Voltammetric studies on mechanisms of dopamine efflux in the presence of 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 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.
Department of Pharmacology, Nanjing Medical University, PRC.
N
N
Chen
C G
CG
Trowbridge
J B
JB
Justice
D018759:Adrenergic Uptake Inhibitors / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003042:Cocaine / Q000494:pharmacology*; D003891:Desipramine / Q000494:pharmacology; D004298:Dopamine / Q000032:analysis / Q000378:metabolism*; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005473:Fluoxetine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D018374:LLC-PK1 Cells / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009638:Norepinephrine / Q000037:antagonists & inhibitors / Q000378:metabolism; D050484:Norepinephrine Plasma Membrane Transport Proteins; D010042:Ouabain / Q000494:pharmacology; D010627:Phenethylamines / Q000494:pharmacology; D011619:Psychotropic Drugs / Q000494:pharmacology; D013552:Swine; D027981:Symporters; D013997:Time Factors; D014439:Tyramine / Q000494:pharmacology; D014634:Valinomycin / Q000494:pharmacology; D014701:Veratridine / Q000494:pharmacology
D018759:Adrenergic Uptake Inhibitors; D002352:Carrier Proteins; D018765:Dopamine Uptake Inhibitors; D004791:Enzyme Inhibitors; D007476:Ionophores; D050484:Norepinephrine Plasma Membrane Transport Proteins; D010627:Phenethylamines; D011619:Psychotropic Drugs; C493227:SLC6A2 protein, human; D027981:Symporters; D005473:Fluoxetine; C011386:nisoxetine; D014634:Valinomycin; C029261:phenethylamine; D010042:Ouabain; D014701:Veratridine; D003042:Cocaine; C028169:3-tyramine; D003891:Desipramine; D004298:Dopamine; D009638:Norepinephrine; D014439:Tyramine