Effect of ergot alkaloids on 3H-flunitrazepam binding to mouse brain GABAA receptors

In vitro effects of dihydroergotoxine, dihydroergosine, dihydroergotamine, alpha-dihydroergocriptine (ergot alkaloids), diazepam, methyl-beta-Carboline-3-carboxilate (beta-CCM), flumazenil (benzodiazepines), gamma-amino butyric acid (GABA) and thiopental (barbiturate) were studied on mouse brain (cerebrum minus cerebral cortex) benzodiazepine binding sites labeled with 3H-flunitrazepam. Specific, high affinity (affinity constant, Kd = 57.7 8.6 nM) binding sites for 3H-flunitrazepam on mouse brain membranes were identified. All benzodiazepine drugs inhibited 3H-flunitrazepam binding with nanomolar potencies. In contrast to benzodiazepines, all ergot drugs, GABA and thiopental produced an enhancement of 3H-flunitrazepam binding to its binding site at the GABAA receptor of the mouse brain. The rank order of potency was: neurotransmitter (GABA) > dihydroergotoxine > thiopental > alpha-dihydroergocriptine > dihydroergosine > dihydroergotamine. The results suggest that dihydrogenated ergot derivatives do not bind to the brain benzodiazepine binding sites labeled with 3H-flunitrazepam. However, an enhancement of 3H-flunitrazepam binding by all ergot drugs tested, clearly identifies an allosteric interaction with the benzodiazepine binding sites of GABAA receptors.     

Enhanced binding of radioligands to receptors of γ-aminobutyric acid and benzodiazepine by a new anticonvulsive agent,LY81067

A diaryltriazine, LY81067, effectively protects against pentylenetetrazole- and picrotoxin-induced convulsions in mice, with ED₅₀ values of 5.7 and 5.8 mg/kg i.p., respectively. LY81067 enhances the binding of both ³H-GABA and ³H-flunitrazepam to specific sites in rat brain membranes. The degree of enhancement by LY81067 varies from one brain region to another and is different for the binding of ³H-GABA and ³H-flunitrazepam. In cortical membranes, LY81067 increases the affinity of ³H-GABA for both high and low affinity sites and increases the number of sites. LY81067 increases the affinity of ³H-flunitrazepam for its binding sites without greatly increasing the number of sites. Like the pyrazolopyridines, the enhancement of ³H-flunitrazepam binding by LY81067 is dependent on chloride or related anions and is reversed by picrotoxin, suggesting that LY81067 exerts its anticonvulsant effects by binding to or near picrotoxin binding sites. The differential effects of LY81067 on the enhancements of ³H-GABA and ³H-flunitrazepam binding in several brain regions suggest extensive multiplicity of GABA/benzodiazepine/picrotoxin/anioin receptor complexes.     

Modulation of benzodiazepine by lysine and pipecolic acid on pentylenetetrazol-induced seizures

L-lysine, an essential amino acid for man and animals, and its metabolite pipecolic acid (PA) have been studied for their effects on pentylenetetrazol (PTZ)-induced seizures in mice. L-Lysine or L-PA i.p. significantly increased clonic and tonic latencies in a dose-dependent manner against 90 mg/kg PTZ-induced seizures. L-Lysine but not L-PA enhanced the anticonvulsant effect of diazepam (DZ) (0.2 mg/kg). L-PA (0.1 mmol/kg) i.c.v. showed a slight decrease in clonic latency; it did not enhance the antiseizure activity of DZ; it caused seizures at 0.6 mmol/kg. D-PA (0.1 mmol/kg) i.c.v. displayed an opposite effect compared to its L-isomer. The anticonvulsant effect of L-lysine in terms of increase in seizure latency and survival was even more amplified when tested with a submaximal PTZ concentration (65 mg/kg). L-Lysine showed an enhancement of specific 3H-flunitrazepam (FZ) binding to mouse brain membranes both in vitro and in vivo. The possibility of L-lysine acting as a modulator for the GABA/benzodiazepine receptors was demonstrated. Since L-PA showed enhancement of 3H-FZ binding only in vitro but not in vivo, the anticonvulsant effect of L-PA may not be linked to the GABA/benzodiazepine receptor.     

Daily rhythms of benzodiazepine receptor numbers in frontal lobe and cerebellum of the rat

Behavioural, biochemical and neurophysiological evidence suggests that gamma-aminobutyric acid (GABA) may play an important role in the neural control of circadian rhythms. Central receptors for benzodiazepines are functionally coupled to GABA receptors and appear to mediate behavioural effects of exogenous benzodiazepines. The binding of 3H-flunitrazepam to synaptic plasma membranes prepared from various regions of rat brain was examined at 6-hour intervals over a 36-hour period. Prominent daily rhythms in receptor number (Bmax) were observed in the frontal lobe and the cerebellum but not in the temporoparietal regions, hypothalamus or medulla/pons. Binding was highest during periods of sleep/low activity with a significant decrease occurring just prior to waking. These results suggest that daily fluctuations in benzodiazepine receptor numbers may be related to the temporal control of sleep/wake and muscle activity cycles.     

Chronic D1 and D2 dopaminomimetic treatment of MPTP-denervated monkeys: effects on basal ganglia GABA(A)/benzodiazepine receptor complex and GABA content.

The effect of various chronic dopaminergic treatments in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys on the brain gamma-aminobutyric acid type A (GABA(A)) /benzodiazepine receptor complex and GABA content was investigated in order to assess the GABAergic involvement in dopaminomimetic-induced dyskinesia. Three MPTP monkeys received for one month pulsatile administrations of the D1 dopamine (DA) receptor agonist SKF 82958 whereas three others received the same dose of SKF 82958 by continuous infusion. A long acting D2 DA receptor agonist, cabergoline, was given to another three animals. Untreated MPTP as well as naive control animals were also included. Pulsatile SKF 82958 relieved parkinsonian symptoms but was also associated with dyskinesia in two of the three animals whereas animals treated continuously with SKF 82958 remained as untreated MPTP monkeys. Chronic cabergoline administration improved motor response with no persistent dyskinesia. MPTP treatment induced a decrease of 3H-flunitrazepam binding in the medial anterior part of caudate-putamen and an increase in the internal segment of globus pallidus (GPi) which was in general unchanged by pulsatile or continuous SKF 82958 administration. Throughout the striatum, binding of 3H-flunitrazepam remained reduced in MPTP monkeys treated with cabergoline but was not significantly lower than untreated MPTP monkeys. Moreover, cabergoline treatment reversed the MPTP-induced increase in 3H-flunitrazepam binding in the GPi. GABA concentrations remained unchanged in the striatum, external segment of globus pallidus and GPi following MPTP denervation. Pulsatile but not continuous SKF 82958 administration decreased putamen GABA content whereas cabergoline treatment decreased caudate GABA. No alteration in GABA levels were observed in the GPe and GPi following the experimental treatments. These results suggest that: (1) D2-like receptor stimulation with cabergoline modulates GABA(A) receptor density in striatal subregions anatomically related to associative cortical afferent and (2) the absence of dyskinesia in dopaminomimetic-treated monkeys might be associated with the reversal of the MPTP-induced upregulation of the GABA(A)/benzodiazepine receptor complex in the Gpi.     

Anticonvulsant effects of some inhibitory neurotransmitter amino acids

The anticonvulsive effects of GABA, taurine, and glycine were investigated on several chemically-induced and genetic seizure models. Intravenous injections of either GABA, taurine, or glycine provided protection against 3-mercaptopropionic acid (MPA)-induced convulsions in adult Swiss mice. GABA was partially effective against isonicotinic acid hydrazide and was without effect against bicuculline-induced convulsions bProlonged administration of glycine prevented MPA-induced convulsions but not electrically induced seizures or seizures induced by strychnine or metrazol.Intragastric glycine protected young audiogenic seizure-susceptible DBA/2 mice against all three phases of sound-induced convulsions (wild running, clonic and tonic seizure), but GABA and taurine provided little or no protection. With increase of glycine, the cerebral levels of glutamine and serine also increased, but that of glutamic acid decreased. The endogenous glutamic and glycine levels were slightly higher in the brains of the audiogenic seizure-susceptible DBA/2 mice than in that of the resistant BALB/Cy strain.     

Anticonvulsant effects of clonazepam on chemically induced convulsions

The anticonvulsant effects of two doses of clonazepam (CZP, Rivotril Roche, 0.1 and 1 mg/kg i.p.) were studied on model motor seizures induced by strychnine, bicuculline, 3-mercaptopropionic acid and metrazol in male laboratory rats (Wistar strain). In the first part the effects of different doses of the convulsants were investigated and for interaction with CZP doses were chosen after which more than 70% of the animals displayed generalized tonic-clonic convulsions (a grand mal seizure). Strychnine induced this type of seizure only: two doses (2 and 3 mg/kg s.c.) were used. CZP reduced the incidence of convulsions only after the larger dose, but plain solvent (propylene glycol, ethanol, water) was equally effective. The other substances first induced a seizure of minimal (mainly clonic) convulsions and only later a grand mal seizure. CZP was highly effective against bicuculline (3 mg/kg s.c.) and metrazol (100 mg/kg s.c.), but was less so against 3-mercaptopropionic acid. The effect on grand mal seizures was more pronounced in every case than on minimal seizures. The decisive role in the anticonvulsant effect of CZP is played by the mechanisms by which the convulsants induce epileptic manifestations. CZP is most effective against substances acting on the supramolecular complex GABA receptor (benzodiazepine receptor) chloride ionophore (bicuculline and probably metrazol).     

Further characterization of benzodiazepine receptor differences in long-sleep and short-sleep mice

Molecular and conformational characteristics of benzodiazepine (BZ) receptors in cortex and cerebellum from long-sleep and short-sleep mice were investigated using heat inactivation and beta-carboline competition techniques. To investigate differences in the allosteric coupling between GABA and BZ receptors, the protection of BZ receptors from heat inactivation, by GABA, was also evaluated. The two genotypes do not differ in the affinity or number of BZ receptors in the cortex or cerebellum. They do, however, appear to differ in the molecular structure and/or regulation of the conformational state of the receptor in the cortex, as indicated by a greater sensitivity of LS mice to both heat inactivation and beta-carboline competition of 3H-flunitrazepam (FNZ) binding in this region. Evidence for differences in the nature of coupling between GABA and BZ receptors is provided by the finding that in both regions, GABA protected BZ receptors from inactivation to a greater degree in LS mice. The relationship between these differences and the multiplicity of expression of BZ receptors is discussed.     

Differences in the Biophysical Properties of the Benzodiazepine/γ-Aminobutyric Acid Receptor Chloride Channel Complex in the Long-Sleep and Short-Sleep Mouse Lines

Significant differences in the thermal stability of benzodiazepine receptors were found in cerebral cortical membranes prepared from the long-sleep (LS) and short-sleep (SS) selected mouse lines. Thus, benzodiazepine receptors from LS mice were heat inactivated (55 degrees C) at a significantly faster rate than those from SS mice. Although gamma-aminobutyric acid (GABA) reduced the rate of heat inactivation in both lines, the more rapid rate of inactivation in the LS line was maintained. Furthermore, the potency of GABA to enhance [3H]flunitrazepam binding decreased threefold in membranes from LS mice as the incubation temperature was increased from 0 degrees to 37 degrees C, but was unaltered in membranes from SS mice. These differences in the biophysical properties of the benzodiazepine/GABA receptor chloride channel complex ("supramolecular complex"), together with a higher KD for t-[35S]butylbicyclophosphorothionate in membranes from LS compared to SS mice, suggest that the supramolecular complex may modulate the differential sensitivity to some depressants and convulsants in these lines.     

Soluble benzodiazepine receptors: Gabaergic regulation

Benzodiazepine receptor binding has been measured in soluble brain extracts with ³H-flunitrazepam as a ligand. Binding to soluble receptors is enhanced by GABAergic agonists with potencies and maximal augmentation essentially the same as on membrane bound benzodiazepine receptors. The GABA induced increase of binding to soluble receptors is reversed by the GABA antagonist bicuculline.     

Glycoprotein as a Constituent of Purified γ-Aminobutyric Acid/Benzodiazepine Receptor Complex: Structures and Physiological Roles of Its Carbohydrate Chain

The effect of treatments with various enzymes and chemically modifying agents on [3H]muscimol binding to a purified gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex from the bovine cerebral cortex was examined. Treatments with pronase, trypsin, guanidine hydrochloride, and urea significantly decreased the binding of [3H]muscimol, but dithiothreitol, N-ethylmaleimide, reduced glutathione, oxidized glutathione, cysteine, and cystine had no significant effect. These results indicate that the GABA receptor indeed consists of protein, but -SH and -S-S- groups in the protein are not involved in the exhibition of the binding activity. On the other hand, column chromatography using concanavalin A-Sepharose eluted protein having [3H]muscimol binding activity and staining of glycoprotein using an electrophoresed slab gel indicated the existence of two bands originating from the subunits of the GABA/benzodiazepine receptor complex. Furthermore, treatments with various glycosidases such as glycopeptidase A, beta-galactosidase, and alpha-mannosidase significantly increased the binding of [3H]muscimol. These results strongly suggest that GABA/benzodiazepine receptor complex is a glycoprotein and that its carbohydrate chain may be a hybrid type. Treatment with beta-galactosidase resulted in the disappearance of the low-affinity site for [3H]muscimol binding and in an increase of Bmax of the high-affinity site, without changing the KD value. These results suggest that the carbohydrate chain in the receptor complex may have a role in exhibiting the low-affinity binding site for GABA. The observation that the enhancement of [3H]muscimol binding by treatments with beta-galactosidase and glycopeptidase A were much higher than that with alpha-mannosidase may also indicate a special importance of the beta-galactosyl residue in the inhibition of GABA receptor binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Independent benzodiazepine and beta-carboline binding sites in the brain of aggressive and anxious-defensive mice

Distribution of specific 3H-flunitrazepam and 3H-beta-carboline-3-carboxylate binding sites in the brain regions of aggressive and timid-defensive mice was investigated before and after subchronic injection of diazepam (5 mg/kg). The absence of differences between the affinity and concentration of 3H-flunitrazepam binding sites in diencephalon and brain cortex in aggressive and defensive mice may be explained by general benzodiazepine receptor reaction on isolation and agonistic interaction stress. Significant predominance of 3H-beta-carboline-3-carboxylate binding sites in the brain cortex, as compared to the concentration of 3H-flunitrazepam binding sites suggests the presence of specific binding sites for beta-carbolines, which have specific distribution in the brain.     

Gamma-aminobutyric acid and alcohol actions: neurochemical studies of long sleep and short sleep mice

Effects of ethanol and pentobarbital on the GABA receptorchloride channel complex were evaluated in mice selected for differential sensitivity to the hypnotic effects of ethanol (long sleep and short sleep lines). 36Cl- influx, [35S]tbutylbicyclophosphorothionate (TBPS) and [3H]muscimol binding were measured in a membrane vesicle suspension (microsacs) from cerebellum or forebrain. Muscimol was found to be a more potent stimulator of 36Cl- flux in the LS cerebellum, as compared to the SS cerebellum, but a similar maximal level of uptake was achieved in the two lines. Muscimol displaced [35S]TBPS (a ligand for the convulsant site) from cerebellar microsacs, and LS mice were also more sensitive than SS mice to this action of muscimol. However, the number or affinity of high affinity [3H]muscimol binding sites did not differ between the lines. Physiologically relevant concentrations of ethanol (15-50 mM) potentiated muscimol stimulation of 36Cl- uptake in LS cerebellum but had no effect in SS cerebellum. Ethanol failed to alter stimulated chloride flux hippocampal microsacs from either line. Both the LS and SS lines responded similarly to pentobarbital potentiation of muscimol stimulated chloride uptake regardless of brain region. The demonstrated difference between the LS and SS mice in muscimol stimulated chloride uptake as well as in muscimol displacement of [35S]TBPS binding offers a biochemical explanation for the line differences in behavioral responses to GABAergic agents. Moreover, the findings suggest that genetic differences in ethanol hypnosis are related to differences in the sensitivity of GABA-operated chloride channels to ethanol.     

Solubilization of γ-aminobutyric acid receptor from rat brain

Gamma-aminobutyric acid (GABA) binding sites were solubilized from rat brain synaptosomal fractions by extraction with a combination of sodium deoxycholate and potassium chloride. Specific ³H-GABA binding to the solubilized fraction was saturable with the apparent dissociation constant, Kd = 23.4 ± 0.2 nM. GABA agonists and an antagonist inhibited the binding. The relative potencies of these drugs in competing for ³H-GABA binding to the solubilized fraction are in good agreement with findings with the membrane fraction, suggesting that the binding sites in the solubilized fraction retain the characteristics of membrane-bound GABA receptor. The sedimentation coefficient value of ³H-GABA binding site was estimated to be 11.3S by sucrose density gradient centrifugation, and this value was identical with that of ³H-flunitrazepam binding site in the same solubilized fraction.     

L-lysine is a barbiturate-like anticonvulsant and modulator of the benzodiazepine receptor

Our earlier observations showed thatl-lysine enhanced the activity of diazepam against seizures induced by pentylenetetrazol (PTZ), and increased the affinity of benzodiazepine receptor binding in a manner additive to that caused by -aminobutyric acid (GABA). The present paper provides additional evidence to show thatl-lysine has central nervous system depressant-like characteristics.l-lysine enhanced [³H]flunitrazepam (FTZ) binding in brain membranes was dose-dependent and stimulated by chloride, bromide and iodide, but not fluoride. Enhancement of [³H]FTZ binding byl-lysine at a fixed concentration was increased by GABA but inhibited by pentobarbital between 10^–7 to 10^–3M. While GABA enhancement of [³H]FTZ binding was inhibited by the GABA mimetics imidazole acetic acid and tetrahydroisoxazol pyridinol, the enhancement by pentobarbital andl-lysine of [³H]FTZ binding was dose-dependently increased by these two GABA mimetics. The above results suggest thatl-lysine and pentobarbital acted at the same site of the GABA/benzodiazepine receptor complex which was different from the GABA binding site. The benzodiazepine receptor antagonist imidazodiazepine Ro15-1788 blocked the antiseizure activity of diazepam against PTZ. Similar to pentobarbital, the anti-PTZ effect ofl-lysine was not blocked by Ro15-1788. Picrotoxinin and the GABA, receptor antagonist bicuculline partially inhibitedl-lysine's enhancement of [³H]FTZ binding with the IC₅₀s of 2 M and 0.1 M, respectively. The convulsant benzodiazepine Ro5-3663 dose-dependently inhibited the enhancement of [³H]FTZ binding byl-lysine. This article shows the basic amino acidl-lysine to have a central nervous system depressant characteristics with an anti-PTZ seizure activity and an enhancement of [³H]FTZ binding similar to that of barbiturates but different from GABA.     

Barbiturate and benzodiazepine modulation of GABA receptor binding and function

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) acts primarily on receptors that increase chloride permeability in postsynaptic neurons. These receptors are defined by sensitivity to the agonist muscimol and the antagonist bicuculline, and are also subject to indirect allosteric inhibition by picrotoxin-like convulsants and enhancement by the clinically important drugs, the benzodiazepines and the barbiturates. All of these drugs modulate GABA-receptor regulated chloride channels at the cellular level assayed by electrophysiological or radioactive ion tracer techniques. Specific receptor sites for GABA, benzodiazepines, picrotoxin/convulsants, and barbiturates can be assayed in vitro by radioactive ligand binding. Mutual chloride-dependent allosteric interactions between the four receptor sites indicate that they are all coupled in the same membrane macromolecular complex. Indirect effects of barbiturates on the other three binding sites define a pharmacologically specific, stereospecific receptor. All of the activities can be solubilized in the mild detergent 3-[(3-cholamidopropyl)-dimethylammonio]propane sulfonate (CHAPS) and co-purify as a single protein complex.     

Actions of pentobarbitone and derivatives with modified 5-butyl substituents on GABA and diazepam binding to rat brain synaptosomal membranes

The effects of a variety of factors known to influence the enhancement of GABA binding by diazepam, were studied upon pentobarbitone stimulation of GABA binding to washed synaptosomal membranes prepared from whole rat brains. The differential kinetics of, and effects of temperature, chloride ions, a benzodiazepine receptor antagonist (Ro15-1788) and picrotoxinin upon pentobarbitone and diazepam enhancement of GABA binding, suggest that these drugs exert their actions upon GABA binding at different loci. The degree of enhancement of diazepam binding and of high affinity GABA binding in chloride-containing media at 25 degrees C by members of a series of twelve side chain methyl substituted and/or unsaturated derivatives of 5-butyl-5-ethyl-barbituric acid (pentobarbitone analogs) correlated significantly. For the sedative members of the series, enhancement of high affinity GABA binding correlated with their anaesthetic but not their anticonvulsant activities. It appears likely that the anaesthetic and anticonvulsant activities of barbiturates arise from different molecular actions.     

Avermectin Bla Modulation of γ-Aminobutyric Acid/Benzodiazepine Receptor Binding in Mammalian Brain

The anthelminthic natural product avermectin B1a (AVM) modulates the binding of gamma-aminobutyric acid (GABA) and benzodiazepine (BZ) receptor ligands to membrane homogenates of mammalian brain. The potent (EC50 = 40 nM) enhancement by AVM of [3H]diazepam binding to rat or bovine brain membranes resembled that of barbiturates and pyrazolopyridines in being inhibited (partially) by the convulsants picrotoxin, bicuculline, and strychnine, and by the anticonvulsants phenobarbital and chlormethiazole. The maximal effect of AVM was not increased by pentobarbital or etazolate. However, AVM affected BZ receptor subpopulations or conformational states in a manner different from pentobarbital. Further, unlike pentobarbital and etazolate, AVM did not inhibit allosterically the binding of the BZ receptor inverse agonist [3H]beta-carboline-3-carboxylate methyl ester, nor did it inhibit, but rather enhanced, the binding of the cage convulsant [35S]t-butyl bicyclophosphorothionate to picrotoxin receptor sites. AVM at submicromolar concentrations had the opposite effect of pentobarbital and etazolate on GABA receptor binding, decreasing by half the high-affinity binding of [3H]GABA and related agonist ligands, and increasing by over twofold the binding of the antagonist [3H]bicuculline methochloride, an effect that was potentiated by picrotoxin. AVM also reversed the enhancement of GABA agonists and inhibition of GABA antagonist binding by barbiturates and pyrazolopyridines. These overall effects of AVM are unique and require the presence of another separate drug receptor site on the GABA/BZ receptor complex.     

Similar properties of [35S]t-butylbicyclophosphorothionate receptor and coupled components of the GABA receptor-ionophore complex in brains of human, cow, rat, chicken and fish

No significant differences are evident in the specific binding characteristics of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) to EDTA/water-dialyzed P2 membranes of human, cow, rat, chicken and fish brain. This species similarity includes dissociation constants of 61-77 nM at 37 degrees C, maximum receptor densities of 3-7 pmol/mg protein, and sensitivity to inhibition or displacement by gamma-aminobutyric acid (GABA), two cage convulsants (picrotoxinin and t-butylbicycloorthobenzoate) and the insecticide [1R,cis, alpha S]-cypermethrin, indicating a constancy during vertebrate evolution of the [35S]TBPS binding site and its coupling with other components of the GABA receptor-ionophore complex. As a possible exception, chicken and fish brain membranes appear to be less sensitive than the others to the insecticide alpha-endosulfan. Human and rat preparations are also essentially identical relative to the inhibition of radioligand binding by two GABA mimetics (muscimol and 3-amino-propanesulfonic acid), six other cage convulsants (including examples of three classes of polychlorocycloalkane insecticides), a potent anthelmintic agent (Ivermectin), dimethylbutylbarbiturate, the convulsant benzodiazepine Ro 5-3663, and ethanol. The findings to date with [35S]TBPS and the GABA receptor-ionophore complex in rat brain membranes are therefore generally applicable to human preparations. Cow brain is an appropriate source for large scale preparations in receptor purification studies since it is essentially identical to human and rat preparations in all parameters examined. Species differences in sensitivity to the toxic effects of the convulsants and polychlorocycloalkane insecticides considered are apparently not attributable to receptor site specificity.     

Modulation of [3H]Diazepam Binding in Rat Cortical Membranes by GABAA Agonists

GABAA receptor agonists modulate [3H]diazepam binding in rat cortical membranes with different efficacies. At 23 degrees C, the relative potencies for enhancement of [3H]diazepam binding by agonists parallel their potencies in inhibiting [3H]gamma-aminobutyric acid [( 3H]GABA) binding. The agonist concentrations needed for enhancement of [3H]diazepam binding are up to 35 times higher than for [3H]GABA binding and correspond closely to the concentrations required for displacement of [3H]bicuculline methochloride (BMC) binding. The maximum enhancement of [3H]diazepam varied among agonists: muscimol = GABA greater than isoguvacine greater than 3-aminopropane sulphonic acid (3APS) = imidazoleacetic acid (IAA) greater than 4,5,6,7-tetrahydroisoxazolo (4,5,6)-pyridin-3-ol (THIP) = taurine greater than piperidine 4-sulphonic acid (P4S). At 37 degrees C, the potencies of agonists remained unchanged, but isoguvacine, 3 APS, and THIP acquired efficacies similar to GABA, whereas IAA, taurine, and P4S maintained their partial agonist profiles. At both temperatures the agonist-induced enhancement of [3H]diazepam binding was reversible by bicuculline methobromide and by the steroid GABA antagonist RU 5135. These results stress the importance of studying receptor-receptor interaction under near-physiological conditions and offer an in vitro assay that may predict the agonist status of putative GABA receptor ligands.