TY - JOUR T1 - Transporter-Mediated Release: A Superfusion Study on Human Embryonic Kidney Cells Stably Expressing the Human Serotonin Transporter JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 870 LP - 878 VL - 293 IS - 3 AU - Petra Scholze AU - Julia Zwach AU - Alexandra Kattinger AU - Christian Pifl AU - Ernst A. Singer AU - Harald H. Sitte Y1 - 2000/06/01 UR - http://jpet.aspetjournals.org/content/293/3/870.abstract N2 - 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. The American Society for Pharmacology and Experimental Therapeutics ER -