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.
Footnotes
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Send reprint requests to: Dr. Harald H. Sitte, Department of Pharmacology, University of Vienna, Waehringerstr. 13a, A-1090 Vienna, Austria. E-mail:harald.sitte{at}univie.ac.at
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↵1 This work was supported by the Austrian Science Foundation, Project P13183. Part of the work was presented at the Summer Meeting of the British and German Pharmacological Societies at the University of Nottingham, UK, July 1999.
- Abbreviations:
- 5-HT
- 5-hydroxytryptamine
- hSERT
- human serotonin transporter
- SERT
- serotonin transporter
- PCA
- p-chloroamphetamine
- MPP+
- N-methyl-4-phenylpyridinium
- Received October 18, 1999.
- Accepted February 4, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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