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Effect of deltamethrin on acetylcholine-operated ionic channels in identified Helix pomatia L. neurons
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
39, 196-204 (1991)
Effect of Deltamethrin on Acetylcholine-Operated Ionic Channels Identified Helix pomatia L. Neurons T. KISS Balaton
Limnological
Research
Institute
N.
OSIPENKO’
of the Hungarian
Academy
AND
OLEG
of Sciences,
Tihany,
in
Hungary
Received July 18, 1990; accepted October 11, 1990 The main action of pyrethroids is to slow the gating kinetics of the Na channel of the axon and soma. However, it was also found that pyrethroids may affect the neurotransmission as well, acting postsynaptically. It can be proposed, therefore, that pyrethroids may have their target at the synaptic site in addition to the axonal or somal Na channels. The aim of the present study was to examine the effect of deltamethrin on acetylcholine (ACh) receptors of snail neurons using a two-microelectrode voltage clamp. Ionophoretic application of ACh on the soma of identified Helix neurons produced inward current of which amplitude was dependent on the holding potential. Deltamethrin (l-100 pM) depressed the ACh-induced current in a concentration-dependent manner. The I-V curves, both in control and in deltamethrin-containing saline, were linear in the potential range of - 100 and - 25 mV. The reversal potential was unchanged. The data suggest a monomolecular binding of deltamethrin to the ACh-activated channel with a KD of 50 pM. Deltamethrin increased the time of recovery from ACh desensitization. It can be concluded, therefore, that deltamethrin may contribute to its toxic effect by affecting the function of the ACh-receptors, although this effect could be secondary. D 1~1 Academic PKSS, IK. INTRODUCTION
The main action of pyrethroids is to slow the gating kinetics of the Na channel of the axon and soma. However, it was also found that pyrethroids may affect the neurotransmission as well, acting postsynaptically. Eldefrawi ef al. (1) have reported that pyrethroid insecticides do interact with nicotinic acetylcholine (ACh) receptors of Torpedo, inhibiting its function at concentrations nearly equivalent to those at which they affect Na channels. Studies on neuroblastoma cells revealed that pyrethroids exert a nonspecific, inhibitory effect on ACh receptor-operated ion channels (2). It can be proposed, therefore, that pyrethroids may have their target at the synaptic site in addition to the axonal or somal Na channels. However, the effect on the ACh receptor could be secondary. Earlier we described the sensitivity of the snail neuronal
Na channels to deltamethrin (3), as well as observing no effect of deltamethrin on Ca channels (unpublished data). The aim of the present study was to examine the effect of deltamethrin on ACh-activated currents of snail neurons. It is well known that ACh could either excite or inhibit gastropoda neurons. Moreover, these two effects could be observed on the same neuron. Based on this observation ACh receptors were classified as D (depolarizing, excitatory) and H (hyperpolarizing, inhibitory) ones since classification in terms of vertebrate muscarinic and nicotinic receptors was not possible (see Refs. (4-6)). The effects of ACh on molluscan neurones have been recently shown to involve changes in Na+, Na+ + Kf , Cl- and Kf (7, 8), or Na+ + Cl- (9) permeabilities. The present paper is concerned with the study of deltamethrin effects on AChevoked D and H responses (DNar DNa+c,, and H,,).
i Permanent address: Department of General Physiology of Nervous System, A. A. Bogomoletz Institute of Physiology, Bobomoletz str., 4, Kiev-24, GSP, 252601, USSR.
MATERIALS
Experiments 196
0048-3575/91 $3.00 Copyrig&
0 1991 by Academic
All tights of
Press, Inc.
reproduction in any form
resented.
AND
METHODS
were performed
on neu-
EFFECT
OF
DELTAMETHRIN
ON
rones: RPal-3, RPll, LPa3, and Vl-9 from subeosephageal ganglia of Helix pomatiu L. The neurons were identified according to the nomenclature of Sakharov and Sakinki m. After removal of the connective tissue using proteinase (Fluka) (1 mg/ml for 30 min), the cells were exposed and ready for experiments. The enzymatic treatment had little effect on the electrical parameters and the ACh sensitivity of the neuronal membrane, but it completely inactivated acetylcholinesterase activity (1). The membrane potential (MP) and membrane currents were recorded using a two-microelectrode voltage clamp. Cells were impaled with double-barrelled or, in part of the experiments, two separate microelectrodes filled with 2.5 M KC1 (1 S-2 Ma). The electrodes were covered with silver paint and grounded. The block scheme of the recording and ionophoretic device is shown in Fig. 1. ACh was applied to the cell surface ionophoretically. The ionophoretic pipette was filled with 10e3 M ACh dissolved in distilled water. External physiological saline had the following cdmposition (in mM): NaCl, 80; KCI, 4; CaCl,, 10; MgCl,, 5; glucose, IO; Tris-HCl, 5, pH 7.4. The application current was lOWOO nA. Between ap-
SNAIL
ACh
RECEPTORS
197
plications of ACh, a retardation current of 10-15 nA was used to prevent leakage of excess ACh from the micropipette. Before each experiment had started, application of ACh was performed several times, with the same duration near the resting membrane potential until the ACh-evoked response became stabilized. The actual concentration of ACh at the cell surface was lower than in the pipette and was related to the time of application. Ganglia were continuously perfused with physiological saline at a constant rate of 1 ml/min. Therefore, in the figures, ACh concentrations are related to the time of application. Deltamethrin was applied by switching of the perfusion system to the deltamethrin-containing saline. Stock solutions of deltamethrin were made in dimethyl sulfoxide (DMSO) at a concentration of 5 x lop3 M. DMSO itself had no detectable effect on the voltageclamped membrane at the same concentration. In some experiments, the effect of deltamethrin was studied on CAMP-induced currents (12). In this case, CAMP was ionophoretized using a triple-barrelled micropipette. The third barrel was filled with 0.5 M CAMP (Calbiochem). All experiments were carried out at room temperature (20-22°C). RESULTS
@
Vh
18V
FIG. 1. Block scheme of the voltage clamp and ionophoretic circuits. The left side shows the voltage clamp amplifiers (A,, A,, A,). The right side represents the ionophoretic device. Switching between resistors (XI-200 Ma), the injecting current could be regulated. In the middle of the figure the perfusion chamber with the preparation and electrodes is shown.
Under voltage clamp conditions, the ionophoretic application of ACh on the soma of identified Helix neurons produced inward (ha + cl or DNa) (Figs. 2A and 8A) and outward (H,,) (Fig. 9A) currents, the amplitude of which depended on the holding potential (HP) and the concentration (Fig. 3A). ACh-induced currents were activated rapidly and successively inactivated to a zero level of the holding current. In most cases, DNa+CI responses have been observed. Pretreatment of the neuron with 1 @4 deltamethrin for 10-15 min led to a substantial decrease of the ACh-induced currents (Fig. 2A). In both salines, the holding current was constant. The currentvoltage (I-V) relationships were linear in
198
KISS
OSIPENKO
1oofiM Deltam
lo oM D&am
-i/----
AND
-1s)
IhAl
B
C
-
FIG. 2. (A) Voltage dependence of the AChactivated (DNn + =,) currents in control saline and in the presence of 1, 10, and 100 JLM concentrations of deltamethrin. Numbers on the left denote the holding potentials at which ACh applications were performed. ACh was applied, here and in all subsequent figures, for periods indicated by horizontal bars above each response. (B) Current-voltage relationships of AChinduced currents in control saline (open symbols) and in the presence of three different concentrations of deltamethrin (filled symbols). Each point fell on a straight line. Neuron V6.
the potential range of - 100 and - 30 mV, with and without deltamethrin. The equilibrium potential (E,,,) was unchanged in the presence of deltamethrin and lies between -30 and -20 mV (Fig. 2B). The ACh-induced currents are shown in Fig. 3B as a function of the agonist concentration (the same cells are presented in Fig. 3A). With increasing ACh concentrations, the response increased and then decreased
FIG. 3. (A) Dose-response relationship for AChinduced (DNo + d res Ponses in control saline and in the presence of three different pyrethroid concentrations. (B) Dose-response curves for currents are presented in A. Symbols as in Fig. 2B. (C) Concentration dependence of the deltamethrin effect on ACh-induced currents. The first point represents current measured in control saline. The duration of ACh application is 4 sec. HP = -100 mV. V6 neuron.
due to the desensitization of the ACh receptors. Therefore, the time of drug application (3-6 set) and the inter-pulse period were constant and selected to allow complete recovery. Experiments were repeated in the presence of three different concentrations of deltamethrin. The dose-effect curves showed that with an increased deltamethrin concentration the ACh-evoked responses gradually decreased (Fig. 3B). The concentration dependence of peak vatues of the ACh-induced currents in deltamethrin solutions is shown in Fig. 3C, with a IO-fold change in the deltamethrin concentration causing 30% suppression of the ACh-activated currents.
EFFECT
OF
DELTAMETHRIN
The summarized dose-effect curves of the ACh-evoked responses in control saline and in the presence of 10 PM deltamethrin are shown in Fig. 4. The hyperbolic relationship in the presence of deltamethrin was shifted to the right along the concentration axis, which means that a higher ACh concentration was necessary to get the same response (Fig. 4B). The doublereciprocal plot suggests a noncompetitive inhibition of ACh-induced currents by deltamethrin (Fig. 4A). The time course of deactivation, i.e., the recovery following ACh application, was fitted by double exponentials, with fast (T,) and slow (TJ time constants, respectively (Fig. 5). It can be noticed that time-to-peak and T, of the relaxing phase remained practically the same. The slow deactivating
ON
SNAIL
ACh
199
RECEPTORS
B 13
1
ACh
c
1
ACh+l@lyhl
10 5
5
klzrr-
0
A
deltam
12
tbec)
40 so
t (set)
FIG. 5. (A) Two current traces induced by the same amount of ACL in control and deltamethrin-containing salines. Pyrethroid substantially decreased the amplitude and the inactivating phase of the ACh-activated current. (B, C) Semilogarithmic plot of the inactivating phase revealed two components with fast (T,) and slow (TJ time constants. HP = -80 mV. Neuron RPll.
TOW l/t(l/sec) 1
B
OACh l ACh
l
10pMdeltam
tlsec)
4. The dose-response curve of ACh-induced 0’~o+cS currents in control saline and in the presence of 10 FM deltamethrin. Data are pooled from 8 to 10 experiments. Vertical bars denote SEM. Continuous lines were drawn by eye. (A) Data plotted in Lineweaver-Burk plot showing noncompetitive of deltamethrin. (B) Dose-response “antagonism” curve in linear scale gives a hyperbolic function, which is shifted to the right by deltamethrin. HP = - 100 mV. Neurons V6, RPa3, and LPa3. FIG.
component (TJ, however, was increased nearly twice in the presence of 100 pit4 deltamethrin (Figs. SB and 5C). It is believed that the two exponentials reflect two binding sites on the ACh-receptor complex. Since the time constants of the relaxing phase reflect the onset of the desensitization on Fig. 6A, the recovery from desensitization is presented. The pulse protocol for studying the recovery from desensitization is shown in Fig. 6B. Both in control saline and in the presence of 10 t.N deltamethrin the recovery proceeds along two exponentials. Plotting the data in a semilogarithmic plot, the time constants of 3.8 and 24.3 min and 7.4 and 18.4 min were obtained, respectively. These results suggest that deltamethrin does interact with the AChreceptor complex. Since a LO-fold change in deltamethrin concentration caused consid-
KISS
200
AND
OSIPENKO
I/lmax
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1
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FIG. 6. Recovery from desensitization in control saline and in the presence of 10 u.M deltamethrin. (A) Experimental data. (B) Protocol for measuring the recovery from desensitization. Current evoked at T,, was taken as a reference amplitude, I,_ was the desensitizing current. Time between pulses was 3 min. HP = -60 mV. Neuron RPal. Curve with open symbols is control, with full symbols with deltamethrin.
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erable suppression of ACh-evoked responses, more concentrations were tested. Fig. 7A shows summarized data obtained at three different pyrethroid concentrations. The plot suggests a monomolecular binding of deltamethrin to the receptorchannel complex with a dissociation constant of 50 pJ4. Moreover, the steep concentration dependence suggests a cooperativity in the pyrethroid action. The transformation of the dose-effect relationship into a Scatchard graph (Fig. 7B) suggests positive cooperativity. The Hill plot of the data (Fig. 7C) shows that in control saline the slope of the straight line equals 2, which means that the ACh-evoked current may be the result of binding of two ACh molecules to the receptor complex. After deltamethrin treatment, the Hill coefftcient was found to equal 1, which suggests that deltamethrin probably competes for the same binding site located on the ACh receptor-channel complex. To study the site of deltamethrin action, d-tubocurarine (dTC), a well-known ACh
. . 'Ok
1
11J B/
.
"ACh l ACh + lOj~Mdeltam
O-%Yi--T-
10
tkied
FIG. 7. (A) Concentration dependence of AChinduced current blockade by deltamethrin. Data are pooled from six experiments. Horizontal bars represent SEM. The continuous solid curve was drawn by eye. Dotted and thin continuous curves correspond to bimolecular (Ii&, = (1 + [x]~/K~~)-’ and monomolecular (Ill, = (1 + [xl/K,,- ’ binding of the pyrethroid to the ACh-operated ionic channel. In both cases the Kd = 50 )r.M. (B) Scatchardplot. Points are experimental data, while the dotted line was calculated from the theoretical cuwe describing the binding of two molecules of the insecticide to one binding site with Kd = 50 a. Experimental points are approximated by a convex cuwe which indicates a positive cooperativity. (C) Hill plot of the experimental data shows that in both control and deltamethrin-containing salines data are linearly distributed with slope factors of 2 (open symbols) and 1 (hill symbols), respectively. HP = -80 mV. NeuronsV6, RPa3,and LPa3.
EFFECT
OF
DELTAMETHRIN
receptor antagonist, was used to block the drug-induced responses. When the neuron was pretreated for 10-15 min with 100 (LM dTC, all ACh-induced currents, D,, (Fig. 8) and H,, (Fig. 9), were decreased but not completely blocked (Figs. 8A and 9A). When 10 pII4 (10 min) deltamethrin was added to this saline an almost complete block of the DNa (Fig. 8A) was achieved as well as a 50% decrease of the Hc, (Fig. 9A).
ON
SNAIL
ACh
201
RECEPTORS
A. Control
-150 I l/f-
100pMdTC+lOpMDeltam .
-T/f-----
1mM
wash
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A
Control
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-
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10pMtkltam
4
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l- 20
o contrd 0 dTClCUptl~de~tam
6
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wash
0 lmMdeltam
-100
-80
-60
-40
-20
0 FIG. 9. (A) dTC (ZOO FM) and deltumethrin (10 PM) induced a 50% block of ACh-evoked (Hc,) responses. After washing out, a repeated application of 1 mM deltamethrin caused a 70% block of ACh-induced currents. (B) The Z-V curves of the currents show a near linear relationship. Neuron VI.
I(nN 0 control
l-z5
0 dTC m dTC +&&tam 9 d&am
FIG. 8. (A) dTC (100 @I) partly depressed the AChevoked (DN,J responses. Adding 10 w deltamethrin caused a full block of the ACh-induced currents which were partly recovered in 10 FM deltamethrincontaining saline. (B) The Z-V curves of the data show a linear relationship and an additive effect of dTC and deltamethrin. Neuron VS.
After washing (15 min) the ganglion with 10 p44 deltamethrin-containing saline, partial recovery of the D,, was observed (Fig. 8A). Washout of the preparation by normal solution caused recovery of the Hc, responses and a further application of I mM deltamethrin caused a larger blocking effect. From the I-V curves, it can be seen that the blocking effects of dTC and deltamethrin were nearly equal and additive, as well as potential independent (Figs. 8B and 9B).
202
KISS
AND
Deltamethrin had no effect on CAMPinduced currents (results are not shown), which suggests that the pyrethroid effect on ACh-induced currents is specific and does not depend on CAMP metabolism. DISCUSSION In this paper we examined the effect of deltamethrin on the ACh-activated D and H responses in snail neurons. We found that in the presence of deltamethrin, all currents activated by ionophoretic application of ACh were decreased. D Na+CI in control saline showed a sigmoid relationship on agonist concentrations with the Hill coefficient equal to 2 (Fig. 2C). This suggests that two molecules of ACh bind to the receptor-ionophore complex and that the binding has a positive cooperativity. These results agree with those described on frog nicotinic ACh receptors (13). Deltamethrin shifted the dose-effect curve of ACh-induced currents right along the concentration axis and suppressed their amplitudes. The blocking effect of deltamethrin was concentration dependent (Fig. 3C), but was potential and use independent (Fig. 2B) and showed noncompetitive inhibition (Fig. 4A). This observation, however, contradicts the earlier studies on frog motor end plate, where the postsynaptic nicotinic ACh receptors were not affected by pyrethroids (14, 15). On the other hand, in Torpedo electric organ, pyrethroids reduced receptor desensitization by binding to the nicotinic type of ACh receptors at a site that is different from the agonist or high-affinity channel sites (16). On neuroblastoma cells, pyrethroids exerted nonspecific, inhibitory effects on the ACh receptor-operated ion channel. It was found that there was no correlation between these effects of pyrethroids and their insecticidal activity. The absence of stereoselectivity suggested that pyrethroids do not bind to the ACh-binding site to produce their blocking effect (2). Several possibilities should be considered when explaining the deltamethrin ef-
OSIPENKO
feet on the ACh-induced current in snail neurons. The first possibility is that the ACh receptor-ion channel complex has at least two binding sites for the agonist: a high and a low affinity one. It has been suggested that deltamethrin binds to the low-affinity binding site, changing the kinetics and the permeability of the receptor-operated channel. This suggestion is supported by the observation that the deltamethrin effect was not use and voltage dependent (Figs. 2B, 8B, and 9B) and that it was independent from the ACh-induced current amplitude (Fig. 3B). Furthermore, since deltamethrin affected the kinetics of the decay of the ACh-induced current (Fig. 6), and also the recovery from desensitization (Fig. 5), it can be concluded that the pyrethroid changed the kinetic parameters of the ACh receptor in snail neuronal membrane. This first possibility is contradicted by the absence of a pyrethroid effect on the ‘251-o-bungarotoxin binding on the electric organ of Torpedo (16). Second, it is well known that pyrethroids are lipid-soluble substances, thus penetrating into the lipid phase of the membrane they may affect channel-protein properties by binding outside to the channel. In addition to the sites that recognize agonists, the ACh receptor has several sites, presumably located at the ionic channel moiety, to which agents can bind allosterically (17, 18). These sites bind to a class of ligands, known as noncompetitive inhibitors, which comprise a large variety of drugs such as alcohols, local anesthetics, and pyrethroids (18-20). The potentiation of the dTC effect by deltamethrin likely suggests that the pyrethroid does not bind to the agonist, highaffinity, noncompetitive blocker sites (Figs. 8 and 9). Rather, deltamethrin may bind the low-affinity site probably located on the channel protein of the ACh-receptor complex. Blocking of the H,, response by deltamethrin (Fig. 9) is similar to the pyrethroid inhibition of the GABA receptor. The GABA-gated Cl- influx is blocked by pyre-
EFFECT OF DELTAMETHRIN
throids (21) and this inhibitory effect is proposed to be specific (22, 23). It was suggested that the GABA receptor is a secondary target for pyrethroids and that the binding site is allosterically located (24,25). The similarity between the deltamethrin effects on the ACh and GABA receptors suggests that different transmitter receptors may be linked to the same ionic channel (26). Thus pyrethroids as antagonists would appear to act at the ionophore level of the transmitter receptor protein, having blocked at this site, all receptors acting through this ionophore would be blocked (27). Since the binding sites for pyrethroids could be different than binding sites of agonists or antagonists in this sense, the pyrethroid effect could be nonspecific (2). On the other hand, our results do not exclude the possibility of a specific effect because deltamethrin had no effect on the holding current and on the CAMP-induced current. It is proposed, therefore, that deltamethrin binds to ACh receptor channel sites, but this point, however, should be verified by further experiments, using different receptor-activated channels and structurallf different pyrethroids. REFERENCES 1. M. E. Eldefrawi, M. A. Abbassy, and A. T. Eldefrawi, Effects of environmental toxicants on nicotinic acethylcholine receptors: Action of pyrethroids, in “Cellular and Molecular Neurotoxicology” (T. Narahashi, Ed.), pp. 177-189, Raven Press, New York, 1984. 2. M. Oortgiesen, R. G. M. van Kleef, and H. P. M. Vijverberg, Effects of pyrethroids on neurotransmitter-operated ion channels in cultured mouse neuroblastoma cells, Pestic. Biochem. Physiol. 34, 164 (1989). 3. T. Kiss, Properties of Na channels of identified snail (Helix pomatiuL.) neurones modified by deltamethrin, Pestic. Biochem. Physiol. 32,247 (1988). 4. H. M. Gerschenfeld, Chemical transmission in invertebrate central nervous system and neuromuscular junction,. Physiol. Rev. 53, 1 (1973). 5. K. S. Rbzsa, The pharmacology of molluscan neurons, Prog. Neurobiol. 23, 79 (1984). 6. R. J. Walker, Transmitters and modulators, in “The Mollusca” (A. 0. D. Willows, Ed.), Vol.
ON SNAIL ACh RECEPTORS
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
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9, Part 2, pp. 279-485, Academic Press, San Diego, 1986. 0. W. Witte, E.-J. Speckmann, and J. Walden, Acetylcholine responses of identified neurons in Helix pornatia, II. Pharmacological properties of acetylcholine responses, Comp. Biothem, Physiol. C 80, 25 (1985). 0. W. Witte, E.-J. Speckmann, and J. Walden, Acetylcholine responses of identified neurons in Helix pomatia, III. Ionic composition of the depolarizing currents by acetylcholine, Camp. Biochem. Physiol. C 80, 37 (1985). S. N. Ayrapetyan, V. L. Arvanov, and S. B. Maginyan, The pharmacological characteristics of two types of cholinoreceptors in the membrane of dialyzed neurons, Camp. Biochem. Physiol. C 90, 29 (1988). D. A. Sakharov and J. Sal&&i, Physiological and pharmacological identification of neurons in the central nervous system of Helix pomatia L., Acta Physiol. Acad. Sci. Hung. 35, 19 (1969). M. A. Kostenko, V. I. Geletyuk, and B. N. Veprintsev, Completely isolated neurones in the mollusc Lymnaea stagnalis: A new objective for nerve cell biology investigation, Camp. Biothem. Physiol. A 49, 89 (1974). N. I. Kononenko, P. G. Kostyuk, and A. D. Scherbatko, The effect of intracellular CAMP injections on stationary membrane conductance and voltage- and time-dependent ionic currents in identified snail neurons, Brain Res. 268, 321 (1983). N. Akaike, N. Tokutomi, and H. Kijima, Kinetic analysis of acetylcholine-induced current in isolated frog sympathetic ganglion cells, J. Neurophysiol. 61, 283 (1989). M. H. Evans, End-plate potentials in frog muscle exposed to a synthetic pyrethroid, Pestic. Biothem. Physiol. 6, 547 (1976). G. S. F. Ruigt and J. van den Bercken, Action of pyrethroids on a nerve-muscle preparation of the dawed frog, Xenopus laevis, Pestic. Biothem. Pharmncol. 25, 176 (1986). S. M. Sherby, A. T. Eldefrawi, S. S. Deshpande, E. X. Albuquuerque, and M. E. Eldefrawi, Effect of pyrethroids on nicotinic acetylcholine receptor binding and function, Pestic. Biochem. Physiol. 26, 107 (1986). J.-P. Changeux, A. Devillers-Thiery, and P. Chemouilli, Acetylcholine receptor: An allosteric protein, Science 225, 1335 (1984). E. K. Krodel, R. A. Beckmann, and J. B. Cohen, Identification of local anesthetic binding site in nicotinic postsynaptic membranes isolated from Torpedo marmorata electric tissue, Mol. Phnrmacol. 15, 294 (1979).
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19. M. A. Abbassy, M. E. Eldefrawi, and A. T. Eldefrawi, Influence of the alcohol moiety of pyrethroids on their interactions with the nicotinic acetylchoiine receptor, J. Toxicol. Environ. ffealrh 12, 575 (1983). 20. E. F. El-Fakahany, E. R. Miller, M. A. Abbassy, A. T. Eldefrawi, and M. E. Eldefrawi, Alcohol modulation of drug binding to the channel sites of the nicotinic acetylcholine receptor, J. Phnrmacol. Exp. Ther. 224, 289 (1983). 21. J. R. Bloonquist and D. M. Soderlund, Neurotoxic insecticides inhibit GABA-dependent chloride uptake by mouse brain vesicles, Biothem. Biophys. Res. Commun. 133, 37 (1985). 22. M. E. Eldefrawi and A. T. Eldefrawi, Action of toxicants on GABAs and glutamate receptors, in “Neurotox 88: Molecular Basis of Drug and
23.
24.
25.
26.
Pesticide Action” (G. G. Lunt, Ed.), pp. 207221, Elsevier, Amsterdam, 1988. L. J. Lawrence and J. E. Casida, Stereospecific action of pyrethroid insecticides on the y-aminobutyric acid receptor-ionophore complex, Science 221, 1399 (1983). A. T. Eldefrawi and M. E. Eldefrawi, Receptors for y-aminobutyric acid and voltage-dependent chloride channels as targets for drugs and toxicants, FASEB J. 1, 262 (1987). A. A. Ramadan, N. M. Marei, H. A. Aly, A. T. Eldefrawi, and M. E. Eldefrawi, Action of pyrethroids on the peripheral benzodiazepine receptor, Pestic. Biochem. Physiol. 32, 97 (1988). D. A. Maccormick, Refinements in the in vitro slice technique and human neuropharmacology, Trends Pharmacof. Sci. 11, 53 (1990).
BIOCHEMISTRY
AND
PHYSIOLOGY
39, 196-204 (1991)
Effect of Deltamethrin on Acetylcholine-Operated Ionic Channels Identified Helix pomatia L. Neurons T. KISS Balaton
Limnological
Research
Institute
N.
OSIPENKO’
of the Hungarian
Academy
AND
OLEG
of Sciences,
Tihany,
in
Hungary
Received July 18, 1990; accepted October 11, 1990 The main action of pyrethroids is to slow the gating kinetics of the Na channel of the axon and soma. However, it was also found that pyrethroids may affect the neurotransmission as well, acting postsynaptically. It can be proposed, therefore, that pyrethroids may have their target at the synaptic site in addition to the axonal or somal Na channels. The aim of the present study was to examine the effect of deltamethrin on acetylcholine (ACh) receptors of snail neurons using a two-microelectrode voltage clamp. Ionophoretic application of ACh on the soma of identified Helix neurons produced inward current of which amplitude was dependent on the holding potential. Deltamethrin (l-100 pM) depressed the ACh-induced current in a concentration-dependent manner. The I-V curves, both in control and in deltamethrin-containing saline, were linear in the potential range of - 100 and - 25 mV. The reversal potential was unchanged. The data suggest a monomolecular binding of deltamethrin to the ACh-activated channel with a KD of 50 pM. Deltamethrin increased the time of recovery from ACh desensitization. It can be concluded, therefore, that deltamethrin may contribute to its toxic effect by affecting the function of the ACh-receptors, although this effect could be secondary. D 1~1 Academic PKSS, IK. INTRODUCTION
The main action of pyrethroids is to slow the gating kinetics of the Na channel of the axon and soma. However, it was also found that pyrethroids may affect the neurotransmission as well, acting postsynaptically. Eldefrawi ef al. (1) have reported that pyrethroid insecticides do interact with nicotinic acetylcholine (ACh) receptors of Torpedo, inhibiting its function at concentrations nearly equivalent to those at which they affect Na channels. Studies on neuroblastoma cells revealed that pyrethroids exert a nonspecific, inhibitory effect on ACh receptor-operated ion channels (2). It can be proposed, therefore, that pyrethroids may have their target at the synaptic site in addition to the axonal or somal Na channels. However, the effect on the ACh receptor could be secondary. Earlier we described the sensitivity of the snail neuronal
Na channels to deltamethrin (3), as well as observing no effect of deltamethrin on Ca channels (unpublished data). The aim of the present study was to examine the effect of deltamethrin on ACh-activated currents of snail neurons. It is well known that ACh could either excite or inhibit gastropoda neurons. Moreover, these two effects could be observed on the same neuron. Based on this observation ACh receptors were classified as D (depolarizing, excitatory) and H (hyperpolarizing, inhibitory) ones since classification in terms of vertebrate muscarinic and nicotinic receptors was not possible (see Refs. (4-6)). The effects of ACh on molluscan neurones have been recently shown to involve changes in Na+, Na+ + Kf , Cl- and Kf (7, 8), or Na+ + Cl- (9) permeabilities. The present paper is concerned with the study of deltamethrin effects on AChevoked D and H responses (DNar DNa+c,, and H,,).
i Permanent address: Department of General Physiology of Nervous System, A. A. Bogomoletz Institute of Physiology, Bobomoletz str., 4, Kiev-24, GSP, 252601, USSR.
MATERIALS
Experiments 196
0048-3575/91 $3.00 Copyrig&
0 1991 by Academic
All tights of
Press, Inc.
reproduction in any form
resented.
AND
METHODS
were performed
on neu-
EFFECT
OF
DELTAMETHRIN
ON
rones: RPal-3, RPll, LPa3, and Vl-9 from subeosephageal ganglia of Helix pomatiu L. The neurons were identified according to the nomenclature of Sakharov and Sakinki m. After removal of the connective tissue using proteinase (Fluka) (1 mg/ml for 30 min), the cells were exposed and ready for experiments. The enzymatic treatment had little effect on the electrical parameters and the ACh sensitivity of the neuronal membrane, but it completely inactivated acetylcholinesterase activity (1). The membrane potential (MP) and membrane currents were recorded using a two-microelectrode voltage clamp. Cells were impaled with double-barrelled or, in part of the experiments, two separate microelectrodes filled with 2.5 M KC1 (1 S-2 Ma). The electrodes were covered with silver paint and grounded. The block scheme of the recording and ionophoretic device is shown in Fig. 1. ACh was applied to the cell surface ionophoretically. The ionophoretic pipette was filled with 10e3 M ACh dissolved in distilled water. External physiological saline had the following cdmposition (in mM): NaCl, 80; KCI, 4; CaCl,, 10; MgCl,, 5; glucose, IO; Tris-HCl, 5, pH 7.4. The application current was lOWOO nA. Between ap-
SNAIL
ACh
RECEPTORS
197
plications of ACh, a retardation current of 10-15 nA was used to prevent leakage of excess ACh from the micropipette. Before each experiment had started, application of ACh was performed several times, with the same duration near the resting membrane potential until the ACh-evoked response became stabilized. The actual concentration of ACh at the cell surface was lower than in the pipette and was related to the time of application. Ganglia were continuously perfused with physiological saline at a constant rate of 1 ml/min. Therefore, in the figures, ACh concentrations are related to the time of application. Deltamethrin was applied by switching of the perfusion system to the deltamethrin-containing saline. Stock solutions of deltamethrin were made in dimethyl sulfoxide (DMSO) at a concentration of 5 x lop3 M. DMSO itself had no detectable effect on the voltageclamped membrane at the same concentration. In some experiments, the effect of deltamethrin was studied on CAMP-induced currents (12). In this case, CAMP was ionophoretized using a triple-barrelled micropipette. The third barrel was filled with 0.5 M CAMP (Calbiochem). All experiments were carried out at room temperature (20-22°C). RESULTS
@
Vh
18V
FIG. 1. Block scheme of the voltage clamp and ionophoretic circuits. The left side shows the voltage clamp amplifiers (A,, A,, A,). The right side represents the ionophoretic device. Switching between resistors (XI-200 Ma), the injecting current could be regulated. In the middle of the figure the perfusion chamber with the preparation and electrodes is shown.
Under voltage clamp conditions, the ionophoretic application of ACh on the soma of identified Helix neurons produced inward (ha + cl or DNa) (Figs. 2A and 8A) and outward (H,,) (Fig. 9A) currents, the amplitude of which depended on the holding potential (HP) and the concentration (Fig. 3A). ACh-induced currents were activated rapidly and successively inactivated to a zero level of the holding current. In most cases, DNa+CI responses have been observed. Pretreatment of the neuron with 1 @4 deltamethrin for 10-15 min led to a substantial decrease of the ACh-induced currents (Fig. 2A). In both salines, the holding current was constant. The currentvoltage (I-V) relationships were linear in
198
KISS
OSIPENKO
1oofiM Deltam
lo oM D&am
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FIG. 2. (A) Voltage dependence of the AChactivated (DNn + =,) currents in control saline and in the presence of 1, 10, and 100 JLM concentrations of deltamethrin. Numbers on the left denote the holding potentials at which ACh applications were performed. ACh was applied, here and in all subsequent figures, for periods indicated by horizontal bars above each response. (B) Current-voltage relationships of AChinduced currents in control saline (open symbols) and in the presence of three different concentrations of deltamethrin (filled symbols). Each point fell on a straight line. Neuron V6.
the potential range of - 100 and - 30 mV, with and without deltamethrin. The equilibrium potential (E,,,) was unchanged in the presence of deltamethrin and lies between -30 and -20 mV (Fig. 2B). The ACh-induced currents are shown in Fig. 3B as a function of the agonist concentration (the same cells are presented in Fig. 3A). With increasing ACh concentrations, the response increased and then decreased
FIG. 3. (A) Dose-response relationship for AChinduced (DNo + d res Ponses in control saline and in the presence of three different pyrethroid concentrations. (B) Dose-response curves for currents are presented in A. Symbols as in Fig. 2B. (C) Concentration dependence of the deltamethrin effect on ACh-induced currents. The first point represents current measured in control saline. The duration of ACh application is 4 sec. HP = -100 mV. V6 neuron.
due to the desensitization of the ACh receptors. Therefore, the time of drug application (3-6 set) and the inter-pulse period were constant and selected to allow complete recovery. Experiments were repeated in the presence of three different concentrations of deltamethrin. The dose-effect curves showed that with an increased deltamethrin concentration the ACh-evoked responses gradually decreased (Fig. 3B). The concentration dependence of peak vatues of the ACh-induced currents in deltamethrin solutions is shown in Fig. 3C, with a IO-fold change in the deltamethrin concentration causing 30% suppression of the ACh-activated currents.
EFFECT
OF
DELTAMETHRIN
The summarized dose-effect curves of the ACh-evoked responses in control saline and in the presence of 10 PM deltamethrin are shown in Fig. 4. The hyperbolic relationship in the presence of deltamethrin was shifted to the right along the concentration axis, which means that a higher ACh concentration was necessary to get the same response (Fig. 4B). The doublereciprocal plot suggests a noncompetitive inhibition of ACh-induced currents by deltamethrin (Fig. 4A). The time course of deactivation, i.e., the recovery following ACh application, was fitted by double exponentials, with fast (T,) and slow (TJ time constants, respectively (Fig. 5). It can be noticed that time-to-peak and T, of the relaxing phase remained practically the same. The slow deactivating
ON
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199
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FIG. 5. (A) Two current traces induced by the same amount of ACL in control and deltamethrin-containing salines. Pyrethroid substantially decreased the amplitude and the inactivating phase of the ACh-activated current. (B, C) Semilogarithmic plot of the inactivating phase revealed two components with fast (T,) and slow (TJ time constants. HP = -80 mV. Neuron RPll.
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4. The dose-response curve of ACh-induced 0’~o+cS currents in control saline and in the presence of 10 FM deltamethrin. Data are pooled from 8 to 10 experiments. Vertical bars denote SEM. Continuous lines were drawn by eye. (A) Data plotted in Lineweaver-Burk plot showing noncompetitive of deltamethrin. (B) Dose-response “antagonism” curve in linear scale gives a hyperbolic function, which is shifted to the right by deltamethrin. HP = - 100 mV. Neurons V6, RPa3, and LPa3. FIG.
component (TJ, however, was increased nearly twice in the presence of 100 pit4 deltamethrin (Figs. SB and 5C). It is believed that the two exponentials reflect two binding sites on the ACh-receptor complex. Since the time constants of the relaxing phase reflect the onset of the desensitization on Fig. 6A, the recovery from desensitization is presented. The pulse protocol for studying the recovery from desensitization is shown in Fig. 6B. Both in control saline and in the presence of 10 t.N deltamethrin the recovery proceeds along two exponentials. Plotting the data in a semilogarithmic plot, the time constants of 3.8 and 24.3 min and 7.4 and 18.4 min were obtained, respectively. These results suggest that deltamethrin does interact with the AChreceptor complex. Since a LO-fold change in deltamethrin concentration caused consid-
KISS
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FIG. 6. Recovery from desensitization in control saline and in the presence of 10 u.M deltamethrin. (A) Experimental data. (B) Protocol for measuring the recovery from desensitization. Current evoked at T,, was taken as a reference amplitude, I,_ was the desensitizing current. Time between pulses was 3 min. HP = -60 mV. Neuron RPal. Curve with open symbols is control, with full symbols with deltamethrin.
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erable suppression of ACh-evoked responses, more concentrations were tested. Fig. 7A shows summarized data obtained at three different pyrethroid concentrations. The plot suggests a monomolecular binding of deltamethrin to the receptorchannel complex with a dissociation constant of 50 pJ4. Moreover, the steep concentration dependence suggests a cooperativity in the pyrethroid action. The transformation of the dose-effect relationship into a Scatchard graph (Fig. 7B) suggests positive cooperativity. The Hill plot of the data (Fig. 7C) shows that in control saline the slope of the straight line equals 2, which means that the ACh-evoked current may be the result of binding of two ACh molecules to the receptor complex. After deltamethrin treatment, the Hill coefftcient was found to equal 1, which suggests that deltamethrin probably competes for the same binding site located on the ACh receptor-channel complex. To study the site of deltamethrin action, d-tubocurarine (dTC), a well-known ACh
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.
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FIG. 7. (A) Concentration dependence of AChinduced current blockade by deltamethrin. Data are pooled from six experiments. Horizontal bars represent SEM. The continuous solid curve was drawn by eye. Dotted and thin continuous curves correspond to bimolecular (Ii&, = (1 + [x]~/K~~)-’ and monomolecular (Ill, = (1 + [xl/K,,- ’ binding of the pyrethroid to the ACh-operated ionic channel. In both cases the Kd = 50 )r.M. (B) Scatchardplot. Points are experimental data, while the dotted line was calculated from the theoretical cuwe describing the binding of two molecules of the insecticide to one binding site with Kd = 50 a. Experimental points are approximated by a convex cuwe which indicates a positive cooperativity. (C) Hill plot of the experimental data shows that in both control and deltamethrin-containing salines data are linearly distributed with slope factors of 2 (open symbols) and 1 (hill symbols), respectively. HP = -80 mV. NeuronsV6, RPa3,and LPa3.
EFFECT
OF
DELTAMETHRIN
receptor antagonist, was used to block the drug-induced responses. When the neuron was pretreated for 10-15 min with 100 (LM dTC, all ACh-induced currents, D,, (Fig. 8) and H,, (Fig. 9), were decreased but not completely blocked (Figs. 8A and 9A). When 10 pII4 (10 min) deltamethrin was added to this saline an almost complete block of the DNa (Fig. 8A) was achieved as well as a 50% decrease of the Hc, (Fig. 9A).
ON
SNAIL
ACh
201
RECEPTORS
A. Control
-150 I l/f-
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0 FIG. 9. (A) dTC (ZOO FM) and deltumethrin (10 PM) induced a 50% block of ACh-evoked (Hc,) responses. After washing out, a repeated application of 1 mM deltamethrin caused a 70% block of ACh-induced currents. (B) The Z-V curves of the currents show a near linear relationship. Neuron VI.
I(nN 0 control
l-z5
0 dTC m dTC +&&tam 9 d&am
FIG. 8. (A) dTC (100 @I) partly depressed the AChevoked (DN,J responses. Adding 10 w deltamethrin caused a full block of the ACh-induced currents which were partly recovered in 10 FM deltamethrincontaining saline. (B) The Z-V curves of the data show a linear relationship and an additive effect of dTC and deltamethrin. Neuron VS.
After washing (15 min) the ganglion with 10 p44 deltamethrin-containing saline, partial recovery of the D,, was observed (Fig. 8A). Washout of the preparation by normal solution caused recovery of the Hc, responses and a further application of I mM deltamethrin caused a larger blocking effect. From the I-V curves, it can be seen that the blocking effects of dTC and deltamethrin were nearly equal and additive, as well as potential independent (Figs. 8B and 9B).
202
KISS
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Deltamethrin had no effect on CAMPinduced currents (results are not shown), which suggests that the pyrethroid effect on ACh-induced currents is specific and does not depend on CAMP metabolism. DISCUSSION In this paper we examined the effect of deltamethrin on the ACh-activated D and H responses in snail neurons. We found that in the presence of deltamethrin, all currents activated by ionophoretic application of ACh were decreased. D Na+CI in control saline showed a sigmoid relationship on agonist concentrations with the Hill coefficient equal to 2 (Fig. 2C). This suggests that two molecules of ACh bind to the receptor-ionophore complex and that the binding has a positive cooperativity. These results agree with those described on frog nicotinic ACh receptors (13). Deltamethrin shifted the dose-effect curve of ACh-induced currents right along the concentration axis and suppressed their amplitudes. The blocking effect of deltamethrin was concentration dependent (Fig. 3C), but was potential and use independent (Fig. 2B) and showed noncompetitive inhibition (Fig. 4A). This observation, however, contradicts the earlier studies on frog motor end plate, where the postsynaptic nicotinic ACh receptors were not affected by pyrethroids (14, 15). On the other hand, in Torpedo electric organ, pyrethroids reduced receptor desensitization by binding to the nicotinic type of ACh receptors at a site that is different from the agonist or high-affinity channel sites (16). On neuroblastoma cells, pyrethroids exerted nonspecific, inhibitory effects on the ACh receptor-operated ion channel. It was found that there was no correlation between these effects of pyrethroids and their insecticidal activity. The absence of stereoselectivity suggested that pyrethroids do not bind to the ACh-binding site to produce their blocking effect (2). Several possibilities should be considered when explaining the deltamethrin ef-
OSIPENKO
feet on the ACh-induced current in snail neurons. The first possibility is that the ACh receptor-ion channel complex has at least two binding sites for the agonist: a high and a low affinity one. It has been suggested that deltamethrin binds to the low-affinity binding site, changing the kinetics and the permeability of the receptor-operated channel. This suggestion is supported by the observation that the deltamethrin effect was not use and voltage dependent (Figs. 2B, 8B, and 9B) and that it was independent from the ACh-induced current amplitude (Fig. 3B). Furthermore, since deltamethrin affected the kinetics of the decay of the ACh-induced current (Fig. 6), and also the recovery from desensitization (Fig. 5), it can be concluded that the pyrethroid changed the kinetic parameters of the ACh receptor in snail neuronal membrane. This first possibility is contradicted by the absence of a pyrethroid effect on the ‘251-o-bungarotoxin binding on the electric organ of Torpedo (16). Second, it is well known that pyrethroids are lipid-soluble substances, thus penetrating into the lipid phase of the membrane they may affect channel-protein properties by binding outside to the channel. In addition to the sites that recognize agonists, the ACh receptor has several sites, presumably located at the ionic channel moiety, to which agents can bind allosterically (17, 18). These sites bind to a class of ligands, known as noncompetitive inhibitors, which comprise a large variety of drugs such as alcohols, local anesthetics, and pyrethroids (18-20). The potentiation of the dTC effect by deltamethrin likely suggests that the pyrethroid does not bind to the agonist, highaffinity, noncompetitive blocker sites (Figs. 8 and 9). Rather, deltamethrin may bind the low-affinity site probably located on the channel protein of the ACh-receptor complex. Blocking of the H,, response by deltamethrin (Fig. 9) is similar to the pyrethroid inhibition of the GABA receptor. The GABA-gated Cl- influx is blocked by pyre-
EFFECT OF DELTAMETHRIN
throids (21) and this inhibitory effect is proposed to be specific (22, 23). It was suggested that the GABA receptor is a secondary target for pyrethroids and that the binding site is allosterically located (24,25). The similarity between the deltamethrin effects on the ACh and GABA receptors suggests that different transmitter receptors may be linked to the same ionic channel (26). Thus pyrethroids as antagonists would appear to act at the ionophore level of the transmitter receptor protein, having blocked at this site, all receptors acting through this ionophore would be blocked (27). Since the binding sites for pyrethroids could be different than binding sites of agonists or antagonists in this sense, the pyrethroid effect could be nonspecific (2). On the other hand, our results do not exclude the possibility of a specific effect because deltamethrin had no effect on the holding current and on the CAMP-induced current. It is proposed, therefore, that deltamethrin binds to ACh receptor channel sites, but this point, however, should be verified by further experiments, using different receptor-activated channels and structurallf different pyrethroids. REFERENCES 1. M. E. Eldefrawi, M. A. Abbassy, and A. T. Eldefrawi, Effects of environmental toxicants on nicotinic acethylcholine receptors: Action of pyrethroids, in “Cellular and Molecular Neurotoxicology” (T. Narahashi, Ed.), pp. 177-189, Raven Press, New York, 1984. 2. M. Oortgiesen, R. G. M. van Kleef, and H. P. M. Vijverberg, Effects of pyrethroids on neurotransmitter-operated ion channels in cultured mouse neuroblastoma cells, Pestic. Biochem. Physiol. 34, 164 (1989). 3. T. Kiss, Properties of Na channels of identified snail (Helix pomatiuL.) neurones modified by deltamethrin, Pestic. Biochem. Physiol. 32,247 (1988). 4. H. M. Gerschenfeld, Chemical transmission in invertebrate central nervous system and neuromuscular junction,. Physiol. Rev. 53, 1 (1973). 5. K. S. Rbzsa, The pharmacology of molluscan neurons, Prog. Neurobiol. 23, 79 (1984). 6. R. J. Walker, Transmitters and modulators, in “The Mollusca” (A. 0. D. Willows, Ed.), Vol.
ON SNAIL ACh RECEPTORS
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
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9, Part 2, pp. 279-485, Academic Press, San Diego, 1986. 0. W. Witte, E.-J. Speckmann, and J. Walden, Acetylcholine responses of identified neurons in Helix pornatia, II. Pharmacological properties of acetylcholine responses, Comp. Biothem, Physiol. C 80, 25 (1985). 0. W. Witte, E.-J. Speckmann, and J. Walden, Acetylcholine responses of identified neurons in Helix pomatia, III. Ionic composition of the depolarizing currents by acetylcholine, Camp. Biochem. Physiol. C 80, 37 (1985). S. N. Ayrapetyan, V. L. Arvanov, and S. B. Maginyan, The pharmacological characteristics of two types of cholinoreceptors in the membrane of dialyzed neurons, Camp. Biochem. Physiol. C 90, 29 (1988). D. A. Sakharov and J. Sal&&i, Physiological and pharmacological identification of neurons in the central nervous system of Helix pomatia L., Acta Physiol. Acad. Sci. Hung. 35, 19 (1969). M. A. Kostenko, V. I. Geletyuk, and B. N. Veprintsev, Completely isolated neurones in the mollusc Lymnaea stagnalis: A new objective for nerve cell biology investigation, Camp. Biothem. Physiol. A 49, 89 (1974). N. I. Kononenko, P. G. Kostyuk, and A. D. Scherbatko, The effect of intracellular CAMP injections on stationary membrane conductance and voltage- and time-dependent ionic currents in identified snail neurons, Brain Res. 268, 321 (1983). N. Akaike, N. Tokutomi, and H. Kijima, Kinetic analysis of acetylcholine-induced current in isolated frog sympathetic ganglion cells, J. Neurophysiol. 61, 283 (1989). M. H. Evans, End-plate potentials in frog muscle exposed to a synthetic pyrethroid, Pestic. Biothem. Physiol. 6, 547 (1976). G. S. F. Ruigt and J. van den Bercken, Action of pyrethroids on a nerve-muscle preparation of the dawed frog, Xenopus laevis, Pestic. Biothem. Pharmncol. 25, 176 (1986). S. M. Sherby, A. T. Eldefrawi, S. S. Deshpande, E. X. Albuquuerque, and M. E. Eldefrawi, Effect of pyrethroids on nicotinic acetylcholine receptor binding and function, Pestic. Biochem. Physiol. 26, 107 (1986). J.-P. Changeux, A. Devillers-Thiery, and P. Chemouilli, Acetylcholine receptor: An allosteric protein, Science 225, 1335 (1984). E. K. Krodel, R. A. Beckmann, and J. B. Cohen, Identification of local anesthetic binding site in nicotinic postsynaptic membranes isolated from Torpedo marmorata electric tissue, Mol. Phnrmacol. 15, 294 (1979).
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19. M. A. Abbassy, M. E. Eldefrawi, and A. T. Eldefrawi, Influence of the alcohol moiety of pyrethroids on their interactions with the nicotinic acetylchoiine receptor, J. Toxicol. Environ. ffealrh 12, 575 (1983). 20. E. F. El-Fakahany, E. R. Miller, M. A. Abbassy, A. T. Eldefrawi, and M. E. Eldefrawi, Alcohol modulation of drug binding to the channel sites of the nicotinic acetylcholine receptor, J. Phnrmacol. Exp. Ther. 224, 289 (1983). 21. J. R. Bloonquist and D. M. Soderlund, Neurotoxic insecticides inhibit GABA-dependent chloride uptake by mouse brain vesicles, Biothem. Biophys. Res. Commun. 133, 37 (1985). 22. M. E. Eldefrawi and A. T. Eldefrawi, Action of toxicants on GABAs and glutamate receptors, in “Neurotox 88: Molecular Basis of Drug and
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Pesticide Action” (G. G. Lunt, Ed.), pp. 207221, Elsevier, Amsterdam, 1988. L. J. Lawrence and J. E. Casida, Stereospecific action of pyrethroid insecticides on the y-aminobutyric acid receptor-ionophore complex, Science 221, 1399 (1983). A. T. Eldefrawi and M. E. Eldefrawi, Receptors for y-aminobutyric acid and voltage-dependent chloride channels as targets for drugs and toxicants, FASEB J. 1, 262 (1987). A. A. Ramadan, N. M. Marei, H. A. Aly, A. T. Eldefrawi, and M. E. Eldefrawi, Action of pyrethroids on the peripheral benzodiazepine receptor, Pestic. Biochem. Physiol. 32, 97 (1988). D. A. Maccormick, Refinements in the in vitro slice technique and human neuropharmacology, Trends Pharmacof. Sci. 11, 53 (1990).