Differential effects of triton X-100 on benzodiazepine and GABA binding in a frozen-thawed synaptosomal fraction of rat brain

The effect of Triton X-100 on 3H-GABA and 3H-diazepam binding was measured in a frozen-thawed synaptosomal fraction of rat brain. Specific binding activity (amount bound per mg protein) of both ligands was increased by the treatment. Diazepam binding capacity in the pellet was progressively decreased, while GABA binding was increased, then decreased by increasing Triton X-100. Diazepam binding affinity was unchanged, while GABA binding affinity increased. Triton X-100 appears to preferentially solubilize benzodiazepine binding sites, indicating GABA and benzodiazepine binding sites are on separate macromolecules.     

Increased benzodiazepine receptor number elicited in vitro by a novel purine, EMD 28422

EMD 28422 (N6-[2-(4-chlorophenyl)-bicyclo-2.2.2.octyl-(3)]-adenosine) was demonstrated to increase the number of binding sites for [3H]diazepam (Bmax) in vitro without an accompanying increase in receptor affinity (KD). The increase in receptor number was observed in both crude synaptosomal preparations (P2) and thrice-washed membrane preparations with and without the addition of 50 microM GABA. Furthermore, this effect appeared to be independent of the concentration of chloride ion, since the increases in Bmax were observed in both Tris-HCl and Tris-maleate buffer. The effects of EMD 28422 were stereospecifically antagonized by the GABA antagonist bicuculline, despite the lack of effect of EMD 28422 on [3H]muscimol binding at concentrations which markedly increased benzodiazepine receptor number. Neither EMD 39011 nor adenosine, the two parent moieties of EMD 28422, increased [3H]diazepam binding at concentrations of up to 1 mM. The increases in benzodiazepine receptor number observed with EMD 28422 in vitro suggests that this compound induces a conformational change in the benzodiazepine receptor which may cause the dissociation of an endogenous noncompetitive inhibitor of [3H]diazepam binding from the membrane, thus 'unmasking' binding sites. The stereospecific antagonism of this effect by bicuculline and the apparent inability of GABA to alter the action of EMD 28422 suggests the presence of a novel type or different functional state of GABA receptor which may play a permissive role in the rapid modulation of benzodiazepine receptor number in vitro.     

Enhancement of benzodiazepine and GABA binding by the novel anxiolytic, tracazolate

Tracazolate (ICI 136,753) 4-butylamine-1-ethyl-6-methyl-1H-pyrazolo[3,4]pyridine-5-carboxylic acid ethyl ester is a non-benzodiazepine with anxiolytic-like activity in animal models. In contrast to the benzodiazepines, it enhances [3H]flunitrazepam binding in rat synaptic membrane fragments. The enhancement is potential by chloride ion and is due to an increase in affinity of the receptor. The enhancement of benzodiazepine binding by gamma-aminobutyric acid (GABA) is additive with that of tracazolate; however, the GABA antagonist bicuculline blocks the enhancement by both compounds. Tracazolate enhances [3H]GABA binding to frozen and thawed Triton X-100-treated membrane fragments. The enhancement is due to an increase in the number of sites and potentiated by chloride. Benzodiazepines also enhanced GABA binding but the effect was due to an apparent change in affinity and not potentiated by chloride. The rank order to chlorodiazepoxide, diazepam and flunitrazepam for enhancement of GABA binding and displacement of [3H]flunitrazepam binding were the same. The enhancement of [3H]GABA binding by flunitrazepam and tracazolate were additive. Possible interactions between these various receptors are discussed.     

Inhibitory effect on 3H-diazepam binding and potentiating action on GABA of ethyl loflazepate, a new minor tranquilizer

A new benzodiazepine compound, ethyl loflazepate (ethyl-7-chloro-2,3-dihydro-5-(2-fluorophenyl)-2-oxo-1H,1,4- benzodiazepine-3-carboxylate; CM6912) was studied using in vitro experimental systems for its displacement activity on 3H-diazepam binding to the synaptosomal membrane fraction of rat cerebrum and potentiating action on GABA. CM6912 inhibited the specific binding of 3H-diazepam by 25%, 75% and 90% at concentrations of 0.01 microM, 0.1 microM and 1 microM, respectively, while its metabolites CM6913 and CM7116, at 0.1 microM, completely inhibited the binding. Concentrations for 50% inhibition (IC50) were 25 nM for CM6912, 3.2 nM for CM6913 and 1.4 nM for CM7116. These results suggest that the metabolite CM7116 is stronger than its parent compound in displacing the 3H-diazepam binding, and they also suggest that the long-lasting anti-anxietic action of CM6912 might be due to the in vivo formation of CM7116. CM6912, CM7116 and diazepam potentiated the suppressive action of GABA on spontaneous spikes of Purkinje cells in guinea pig cerebellar slices in a dose-dependent manner. Concentrations for 50% suppression (IC50) were 96.0 microM for GABA alone, 75.0 microM for GABA plus diazepam (5 microM), 78.9 microM for GABA plus CM6912 (5 microM) and 60.8 microM for GABA plus CM7116 (5 microM). These findings suggest that CM6912 and CM7116 may potentiate the postsynaptic inhibitory action of GABA in a manner similar to and probably more strongly than diazepam.     

GABA and benzodiazepine receptors in the offspring of dams receiving diazepam: ontogenetic studies

Ontogenesis of the regulation of ³H-GABA and ³H-diazepam binding to rat brain plasma membranes treated with 0.05% Triton X-100 has been studied. The density of ³H-diazepam and ³H-GABA binding in cortex, cerebellum and corpus striatum at birth was approximately one third of the adult values. They increased at the same rate and reached the adult values between 14–21 days after birth. Study of the binding characteristics showed that the K_D for high and low affinity for ³H-GABA, and for ³H-diazepam did not change during ontogenesis and the increase reflects only an increase of Bmax. The number of Triton X-100 treatments of crude synaptic membrane (CSM) required to maximize ³H-GABA for the high affinity component were different at various postnatal days: only one treatment was required in 1-day old rats, two in 7- and 14-day old rats and three in adult animals. In addition, the capability of muscimol to stimulate ³H-diazepam binding in both frozen-thawed and Triton X-100 treated membrane preparations decreased with increasing age. Binding of ³H-GABA and ³H-diazepam to brain of newborn rats whose dams received diazepam throughout pregnancy (100 mg/kg, × os, bid) was also studied. No significant differences were observed in the ontogenetic development of both bindings. However, in the cortex of these newborn rats the capability of muscimol to stimulate ³H-diazepam binding was greatly reduced in Triton X-100-treated membranes.     

Regional rat brain benzodiazepine receptor number and gamma-aminobutyric acid concentration following a convulsion.

1 Following administration to rats of an electroconvulsive shock (ECS) which resulted in a major tonic-clonic seizure, no changes in [3H]-diazepam binding characteristics were observed in cortex or hippocampus with either a well washed membrane preparation or a crude synaptosomal preparation. 2 No changes were observed in [3H]-diazepam binding in any other brain region examined 30 min after an ECS. 3 Thirty min following an ECS, regional brain gamma-aminobutyric acid (GABA) concentrations increased in hippocampus, cortex and hypothalamus. Only in the hippocampus did the increase occur within 5 min of the seizure. 4 Similar increases in GABA concentration were seen after a bicuculline-induced seizure but not after seizure induced by flurothyl; both treatments produced a tonic-clonic seizure. 5 Pretreatment of the rats with (+)-propranolol 5 min before the ECS abolished the tonic extension and prevented the brain GABA concentration changes that occur 30 min after the seizure. 6 No increase in GABA concentration was seen in hippocampus, cortex and hypothalamus 30 min after the final ECS of a course of 10 ECS given once daily for 10 days. In contrast a marked increase in striatal GABA concentration was observed. 7 These changes in GABA biochemistry following a seizure are discussed in relation to the post-ictal rise in seizure threshold that is occurring at the same time.     

A steroid derivative,R 5135, antagonizes the GABA/benzodiazepine receptor interaction

A novel steroid derivative, R 5135 (3α-hydroxy-16-imino-5β-17-aza-androstan-11-one) showed high affinity for both the GABA and glycine receptors in vitro. It also displaced [³H]diazepam from the benzodiazepine receptor in a rat cortex membrane preparation, but in this case a plateau occurred in the displacement curve at a concentration of R 5135 between 10^?7 and 10^?6 M, where binding was decreased by about 50 %. The “high affinity” component of R 5135 inhibition no longer appeared when the endogenous GABA concentration was reduced by extensive washing of the membrane preparation and it reappeared when GABA was re-introduced. Thus R 5135 behaves as a powerful antagonist of the GABA stimulation of [³H]diazepam binding, being 500 times more active than the GABA-antagonist bicuculline. The dual interaction between R 5135 and GABA and glycine receptors suggests that these may share some common structural feature or that they have overlapping specificity.     

Aminooxyacetic acid induced accumulation of GABA in the rat brain

The effect of aminooxyacetic acid (AOAA, 90 mg/kg i.v.) on bicuculline, picrotoxin and 3-mercaptopropionic acid (3-MPA) induced convulsions and on GABA concentrations in cerebellum, whole brain and a synaptosomal fraction of whole brain was investigated. At various intervals after AOAA the rats were either injected with one of the convulsive drugs or sacrificed for analysis of the GABA concentration. AOAA caused a rapid initial (0-30 min) and a later slower increase of GABA in cerebellum and whole brain. In the synaptosomal fraction the GABA accumulation was delayed and less pronounced when compared to the whole brain. The bicuculline induced convulsions were markedly potentiated during the first hour but completely blocked from 2-6 h after AOAA. Picrotoxin showed a somewhat different pattern to bicuculline in the interactions with AOAA. The initial strong potentiation was not observed but the later phase of protection was present. In the interactions with 3-MPA, the effect of AOAA was always protective. The time to onset of convulsions was gradually increased during the first 30 min after AOAA. This protective effect remained practically unchanged up to 6 h after AOAA. However, once started, the convulsions were generally of the same duration and intensity. The results can be interpreted as GABA accumulating after AOAA stimulates GABA receptors to a degree more or less proportional to the whole brain GABA concentration and further that GABA synthetized in neurons is liberated, stimulates inhibitory bicuculline sensitive (predominant) and excitatory bicuculline insensitive receptors and is captured to a large extent by non-neuronal cells.     

Bicuculline-sensitive GABA receptors in rat ovary

A specific [3H]GABA binding site, characterized by a kinetic constant of 52 nM and by a maximal binding capacity of 17 fmol/mg protein was identified in a membrane preparation of rat ovary. [3H]GABA binding was displaced by muscimol, unlabelled GABA or bicuculline. The accumulation of cyclic AMP in the slices of rat ovary was strongly increased by GABA or muscimol and these effects were antagonized by picrotoxin or bicuculline. Our data suggest a possible role of GABA receptors in the regulation of ovarian function in the rat.     

Characteristics of type 1 and type 2 benzodiazepine receptors in the ovine brain

Analysis of the displacement of 3H-diazepam binding to membranes prepared from the ovine frontal cortex by the triazolopyradiazine CL218,872 yielded a Hill coefficient significantly below unity. By analogy with similar studies of this drug in rat brain this suggested the existence of Types 1 and 2 benzodiazepine receptors. The degree of displacement of 3H-diazepam by CL218,872 (200 nM, Type 1; 800 nM, Type 2) in homogenates of brain regions differed, the rank order being cerebellum greater than parietal cortex greater than frontal cortex congruent to temporal cortex congruent to hippocampus greater than striatum. Displacement of 3H-diazepam by CL218,872 was enhanced by 10(-5) M GABA in the striatum (at 200 nM and 800 nM CL218,872) and cerebellum (at 200 nM CL218,872). Benzodiazepine receptors in the ovine frontal cortex were least sensitive to CL218,872 (200 nM) in young fetuses (54-68 days gestation) and achieved adult levels of sensitivity by late gestation. Finally, the potency of CL218,872 to displace 3H-diazepam was not effected by the 3H-ligand concentration (0.5 nM or 5.0 nM), suggesting that Types 1 and 2 benzodiazepine receptors are not identical to the high and low affinity 3H-diazepam binding sites we have previously identified in the ovine brain.     

Characterization of L-[3H]glutamate binding sites in bovine brain coated vesicles.

Clathrin-coated vesicles isolated from bovine brain exhibit an L-[3H]glutamate-specific binding. Coated vesicles were purified from bovine brain by differential centrifugation and gel filtration. High purity of coated vesicles was established previously by several enzyme markers and electron microscopy. The binding activity was performed in the absence of Na+, Ca2+, and Cl- ions to avoid binding and/or uptake to uptake sites. Coated vesicles were frozen, thawed, treated with 0.04% Triton X-100 and washed before incubation with L-[3H]glutamate. Saturation binding experiments revealed a single binding site with a Kd = 439 +/- 87 nM and a Bmax = 11.74 +/- 3.4 pmol/mg protein, consistent with kinetics characteristic for glutamate receptors. The glutamate-specific binding was stereospecific for glutamate and aspartate, showing higher affinity for L-forms than D-forms. Pharmacological characterization indicated that specific binding was sensitive to quisqualate and almost insensitive to kainate and N-methyl-D-aspartate. 200 microM guanosine triphosphate (GTP) produced a decrease of 50% in L-[3H]glutamate binding activity and competition experiments produced an affinity shift to the right of the glutamate dose-response curve. These results support the evidence that glutamate receptors are present in bovine brain coated vesicles and, at least in part, are associated to a G-protein.     

Functionally relevant gamma-aminobutyric acidA receptors: equivalence between receptor affinity (Kd) and potency (EC50)?

Many neurotransmitter receptors bind agonists with high affinity (Kd in the nanomolar range), whereas micromolar concentrations of the same agonists are required to elicit a functional effect. We have identified low affinity agonist binding sites for the gamma-amino-butyric acidA (GABAA) receptor-chloride channel under conditions normally used in 36Cl- uptake assays (a measure of receptor function). The GABAA agonist [3H]muscimol bound to a population of receptors with a Kd (2 microM) similar to its EC50 value for 36Cl- uptake. Binding was inhibited by the GABA agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol and by the GABA antagonist bicuculline methiodide. A reduction in the number of [3H]muscimol binding sites (Bmax) by a thiol-modifying reagent produced a corresponding decrease in the Emax for muscimol. The benzodiazepine diazepam enhanced the potency of muscimol in ion flux experiments but did not alter the affinity of [3H]muscimol binding sites. We propose that benzodiazepines enhance GABAergic function by increasing receptor-ion channel coupling, rather than by increasing GABAA receptor affinity. These studies suggest that the study of physiologically relevant (low affinity) binding sites is necessary when examining regulation of receptors by cellular processes, drugs, and disease.     

Benzodiazepine binding sites in human pineal gland

The high affinity binding of [3H]flunitrazepam (FNZP) to crude membrane preparations was examined in human pineal glands. Scatchard analysis of the data at equilibrium revealed a single population of binding sites with dissociation constant = 2.36-2.53 nM and binding site concentration = 59-108 fmol/mg protein. When various benzodiazepine (BZP) analogues were tested for their ability to inhibit [3H]FNZP binding the following Ki (nM) were found: clonazepam (0.13), RO 15-1788 (0.60), FNZP (2.14), diazepam (13.5), Ro 5-4864 (greater than 10 000). In both human pineal gland and cerebral cortex 10-100 microM gamma-aminobutyric acid (GABA) increased BZP binding by about 30%, an effect inhibited by the GABA receptor blocker bicuculline. The stimulatory effect of GABA on [3H]FNZP binding in rat cerebral cortex (about 60%) decreased as a function of time elapsed postmortem at room temperature to reach values similar to those observed in human brains. These results suggest the existence of central type BZP receptors in the human pineal glands.     

Heterocyclic GABA analogues. Displacement of radiolabelled 3H-GABA from rat brain membrane preparations

2,5-Dimethyl-1H-pyrrol-1-yl-butanoic acid (I) and 2,5-diethyl-1H-pyrrol-1-yl-butanoic acid (V) were synthesised as non basic analogues of 4-aminobutanoic acid (GABA) to investigate the influence of the pKa of the 4-nitrogen on the in vitro binding to GABA receptors. (I) displaced 3H-GABA from specific binding sites of synaptosomal membrane preparations from rat cerebellum with an IC50 of 0.5 microM and (V) with an IC50 of 0.4 microM. (I) was inactive in vivo in the bicuculline anticonvulsant test (mice i.p.). The authors conclude that a basic nitrogen is not necessary for the binding to the GABAergic receptors although the ensuing complex is likely to be pharmacologically ineffective.     

Long-term treatment with different calcium- and calmodulin-antagonists induces changes in rat brain alpha-adrenoceptors.

1. The binding characteristics (Bmax and Kd) of the alpha-adrenoceptor radioligand [3H] WB4101 in crude membrane fraction (fraction P2) from cerebral cortex were studied after 13-day oral treatment of male Wistar rats with the Ca(2+)-antagonists nifedipine (20 mg/kg), verapamil (50 mg/kg), flunarizine (10 mg/kg) and with the calmodulin-antagonist trifluoperazine (TFP) (3 mg/kg). 2. A significant reduction of the binding sites (Bmax) for [3H] WB4101 was established after the three Ca(2+)-antagonists as well as after TFP treatment. 3. Different changes in the affinity constant (Kd) of brain adrenoceptors were observed depending on the type of the Ca2+ or CaM-antagonist used: nifedipine did not change the Kd value, verapamil and TFP decreased whereas flunarizine increased the Kd value. 4. Relationships between Ca ions and alpha-adrenoceptor functions are suggested.     

[3H]-lifarizine,a high affinity probe for inactivated sodium channels.

1. [3H]-lifarizine bound saturably and reversibly to an apparently homogeneous class of high affinity sites in rat cerebrocortical membranes (Kd = 10.7 +/- 2.9 nM; Bmax = 5.10 +/- 1.43 pmol mg-1 protein). 2. The binding of [3H]-lifarizine was unaffected by sodium channel toxins binding to site 1 (tetrodotoxin), site 3 (alpha-scorpion venom) or site 5 (brevetoxin), Furthermore, lifarizine at concentrations up to 10 microM had no effect on [3H]-saxitoxin (STX) binding to toxin site 1. Lifarizine displaced [3H]-batrachotoxinin-A 20-alpha-benzoate (BTX) binding with moderate affinity (pIC50 7.31 +/- 0.24) indicating an interaction with toxin site 2. However, lifarizine accelerated the dissociation of [3H]-BTX and decreased both the affinity and density of sites labelled by [3H]-BTX, suggesting an allosteric interaction with toxin site 2. 3. The binding of [3H]-lifarizine was voltage-sensitive, binding to membranes with higher affinity than to synaptosomes (pIC50 for cold lifarizine = 7.99 +/- 0.09 in membranes and 6.68 +/- 0.14 in synaptosomes). Depolarization of synaptosomes with 130 mM KCl increased the affinity of lifarizine almost 10 fold (pIC50 = 7.86 +/- 0.25). This suggests that lifarizine binds selectively to inactivated sodium channels which predominate both in the membrane preparation and in the depolarized synaptosomal preparation. 4. There was negligible [3H]-lifarizine and [3H]-BTX binding to solubilized sodium channels, although [3H]-STX binding was retained under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Equilibrium analysis of [3H]TCP binding: effects of glycine, magnesium and N-methyl-D-aspartate agonists

It has been reported that glutamate can increase the binding of [3H]TCP to phencyclidine (PCP) receptors by an action on receptors which are selective for N-methyl-D-aspartate (NMDA). Recently this laboratory has reported that glycine and magnesium can amplify this effect of NMDA agonists in well-washed, lysed cortical membranes. Here we report that maximally effective concentrations of glutamate (10 microM), NMDA (300 microM), MgCl2 (300 microM) and glycine (10 microM) increase the affinity of the PCP receptor for [3H]TCP by approximately 4-fold in the absence of any change in the density of PCP receptors. However, in combination with glutamate, magnesium had the further effect of increasing the Bmax by about 75%. Finally, a synaptosomal P2 preparation, which had not been washed to minimize the concentration of endogenous effectors had a Bmax value similar to the well-washed preparation, but had a KD value 8-fold lower. These data indicate that the primary effect of NMDA agonists, glycine, and low concentrations of magnesium ions is to convert the PCP receptor from a low-affinity to a high-affinity state. These data are discussed in relation to the functional regulation of the NMDA ionophore.     

Solubilization and characterization of GABAB receptor binding sites from porcine brain synaptic membranes.

1. The characteristics of membrane bound GABAB receptors in pig brain are similar to those in rat brain as judged by in vitro binding experiments and sensitivity to GTP. The rank order of affinity of GABAB receptor ligands was CGP 54626 > GABA approximately (-)-baclofen >> CGP 35348 = CGP 36742 > (+)-baclofen in membranes from both species. 2. For solubilization of GABAB receptors from pig brain, washed membranes were preincubated with 5 mM MgSO4 and subsequently incubated with various detergents. 3-[(3-Cholamidopropyl)dimethyl-ammoniol]-1-propane sulphonate (CHAPS) (0.5%) proved to be the most successful, solubilizing 22.7 +/- 4.7% (mean +/- s.e. mean, n = 6) of GABAB receptors. 3. Binding of [3H]-GABA to GABAB receptors solubilized with 0.5% CHAPS exhibited similar characteristics to the binding at membrane bound receptors since, firstly, the Kd and Bmax values (around 30 nM and 450 fmol mg-1 protein, respectively) were comparable; secondly, stereospecific binding for baclofen was obtained in both forms; thirdly, the affinity for the agonists GABA and (-)-baclofen and the antagonists CGP 35348, CGP 36742 and CGP 54626 were the same; fourthly, comparable sensitivity to Ca2+ (2.5 mM) was observed and finally, a similar sensitivity to GTP was apparent. 4. Saturation experiments performed with the GABAB antagonist, [3H]-CGP 54626, indicated a higher Kd value and a lower Bmax value for solubilized (7.7 +/- 2.6 nM and 1033 +/- 41 fmol mg-1 protein, mean +/- s.e. mean, n = 3) than for membrane bound receptors (1.35 +/- 0.08 nM, 1171 +/- 20 fmol mg-1 protein, n = 3).     

Radiation inactivation of brain [35S]t-butylbicyclophosphorothionate binding sites reveals complicated molecular arrangements of the GABA/benzodiazepine receptor chloride channel complex

[35S]t-Butylbicyclophosphorothionate ([35S]TBPS), a bicyclic cage convulsant, binds to the anion gating mechanism of the GABA/benzodiazepine receptor chloride channel complex. Using a carefully calibrated radiation inactivation technique, the molecular weight of [35S]TBPS binding complexes from frozen rat cerebral cortex was estimated to be 137,000 daltons. The GABA agonist muscimol reduced [35S]TBPS binding to 0-10% of the control value, in a way which is independent of the radiation dose. This shows that the GABA receptor (Mw = 55,000 daltons) is included in the 137,000-dalton [35S]-TBPS binding complex; the [35S]TBPS binding protein alone accounts for 137,000-55,000 = 82,000 daltons. The pyrazolopyridazine etazolate (SQ 20.009) and etomidate in appropriate concentrations both reduced specific binding of [35S]TBPS. The ability of SQ 20.009 and etomidate to reduce [35S]TBPS binding was greatly reduced by exposure to low radiation doses, suggesting that SQ 20.009 and etomidate reduce [35S]TBPS binding by an allosteric mechanism requiring a molecular structure of 450,000-500,000 daltons. Benzodiazepine agonists (ethyl 4-methoxymethyl-6-benzyloxy-beta-carboline-3-carboxylate, ZK 93423) and inverse agonists (methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, DMCM) enhance and reduce [35S]TBPS binding, respectively, in repeatedly frozen and washed membrane preparations. The effects of ZK 93423 and DMCM on [35S]TBPS binding disappeared upon exposure of membranes to low radiation doses. This suggests that the benzodiazepine receptor site interacts allosterically with the [35S]TBPS binding site, requiring a molecular complex of at least c. 400,000 daltons. The [35S]TBPS site alone in these latter conditions of membrane preparation (repeatedly frozen/washed) revealed a molecular weight of 221,000 daltons (TBPS-site + GABA receptor + unknown structures). The number of binding sites for [35S]TBPS (145 pmol/g tissue) was only slightly higher than for [3H]flunitrazepam (130 pmol/g tissue) in cerebral cortex. These results are all consonant with the conclusion that the GABA/BZ receptor chloride channel complex is composed of highly integrated multimeric subunits, tentatively accounted for by a tetramic complex of molecular weight 548,000 daltons.     

Influence of GABA receptor agonists and antagonists on the binding of 3H-diazepam to the benzodiazepine receptor

The GABA receptor agonists, GABA and muscimol, increased, while the GABA receptor antagonists, (+)-bicuculline, decreased tha affinity of the benzodiazepine receptor for ³H-diazepam. The effect was seen at both 0 and 25°C in spite of a large difference of affinity for ³H-diazepam at the two temperatures.