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

Roviezzo F, Bucci M, , Delisle C*, Brancaleone V, Di Lorenzo A, Gratton JP*, Cirino G. Department of Experimental Pharmacology, Faculty of Pharmacy, University of Naples -Federico II-; * Laboratory of Endothelial Cell Biology, Institut de Recherches Cliniques de Montreal (IRCM), Montreal, Quebec H2W 1R7, Canada

Sphingosine-1-phospahte (S1P) is a bioactive sphingolipid that acts both as an extracellular ligand for endothelial differentiation gene receptor family and as an intracellular 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 receptor-mediated of S1P . Here we have studied the possible interplay among eNOS, S1P and hsp90 whose recruitment is critical for eNOS activation by using isolated rat aortic rings. Thoracic aorta was rapidly excised from male Wistar rats ( Charles River, 200- 250 g) sacrificed by exanguination and cleaned from fat. Rings of 2- 3 mm width were cut and placed in organ baths filled with oxygenated Krebs solution at 37°C and connected to an isometric transducer under resting tension of 0.5 g. S1P (10 nM-50 µM) causes an endothelium-dependent vasorelaxation in rat aorta (max relaxation 72±14.4%, n=6) which is PTX sensitive (max relaxation 23±1.2%, n=6 p<0.001), inhibited by L-NAME (max relaxation 20±2.9%, n=6 p<0.001) and mainly dependent on hsp90. When rat aorta rings were incubated with the SPKI inhibitor DL-threo-dihydrosphingosine (DTD) there was a concentration dependent reduction of Ach-induced vasorelaxation (10 nM-50 µM) (from max relaxation 87±2.3% to 32±7.6%, n=6 p<0.001) implying a consistent contribution of sphingolipid pathway in Ach-induced vasorelaxation, through sphingosine release and phosphorylation. Co-immunoprecipitation experiments consistently showed increased association of hsp90 with eNOS following 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 pre-treatment with the SPK inhibitor DTD. In conclusion our data demonstrate that exists an interplay between eNOS, S1P and hsp90 where coupling to hsp90 plays a major role as confirmed by functional and molecular studies.