Sphingosine 1-phospbate and activation of endothelial nitric-oxide synthase - Differential regulation of Akt and MAP kinase pathways by EDG and bradykinin receptors in vascular endothelial cells

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that elici ts numerous biological responses in endothelial cells mediated by a family of G protein-coupled EDG receptors, Stimulation of EDG receptors by S1P has been shown to activate the endothelial isoform of nitric-oxide synthase (e NOS) in heterologous expression systems (Igarashi, J,, and Michel, T. (2000 ) J. Biol Chem. 275, 32363-32370), However, the signaling pathways that mod ulate eNOS regulation by S1P/EDG in vascular endothelial cells remain less well understood. We now report that SIP treatment of bovine aortic endothel ial cells (BAEC) acutely increases eNOS enzyme activity; the EC50 for S1P a ctivation of eNOS is similar to 10 nar. The magnitude of eNOS activation by S1P in BAEC is equivalent to that elicited by the agonist bradykinin. S1P treatment activates Akt, a protein kinase implicated in phosphorylation of eNOS. S1P treatment of BAEC leads to eNOS phosphorylation at Ser(1179), a r esidue phosphorylated by Akt; an eNOS mutant in which this Akt phosphorylat ion site is inactivated shows attenuated S1P-induced eNOS activation. S1P-i nduced activation both of Akt and of eNOS is inhibited by pertussis toxin, by the phosphoinositide 3-kinase inhibitor wortmannin, and by the intracell ular calcium chelator BAPTA (1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraace tic acid). By contrast to S1P, activation of G protein-coupled bradykinin B 2 receptors neither activates kinase Akt nor promotes Ser1179 eNOS phosphor ylation despite robustly activating eNOS enzyme activity. Understanding the differential regulation of protein kinase pathways by S1P and bradykinin m ay lead to the identification of new points for eNOS regulation in vascular endothelial cells.