Actions of pyrethroids on the peripheral benzodiazepine receptor

PESTICIDE

BIOCHEMISTRY

AND

PHYSIOLOGY

32, 106-l 13 (1988)

Actions of Pyrethroids on the Peripheral Benzodiazepine Receptor ADEL

A. RAMADAN,*NABILA M. BAKRY,?ABDEL-SALAM M. MAREI,? AMIRA T. ELDEFRAWI,* ANDMOHYEE E. ELDEFRAWI*,'

*Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21201, and fDepartment of Plant Protection, Faculty of Agriculture, University of Alexandria, Alexandria, Egypt Received March 8, 1988; accepted June 23, 1988 The interactions of 14 pyrethroids, as well as 2 permethrin isomers and 8 pure geometric cypermethrin isomers, with the peripheral benzodiazepine (PBZ) receptor of rat brain were studied. This receptor, which is located in the outer membrane of mitochondria, was identified by its specific binding of 3H-labeled 7-chloro-l,3-dihydro-l-methyl-5-~-chlorophenyl)-2~-1,4-benzodiazepine-2-one (r3H]Ro5-4864) (K, 7.5 nM). Pyrethroids that do not contain a-cyano3-phenoxybenzyl alcohol (i.e., type I), as well as those that generally do (i.e., type II), inhibited the binding with IC,, values ranging from 0.15 to >lOO PM with decreasing potency as follows: deltamethrin > flucythrinate > pyrethrins > cypennethrin = cyfluthrin > tetramethrin > allethrin > tralomethrin > bioallethrin = trans-permethrin > S-bioallethrin = resmethrin > fenvalerate = permethrin and cis-permethrin > fluvalinate. Except for fluvalinate, and possibly fenvalerate, type II pyrethroids were in general more potent inhibitors than type I pyrethroids. Of the eight cypermethrin isomers tested at 1 p&f, only the lR,cis,uS inhibited [3H]Ro5-4864 binding, and its potency was unaffected by the nontoxic isomers. It is suggested that pyrethroids bind to the PBZ receptor, which for certain pyrethroids may contribute to their toxicities. However, the poor correlation between the potencies of either or both types of pyrethroids as inhibitors of [3H]Ro5-4864 binding and their toxicities suggests that the PBZ receptor is not a primary target that is critical for pyrethroid toxicity. 0 1988 Academic Press. Inc.

INTRODUCTION

Central benzodiazepine(BZ)2 receptors have high affinities (Kd in nanomolars)for BZ anticonvulsants such as diazepam and clonazepam. These receptors are found mainly in the central nervous system and are part of the y-aminobutyric acid (GABA) receptor/chloride channel protein. It is responsible for the many well-known centrally mediated effects of BZ including antianxiety and muscle relaxant actions (1). Another classof BZ receptorsis the peripheral benzodiazepine(PBZ) receptor, which is abundantin peripheraltissues,but is also found centrally (2). It differs from the ceni To whom reprint requests should be addressed. 2 Abbreviations used: BZ, benzodiazepine; PBZ, peripheral benzodiazepine; TBPS, t-butylbicyclophosphorothionate; PK-11195, I-(2-chlorophenyl) N-methyl-(l-methylpropyl)-3-isoquinoline carboxamide; RoS-4864, 7-chloro-1,3-dihydro-l-methyl5-@-chlorophenyl)-2H-1 ,Cbenzodiazepi ne-2-one.

tral-type BZ receptor in phylogenetic and ontogenetic appearance, tissue distribution, and drug specificity (3). The PBZ receptor doesnot bind clonazepam,but binds with high affinity the convulsant BZ Ro54864(& 1.1 nM) (which differs from diazepam only by a p-chloro substituent) and the non-BZ isoquinoline carboxamidecompound PK-11195 (Fig. 1). On the other hand, RoS-4864is IOOO-foldless potent on the central BZ receptor (i.e., GABA, receptor) (4). Binding studies using [3H]Ro5-4864revealed PBZ receptors in brain as well as kidney with different distributions from [3H]benzodiazepinebinding sites (5). The PBZ receptorsare found in high concentrations in adrenalcortex and skin, with substantiallevels also in heart, salivary glands, nasalepithelium, and lung (6). Their highest concentration correlates with high mitochondrial concentrations and cytochrome oxidase activity. Binding of [3H]Ro5-4864

106 0048-3575188 $3.00 Copyright 6 1988 by Academic Press, Inc. AU rights of reproduction in any form reserved.

ACTION

OF

Diazepam

PYRETHROIDS

ON

PERIPHERAL

Clanazcpam

Cl Ro 5-4064 FIG. 1. Chemical ligands.

PK 11195

structures

of benzodiazepine

(6) and [3H]PK-11195(7) (which has equal or higher affinity for the PBZ receptor (8)) was found associatedwith the mitochondrial outer membrane, suggestingthat it is the site of the PBZ receptor. Porphyrins are suggestedto be the endogenousligands for the PBZ receptor, basedon their very high affinity for it and similar cellular localizations (2, 9). Accordingly, the PBZ receptor may be a regulator of mitochondrial functions and may affect cell growth and differentiation (10). It has been speculatedthat the PBZ receptor might be the mitochondrial pore protein (lo), but no biological function has as yet beenlinked to the pore protein. A role for the PBZ receptorin steroid metabolism or hormonal responsiveness is also implicated by its high concentration in interstitial tissue of the testis and its stimulation of testosteroneproduction in decapsulatedtestes by BZ. Also, porphyrin-containing mitochondrial enzymes are required for steroidogenesis(11). Ro5-4864 has convulsant and proconvulsant3activities in experimental animals (12, 13), as do pyrethroids, which lower the dose of pentylenetetrazole required to elicit a seizure(14). Maximal proconvulsant effects of pyrethroids are produced by concentrations that are asymp3 A proconvulsant is a action of convulsants.

drug

which potentiates the

BENZODIAZEPINE

RECEPTOR

107

tomatic with intoxication (14), which suggeststhat PBZ may not constitute the primary lesion in pyrethroid neurotoxicity . However, their action on PBZ receptor as convulsants may contribute to their toxicity. Administration (icv) in mice of Ro54864,type II pyrethroids, or cage convulsants,elicited generallysimilar signsof toxicity (13, 15).Type II pyrethroids generally contain a-cyano-3-phenoxybenzylalcohol and produce choreoathetosis,convulsions, and salivation, while type I pyrethroids do not have the cr-cyanoconstitutent and produce tremors and convulsions. The poisoning signs of the type II pyrethroids more closely resemble those of Ro5-4864than picrotoxinin. Furthermore, deltamethrin and lR,cis,oS cypermethrin were 60 times more potent inhibitors of [3H]Ro5-4864 binding to rat brain membranes, and with higher Hill coefficients, than of r-[35S] butylbicyclophosphorothionate (TBPS) binding (16),the potent label of the GABA, receptor’s chloride channel.Also, pretreatment of rats with PK-11195,an antagonist of the PBZ receptor, elicited complete reversal of the stereospecific proconvulsant activities of pyrethroids (14).All thesefindings suggestedthat the PBZ receptor may be an important molecular target for the action of type II pyrethroids. The present study was initiated to evaluatethis hypothesis by comparing the affinities of several type I and type II pyrethroids for PBZ receptors of rat brain, as identified by [3H]Ro5-4864binding, and by correlating pyrethroid potenciesin inhibiting the binding with toxicities and symptoms. MATERIALS

AND

METHODS

Membrane preparation. Whole brains of male Sprague-Dawleyrats were rapidly removed after sacrificing the animals by guillotine and rinsed in ice-cold 0.32 M sucrose. The brain was cut into small pieces and homogenizedin 0.32 M sucrose (v/w ratio = 10)using a Wheaton overheadstirrer set at 900t-pmin a glass-Teflon homogenizer (12 strokes). The homogenatewas centrifugedat 1OOOg for 10 min in a Sorvall

108

RAMADAN

RC-SW refrigerated centrifuge. The pellet was discardedand the supernatantfraction centrifugedat 9000gfor 20 min. The supernatant was discarded and the pellet suspendedin 10 vol of phosphate-bufferedsaline (i.e., 50 mM Naf-K+ phosphate buffer containing 200 mM NaCl, pH 7.4). This membrane preparation was washed twice with the same buffer by centrifugation at 9000gfor 20 min each.The final pellet was suspendedin the same buffer to yield -5 mg protein/ml. [3H]Ro5-4864 binding assay. PBZ receptors were identified by their specific binding of [3H]Ro5-4864 as described by Lawrence et al. (16).E3H]Ro5-4864 (78.9Ci/ mmol, from New EnglandNuclear) at 1 nM was incubatedin 1 ml final volume of phosphate-buffered saline with 0.5 mg membrane proteins (-100 ~1) and either unlabeled Ro5-4864 (kindly provided by Dr. L. J. Lawrence; PharmaceuticalResearch Lab., Lexington, KY) or pyrethroid added. Pyrethroidswere addedin 10p,Iof dimethyl sulfoxide which did not affect the binding. The membranepreparationwas added last to initiate the assay and after 120min. at 0°C it was filtered through a 2.l-cm Whatman GF/B glass fiber filter pretreatedwith 0.05% polyethyleneimine. The filter was then washed with 8 ml of ice-cold buffer, placed in a mini vial with 4.5 ml of Beckman Ready-Solv solution, and its radioactivity measured by liquid scintillation spectroscopy at 45% efficiency. Nonspecific binding was determined routinely in the presence of 10 pit4 unlabeledRoS-4864. Pyrethroids

and binding

ET

AL.

saturable. Scatchard analysis of the displacement of 1 ti [3H]Ro5-4864binding with unlabeled RoS-4864indicated that it bound to a single homogenous class of binding sites with high affinity (Kd = 7.5 nM) (Fig. 2). This specific binding of [3H]Ro5-4864was totally insensitive to the convulsantspicrotoxinin and TBPS (Fig. 3) as well as GABA up to 100l&f, all of which affect GABAA receptor binding. However, [3H]Ro5-4864binding was inhibited by the GABA, receptor antagonist bicuculline, but only at high concentrations above 10 p& (Fig. 3). As expectedof a PBZ receptor (7), the binding was inhibited by the benzodiazepinesdiazepam (Fig. 3) and clonazepam aswell as PK-11195with ICsOvaluesof 2 t&, 15 p&f, and 15 nM, respectively. Inhibition pyrethroids.

of [3HjRo5-4864

binding

by

Type I pyrethroids inhibited the specific binding of [3H]Ro5-4864significantly when present at concentrations above 1 $V (Fig. 4). Hill coefficients of the displacementcurves were all
data analysis.

The sourcesand purities of the pyrethroids studied are as mentioned previously (17). The Equilibrium Binding Data Analysis program was used to analyze the binding data on an IBM PC (18). Student’s t test was used to determine the level of significance. RESULTS

Binding of [3Z-fjRo5-4864 and the effect of GABAergic drugs. Binding of [3H]Ro5-4864

to rat brain membraneswas reversible and

FIG. 2. Displacement of [3H]Ro5-4864 from rat brain membranes by Ro5-4864. Inset. The Scatchard plot of the data. B, specific binding of [3H]Ro54864 in picomoles per milligram protein; F, concentration of free [3H]Ro54864 in nanomolar.

ACTION

OF

PYRETHROIDS

ON

PERIPHERAL

BENZODIAZEPINE

109

RECEPTOR

. Diazspam O(+)Bicucullim m TBPS 0 Picroioxinin n ‘I

I-

F

)-

Jv

FL IO

“-: ”

6 5 4 -LOP drug concentration

7 -Log wrethroid (Ml

FIG. 3. The effect

of increasing concentrations of GABAergic drugs on the specific binding of I nM r3H]RoS-4864 to rat brain membranes. Symbols and bars are means *SD of three experiments.

of the binding only at the highest concentration tested (i.e., 10 FM), while others effectively displaced the binding at submicromolar concentrations (Fig. 5, Table 1). The most potent type II pyrethroid was deltamethrin (I&, = 0.15 l&Q (Fig. 6, Table 1). The correlation coeffkients between I&, values of [3H]Ro5-4864 binding and toxicity for type I, type II, and both types of pyrethroids were -0.38, 0.08, and -0.11, respectively. Hill coeffkients of the displacement functions of both allethrin and deltamethrin were much lower than one. Stereospecificity [3HJRo5-4864 binding

of inhibition by pyrethroids.

of

The lR,cis,o.S isomer was the only one of eight cypermethrin isomers tested at 1 PM that inhibited the binding significantly (P < 0.01) (Fig. 7). The dose-dependent function of displacing [3H]Ro5-4864 binding by the lR,cis,aS isomer of cypermethrin indicated that it was more effective than the mixture of four isomers (lR,cis,aS; lR,cis,aR; lR,trans,aS; and lS,trans,aS) (Fig. 8). Interestingly, the difference was not significant suggesting that the less potent isomers were not interfering with binding of the more potent lR,cis,oS isomer. The cis and tram isomers of the type I pyrethroid permethrin were similar in their effect on L3H]Ro5-4864 binding over a wide range of concentrations (Fig. 9). The mixture of the

FIG. 4. Inhibition [3H]Ro5-48@ to rat pyrethroids. Symbols three experiments.

6 concsnlrot~on

5 (M)

of the specific binding of I nM bruin membranes by five type I and bars are means *SD of

two isomers was not significantly from either isomer alone.

different

DISCUSSION

Several characteristics of [3H]Ro5-4864 binding to rat brain membranes suggest that it is binding to PBZ receptor as reported by others (2, 14): the high affinity lO and >lOO @4, respectively), even less effective than most type I pyrethroids (Fig. 5. Table 1).

110

RAMADAN

TABLE 1 of the Potencies of Pyrethroids as Inhibitors of [31ilRo54864 with Their Mammalian Toxicities

Comparison

Pyrethroid

Type1 Allethrin Bioallethrin S-Bioallethrin Permethrin cis-Permethrin trans-permethrin Pyrethrins Resmethrin Tetramethrin Type II Cyfluthrin Cypermethrin Deltamethrin Fenvalerate Flucythrinate Fluvalinate Tralomethrin 0 Data Industrial b I&,, ’ Data

ET AL.

Binding of [3H]Ro5-4864 Go (Pw

Mammalian Toxicity LDso (oral-rat; mg/kg)

Potency ratiob

4 k 0.3 7 2 0.5 10 ? 0.8 >lO >lO 7 2 0.6 1.2 2 0.1 10 2 0.9 3 ” 0.2

680 425 430 410 85’ 3,100’ 750 1,500 >20,000

5 2.9 8 Cl.4 <1 1.4 2.5 4 5

2 2 0.1 2 2 0.1 0.15 2 0.01 >lO 1 f 0.1 >lOO 6 2 0.5

590 251 100 451 81 282 99

0.4 0.6 4.7 CO.8 0.4 <0.003 0.03

from EPA 6004-84-082 “Analytical Reference Standards and Supplemental Data: The Pesticides and Chemicals Repository” and supplements. of GABA receptor function (Ref. (17)) divided by I& of r3H]Ro5-4864 binding. (oral-mouse) from same reference as a.

On the other hand, pyrethrins are the third most potent compoundstestedwith an I&, of only 1.2 PM. There is very poor correlation between mammalian toxicities of all 16pyrethroids tested, or the 7 type II pyrethroids alone, and their potencies in inhibiting [3H]Ro5-4864(Table 1). 0 . a 0 0

Z h8 IOO-

f

Of the eight cypermethrin isomers tested at 1 p,M, only lR,cis,aS is effective significantly in displacing [3H]Ro5-4864(Fig. 7), which agrees with an earlier report (16). However, the lR,trans,aS isomer, which is also toxic, at 1 PM does not inhibit [3H]Ro5-4864binding (Fig. 7). The lack of stereospecific effect for pyrethroid action

Fluvollnote Fenval.,ate Cytluthrin Flucythrinata Tmlomcthrin Pc

”

z P 3 00

8 B z P

50-

t

3

% : &J B P -2

s % E 25

IOI

6 -Lop

7 pyrdhroid

6 concentration

5 (Ml

5. Inhibition of the specific binding of 1 nM [3Z!ZlRo54864 to rat brain membranes by five type II pyrethroids. Symbols and bars are means *SD of three experiments. FIG.

Binding to Rat Brain Membranes

100

(~)deltamethrin

50

IO

-109

0 7 pyrethroid

6 COnCW,t,OtiOn

5 (M)

6. Inhibition of the specific binding of I nM [3H]Ro5-4844 to brain membranes by two different types of pyrethroids. Symbols and bars are means *SD of three experiments. FIG.

ACTION

OF

PYRETHROIDS

ON

PERIPHERAL

on the PBZ receptor is also reflected in the similar effects on [3H]Ro5-4864binding of the toxic trans-permethrin as well as the nontoxic cis permethrin (Fig. 9). These findings, as well as the ineffectivenessof fenvalerate and fluvalinate in these assays (Table 1) and the poor correlation with pyrethroid toxicity, provide persuasive evidencethat the PBZ receptordoesnot play a critical role in the intoxication by pyrethroids. PK-11195is an antagonistof the PBZ receptor. It inhibits [3H]Ro5-4864binding to kidney, heart, and brain (22) and antagonizes the effects of Ro5-4864on the heart (23) as well as the pyrethroid-inducedproconvulsantactivity (15, 18).Thus, it may be assumedthat RoS-4864and pyrethroids are agonistsor potentiators of the PBZ receptor, while PK-11195is an antagonist.This is oppositeto the action of pyrethroids on the GABA, receptor, which is inhibitory (14). Since the displacement curves of [3H] RoS-4864binding by pyrethroids are shallow with Hill coefficientswell below one, it is suggestedthat pyrethroids bind to an allosteric site on the PBZ receptor, similar to their action on the voltage-dependentsodium channel.Pyrethroidsmay act as allos-

BENZODIAZEPINE

RECEPTOR

111

teric modulators, potentiating the action of an endogenousagonist. Becausethere is no known functional assayfor the PBZ receptor, nor knowledgeof the functional consequencesof its occupancy by pyrethroids, their effects cannot be easily comparedwith those on GABA, receptor function. Nor can pyrethroid potencies as inhibitors of binding of [3H] TBOB to GABAA receptor be compared with their inhibition of [3H]Ro5-4864binding to the PBZ receptor, sincethe inhibition appearsto be noncompetitive and the afflnity of GABA, receptor is greatly affected by receptor conformation, being enhanced when the receptor is activated or desensitized (17).Taking this into consideration,it appearsthat all type I pyrethroids, except for permethrin and its isomers, are more potent (1.4- to S-fold) inhibitors of [3H]Ro5-4864binding than GABAA receptor function (17). On the other hand, all type II pyrethroids, except for deltamethrin, appear to be more potent inhibitors of GABAA receptor function (17) than displacing [3H]Ro5-4864binding (Table 1). Considering that the binding of [3H] Ro5-4864is measuredat 1 n&f, which represents
Cyparmethrm l Itt, ClS.aS0 t,,xture

Cypermathrin

-Lop cyparmethr~n

MOTWS (IuM)

7. The effects of eight cypermethrin isomers at 1 JLM on the specific binding of 1 nM [3H]Ro54864 to brain membranes. Bars are *SD of three experiments. The asterisk marks the only isomer which produces signijicant inhibition. FIG.

concentration(M)

8. Inhibition of the specific binding of 1 nM [3HjRo5-4864 to brain membranes by the active isomer of cypermethrin and a mixture of four isomers. Symbols and bars are means *SD of three experiments. FIG.

112

RAMADAN

ET AL. mers, by Dr. L. J. Lawrence (Pharmaceutical Research Laboratory, Lexington, KY) of RoS-4864, and by the U.S. Environmental Protection Agency of the other pyrethroids studied. We also thank Ms. Sharon Boardley for word processing. REFERENCES

-Log

permathrln

concsntrotion

(M)

9. Inhibition of the specific binding of 1 nM [‘H~Ro5-4864 to brain membranes by permethrin isomers and a mixture of both. FIG.

ing to the samesite as [3H]RoS-4864(which is not true), would be close to the I&,, values reported in Table 1. On the other hand, the 36C1- influx measured is induced by 100 @l4 GABA, which results in total receptor occupancy; thus, the Ki values would be at least lo-fold lower than the I&,, values. Accordingly, we may concludethat most type II pyrethroids are more potent on GABA, receptor than they are on the PBZ receptor. Since PBZ receptorsare abundantin salivary glandsandlung, it is tempting to speculate that excessive salivation and persistent edema in lungs of mice intoxicated with type II pyrethoids (24) may be an expressionof the action of pyrethroids on the PBZ receptor. In summary, it is suggested that the actions of pyrethroids at the PBZ receptor do not constitute the primary mechanism of neurotoxicity. In addition, since activation of PBZ receptor increases neural excitability (25) and inhibition of GABA, receptor also results in increased excitation, the action of pyrethroids at these two targets is expectedto contribute to the massive neuroexcitability originating from the action of pyrethroids on sodium channels. ACKNOWLEDGMENTS

This research was supported in part by NIH Grant ES 02594 (A.T.E.) and Amideast Peace Fellowship (A.A.I.R.). We are grateful for the donation by CibaGeigy Ltd. Basel, Switzerland of cypermethrin iso-

1. D. Rampe and D. J. Triggle, Benzodiazepines and calcium channel function, Trends Pharmacol. Sci. 7, 461-463 (1986). 2. S. H. Snyder, A. Verma, and R. R. Trifiletti, The peripheral-type benzodiazepine receptor: A protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligand, FASEB J. 1, 282-288 (1987). 3. R. R. H. Anholt, Mitochondrial benzodiazepine receptors as potential modulators of intermediary metabolism, Trends Pharmacol. Sci. 7, 506-511 (1986). 4. C. Braestrup and R. F. Squires, Brain specific benzodiazepine receptors, Brit. J. Psychiatry 133, 249-268 (1978). 5. H. Schoemaker, R. G. Boles, W. D. Horst, and H. I. Yamamura, Specific high-affinity binding sites for [3H]Ro5-4864 in rat brain and kidney, J. Pharmacol. Exp. Ther. 225, 6149 (1983). 6. R. R. H. Anhoit, E. B. DeSouza, M. L. Ostergranite, and S. H. Snyder, Peripheral-type benzodiazepine receptors: Autoradiographic localization in whole-body sections of neonatal rats, J. Pharmacol. Exp. Ther. 233, 517-526 (1985). 7. R. R. H. Anholt, P. L. Pedersen, E. B. DeSouza, and S. H. Snyder, The peripheral-type benzodiazepine receptor: Location to the mitochondrial outer membrane, J. Biol. Chem. 261, 576 583 (1986). 8. G. Le Fur, M. L. Perrier, N. Vaucher, F. Imbault, A. Flanier, J. Benavides, A. Uzan, C. Renault, M. C. Dubroeucq, and C. Gueremy, Peripheral benzodiazepine binding sites: Effect of PK11195, I-(2-chlorophenyl)-N-methyl-N(I-methylpropyl)-3-isoquinoline carboxamide, Life Sci. 32, 1839-1847 (1983). 9. A. Verma, J. S. Nye, and S. H. Snyder, Porphyrins are endogenous ligands for the mitochondrial (peripheral type) benzodiazepine receptor, Proc. Natl. Acad. Sci. USA 84, 2256-2260 (1987). 10. R. R. H. Anholt, Mitochondrial benzodiazepine receptors as potential modulators of intermediary metabolism, Trends Pharmacol. Sci. 7, 506-511 (1986). 11. M. N. Ritta, M. B. Campos, and R. S. Calandra, Effect of GABA and benzodiazepine on testicular androgen production, Life Sci. 40,791-798 (1987). 12. B. A. Weissman, J. Cott, S. M. Paul, and P. C. Skolnick, Ro5-4864: A potent benzodiazepine

ACTION

13.

14.

15.

16.

17.

OF PYRETHROIDS

ON

PERIPHERAL

convulsant, Eur. .I. Pharmacol. 90, 149-150 (1983). S. Pellow and S. E. File, Pro- and anti-convulsant drug effects in combination with the convulsant benzodiazepine Ro5-4864, J. Pharm. Pharmacol. 37, X0-563 (1985). L. L. Devaud, P. Szot, and T. F. Murray, PK11195 antagonism of pyrethroid-induced proconvulsant activity, Eur. J. Pharmacol. 120, 269-273 (1986). L. J. Lawrence and J. E. Casida, Pyrethroid toxicology: Mouse intracerebral structure-toxicity relationships, Pestic. Biochem. Physiol. 18, 914 (1982). L. J. Lawrence, K. W. Gee, and H. I. Yamamura, Interactions of pyrethroid insecticides with chloride ionophore-associated binding sites, Neurotoxicology 6, 87-98 (1985). A. A. Ramadan, N. M. Bakry , A. S. M. Marei, A. T. Eldefrawi, and M. E. Eldefrawi, Action of pyrethroids on GABA, receptor function, Pestic.

Biochem.

Physiol.

32, 116122

(1988).

18. G. A. McPherson, A practical computer-based approach to the analysis of radioligand binding, Comput. Programs Biomed. J. 17, 107-114 (1983). 19. H. Schoemaker, M. Bliss, and H. I. Yamamura, Specific high-affinity saturable binding of [‘H]Ro5-4864 to benzodiazepine binding sites in the rat cerebral cortex, Eur. J. Pharmacol. 71, 173-175 (1981).

BENZODIAZEPINE

RECEPTOR

113

20. 3. K. T. Wang, T. Taniguchi, and S. Spector, Structural requirements for the binding of benzodiazepines to their peripheral type sites, Mol. Pharmacol. 25, 349-351 (1984). 21. M. K. Ticku and R. Ramanjaneujulu, RoS-4864 inhibits the binding of [35S]r-butylbicyclophosphorothionate to rat brain membranes, Life Sci. 34, 631-638 (1984). 22. G. Le Fur, F. Guilloux, P. Rufat, J. Benavides, A. Uzan, C. Renault, M. C. Dubroeucq, and C. Gueremy, Peripheral benzodiazepine binding sites: Effect of PK 11195, 1-(2-chlorophenyl)N-methyl-(I-metbylpropyl)-3-isoquinoliiecarboxamide, Life Sci. 32, 1849-1856 (1983). 23. M. Mestre, T. Carriot, G. Neliat, A. Uzan, C. Renault, M. C. Dubroeucq, C. Gueremy, A. Doble, and G. Le Fur, PK 11195, an antagonist of peripheral benzodiazepine receptors, modulates BAY K8644 sensitive but not p- or H, receptor sensitive voltage operated calcium channels in the guinea pig heart, Life Sci. 39, 329-339 (1986). 24. A. J. Grey, Pyrethroid structure-toxicity relationships in mammals, Neurotoxicology 6, 127-138 (1985). 25. M. Massati and D. Lucautoni, The peripheral benzodiazepine receptor ligand RoS-4864 induces supraspinal convulsions in rabbits: Reversal by the central benzodiazepine antagonist RolS1788, Psychopharmacology 88, 336-340 (1986).