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We investigated the effect of sphingosine 1-phosphate (S1P) on intracellular Ca(2+) dynamics in rat vascular smooth muscle cells (VSMCs).,Intracellular Ca(2+) concentration ([Ca(2+) ]i) was determined using a fluorescence dye fura-2/AM. Small interfering RNAs (siRNA) were transfected into VSMCs to deplete the expression of S1P2 and S1P3 receptors.,S1P induced a rapid and transient elevation in [Ca(2+) ]i, which was maximal 1min after the stimulation, followed by a sustained increase. When extracellular Ca(2+) was removed, a decrease in resting level and a small and transient increase in [Ca(2+) ]i by S1P stimulation were observed. siRNA targeted for the S1P3 receptor almost completely inhibited the S1P-induced increase in [Ca(2+) ]i. The rapid and transient increase in [Ca(2+) ]i was significantly inhibited by diltiazem at a high concentration. Pertussis toxin and a phospholipase C (PLC) inhibitor inhibited the S1P-induced increase in [Ca(2+) ]i regardless of the presence of extracellular Ca(2+) . Furthermore, S1P activated store-operated and receptor-operated Ca(2+) entry.,These results suggest that S1P increases [Ca(2+) ]i via the S1P3 receptor by inducing an influx of extracellular Ca(2+) partially through the voltage-dependent Ca(2+) channels, as well as by mobilizing Ca(2+) from its intracellular stores. S1P3 receptor-coupled Gi/o protein and PLC activation mediate the mechanisms.