Autoradiographic Localization of [3H]Zolpidem Binding Sites in the Rat CNS: Comparison with the Distribution of [3H]Flunitrazepam Binding Sites

The regional distribution of [3H]zolpidem, a novel imidazopyridine hypnotic possessing preferential affinity for the BZD1 (benzodiazepine subtype 1) receptor, has been studied autoradiographically in the rat CNS and compared with that of [3H]flunitrazepam. The binding of [3H]zolpidem to rat brain sections was saturable, specific, reversible, and of high affinity (KD = 6.4 nM). It occurred at a single population of sites whose pharmacological characteristics were similar to those of the benzodiazepine receptors labeled with [3H]flunitrazepam. However, ethyl-beta-carboline-3-carboxylate and CL 218,872 were more potent displacers of [3H]zolpidem than of [3H]flunitrazepam. The autoradiographic brain distribution of [3H]zolpidem binding sites was qualitatively similar to that previously reported for benzodiazepine receptors. The highest levels of [3H]-zolpidem binding sites occurred in the olfactory bulb (glomerular layer), inferior colliculus, ventral pallidum, nucleus of the diagonal band of Broca, cerebral cortex (layer IV), medial septum, islands of Calleja, subthalamic nucleus, and substantia nigra pars reticulata, whereas the lowest densities were found in parts of the thalamus, pons, and medulla. Comparative quantitative autoradiographic analysis of the binding of [3H]zolpidem and [3H]flunitrazepam [a mixed BZD1/BZD2 (benzodiazepine subtype 2) receptor agonist] in the CNS revealed that the relative density of both 3H-labeled ligands differed in several brain areas. Similar levels of binding for both ligands were found in brain regions enriched in BZD1 receptors, e.g., substantia nigra pars reticulata, inferior colliculus, cerebellum, and cerebral cortex lamina IV. The levels of [3H]zolpidem binding were five times lower than those of [3H]flunitrazepam binding in those brain regions enriched in BZD2 receptors, e.g., nucleus accumbens, dentate gyrus, and striatum. Moreover, [3H]zolpidem binding was undetectable in the spinal cord (which contains predominantly BZD2 receptors). Finally, like CL 218,872 and ethyl-beta-carboline-3-carboxylate, zolpidem was a more potent displacer of [3H]flunitrazepam binding in brain regions enriched in BZD1 receptors than in brain areas enriched in BZD2 receptors. The present data add further support to the view that zolpidem, although structurally unrelated to the benzodiazepines, binds to the benzodiazepine receptor and possesses selectivity for the BZD1 receptor subtype.     

Early Undernutrition and [3H]γ-Aminobutyric Acid Binding in Rat Brain

The effect of early undernutrition and dietary rehabilitation on [3H]gamma-aminobutyric acid ([3H]GABA) binding in rat brain cerebral cortex and hippocampus was examined. Undernourished animals were obtained by exposing their mothers to a protein-deficient diet during both gestation and lactation. Saturation analysis of [3H]GABA binding in the cerebral cortex and hippocampus revealed high- and low-affinity components in the undernourished group, whereas control animals possessed only a low-affinity site. The concentration of low-affinity binding sites was greater in the undernourished animals. Rehabilitation of undernourished animals completely abolished the binding site differences. Treatment of brain membranes with Triton X-100 yielded two binding components in both the undernourished and control animals, although the concentration of lower affinity sites was still greater in the undernourished group. Neither the efficacy nor the potency of GABA to activate benzodiazepine binding in cerebral cortex was modified by undernutrition. These data suggest that early undernourishment modifies the characteristics of [3H]GABA binding, perhaps by reducing the brain content of endogenous inhibitors of the higher affinity binding site. The lack of effect on GABA-activated benzodiazepine binding suggests the possibility that neither the high- nor the low-affinity GABA binding sites are coupled to this receptor component.     

Zolpidem Displays Heterogeneity in Its Binding to the Nonhuman Primate Benzodiazepine Receptor In Vivo

Abstract: The distinctive pharmacological activity of zolpidem in rats compared with classical benzodiazepines has been related to its differential affinity for benzodiazepine receptor (BZR) subtypes. By contrast, in nonhuman primates the pharmacological activity of zolpidem was found to be quite similar to that of classical BZR agonists. In an attempt to explain this discrepancy, we examined the ability of zolpidem to differentiate BZR subtypes in vivo in primate brain using positron emission tomography. The BZRs were specifically labeled with [¹¹C]flumazenil. Radiotracer displacement by zolpidem was monophasic in cerebellum and neocortex, with in vivo Hill coefficients close to 1. Conversely, displacement of [¹¹C]flumazenil was biphasic in hippocampus, amygdala, septum, insula, striatum, and pons, with Hill coefficients significantly smaller than 1, suggesting two different binding sites for zolpidem. In these cerebral regions, the half-maximal inhibitory doses for the high-affinity binding site were similar to those found in cerebellum and neocortex and ～100-fold higher for the low-affinity binding site. The low-affinity binding site accounted for <32% of the specific [¹¹C]-flumazenil binding. Such zolpidem binding characteristics contrast with those reported for rodents, where three different binding sites were found. Species differences in binding characteristics may explain why zolpidem has a distinctive pharmacological activity in rodents, whereas its pharmacological activity in primates is quite similar to that of classical BZR agonists, except for the absence of severe effects on memory functions, which may be due to the lack of substantial zolpidem affinity for a distinct BZR subtype in cerebral structures belonging to the limbic system.     

Age-Related Loss of t-[35S]Butylbicyclophosphorothionate Binding to the γ-Aminobutyric AcidA Receptor-Coupled Chloride Ionophore in Rat Cerebral Cortex

Muscimol and t-butylbicyclophosphorothionate (TBPS) are known to label two distinct sites within the gamma-aminobutyric acidA (GABAA) receptor complex, i.e., the GABA recognition site and the chloride ionophore, respectively. Age-dependent changes in the specific binding of [3H]muscimol and [35S]TBPS were compared in membranes prepared from the cerebral cortex of rats, 2-800 days old. Perinatal (day 2) binding of muscimol and TBPS represented 8 and 20% of the respective values for adults (day 180). After the first week, muscimol binding increased more rapidly than TBPS binding. Levels near those of adults were reached at day 20 and remained practically unchanged in adulthood (day 180). In aged (780-day-old) rats, the binding of TBPS was significantly reduced, whereas muscimol binding did not change compared with adult values. This decrease of TBPS binding derived from a reduced density of binding sites, rather than from affinity changes. The allosteric responsiveness of TBPS binding to exogenous GABA was also reduced in aged animals. These findings indicate an age-related change in the molecular (structural) organization of the GABAA receptor-chloride ionophore complex in rat cerebral cortex.     

γ-Aminobutyric AcidB Receptor Activation Modifies Agonist Binding to β-Adrenergic Receptors in Rat Brain Cerebral Cortex

The interaction of isoproterenol with beta-adrenergic receptor (beta AR) binding sites was measured in membranes prepared from rat brain cerebral cortical slices previously incubated in the presence or absence of gamma-aminobutyric acid (GABA) receptor agonists. Both GABA and baclofen, but not isoguvacine, altered beta AR agonist binding by increasing the affinity of both the low- and high-affinity binding sites and by increasing the proportion of low-affinity receptors. The response to baclofen was stereoselective, and the effect of GABA was not inhibited by bicuculline. The results suggest that GABAB, but not GABAA, receptor activation modifies the coupling between beta AR and stimulatory guanine nucleotide-binding protein, which may in part explain the ability of baclofen to augment isoproterenol-stimulated cyclic AMP accumulation in brain slices.     

Ion and Temperature Effects on the Binding of γ-Aminobutyrate to Its Receptors and the High-Affinity Transport System

A set of procedures was developed to study the binding of gamma-[3H]aminobutyric acid ([3H]GABA) to GABAA and GABAB receptors, and to the Na(+)-dependent transport carrier, at 25 and 37 degrees C in the presence of physiological concentrations of Na+. The membrane preparation used in these procedures was not subjected to freeze-thawing or treatment with Triton X-100. Isoguvacine, (-)-baclofen, and (-)-nipecotate were used to block selectively the binding to GABAA receptors, GABAB receptors, and the transport site, respectively. Analysis of the binding characteristics of [3H]GABA to the GABAA receptor suggested the existence of high-(KD less than 30 nM), middle- (KD = 100-500 nM), and low-affinity (KD greater than 5 microM) binding sites. However, the binding data in the middle-affinity region (100-1,000 nM) were often indicative of cooperativity. The affinity between GABA and the GABAA receptor was reduced modestly by increases in temperature and by the presence of Cl- at physiological concentrations. Binding to the GABAB receptor required Ca2+ and Cl-. Apparent binding to the transport carrier required both Na+ and Cl-. A comparison of Bmax values in three brain regions revealed an inverse relationship between the high-affinity site of the GABAA receptor and the transport binding site.     

CL 218,872 Binding to Benzodiazepine Receptors in Rat Spinal Cord: Modulation by γ-Aminobutyric Acid and Evidence for Receptor Heterogeneity

The binding of the triazolopyridazine CL 218,872 to central benzodiazepine receptors identified with [3H]Ro 15-1788 was studied in extensively washed homogenates of rat spinal cord and cerebral cortex. CL 218,872 displacement curves were shallow in both spinal cord (nH = 0.67) and cortex (nH = 0.54), suggesting the presence of type 1 and type 2 benzodiazepine receptors in both tissues. CL 218,872 had lower affinity in spinal cord (IC50 = 825 nM) than cortex (IC50 = 152 nM), possibly reflecting the presence of fewer type 1 sites in the cord. Activating gamma-aminobutyric acid (GABA) receptors with 10 microM muscimol resulted in a two- to threefold increase in CL 218,872 affinity in both tissues without changes in the displacement curve slope. This indicates that GABA enhances CL 218,872 affinity for both type 1 and type 2 sites in both spinal cord and cerebral cortex.     

Type I and Type II γ-Aminobutyric Acid/Benzodiazepine Receptors: Purification and Analysis of Novel Receptor Complex from Neonatal Cortex

The gamma-aminobutyric acid (GABA) type A receptor was purified several thousandfold by affinity chromatography from rat cerebellum, adult cortex, and neonatal cortex. Competition for the benzodiazepine binding site by CL 218872 indicated that cerebellar receptors were predominantly type I, adult cortical receptors were a mixture of subtypes, and neonatal cortex was enriched in type II receptor. The receptor purified from neonatal cortex contained predominantly a 54-kilodalton (kDa), beta-subunit-like protein, whereas receptors from cerebellum and adult cortex contained nearly equal amounts of a 50-kDa, alpha-subunit-like protein and a 54-kDa polypeptide. Peptide maps of trypsin-digested 54-kDa subunits from cerebellum, adult cortex, and neonatal cortex exhibited very similar profiles, a result indicating considerable homology between these proteins in the receptor subtypes. A 59-kDa subunit protein was detected in the receptor complex purified from neonatal cortex. Like the 50-kDa, alpha-subunit of the type I receptor, this protein was photolabeled with [3H]flunitrazepam. The photolabeled peptide fragments, produced by trypsin digestion of these alpha 50- and alpha 59-subunits, exhibited the same retention times on reverse-phase HPLC. A less highly purified GABAA receptor preparation from adult rat spinal cord possessed characteristics that were very similar to those of the receptors purified from neonatal cortex.     

Increased γ-Aminobutyric Acid Receptor Function in the Cerebral Cortex of Myoclonic Calves with an Hereditary Deficit in Glycine/Strychnine Receptors

Inherited congenital myoclonus (ICM) of Poll Hereford cattle is a neurological disease in which there are severe alterations in spinal cord glycine-mediated neurotransmission. There is a specific and marked decrease, or defect, in glycine receptors and a significant increase in neuronal (synaptosomal) glycine uptake. Here we have examined the characteristics of the cerebral gamma-aminobutyric acid (GABA) receptor complex, and demonstrate that the malfunction of the spinal cord inhibitory system is accompanied by a change in the major inhibitory system in the cerebral cortex. In synaptic membrane preparations from ICM calves, both high-and low-affinity binding sites for the GABA agonist [3H]muscimol were found (KD = 9.3 +/- 1.5 and 227 +/- 41 nM, respectively), whereas only the high-affinity site was detectable in controls (KD = 14.0 +/- 3.1 nM). The density and affinity of benzodiazepine agonist binding sites labelled by [3H]diazepam were unchanged, but there was an increase in GABA-stimulated benzodiazepine binding. The affinity for t-[3H]butylbicyclo-o-benzoate, a ligand that binds to the GABA-activated chloride channel, was significantly increased in ICM brain membranes (KD = 148 +/- 14 nM) compared with controls (KD = 245 +/- 33 nM). Muscimol-stimulated 36Cl- uptake was 12% greater in microsacs prepared from ICM calf cerebral cortex, and the uptake was more sensitive to block by the GABA antagonist picrotoxin. The results show that the characteristics of the GABA receptor complex in ICM calf cortex differ from those in cortex from unaffected calves, a difference that is particularly apparent for the low-affinity, physiologically relevant GABA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of γ-Aminobutyric Acid/Benzodiazepine/β-Carboline Receptor Complex in Rat Spinal Cord

The properties of muscimol, beta-carboline (BC), and benzodiazepine (BZD) binding to crude synaptic membranes were studied in the spinal cord and cerebellum of rats. In cerebellar membranes, the density of high-affinity [3H]muscimol and [3H]6,7-dimethoxy-4-ethyl-beta-carboline ([3H]BCCM) binding sites is almost identical to that of [3H]flunitrazepam ([3H]FLU) or [3H]flumazenil (Ro 15-1788; ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a] [1-4]benzodiazepine-3-carboxylate). In contrast to the cerebellum, the number of muscimol and BC binding sites in rat spinal cord is approximately 20-25% of the number of FLU or flumazenil binding sites. Moreover, in spinal cord membranes, BC recognition site ligands displace [3H]-flumazenil bound to those sites, with low affinity and a Hill slope significantly less than 1; the potency of the different BCs in displacing [3H]flumazenil is 20-50-fold lower in the spinal cord than in the cerebellum. [3H]Flumazenil is not displaced from spinal cord membranes by the peripheral BZD ligand Ro 5-4864 (4'-chlorodiazepam), whereas it is displaced with low affinity and a Hill slope of less than 1 (nH = 0.4) by CL 218,872 (3-methyl-6-(3-trifluoromethylphenyl)-1,2,4-triazolol[4,3-b] pyridazine). These data suggest that a large number of BZD binding sites in spinal cord (approximately 80%) are of the central-type, BZD2 subclass, whereas the BZD binding sites in cerebellum are predominantly of the central-type, BZD1 subclass.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the Binding of [3H]SR 95531, a GABAA Antagonist, to Rat Brain Membranes

A synthetic derivative of gamma-aminobutyric acid (GABA), SR 95531 [2-(3'-carboxy-2'-propyl)-3-amino-6-p-methoxyphenylpyridazinium bromide], has recently been reported, on the basis of biochemical and in vivo microiontophoretic studies, to be a potent, selective, competitive, and reversible GABAA antagonist. In the present study, the binding of [3H]SR 95531 to washed, frozen, and thawed rat brain membranes was characterized. Specific binding was linear with tissue concentrations, had a pH optimum at neutrality, and was maximal at 4 degrees C after 30 min of incubation. Pretreatment of the membranes with Triton X-100 resulted in a 50% decrease of specific binding. Addition of iodide, thiocyanate, or nitrate to the incubation mixture decreased the affinity of [3H]SR 95531 for its binding site; Na+ had no effect. Subcellular fractionation showed that 74% of the P2 binding was in synaptosomes; 31% of the total homogenate binding was in P2 and 50% in P3. The binding of [3H]SR 95531 was saturable; Scatchard analysis of the saturation isotherm revealed two apparent populations of binding sites (KD of 6.34 nM and Bmax of 0.19 pmol/mg of protein; KD of 32 nM and Bmax of 0.81 pmol/mg of protein). The binding of [3H]SR 95531 was reversible, and association and dissociation kinetics confirmed the existence of two binding sites. Only GABAA ligands were effective displacers of [3H]SR 95531. GABAA antagonists were relatively more potent in displacing [3H]SR 95531 than [3H]GABA; the inverse was true for GABAA agonists. There were marked regional differences in the distribution of binding sites: hippocampus = cerebral cortex greater than thalamus = olfactory bulb = hypothalamus = amygdala = striatum greater than pons-medulla and cerebellum. The surprisingly low density of binding sites in the cerebellum was owing to a marked reduction of Bmax values at both the high- and the low-affinity binding sites. In conclusion, the present results demonstrate specific, high-affinity, saturable, and reversible binding of [3H]SR 95531 to rat brain membranes and strongly suggest that this radioligand labels the GABAA receptor site in its antagonist conformation.     

n-[3H]Butyl-β-Carboline-3-Carboxylate, a Putative Endogenous Ligand, Binds Preferentially to Subtype 1 of Central Benzodiazepine Receptors

Synthetic n-butyl beta-carboline-3-carboxylate, an endogenous central benzodiazepine receptor inhibitor found in brain, was tritium-labeled from the butenyl ester. Binding of this [3H]beta-carboline was concentrated particularly in the synaptosomal membrane fraction of the cerebral cortex; this fraction showed a single type of high-affinity site (KD = 2.7 +/- 0.1 nM) with a Bmax of 1.16 +/- 0.08 pmol/mg of protein. The number of sites labeled was about half of that obtained with [3H]flunitrazepam binding (Bmax = 2.36 +/- 0.06 pmol/mg of protein). On the other hand, in the cerebellum, both ligands bound to practically the same number of sites. When [3H]flunitrazepam binding was done in the presence of 10(-11)-10(-5) M butyl beta-carboline, the differences between the two brain regions were more apparent. In cerebellar membranes the data fitted a straight line in the Eadie-Hofstee plot; this finding and a Hill number near unity suggest a single type of binding site. In the cortical membranes the data of binding fitted a concave curve, and the Hill number was 0.6. These are characteristics of two types of binding sites with different affinities (KD1 = 0.6-1.5 nM and KD2 = 12-18 nM). The differentiation of a high- and low-affinity site in the cerebral cortex was corroborated by experiments in which [3H]butyl beta-carboline binding was displaced by the triazolopyridazine CL 218,872. These results demonstrate that in the cerebral cortex there are two subtypes of sites (1 and 2) of central benzodiazepine receptors and that CL 218,872 binds preferentially to subtype 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycine Binding to Rat Cortex and Spinal Cord: Binding Characteristics and Pharmacology Reveal Distinct Populations of Sites

Glycine is the principal inhibitory neurotransmitter in posterior regions of the brain. In addition, glycine serves as an allosteric regulator of excitatory neurotransmission mediated by the N-methyl-D-aspartate (NMDA) acidic amino acid receptor subtype. The studies presented here characterize [3H]glycine binding to washed membranes prepared from rat spinal cord and cortex, areas enriched in glycine inhibitory and NMDA receptors, respectively, in an attempt to define the glycine recognition sites on the two classes of receptors. Specific binding for [3H]glycine was seen in both cortex and spinal cord. Saturation analyses in cortex were best fitted by a two-site model with respective equilibrium dissociation constants (KD values) of 0.24 and 5.6 microM and respective maximal binding constants (Bmax values) of 3.4 and 26.7 pmol/mg of protein. Similar analyses in spinal cord were best fitted by a one-site model with a KD of 5.8 microM and Bmax of 20.2 pmol/mg of protein. Na+ had no effect on [3H]glycine binding to cortical membranes but increased the binding to spinal cord membranes by greater than 15-fold. This Na+-dependent binding may reflect glycine binding to the recognition site of the high-affinity, Na+-dependent glycine uptake system. Several short-chain, neutral amino acids displaced [3H]glycine binding from both cortical and spinal cord membranes. The most potent displacers of [3H]glycine binding to cortical membranes were D-serine and D-alanine, followed by the L-isomers of serine and alanine and beta-alanine. In contrast, D-serine and D-alanine were similar in potency to L-serine in spinal cord membranes. Compounds active at receptors for the acidic amino acids had disparate effects on the binding of [3H]glycine. At 10 microM, NMDA resulted in a 25% increase, whereas D- and L-2-amino-5-phosphonovaleric acid at 100 microM resulted in a 30% decrease, in [3H]glycine binding to cortical membranes. Kynurenic acid was the most potent of the acidic amino acid-related compounds at displacing [3H]glycine binding. In cortical membranes, kynurenic acid displacement was resolved into a high- and a low-affinity component; the high-affinity component displaced the high-affinity component of [3H]glycine binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regional Variations in α1-Adrenergic Receptor Subtypes in Rat Brain

alpha 1-Adrenergic receptor subtypes were differentiated by their affinities for the competitive antagonist WB 4101 and their sensitivities to inactivation by chlorethylclonidine (CEC) in eight rat brain regions. WB 4101 showed low Hill coefficients for inhibition of specific 125I-[2-beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone (125IBE) binding in all regions. Nonlinear regression analysis showed that there were two binding sites with different affinities for WB 4101 in each region. The proportions of these sites varied among regions, although the affinity of WB 4101 for each site remained constant. Thalamus and cerebral cortex had the highest proportion of low-affinity sites, whereas hippocampus and pons-medulla had the highest proportion of high-affinity sites. Pretreatment with CEC in hypotonic buffer significantly reduced the density of 125IBE binding sites in all brain regions. Cerebral cortex and cerebellum had the highest proportion of CEC-sensitive sites, whereas hippocampus and spinal cord had the highest proportion of CEC-insensitive sites. There was a significant correlation between the proportion of binding sites with a low affinity for WB 4101 and those sensitive to inactivation by CEC.     

γ-Aminobutyric AcidA Receptor α5-Subunit Creates Novel Type II Benzodiazepine Receptor Pharmacology

A cDNA encoding a protein with 70% amino acid identity to the previously characterized gamma-aminobutyric acidA (GABAA) receptor alpha-subunits was isolated from a rat brain cDNA library by homology screening. As observed for alpha 1-, alpha 2-, and alpha 3-subunits, coexpression of this new alpha-subunit (alpha 5) with a beta- and gamma 2-subunit in cultured cells produces receptors displaying high-affinity binding sites for both muscimol, a GABA agonist, and benzodiazepines. Characteristic of GABAA/benzodiazepine type II sites, receptors containing alpha 2-, alpha 3- or alpha 5-subunits have low affinities for several type I-selective compounds. However, alpha 5-subunit-containing receptors have lower affinities for zolpidem (30-fold) and Cl 218 872 (three-fold) than measured previously using recombinantly expressed type II receptors containing either alpha 2- or alpha 3-subunits. Based on these findings, a reclassification of the GABAA/benzodiazepine receptors is warranted.     

Dissociation of Muscimol, SR 95531, and Strychnine from GABAA and Glycine Receptors, Respectively, Suggests Similar Cooperative Interactions

Binding to gamma-aminobutyric acid-A (GABAA) receptors was studied in synaptosomal membranes of rat brain. Dissociation of [3H]muscimol and the GABAA antagonist [3H]2-(3-carboxypropyl)-3-amino-6-p-methoxyphenylpyridazinium bromide ([3H]SR 95531) binding elicited by 100-fold dilution was accelerated by excess of GABA or SR 95531. Control dissociation might be retarded by rebinding. The contribution of a rapid first phase of dissociation of the agonist [3H]muscimol was preferentially enhanced by SR 95531. In contrast, the dissociation of [3H]SR 95531 binding was preferentially accelerated by GABA. These opposite preferential accelerations can be explained by negative heterotropic cooperativity and a reversed affinity relationship of agonists and antagonists to GABAA binding sites with different affinities. Modification of the membranes by p-diazobenzenesulfonic acid (DSA) selectively decreased the accelerating effect of GABA on the dissociation of [3H]SR 95531 binding. [3H]Strychnine binding was studied in a membrane preparation of rat spinal cord. The dissociation of the antagonist [3H]strychnine elicited by dilution was preferentially accelerated by glycine. Again, pretreatment with DSA decreased selectively this negative heterotropic (i.e., allosteric) interaction. Chemical modification by DSA might be attributed to tyrosine residues responsible for similar allosteric interactions for the GABA- and glycine-gated chloride channels.     

Antibodies Recognising the GABAA/Benzodiazepine Receptor Including Its Regulatory Sites

Polyclonal antibodies have been raised against the GABA/benzodiazepine receptor purified to homogeneity from bovine cerebral cortex in deoxycholate and Triton X-100 media. Radioimmunoassay was applied to measure specific antibody production using the 125I-labelled gamma-aminobutyric acid (GABA)/benzodiazepine receptor as antigen. The antibodies specifically immunoprecipitated the binding sites for [3H]muscimol and for [3H]flunitrazepam from purified preparations. In addition, when a 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulphonate (CHAPS) extract of bovine brain membranes was treated with the antibodies, those sites as well as the [3H]propyl-beta-carboline-3-carboxylate binding, the [35S]t-butylbicyclophosphorothionate binding (TBPS), the barbiturate-enhanced [3H]flunitrazepam binding, and the GABA-enhanced [3H]flunitrazepam binding were all removed together into the immunoprecipitate. Western blot experiments showed that these antibodies recognise the alpha-subunit of the purified GABA/benzodiazepine receptor. These results further support the existence in the brain of a single protein, the GABAA receptor, containing a set of regulatory binding sites for benzodiazepines and chloride channel modulators.     

Photoaffinity Labeling of Benzodiazepine Receptors in Rat Brain with Flunitrazepam Alters the Affinity of Benzodiazepine Receptor Agonist But Not Antagonist Binding

Abstract: Recently, it was proposed that β-carbolines interact with a subset of benzodiazepine (BZD) binding sites in mouse brain. This postulate was based upon evidence showing changes in binding properties of the BZD receptor following photoaffinity labeling of membranes with flunitrazepam (FLU). Under conditions in which 80% of specific [³H]diazepam binding was lost in photolabeled membranes, specific [³H]propyl β-carboline-3-carboxylate ([³H]PCC) binding was spared. In this study, the binding of the BZD antagonists [³H]PCC, [³H]Ro15 1788 and [³H]CGS 8216 was examined in rat brain membranes following photoaffinity labeling with FLU. No significant changes in the apparent K_D and small reductions in the B_max of ³H antagonist binding were observed. However, in the same membranes, up to 89% of specific [³H]FLU binding was lost. When [³H]PCC (0.05 nM) was used to label the receptors in control and photolabeled membranes, the ability of BZD receptor agonists to inhibit [³H]PCC binding was greatly diminished in the photolabeled membranes. In contrast, the potency of BZD antagonists remained the same in both control and treated membranes. Based upon PCC/[³H]Ro15 1788 competition experiments, the ability of PCC to discriminate between BZD receptor subtypes was unaffected by photoaffinity labeling of cortical membranes. Overall, these findings suggest that β-carbolines do not interact with a subset of BZD binding sites per se, but may be a consequence of the differential interaction of BZD agonists and antagonists with BZD binding sites that have been photoaffinity labeled with FLU. A possible mechanism underlying this phenomenon is discussed. The ability of photolabeled membranes to differentiate between BZD agonists and antagonists provides a potential screen for agonist and antagonist activity in compounds that interact with the BZD receptor.     

Competitive Inhibition of γ-Aminobutyric Acid Receptor Binding by N-2-Hydroxyethylpiperazine-N''-2- Ethanesulfonic Acid and Related Buffers

Several Good buffers (MOPS, ACES, BES, HEPES, ADA, and PIPES) competitively inhibited both high-affinity and low-affinity [3H]gamma-aminobutyric acid receptor binding to rat brain synaptic membranes. The most potent inhibitor was MOPS, which had Ki values of 180 nM and 79 nM for the high- and low-affinity binding sites, respectively. HEPES had Ki values of 2.25 mM and 115 microM. The buffers had no appreciable effect on sodium-dependent GABA binding or on gamma-aminobutyrate aminotransferase activity. Surprisingly, the buffers were extremely ineffectual as inhibitors of either high- or low-affinity [3H]muscimol binding. Indeed, they were of the order of 10(5) times less effective in this case than against [3H]GABA binding. These results clearly show (a) that the use of such buffers as MOPS or HEPES should be avoided in studying the interaction of GABA with its receptor, and (b) the binding sites of [3H]GABA and [3H]muscimol are not identical.     

Neuronal [3H]Benzodiazepine Binding and Levels of GABA, Glutamate, and Taurine Are Normal in Huntington''s Disease Cerebellum

Alterations in one subunit of the proposed GABA receptor complex, namely, the GABA receptor, have been observed in Huntington's disease cerebellum. We measured binding to a second subunit, the benzodiazepine binding site, in the autopsied cerebellum of 12 patients dying with adult-onset Huntington's disease. Neuronal benzodiazepine ([3H]flunitrazepam) binding density (Bmax) and affinity in cerebellar cortex of the Huntington's disease patients were not significantly different from control values. Similarly, maximal GABA stimulation of benzodiazepine binding was normal in the Huntington's disease cerebellum. In addition, no significant changes were observed in the concentrations of GABA, glutamate, and taurine in cerebellar cortex, nor of GABA in the dentate nucleus.