[H-3]SUBSTRATE-SPECIFIC AND CELL-SPECIFIC EFFECTS OF UPTAKE INHIBITORS ON HUMAN DOPAMINE AND SEROTONIN TRANSPORTER-MEDIATED EFFLUX

Drug-induced efflux of substrates was characterized in C6 rat glioma c ells stably expressing a recombinant human dopamine (DA) or serotonin (5-HT) transporter (CG-hDAT and CG-hSERT, respectively). In the absenc e of Ca2+, these cells spontaneously and rapidly released preloaded [H -3]DA or [H-3]5-HT, respectively, but maintained constant levels of [H -3]N-methy-4-phenylpyridinium (MPP+) for up to 90 minutes. In C6-hSERT cells, transporter substrates such as methamphetamine, amphetamine, a nd dopamine induced relatively rapid release of [H-3]MPP+, with t(1/2) values of approximately 15 minutes, while the t(1/2) value for seroto nin was about 30 minutes. Similar results were obtained with C6-hDAT c ells. Uptake blockers that are not substrates at the transporters had considerably greater t(1/2) values, as compared to substrates, suggest ing different mechanisms for altering transporter function. Dose-respo nse curves for each drug, conducted at each drug's t(1/2), indicated c onsiderable differences in potency (EC50) at stimulating [H-3]MPP+ rel ease from C6hSERT cells [3 beta-(4-iodophenyl)tropane-2 beta-carboxyli c acid methyl ester (RTI-55) > imipramine > 1- iphenylmethoxy]ethyl-4- (3-phenylpropyl)-piperazine (GBR-12935) threo-(+/-)methylphenidate > c ocaine > mazindol > 2-beta-carbometl loxy-3 beta-(4-fluorophenyl)tropa ne (CFT) > (+)methamphetamine > amphetamine > DA > fenfluramine > nore pinephrine (NE) > 5-HT].A different rank order of potency was observed for the effects of drugs on [H-3]MPP+ release from C6-hDAT cells [imi pramine > RTI-55 > cocaine > mazindol > CFT > GBR-12935 > threo-(+/-)- methylphenidate > amphetamine > (+)methamphetamine > fenfluramine > DA > NE > 5-HT]. Based on efficacies for stimulating [H-3]MPP+ release f rom C6-hDAT cells, drugs could be grouped into three categories, with substrates causing release of similar to 75% of loaded [H-3]MPP+, coca ine analogues causing -50% release, and other drugs causing an average release of similar to 25% of loaded [H-3]MPP+. The results, taken tog ether with results from previous reports, suggest that the transfected cell type contributes to the characteristics of transporter-mediated release, that drugs interact with different sites on the transporters in the uptake and release process, and that the mechanism of transport er-mediated release may not be a simple reversal of substrate uptake. Synapse 30:97-106, 1998. (C) 1998 Wiley-Liss, Inc.