SPHINGOSINE KINASE-MEDIATED CA2-PROTEIN-COUPLED RECEPTORS( SIGNALING BY G)

Formation of inositol 1,4,5-trisphosphate (IP3) by phospholipase C (PL C) with subsequent release of Ca2+ from intracellular stores, is one o f the major Ca2+ signalling pathways triggered by G-protein-coupled re ceptors (GPCRs), However, in a large number of cellular systems, Ca2mobilization by GPCRs apparently occurs independently of the PLC-IP3 p athway, mediated by an as yet unknown mechanism. The present study inv estigated whether sphingosine kinase activation, leading to production of sphingosine-1-phosphate (SPP), is involved in GPCR-mediated Ca2+ s ignalling as proposed for platelet-derived growth factor and Fc epsilo n RI antigen receptors, Inhibition of sphingosine kinase by DL-threo-d ihydrosphingosine and N,N-dimethylsphingosine markedly inhibited [Ca2](i) increases elicited by m2 and m3 muscarinic acetylcholine receptor s (mAChRs) expressed in HEK-293 cells without affecting mAChR-induced PLC stimulation. Activation of mAChRs rapidly and transiently stimulat ed production of SPP in HEK-293 cells. Finally, intracellular injectio n of SPP induced a rapid and transient Ca2+ mobilization in HEK-293 ce lls which was not antagonized by heparin. We conclude that mAChRs util ize the sphingosine kinase-SPP pathway in addition to PLC-IP3 to media te Ca2+ mobilization, As Ca2+ signalling by various, but not all, GPCR s in different cell types was likewise attenuated by the sphingosine k inase inhibitors, we suggest a general role for sphingosine kinase, be sides PLC, in mediation of GPCR-induced Ca2+ signalling.