Voltammetric Studies on Mechanisms of Dopamine Efflux in the Presence of Substrates and Cocaine from Cells Expressing Human Norepinephrine Transporter

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Abstract
The effects of substrates m‐tyramine and β‐phenethylamine, as well as cocaine, on the DA efflux from a cell line stably expressing the human norepinephrine transporter (hNET) were investigated by using rotating disk electrode voltammetry. Both the substrates and cocaine induced apparent DA efflux in a concentration‐dependent manner. Their EC50 values for inducing DA efflux were similar to their IC50 values for inhibiting DA uptake. The substrate‐induced DA efflux was inhibited by various NET blockers, enhanced by raising the internal [Na+] with Na+,K+‐ATPase inhibition, but was insensitive to membrane potential‐altering agents valinomycin, veratridine, and high [K+]. 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 the presence of cocaine displayed a much slower efflux rate, lower efficacy, was not stimulated by elevated internal [Na+], and was nonsaturable with preloaded [DA]. Single exponential kinetic analysis of the entire time 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 cocaine was unchanged with varying preloaded [DA]. These results suggest that the substrates stimulate the NET‐dependent DA efflux by increasing the accessibility of the NET to internal DA, whereas cocaine “uncovers” NET‐independent DA efflux by reducing the accessibility of diffused/leaked external DA to the NET.

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