Characterisation of a sphingosine 1-phosphate-activated Ca2+ signalling pathway in human neuroblastoma cells.

Sphingosine 1-phosphate (S1 P) has assumed great importance within neuroscience research because of putative links between S1P-sensitive Edg receptors and neuroregeneration, cell survival, and alterations in neurite outgrowth. In the present study, we examined the mechanisms by which the endogenous complement of S1 P-sensitive human Edg receptors can elevate Ca 2 + in the human neuroblastoma cell line, SH-SY5Y. Reverse transcriptase-polymersase chain reaction (RT-PCR) confirmed the expression of mRNA for Edg 3, 5, and 8 subtypes of S1P-responsive Edg receptors in SH-SY5Y cells. Neither S1 P nor the muscarinic agonist methacholine were able to cause a change in SH-SY5Y cell morphology, whereas retinoic acid caused a range of changes, including an increase in neurite outgrowth, under similar test conditions. Stimulation with S1 P resulted in a slowly rising increase in cytosolic Ca 2 + levels. These responses were dependent upon inositol-1,4,5-trisphosphate receptors, thapsigargin-sensitive endo-plasmic reticulum, and also intact functional mitochondria. S1P-evoked Ca 2 + responses were similar in mechanism to those of methacholine, which activated a much faster responding, larger amplitude Ca 2 + response. These studies indicate that in an endogenous human expression system, S1 P appears to be an efficacious agonist of Edg receptors. Despite its slow time course of response, S1 P appears to activate the same single Ca 2 + store in SH-SY5Y cells as is activated by methacholine and other G protein coupled receptors.