The Role of Protease Activated Receptors in the Phosphorylation of eNOS Thr495

Protease Activated Receptors (PARs) are G protein‐coupled receptors known to regulate endothelial nitric oxide synthase (eNOS) activity by phosphorylating the enzyme at various sites. Ser1179 is a positive regulatory site when phosphorylated, whereas, Thr495 phosphorylation negatively regulates eNOS activity. We have shown that thrombin, a PAR agonist, mediates eNOS phosphorylation through a Ca 2+ and PKCδ sensitive, but, PI3K/AKT‐independent pathway. However, the mechanism for eNOS Thr495 activation by PAR agonists is unknown. In this study, we used a specific synthetic PAR‐1 activating peptide, TFLLR, and thrombin to assess the role of PAR‐1 involvement in the phosphorylation of eNOS Thr495 in human umbilical vein endothelial cells (HUVECs). We found that thrombin induces phosphorylation of Thr495 in a time and dose‐dependent manner. Maximal phosphorylation of eNOS Thr495 occurred at 5 min in cells stimulated with thrombin (10U/ml). HUVECs stimulated with TFLLR (10–20μM) showed maximal phosphorylation at 5 min. We also showed that cells pretreated with the PAR‐1 inhibitor, SCH79797 , caused a significant decrease in thrombin‐ and TFLLR‐induced phosphorylation of eNOS Thr495. Our data suggest that PAR‐1 plays an important role in the phosphorylation of eNOS Thr495, which may be beneficial in understanding the pathways responsible for eNOS‐induced nitric oxide production. NIH‐NCRR 2G12RR03032.