Abstract

The inhibition of [³H]flunitrazepam binding by a novel anxiolytic agent [CL 218,872; 3-methyl-6-[3-trifluoromethyl)phenyl]-l,2,4-triazolo-[4,3-b]pyridazine] was studied in membranes prepared from bovine retina and rat cerebral cortex, cerebellum, and kidney. The order of potency for the inhibition of [³H]fluintrazepam binding by CL 218,872 in these tissues was cerebellum > retina ≅ cerebral cortex » kidney. The slope factors (Hill coefficients) for CL 218,872 inhibition of [³H]flunitrazepam were approximately 1.0 for kidney, 0.9 for cerebellum, and 0.7 for cerebral cortex and retina. In thoroughly washed membrane preparations from all of the central tissues, K_i values were significantly decreased an average of 60% in the presence of 100 µM γ-aminobutyric acid (GABA) (p < 0.01). With kidney membranes there was no apparent affect of GABA on the K_i of CL 218,872. (+)-Bicuculline (100 µM) could antagonize the effect of GABA on membranes from central tissues. Nonlinear least-squares regression analyses were used to reanalyze these data in terms of receptor models describing the interaction of a ligand with either one or two classes of independent binding sites. A two-site regression model resulted in a highly significant improvement in the fit of data obtained from retina, cerebral cortex, and cerebellum (p < 0.01), but not with data from kidney (p > 0.05). GABA was found to enhance the affinity of CL 218,872 for both of the sites without changing the proportion of sites. The results of these studies show that GABA enhances the affinity of CL 218,872 for the central benzodiazepine receptor(s) and that the inhibition of [³H]flunitrazepam binding by CL 218,872 in bovine retina, rat cerebellum, and cerebral cortex may be explained by interactions with two classes of independent binding sites.