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Keywords:

  • Cocaine;
  • Dopamine;
  • Human norepinephrine transporter;
  • β-Phenethylamine;
  • m-Tyramine;
  • Rotating disk electrode voltammetry

Abstract

  1. Top of page
  2. Abstract

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.

Abbreviations used: DA, dopamine; DAT, dopamine transporter; hNET, human norepinephrine transporter; LLC-hNET, LLC-PK1 cells stably expressing the hNET; NE, norepinephrine; NET, NE transporter; RDEV, rotating disk electrode voltammetry.