Photoaffinity labeling of the benzodiazepine receptor in bovine cerebral cortex

Clonazepam, nitrazepam and flunitrazepam were found to engage in an irreversible interaction with benzodiazepine binding sites in bovine cerebral cortex homogenates upon irradiation with ultraviolet light. Photoaffinity labeling with [³H]flunitrazepam could be substantially (approx. 85%) inhibited by a number of different benzodiazepines, including clonazepam, lorazepam, Ro5-3027, and non-radioactive flunitrazepam. Spiroperidol, atropine, naltrexone, propranolol and GABA had no effect on irreversible [³H]flunitrazepam binding, indicating that this binding is to the benzodiazepine receptor as defined in previous studies.     

High affinity renal [3H]flunitrazepam binding: Characterization,localization, and alteration in hypertension

The binding of [³H]flunitrazepam was studied in membranes prepared from the kidney and cerebral cortex of unilaterally nephrectomized rats made hypertensive by simultaneous deoxycorticosterone acetate (DOCA) and NaCl administration. A significant 35–43% increase in the number of [³H]flunitrazepam binding sites (Bmax) was found in the renal membranes prepared from the hypertensive rats; there was no change in the density of binding sites in the membranes obtained from the cerebral cortex. The K_d of [³H]flunitrazepam binding did not change either in the renal or in the cerebral membranes (～ 12 nM in the kidney and ～2.0 nM in the brain). Drug specificity studies with renal membranes showed that the inhibition of [³H]flunitrazepam binding by various benzodiazepines did not jibe with their pharmacologic potency as anxiolytic agents. An intrarenal distribution of specific [³H]flunitrazepam binding was found in the bovine kidney; specific binding was greatest in the outer cortex and virtually absent in the medulla, the minor calyx and the renal artery. The evidence that the renal benzodiazepine binding site is of high affinity, is specific, has a unique distribution, and is regulated during hypertension suggests that it may be associated with an important pathophysiologic structure.     

Acute stress induces an increase in rat cerebral cortex levels of n-butyl-β-carboline-3-carboxylate,an endogenous benzodiazepine binding inhibitor

The effect of an acute swimming stress in rats on the amount of n-butyl-β-carboline-3-carboxylate, an endogenous benzodiazepine receptor binding inhibitor, was investigated. In 15 min this substance increased two fold in the cerebral cortex of the stressed rat and this increase was blocked by the previous injection of diazepam; however, no changes were observed in the cerebellum with stress. These results are discussed in relation to previous findings that, after the acute stress, [³H]flunitrazepam binding decreases in cerebral cortex and hippocampus, but not in cerebellum. A possible relationship between this benzodiazepine receptor binding inhibitor and the state of “anxiety” produced by stress is postulated.     

Rat retinal benzodiazepine receptors are controlled by visual cortical mechanisms

[³H]flunitrazepam binding was assayed in retinae of 25-day-old rats subjected either to unilateral enucleation at day 15, to intracranial unilateral cutting of the optic nerve at day 17, or to unilateral ablation of the visual cortex at day 17 postnatally.Unilateral enucleation resulted in an enhanced [³H]flunitrazepam binding in the retina of the remaining eye by 23% (P < 0.002, two-tailed Student t-test) as compared to unoperated controls.In rats with one optic nerve cut shortly before the optic chiasm, benzodiazepine binding in the retina of the lesioned side was significantly higher by 20.4 ± 7.6% (P < 0.02, N = 10, paired test) in comparison to that in the retina with the intact optic nerve.Unilateral visual cortex ablation resulted in a 13% decrease (P < 0.02) in [³H]flunitrazepam binding in the retina contralateral to the brain lesion.In the lesioned rats of all three groups, the retinal benzodiazepine receptors were no longer capable of being modified by light/dark adaptation as is observed in normal rats. Our data suggest that (i) rat retinal benzodiazepine receptors are under a control from the visual cortex, and (ii) the benzodiazepine receptors of both eyes seem to be mutually tuned, presumably via a cortico-retinal feedback loop and an interhemispheric cortico-cortical information transfer.     

Photoaffinity labeling of [3H]flunitrazepam- and [3H]Ro15-4513-bound pellets in rat cerebral cortex and cerebellum

Irreversible incorporation of [3H]flunitrazepam and [3H]Ro15-4513 into GABA/benzodiazepine receptor subunits was studied by UV irradiation using ligand-bound membrane pellets from rat cerebral cortical and cerebellar synaptic membranes. Specific incorporation for [3H]flunitrazepam was greater in the pellet than in the suspension. The incorporation was identical for [3H]Ro15-4513 in both pellet and suspension. With the ligand-bound pellets, 50% of the available binding sites were photolabeled by both ligands in cortex and cerebellum. SDS polyacrylamide gel electrophoresis and fluorography of [3H]flunitrazepam photo-labeled receptor revealed the same number of major sites in both brain regions. In contrast, [3H]Ro15-4513 appears to label fewer sites in cortex and cerebellum. Photoaffinity labeling with [3H]flunitrazepam in ligand-bound membrane pellet provides a more selective and reliable method for studying the subunit structure of GABA/benzodiazepine receptor complex.     

Changes in [3H]flunitrazepam binding in the brain of rats made tolerant to and dependent upon pentobarbital

Changes in benzodiazepine binding sites labeled by [³H]flunitrazepam (FNZ) in twenty discrete brain regions of rats made tolerant to and dependent upon pentobarbital were examined. Animals were rendered tolerant by intracerebroventricular (i.c.v) infusion with pentobarbital (300 μg/ 10 μ1/ hr for six days) through pre-implanted cannulae connected to osmotic mini-pumps. The pentobarbital dependence was assessed 24 hr after abrupt withdrawal from pentobarbital. In the tolerant rats, a significant increase in [³H]FNZ binding sites was found in layer IV of frontal cortex and the molecular layer of olfactory bulb. [³H]FNZ binding sites in the pentobarbital dependent rats were significantly increased in layers I-III and V-VI of frontal cortex, caudate-putamen, olfactory tubercle, globus pallidus and ventral pallidum, in addition to those observed in the tolerant group. There was, however, no significant difference in the hippocampus and several regions in the hindbrain in either pentobarbital-treated group. Taken together with characteristics of subtypes of benzodiazepine receptors and changes in GABA-benzodiazepine receptor complexes elucidated in our previous studies, these findings suggest that both types of benzodiazepine receptors are involved in the development of pentobarbital intoxication mediated by GABA_A receptors.     

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.     

Effects of chronic ethanol exposure on the gaba-benzodiazepine receptor complex in rat brain

Chronic treatment of male Wistar rats with ethanol by inhalation did not affect the binding of [³H]flunitrazepam, [³H]GABA or [³H]muscimol to extensively washed synaptic membranes. Neither the affinity (K_d) nor the number of binding sites (Bmax) for these ligands was changed. However, GABA enhancement of [³H]flunitrazepam binding was significantly decreased by approx. 40% in ethanol-treated animals (172% compared to 215%). Acute treatment with ethanol did not produce changes in the binding of [³H]flunitrazepam or [³H]muscimol. These findings suggest that chronic ethanol treatment leads to uncoupling of the various receptor sites on the GABA—benzodiazepine receptor ionophore-complex in the brain.     

Increased Binding of [3H]Muscimol and [3H]Flunitrazepam in the Rat Brain Under Hypoxia

We examined the effects of in vivo hypoxia (10% O2/90% N2) on the gamma-aminobutyric acid (GABA)/benzodiazepine receptors and on glutamic acid decarboxylase (GAD) activity in the rat brain. Male Wistar rats were exposed to a mixture of 10% O2 and 90% N2 in a chamber for various periods (3, 6, 12, and 24 h). The control rats were exposed to room air. The brain regions examined were the cerebral cortex, striatum, hippocampus, and cerebellum. GABA and benzodiazepine receptors were assessed using [3H]muscimol and [3H]flunitrazepam, respectively. Compared with control values, GAD activity was decreased significantly following a 6-h exposure to hypoxia in all four regions studied. On the other hand, the numbers of both [3H]muscimol and [3H]flunitrazepam binding sites were increased significantly. The increase in receptor number tended to return to control values after 24 h. Treatment of the membrane preparations with 0.05% Triton X-100 eliminated the increase in the binding capacity. These results may represent an up-regulation of postsynaptically located GABA/benzodiazepine receptors corresponding to the impaired presynaptic activity under hypoxia.     

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.     

Autoradiographic study on [3H]flunitrazepam binding in rat cortex and hippocampus after chronic ethanol treatment

Ethanol alters almost all membrane functions, but it behaves essentially like a benzodiazepine-type GABAergic agonist. The mechanism by which ethanol affects the GABA/benzodiazepine complex is not clear. We studied the possible changes in [³H]flunitrazepam binding induced by chronic ethanol treatment, using light microscopic autoradiography, to try to elucidate the controversy underlying this topic. This technique allows us to measure densities of benzodiazepine receptors in different anatomical brain areas—visual cortex and hippocampus—which seem to constitute the anatomical support for the behavioral and physiological responses affected by ethanol. Autoradiographic studies on the visual cortex and hippocampus from rats chronically treated with ethanol do not show statistically significant differences in the binding of, [³H]flunitrazepam with respect to control animals. Furthermore, we did not find either rostro-caudal or medio-lateral differences, in benzodiazepine receptor densities in each layer of the visual cortex.     

Alterations in central neurotransmitter receptor binding sites following estradiol implantation in female rats

The subcutaneous implantation of an estradiol pellet (10 mg) into female rats induced a hypophyseal hyperplasia with hyperprolactinaemia. Examination of neurotransmitter receptors in the hippocampus, striatum and cerebral cortex one month after the implantation revealed that estrogenization was associated with: an increased density of ³H-domperidone binding sites (D₂ receptors) in the striatum and reduced numbers of ³H-serotonin high affinity sites (5-HT₁ receptors) in the hippocampus and of ³H-muscimol binding sites (GABA receptors) in the hippocampus, striatum and cerebral cortex. In contrast, the characteristics of ³H-spiperone binding to 5-HT₂ receptors (in the cerebral cortex) and those of ³H-flunitrazepam binding to benzodiazepine sites (in the three brain regions examined) were not significantly different in estrogenized and in control female rats. However, the enhancing effect of GABA on ³H-flunitrazepam binding was markedly reduced in brain membranes from estrogenized animals. The respective roles of estradiol and prolactin in mediating these changes in neurotransmitter receptors are discussed notably with regard to the regional heterogeneity of estradiol binding capacity in the rat brain.     

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.     

6,3'-dibromoflavone and 6-nitro-3'-bromoflavone: new additions to the 6,3'-disubstituted flavone family of high-affinity ligands of the brain benzodiazepine binding site with agonistic properties

6,3'-dibromoflavone and 6-nitro-3'-bromoflavone inhibited [(3)H]flunitrazepam binding to the benzodiazepine binding site of the gamma amino butyric acid receptor complex with K(i) values between 17 and 36 nM in different brain regions. Their gamma amino butyric acid ratio for [(3)H]flunitrazepam binding to cerebral cortex membranes indicated partial agonistic properties. Both compounds had similar pharmacological effects: they produced anxiolytic-like effects at low doses but did not alter locomotor activity or muscle tonicity; sedation was caused only at doses higher than 30 mg/kg in mice. These synthetic flavone derivatives join an existing family of 6,3'-disubstituted flavone compounds with high affinity for the benzodiazepine binding site and partial agonistic profiles.     

Aging: Effect on ex-vivo benzodiazepine binding after a diazepam injection

Ex vivo [³H]flunitrazepam receptor occupation was determined in the brain of young, mature and old male Fischer 344 rats after a single intravenous injection of a low dose of diazepam. The two benzodiazepine receptor subtypes or conformations (BZ₁ and BZ₂) were differentiated by the displacement of [³H]flunitrazepam specific binding with the triazolopyridazine, CL 218,872. The acute diazepam injection decreased ex vivo [³H]flunitrazepam binding in only the senescent rats. The [³H]flunitrazepam binding at both the BZ₁ and BZ₂ receptor or receptor conformation was significantly reduced in the old rats.     

Uptake and effects of melatonin on the synthesis of proteins by the rat cerebral cortex

Slices of rat parietal cerebral cortex took up and retained [³H] melatonin up to a tissue concentration about 4-fold to that present in the incubation medium. This phenomenon was time-dependent, maxima being observed after 180 min-incubations Eighty to 93% of the radioactivity present in the cerebral cortex slices was chromatographically identified as melatonin. Even at the highest melatonin concentration that could be dissolved in the incubation media, a constant proportion of [³H] melatonin was bound to cortical slices, indicating that within this concentration range, melatonin binding is independent of its concentration. Melatonin effects on protein synthesis in the rat cerebral cortex were investigated by studying the incorporation of [³H] L-leucine into proteins in cerebral cortex of rats injected s.c. with 10 or 100 μg/day of melatonin for 5 to 10 days. Both treatments caused leucine incorporation into proteins to increase significantly by about 50 to 60%.     

The specific binding of the platelet-activating factor (PAF) receptor antagonist WEB 2086 and the benzodiazepine flunitrazepam to rat hepatocytes

Thieno-triazolodiazepines WEB 2086 and BN 50739 have been described as the potent PAF receptor antagonists. Binding of radiolabeled [³H]WEB 2086 has been widely employed to characterize PAF receptors in different cells. In a search for a PAF receptor in isolated rat hepatocytes, we discovered that the binding of [³H]WEB to rat hepatocytes was highly specific but had a relatively low affinity with a K_d of 113 nM and B_max of 0.65 pmol/10⁶ cells in freshly isolated cell suspension and K_d of 1.65 μM and B_max of 2.0 pmol/plate in cultured hepatocytes. No consistent specific binding of [³H]PAF itself was found in the same cell preparations. The binding of [³H]flunitrazepam in the presence of the peripheral type of benzodiazepine receptor antagonist Ro 5-4864 was saturated and exhibited a K_i of 3.8 nM and B_max of 3.5 pmol/plate. The central type of benzodiazepine receptor antagonist clonazepam also competed for the [³H]flunitrazepam binding, however with a much lower affinity. Various antagonists inhibited the binding of [³H]WEB 2086 with a rank order BN 50739⪢Ro 5-4864≥clonazepam. Interestingly, bicuculline, a specific antagonist of GABA(A) recognition sites, also significantly reduced the binding of [³H]WEB 2086. The binding of [³H]flunitrazepam was inhibited with a rank potency BN 50739⪢WEB 2086. Taken together, these findings suggest that the specific binding of PAF receptor antagonists WEB 2086 and BN 50739 in rat hepatocytes does not involve PAF receptors and occurs via peripheral benzodiazepine and, possibly GABA(A) receptor sites.     

[3H]Propyl β-Carboline-3-Carboxylate as a Selective Radioligand for the BZ1 Benzodiazepine Receptor Subclass

Abstract: Ethyl β-carboline-β-carboxylate (β-CCE) is a mixed-type inhibitor of [³H]flunitrazepam ([³H]FNM) binding to benzodiazepine receptors in noncerebellar regions of rat brain. These findings may represent the presence of either receptor multiplicity or negative cooperativity among benzodiazepine receptors. [³H]Propyl β-carboline-3-carboxylate ([³H]PrCC) has previously been shown to bind specifically to benzodiazepine receptors of rat cerebellum. In the present study we found no indication of the presence of true negative cooperativity among benzodiazepine receptors when [³H]PrCC was used as radioligand. However, we observed that [³H]PrCC labelled only 57% of [³H]FNM binding sites in rat hippocampus (B_max values) and 71% in rat cerebral cortex, whereas the number of receptors labelled by both ligands was equal in the cerebellum. Hofstee analyses of the shallow inhibition curves seen in hippocampus and cerebral cortex when [³H]FNM binding was inhibited by β-CCE indicate that β-CCE and some other β-carboline-3-carboxylate derivatives interact preferentially with a subclass of receptors, and that the percentage of this subclass is equivalent to the number of receptors labelled by [³H]PrCC. We conclude that [³H]PrCC at low concentration (0.3–0.4 × 10^-9 M) labels a subclass of benzodiazepine receptors, BZ₁, while another class, BZ₂ receptors, are not labelled by [³H]PrCC when filtration assays are used. By parallel determinations of the proportion between [³H]FNM and [³H]PrCC binding we calculated the percentage of BZ₁ receptors in several regions of rat, guinea pig and calf brain and in mouse forebrain. The values ranged from approximately 50% in hippocampus to 90% in the guinea pig pons.     

Postnatal development and GABA allosteric modulation of benzodiazepine receptor binding in the vitamin B-6 deficient rat brain

We have measured the postnatal development and GABA modulation of benzodiazepine receptors in neuronal membranes from vitamin B-6 deficient and normal rats. In rats fed vitamin B-6 adequate and deficient diets there were age-dependent changes in [³H]flunitrazepam binding site affinity and in the number of binding sites. Vitamin B-6 deficiency produced a significant reduction in the potency of GABA to enhance [³H]flunitrazepam binding to cortical membranes prepared from 14 day old rats. These results suggests an uncoupling of the GABAa/benzodiazepine receptor at a developmental period when the animals are most susceptible to spontaneous seizures.     

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.