Dihydropyridine and peripheral type benzodiazepine binding sites: subcellular distribution and molecular size determination

Electrophysiological and pharmacological studies have shown that peripheral-type benzodiazepine receptors modulate voltage-sensitive calcium channels in the heart. We have compared these binding sites with binding sites for [3H]dihydropyridines, which are believed to label such channels. Although no direct or allosteric interaction could be demonstrated between the two sites, their subcellular distribution--sarcolemma and ryanodine-sensitive sarcoplasmic reticulum--was parallel. Size determination of the two sites suggests that the receptors for these two classes of compounds are separate molecules packaged in the same membrane compartment.     

GABA and benzodiazepine binding sites in spontaneously hypertensive rat

The binding of GABA to its central receptor sites and of benzodiazepines to central and peripheral types of binding sites was compared in age-matched spontaneously hypertensive rat (SHR) and normotensive (WKY) rat. The KD and Bmax values of GABA agonist binding were not significantly different between SHR and WKY rat in the forebrain, cerebellum or pons-medulla. The binding of [3H]flunitrazepam to central types of binding sites was also not different in 12 week-old SHR and WKY rat. However, the binding of [3H]flunitrazepam and [3H]RO5-4864 was significantly lower in the SHR kidney, as compared to WKY rat kidney. This decrease was due to a lower Bmax in SHR kidney. In contrast, the binding of these radioligands in the hearts of the two strains was not different. These results indicate that the SHR has lower peripheral benzodiazepine binding sites in the kidney as compared to age-matched normotensive WKY rat kidney.     

Correlation between exploratory activity in an elevated plus-maze and number of central and peripheral benzodiazepine binding sites

Summary Two groups of rats were selected from a small animal population on the basis of their exploratory activity in an elevated plus-maze model of anxiety. One group had a considerably lower and the other one a higher exploratory activity than the average total population. These subgroups were termed anxious and nonanxious, respectively. In both groups central benzodiazepine binding sites in various brain structures were labelled with ³H-flunitrazepam. Peripheral benzodiazepine binding sites labelled in vitro with different tritiated ligands were also studied in several peripheral organs including blood platelets and lymphocytes. Anxious animals had a significantly lower number of ³H-flunitrazepam binding sites in the cerebral cortex but not in the hippocampus and cerebellum. In this subgroup ³H-Ro 5-4864 binding to peripheral benzodiazepine recognition sites was also lower than in the other one in adrenals, kidneys, platelets and lymphocytes. In the heart no differences of ³H-Ro 5-4864 binding between subgroups studied were found. Although in anxious rats ³H-diazepam and ³H-PK 11195 binding was significantly lower only in lymphocytes, a somewhat decreased binding to these ligands was also present in platelets. No significant differences in the affinity were found between the two groups throughout the experiments described. The results indicate that behavioral anxiety in rats is correlated not only with the lower number of central benzodiazepine receptors but also with a lower density of peripheral benzodiazepine binding sites in several peripheral organs including platelets and lymphocytes.     

Biological actions in vivo and in vitro of two gamma-aminobutyric acid (GABA) analogues: beta-chloro GABA and beta-phenyl GABA

1 The synthesis of two analogues of gamma-aminobutyric acid (GABA), beta-chloro GABA and beta-phenyl GABA is described.2 The activity of brain GABA aminotransferase was inhibited by beta-chloro GABA (5.7 x 10(-5)M) and beta-phenyl GABA (4.6 x 10(-3)M) in a competitive manner with GABA.3 beta-Chloro GABA exhibited 50% of the inhibitory activity of GABA in blocking the discharge of the crayfish stretch receptor neurone; beta-phenyl GABA had no detectable effect.4 Injection of beta-phenyl GABA (200 mg/kg) into normal or epileptic cats (cobalt) caused the appearance of synchronized slow-wave EEG activity.5 Administration of beta-chloro GABA (200 mg/kg) to epileptic cats (cobalt) produced a temporary diminution or abolition of epileptic discharges while causing no alteration in normal EEG activity.6 beta-Chloro GABA and beta-phenyl GABA had no effect on the concentrations of catecholamines or of amino acids in mouse brain.7 The results suggest that both beta-chloro GABA and beta-phenyl GABA may pass the blood-brain barrier.     

Characterization of peripheral benzodiazepine binding sites in human term placenta

Peripheral benzodiazepine binding sites were characterized in human term placental membranes using [3H]PK 11195, which is a ligand specific for peripheral benzodiazepine binding sites. Binding of [3H]PK 11195 to human term placental membranes was found to be saturable. Scatchard analysis revealed a single population of binding sites (r = 0.98). Equilibrium dissociation constant (KD) was 2.1 +/- 0.3 nM, and density of binding sites (Bmax) was 920 +/- 105 fmol/mg protein. The KD value calculated from kinetic experiments was 3.6 +/- 0.2 nM. The ability of various drugs to displace [3H]PK 11195 from human term placental binding sites was tested: the inhibition constants (KI) for PK 11195, Ro 5-4864, and diazepam were 2.9, 11.8, and 177 nM, respectively, whereas clonazepam, methyl-beta-carboline-3-carboxylate, Ro 15-1788, chlordiazepoxide, atropine, and estradiol were inefficient in displacing [3H]PK 11195 (KI greater than 10(-5) M).     

Modulation of peripheral benzodiazepine binding sites following chronic estradiol treatment

A 10 day estradiol treatment of rats resulted in a marked reduction (43%) in peripheral benzodiazepine (BZ) binding sites in the testis and an up-regulation (27%) of these sites in the kidney. No significant alterations in brain central BZ receptors and heart peripheral binding sites were observed. The down-regulation of BZ binding sites in the testis may be related to or accompany estrogen-induced testicular atrophy. The physiological and pharmacological implications of the alterations obtained in the kidney are as yet unclear.     

Decreased peripheral benzodiazepine binding sites in platelets of neuroleptic-treated schizophrenics

A decrease of 30% was observed in the density of peripheral-type benzodiazepine binding sites in platelets of nine schizophrenics maintained on chronic neuroleptic treatment as compared to platelets of six untreated schizophrenics and 15 healthy volunteers. The equilibrium dissociation constant for binding by [3H]PK 11195, which is a ligand specific for peripheral benzodiazepine binding sites, was similar in all three groups. The relevance of the reduction in peripheral-type benzodiazepine binding sites to central or peripheral side-effects induced by long-term neuroleptic treatment merits further investigation.     

Presence of peripheral benzodiazepine binding sites on primary rat skeletal fibroblasts

Employing [3H]Ro5-4864 as a radioligand, the existence of peripheral benzodiazepine binding sites in rat hind limb skeletal fibroblasts was explored. Saturable, high affinity binding of this ligand is described for fibroblasts and cell membrane preparations, demonstrating a pharmacological profile characteristic for such receptors. The binding of [3H]Ro5-4864 exhibited higher affinity and density in fibroblasts compared to membrane preparations. A quaternaryamine derivative of PK 11195 showed a similar potency as a displacer of [3H]Ro5-4864 in both preparations. The data suggest that peripheral benzodiazepine receptors are present in the plasma membrane of primary mammalian fibroblasts.     

Reduced affinity of peripheral benzodiazepine binding sites in elderly insomniac patients

Summary Peripheral-type benzodiazepine binding sites on intact platelets from untreated chronic insomniac patients and those chronically treated with benzodiazepine hypnotics were investigated to evaluate their putative involvement in sleep pathology and the influence of treatment. There were 34 elderly subjects in the study, 14 controls (80.7 years) and 20 insomniac patients, of whom 7 were untreated (61.1 years) and 13 were treated (84.4 years). There was an equivalent number of peripheral-type benzodiazepine ³H-PK 11195 binding sites on platelets from untreated (7.61 pmol/mg protein) and treated insomniacs (6.39 pmol/mg protein) and on platelets from the controls (6.21 pmol/mg protein). However, there was a twofold reduction in the affinity of these sites in untreated (Kd=8.02 nM) and treated (Kd=7.40 nM) insomniacs compared to controls (3.79 nM). This difference raises the possibility that peripheral-type benzodiazepine sites are involved in abnormal sleep.     

Identification and distribution of peripheral benzodiazepine binding sites in male rat genital tract

In the present study we identified and characterized the distribution of high-affinity peripheral benzodiazepine binding sites (PBzS) in male rat vas deferens (whole, and prostatic and epididymal portions), prostate, seminal vesicles, and Cowper's glands. [3H]PK 11195, an isoquinoline carboxamide derivative, was used as a radioligand specific for PBzS. Scatchard analysis of saturation curves of [3H]PK 11195 binding in the whole vas deferens, the prostatic and epididymal portions of the vas deferens, the prostate, the seminal vesicles, and Cowper's glands yielded mean maximal numbers of binding sites of 1211 +/- 158, 1012 +/- 311, 1451 +/- 156, 1805 +/- 86, 865 +/- 51, and 2251 +/- 135 fmol/mg protein, respectively. The equilibrium dissociation constant values ranged between 1 and 3 mM in all the above tissues. The ability of various drugs to displace the specific binding of [3H]PK 11195 from PBzS in Cowper's gland membranes was also tested. The inhibition constants for Ro 5-4864, diazepam, and PK 11195 were 28, 330, and 4 nM, respectively, whereas clonazepam, Ro 15-1788, and testosterone were inefficient in displacing [3H]PK 11195. The presence of high densities of PBzS in the male genital tract suggests a functional role in these hormone-dependent organs.     

Compared properties of central and peripheral binding sites for phencyclidine

Phencyclidine and its derivatives bind specifically and reversibly to rat brain and peripheral organs. Binding characteristics are different in brain, lung, kidney, heart and liver. Affinities of phencyclidines for the brain receptor but not those for peripheral organs are correlated with the pharmacological activities of phencyclidines as measured in the rotarod test.     

Anxioselective compounds acting at the GABA(A) receptor benzodiazepine binding site

With the exception of obsessive compulsive disorder, benzodiazepines (BZs) remain a major first line treatment for anxiety disorders. However, as well as being anxiolytic, BZs also cause sedation acutely, related to the fact that BZs are also used as hypnotics, and chronically may have abuse potential as well as cause physical dependence which manifests itself as the demonstration of a number of adverse events upon discontinuation. The molecular mechanisms of BZs are now well defined in that they enhance the actions of the inhibitory neurotransmitter GABA by binding to a specific recognition site on GABA(A) receptors containing alpha1, alpha2, alpha3 and alpha5 subunits. Compounds that bind at this modulatory site and enhance the inhibitory actions of GABA are classified as agonists, those that decrease the actions of GABA are termed inverse agonists whereas compounds which bind but have no effect on GABA inhibition are termed antagonists. The clinically used BZs are full agonists and between the opposite ends of the spectrum, i.e. full agonist and full inverse agonist, are a range of compounds with differing degrees of efficacy, such as partial agonists and partial inverse agonists. Attempts have been made to develop compounds which are anxioselective in that they retain the anxiolytic properties of the full agonist BZs but have reduced sedation and dependence (withdrawal) liabilities. Such compounds may interact with all four (i.e. alpha1-, alpha2-, alpha3- and alpha5-containing) GABA(A) receptor subtypes and have partial rather than full agonist efficacies. Examples of nonselective partial agonists include bretazenil, imidazenil, FG 8205, abecarnil, NS 2710, pagoclone, RWJ-51204 and (S)-desmethylzopiclone. Alternatively, a compound might have comparable binding affinity but different efficacies at the various subtypes, thereby preferentially exerting its effects at subtypes thought to be associated with anxiety (alpha2- and/or alpha3-containing receptors) rather than the subtype associated with sedation (alpha1-containing receptors). Examples of efficacy selective compounds include L-838417, NGD 91-3 and SL651498. For each compound, preclinical and where available clinical data will be reviewed. Emerging themes include the lack of definitive intrinsic efficacy data for certain compounds (e.g. abecarnil, ocinaplon, pagoclone) and the difficulty in translating robust anxiolysis and a separation between anxiolytic and sedative doses of non-selective partial agonists in preclinical species into consistent clinical benefit in man (e.g. bretazenil, abecarnil, pagoclone). With respect to efficacy selective compounds, NGD 91-3 was not anxiolytic in man but in the absence of efficacy data, these results are difficult to interpret. Nevertheless, efficacy selective compounds represent a novel approach to targeting specific subtypes of the GABA(A) receptor, the ultimate test of which will be evaluation in the clinic.     

Interaction of histamine H2-receptor antagonists with GABA and benzodiazepine binding sites in the CNS

The histamine H2-receptor antagonist cimetidine potently inhibited [3H]muscimol and enhanced [3H]flunitrazepam binding in membranes prepared from several brain regions in the rat, including the dorsal raphe nucleus. As further examined in cortical membranes, this effect on both GABA and benzodiazepine binding sites was specific for imidazole-derived H2-receptor antagonists (potency: cimetidine greater than metiamide greater than tiotidine) and not observed with either several H1-receptor antagonists or histamine. These data indicate a striking similarity between the actions of cimetidine (and other imidazole-derived H2-receptor antagonists) and GABA on binding parameters at the GABA receptor complex.     

Inactivation of GABA receptors by phenoxybenzamine: Effects on GABA-stimulated benzodiazepine binding in the central nervous system

Treatment of particulate fractions of rat forebrain with phenoxybenzamine produced dose related inhibition of specific muscimol binding. Scatchard analysis revealed a significant reduction of binding sites but no change in affinity constant. The inhibition was not reversed by washing or incubation at 37°C suggesting that phenoxybenzamine alkylates GABA receptors, i.e. acts as a non-competitive, non-equilibrium inhibitor. Phenoxybenzamine inhibited the GABA-induced increase in specific diazepam binding but had little direct effect on diazepam binding. Preincubation of synaptic membranes with GABA or muscimol, but not diazepam, protected the muscimol binding site from inactivation by phenoxybenzamine.     

Functional coupling of gamma-aminobutyric acid (GABA)A and benzodiazepine type II receptors: analysis using purified GABA/benzodiazepine receptor complex from bovine cerebral cortex

Possible functional coupling between gamma-aminobutyric acid (GABA) and benzodiazepine receptors was examined using a purified GABA/benzodiazepine receptor complex. The purified receptor complex was obtained by 1012-S-acetamide adipic hydrazide Sepharose 4B affinity column chromatography, following the solubilization of synaptic membrane from the bovine cerebral cortex with Nonidet P-40. The binding of [3H]GABA to the purified GABA receptor was displaced significantly by muscimol and bicuculline, GABAA receptor agonists and antagonists, respectively, but not by baclofen, a GABAB receptor agonist. On the other hand, the binding of [3H]flunitrazepam to the purified benzodiazepine receptor was found to be displaced by microM ranges of CL 218,872, which is known to be sensitive to the benzodiazepine type II receptor. Furthermore, it was found that the binding of [3H]muscimol to these purified GABAA receptors was enhanced by benzodiazepines, while the binding of [3H]flunitrazepam to these benzodiazepine type II receptors was increased by GABA receptor agonists. These enhancements by GABA agonists and benzodiazepine agonists were found to be blocked by bicuculline and a benzodiazepine receptor antagonist, Ro15-1788, respectively. These results strongly suggest that the purified receptor may consist of GABAA and benzodiazepine type II receptors and possess a functional coupling of these sites, as shown in cerebral synaptic membranes.     

Facilitation of benzodiazepine binding by sodium chloride and GABA

An endogenous substance was found which facilitated benzodiazepine binding and was subsequently identified as GABA. The facilitation of benzodiazepine binding produced by GABA obeyed Michaelis — Menten Kinetics and Scatchard analysis revealed that this facilitation was due to an increased affinity of the benzodiazepine receptor. Sodium chloride itself increased benzodiazepine binding and also markedly enhanced the facilitation caused by GABA, by a further increase in the affinity of the benzodiazepine receptor. The enhancement of the GABA effect produced by sodium chloride showed sigmoidal kinetics indicating cooperativity, no evidence could be found for the involvement of sodium-dependent GABA binding in this interaction.     

The responsiveness of central benzodiazepine and GABA(A) receptors in vasopressin and spontaneously hypertensive rats

Administration of diazepam, an agonist of benzodiazepine receptors, and muscimol, an agonist of GABA(A) receptors, resulted in an increase in cytosol GABA-modulin in hypothalamus and cerebellum. Low dose of muscimol, ineffective by itself, completely antagonized the isoniazid, bicuculline and picrotoxin-induced decrease in GABA-modulin activity. In contrast, low dose of diazepam was able to block only the action of isoniazid. That confirms different mechanism of action of muscimol and diazepam. The increase in cytosol GABA-modulin activity in the anterior and posterior hypothalamus of spontaneously hypertensive rats (SHR) and of vasopressin hypertensive rats required much higher doses of muscimol than in normotensive animals showing subsensitivity of GABA(A) receptors in these brain structures. In contrast, the sensitivities of GABA(A) receptors in the cerebellum and of benzodiazepine receptors in the hypothalamus and cerebellum were equal to hypertensive and normotensive rats.     

Peripheral-type benzodiazepine binding sites in a renal epithelial cell line (MDCK)

Madin-Darby canine kidney (MDCK) cells express a high density of binding sites (Bmax = 0.67 pmol/10(6) cells) for [3H]RO 5-4864, a peripheral-type benzodiazepine (BZD) receptor ligand. Receptor affinity (Kd = 45 nM) in MDCK cells is 30-50 fold lower than in rat kidney, but its pharmacological specificity is identical to that of peripheral-type BZD receptors in the rat kidney (PK 11195 greater than RO 5-4864 greater than diazepam = flunitrazepam greater than clonazepam). The MDCK cell line should provide a useful model system for studying the role of peripheral type BZD receptors in renal function.