Serotonin regulation of serotonin uptake in RN46A cells.
OBJECTIVE
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.
Koldzic-Zivanovic
Nina
N
Department of Microbiology and Immunology, University of Texas Medical Branch, 301 University Blvd., Galveston 77555-1019, USA.
Seitz
Patricia K
PK
Cunningham
Kathryn A
KA
Thomas
Mary L
ML
Hughes
Thomas K
TK
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D008526:Medulla Oblongata / Q000196:embryology / Q000378:metabolism; D011903:Raphe Nuclei / Q000196:embryology / Q000378:metabolism*; D051381:Rats; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000502:physiology*
D050486:Serotonin Plasma Membrane Transport Proteins; D012701:Serotonin; D000242:Cyclic AMP; D002118:Calcium