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Serotonin regulation of serotonin uptake in RN46A cells. | LitMetric
Aim : The role of the serotonin transporter (SERT) is to remove serotonin (5-HT) from the synaptic space. In vitro studies have shown that 5-HT uptake via SERT is influenced by the availability of its substrate, 5-HT. We used RN46A cells, a line that expresses SERT, to investigate 5-HT regulation of 5-HT uptake and the intracellular signaling pathways involved. RN46A cells also express mRNAs for 5-HT receptors (5-HT(1A), 5-HT(1B), 5-HT(2A), and 5-HT(2C)) and as cAMP and intracellular Ca(2+) are modulated by different 5-HT receptors, we studied both pathways.Methods : 5-HT uptake was determined as imipramine-inhibitable uptake of [(3)H]5-HT, intracellular cAMP was measured by RIA and intracellular Ca(2+) changes were determined using the ratiometric method of intracellular Ca(2+) imaging.Results : For uptake experiments, cells were kept for 30 min either with or without 1 microM 5-HT in the medium before measuring uptake. Removal of 5-HT for 30 min significantly decreased [(3)H]5-HT uptake. The absence of 5-HT for 15 min failed to induce any changes in intracellular cAMP levels. Removal of 5-HT from the medium did not change intracellular Ca(2+) levels either; however, adding 1 microM 5-HT after 5 min in 5-HT-free conditions rapidly increased intracellular Ca(2+) levels in 50% of the cells. The remaining cells showed no changes in the intracellular Ca(2+) levels.Conclusions : We have shown that in RN46A cells, that endogenously express SERT and mRNAs for several 5-HT receptors, changes in 5-HT levels influence 5-HT uptake rate as well as induce changes in intracellular Ca(2+) levels. This suggests that 5-HT may utilize intracellular Ca(2+) to regulate 5-HT uptake.
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