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‐[ 3 H]hydroxytryptamine ([ 3 H]5‐HT) and superfused. Substrates of the rSERT, such as p ‐chloroamphetamine (PCA) or methylenedioxymethamphetamine, concentration‐dependently increased basal efflux of [ 3 H]5‐HT. 5‐HT reuptake blockers (e.g., imipramine, citalopram) also caused an enhancement of [ 3 H]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 ( r 2 = 0.92). Addition of the Na + ,K + ‐ATPase inhibitor ouabain (100 μ M ) to or the omission of K + from the superfusion buffer accelerated basal efflux. The effect of PCA (10 μ M ) was markedly enhanced by both measures, whereas the effect of uptake inhibitors remained unchanged. When [ 3 H]MPP + , a substrate with low affinity for the rSERT, was used instead of [ 3 H]5‐HT for labeling the cells, uptake inhibitors failed to augment efflux. By contrast, PCA accelerated [ 3 H]MPP + efflux, and its effect was strongly enhanced in the presence of ouabain. The results suggest that the [ 3 H]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.