Abstract 1913: Distinct eNOS Regulation by Protease-Activated Receptor 1 and 2 Involving Gq, G12/13 and Y27632-Sensitive Non-Rho Kinase

Abstract

Protease-activated receptors (PARs), such as PAR1 and PAR2 have been implicated in the regulation of endothelial nitric oxide (NO) production. We hypothesized that PAR1 and PAR2 distinctly regulate the activity of endothelial NO synthase (eNOS) through the selective phosphorylation of a positive regulatory site, Ser 1179 and a negative regulatory site, Thr 497 . In bovine aortic endothelial cells (BAECs), a PAR1 ligand, TFLLR, phosphorylated eNOS at Thr 497 . It had no effect on Ser 1179 phosphorylation or cyclic GMP (cGMP) production. In contrast, a PAR2 ligand, SLIGRL, phosphorylated Ser 1179 with no noticeable effect on Thr 497 . SLIGRL stimulated cGMP production that was blocked by L-NAME. Neither eNOS phosphorylation nor cGMP production was observed by a PAR4 agonist, AY-NH2. Thrombin has been shown to transactivates PAR2 through PAR1. Thus, thrombin stimulates eNOS phosphorylation at both sites as well as cGMP production in BAECs. Importantly, SLIGRL-induced Ser 1179 phosphory-lation and cGMP production were inhibited by a G q inhibitor, YM-254890. In addition, eNOS phosphorylation was not affected by over-expression of GRK2 C-tail or pertusis toxin pretreatment. By contrast, TFLLR-induced Thr 497 phosphorylation was selectively inhibited by a Rho-kinase inhibitor, Y27632, and over-expression of p115RhoGEF RGS domain, a selective G 12/13 inhibitor. However, other inhibitors (H-1152 and dnRho) that target Rho-kinase or a PKC inhibitor, GF109203X, failed to block TFLLR-induced Thr 497 phosphorylation. Taken together, these data suggest that PAR1-induced phosphorylation of eNOS at Thr 497 requires G 12/13 and subsequent activation of a Y27632-sensitive eNOS Thr 497 kinase that is distinct from Rho-kinase or PKC. We conclude that PAR1 and PAR2 distinctly regulate eNOS activity through G q and G 12/13 , respectively, delineating the novel signaling pathways by which proteases regulate eNOS activity. This research has received full or partial funding support from the American Heart Association, AHA National Center.