Characterization of diazepam-insensitive [3H]Ro 15-4513 binding in rodent brain and cultured cerebellar neuronal cells

Abstract

Experiments were performed to characterize diazepam-insensitive [3H]Ro 15-4513 binding sites in discrete regions of rodent brain and cultured rat cerebellar granule cells. Scatchard analysis of [3H]Ro 15-4513 binding in the presence of 10 microM diazepam revealed that diazepam-insensitive binding sites in the rat brain were most abundant in the cerebellum, followed by the hippocampus, cerebral cortex and olfactory bulb. Diazepam-insensitive sites represented approximately 80% of the total [3H]Ro 15-4513 binding sites in the membranes of cultured rat cerebellar granule cells. The Bmax values for total [3H]Ro 15-4513 and [35S]TBPS are almost identical, and 5-6 times larger than that for [3H]diazepam in this preparation. Although some annelated [1,5-a]benzodiazepine analogues such as Ro 15-4513, ro 16-6028, flumazenil and Ro 15-3505, and an imidazothieno-diazepine, Ro 19-4603, showed high affinity for cortical and cerebellar diazepam-insensitive sites, all the annelated benzodiazepine compounds tested showed higher affinity for cerebellar diazepam-insensitive sites than cortical ones. In contrast, a pyrazoloquinoline compound, CGS 8216, and beta-carboline analogues such as beta-carboline-3-carboxylate ethyl ester (beta-CCE) and beta-carboline-3-carboxylate methyl ester (beta-CCM) exhibited higher affinity for cortical than cerebellar sites. These results suggest that diazepam-insensitive sites are heterogeneous in brain areas with respect to ligand specificity.

Chronic pentobarbital administration alters ?-aminobutyric acidA receptor ?6-subunit mRNA levels and diazepam-insensitive [3H]Ro15-4513 binding

“…This is in accordance with our previous results that show no significant change in the flunitrazepam binding (i.e., diazepamsensitive binding) in the granule cells after chronic pen-tobarbital treatment . The diazepam-insensitive binding accounts for 80% of the total Ro15-4513 binding in the granule cells (Ito et al, 1994). The percentage of diazepam-insensitive sites detected in our experiments (about 92% of the total binding in the control group) is larger than that previously reported, which we believe is due to differences in the experimental protocols.…”

GABAA Receptors Containing the α4‐Subunit: Prevalence, Distribution, Pharmacology, and Subunit Architecture In Situ

“…However, diazepaminsensitive [ 3HIR0 15-4513 binding is also discerned in other brain areas ( Fig. 3; Turner et al, 1991;Ito et al, 1994), where it appears to be represented by receptors containing the cr4-subunit. The latter conclusion is based on several lines of evidence.…”

“…Immunoprecipitation with a6-subunitspecific antisera provided proof that receptors containing the a6-subunit contain diazepam-insensitive [3HIIR0 15-45 13 binding sites Quirk et al, 1994;Khan et al, 1996a). However, diazepam-insensitive receptors are also present at low abundance in additional brain areas, as shown by diazepam-insensitive binding of~3H]Ro 15-45 13 in brain sections and membranes (Sieghart et al, 1987;Turner et al, 1991;Wong and Skolnick, 1992;Korpi et al, 1993;Ito et al, 1994). In these brain areas, the presence of the a4-subunit mRNA points to diazepam-insensitive GABAA receptors characterized by the a4subunit.…”

“…In situ, diazepam-insensitive receptors are expressed with high abundance in the granule cell layer of the cerebellum, where they contribute to about one-third of [ 3HIR0 15-4513 binding sites, as detected by radioligand binding (Turner et al, 1991;Wong and Skolnick, 1992;Korpi et al, 1993;Ito et al, 1994). Immunoprecipitation with a6-subunitspecific antisera provided proof that receptors containing the a6-subunit contain diazepam-insensitive [3HIIR0 15-45 13 binding sites Quirk et al, 1994;Khan et al, 1996a).…”

Identification of a residue in the γ‐aminobutyric acid type A receptor α subunit that differentially affects diazepam‐sensitive and ‐insensitive benzodiazepine site binding

“…However, other ligands from diverse classes retain affinity for these diazepam‐insensitive (DI) receptors and some display differential affinity for those containing the α4 or α6 isoform. Certain β‐carboline and pyrazoloquinolinone ligands, for example, have higher affinity for DI receptors in the cortex compared with the cerebellum (Ito et al . 1994).…”