Essential requirement for sphingosine kinase activity in eNOS-dependent NO release and vasorelaxation.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that acts both as an extracellular ligand for endothelial differentiation gene receptor family and as an intracellular second messenger. Cellular levels of S1P are low and tightly regulated by sphingosine kinase (SPK). Recent studies have suggested that eNOS pathway may function as a downstream target for the biological effects of receptor-mediated S1P. Here we have studied the possible interplay between intracellular SIP generation and the eNOS activation pathway. S1P causes an endothelium-dependent vasorelaxation in rat aorta that is PTX sensitive, inhibited by L-NAME that involves eNOS phosphorylation, and mainly dependent on hsp90. When rat aorta rings were incubated with the SPK inhibitor DL-threo-dihydrosphingosine (DTD), there was a concentration-dependent reduction of Ach-induced vasorelaxation, implying a consistent contribution of sphingolipid pathway through intracellular sphingosine release and phosphorylation. Co-immunoprecipitation experiments consistently showed increased association of hsp90 with eNOS after exposure of cells to S1P as well to BK or calcium ionophore A-23187. Interestingly, as opposite to A-23187, BK and S1P effect were significantly inhibited by pretreatment with the SPK inhibitor DTD. In conclusion, our data demonstrate that an interplay exists among eNOS, hsp90, and intracellularly generated S1P where eNOS coupling to hsp90 is a major determinant for NO release as confirmed by our functional and molecular studies.