Characterization of carrier-mediated efflux in human embryonic kidney 293 cells stably expressing the rat serotonin transporter: a superfusion study.
Human embryonic kidney 293 cells stably transfected with the rat plasmalemmal serotonin transporter (rSERT) were incubated with 5-[3H]hydroxytryptamine ([3H]5-HT) and superfused. Substrates of the rSERT, such as p-chloroamphetamine (PCA) or methylenedioxymethamphetamine, concentration-dependently increased basal efflux of [3H]5-HT. 5-HT reuptake blockers (e.g., imipramine, citalopram) also caused an enhancement of [3H]5-HT efflux, reaching about half the maximal effect of the rSERT substrates. In uptake experiments, both groups of substances concentration-dependently inhibited 5-HT uptake. EC50 values obtained in superfusion experiments significantly correlated with IC50 values from uptake studies (r2 = 0.92). Addition of the Na+,K(+)-ATPase inhibitor ouabain (100 microM) to or the omission of K+ from the superfusion buffer accelerated basal efflux. The effect of PCA (10 microM) was markedly enhanced by both measures, whereas the effect of uptake inhibitors remained unchanged. When [3H]MPP+, a substrate with low affinity for the rSERT, was used instead of [3H]5-HT for labeling the cells, uptake inhibitors failed to augment efflux. By contrast, PCA accelerated [3H]MPP+ efflux, and its effect was strongly enhanced in the presence of ouabain. The results suggest that the [3H]5-HT efflux caused by substrates of rSERT is carrier-mediated, whereas efflux induced by uptake inhibitors is a consequence of interrupted high-affinity reuptake that is ongoing even under superfusion conditions.
Sitte
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HH
Department of Pharmacology, University of Vienna, Austria.
Scholze
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Schloss
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Pifl
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Singer
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D015655:1-Methyl-4-phenylpyridinium / Q000493:pharmacokinetics; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism* / Q000502:physiology*; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010042:Ouabain / Q000494:pharmacology; D010477:Perfusion; D051381:Rats; D012701:Serotonin / Q000493:pharmacokinetics; D018490:Serotonin Agents / Q000494:pharmacology; D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors; D014162:Transfection; D010133:p-Chloroamphetamine / Q000494:pharmacology
D015655:1-Methyl-4-phenylpyridinium; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010042:Ouabain; D012701:Serotonin; D018490:Serotonin Agents; D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors; D000254:Sodium-Potassium-Exchanging ATPase; D010133:p-Chloroamphetamine; C493230:SLC6A4 protein, human; C493232:Slc6a4 protein, rat