Involvement of proteinase activated receptor-2 in vascular response to sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) represents one of the key latest additions to the list of vasoactive substances that modulate vascular tone. Proteinase activated receptor-2 (PAR-2) has been shown to be involved in cardiovascular function. In the present study we investigated the involvement of PAR-2 in S1P-induced effect on vascular tone. This study has been performed by using isolated mouse aortas. Both S1P and PAR-2 agonists induce endothelium-dependent vasorelaxation. L-NAME and wortmannin abrogate S1P-induced vasorelaxatioin, while significantly inhibit PAR-2 mediated effect. Either ENMD1068, a PAR-2 antagonist or gabexate, a serine protease inhibitor, significantly inhibit S1P-induced vasorelaxation. Aortic tissues harvested from mice overexpressing PAR-2 display a significant increased vascular response to S1P as opposite to PAR-2 null mice. Immunoprecipitation and immunofluorescence studies demonstrate that S1P1 interacts with PAR-2 and co-localizes with PAR-2 on the vascular endothelial surface. Furthermore S1P administration to vascular tissues triggers PAR-2 mobilization from the plasma membrane to the perinuclear area; S1P-induced translocation of PAR-2 is abrogated when aortic rings were pretreated with ENMD1068 or when caveolae dysfunction occurs. Similarly, experiments performed in cultured endothelial cells (HUVEC) show a colocalization of S1P1 and PAR2, as well as S1P ability to induce PAR-2 trafficking. Our results suggest that S1P induces endothelium dependent vasorelaxation mainly through S1P1 and involves PAR-2 transactivation