Pharmacology of pyrazolopyridines

Pyrazolopyridines (PZP's) in general represent a chemically unique class of non-sedative anxiolytic agents. Tracazolate (ICI 136,753) is a member of pyrazolopyridine series that has shown anxiolytic properties in animal models. Tracazolate demonstrates a wider separation between sedative and therapeutic doses than do benzodiazepines. In addition, tracazolate appears to cause fewer adverse interactions than the benzodiazepines in combination with barbiturates and alcohol. In interaction studies, tracazolate potentiated both the antimetrazol and anticonflict effects of chlordiazepoxide. Pyrazolopyridines cause enhancement of both 3H-flunitrazepam (3H-FLU) and 3H-GABA to their binding sites in brain. The enhancement of 3H-FLU binding by PZP's and GABA are additive and reversed by bicuculline. The enhancement of 3H-GABA binding by PZP's and benzodiazepines are additive and reversed by picrotoxin. It is hypothesized that the action of PZP's, and particularly tracazolate, may be related to their effects upon a GABA-stimulated chloride ionophore site. Finally, benzodiazepine antagonists (e.g., RO-15 1788) fail to reverse either the anxiolytic properties of 3H-FLU enhancers or their 3H-GABA binding enhancement effects. In contrast, benzodiazepine antagonists readily reverse the anxiolytic effects of benzodiazepines and non-benzodiazepines which cause 3H-FLU displacement. These data suggest that tracazolate, a non-benzodiazepine, has a pharmacological profile suggestive of novel anxiolytic activity.     

Enhancement of benzodiazepine binding by a diaryltriazine,LY81067

Ly81067, a diaryltriazine, represents a new class of compounds to enhance [³H]flunitrazepam binding to membranes of rat cerebral cortex. The enhancement induced by LY81067 exceeds that induced by γ-aminobutyric acid (GABA) and is partially abolished by the GABA antagonists bicuculline and picrotoxin. Scatchard analysis on the saturable binding of [³H]flunitrazepam reveals that LY81067 at 10 μM increases mainly the affinity for the [³H]ligand, due to a reduced rate of dissociation of the receptor-bound [³H]flunitrazepam. A dependence on chloride anions was demonstrated in the enhancement of [³H]flunitrazepam binding by LY81067. These findings suggest that the diaryltriazine LY81067 enhances [³H]flunitrazepam binding by exerting its effect at or near the picrotoxinsensitive anion recognition sites of the GABA/benzodiazepine receptor complex.     

Pharmacological properties of tracazolate: a new non-benzodiazepine anxiolytic agent

Tracazolate (ICI 136,753, 4-butylamino-1-ethyl-6-methyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid ethyl ester) demonstrated dose-related anticonflict activity in rats and mice. The potency of tracazolate appears to be one-quarter to one-half that of chlordiazepoxide. No tolerance to the anticonflict activity of either tracazolate or chlordiazepoxide was evident following 12 consecutive days of treatment. Tracazolate exhibits a much greater separation between sedative and therapeutic doses than does chlordiazepoxide. Furthermore, based on rodent studies, tracazolate should be much less likely than the benzodiazepines to potentiate the actions of barbiturates and ethanol in man. Tracazolate potentiated both the anticonvulsant and anxiolytic effects of chlordiazepoxide in rodents. Unlike benzodiazepines, tracazolate enhances the binding of benzodiazepines to its receptor site. These results suggest that tracazolate is a novel agent with potential clinical utility as an anxiolytic drug.     

Tracazolate: A novel nonsedative anxiolytic

Tracazolate (ICI 136,753; 4-butylamino-1-ethyl-6-methyl-1H-pyrazolo [3,4-b] pyridine-5-carboxylic acid ethyl ester) exhibited dose-related anticonflict activity in mice, rats, and squirrel monkeys. The potency of tracazolate appears to be one-quarter to one-half that of chlordiazepoxide, and their durations of activity are similar in rats. No tolerance to the anticonflict activity of either chlordiazepoxide or tracazolate was evident following 12 consecutive days of treatment. Tracazolate exhibits a much greater separation between sedative and therapeutic doses than does chlordiazepoxide; thus, it is predicted that tracazolate will not be sedative at anxiolytic doses in man. Furthermore, based upon studies in rodents, tracazolate should be much less likely than the benzodiazepines to potentiate the actions of barbiturates and ethanol in man. Although tracazolate is an anticonvulsant in rodents, it is considerably less potent in this respect than chlordiazepoxide. In contrast to benzodiazepine anxiolytics, tracazolate enhances the binding of ³H-benzodiazepines to their binding sites in brain. Consistent with this finding, tracazolate potentiated both the anticonvulsant and anxiolytic effects of chlordiazepoxide in rodents. Although the benzodiazepine antagonists (RO 15-1788 and CGS 8216) clearly antagonize the anticonflict activity of chlordiazepoxide, the activity of tracazolate was not significantly altered by either antagonist. Both chlordiazepoxide and tracazolate significantly enhanced ³H-γ-aminobutyric acid (GABA) binding. These results suggest that tracazolate is a novel psychoactive agent that should be a useful anxiolytic drug in man.     

Enhancement of benzodiazepine binding by methaqualone and related quinazolinones

Methaqualone and mecloqualone were found to inhibit [³H]diazepam binding to rat cortical membranes, whereas a related quinazolinone, piriqualone (3-(2-methylphenyl)-2-[2-(2-pyridinyl)ethenyl]-4(3H)-quinazolinone), elicited an increase in the binding. Irrespective of their in vitro effects on benzodiazepine binding, all three quinazolinones enhanced the amount of intravenously administered [³H]flunitrazepam bound to mouse brain in vivo. Ex vivo experiments indicated that the enhanced binding induced by methaqualone and piriqualone, as well as that elicited by the pyrazolopyridine binding enhancers cartazolate and tracazolate, involved an increase in receptor density. This ex vivo effect differed from the in vitro enhancement of [³H]diazepam binding by piriqualone, cartazolate, and tracazolate, which was caused by an increase in binding affinity, and the in vitro inhibition of binding by methaqualone. The quinazolinones did not appear to affect [³H]GABA binding, but GABA-like activity was suggested by their potent reversal of the cerebellar cyclic GMP accumulation induced by isoniazid. The benzodiazepinelike actions (anticonvulsant, hypnotic, anxiolytic) exerted by methaqualone and related quinazolinones may be mediated via GABA/benzodiazepine/barbiturte receptor complexes.     

Evidence for association of a high affinity avermectin binding site with the benzodiazepine receptor

Avermectin B1a concentrations corresponding to the KD value of a specific high affinity binding site for [3H]avermectin B1a are sufficient to stimulate the specific high affinity binding of [3H]flunitrazepam to its receptors. This stimulation of [3H]flunitrazepam binding by low concentrations of avermectin B1a was enhanced by chloride ions and picrotoxinin and reduced by the GABA agonists THIP and PSA. The similar modulation by chloride ions, GABA agonists and picrotoxinin and the resistance against washing of membranes of avermectin B1a bound to its specific high affinity binding site or to the site modulating [3H]flunitrazepam binding indicate a close association of the specific high affinity binding site for [3H]avermectin B1a with the GABA-benzodiazepine receptor complex.     

Action of pyrazolopyridines as modulators of [3H]flunitrazepam binding to the gaba/benzodiazepine receptor complex of the cerebellum

The pyrazolopyridines etazolate (SQ 20009) and cartazolate (SQ 65396) have strong modulatory effects on the GABA/benzodiazepine receptor complex of rate cerebellum. Thus, etazolate and cartazolate directly stimulate [3H]flunitrazepam binding (with EC50 values of 1.2 microM and 0.3 microM respectively) by increasing the apparent affinity of [3H]flunitrazepam for its binding sites. Stimulation of [3H]flunitrazepam binding by pyrazolopyridines is dependent on the presence of certain anions like chloride, bromide, iodide, nitrite, nitrate but not fluoride, acetate, formate or sulfate. If is inhibited by bicuculline-methiodide, and by the "chloride channel drugs' picrotoxinin and IPTBO. isoTHAZ, a GABA analogue with GABA antagonist properties in vivo, fails to inhibit binding stimulated by etazolate but antagonizes [3H]flunitrazepam binding stimulated by GABA. The pyrazolopyridines have also indirect effects on benzodiazepine receptor binding since they enhance the apparent sensitivity of those GABA recognition sites which are coupled to benzodiazepine binding sites. Thus, in the presence of 10 microM etazolate, GABA and muscimol enhance [3H]flunitrazepam binding, with EC50 values of 109 nM and 12 nM respectively. This sensitization effect is partially dependent on the presence of chloride ions. The pyrazolopyridines facilitate also the stimulation of benzodiazepine receptor binding by beta-alanine and taurine and by the rigid and flattened GABA analogues THIP and piperidine-4-sulfonic acid. Taken together, these results suggest that the pyrazolopyridines modulate [3H]flunitrazepam binding by acting at a site closely related to GABA receptor-regulated chloride ion channels.     

Modulation of benzodiazepine receptor binding: insight into pharmacological efficacy

The effects of GABA on the binding of analogues of benzodiazepines, triazolopyridazines, beta-carbolines and imidazodiazepines were examined in ligand/[3H] flunitrazepam competition experiments. GABA increased the potency of anxiolytics, like flunitrazepam, whereas the potency of benzodiazepine antagonists, like Ro15-1788, was largely insensitive to the influence of GABA. Several other agents including pyrazolopyridines, barbiturates and etomidate caused a chloride dependent enhancement of [3H] flunitrazepam binding but not an enhancement of [3H] propyl-beta-carboline-3-carboxylate binding.     

Enhancement of benzodiazepine receptor binding by L-lysine is chloride-dependent and due to increase in binding affinity

L-Lysine enhanced specific [3H]flunitrazepam binding dose dependently on extensively washed bovine brain membrane in vitro. This enhancement was stimulated by chloride ions dose dependently. Scatchard analysis indicated this enhancement by L-lysine to be due to increase in binding affinity (KD) with no change in receptor density (Bmax). Since enhancement of [3H]flunitrazepam binding by L-lysine was partially inhibited by picrotoxinin, L-lysine may act on a distinct picrotoxinin-sensitive site which was distinct from the gamma-aminobutyric acid receptor site. This binding site, however, appears to have some features resembling that of the central nervous system-depressant barbiturates.     

Chronic agonist exposure induces down-regulation and allosteric uncoupling of the gamma-aminobutyric acid/benzodiazepine receptor complex

Using [3H]flunitrazepam as a probe for the benzodiazepine-sensitive modulator site located on the gamma-aminobutyric acid (GABA)A receptor complex, we have investigated the cellular regulation of the GABAA receptor in neuronal cultures derived from embryonic chick brain. Treatment of cultures with 1 mM GABA for 48 hr causes a reversible 35% decrease in the number of [3H]flunitrazepam binding sites with no change in affinity. The EC50 for chronic GABA-induced down-regulation is 94 microM and the half-time is 25 hr. The effect of GABA is blocked by SR-95531, a GABAA receptor antagonist, and mimicked by muscimol but not baclofen. Consistent with the decrease in [3H]flunitrazepam binding, chronic GABA exposure causes a 43% decrease in the binding of [35S]t-butylbicyclophosphorothionate, a ligand for the receptor-associated chloride ionophore. In addition to chronic GABA-induced down-regulation, allosteric interactions between GABA and benzodiazepine recognition sites are uncoupled by 34%. The half-time and pharmacology for chronic GABA-induced uncoupling is indistinguishable from that for GABA-induced down-regulation, consistent with the hypothesis that the action of GABA at a common site induces both down-regulation and uncoupling.     

Effect of chronic treatment of ethanol on benzodiazepine and picrotoxin sites on the GABA receptor complex in regions of the brain of the rat

Ethanol has been shown to enhance gamma-aminobutyric acid (GABA)ergic transmission. In this study an examination was made of the effect of chronic treatment with ethanol and its withdrawal at 24 h on the binding of [3H]flunitrazepam and [35S]t-butylbicyclophosphorothionate (TBPS) to brain regions in rat. Rats were rendered tolerant to, and dependent on, ethanol by an intragastric intubation method. The affinity (KD) or the binding capacity (Bmax) of [3H]flunitrazepam or [35S]TBPS was not altered by chronic treatment with ethanol or during withdrawal from ethanol. Neither the enhancing effect of GABA on the binding of [3H]flunitrazepam nor its inhibitory effect on the binding of [35S]TBPS were affected by chronic treatment with ethanol or its withdrawal at 24 h. These results suggest that the sensitivity of benzodiazepine and picrotoxin sites on the oligomeric GABA receptor complex is not affected during tolerance to, or withdrawal from ethanol. It is suggested that the effects of ethanol on GABAergic transmission may be produced at the level of coupled chloride ion channels.     

Anxiolytic cyclopyrrolones zopiclone and suriclone bind to a novel site linked allosterically to benzodiazepine receptors

The interactions of zopiclone and suriclone, representatives of nonbenzodiazepine cyclopyrrolone anxiolytics, with central-type benzodiazepine receptors have been characterized in rat and bovine brain. While zopiclone potently (IC50 approximately 50 nM) inhibits [3H]Ro-15-1788 binding in an apparent mass action fashion, suriclone and its metabolite 35,489 RP are extremely potent (IC50 approximately 350 pM and 1 nM, respectively) and display Hill coefficients of approximately 2.0. Like classical benzodiazepines, none of the cyclopyrrolones studied display selectivity for type I or type II benzodiazepine receptors. Using [3H]suriclone, saturable high affinity sites for cyclopyrrolone anxiolytics were directly labeled in rat and bovine brain. The regional distribution and pharmacologic specificity of [3H]suriclone and [3H]Ro-15-1788 binding sites are similar, suggesting that [3H]suriclone recognition sites reside on the benzodiazepine receptor complex. Unlike classical benzodiazepine agonists, such as diazepam, the binding of [3H]suriclone is not modulated by GABA, Cl-, pentobartibal, or tracazolate. Unlike those of [3H]diazepam, [3H]suriclone-binding sites are only minimally affected by photoaffinity labeling with flunitrazepam. Whereas the binding affinities of [3H]Ro-15-1788, [3H]flunitrazepam, and [3H]ethyl beta-carboline 3-carboxylate increase at lower temperatures, [3H]suriclone binds with higher affinity at higher temperatures. Scatchard analysis of [3H]flunitrazepam, [3H]ethyl beta-carboline 3-carboxylate, and [3H]Ro-15-1788 binding in the presence of all cyclopyrrolones studied reveals an apparent noncompetitive pattern of inhibition of binding in each case; by contrast, inhibition of [3H]suriclone binding by Ro-15-1788 flunitrazepam, methyl beta-carboline 3-carboxylate and all of the cyclopyrrolones studied appears competitive. The dissociation kinetics of [3H]Ro-15-1788 indicate that cyclopyrrolones, but not benzodiazepines, increase the dissociation rate of [3H]Ro-15-1788 from its membrane receptors; the converse is true for [3H]suriclone dissociation kinetics. The association kinetics of [3H]suriclone suggest that suriclone induces a conformational change upon binding to receptors. Taken together, these results indicate that [3H]suriclone labels a site on the benzodiazepine receptor complex allosteric to the recognition site for benzodiazepines. A model is proposed to describe the interaction between benzodiazepines and cyclopyrrolones.     

Enhancement of benzodiazepine binding sites following chronic treatment with flumazenil

The aim of this study was to improve our knowledge of the mechanisms leading to adaptive changes in gamma-aminobutyric acid(A) (GABA(A)) receptors following chronic drug treatment. Exposure (48 h) of human embryonic kidney (HEK 293) cells stably expressing recombinant alpha1beta2gamma2S GABA(A) receptors to the antagonist of benzodiazepine binding sites, flumazenil (5 microM), enhanced the maximum number (B(max)) and the equilibrium dissociation constant (K(d)) of [3H]flunitrazepam binding sites. The flumazenil-induced enhancement in B(max) was potentiated by GABA (50 microM) and reduced by the GABA(A) receptor antagonist, bicuculline (100 microM). Flumazenil-induced enhancement in K(d) was affected by neither of these treatments. GABA (50 microM) enhanced the density of [3H]flunitrazepam binding sites, and this enhancement was greater in the presence of diazepam (1 microM). The results suggest that chronic flumazenil treatment up-regulates in a bicuculline-sensitive manner benzodiazepine binding sites at stably expressed GABA(A) receptors.     

Benzodiazepine interactions with GABAA receptors on chick ciliary ganglion neurons

gamma-Aminobutyric acidA (GABAA) receptors on chick ciliary ganglion neurons can be modulated by benzodiazepines and identified by radiolabeled benzodiazepine binding. Enhancement of submaximal GABA responses by benzodiazepines was demonstrated using a multibarrel pipette to construct complete benzodiazepine dose-response curves for single cells in culture. EC50 values of 22 +/- 5 nM, 1.1 +/- 0.3 microM, and 4.6 +/- 0.5 microM were obtained for flunitrazepam, clonazepam, and chlordiazepoxide, respectively. Chlordiazepoxide shifted the GABA dose-response curve to lower GABA concentrations without increasing the maximal response to GABA, demonstrating that benzodiazepines enhance the GABA response by increasing the receptor affinity for GABA. The imidazodiazepine Ro15-1788 potentiated the GABA response with an EC50 of 250 +/- 70 nM, and Ro5-4864 (chlorodiazepam) partially blocked the GABA response both in the presence and absence of chlordiazepoxide. Scatchard analysis of data from binding studies with [3H]flunitrazepam to ganglion membrane homogenates was consistent with the presence of a single class of high affinity sites with a KD of 34 +/- 6 nM and a Bmax of 145 +/- 26 fmol/mg of protein. Several lines of evidence indicated that the sites were associated with GABAA receptors. The KD of [3H]flunitrazepam binding was similar to the EC50 for flunitrazepam modulation of the GABA response. The level of [3H]flunitrazepam binding was enhanced approximately 50% over control levels by GABA. The binding was decreased both by clonazepam and by Ro5-4864 at concentrations similar to those required for the compounds to modulate the GABA response. These studies demonstrate that ciliary ganglion GABAA receptors are similar in major respects to GABAA receptors in the central nervous system but may differ in minor pharmacological properties.     

Flunitrazepam photoaffinity labeling of the GABA(A) receptor reduces inhibition of [3H]Ro15-4513 binding by GABA

The benzodiazepine drugs modulate gamma-aminobutyric acid (GABA)-mediated synaptic transmission via a high-affinity binding site that is part of the GABA(A) receptor complex, but which is distinct from the GABA binding site. Ro15-4513 is a benzodiazepine negative modulator of GABA action that displays unique anti-ethanol properties both in vivo and in vitro. Ro15-4513 has been reported to photoaffinity label nearly 100% of the benzodiazepine binding sites in rat brain homogenates. In contrast, the benzodiazepine positive modulator flunitrazepam photoaffinity labels only 25% of the sites. Here, we have examined the reversible binding of [3H]Ro15-4513, [3H]flumazenil (Ro15-1788), and [3H]flunitrazepam to embryonic chick brain membranes, and to membranes that have been photoaffinity labeled with nonradioactive flunitrazepam. Photoaffinity labeling with flunitrazepam decreased the subsequent reversible binding of [3H]flunitrazepam and [3H]flumazenil, but increased the binding of [3H]Ro15-4513. The increase in [3H]Ro15-4513 binding after flunitrazepam photoaffinity labeling was due to a decrease in the apparent Kd, with no change in Bmax. Following photoaffinity labeling, negative modulation of [3H]Ro15-4513 binding by GABA was lost, whereas positive modulation of residual [3H]flunitrazepam binding was retained. We conclude that the site photoaffinity labeled by flunitrazepam is distinct from the site responsible for reversible binding of [3H]Ro15-4513.     

Potentiated modulation of pregnolone on GABAA receptors in behaviorally stressed borderline-hypertensive rats

The modulatory effects of behavioral stress on [(3)H]flunitrazepam, an agonist for the central-type benzodiazepine receptor binding to the GABA(A)-benzodiazepine receptor complex, in borderline hypertensive rats (BHR) were examined. In repeatedly immobilized (for 2 weeks, for 2 h/d) BHR, enhancement of [(3)H]flunitrazepam binding to the receptor was observed to be potentiated. The percent enhancement of [(3)H]flunitrazepam binding in BHR was higher than that in normotensive control Wistar-Kyoto rats. Pregnanolone, a neuroactive steroid that has been reported to be a putative endogenous modulator in the stress response, concentration dependently enhanced [(3)H]flunitrazepam binding to the receptor. Enhancement of [(3)H]flunitrazepam binding was observed to be potentiated by the same immobilized stress, and the EC(50) values of pregnanolone in BHR was significantly lower than those in controls and E(max) values were higher. From the above results, it can be concluded that neural modulation to behavioral stress, especially in GABAergic neurotransmission, is exaggerated in BHR. We propose strain-specific differences of stress reactivity as an important pathogenetic factor in psychosomatic disorders including stress-induced hypertension. This is supported by reports showing exaggerated cardiovascular and symathoadrenal responses to stress in BHR.     

Solubilization and characterization of benzodiazepine receptors in frontal cortex of rabbit brain

[3H]Flunitrazepam binding to benzodiazepine receptors solubilized by the detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-l-propanesulfonate (CHAPS) was saturable and showed non-linear Scatchard plot with KD1 0.31 nmol/L and KD2 6.7 nmol/L. The affinities of soluble receptors to benzodiazepine were consistent with P2 membrane. One radioactive zone was found by SDS-PAGE after photoaffinity labelling of soluble membrane and the apparent molecular weight of 55,000 was reported. [3H]Flunitrazepam binding to soluble receptors was enhanced by GABA, NaCl or KCl and barbiturates, but inhibited by bicuculline and picrotoxinin. The enhancement of GABA on [3H]flunitrazepam binding was amplified by NaCl or KCl and antagonized by bicuculline and picrotoxinin. These results suggest that the benzodiazepine receptors solubilized by CHAPS have their pharmacological properties and are still associated with GABA receptors and chloride channel.     

Enhancement of the in vivo binding of [3H]flunitrazepam by the atypical neuroleptic, clozapine

Modulation of the benzodiazepine receptor/GABA receptor/chloride ionophore complex in vivo involves a number of intricate regulatory interactions between the three components of the receptor complex. One way to assess these potential interactions involves the in vivo labelling of the benzodiazepine receptor with [3H]flunitrazepam. In these studies, we used this approach to demonstrate that the neuroleptic, clozapine, increases [3H]flunitrazepam binding in mouse brain in a bicuculline-reversible manner. This potentiation of benzodiazepine binding was not antagonized by picrotoxin and was found to result from a slower dissociation of [3H]flunitrazepam from the benzodiazepine receptor. These data suggest that clozapine acts to increase [3H]flunitrazepam binding via a GABAergic mechanism, independent of the chloride channel.     

GABA induces down-regulation of the benzodiazepine-GABA receptor complex in the rat cultured neurons

Cultured neurons from embryonic rat brain display central type benzodiazepine receptors characterized by high-affinity binding of [3H]flunitrazepam which is allosterically enhanced in the presence of gamma-aminobutyric acid (GABA). A 48 h treatment of the cultured neurons with 1 microM diazepam, 0.1 microM clonazepam or 0.1 microM beta-carboline ester derivatives did not change either Bmax or KD values of the [3H]flunitrazepam specific binding. A 48 h incubation in the presence of GABA (1 mM) or muscimol (0.1 mM) induced a 30% decrease of the Bmax value of [3H]flunitrazepam specific binding without change of the KD value. The down-regulation was dependent on GABA concentrations and temperature, and was partially inhibited by bicuculline but not by the benzodiazepine antagonist Ro 15-1788. The other subunits of the benzodiazepine-GABA-chloride channel receptor complex also seemed to be down-regulated by GABA since there was a decrease of the specific binding of [3H]muscimol and [35S]t-butylbicyclophosphorothionate (TBPS) to the GABAA and chloride channel sites respectively. The GABA-induced down-regulation of the GABA-benzodiazepine receptor seems to be selective since the specific binding of ligands to other receptors was not affected. Our results suggests that activation of the low-affinity GABA subunit which is involved in cellular electrophysiological responses, induced the receptor down-regulation.     

Mercury interaction with the GABA(A) receptor modulates the benzodiazepine binding site in primary cultures of mouse cerebellar granule cells.

Mercury compounds are neurotoxic compounds with a great specificity for cerebellar granule cells. The interaction of mercury compounds with proteins in the central nervous system may underlie some of their effects on neurotransmission. In this work we study the interaction of mercuric chloride (HgCl2) and methylmercury (MeHg) with the GABA(A) receptor in primary cultures of cerebellar granule cells. Both compounds increased, dose dependently, the binding of [3H]flunitrazepam to the benzodiazepine recognition site. EC50 values for this effect were 3.56 and 15.24 microM for HgCl2 and MeHg, respectively, after 30 min exposure of intact cultured cerebellar granule cells. The increase of [3H]flunitrazepam binding by mercury compounds was completely inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxinin, and by the organochlorine pesticide alpha-endosulfan. It was also partially inhibited by the anion transporter blocker DIDS, however this effect could be due to a possible chelation of mercury by DIDS. Intracellular events, like intracellular calcium, kinase activation/inactivation or antioxidant conditions did not affect [3H]flunitrazepam binding or its increase induced by mercury compounds. The sulfhydryl alkylating agent N-ethylmaleimide mimicked the effect of mercury compounds on [3H]flunitrazepam binding suggesting a common mechanism. We conclude that mercury compounds interact with the GABA(A) receptor by the way of alkylation of SH groups of cysteinyl residues found in GABA(A) receptor subunit sequences.