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 Gi-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 Cmediated Ca 2+ release from the InsP 3 -sensitive Ca 2+ pool in cat esophageal smooth muscle cells.