Camp. Bioe~~~. Physiol. Vol. 82C, No. 1, pp. 147-154, 1985 0306-~92~85 $3.00 + 0.00 Printed in Great Britain Q 1985 Pergamon Press Ltd

EFFECT OF NEW ORGANOPHOSPHATES ON THE MEMBRANE OF IDENTIFIED CENTRAL NEURONS OF

HELIX AUNT TIA L. ~MOLLUSCA, GASTROPODA)

I. KISS* and KATALIN S.-R&sA~ *Research Institute for Heavy Chemical Industries, Veszpr&m, Hungary, H-8200 and tBiologica1 Research

Institute of the Hungarian Academy of Sciences, Tihany, Hungary, H-8237

(Received I4 January 1985)

Abstract-l. Effects of two newly synthesised organophosphates were studied on identified neurons of H&x p~~~riff by microelectrophysiological methods.

2. The single intracellular spikes were processed by computer using a “phase-plane trajectory” method. Dimethoate was also involved as reference substance.

3. The mechanism of action of the substance NE-79297 was found to be similar to that of dimethoate resulting in prolongation of action potentials due to a delayed rectification of the outward current.

4. Phosmethylan (NE-79168), a much more selective compound, altered the membrane parameters in a different way: it affected the slow---mainly calcium-mediated-inward current.

INTRODUCTION

Organophosphates (OP) are known as one of the main groups of insecticides presently used. Their basic biological effects are inhibition of cholinesterase (ChE) (O’Brien, 1967) and cholinergic receptors (Donnellan et al., 1980; Pichon, 1974). However, it has become more and more evident that for a better understanding of the mechanism underlying the toxic action of pesticides a detailed study of the processes taking place on the cellular membranes has to be involved (Fossier et al., 1983). Chemically different groups of insecticides, such as DDT-analogs and pyrethroids, have been proved to exert their neu- rotoxic effect by binding to the same active site of neuronat membranes (Beeman, 1982), and con- sequently by modifying the ionic channels (Nar- ahashi, 1976, 1979; Bercken and Vijverbcrg, 1980).

Adequate knowledge of the direct effects of these pesticides at the membrane level may facilitate the understanding of the mechanism of toxicity in mam- mals, as well as the correct antidote in case of human intoxication.

The new generation of OP pesticides has a most sophisticated structure, while the requirement for selectivity has been greatly increased. In addition, development of resistance against them is a con- tinuously increasing problem (Sawicki et al., 1978). Most questions cannot be answered simply by ChE inhibition any more. There are even special types of OPs having other sites of action than the ChE enzyme (Beeman, 1982). Selectivity and synergism also in- volved quite complicated mechanisms in the nervous system which are not yet completely understood.

$The molecule NE-79168 has been developed as a new pesticide in NEVIKI with the common name phos- methylan [0,0-diethyl-S-(N-isopropyl-amino-carboxyl- methyl)-dithio-phosphate]. Registration number: 181202.

In the last few years a series of new or- ganophosphate molecules with insecticide and acar- icide effects have been developed in NEVIKI. Their characteristics as ChE inhibitors are more or less known (Kovbcsn&BuzBs et al., 1983), but not any other biochemical and biophysical effects on the nervous system. In the present paper the direct membrane effect of two new OP molecules will be described.

MATERIALS AND METHODS

In the experiments the molecules NE-79297 and NE- 79168$ were used in comparison with dimethoate, which appears to be a well known pesticide ingredient and is a reference substance in our biological screening. Both test substances were supplied by the manufacturer, as pure ingredients, while dimethoate was the commercial product named Bi-58.

As our molecules are not water-soluble, they were solu- bilized for experimental purposes in a xylol-sorbol (4: 1) mixture. It was considered as 100% stock solution. The latter was diluted with distilled water to obtain lO-‘-lO-s:/, suspensions. The concentrations should be considered as relative values, not suitable for obtaining absolute dose-response curves. The effect of the solvent was also examined, and all the test results were evaluated as devi- ations from the effect of the solvent.

Examinations were performed on identified central neu- rons of Helixpomatiu, using the heart-nerve-brain prepara- tion developed earlier (S.-R&a and Salinki, 1973; S.-Rbzsa, 1976). Identification of the neurons was as pre- viously described (S.-R6zsa, 1976, 1979). In the present work neurons RPa2, RPal (Br) and V21 were involved.

Activity of the central neurons was recorded by con- ventional glass microelectrodes. For displaying and record- ing a four-channel Tektronix oscilloscope (RSI03N) and a Gould-Brush recorder were used. Spike activity was recorded on magnetic tape for computer processing, where the phase-plane trajectory method was employed (Jenerick, 1963; Sperelakis and Shumaker, 1968; Winlow et al,, 1982). According to this method some basic parameters of the membrane are calculated by functions derived from the

147

148 I. KISS and KATALIN S.-R&~A

Hodgkin-Huxley equations. Action potentials were fed into an R10 computer in digitalizcd form. The phase-plane trajectory, as well as the total membrane current in relation to time and voltage, was drawn on an X-Y recorder connected to the output of the computer. From this total membrane current, g,, and g,,, conductance values can be determined in a graphic way. The application of the sub- stances was performed in two different ways.

(I) Drop-application: the desired volume of a solution of given concentration several hundred ~1 was added by automatic micropipette to the ganglionic surface, as close to the cell as possible.

(2) Pressure application: micropipettes of 24flm tip- diameter were filled with a solution of the test substances of known concentration. Then the micropipette was placed to the cell surface by microamanipulator and the substances were applied by pressure.

In both cases the substances were washed out by perfusion with Meng’s solution (Meng, 1958). All the test solutions were diluted at the same saline.

RESULTS AND DISCUSSION

1. Testing of solvent

The solvent concentration levels, together with test substance used in our study, did not influence significantly either the spontaneous activity of neu- rons or the measured membrane parameters. In the case of solvent concentrations exceeding 10e4% the inward current and conductance were obviously changed; namely, the solvent caused a non-significant increase in these parameters (Fig. 1).

2. Eflect of tesf sabsrai~&es on the sponta~~eous neu- ronal activity

(a) Dimethoate and NE-79297. These two com- pounds will be discussed together, as they showed almost the same effect, a concentration-dependent, two- or three-phase variation in the spontaneous activity. At the lower concentration (10-40k), corre- sponding to the threshold concentration for the neu- ron RPa2, an inhibition of activity predominated, while the higher concentrations (lo-‘, lo-‘%) and pressure application caused an inhibition with tran- sient excitation (Figs 2 and 3A). At concentrations of 10-l and lo-?% the effect terminated in an irre- versible paralysis at the depolarized phase. This last phase developed within l-2 min at lo-‘%, but the same effect occurred later at a concentration of lo-*% (Figs 2G, 3C and 4). The type of effect depends on the neuron examined, as shown in the figures.

The most characteristic changes in the pattern began with the generation of double spikes, followed by development of burst activity (Fig. 4B), then the alteration in the repolarization phase resulted in a long-lasting plateau phase with abortive action po- tentials superposed on it (Figs 3B and C).

(b) Phosmethylan. In the case of the cell RPa2 the threshold concentration was lo-‘% (Fig. 6). At con- centrations of lo-’ and 10-40/:, the effect developed within 1 min, resulting in a fast depolarization, an increase in the spike amplitude and an irreversible biock of potential generation at the depolarization level. At the threshold concentration the development of this effect was prolonged (up to 3 min), and the

Fig. I. Effect of the examined substances on the functional parameters of the neuron RPa2. Zero level represents the controt, and all changes are given as percentage alteration from this level. U-amplitude of action potential, T,,-50% repolarization time, T,,-900/, repolarization time, 4, and I,,,---total inward and outward current, respectively, g,, and g,,,---total inward and outward conductance, respectively.

effect was reversible after washing out; nevertheless, it was of the same type as at higher concentrations.

3. Eflect on the single action potentials and ionic carrent

The resuks with dimethoate may partially be expla- ined by the effect on the whole neural network. Compounds of this type are known as ChE in- hibitors; consequently, the efficacy of cholinergic synapses converging onto the examined neuron may increase. In addition, the direct effect on the mem- brane obviously has importance, as it was supported by pressure-application of substances to the surface of the soma. In a number of cases development of burst activity through double action potentials was clearly shown, where the first phenomenon was al- ways a great increase in the impufse duration (Fig. 7).

Computer processing of the phase-plane trajectory showed practically no change either in inward con- ductance, or inward current following the application of dimethoate and NE-79297. In contrast, the values of outward conductance and current dramatically changed (Fig. 1). These results suggest that pro- longation of the action potential can be explained completely by a delayed rectification of the outward current underlying the repolarization process. As a next step, the depolarization shift developed due to the delayed rectification allows the initiation of a new

Effect of OP on Helix neurons 149

6

Fig. 2. Neuron IWa2. Effect of Bi-58, 2 x 200 ~1 drop-application. (A) Control + 3.8 x 10m4% Bi-58. Progressive development of partial inhibition. (B, C) Control + 3.8 x 10m3% Bi-58. Complete inhibition, washing out takes a long time. (D, E and F) 3.8 x lo-‘% Bi-58, effect and partial restoration of the control activity. (G) Effect of 3.8 x lo-‘% Bi-58 resulted in a multi-phase response. At 1 set following the

drop-application a fast depolarization, increase in tiring rate and irreversible paralysis occurred.

action potential superposed on the preceding spike’s repolarization phase (Fig. 8). It can be seen that the inward current required for this latter process is only a small percentage of that observed during the rising phase of the preceeding spike, but it flows through an unaltered membrane resistance (Fig. 9). Repetition of this process results in the paroxysmal depolarization shift. It can be supposed that the prolonged action potentials and/or the long-lasting paroxysmal depo- larization might activate the Ca-dependent pre- synaptic transmitter release initiating a feed-back reverberation process in the whole neural network.

The experiments with the compound phosmethylan suggested a completely different mechanism for its effect on the membrane. Figure 1 shows that changes in all of the parameters except g,,, were opposite, compared to those of dimethoate. It is especially true for the impulse duration. In contrast to the extreme prolongation of spike by dimethoate and NE-79297, phosmethylan did not induce any increase in the pulse duration, when measuring the 50% re- polarization time. On the contrary, its effect resulted in an apparent decrease in the 90% repolarization time (Fig. 10). A similar pharmacological effect has been described for heart muscle, connected to the activation of the slow inward calcium-channel. (For detailed discussion of this question see Salata and Jalife, 1982.) A similar slow Ca-channel is known to exist in snail neurons (Heyer and Lux, 1978); there- fore, phosmethylan might use this mechanism. The

inward currents were examined specially according to this point of view.

Dimethoate did not alter the time course of I,,, while a 10m3% solution of phosmethylan resulted in a shift of the inward current time curve to the positive direction. In Lymnaea neurons it was proved (Kiss, 1977) that curves like that shown in Fig. 11 represent mixed current where the slow Ca-current predom- inates. Considering that the present method gives total inward currents, it must be concluded that in spite of the quantitatively unchanged net current values there must be a significant qualitative alter- ation. It appeared to be that the substance caused an almost complete block of the fast sodium-channel (and even antagonized the increased Na-influx due to the solvent), and activated the slow Cai, current at the same time. In this manner, though no significant decrease can be found in the total inward current, the amplitude of the action potential is continuously decreasing, as a result of the blockage of the fast sodium-current and a small decrease in the outward conductance. At last, the membrane will not be able to generate an action potential any more.

CONCLUSION

The present results help in studying the direct membrane effects of organophosphate insecticides. However, our findings on snail neurons must be extrapolated cautiously to other species, including

150 I. KM and KATALIN S.-R&W

Fig. 3. (A) Effect of Bi-58 following pressure-application, where the multiphase response is clearly shown. The concentration of test substance within the micropipette is 3.8 x IO-‘%. (B) Effect of Bi-58 on the Br-neuron. Drop-application, two times 2OOpl of 3.8 x IO-*% solution. In the continuous record note the initiation of the plateau phase. (C) Bi-58 application to the Br-neuron by pressure. The substance injected onto the membrane surface caused damage to the repolarization process, and during the long-lasting plateau phase abortive spikes appeared, the membrane was strongly depolarized, then the

potential generation ceased.

Effect of OP on Helix neurons 1.51

NE

Fig. 4. Effect of NE-79297 (drop-application). (A) Upper record: neuron V21, two times 200 pi 10-3x solution, inhibitory effect. Lower record: irreversible inhibition at depolatized level caused by 0.1% solution. (B) Neuron RPa2, continuous recording. Application of 200 ~1 0.1% solution at the arrow. Development of burst activity through double-spiking and progressive elongation of the plateau phase.

W = washing out.

Fig. 5. Effect of 10e4% phosmethylan on the neuron RPa2. Continuous recording. The cell function is inhibited within I min, and IPSPs appear. After 3 min treatment the control activity cannot be restored

any more, despite washing several times.

152 I. KISS and KATALIN S.-R~ZSA

Fig. 6. Erect of 10m50/, phosmethylan on the neuron RPa2. From (A) to (D) continuous recording. a = heart activity. An inhibition of potential generation is shown over 3 min. (E) Restoration of electrical

activity after washing out several times,

either insects or mammals; nevertheless, they may’ be phosmethylan appears to be unexpected, as their useful, especially because the effect of substances like biological effectiveness in conventional screening US-

those examined here on the membrane currents is ing sensitive species was quite similar. The only hardly well known. The pronounced difference be- difference was found in their selectivity: NE-79297 is tween the direct membrane effects of NE-79297 and toxic to practically all species examined, while the

-20 I Fig. 7. Increase in the impulse duration of action potential elicited by dimethoate.

Effect of OP on Helix neurons 153

Fig. 8. Double spikes elicited by dimethoate as drawn on the computer X-Y terminal (all the following figures are

obtained in a similar way using this terminal).

species-selectivity of phosmethyIan is signi~cant~y higher (personal info~ation from the manu- facturer). The mechanism of action of the molecule has not been studied. On the basis of the present

------con,rot -NE-79168

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70 t(m5.s)

Fig. 11. Effect of phosmethylan on the inward current.

results further biochemical studies of this mechanism are in progress, both in insects and in mammals, with special regard to the role of calcium transport.

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l(XlIT7A)

.2

.l

o- ), -1 - \ lo 20 30 60 ?a 80 90 xx) rbn5ed

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-3-

4-

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-?-

k

9

a- c

-11 1 Fig. 9. Total ionic currents underlying a double spike.

------ control --NE-79164 ao-

- 250tlJnsec1

U(mV)

Fig. 10. Effect of phosmethylan on the impulse duration and repolarization time.

154 I. KISS and KATALIN S.-R~ZSA

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