[3H]Substrate- and cell-specific effects of uptake inhibitors on human dopamine and serotonin transporter-mediated efflux

Robert A. Johnson, Amy J. Eshleman, Toni Meyers, Kim A. Neve, Aaron Janowsky

Research output: Contribution to journalArticlepeer-review

40 Scopus citations


Drug-induced efflux of substrates was characterized in C6 rat glioma cells stably expressing a recombinant human dopamine (DA) or serotonin (5- HT) transporter (C6-hDAT and C6-hSERT, respectively). In the absence of Ca2+, these cells spontaneously and rapidly released preloaded [3H]DA or [3H]5-HT, respectively, but maintained constant levels of [3H]N-methy-4- phenylpyridinium (MPP+) for up to 90 minutes. In C6-hSERT cells, transporter substrates such as methamphetamine, amphetamine, and dopamine induced relatively rapid release of [3H]MPP+, with t 1/4 values of approximately 15 minutes, while the t 1/4 value for serotonin was about 30 minutes. Similar results were obtained with C6-hDAT cells. Uptake blockers that are not substrates at the transporters had considerably greater t 1/4 values, as compared to substrates, suggesting different mechanisms for altering transporter function. Dose-response curves for each drug, conducted at each drug's t 1/4 , indicated considerable differences in potency (EC50) at stimulating [3H]MPP+ release from C6-hSERT cells [3β(4- iodophenyl)tropane-2β-carboxylic acid methyl ester (RTI-55) > imipramine > 1-[2-diphenylmethoxy]ethyl-4-(3-phenylpropyl)-piperazine (GBR-12935) threo- (±)methylphenidate > cocaine > mazindol > 2-β-carbomethoxy-3β-(4- fluorophenyl)tropane (CFT) > (+)methamphetamine > amphetamine > DA > fenfiuramine > norepinephrine (NE) > 5-HT]. A different rank order of potency was observed for the effects of drugs on [3H]MPP+ release from C6-hDAT cells [imipramine > RTI-55 > cocaine > mazindol > CFT > GBR-12935 > threo- (±)-methylphenidate > amphetamine > (+)methamphetamine > fenfluramine > DA > NE > 5-HT]. Based on efficacies for stimulating [3H]MPP+ release from C6- hDAT cells, drugs could be grouped into three categories, with substrates causing release of ~75% of loaded [3H]MPP+, cocaine analogues causing ~50% release, and other drugs causing an average release of ~25% of loaded [3H]MPP+. The results, taken together 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 transporter-mediated release may not be a simple reversal of substrate uptake.

Original languageEnglish (US)
Pages (from-to)97-106
Number of pages10
Issue number1
StatePublished - Sep 1998


  • Amphetamine
  • Cocaine
  • Fenfluramine
  • GBR12935
  • MPP
  • Mazindol
  • Release

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience


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