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) were grown on coverslips, preincubated with [(3)H]5-hydroxytryptamine (5-HT), and superfused. Substrates of the hSERT [e.g., p-chloroamphetamine (PCA)], increased the basal efflux of [(3)H]5-HT in a concentration-dependent manner. 5-HT reuptake blockers (e.g., imipramine, paroxetine) also raised [(3)H]5-HT efflux, reaching approximately one-third of the maximal effect of the hSERT substrates. In uptake experiments, both groups of substances inhibited [(3)H]5-HT uptake. Using the low-affinity substrate [(3)H]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 [(3)H]MPP(+) efflux in both types of cells, and its effect was inhibited by paroxetine. The addition of the Na(+),K(+)-ATPase inhibitor ouabain (100 microM) to the superfusion buffer enhanced basal efflux of [(3)H]5-HT-loaded hSERT cells by approximately 2-fold; the effect of PCA (10 microM) was strongly augmented by ouabain, whereas the effect of imipramine was not. The Na(+)/H(+) ionophore monensin (10 microM) also augmented the effect of PCA on efflux of [(3)H]5-HT as well as on efflux of [(3)H]MPP(+). In [(3)H]5-HT-labeled cells, the combination of imipramine and monensin raised [(3)H]5-HT efflux to a greater extent than either of the two substances alone. In [(3)H]MPP(+)-labeled cells, imipramine had no effect on its own and fully reversed the effect of monensin. The results suggest that the [(3)H]5-HT efflux caused by uptake inhibitors is entirely due to interrupted high-affinity reuptake, which is ongoing even under superfusion conditions.
Scholze
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Department of Pharmacology, University of Vienna, Austria. harald.sitte@univie.ac.at
Zwach
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J
Kattinger
A
A
Pifl
C
C
Singer
E A
EA
Sitte
H H
HH
D015655:1-Methyl-4-phenylpyridinium / Q000378:metabolism; D002352:Carrier Proteins / Q000502:physiology*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005277:Fenfluramine / Q000494:pharmacology; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000502:physiology*; D026901:Membrane Transport Proteins; D008985:Monensin / Q000494:pharmacology; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology; D009419:Nerve Tissue Proteins; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors / Q000494:pharmacology; D014162:Transfection; D010133:p-Chloroamphetamine / Q000494:pharmacology
D015655:1-Methyl-4-phenylpyridinium; D002352:Carrier Proteins; D005277:Fenfluramine; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008985:Monensin; D008996:Monoamine Oxidase Inhibitors; D009419:Nerve Tissue Proteins; D012701:Serotonin; D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors; D010133:p-Chloroamphetamine; C493230:SLC6A4 protein, human