[3H]substrate- 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 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 t1/2 values of approximately 15 minutes, while the t1/2 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 t1/2 values, as compared to substrates, suggesting different mechanisms for altering transporter function. Dose-response curves for each drug, conducted at each drug's t1/2, indicated considerable differences in potency (EC50) at stimulating [3H]MPP+ release from C6-hSERT cells [3beta-(4-iodophenyl)tropane-2beta-carboxylic acid methyl ester (RTI-55) > imipramine > 1-[2-diphenylmethoxy]ethyl-4-(3-phenylpropyl)-piperazine (GBR-12935) threo-(+/-)-methylphenidate > cocaine > mazindol > 2-beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) > (+)methamphetamine > amphetamine > DA > fenfluramine > 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 approximately 75% of loaded [3H]MPP+, cocaine analogues causing approximately 50% release, and other drugs causing an average release of approximately 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.
Johnson
R A
RA
Research Service, Veterans Affairs Medical Center, Portland, Oregon 97201, USA.
Eshleman
A J
AJ
Meyers
T
T
Neve
K A
KA
Janowsky
A
A
D015655:1-Methyl-4-phenylpyridinium / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D003042:Cocaine / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism* / Q000494:pharmacology; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology*; D005910:Glioma; D006207:Half-Life; D006801:Humans; D007099:Imipramine / Q000494:pharmacology; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008694:Methamphetamine / Q000494:pharmacology; D009419:Nerve Tissue Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism* / Q000494:pharmacology; D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors / Q000494:pharmacology*; D014162:Transfection; D014316:Tritium
D015655:1-Methyl-4-phenylpyridinium; D002118:Calcium; D002352:Carrier Proteins; D003042:Cocaine; D004298:Dopamine; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D007099:Imipramine; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008694:Methamphetamine; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D012701:Serotonin; D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors; D014316:Tritium; C493230:SLC6A4 protein, human; C493232:Slc6a4 protein, rat