Lysophosphatidic acid-induced Ca2+ mobilization requires intracellular sphingosine 1-phosphate production - Potential involvement of endogenous Edg-4receptors

Lysophosphatidic acid (LPA)-mediated Ca2+ mobilization in human SH-SY5Y neu roblastoma cells does not involve either inositol 1,4,5-trisphosphate (Ins( 1,4,5)P-3)- or ryanodine-receptor pathways, but is sensitive to inhibitors of sphingosine kinase. This present study identifies Edg-4 as the receptor subtype involved and investigates the presence of a Ca2+ signaling cascade based upon the lipid second messenger molecule, sphingosine I-phosphate. Bo th LPA and direct G-protein activation increase [H-3]sphingosine l-phosphat e levels in SH-SY5Y cells. Measurements of Ca-45(2+) release in premeabiliz ed SH-SY5Y cells indicates that sphingosine I-phosphate, sphingosine, and s phingosylphosphorylcholine, but not N-acetylsphingosine are capable of mobi lizing intracellular Ca2+. Furthermore, the effect of sphingosine was atten uated by the sphingosine kinase inhibitor dimethylsphingosine, or removal o f ATP. Confocal microscopy demonstrated that LPA stimulated intracellular C a2+ "puffs," which resulted from an interaction between the sphingolipid Ca 2+ release pathway and Ins(1,4,5)P-3 receptors. Down-regulation of Ins(1,4, 5)P-3 receptors uncovered a Ca2+ response to LPA, which was manifest as a p rogressive increase in global cellular Ca2+ with no discernible foci. We su ggest that activation of an LPA-sensitive Edg-4 receptor solely utilizes th e production of intracellular sphingosine l-phosphate to stimulate Ca2+ mob ilization in SB-SY5Y cells. Unlike traditional Ca2+ release processes, this novel pathway does not require the progressive recruitment of elementary C a2+ events.