VOLTAMMETRIC STUDIES ON MECHANISMS OF DOPAMINE EFFLUX IN THE PRESENCEOF SUBSTRATES AND COCAINE FROM CELLS EXPRESSING HUMAN NOREPINEPHRINE TRANSPORTER

The effects of substrates m-tyramine and beta-phenethylamine, as well as cocaine, on the DA efflux from a cell line stably expressing the hu man norepinephrine transporter (hNET) were investigated by using rotat ing disk electrode voltammetry. Both the substrates and cocaine induce d apparent DA efflux in a concentration-dependent manner. Their EC50 v alues for inducing DA efflux were similar to their IC50 values for inh ibiting DA uptake. The substrate-induced DA efflux was inhibited by va rious NET blockers, enhanced by raising the internal [Na+] with Na+, K +-ATPase inhibition, but was insensitive to membrane potential-alterin g agents valinomycin, veratridine, and high [Ki],The initial rate of m -tyramine-induced DA efflux was related to preloaded [DA] in a manner defined by a Michaelis-Menten expression. In contrast, DA efflux in th e presence of cocaine displayed a much slower efflux rate, lower effic acy, was not stimulated by elevated internal [Na+], and was nonsaturab le with preloaded [DA], Single exponential kinetic analysis of the ent ire lime course of the DA efflux showed that the apparent first-order rate constant for m-tyramine-induced DA efflux declined with increased preloaded [DA],whereas that for the DA efflux in the presence of coca ine was unchanged with varying preloaded [DA], These results suggest t hat the substrates stimulate the NET-dependent DA efflux by increasing the accessibility of the NET to internal DA, whereas cocaine ''uncove rs'' NET-independent DA efflux by reducing the accessibility of diffus ed/leaked external DA to the NET.