title: Characterization of two cerebellar binding sites of [3H]Ro 15-4513.
abstract: Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H- imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate), a partial inverse agonist of central benzodiazepine receptors, binds to two distinct sites in the cerebellum. The binding to diazepam-sensitive (DZ-S) sites is displaced by different benzodiazepine receptor ligands, whereas the other site is insensitive to benzodiazepine agonists [diazepam-insensitive (DZ-IS)]. The binding of [3H]Ro 15-4513 was studied in pig cerebellar membranes and in receptors solubilized and purified from these. Micromolar concentrations of gamma-aminobutyric acid (GABA) decreased DZ-S binding at both 0 and 37 degrees C, whereas it had no effect on DZ-IS binding at 0 degrees C and was stimulatory at 37 degrees C. The pH profiles of [3H]Ro 15-4513 binding were quite similar in both binding sites in the pH range of 5.5-10.5 but differed at acidic pH values from those reported for flunitrazepam and Ro 15-1788 (flumazenil; ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H- imidazol[1,5-a][1,4]benzodiazepine-3-carboxylate) binding in DZ-S sites, suggesting that [3H]Ro 15-4513 does not interact with a histidine residue apparently present in the binding site. Zn2+, Cu2+, Co2+, and Ni2+ enhanced the binding to DZ-S sites, and the first three mentioned also enhanced the binding to DZ-IS sites. [3H]Ro 15-4513 binding activity was solubilized by various detergents. All detergents tested were more efficient in solubilizing DZ-S binding activity. High ionic strength improved especially the solubility of DZ-IS binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Tampere Brain Research Center, Department of Biomedical Sciences, University of Tampere, Finland.
authors.forename: M
authors.initials: M
authors.lastname: Uusi-Oukari
mesh_terms: D000818:Animals; D001386:Azides / Q000378:metabolism*; D001569:Benzodiazepines / Q000378:metabolism*; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002531:Cerebellum / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D002846:Chromatography, Affinity; D003975:Diazepam / Q000378:metabolism / Q000494:pharmacology; D005445:Flunitrazepam / Q000378:metabolism; D011963:Receptors, GABA-A / Q000032:analysis*; D013552:Swine; D014316:Tritium
chemical_list: D001386:Azides; D011963:Receptors, GABA-A; D014316:Tritium; D001569:Benzodiazepines; D005445:Flunitrazepam; C042957:Ro 15-4513; D003975:Diazepam