Sphingosine 1-phosphate (S1P) can prevent endothelial cell apoptosis. We investigated the molecular mechanisms and signaling pathways by which S1P protects endothelial cells from serum deprivation-induced apoptosis. We show here that human umbilical vein endothelial cells (HUVECs) undergo apoptosis associated with increased DEVDase activity, caspase-3 activation, cytochrome c release, and DNA fragmentation after 24 h of serum deprivation. These apoptotic markers were suppressed by the addition of S1P, the NO donor Snitroso-N-acetylpenicillamine (100 M), or caspase-3 inhibitor z-VAD-fmk. The protective effects of S1P were reversed by the nitric-oxide synthase (NOS) inhibitor N-monomethyl-L-arginine, but not by the soluble guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo[4,3-a]-quanoxaline-1-one, suggesting that NO, but not cGMP, is responsible for S1P protection from apoptosis. Furthermore, S1P increased NO production by enhancing Ca 2؉ -sensitive NOS activity without changes in the eNOS protein level. S1P-mediated cell survival and NO production were suppressed significantly by pretreatment with antisense oligonucleotide of EDG-1 and partially by EDG-3 antisense. S1P-mediated NO production was suppressed by the addition of pertussis toxin, an inhibitor of G i proteins, the specific inhibitor of phospholipase C (PLC), U73122, and the Ca 2؉ chelator BAPTA-AM. These findings indicate that S1P protects HUVECs from apoptosis through the activation of eNOS activity mainly through an EDG-1 and -3/G i /PLC/Ca 2؉ signaling pathway.Sphingosine 1-phosphate (S1P), 1 generated from sphingosine by sphingosine kinase, is a recently described lipid mediator regulating cellular responses, including cell proliferation (1), cell motility (2, 3), and morphological changes (2, 4) in endothelial cells. In addition, S1P was also demonstrated to protect endothelial cells from apoptosis induced by C 2 -ceramide, TNF␣, and anti-Fas antibody (1, 4, 5). All these effects of S1P on endothelial functions are thought to correlate with the intracellular signaling pathway linked to the EDG-1 (endothelial differentiation gene-1), EDG-3, and EDG-5 subtypes of G protein-coupled receptors (1-4, 6). Of these receptors, EDG-1 is exclusively expressed in HUVECs (2, 4), and S1P induces a robust calcium response mainly via the G i -coupled S1P receptors EDG-1 and -3 in HUVECs (4). Consistent with this view, we have recently observed that intracellular Ca 2ϩ mobilization evoked by the G i -PLC system is responsible for the signaling pathway of mitogen-activated protein kinases, focal adhesion kinase (p125 FAK ), and chemotaxis in response to S1P in HUVECs (6). It is likely that intracellular signaling events of S1P in endothelial cells may be directly associated with increase in [Ca 2ϩ ] i . Endogenous nitric oxide (NO) is synthesized from L-arginine by catalytic reaction of three isotypes of NO synthases (NOS), the neuronal or type I isoform (nNOS), the inducible or type II isoform (iNOS), and the endothelial or type III isoform (eNOS) (7,8). Endoth...