Benzodiazepine antagonist Ro 15-1788: binding characteristics and interaction with drug-induced changes in dopamine turnover and cerebellar cGMP levels

The recently discovered benzodiazepine antagonist Ro 15-1788 was characterized in binding studies, and its potency and selectivity were determined in vivo by interaction with drug-induced changes in dopamine turnover and cerebellar cGMP level. Ro 15-1788 reduced [3H]flunitrazepam binding in the brain in vivo with a potency similar to that of diazepam and effectively inhibited [3H]diazepam binding in vitro (IC50 = 2.3 +/- 0.6 nmol/liter). [3H]Ro 15-1788 bound to tissue fractions of rat cerebral cortex with an apparent dissociation (KD) of 1.0 +/- 0.1 nmol/liter. The in vitro potency of various benzodiazepines in displacing [3H]Ro 15-1788 from its binding site was of the same rank order as found previously in [3H]diazepam binding. Autoradiograms of [3H]Ro 15-1788 binding in sections of rat cerebellum showed the same distribution of radioactivity as with [3H]flunitrazepam. The attenuating effect of diazepam on the chlorpromazine- or stress-induced elevation of homovanillic acid in rat brain was antagonized by Ro 15-1788. Among a series of compounds which either decreased or increased the rat cerebellar cGMP level, only the effect of benzodiazepine receptor ligands (diazepam, zopiclone, CL 218 872) was antagonized by Ro 15-1788. Thus, Ro 15-1788 is a selective benzodiazepine antagonist acting at the level of the benzodiazepine receptor in the central nervous system. Peripheral benzodiazepine binding sites in kidney and schistosomes were not affected by Ro 15-1788.     

Benzodiazepine Antagonist Ro 15–1788: Binding Characteristics and Interaction with Drug-Induced Changes in Dopamine Turnover and Cerebellar cGMP Levels

Abstract: The recently discovered benzodiazepine antagonist Ro 15-1788 was characterized in binding studies, and its potency and selectivity were determined in vivo by interaction with drug-induced changes in dopamine turnover and cerebellar cGMP level. Ro 15-1788 reduced [³H]flunitrazepam binding in the brain in vivo with a potency similar to that of diazepam and effectively inhibited [³H]diazepam binding in vitro (IC₅₀= 2.3 ± 0.6 nmol/liter). [³H]Ro 15-1788 bound to tissue fractions of rat cerebral cortex with an apparent dissociation constant ( K _D) of 1.0 ± 0.1 nmol/liter. The in vitro potency of various benzodiazepines in displacing [³H]Ro 15-1788 from its binding site was of the same rank order as found previously in [³H]diazepam binding. Autoradiograms of [³H]Ro 15-1788 binding in sections of rat cerebellum showed the same distribution of radioactivity as with [³H]flunitrazepam. The attenuating effect of diazepam on the chlorpromazine- or stress-induced elevation of homovanillic acid in rat brain was antagonized by Ro 15-1788. Among a series of compounds which either decreased or increased the rat cerebellar cGMP level, only the effect of benzodiazepine receptor ligands (diazepam, zopiclone, CL 218 872) was antagonized by Ro 15-1788. Thus, Ro 15-1788 is a selective benzodiazepine antagonist acting at the level of the benzodiazepine receptor in the central nervous system. Peripheral benzodiazepine binding sites in kidney and schistosomes were not affected by Ro 15-1788.     

3H]Ro 15-1788 binding sites to brain membrane of the saltwater Mugil cephalus

The equilibrium binding parameters of the benzodiazepine antagonist [3H]Ro 15-1788 (8-fluoro-3-carboethoxy-5,6-dihydro-5-methyl-6-oxo-4H-imidazol-[1,5-a]-1,4 benzodiazepine) were evaluated in brain membranes of the saltwater teleost fish, Mugil cephalus. To test receptor subtype specificity, displacement studies were carried out by competitive binding of [3H]Ro 15-1788 against six benzodiazepine receptor ligands, flunitrazepam [5-(2-fluoro-phenyl)-1,3-dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepin-2-one], alpidem [N,N-dipropyl-6-chloro-2-(4-chlorophenyl)imidazo[1,2-a]pyridine-3-acetamide], zolpidem [N,N-6 trimethyl-2-(4-methyl-phenyl)imidazo[1,2-a]pyridine-3-acetamide hemitartrate], and beta-CCM (methyl beta-carboline-3-carboxylate). Saturation studies showed that [3H]Ro 15-1788 bound saturatably, reversibly and with a high affinity to a single class of binding sites (Kd value of 1.18-1.5 nM and Bmax values of 124-1671 fmol/mg of protein, depending on brain regions). The highest concentration of benzodiazepine recognition sites labeled with [3H]Ro 15-1788 was present in the optic lobe and the olfactory bulb and the lowest concentration was found in the medulla oblongata, cerebellum and spinal cord. The rank order of displacement efficacy of unlabelled ligands observed suggested that central-type benzodiazepine receptors are present in one class of binding sites (Type I-like) in brain membranes of Mugil cephalus. Moreover, the uptake of 36Cl- into M. cephalus brain membrane vesicles was only marginally stimulated by concentrations of GABA that significantly enhanced the 36Cl- uptake into mammalian brain membrane vesicles. The results may indicate a different functional activity of the GABA-coupled chloride ionophore in the fish brain as compared with the mammalian brain.     

Subcellular Localization of "Peripheral-Type" Binding Sites for Benzodiazepines in Rat Brain

The binding of [3H]Ro 5-4864, a specific ligand for "peripheral-type" benzodiazepine binding sites and [3H]Ro 15-1788, a specific ligand for the central benzodiazepine receptors, was determined in subcellular fractions of rat brain. As previously reported, the highest levels of "peripheral-type" benzodiazepine binding sites and benzodiazepine receptors were found in the crude P1 and P2 fractions, respectively. Purification of these crude fractions revealed that high levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding were present in the mitochondrial and synaptosomal fractions. In contrast, the purified nuclei and myelin contained low levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding.     

Quantitative Immunoprecipitation Studies with Anti-γ-Aminobutyric AcidA Receptor γ2 1–15 Cys Antibodies

Antibodies raised against the synthetic peptide NH2-QKSDDDYEDYASNKTC-COOH (gamma 2 1-15 Cys), which corresponds to the N-terminal amino acid sequence with a C-terminal cysteine of the human gamma 2 subunit of the gamma-aminobutyric acidA (GABAA) receptor, were used to study the quantitative immunoprecipitation of agonist benzodiazepine binding sites from bovine brain. Anti-gamma 2 1-15 Cys antibodies were found to immunoprecipitate specifically in parallel [3H]flunitrazepam- and [3H]muscimol-reversible binding sites in a dose-dependent manner. The maximum percentages of [3H]flunitrazepam binding sites immunoprecipitated from detergent extracts of bovine cerebral cortex, cerebellum, and hippocampus were 68, 77, and 83%, respectively. Immunoprecipitation studies with anti-alpha 1 324-341 antibodies carried out in parallel with anti-gamma 2 1-15 Cys antibodies provided evidence for the promiscuity of the gamma 2 subunit within native GABAA receptors. These results substantiate the association of the gamma 2 polypeptide with native GABAA receptors.     

Amoxapine inhibition of GABA-stimulated chloride conductance: investigations of potential sites of activity

Amoxapine inhibits GABA-stimulated chloride conductance by acting on the GABAA-receptor chloride-ionophore complex which can be studied using membrane vesicles prepared from rat cerebral cortex. Amoxapine produces a right shift in the GABA concentration-response curve for the stimulation of 36Cl- uptake into these vesicles with no apparent change in the maximum response. Schild analysis of these data gave a pA2 value of 5.52 with a slope of 0.79. Amoxapine inhibits the binding of the GABAA receptor selective antagonist [3H]SR 95531 with an IC50 value of 3.45 microM and a pseudo Hill coefficient of 0.83. In contrast, 10 microM amoxapine inhibits [3H]flunitrazepam binding by less than 25% while the benzodiazepine antagonist Ro 15-1788 reduces the amoxapine inhibition of GABA-stimulated chloride conductance only at high concentrations. These data suggest that amoxapine does not inhibit chloride conductance by acting as a benzodiazepine inverse agonist and either acts directly on the GABAA receptor as an antagonist or blocks GABA activity at a site closely coupled to it. The ability of amoxapine to inhibit GABA-stimulated chloride conductance is a likely explanation for its proconvulsant activity observed at high doses.     

Photoaffinity Labeling of Benzodiazepine Receptor Proteins with the Partial Inverse Agonist [3H]Ro 15–4513: A Biochemical and Autoradiographic Study

Photolabeling of the benzodiazepine receptor, which to date has been done with benzodiazepine agonists such as flunitrazepam, can also be achieved with Ro 15-4513, a partial inverse agonist of the benzodiazepine receptor. [3H]Ro 15-4513 specifically and irreversibly labeled a protein with an apparent molecular weight of 51,000 (P51) in cerebellum and at least two proteins with apparent molecular weights of 51,000 (P51) and 55,000 (P55) in hippocampus. Photolabeling was inhibited by 10 microM diazepam but not by 10 microM Ro 5-4864. The BZ1 receptor-selective ligands CL 218872 and beta-carboline-3-carboxylate ethyl ester preferentially inhibited irreversible binding of [3H]Ro 15-4513 to protein P51. Not only these biochemical results but also the distribution and density of [3H]Ro 15-4513 binding sites in rat brain sections were similar to the findings with [3H]flunitrazepam. Thus, the binding sites for agonists and inverse agonists appear to be located on the same proteins. In contrast, whereas [3H]flunitrazepam is known to label only 25% of the benzodiazepine binding sites in brain membranes, all binding sites are photolabeled by [3H]Ro 15-4513. Thus, all benzodiazepine receptor sites are associated with photolabeled proteins with apparent molecular weights of 51,000 and/or 55,000. In cerebellum, an additional protein (MW 57,000) unrelated to the benzodiazepine receptor was labeled by [3H]Ro 15-4513 but not by [3H]flunitrazepam. In brain sections, this component contributed to higher labeling by [3H]Ro 15-4513 in the granular than the molecular layer.     

Effect of some potent adenosine uptake inhibitors on benzodiazepine binding in the CNS

A series of nucleoside transport inhibitors has been tested for their ability to displace [³H]diazepam binding to CNS membranes. No correlation between their potency as [³H]adenosine uptake blockers and as inhibitors of [³H]diazepam binding was found, either in rat or guinea-pig brain tissue. Dipyridamole, a potent adenosine transport inhibitor interacted strongly (K_i = 54 nM) with peripheral-type benzodiazepine binding sites (“acceptor sites”) and was 4–5 fold weaker in displacing [³H]methylclonazepam and [³H]Ro15-1788, ligands selective for the specific central benzodiazepine “receptor”. Unlike the benzodiazepines, dipyridamole had no anticonvulsant action against metrazole-induced convulsions in mice. Ro5-4864, a benzodiazepine which selectively interacts with the peripheral-type benzodiazepine binding site, was approximately equipotent with diazepam in inhibiting [³H]adenosine uptake in brain tissue. These results do not support the idea of a very close link between high-affinity central binding sites for clinically-active benzodiazepines and the adenosine uptake site. The possibility of a connection between benzodiazepine “acceptor” sites and the membrane nucleoside transporter is discussed.     

Benzodiazepine Receptor Binding of Triazolobenzodiazepines In Vivo: Increased Receptor Number with Low-Dose Alprazolam

Abstract: Triazolobenzodiazepines are in clinical use as hypnotics and anxiolytics. We analyzed in vivo receptor binding and brain concentrations of alprazolam, triazolam, and estazolam. Drug concentrations measured in the cerebral cortex 1 h after administration were directly proportional to dose for all three compounds. In vivo receptor binding, as defined by the specific uptake of [³H]Ro 15–1788, decreased with increasing doses of estazolam and triazolam, a finding indicating dose-related increases in receptor occupancy due to these compounds. Triazolam was substantially more potent, with an IC₅₀ value of 16 ng/g, compared with 117 ng/g for estazolam. At higher doses of alprazolam (>0.2 mg/kg), receptor binding by [³H]Ro 15–1788 likewise decreased with increasing dose of the former drug. However, at lower doses of alprazolam (0.02–0.05 mg/kg), which resulted in cortex concentrations of 2–7 ng/g, receptor binding was increased above control values in cortex, hypothalamus, and hippocampus but not in several other brain regions. Binding returned to control values at doses of ≤0.01 mg/kg. Similar results were obtained in time course studies. At 8 and 10 h after a dose of 1 mg/kg i.p., corresponding to cortex concentrations of 2.7–7 ng/g, receptor binding was increased compared with controls. Similarly, at 1, 2, and 3 h after a single dose of 0.05 mg/kg, corresponding to cortex concentrations of 3.7–5.8 ng/g, receptor binding was also increased. The apparent affinity of benzodiazepine receptors for clonazepam in mice receiving alprazolam (0.05 mg/kg) was unchanged from that in untreated control mice, an observation suggesting that low doses of alprazolam increased receptor number. The brain concentration vs. receptor occupancy relationships for triazolam and estazolam resemble those for other benzodiaze-pines, but alprazolam appears to be anomalous in that low brain concentrations increase benzodiazepine receptor number.     

Benzodiazepine Binding Characteristics of Embryonic Rat Brain Neurons Grown in Culture

The binding of [3H]diazepam to cell homogenates of embryonic rat brain neurons grown in culture was examined. Under the conditions used to prepare and maintain these neurons, only a single, saturable, high-affinity binding site was observed. The binding of [3H]diazepam was potently inhibited by the CNS-specific benzodiazepine clonazepam (Ki = 0.56 +/- 0.08 nM) but was not affected by the peripheral-type receptor ligand Ro5-4864. The KD for [3H]diazepam bound specifically to cell homogenates was 2.64 +/- 0.24 nM, and the Bmax was 952 +/- 43 fmol/mg of protein. [3H]Diazepam binding to cell membranes washed three times was stimulated dose-dependently by gamma-aminobutyric acid (GABA), reaching 112 +/- 7.5% above control values at 10(-4) M. The rank order for potency of drug binding to the benzodiazepine receptor site in cultured neurons was clonazepam greater than diazepam greater than beta-carboline-3-carboxylate ethyl ester greater than Ro15-1788 greater than CL218,872 much greater than Ro5-4864. The binding characteristics of this site are very similar to those of the Type II benzodiazepine receptors present in rat brain. These data demonstrate that part, if not all, of the benzodiazepine-GABA-chloride ionophore receptor complex is being expressed by cultured embryonic rat brain neurons in the absence of accompanying glial cells and suggest that these cultures may serve as a model system for the study of Type II benzodiazepine receptor function.     

γ-Aminobutyric AcidA Receptor Pharmacology in Rat Cerebral Cortical Synaptoneurosomes

Equilibrium binding interactions at the gamma-aminobutyric acid (GABA) and benzodiazepine recognition sites on the GABAA receptor-Cl- ionophore complex were studied using a vesicular synaptoneurosome (microsacs) preparation of rat brain in a physiological HEPES buffer similar to that applied successfully in recent GABAergic 36Cl- flux measurements. NO 328, a GABA reuptake inhibitor, was included in the binding assays to prevent the uptake of [3H]muscimol. Under these conditions, the equilibrium dissociation constant (KD) values for [3H]muscimol and [3H]diazepam bindings are 1.9 microM and 40 nM, respectively. Binding affinities for these and other GABA and benzodiazepine agonists and antagonists correlate well with the known physiological doses required to elicit functional activity. This new in vitro binding protocol coupled with 36Cl- flux studies should prove to be of value in reassessing the pharmacology of the GABAA receptor complex in a more physiological environment.     

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.     

Purification and Characterization of Naturally Occurring Benzodiazepine Receptor Ligands in Rat and Human Brain

Chemicals that are active at the benzodiazepine receptor (endozepines) are naturally present in the CNS. These substances are present in tissue from humans and animals and in plants and fungi. Using selective extraction protocols, HPLC purification, receptor binding displacement studies, and selective anti-benzodiazepine antibodies, we have identified six or seven peaks of endozepines in rat and human brain. All material could competitively displace [3H]flunitrazepam binding to cerebellar benzodiazepine binding sites. Two peaks also competitively displaced Ro 5-4864 binding to the mitochondrial benzodiazepine binding site. Total amounts of brain endozepines were estimated to be present in potentially physiological concentrations, based on their ability to displace [3H]flunitrazepam binding. Although endozepine peaks 1 and 2 had HPLC retention profiles similar to those of nordiazepam and diazepam, respectively, gas chromatography-mass spectrometry as well as high-performance TLC revealed biologically insignificant amounts of diazepam (less than 0.02 pg/g) and nordiazepam (less than 0.02 pg/g) in the purified material. Electrophysiologically, some purified endozepines positively modulated gamma-aminobutyric acid (GABA) action on Cl- conductance, monitored in patch-clamped cultured cortical neurons or in mammalian cells transfected with cDNA encoding various GABAA receptor subunits. These studies demonstrate that mammalian brains contain endozepines that could serve as potent endogenous positive allosteric modulators of GABAA receptors.     

Kinetics of [3H]Ro 15–1788 Binding to Membrane-Bound Rat Brain Benzodiazepine Receptors

Abstract: Ro 15–1788 is thought to interact specifically with the benzodiazepine receptor population in the mammalian CNS as an antagonist. We have compared the kinetics of interaction of this ligand with the benzodiazepine agonist flunitrazepam in the rat cerebellum. The association of [³H]Ro 15–1788 with the benzodiazepine receptor in this brain region is monoexponential, and the dissociation, initiated either by dilution or by displacement with a number of different ligands, also obeys simple monoexponential kinetics. There is no evidence of cooperative interactions with this ligand, and its dissociation is unaffected by the presence of 100 μ M γ-aminobutyric acid and/or 150 m M sodium chloride. In contrast, the dissociation of the agonist [³H]flunitrazepam is biphasic, and the possible interpretation of this data in terms of agonist-induced conformational change is discussed. Evidence is also presented that the mechanism of interaction of Ro 15–1788 with the benzodiazepine receptor population in hippocampal membranes is distinct from that found in the cerebellum.     

Effects of Guanyl Nucleotides on [3H]Flunitrazepam Binding to Rat Hippocampal Synaptic Membranes: Equilibrium Binding and Dissociation Kinetics

The effects of guanyl nucleotides on the binding of [3H]flunitrazepam to rat hippocampal synaptic membranes were studied. In equilibrium binding studies, gamma-amino-n-butyric acid (GABA) increased and GTP decreased the binding affinity of [3H]flunitrazepam; GTP also caused a decrease in binding capacity. The effect, however, is variable. In studies of the dissociation kinetics of [3H]flunitrazepam using diazepam and the antagonist Ro 15-1788 as the displacers, there was evidence of two dissociation rate constants. GTP increased both the fast- and slow-dissociation rate constants and increased the ratio of the slow-dissociation binding state. The effect of GTP was mimicked by its nonhydrolyzable analogue 5'-guanylylimidodiphosphate but not by ATP and occurred when diazepam, but not when Ro 15-1788, was used as the displacer. GABA antagonized the effect of GTP on the dissociation of [3H]flunitrazepam. The nature of the benzodiazepine receptor, its actions, and the possible role of cyclic AMP as a second messenger are discussed.     

Nonuniform effect of prolonged haloperidol administration on the GABA(A) and benzodiazepine receptors in different parts of the brain

The experiments on male mice and rats have revealed reversed behavioral effects of muscimol and Ro 15-1788 after 15 days of haloperidol (0.25 mg/kg, twice daily) treatment. Muscimol (0.75 mg/kg), which depressed motor activity in saline-pretreated mice, stimulated it after discontinuation of long-term haloperidol administration. Ro 15-1788 stimulating effect in saline-pretreated rats gave way to sedative effect following haloperidol withdrawal. Simultaneously, the number of 3H-muscimol and 3H-flunitrazepam binding sites was decreased in forebrain, but increased in hindbrain. It was suggested that GABAA and benzodiazepine receptors in forebrain and hindbrain play opposite (inhibiting and stimulating, respectively) functional roles in the regulation of behaviour.     

Binding of the benzodiazepine ligand [H]-Ro 15–1788 to brain membrane of the saltwater fish Mullus surmuletus

The distribution and the pharmacological properties of the binding of the benzodiazepine receptor antagonist [³H]-Ro 15–1788 (8-fluoro-3-carboethoxy-5,6-dihydro-5-methyl-6-oxo-4H imidazol [1,5-a] 1,4 benzodiazepine) were compared in some brain membranes of the saltwater teleost fish, Mullus surmuletus: only a single population of [³H]-Ro 15–1788 binding sites was detected. The binding was saturable and reversible with a high affinity, revealing a significant population of binding sites (K_d value of 2.1 ± 0.2 nM and B_max value of 1400-900 fmol mg⁻¹ of protein, depending on fish length). The highest concentration of benzodiazepine recognition sites labelled with [³H]-Ro 15–1788 was present in the optic lobe and the olfactory bulb and the lowest concentration was found in the medulla oblongata, cerebellum and spinal cord. In order to explore behavioural selectivity as a consequence of multiple receptor subtypes, six benzodiazepine receptor ligands, flunitrazepam (5-(2-fluoro-phenyl)-1,3,dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepine-2-one), alpidem, (N,N-dipropyl-6-chloro-2-(4-chlorophenyl) imidazo [1,2-a] pyridine-3-acetamide) zolpidem {N,N,6, trimethyl-2-(4-methyl-phenyl) imidazo [1,2-a] pyridine-3-acetamide hemitartrate}, methyl β carboline-3-carboxylate (βCCM), Ro 15–1788 and Ro 5–4864 (4′-chlorodiazepam), were tested in vitro by binding of [³H]-Ro 15–1788 to membrane preparations from various brain areas of Mullus surmuletus. Displacement studies showed a similar rank order of efficacy of various unlabelled ligands. In all regions of the brain and in the spinal cord, GABA potentiate [³H]-flunitrazepam binding in a similar order, suggesting that the BDZ recognition sites are part of the GABA_A receptor structure. These results suggest that central-type benzodiazepine receptors are present in one class of benzodiazepine binding sites in the saltwater teleost fish brain of Mullus surmuletus (type I-like). Here we report initial evidence of homogeneity of subtypes of central benzodiazepine receptors in the spinal cord of the saltwater teleost fish, Mullus surmuletus.     

Enhancement of γ-Aminobutyric Acid Binding by the Anxiolytic β-Carbolines ZK 93423 and ZK 91296

The effects of two anxiolytic beta-carboline derivatives, ZK 93423 and ZK 91296, on the binding of gamma-[3H]aminobutyric acid ([3H]GABA) to brain membrane preparations from rat cerebral cortex were examined. ZK 93423 concentration-dependently enhanced the specific binding of [3H]GABA, with a maximal increase of 45% above control at a 50 microM concentration. A less pronounced increase was induced by diazepam and by the partial agonist ZK 91296. Scatchard plot analysis revealed that the effect of ZK 93423 was due to an increase in the total number of high- and low-affinity GABA binding sites. The action of ZK 93423 was mediated by benzodiazepine recognition sites since it was blocked by the benzodiazepine antagonists Ro 15-1788 and ZK 93426 at concentrations that failed to modify [3H]GABA binding on their own. Moreover the stimulatory effect of ZK 93423 on [3H]GABA binding was also blocked by the beta-carboline inverse agonist ethyl beta-carboline-3-carboxylate. These results are consistent with the view that ZK 93423 and ZK 91296, similarly to benzodiazepines, exert their pharmacological effects by enhancing the GABAergic transmission at the level of the GABA/benzodiazepine receptor complex.     

Photoaffinity Labelling of Benzodiazepine Receptors: Lack of Effect on Ligand Binding to the Nucleoside Transport System

This study was undertaken to investigate the possibility of an allosteric interaction between benzodiazepine receptors and the CNS nucleoside transport system. Irreversible (photoaffinity) labelling of the benzodiazepine receptors in guinea pig cortical membranes resulted in a marked reduction in the binding (Bmax) of both [3H]flunitrazepam (71%) and [3H]ethyl-beta-carboline-3-carboxylate (22%) to the benzodiazepine receptors but had no effect on the binding of [3H]nitrobenzylthioinosine to the nucleoside transport system. Furthermore, although photoaffinity labelling resulted in a significant decrease in the affinities of flunitrazepam (approximately equal to 16-fold) and dipyridamole (approximately equal to sevenfold) for the [3H]Ro 15-1788 binding site of the benzodiazepine receptor complex, the affinities of these compounds for the nucleoside transport system were unaltered. These results suggest that the CNS nucleoside transport system and the benzodiazepine receptor complex are distinct, noninteractive ligand recognition sites.     

Changes in the characteristics of low affinity GABA binding sites elicited by Ro15-1788

3H-GABA binding was studied in cortical membranes from cerebral cortex of handling-habituated and naive rats after the in vitro addition of Ro15-1788. At low concentrations (10(-8), 10(-9) M) Ro15-1788 increased the total number of low affinity 3H-GABA binding sites in brain tissue from naive rats but failed to modify 3H-GABA binding in tissue from handling-habituated ones. On the contrary, Ro15-1788 at higher concentrations (10(-5), 10(-6)M) decreased the total number of low affinity 3H-GABA binding sites in tissue from handling-habituated rats but failed to modify 3H-GABA binding in tissue from naive animals. Ro15-1788 (10(-7)M) failed to modify significantly low affinity 3H-GABA binding in membranes from both naive and handling-habituated rats. However, this concentration abolished the effect of beta-carbolines and diazepam on 3H-GABA binding in membranes from naive and handling-habituated rats, respectively. The changes in the affinity of 3H-GABA binding were inversely related to the changes in the number. The results suggest that: a) the action "in vitro" of Ro15-1788 on low affinity 3H-GABA binding depends from its concentration at the benzodiazepine recognition sites; b) the benzodiazepine recognition site has a modulatory role in the control of the function of GABA-ergic receptor. Our data might explain the conflicting results obtained with this compound "in vivo".