Neuronal Target Sites of Insecticides - American Chemical Society

Chapter 17

Neuronal Target Sites of Insecticides

Downloaded by EAST CAROLINA UNIV on January 5, 2018 | http://pubs.acs.org Publication Date: November 9, 1987 | doi: 10.1021/bk-1987-0356.ch017

Toshio Narahashi Department of Pharmacology, Northwestern University Medical School, Chicago, IL 60611

Pyrethroids have been shown to act on several target sites in the nervous system including the sodium channel, calcium channel and GABA receptor-channel complex. This chapter gives highlights of our recent studies of the mechanisms of action of pyrethroids, DDT and lindane. The sodium channel is the major, if not the sole, target site of type I and type II pyrethroids and DDT, and changes in nerve activity as manifested by repetitive discharges, membrane depolarization, conduction block and synaptic facilitation can be explained on the basis of modification of the sodium channel gating kinetics including prolonged openings. Some effects of type I pyrethroids on the calcium channel are noted, but the toxicological significance remains to be seen. The GABA receptor-channel complex, which was claimed by some investigators to be suppressed by type II pyrethroids, has recently been shown to be totally unaffected by deltamethrin while the sodium channel undergoes drastic modification. Lindane effectively blocks one of the components of the GABA-activated chloride channel current, and this action appears to be responsible for synaptic hyperactivity. whereas the mechanism of action of i n s e c t i c i d e s has been studied f o r many years since the development of synthetic i n s e c t i c i d e s such as DDT, lindane and parathion during and a f t e r World War I I , i t was not u n t i l around the mid-1960's that t h e i r actions on the nervous system were understood at the c e l l u l a r and membrane l e v e l . Since these neuroactive i n s e c t i c i d e s are known to a l t e r membrane e x c i t a t i o n which takes place with a time course of milliseconds, the study o f t h e i r mechanisms of action can best be performed with the a i d of advanced electrophysiological techniques such as voltage clamp which allows us to measure the i o n i c permeabilities of excitable membranes. Studies along t h i s l i n e unveiled a v a r i e t y of important features concerning the

0097-6156/87/0356-0226$07.25/0 © 1987 American Chemical Society Hollingworth and Green; Sites of Action for Neurotoxic Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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interactions o f i n s e c t i c i d e s with ion channels and receptors i n the nervous system. The present chapter gives a b r i e f summary of the h i s t o r i c development of t h i s f i e l d and h i g h l i g h t s of recent discoveries made i n our laboratory.


Pvrethroids Certain pyrethroids such as pyrethrins, a l l e t h r i n and tetramethrin stimulate and then paralyze insects. Various nerves were stimulated to produce r e p e t i t i v e discharges e i t h e r spontaneously or i n response to a single stimulus (1-5). The depolarizing a f t e r - p o t e n t i a l was elevated by the pyrethroids and reached the threshold for r e p e t i t i v e after-discharges ( 6 , 2 ) . At high concentrations of pyrethroids the membrane was gradually depolarized and impulse conduction was eventually blocked ( 6 , 2 ) · Repetitive responses i n the postsynaptic element i n the pyrethroid-poisoned preparations were induced at the presynaptic nerve terminals (fi-lQ). Thus the nerve membrane appears to be the major target s i t e of pyrethroids. E a r l i e r voltage clamp experiments c l e a r l y demonstrated that prolongation o f sodium current and p a r t i a l i n h i b i t i o n of potassium current were responsible f o r the increase i n depolarizing a f t e r - p o t e n t i a l caused by the pyrethroid a l l e t h r i n (11-12). The peak amplitude of sodium current was suppressed r e s u l t i n g i n conduction block. Despite these multiple actions, the prolongation of sodium current was produced at low concentrations o f the pyrethroid, and was d i r e c t l y responsible for the elevation of the depolarizing a f t e r - p o t e n t i a l which triggered r e p e t i t i v e discharges, the major cause o f the symptoms of poisoning i n animals (14.15). Pyrethroids may conveniently be c l a s s i f i e d into two groups based on the chemical structure and t o x i c action ( 1 6 - 1 8 ) . Type I pyrethroids do not possess an alpha-cyano group and include many conventional ones such as a l l e t h r i n , tetramethrin, phenothrin and permethrin. Type I I pyrethroids possess a cyano group at the α p o s i t i o n and include cyphenothrin, cypermethrin, deltamethrin and fenvalerate. E f f e c t s o f Pyrethroids on Resting and Action Potentials The e f f e c t s o f type I pyrethroids such as tetramethrin and a l l e t h r i n on the giant axons of the c r a y f i s h and squid were characterized by an increase and prolongation o f the depolarizing a f t e r - p o t e n t i a l which, at the threshold f o r action p o t e n t i a l generation, l e d to r e p e t i t i v e after-discharges. This occurred at low concentrations i n the nanomolar range, without much change i n the r e s t i n g and action p o t e n t i a l amplitude ( 1 4 ) . Type I I pyrethroids such as fenvalerate and cyphenothrin caused a membrane depolarization and a decrease i n action p o t e n t i a l amplitude. No large change i n the depolarizing a f t e r - p o t e n t i a l occurred, and no r e p e t i t i v e after-discharges were induced ( 1 2 ) . E f f e c t s o f Tvoe I Pyrethroids on Sodium Channels In normal c r a y f i s h and squid giant axons perfused i n t e r n a l l y with

Hollingworth and Green; Sites of Action for Neurotoxic Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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potassium-free cesium solutions, the membrane i o n i c current associated with a step depolarization (e.g. to -20 mV) from a holding p o t e n t i a l (e.g. -100 mV) under voltage clamp conditions was composed of a peak transient inward current (Ip) which was followed by a small slow current ( I ) . The t a i l current ( I t a i l ) associated with a step r e p o l a r i z a t i o n decayed quickly. When perfused i n t e r n a l l y with (+)-trans tetramethrin (20 /iM), the peak current was not changed but the slow current was greatly increased i n amplitude. The t a i l current was also markedly increased i n amplitude (Figure 1). Both the slow current and t a i l current decayed very slowly (14,15). K i n e t i c analyses of the peak sodium current have revealed that the time constants of a c t i v a t i o n ( r ) , i n a c t i v a t i o n (r^) , and the i n i t i a l phase of the t a i l current (*"tail) affected by tetramethrin (14,15). However, i n the presence of tetramethrin the slow component of t a i l current and the slow steady-state i n a c t i v a t i o n curve were both s h i f t e d i n the hyperpolarizing d i r e c t i o n . The amplitude of the slow t a i l current was dose-dependent, whereas i t s time constant was not. These observations l e d to the suggestion that tetramethrin modifies a population of sodium channels to give r i s e to slow a c t i v a t i o n and i n a c t i v a t i o n k i n e t i c s . The normal peak transient current i n tetramethrin simply represents the a c t i v i t y of the unmodified channels. Calculations show that only a very small f r a c t i o n of sodium channels, less than 1%, needs to be modified by tetramethrin i n order to elevate the depolarizing a f t e r - p o t e n t i a l to the l e v e l of threshold f o r r e p e t i t i v e after-discharges (19). This represents a unique a m p l i f i c a t i o n of the t o x i c o l o g i c a l e f f e c t from channel modulation to the symptoms of poisoning i n animals, and accounts i n part f o r the high potency of the pyrethroids. Development of the slow t a i l current during a depolarizing pulse was taken as a measure of the rate at which the sodium channels are modified. I t had a fast and a slow phase, and the l a t t e r disappeared a f t e r removal of sodium channel i n a c t i v a t i o n with pronase. Based on these and other r e s u l t s , a k i n e t i c scheme was developed (Figure 2). Tetramethrin modifies the sodium channel i n both closed and open states, and the modified channel opens and i s inactivated much more slowly than the normal channel (15 ) . However, we have very recently shown that the apparent i n a c t i v a t i o n of the modified sodium channel i s a r e s u l t of depletion of sodium ions i n the periaxonal space (20). Figure 2 incorporates the revised version of the k i n e t i c scheme.


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Effects of Type II Pyrethroids on Sodium Channels Type II pyrethroids also modify the sodium channel k i n e t i c s (20-24). In a squid axon i n t e r n a l l y perfused with 10 μΜ deltamethrin a step depolarization from a holding membrane p o t e n t i a l of -80 mV to -20 mV produced a peak transient sodium current which was followed by a slow current (Figure 3). With a prolonged, 510 msec depolarization the slow component of sodium current was hardly inactivated. The t a i l current associated with step r e p o l a r i z a t i o n of the membrane decayed very slowly with a dual time constant of 33 msec and 1074 msec. Like the peak


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Neuronal Target Sites of Insecticides I,


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Figure 1. Membrane sodium currents i n a squid giant axon before (a) and during (b) internal perfusion with 1 μΜ (+)-trans a l l e t h r i n . Sodium current associated with a step depolarization from -100 mV to -20 mV was recorded a f t e r cesium and tetramethylammonium had been substituted f o r i n t e r n a l K and external K , respectively, to eliminate the potassium current. In the control record (a), the peak sodium current (Ip) i s followed by a small slow sodium current ( I ) during a depolarizing pulse, and the t a i l current ( I t a i l ) associated with step r e p o l a r i z a t i o n decays quickly. In the presence of a l l e t h r i n (b), I remains unchanged while I i s greatly increased i n amplitude. Itail increased i n amplitude and decays very slowly. (Reproduced with permission from r e f . 73. Copyright 1980 Society of Chemical Industry.) +

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Figure 3. Sodium currents recorded from the squid giant axons before (A) and a f t e r (B) i n t e r n a l a p p l i c a t i o n of 10 μΜ deltamethrin. External and i n t e r n a l sodium concentrations were 111 mM and 50 mM, respectively. A, a depolarizing pulse from the holding p o t e n t i a l (V^) of -80 mV to -20 mV e l i c i t e d the normal transient inward sodium current which decayed within 10 msec. Depolarization to a second depolarizing pulse (500 msec) to the sodium reversal p o t e n t i a l (Ejj - +20 mV) y i e l d e d a n e g l i g i b l e current. Repolarization to the holding p o t e n t i a l (-80 mV) produced a very small inward sodium t a i l current. B, the same pulse protocol as that for A but i n the presence of deltamethrin i n another axon*Note a large and prolonged t a i l current upon r e p o l a r i z a t i o n from +20 mV to -80 mV. (Reproduced with permission from r e f . 21. Copyright 1987 Academic Press.) a



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transient current, the slow and t a i l currents were both blocked by 1 μΜ tetrodotoxin (TTX) i n d i c a t i n g that they flowed through the sodium channel. These and other observations are compatible with the k i n e t i c model o r i g i n a l l y developed for type I pyrethroids (Figure 2). Fenvalerate, another type II pyrethroid prolongs the sodium current i n a manner very s i m i l a r to that of deltamethrin. In the presence of fenvalerate, the sodium channel opens normally and i s modified by the pyrethroid. Fenvalerate also binds to the sodium channel at i t s closed state causing a modified closed channel. The l a t t e r opens slowly upon depolarization to generate a slow current. The voltage dependence of a c t i v a t i o n of closed modified channels was s h i f t e d 20-30 mV i n the d i r e c t i o n of hyperpolarization. This, together with the absence of i n a c t i v a t i o n , causes a depolarization. I t appears that type I and type II pyrethroids act on the nerve membrane sodium channel i n a q u a l i t a t i v e l y s i m i l a r manner. However, the opening and c l o s i n g k i n e t i c s of the modified channel are much slower for type II than f o r type I pyrethroids. Effects of Pyrethroids on Gating

Currents

Gating current, which i s generated by channels as they change t h e i r conformation i n response to changes i n membrane p o t e n t i a l , i s an important t o o l for the study of sodium channels, because i t can give more d i r e c t information about the conformational changes of channels than can ionic current. The prolonged sodium current i n pyrethroid-poisoned axons shows that the conducting pore of the channel i s held open, but i t i s not known whether the gating charges themselves are held i n the open p o s i t i o n . When sodium channels of c r a y f i s h axons were put into the modified open state by r e p e t i t i v e stimulation i n the presence of fenvalerate, the intermediate component of the ON gating current (r - 150 /is at +20 mV), which corresponds to sodium channel a c t i v a t i o n , and the f a s t component of the OFF gating current (r - 50 με at -160 mV), which corresponds to sodium channel deactivation, where i n h i b i t e d i n p a r a l l e l with a c t i v a t i o n and deactivation of the sodium current. There was a slow component of ON gating current with the same time constant as sodium i n a c t i v a t i o n (r - 600 με at +20 mV). This component and sodium i n a c t i v a t i o n were both abolished by fenvalerate, suggesting that t h i s gating current component i s associated with sodium i n a c t i v a t i o n . The f a s t component of the ON gating current (r - 45 at +20 mV) was not affected by fenvalerate, suggesting that i t i s not associated with sodium channel gating. I t was concluded that when sodium channels are i n the fenvalerate-modified open state, t h e i r a c t i v a t i o n and i n a c t i v a t i o n gating charges are immobilized. I t appears that the a c t i v a t i o n gating charges are stuck i n the open configuration while the i n a c t i v a t i o n gating charge i s stuck i n the r e s t i n g or open configuration. Effects of Pyrethroids on Single Sodium Channels The voltage clamp experiments outlined above permit recording of opening and c l o s i n g of many sodium channels contained i n a large nerve membrane area. A question arises as to how i n d i v i d u a l


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sodium channels behave i n response to depolarizing stimulation and how they are modified by pyrethroids. Neher and Sakmann (25) developed a technique whereby the opening and c l o s i n g of i n d i v i d u a l ion channels could be recorded from a l i m i t e d area of the membrane by means of a glass c a p i l l a r y electrode. The technique, c a l l e d patch clamp, was l a t e r improved so that the a c t i v i t y of i n d i v i d u a l channels could be distinguished more c l e a r l y (26). This method, referred to as gigaohm s e a l patch clamp, was combined with a technique by which a small patch was p u l l e d o f f the c e l l membrane and attached to the t i p of the recording c a p i l l a r y electrode (22). We started working on patch clamp techniques as applied to cultured neuroblastoma c e l l s (NIE-115 l i n e ) i n 1980, and have since then studied the i n t e r a c t i o n of pyrethroids with single sodium channels (28-32). P r i o r to a p p l i c a t i o n of tetramethrin, i n d i v i d u a l sodium channels opened for a short period of time during a depolarizing step as seen i n the records as inward-going current pulses (downward deflections) (Figure 4A). A f t e r exposure to tetramethrin, i n d i v i d u a l sodium channels opened f o r a much longer period of time (Figure 4B; note change i n time scale compared to 4A). The currents before and after a p p l i c a t i o n of tetramethrin were both blocked by TTX, i n d i c a t i n g that we were observing the sodium channels. The amplitude histograms reveal no change i n the d i s t r i b u t i o n of single channel currents following a p p l i c a t i o n of tetramethrin (Figures 4C and 4D). The open times (lifetimes) of single channels follow the Poisson d i s t r i b u t i o n with a time constant of 1.7 msec i n the control (Figure 4E). A f t e r exposure to tetramethrin, however, the open time d i s t r i b u t i o n i s expressed by two exponential functions: One has a time constant of 1.8 msec (inset) which i s s i m i l a r to that of the control, and the other has a constant of 16.6 msec, much longer than the control (Figure 4F). Thus the open time d i s t r i b u t i o n shows c l e a r l y that i n the tetramethrin-poisoned membrane there are two populations of sodium channels, one having the normal c h a r a c t e r i s t i c s and the other having modified c h a r a c t e r i s t i c s . The former represents the sodium channels not bound by tetramethrin, and the l a t t e r those bound by tetramethrin. This indicates that i n d i v i d u a l sodium channels are modified by tetramethrin i n an all-or-none manner. The e f f e c t s of fenvalerate and deltamethrin on single sodium channels were also studied using the patch clamp method. The fenvalerate-modified channel, once opened, remained open throughout the e n t i r e depolarizing pulse (200 msec) and long a f t e r r e p o l a r i z a t i o n (30). The mean open time was about 1 sec at -80 mV and exhibited a voltage dependence s i m i l a r to that of macroscopic t a i l currents. This type of modified channel a c t i v i t y , designated mode I, can account f o r the prolongation of sodium current during and a f t e r a depolarizing pulse under macroscopic voltage clamp conditions. Occasionally, the channel exhibited spontaneous openings at the holding p o t e n t i a l . These openings had a mean open time of about 50 msec at -80 mV and occurred i n c l u s t e r s l a s t i n g about 10 sec. This type of a c t i v i t y , designated mode I I , may explain the increase i n holding current seen i n macroscopic studies. The two modes of a c t i v i t y do not occur simultaneously. The opening and c l o s i n g behavior of


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the modified channel i s compatible with the notion that fenvalerate and deltamethrin s t a b i l i z e a v a r i e t y of channel states by reducing the t r a n s i t i o n rates between them (29). Similar conclusions were obtained by measurement of sodium channel gating current as described above.


Sites of Action of Pyrethroids on Sodium Channels Two questions were raised regarding the s i t e of action of pyrethroids on the sodium channels, i . e . 1) whether various isomers of a pyrethroid act on the same s i t e , and 2) whether pyrethroids act on the same s i t e as that of other chemical agents known to interact with the sodium channel. The r e s u l t s to be described below are compatible with the notion that the s i t e s of pyrethroid action are not located inside the sodium channel. This l e d to the hypothesis that the properties of the open sodium channels are not changed by pyrethroids. Large differences i n potency have been found among geometric and optic isomers of pyrethroids. This provides us with an excellent opportunity to study the s t e r e o s p e c i f i c i t y of pyrethroid binding s i t e s (19). Four isomers of tetramethrin were used and the amplitude of the t a i l current associated with step r e p o l a r i z a t i o n was taken as a measure of a c t i v i t y , whereas the (+)-trans (lR-3-trans) and (+)-cis (lR-3-cis) forms of tetramethrin were e f f e c t i v e i n increasing the t a i l current amplitude at concentrations as low as 3 χ 10"^M and 5 χ 10"%, respectively, the (-)-trans (IS-3-trans) and (-)-cis (IS-3-cis) forms were i n e f f e c t i v e even at 3 χ 10"^M, the highest concentration used. When pretreated with either (-)-trans or (-)-cis tetramethrin, the effects of the subsequently applied (+)-trans or (+)-cis tetramethrin were diminished greatly. (-)-trans tetramethrin decreased the e f f e c t of (+)-trans tetramethrin largely i n a non-competitive manner. Similar non-competitive antagonism was observed i n the combinations of (-)-trans and (+)-cis forms, and (-)-cis and (+)-trans forms. However, (-)-cis tetramethrin antagonized the action of (+)-cis tetramethrin i n a competitive manner. Tetrodotoxin antagonized (+)-trans tetramethrin i n a non-competitive manner. A l l of these results can be interpreted by a scheme i l l u s t r a t e d i n Figure 5. There are a trans s i t e and a c i s s i t e i n the sodium channel to which the (+)-trans form and (+)-cis form bind respectively with a high a f f i n i t y causing modification of the sodium channel. The (-)-cis form binds to the c i s s i t e with a high a f f i n i t y thereby antagonizing the e f f e c t of (+)-cis form i n a competitive manner. However, the (-)-trans form binds to the trans s i t e only with a low a f f i n i t y ; i t s major binding occurs at a negative a l l o s t e r i c s i t e , modification of which causes an i n h i b i t o r y e f f e c t on the trans and c i s s i t e s . The negative a l l o s t e r i c s i t e i s also bound by the (-)-cis compound but only with a low a f f i n i t y . Tetrodotoxin binds to another s i t e causing a non-competitive antagonism against the active (+) forms. Since c e r t a i n sodium channel agents have been shown to bind to a s i t e inside the channel, they can be used as a useful probe to determine the s i t e of action of pyrethroids i n the sodium


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Figure 4. E f f e c t s of 60 μΜ (+)-trans tetramethrin on single sodium channels i n an inside-out membrane patch excised from a neuroblastoma c e l l (N1E-115 l i n e ) . A, sample records of sodium channel currents (inward deflections) associated with step depolarizations from -90 mV to -50 mV. B, as i n A, but after application of tetramethrin to the i n t e r n a l surface of the membrane. C, current amplitude histogram i n the c o n t r o l . D, as i n C, but after a p p l i c a t i o n of tetramethrin. (Reproduced with permission from r e f . 31. Copyright 1983 Elsevier.) Continued on next page.


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channel. Batrachotoxin (BTX), a sodium channel modulator, has been demonstrated to bind to the " i n a c t i v a t i o n receptor" s i t e (33). BTX was f i r s t perfused i n t e r n a l l y through the squid giant axon, and r e p e t i t i v e stimulations were applied u n t i l the t y p i c a l BTX modification of the sodium channel occurred. This was i l l u s t r a t e d by the development of non-inactivating sodium currents during step depolarization and by the appearance of these modified currents at large negative membrane p o t e n t i a l s . No d r a s t i c change occurred i n t a i l sodium current. When tetramethrin was added to the BTX-treated axon, a large and prolonged t a i l current c h a r a c t e r i s t i c of the tetramethrin modified sodium channel developed. Thus tetramethrin binds to a s i t e d i f f e r e n t from the binding s i t e of BTX which i s located inside of the channel. This r e s u l t i s compatible with the hypothesis that the pyrethroid molecules bind to the channel gating machinery v i a the membrane l i p i d phase thereby a l t e r i n g the k i n e t i c s of channel gating. whereas the pyrethroids modify the gating k i n e t i c s of sodium channels d r a s t i c a l l y , i t remains to be seen whether they modify the properties of the sodium channel when i t i s i n the open configuration. I f the pyrethroid molecules bind to the membrane l i p i d phase i n the v i c i n i t y of the channel thereby a f f e c t i n g the gating k i n e t i c s , the properties of the open channel may not be altered. Experiments were performed with the i n t e r n a l l y perfused, voltage clamped squid giant axon to examine the open sodium channel properties (34»1S)· The permeability r a t i o % a : ^ L i : ^NH4 : ^guanidine : **formamidine i n the presence of 300 mM Na or test cations i n the external s o l u t i o n was 1:1.13:0.27:0.34:0.23 i n control and 1:0.93:0.29:0.21:0.21 i n 50 μΗ tetramethrin. In the presence of 600 mM Na*or test cations, the r a t i o was 1:1.19:0.21:0.28:0.20 i n control, and 1:1.18:0.29:0.29:0.25 i n 50 μΜ tetramethrin. Thus there was no difference between normal and tetramethrin modified channels i n t h e i r s e l e c t i v e permeability to cations. The instantaneous current-voltage (I-V) curves f o r channels were also examined. In Na solution, the instantaneous I-V curve showed a U-shape, r e f l e c t i n g the voltage-dependent block of sodium channels by external calcium ions at hyperpolarizing p o t e n t i a l s . The instantaneous I-V curve f o r the tetramethrin-modified channels i n various solutions was p r a c t i c a l l y i d e n t i c a l to those f o r normal channels (Figure 6). Patch clamp single channel recording experiments with neuroblastoma (NIE-115) c e l l s provided a d d i t i o n a l support to the above hypothesis (32). The single sodium channel conductance of neuroblastoma c e l l s was reduced by increasing the external calcium concentration from 0.18 to 9.0 mM. The d i s s o c i a t i o n constant for calcium block was estimated to be 32.4 + 1.05 mM. The block was i n t e n s i f i e d by hyperpolarization. The voltage dependence of block indicates that calcium ions bind to sodium channels at a s i t e located 37 ± 2% of the e l e c t r i c a l distance from the outside. Sodium ions also blocked sodium channels i n a voltage-dependent manner, with d i s s o c i a t i o n constants of 185 and 204 mM at -50 and 0 mV, respectively. A model c o n s i s t i n g of a one-ion pore with four b a r r i e r s and three wells can account f o r the observations that deviate from the independence p r i n c i p l e ,



17, NARAHASHI

A

-0.4

E (mV) m

ι I

—

•

ι -80

-120

-40


.-04 X.

W//J 80

lOOmMCa

(AZ

50mMCa\

^

.-1.6

J J°

• -2D

NL

lOmMCa

V

I (mA/cm ) Na

2

--2A Figure 6. Instantaneous current-voltage relationships i n 10, 50, and 100 mM Ca + solutions i n a normal axon (A). The control and tetramethrin data weœ obtained from d i f f e r e n t axons. (Reproduced with permission from r e f . 35. Copyright 1986 Elsevier.) Continued on next page. 2



SITES OF ACTION FOR


Figure 6.—Continued. Instantaneous current-voltage relationships i n 10, 50, and 100 mM C a solutions i n 20, 50, and 100 mM C a solutions i n an axon i n t e r n a l l y perfused with 50 μΜ tetramethrin (B). The control and tetramethrin data were obtained from d i f f e r e n t axons. (Reproduced with permission from r e f . 35. Copyright 1986 Elsevier.) 2 +

2 +


17. NARAHASHI


239

namely, the saturation of current, block by calcium ions, and r e c t i f i c a t i o n i n current-voltage r e l a t i o n s h i p . Tetramethrin at a concentration of 50 μΜ had no e f f e c t on any of these parameters examined. Thus i t can be concluded that despite the d r a s t i c prolongation of open time no modification i s brought about by tetramethrin i n the channel properties that control i o n permeation. These r e s u l t s are compatible with our hypothesis that the pyrethroid molecules do not bind to the inside of the sodium channel but bind to the membrane l i p i d phase thereby modifying the gating k i n e t i c s .


Structure-Activity Relationship of Pyrethroids and DDT Intoxication with DDT r e s u l t s i n ataxia, loss of coordination, convulsions, and hyperexcitation of insects and mammals. Various regions of the nervous system were stimulated to discharge r e p e t i t i v e l y either i n response to a single stimulus or spontaneously. These included sensory c e l l s , synapses, and nerve f i b e r s (2,3,36,37). Repetitive after-discharges i n nerve f i b e r s were due to an increase i n the depolarizing (negative) a f t e r - p o t e n t i a l by DDT (38,22)· Repetitive responses i n neuromuscular junctions have been shown to originate i n presynaptic nerve terminals (40). I t appears that DDT and pyrethroids exert s i m i l a r e f f e c t s on the nerve membrane sodium channel (41). Detailed analyses as described below c l e a r l y show that t h i s i s a c t u a l l y the case. Comparison of Action of Pyrethroids and DDT at the Sodium Channel Level Any s t r u c t u r e - a c t i v i t y study must c a r e f u l l y define " a c t i v i t y " so that only those pyrethroids with the same type of a c t i v i t y are compared with respect to potency. The e f f e c t s of a wide range of pyrethroids and DDT analogs on the membrane p o t e n t i a l and membrane sodium currents were studied i n c r a y f i s h , lobster and squid giant axons (17,42,43). DDT, p l i f e n a t e (2, 2, 2 - t r i c h l o r o - l - ( 3 , 4-dichlorophenyl)-ethanol acetate), and EDO (2,2-big.(p-ethoxyphenyl)-3, 3-dimethyloxetane) produced depolarizing a f t e r - p o t e n t i a l s which developed quickly and decayed quickly, and had l i t t l e e f f e c t on the r e s t i n g p o t e n t i a l . Tetramethrin, a l l e t h r i n , resmethrin and proparthrin produced depolarizing a f t e r - p o t e n t i a l s which were more c l e a r l y detectable during multiple a f t e r - p o t e n t i a l s which developed more slowly but lasted longer. The phenoxybenzyl compounds NRDC 157, permethrin and phenothrin produced very persistent depolarizing a f t e r - p o t e n t i a l s and p a r t i a l l y depolarized the r e s t i n g membrane. F i n a l l y , the a-substituted compounds cyphenothrin, deltamethrin and fenvalerate produced very small depolarizing a f t e r - p o t e n t i a l s which were more c l e a r l y detectable during multiple stimulation. The r e s u l t i n g depolarization was so persistent that i t caused a change i n the r e s t i n g p o t e n t i a l . Eventually the depolarization became so large that further conduction was blocked. No r e p e t i t i v e f i r i n g was observed with these compounds i n giant axons. The depolarizing after-potentials, the use-dependent



240

SITES OF ACTION FOR


depolarization and the membrane depolarization caused by various pyrethroids and DDT analogs could a l l be explained by a prolonged increase i n sodium conductance. The o-cyano pyrethroids fenvalerate, cyphenothrin and deltamethrin were slow to modify channels (type II e f f e c t ) . A r e l a t i v e l y small f r a c t i o n of the sodium channels was retained i n the modified open state with each depolarization, but the l i f e t i m e of the channels i n the modified open state was very long. Thus, axons poisoned with these compounds were depolarized u n t i l the action p o t e n t i a l was blocked. In the course of slow depolarization, some neurons f i r e r e p e t i t i v e l y , e s p e c i a l l y those i n the sensory nervous system (44,45). Repetitive discharges from sensory neurons appear to be the cause of t i n g l i n g sensation of the skin when exposed to type II pyrethroids. Membrane depolarization at nerve terminals caused by type II pyrethroids would cause an increase i n transmitter release which i n turn disturbs synaptic transmission (46,4Z). By comparison, DDT and p l i f e n a t e modified sodium channels r a p i d l y but the l i f e t i m e of the channels i n the modified open state was much shorter (10-20 msec) (type I e f f e c t ) . Thus, axons poisoned with these compounds showed long trains of potentials with very l i t t l e e f f e c t on the resting p o t e n t i a l at low concentrations. These two groups of compounds apparently represent two extreme cases of a continuous v a r i a t i o n i n the rates at which sodium channels reach the modified open state and return to the r e s t i n g state. Most of the other compounds tested were intermediate and showed r e p e t i t i v e a c t i v i t y as well as some decrease i n the r e s t i n g p o t e n t i a l (Table I ) . Since the k i n e t i c s Table I.

Time Constants (msec) of T a i l Currents Associated with Step Repolarizations of the Membrane to the Levels Indicated i n Crayfish Giant Axons Treated with Various Compounds

Compound

Cone. (M)

DDT

1

X

ΙΟ**

Plifenate

3

X

ΙΟ"

EDO

1

X

ίο-*

Tetramethrin

2

X

Phenothrin

3

GH401

T a i l Current Time Constant (msec) at -160 mV -120 mV -100 mV 3.0

6.1

9.5

9.4

14.8

17.0

16

44

86

10-5

30

225

620

X

10-5

200

750

1340

1

X

ίο-*

700

1450

Cyphenothrin

1

X

10"

6

(min)

(min)

(CO)

Fenvalerate

1

X

10"

6

(min)

(min)

(«)

10"

6

(min)

(min)

(CO)

Deltamethrin

1

X

6

2220

SOURCE: Reproduced with permission from r e f . 17. Copyright 1983 Academic Press.


17.

NARAHASHI


241

of sodium channel modification vary continuously with the type of i n s e c t i c i d e , s t r u c t u r e - a c t i v i t y studies must attempt to measure the interactions between the insecticides and sodium channels. The most precise measure of t h i s interaction requires the voltage clamp technique, but a q u a l i t a t i v e i n d i c a t i o n can be obtained using conventional i n t r a c e l l u l a r p o t e n t i a l recording.


Effects of Pyrethroids on Calcium Channels In view of the very c r i t i c a l role calcium channels play i n a v a r i e t y of nerve and muscle function, possible e f f e c t s of various i n s e c t i c i d e s on calcium channels are too important to be overlooked. I t was indeed shown that neurosecretory c e l l s of the s t i c k insect generate action potentials by inward calcium currents (48), and that permethrin increases the e l e c t r i c a l a c t i v i t y of these c e l l s at a concentration as low as 5 χ 1 0 - M (49). Although d i r e c t demonstration s t i l l remains to be seen, i t i s possible that pyrethroids act on calcium channels to exert t h e i r toxic e f f e c t s (48,50). We have launched an extensive study of calcium channels i n connection with i n s e c t i c i d a l action. F i r s t , we had to characterize the normal physiological properties of calcium channels i n neuroblastoma c e l l s as they were largely unknown. Whole c e l l patch clamp technique as combined with i n t e r n a l perfusion of neuroblastoma c e l l s (N1E-115) (51) has proved highly successful as described below. Patch clamp techniques as applied to the whole c e l l calcium channels l e d to the discovery of two types of calcium channels i n neuroblastoma c e l l s (52-56). These two types exhibited d i f f e r e n t physiological and pharmacological properties. Barium (50 mM) was used i n the external solution as the c a r r i e r of current through calcium channels. Step depolarizations from a holding p o t e n t i a l of -80 mV to potentials more p o s i t i v e than -50 mV evoked transient inward B a currents which reached a maximum amplitude at -20 mV (type I calcium channel). A second component of the inward current appeared around -20 mV and reached i t s maximum at 0 to +10 mV. This component was not inactivated during prolonged depolarizing steps l a s t i n g more than 200 msec (type II calcium channel). When the holding p o t e n t i a l was changed to -50 mV, step depolarizations f a i l e d to evoke the fast, transient component due to i n a c t i v a t i o n . However, they induced the slow, non-inactivating component i n the i s o l a t e d form. Both components of the inward current was abolished by 1 mM L a , indicating that the non-inactivating component of the current also flowed through calcium channels. Dibutyryl c y c l i c AMP (1 mM) caused an increase i n the amplitude of the non-inactivating component by 30-50%, but f a i l e d to a l t e r the transient component s i g n i f i c a n t l y . The two types of channels also d i f f e r e d i n t h e i r ionic s e l e c t i v i t y as estimated from the peak current amplitude; B a : S r : C a 1.0:1.0:0.6 f o r type I channel, and B a : S r : C a - 1.0:0.7:0.3 for type I I channel. Replacement of B a with C a caused a p o s i t i v e voltage s h i f t i n the I-V relationship f o r type I I , but not f o r type I channels. L a , C d , N i and C o blocked both types of calcium channels i n a dose-dependent manner with one-to-one stoichiometry. For the type I channel, the sequence n

2 +

3 +

2 +

2 +

2 +

2 +

3 +

2+

2 +

2 +

2 +

2 +

2 +

2 +



242

of blocking potency as estimated from the apparent (/xM) was La (1.5) » N i (47) > C d (160) - G o (160). For the type II channel, the sequence was L a (0.9) > C d (7.0) » Ni (280) > C o (560). These results strongly suggest that the two types of calcium channels found i n neuroblastoma c e l l s represent different entities. Tetramethrin (50 μΜ) caused a progressive block of type I and type II calcium channel currents over a 10-15 min period (56). When a steady state was achieved, type I channel current was blocked by 75%, while type II current was blocked only by 30%. The tetramethrin block of both channel types was time-dependent, being enhanced during a 400 msec depolarizing pulse. The time-dependent component of block was e a s i l y reversible a f t e r washing with drug-free solution, while the time-independent component or resting block persisted f o r at l e a s t 40 min a f t e r washing. Deltamethrin and fenvalerate (10 μΜ) had no e f f e c t on either type of calcium channel currents during 30 min of exposure. The results indicate that type I pyrethroids are calcium channel blockers as well as sodium channel modulators. The two components of calcium channel block, a time-dependent reversible block and a time-independent i r r e v e r s i b l e block, suggest two separate s i t e s of action of tetramethrin i n calcium channels. However, type II pyrethroids modify sodium channels only. This difference i n action between type I pyrethroids and α-cyano type II pyrethroids may be p a r t i a l l y responsible f o r the d i f f e r e n t symptoms of poisoning i n animals. 3 +

2 +

2 +

2 +

3 +

2 +

2 +


2 +

Neuroreceptors as Target Sites of Pyrethroid and DDT-like Insecticides I t has recently been shown that the active isomers of type II pyrethroids bind to a s i t e c l o s e l y associated with the GABA receptor-ionophore complex (52). Binding of sulfur-35-labeled t-butylbicyclophosphorothionate (TBPS), a ligand f o r the p i c r o t o x i n i n binding s i t e , was i n h i b i t e d by type II pyrethroids, but not by type I pyrethroids. Measurements of input resistance of c r a y f i s h muscle f i b e r s also supported the antagonism between type II pyrethroids and GABA receptor-ionophore complex ( 5 8 ) . Further support to the hypothesis was provided by the observation that diazepam protected cockroaches against the action of type II pyrethroids (59). However, the potency of deltamethrin, a type II pyrethroid, was 100 times lower on GABA receptors than on sodium channels (60). I t has also been suggested that type II pyrethroids may interact with the brain binding s i t e s f o r dihydropicrotoxinin (61) or kainic acid (62). Our recent study c l e a r l y excludes the p o s s i b i l i t y that the GABA receptor-channel complex i s a major target s i t e of deltamethrin, a type II pyrethroid (63). Patch clamp experiments were performed with the primary cultured neurons i s o l a t e d from the r a t dorsal root ganglion. These neurons are endowed with GABA receptor-channels. Both GABA-induced inward chloride current and voltage-activated sodium channel current were recorded from the same c e l l . The GABA-induced chloride current was unaffected by a p p l i c a t i o n of 10 μΜ deltamethrin, while the sodium current was greatly prolonged indicating drastic modification of the


17. NARAHASHI


channel gating k i n e t i c s . Thus, deltamethrin does not a f f e c t the mammalian GABA receptor-channel complex. I t remains to be seen whether the same conclusion i s applied to the insect GABA receptor-channel complex. N i c o t i n i c acetylcholine (ACh) receptors from Torpedo e l e c t r i c organ were also affected by pyrethroids (64-66) · The binding of H-perhydrohistrionicotoxin, but not of H-ACh, was i n h i b i t e d by type I pyrethroids. Type I I pyrethroids were less potent. The i n h i b i t o r y e f f e c t had a negative temperature c o e f f i c i e n t . The receptor-regulated calcium flux was also i n h i b i t e d . Thus the channels associated with n i c o t i n i c ACh receptors may be another s i t e of action of pyrethroids. However, e l e c t r o p h y s i o l o g i c a l experiments d i d not support t h i s idea. The amplitude of the end-plate potentials i n frog s k e l e t a l muscles was unaffected by type I pyrethroids (8,10). This controversy remains to be solved. 3


243

3

EDO and Glutamate Receptors The e f f e c t s of EDO, a biodegradable DDT analog, on glutamate receptor-channels were examined using c r a y f i s h neuromuscular junctions as a model (40). EDO at a concentration of 40 nM greatly augmented the excitatory junctional potentials (EJPs). Repetitive EJPs were evoked by a single nerve stimulus. However, f o c a l recording by means of an e x t r a c e l l u l a r microelectrode placed i n the immediate v i c i n i t y of the junction revealed that each excitatory j u n c t i o n a l current (EJC) was preceded by a nerve terminal action current (Figure 7). Thus, i n the presence of EDO, the nerve generated r e p e t i t i v e discharges i n response to a single stimulus, thereby augmenting the EJPs. Junctional depolarization evoked by d i r e c t iontophoretic a p p l i c a t i o n of L-glutamate was not affected by EDO at a l l (Figure 8). Thus i t was concluded that EDO had no e f f e c t on the glutamate receptor-channel complex. Pyrethroids and Glutamate Receptors The e f f e c t s of type I and type II pyrethroids on the evoked p o t e n t i a l were studied i n v i t r o using guinea p i g o l f a c t o r y cortex s l i c e s (61). A p a i r of s t i m u l i , 50 msec apart, was applied at a frequency of 0.4 Hz, and the f i e l d potentials were recorded by means of a glass c a p i l l a r y microelectrode f i l l e d with 0.9% NaCl. In normal preparations, the amplitude of the second evoked p o t e n t i a l was s l i g h t l y larger than that of the f i r s t by approximately 15%. After a p p l i c a t i o n of 10 μΜ deltamethrin (R,S-isomer), fenvalerate (S,S-isomer), or tetramethrin ((+)-cis-isomer), the second evoked p o t e n t i a l became smaller than the f i r s t by 10-25%. The f i r s t response was not markedly changed i n amplitude, but was s l i g h t l y prolonged i n duration. The change i n the second response was abolished by a decrease i n calcium concentration from 2 to 1 mM. These r e s u l t s are compatible with the notion that the decrease i n the second response by pyrethroids i s due to suppression of part of the mechanisms responsible f o r transmitter release. The glutamate receptor-channel complex i n t h i s preparation i s not affected by pyrethroids.


244

SITES O F ACTION F O R N E U R O T O X I C PESTICIDES

2xlO"*M EDO SA


j! ',ί

20 msec

Figure 7. A f o c a l recording of multiple EPPs induced by a single stimulus of the excitatory nerve i n the presence of 2 χ 10""M EDO. The i n i t i a l upward d e f l e c t i o n i s the stimulus a r t i f a c t (SA). The small deflections indicated by the arrows p r i o r to each EPP are nerve terminal potentials. Note the f a i l u r e of the f i r s t nerve terminal p o t e n t i a l to produce an EPP. (Reproduced with permission from r e f . 40. Copyright 1979 Intox Press.)

A. CONTROL

a 2xlO" M EDO 30 min 6

200msec 6

Figure 8. E f f e c t of 2 χ 10" M EDO on the iontophoretically induced glutamate p o t e n t i a l . A, glutamate p o t e n t i a l i n van Harreveld's saline. B, glutamate potential a f t e r 30 min of exposure to EDO. The p o t e n t i a l i n Β was obtained at a time when spontaneous nerve a c t i v i t y had occurred i n the nerve. Both records are from the same neuromuscular junction. (Reproduced with permission from r e f . 40. Copyright 1979 Intox Press.)


17. NARAHASHI


245


Lindane on Acetylcholine and GABA Receptors I t has been well established that synapses are the major s i t e of action of lindane. Synaptic transmission i s greatly f a c i l i t a t e d by the action of lindane i n both insects and vertebrates (68-Z1) · However, l i t t l e i s known about the mechanism whereby synaptic transmission i s f a c i l i t a t e d by lindane. An example of the lindane-induced synaptic f a c i l i t a t i o n i s i l l u s t r a t e d i n Figure 9 (21). Synaptic transmission across the s i x t h ( l a s t ) abdominal ganglion of the cockroach was studied using e x t r a c e l l u l a r electrodes. The cereal nerve was stimulated and the postsynaptic response was recorded from the abdominal nerve cord. P r i o r to lindane application, a s i n g l e presynaptic stimulus induced a postsynaptic response which was composed of the i n i t i a l large spikes followed by after-discharges of small amplitudes (Figure 9A). A f t e r a p p l i c a t i o n of 10 μΜ lindane, the postsynaptic after-discharges were greatly augmented and prolonged (Figures 9B and 9C) and eventually a s i n g l e presynaptic stimulus evoked bursts of postsynaptic responses (Figure 9D). More d e t a i l e d analyses of the e f f e c t s of lindane on synaptic transmission were made using the frog neuromuscular j u n c t i o n (72). Lindane (100 μΜ) greatly increased the frequency of spontaneous miniature end-plate potentials (MEPPs) while decreasing t h e i r amplitudes. End-plate depolarization evoked by iontophoretic a p p l i c a t i o n of ACh was also decreased by lindane. The quantal content of end-plate p o t e n t i a l s was increased to 180% of control by lindane. When t h i s factor was taken into consideration, the degree of end-plate block by lindane was estimated to be 30%. No detectable e f f e c t of lindane on transmitter release could be shown i n normal Ringer's s o l u t i o n . However, when the mean quantal content was small (e.g. i n low C a - h i g h M g ) , transmitter release was greatly enhanced. Thus lindane has two e f f e c t s ; one i s to suppress the end-plate response to ACh, and the other i s an e f f e c t consistent with a small increase i n i n t r a c e l l u l a r free C a . The calcium channels of neuroblastoma c e l l s , both i n a c t i v a t i n g (type I) and non-inactivating (type I I ) , were not affected by lindane (72). Our recent study using the primary cultured neurons i s o l a t e d from the r a t dorsal root ganglion has thrown l i g h t on the mechanism of action of lindane (6£). Bath a p p l i c a t i o n of GABA induced two components of inward chloride current. One was a transient component which was desensitized with time, and the other was a non-desensitizing component. The d e s e n s i t i z i n g component of chloride current was completely blocked by 10 μΜ lindane, while the non-desensitizing component remained unaffected. Thus lindane blocks one of the GABA-activated receptor-channel complexes leading to hyperexcitâtion. This accounts for synaptic f a c i l i t a t i o n observed e a r l i e r with the lindane-treated cockroach ganglion and vertebrate preparations. 2+

2+

2 +

Conclusion The r e s u l t s of experiments so f a r obtained i n our laboratory and



246

SITES OF

ACTION FOR

NEUROTOXIC

PESTICIDES

Figure 9. After-discharges of the giant f i b e r s induced by a single supramaximal stimulus to the cereal (presynaptic) nerve of the American cockroach before and a f t e r treatment with 1 χ 1(Γ Μ lindane. 17°C, time marker 50 c.p.s. A, before treatment; very short after-discharge (100 msec). Β, 1 hour and 25 minutes after treatment; s l i g h t after-discharge (380 msec). C, 4 hours and 10 minutes after treatment; prolonged after-discharge (940 msec). D, 6 hours and 10 minutes a f t e r treatment. The three l i n e s are continuous. Very prolonged after-discharge (2380 msec). (Reproduced with permission from r e f . 71. Copyright 1957 Kyoto University for the WHO.) 5



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elsewhere c l e a r l y indicate that the nerve membrane sodium channel i s the major target s i t e of type I and type II pyrethroids and DDT. The gating k i n e t i c s of single sodium channels are modified by pyrethroids and DDT r e s u l t i n g i n marked prolongation of channel open time. This change causes a prolonged sodium current to flow across the nerve membrane which i n turn increases and prolongs the depolarizing a f t e r - p o t e n t i a l i n the presence of type I pyrethroids and DDT, or depolarizes the membrane i n the presence of type II pyrethroids. Thus r e p e t i t i v e discharges are evoked i n nerve f i b e r s (type I pyrethroids and DDT) or i n sensory neurons (type II pyrethroids and DDT). In either case, the end product i s represented by synaptic disturbances of various types which explain the symptoms of poisoning i n animals. In c e r t a i n neurons, pyrethroids have been shown to a f f e c t calcium channels, but the exact r o l e of the channel i n poisoning remains to be seen. Our recent study has shown that the GABA receptor-channel complex, which was claimed by some investigators to be a target of type II pyrethroids, i s not affected at a l l by deltamethrin while the sodium current i s greatly prolonged. Thus the GABA receptor-channel complex i s u n l i k e l y to be the major target s i t e of type II pyrethroids. Lindane has recently been shown to e f f e c t i v e l y block one of the components of the GABA-induced chloride channel current, and t h i s action appears to be responsible for synaptic hyperexcitation. Acknowledgments Our studies quoted i n this chapter were supported by NIH grants NS14143 and NS14144. I thank Vicky James-Houff for s e c r e t a r i a l assistance.

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Neuronal Target Sites of Insecticides - American Chemical Society

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