Kinetic properties of [3H]2-nitroimipramine binding to human platelets

2-Nitroimipramine has previously been reported to be a slowly dissociating ligand at [3H]imipramine binding sites in rat brain. This binding site has been tentatively identified as the recognition site for the serotonin transport mechanism. Since we have previously solubilized imipramine binding sites from platelets, the slowly dissociating nature of this compound was of interest in the continuance of our molecular characterisation studies. Association of [3H]2-nitroimipramine to human platelets was complete within 2 h and saturation analyses implied that the affinity of this ligand was similar to that of [3H]imipramine. Moreover, the ligand binding was inhibited by non-radioactive compounds in a manner consistent with reversible, competitive kinetics. However the dissociation rate, after only 20 min association, was slow (t1/2 = 8.3 h) as compared with [3H]imipramine. After prolonged incubation with membranes (16-20 h) the dissociation rate for [3H]imipramine was essentially unaltered whereas that for [3H]2-nitroimipramine decreased approximately three fold. I conclude that [3H]2-nitroimipramine gradually becomes more slowly reversible upon prolonged incubation, but following short incubation periods it behaves essentially like [3H]imipramine.