the hormone store so it is probable that in these experiments a mechanism other than depletion inhibited release. Consideration of the results with resmethrin support this suggestion since the highest dose (50 mg/kg) neither significantly reduced hormone store nor the release provoked by potassium depolarization. Further, intentional depletion of stored hormone, although it reduced initial release, did not prevent the inactivation of release with time during prolonged stimulation. Both decamethrin and resmethrin, in appropriate doses, prevented inactivation of release. Similarly it is unlikely that calciuminduced exocytosis was influenced by the

compounds because potassium-induced depolarization released as much hormone in the presence of resmethrin as without it. There thus remains interference with action potential-induced Ca influx and with cell excitability in general, although these two latter processes may not be easily separable. In vertebrates, as in invertebrates, pyrethroids frequently induced repetitive firing after stimulation (for references see Wouters and Van den Bercken (7)) and the effects seem particularly marked on neurosecretory cells in insects (15). If the compounds exert the same effects on sodium and potassium channels in rats as they do in the invertebrate they would delay the activation of Na channels, decrease the amplitude of the action potentials, decrease the opening of the potential sensitive Ca channels, and thus reduced hormone release (16). Delayed inactivation of the Na channels would oppose this effect by holding open potential-sensitive calcium channels for longer but an accompanying delay in potassium-channel inactivation would hyperpolarize and close the calcium channels and the two effects would tend to cancel each other out. Thus the most likely explanation of the effect of pyrethroids on initial release is that the compounds act on the neural lobe by a mechanism similar to that described in invertebrates. The reduced inactivation during the course of stimulation may be explained by the experiments in 150 m/t4 sodium which also showed delayed inactivation of hormone release with electrical stimulation. In the untreated preparation it is thought that release is terminated at the end of an action potential by a number of mechanisms including Na-Ca exchange which reduces intracellular free [Ca”‘]. The raised extracellular [Na+] would increase Na- Ca exchange and reduce intracellular [Ca”‘] contributing to the observed reduction in the initial release rate. At the same time it is reasonable to suggest that intracellular [Na+] would be higher as stimulation con-

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tinued. There is evidence that at the neuromuscular junction raising intracellular [Na+] potentiates transmitter release possibly by displacing bound intracellular calcium (17) so that release may have been maintained in a medium with 150 mA4 Na+ but inactivated in a medium with 100 mit4 Na+ because in the former case intracellular [Na+] was higher. Despite its effect on reducing calcium influx the raised sodium concentration may thus have raised the effective intracellular calcium concentration as stimulation was continued. The pyrethroids may have decreased initial release by interfering with Na-channel activation. They may also have helped to maintain release by delaying Na-channel inactivation and thus raising intracellular sodium concentration which in turn tended to raise intracellular calcium concentration. With potassium stimulation in the presence of resmethrin, depolarization was maintained so that, even if slightly delayed in onset, the inactivation of the Na channel was also maintained. Thus no additional Na+ ions entered the endings to inhibit the mechanisms which normally lower intracellular [Ca”] and inactivation of hormone release occurred as expected. It may be concluded that decamethrin and resmethrin both reduced neurohypophysial hormone release in response to electrical stimulation in vitro and that the effect is unlikely to have been due to a reduction of stored hormone or to an interference with the calcium-induced exocytosis mechanism. It is more likely to have been due to an alteration in the action potential-induced increase in intracellular [Caz+]. ACKNOWLEDGMENTS

The work was supported by a generous grant from Shell Research Ltd. The decamethrin was a gift from Dr. R. D. Verschoyle of the MRC Toxicology Unit, Carshalton, and the resmethrin from Mitchell Cotts Chemicals.

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2.

3. 4. 5.

6.

7. 8.

9. 10.

11.

Vol. 42, p.1, Amer. Chem. Sot.. Washington, 1977. J. Van Den Bercken, L. M. A. Akkermans, and J. M. Van Der Zalm, DDT-like action of allethrin in the sensory nervous system of Xenopu.y laevis, Eur. J. Pharmacol. 21, 95 (1973. R. D. Verschoyle and J. M. Barnes, Toxicity of natural and synthetic pyrethrins to rats, Pesric. Biochem. Physiol. 2, 308 (1972). M. Carlton, Some effects of cismethrin on the rabbit nervous system. Pestic. Sci. 8. 700 (1977). T. Narahashi, Effects of insecticides on nervous conduction and synaptic transmission, in “Insecticide Biochemistry and Physiology” (C. F. Wilkinson, Ed.), p.327, Plenum, New York, 1976. A. N. Clements and T. E. May, The actions of pyrethroids on the peripheral nervous system and associated organs of the locust, Pestic S(,i. 8, 661 (1977). W. Wouters and J. Van Den Bercken, Action of pyrethroids, Gen. Pharmacol. 9, 387 (1978). M. H. Evans, End plate potentials in frog muscle exposed to a synthetic pyrethroid, Pestic. Biochem. Physiol. 6, 547 (1976). S. H. P. Maddrell and J. J. Nordmann, “Neurosecretion,” Blackie, Glasgow, 1979. W. W. Douglas, Mechanism of release of neurohypophysial hormones: Stimulus-secretion coupling, in “Handbook of Physiology” (R. 0. Creep and E. B. Astwood, Eds.). Sect. 7, Vol. 4, p. 191, Amer. Physiol. Sot.. Washington. DC., 1974. J. J. Nordmann, Evidence for calcium inactivation during hormone release in the rat neurohypophysis. J. Exp. Biol. 65, 669 (1976).

12. J. J. Nordmann and R. E. J. Dyball, New calcium mobilising agent, Nature (London) 255. 414 (1975). 13. A. Dutton and R. E. J. Dyball, Phasic firing enhances vasopressin release from the rat neurophypophysis, J. Physiol. London 290,433 (1979). 14. G. W. Bisset, B. J. Clark, J. Haldar, M. C. Harris, G. P. Lewis, and M. Rocha e Silva, The assay of milk ejecting activity in the lactating rat, &it. J. Pharmacol. 31, 537 (1967). 15. I. Orchard, The effects of pyrethroids on the electrical activity of neurosecretory cells from the brain of Rhodnius prolixus, Pestic. Biochem. Physiol. 13, 220 (1980). 16. P. F. Baker, Calcium and the control of secretion. Sci. Prog.

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64, 95 (1977).

17. R. I. Birks and M. Cohen, The influence of internal sodium on the behaviour of motor nerve endings, Proc. R. Sot. B 170, 401 (1%8).