Stimulation of intracellular sphingosine-1-phosphate production by G-protein-coupled sphingosine-1-phosphate receptors

Recently, a family of G-protein-coupled receptors named endothelial differe ntiation gene (Edg) receptor family has: been identified, which are specifi cally activated by the two serum lipids, sphingosine-1-phosphate and lysoph osphatidic acid. Sphingosine-1-phosphate can also act intracellularly to re lease Ca2+ from intracellular stores. Since in several cell types, G-protei n-coupled lysophosphatidic acid or sphingosine-1-phosphate receptors mobili ze Ca2+ in the absence of a measurable phospholipase C stimulation, it was analysed here whether intracellular sphingosine-1-phosphate production was the signalling mechanism used by extracellular sphingosine-1-phosphate for mobilization of stored Ca2+. Sphingosine-1-phosphate and the low affinity s phingosine-1-phosphate receptor agonist. sphingosylphosphorylcholine. induc ed a rapid, transient and nearly complete pertussis toxin-sensitive Ca2+ mo bilization in human embryonic kidney (HEK-293) cells. The G-protein-coupled sphingosine-1-phosphate receptors, Edg-1, Edg-3 and Edg-5, were found to b e endogenously expressed in these cells. Most interestingly, sphingosine-1- phosphate and sphingosylphosphorylcholine did not induce a measurable produ ction of inositol-1,4,5-trisphosphate or accumulation of inositol phosphate s. Instead, sphingosine-1-phosphate and sphingosylphosphorylcholine induced a rapid and transient increase in production of intracellular sphingosine- 1-phosphate with a maximum of about 1.4-fold at 30 s. Stimulation of sphing osine-1-phosphate formation by sphingosine-1-phosphate and sphingosylphosph orylcholine was fully blocked by pertussis toxin, indicating that extracell ular sphingosine-1-phosphate via endogenously expressed G(i)-coupled recept ors induces a stimulation of intracellular sphingosine-1-phosphate producti on. As sphingosine-1-phosphate- and sphingosylphosphorylcholine-induced inc reases in intracellular Ca2+ were blunted by sphingosine kinase inhibitors, this sphingosine-1-phosphate production appears to mediate Ca2+ signalling by extracellular sphingosine-1-phosphate and sphingosylphosphorylcholine i n HEK-293 cells. (C) 2001 Elsevier Science B.V. All rights reserved.