[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: Research - peer-reviewArticle

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Abstract

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.

LanguageEnglish (US)
Pages97-106
Number of pages10
JournalSynapse
Volume30
Issue number1
DOIs
StatePublished - Sep 1998

Fingerprint

Serotonin Plasma Membrane Transport Proteins
Dopamine Plasma Membrane Transport Proteins
Serotonin
Dopamine
Pharmaceutical Preparations
Methamphetamine
Amphetamine
Cocaine
GBR 12935
Mazindol
Tropanes
Methylphenidate
Imipramine
Norepinephrine
RTI 55
Drug Liberation
Fenfluramine
Carboxylic Acids
Glioma
Esters

Keywords

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

ASJC Scopus subject areas

  • Physiology
  • Neuroscience(all)
  • Pharmacology
  • Advanced and Specialized Nursing

Cite this

[3H]Substrate- and cell-specific effects of uptake inhibitors on human dopamine and serotonin transporter-mediated efflux. / Johnson, Robert A.; Eshleman, Amy J.; Meyers, Toni; Neve, Kim A.; Janowsky, Aaron.

In: Synapse, Vol. 30, No. 1, 09.1998, p. 97-106.

Research output: Research - peer-reviewArticle

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N2 - 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.

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