Characterization of functional responses in A9 cells transfected with cloned rat 5-HT(1C) receptors

Functional responses to stimulation of rat 5-HT(1C) receptors expressed in A9 cells were studied using whole cell voltage clamp and calcium recording techniques. Stimulation of 5-HT(1C) receptors evoked outward currents clamped at -50 mV. The outward currents were reduced when GTP was excluded from the intracellular recording solution or when GDP-β-S was added. 8-Bromo cyclic AMP (5 mmol/l) neither produced an effect per se nor affected the 5-HT-induced outward current in A9 cells, thus excluding cAMP as a second messenger involved in 5-HT(1C) receptor activation. Phorbol myristic acetate (PMA; 10 μmol/l) did not affect the electrical activity of the transfected A9 cells but reduced the 5-HT-induced current amplitude to 71 ± 9% of the control value (n = 12). This indicates that activation of protein kinase C does not play a direct role in the 5-HT-induced response in these cells. The 5-HT induced currents mainly involved potassium ions, although a small contribution of chloride ions was also observed. The 5-HT-induced current was inhibited by the K+channel blocking agents tetraethylammonium (1 mmol/l), apamin (0,5 μmol/l) and 4-amino-pyridine (5 mmol/l). The 5-HT-induced currents recorded at -50 mV were unaffected by removal of extracellular calcium, but inclusion of the calcium chelator BAPTA (5 mmol/l) in the intracellular solutions abolished the current. Measurement with the calcium indicator Fluo-3 revealed a 5-HT-induced increase in intracellular calcium which was not affected by removal of extracellular calcium but declined after repeated stimulation. Determinated of pD2and K(B) values of several 5-HT ligands using Fura-2 calcium measurements confirmed the pharmacology of the 5-HT(1C) receptor. The results show that cloned rat 5-HT(1C) receptors expressed in A9 cells activate a calcium-dependent potassium conductance