Abstract 329: Reciprocal eNOS Regulation by Protease-Activated Receptors involving G12/13, Rho and Rho-kinase
Protease-activated receptors (PARs), such as PAR-1 and PAR-2 have been implicated in the regulation of endothelial nitric oxide (NO) production. We hypothesized that PAR-1 and PAR-2 distinctly regulate the activity of endothelial NO synthase (eNOS) through the selective phosphorylation of a positive regulatory site, Ser1179 and a negative regulatory site, Thr497. We used selective synthetic peptide ligands for these receptors to see if they phosphorylated eNOS in bovine aortic endothelial cells (BAECs). The PAR-1 ligand, TFLLR, phosphorylated eNOS at Thr497. It had no effect on Ser1179 phosphorylation or cyclic GMP (cGMP) production. In contrast, the PAR-2 ligand, SLIGRL, phosphorylated Ser1179 with no noticeable effect on Thr497. SLIGRL stimulated cGMP production that was blocked by L-NAME. Neither eNOS phosphorylation nor cGMP production was observed by the PAR-4 agonist, AY-NH2. Thrombin has been shown to transactivates PAR-2 through PAR-1. Thus, thrombin stimulates eNOS phosphorylation at both sites as well as cGMP production in BAECs. Importantly, SLIGRL-induced Ser1179 phosphorylation and cGMP production were inhibited by a Gq inhibitor, YM-254890. YM-254890 also blocked thrombin-induced cGMP production and eNOS phosphorylation at Ser1179 but not Thr497. In addition, eNOS phosphorylation was not affected by Gi inhibitors (over-expression of GRK2 C-tail and pertussis toxin pretreatment). By contrast, TFLLR-induced Thr497 phosphorylation was selectively inhibited by a Rho-kinase (ROCK) inhibitor, Y-27632 and over-expression of p115RhoGEF RGS domain, a selective G12/13 inhibitor. Thus, TFLLR but not SLIGRL stimulated Y-27632-inhibitable phosphorylation of MYPT and MLC, the downstream components of Rho/ROCK in BAECs. We also confirmed TFLLR-induced activation of G12/13 and Rho by pull-down assays. From these data, we conclude that PAR-1 and PAR-2 distinctly regulate eNOS activity through Gq and G12/13/ROCK, respectively, delineating the novel signaling pathways by which proteases regulate eNOS activity.