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 plasmalemm al serotonin transporter (rSERT) were incubated with 5-[H-3]hydroxytryptami ne (H-3]5-HT) and superfused. Substrates of the rSERT, such as p-chloroamph etamine (PCA) or methylenedioxymethamphetamine, concentration-dependently i ncreased basal efflux of [H-3]5-HT. 5-HT reuptake blockers (e.g., imipramin e citalopram) also caused an enhancement of [H-3]5-HT efflux: reaching abou t half the maximal effect of the rSERT substrates, In uptake experiments, b oth groups of substances concentration-dependently inhibited 5-HT uptake. E C50 values obtained in superfusion experiments significantly correlated wit h IC50 values from uptake studies (r(2) = 0.92). Addition of the Na+,K+-ATP ase inhibitor ouabain (100 mu M) to or the omission of K+ from the superfus ion buffer accelerated basal efflux, The effect of PCA (10 mu M) was marked ly enhanced by both measures, whereas the effect of uptake inhibitors remai ned unchanged. When [H-3]MPP+, a substrate with low affinity for the rSERT, was used instead of [H-3]5-HT for labeling the cells, uptake inhibitors fa iled to augment efflux. By contrast, PCA accelerated [H-3]MPP+ efflux, and its effect was strongly enhanced in the presence of ouabain, The results su ggest that the [H-3]5-HT efflux caused by substrates of rSERT is carrier-me diated, whereas efflux induced by uptake inhibitors is a consequence of int errupted high-affinity reuptake that is ongoing even under superfusion cond itions.