Transporter-mediated release: A superfusion study on human embryonic kidney cells stably expressing the human serotonin transporter

HEK 293 cells stably expressing the human serotonin transporter (hSERT) wer e grown on coverslips, preincubated with [H-3]5-hydroxytryptamine (5-HT), a nd superfused. Substrates of the hSERT [e.g., p-chloroamphetamine (PCA)], i ncreased the basal efflux of [H-3]5-HT in a concentration-dependent manner. 5-HT reuptake blockers (e.g., imipramine, paroxetine) also raised [H-3]5-H T efflux, reaching approximately one-third of the maximal effect of the hSE RT substrates. In uptake experiments, both groups of substances inhibited [ H-3]5-HT uptake. Using the low-affinity substrate [H-3]N-methyl-4-phenylpyr idinium (MPP+) to label the cells in superfusion experiments, reuptake inhi bitors failed to enhance efflux. Similar results were obtained using human placental choriocarcinoma (JAR) cells that constitutively express the hSERT at a low level. By contrast, PCA raised [H-3]MPP+ efflux in both types of cells, and its effect was inhibited by paroxetine. The addition of the Na+, K+-ATPase inhibitor ouabain (100 mu M) to the superfusion buffer enhanced b asal efflux of [H-3]5-HT-loaded hSERT cells by approximately 2-fold; the ef fect of PCA (10 mu M) was strongly augmented by ouabain, whereas the effect of imipramine was not. The Na+/H+ ionophore monensin (10 mu M) also augmen ted the effect of PCA on efflux of [H-3]5-HT as well as on efflux of [H-3]M PP+. In [H-3]5-HT-labeled cells, the combination of imipramine and monensin raised [H-3]5-HT efflux to a greater extent than either of the two substan ces alone. In [H-3]MPP+-labeled cells, imipramine had no effect on its own and fully reversed the effect of monensin. The results suggest that the [H- 3]5-HT efflux caused by uptake inhibitors is entirely due to interrupted hi gh-affinity reuptake, which is ongoing even under superfusion conditions.