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Vol. 293, Issue 3, 870-878, June 2000
Department of Pharmacology (P.S., J.Z., E.A.S., H.H.S.) and Brain
Research Institute (A.K., C.P.), University of Vienna, Vienna, Austria
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.
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