Evidence that pure uptake inhibitors including cocaine interact slowly with the dopamine neuronal carrier.

We have studied the ability of various uptake blockers to protect the dopamine neuronal carrier labeled with [3H]GBR 12783 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-(propenyl)-piperazine) against N-ethylmaleimide-induced alkylation, using membrane preparations obtained from rat striatum. Pure uptake inhibitors such as mazindol, pyrovalerone, nomifensine and methylphenidate, and substrates (dopamine, d-amphetamine, m-tyramine) protected the [3H]GBR 12783 binding site in a concentration-dependent manner. Preincubation of the membranes with these agents prior to N-ethylmaleimide treatment did not modify the protecting ability of substrates, whereas it significantly improved that of pure uptake inhibitors including cocaine. When the preincubation was omitted, the concentration dependence of the protection observed with pure uptake inhibitors decreased and a maximal 40% protection was observed for 10 microM to 1 mM cocaine concentrations. Effective protecting concentrations of blockers are correlated with their Ki determined in standard binding studies. These results reveal that all pure uptake inhibitors bind slowly to the dopamine neuronal carrier whereas substrates interact with it rapidly.