5-Hydroxytryptamine-stimulated calcium mobilization in cultured canine tracheal smooth muscle cells

Abstract

5-Hydroxytryptamine (5-HT)-induced increase of intracellular Ca 2+ concentration ([Ca 2+] i) was monitored in cultured canine tracheal smooth muscle cells (TSMCs) using a fluorescent Ca 2+ indicator Fura-2. Stimulation of TSMCs by 5-HT produced an initial transient peak followed by a sustained, concentration-dependent elevation of [Ca 2+] i. The log (EC 50) values of 5-HT for the peak and sustained plateau responses were −7.43 and −7.60 M, respectively. 5-HT 1A and 5-HT 3 receptor antagonists, NAN-190 and metoclopramide, inhibited the 5-HT-stimulated increase in [Ca 2+] i with pK B values of 6.3 and 6.2, respectively, indicating that the 5-HT receptors mediating Ca 2+ signal had low affinity for these receptor antagonists. In contrast, 5-HT 2A receptor antagonists, ketanserin and mianserin, had high affinity in antagonizing the changes in [Ca 2+] i response to 5-HT with pK B values of 8.3 and 8.3, respectively. The sustained elevation of [Ca 2+] i was dependent on the presence of extracellular Ca 2+. Removal of extracellular Ca 2+ by addition of 2 mM EGTA during the sustained phase caused a rapid decline in [Ca 2+] i to the resting level. In the absence of extracellular Ca 2+, only an initial peak was observed which then declined to the resting level; the sustained elevation of [Ca 2+] i could then be evoked by addition of 1.8 mM Ca 2+ in the continued presence of 5-HT. Ca 2+ influx was required for the changes of [Ca 2+] i, since the Ca 2+-channel blockers, diltiazem, verapamil, and Ni 2+, decreased both the initial and sustained elevation of [Ca 2+] i in response to 5-HT. These Ca 2+-channel blockers also decreased the sustained elevation of [Ca 2+] i when applied during the plateau phase. In conclusion, these findings indicate that the initial increase in [Ca 2+] i stimulated by 5-HT acting on 5-HT 2A receptors is due to the release of Ca 2+ from internal stores, followed by the influx of external Ca 2+ into the cells. The influx of extracellular Ca 2+ partially involves a diltiazem and verapamil sensitive Ca 2+ channel.