Characterization of the Solubilized GABA and Benzodiazepine Receptors from Various Regions of Bovine Brain

Abstract

GABA and benzodiazepine receptors were solubilized from bovine cerebral cortex, cerebellum and hippocampus and then partially purified by gel filtration and characterized. The apparent MW of all these receptors were determined to be 600,000-650,000 by gel filtration, the sedimentation coefficients being 11.0-11.3 S by sucrose density gradient centrifugation. [3H]Muscimol was bound to 2 classes of sites in fractions from all 3 regions, and [3H]flunitrazepam bound to 1 class of sites. A comparison of the ratio of Bmax [maximum bound concentration] for flunitrazepam binding to Bmax for muscimol binding revealed that the fractions from the hippocampus exhibited a much higher ratio of benzodiazepine binding sites than were detected in fractions from the cortex and cerebellum. GABA agonist and antagonist inhibited [3H]muscimol binding to the fractions from these 3 regions, at similar concentrations. Benzodiazepine agonists and antagonists also inhibited [3H]flunitrazepam binding in these 3 fractions, with similar potency. CL 218,872 [3-methyl-6-[3-trifluoromethylphenyl]-1,2,4-triazolo-[4,3b]-pyridazine] inhibited [3H]flunitrazepam binding in the cerebellar fraction with the lowest IC50 [median inhibitory concentration] value and that in the hippocampal fraction with the highest IC50 value. Hill coefficients for CL 218,872 inhibition were 0.98, 0.64 and 0.58 for cerebellum, cortex and hippocampus, respectively.