1. 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-HT1A, 5-HT1B, 5-HT2A, and 5-HT2C) and as cAMP and intracellular Ca 2+ are modulated by different 5-HT receptors, we studied both pathways. 2. Methods: 5-HT uptake was determined as imipramine-inhibitable uptake of [3H]5-HT, intracellular cAMP was measured by RIA and intracellular Ca2+ changes were determined using the ratiometric method of intracellular Ca2+ imaging. 3. Results: For uptake experiments, cells were kept for 30 min either with or without 1 μM 5-HT in the medium before measuring uptake. Removal of 5-HT for 30 min significantly decreased [3H]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 Ca2+ levels either; however, adding 1 μM 5-HT after 5 min in 5-HT-free conditions rapidly increased intracellular Ca2+ levels in 50% of the cells. The remaining cells showed no changes in the intracellular Ca2+ levels. 4. 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 Ca2+ levels. This suggests that 5-HT may utilize intracellular Ca2+ to regulate 5-HT uptake.
- RN46A cells
- Serotonin transporter
ASJC Scopus subject areas
- Cellular and Molecular Neuroscience
- Cell Biology