Abstract

HEK 293 cells stably expressing the human serotonin transporter (hSERT) were grown on coverslips, preincubated with [³H]5-hydroxytryptamine (5-HT), and superfused. Substrates of the hSERT [e.g., p-chloroamphetamine (PCA)], increased the basal efflux of [³H]5-HT in a concentration-dependent manner. 5-HT reuptake blockers (e.g., imipramine, paroxetine) also raised [³H]5-HT efflux, reaching approximately one-third of the maximal effect of the hSERT substrates. In uptake experiments, both groups of substances inhibited [³H]5-HT uptake. Using the low-affinity substrate [³H]N-methyl-4-phenylpyridinium (MPP⁺) to label the cells in superfusion experiments, reuptake inhibitors 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 [³H]MPP⁺ efflux in both types of cells, and its effect was inhibited by paroxetine. The addition of the Na⁺,K⁺-ATPase inhibitor ouabain (100 μM) to the superfusion buffer enhanced basal efflux of [³H]5-HT-loaded hSERT cells by approximately 2-fold; the effect of PCA (10 μM) was strongly augmented by ouabain, whereas the effect of imipramine was not. The Na⁺/H⁺ionophore monensin (10 μM) also augmented the effect of PCA on efflux of [³H]5-HT as well as on efflux of [³H]MPP⁺. In [³H]5-HT-labeled cells, the combination of imipramine and monensin raised [³H]5-HT efflux to a greater extent than either of the two substances alone. In [³H]MPP⁺-labeled cells, imipramine had no effect on its own and fully reversed the effect of monensin. The results suggest that the [³H]5-HT efflux caused by uptake inhibitors is entirely due to interrupted high-affinity reuptake, which is ongoing even under superfusion conditions.