Sphingosine kinase-mediated Ca2+ signalling by G-protein-coupled receptors

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Abstract
Formation of inositol 1,4,5‐trisphosphate (IP3) by phospholipase C (PLC) with subsequent release of Ca2+ from intracellular stores, is one of the major Ca2+ signalling pathways triggered by G‐protein‐coupled receptors (GPCRs). However, in a large number of cellular systems, Ca2+ mobilization by GPCRs apparently occurs independently of the PLC–IP3 pathway, mediated by an as yet unknown mechanism. The present study investigated whether sphingosine kinase activation, leading to production of sphingosine‐1‐phosphate (SPP), is involved in GPCR‐mediated Ca2+ signalling as proposed for platelet‐derived growth factor and FcϵRI antigen receptors. Inhibition of sphingosine kinase by dl‐ threo ‐dihydrosphingosine and N,N ‐dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by m2 and m3 muscarinic acetylcholine receptors (mAChRs) expressed in HEK‐293 cells without affecting mAChR‐induced PLC stimulation. Activation of mAChRs rapidly and transiently stimulated production of SPP in HEK‐293 cells. Finally, intracellular injection of SPP induced a rapid and transient Ca2+ mobilization in HEK‐293 cells which was not antagonized by heparin. We conclude that mAChRs utilize the sphingosine kinase–SPP pathway in addition to PLC–IP3 to mediate Ca2+ mobilization. As Ca2+ signalling by various, but not all, GPCRs in different cell types was likewise attenuated by the sphingosine kinase inhibitors, we suggest a general role for sphingosine kinase, besides PLC, in mediation of GPCR‐induced Ca2+ signalling.