Sphingosine 1-phosphate increases an intracellular Ca2+ concentration via S1P3 receptor in cultured vascular smooth muscle cells

Objective We investigated the effect of sphingosine 1-phosphate (S1P) on intracellular Ca2+ dynamics in rat vascular smooth muscle cells (VSMCs). Methods Intracellular Ca2+ concentration ([Ca2+]i) was determined using a fluorescence dye fura-2/AM. Small interfering RNAs (siRNA) were transfected into VSMCs to deplete the expression of S1P(2) and S1P(3) receptors. Key findings S1P induced a rapid and transient elevation in [Ca2+]i, which was maximal 1 min after the stimulation, followed by a sustained increase. When extracellular Ca2+ was removed, a decrease in resting level and a small and transient increase in [Ca2+]i by S1P stimulation were observed. siRNA targeted for the S1P(3) receptor almost completely inhibited the S1P-induced increase in [Ca2+]i. The rapid and transient increase in [Ca2+]i was significantly inhibited by diltiazem at a high concentration. Pertussis toxin and a phospholipase C (PLC) inhibitor inhibited the S1P-induced increase in [Ca2+]i regardless of the presence of extracellular Ca2+. Furthermore, S1P activated store-operated and receptor-operated Ca2+ entry. Conclusions These results suggest that S1P increases [Ca2+]i via the S1P(3) receptor by inducing an influx of extracellular Ca2+ partially through the voltage-dependent Ca2+ channels, as well as by mobilizing Ca2+ from its intracellular stores. S1P(3) receptor-coupled G(i/o) protein and PLC activation mediate the mechanisms.