Effects of Guanyl Nucleotides on [3H]Flunitrazepam Binding to Rat Hippocampal Synaptic Membranes: Equilibrium Binding and Dissociation Kinetics

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Abstract
The effects of guanyl nucleotides on the binding of [3H]flunitrazepam to rat hippocampal synaptic membranes were studied. In equilibrium binding studies, GABA increased and GTP decreased the binding affinity of [3H]flunitrazepam: GTP also caused a decrease in binding capacity. The effect is variable. In studies of the dissociation kinetics of [3H]flunitrazepam using diazepam and the antagonist Ro 15-1788 [ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazol-15a-1,4-benzodiazepine-3-carboxylate] as the displacers, there was evidence of 2 dissociation rate constants. GTP increased both the fast- and slow-dissociation rate constants and increased the ratio of the slow-dissociation binding state. The effect of GTP was mimicked by its nonhydrolyzable analog 5''-guanylylimidodiphosphate but not by ATP and occurred when diazepam, but not when Ro 15-1788, was used as the displacer. GABA antagonized the effect of GTP on the dissociation of [3H]flunitrazepam. The nature of the benzodiazepine receptor, its actions and the possible role of cAMP as a 2nd messenger are discussed.