Effect of Sphingosine-1-Phosphate on Intracellular Free Ca(2+) in Cat Esophageal Smooth Muscle Cells

A comprehensive collection of proteins senses local changes in intracellular Ca(2+) concentrations ([Ca(2+)](i)) and transduces these signals into responses to agonists. In the present study, we examined the effect of sphingosine-1-phosphate (S1P) on modulation of intracellular Ca(2+) concentrations in cat esophageal smooth muscle cells. To measure [Ca(2+)](i) levels in cat esophageal smooth muscle cells, we used a fluorescence microscopy with the Fura-2 loading method. S1P produced a concentration-dependent increase in [Ca(2+)](i) in the cells. Pretreatment with EGTA, an extracellular Ca(2+) chelator, decreased the S1P-induced increase in [Ca(2+)](i), and an L-type Ca(2+)-channel blocker, nimodipine, decreased the effect of S1P. This indicates that Ca(2+) influx may be required for muscle contraction by S1P. When stimulated with thapsigargin, an intracellular calcium chelator, or 2-Aminoethoxydiphenyl borate (2-APB), an InsP(3) receptor blocker, the S1P-evoked increase in [Ca(2+)](i) was significantly decreased. Treatment with pertussis toxin (PTX), an inhibitor of G(i)-protein, suppressed the increase in [Ca(2+)](i) evoked by S1P. These results suggest that the S1P-induced increase in [Ca(2+)](i) in cat esophageal smooth muscle cells occurs upon the activation of phospholipase C and subsequent release of Ca(2+) from the InsP(3)-sensitive Ca(2+) pool in the sarcoplasmic reticulum. These results suggest that S1P utilized extracellular Ca(2+) via the L type Ca(2+) channel, which was dependent on activation of the S1P(4) receptor coupled to PTX-sensitive G(i) protein, via phospholipase C-mediated Ca(2+) release from the InsP(3)-sensitive Ca(2+) pool in cat esophageal smooth muscle cells.