title: Role of protease-activated receptor-1 in endothelial nitric oxide synthase-Thr495 phosphorylation.
abstract: Protease activated receptors (PARs) are G protein-coupled receptors that are known to regulate endothelial nitric oxide synthase (eNOS) activity in part by phosphorylating the enzyme at various sites. Ser1177 is a positive regulatory site, which leads to the enhanced production of nitric oxide (NO), a vasodilator of arteries. Thr495 is a negative regulatory site, which inhibits NO production. We have shown that thrombin, a PAR agonist, mediates eNOS-Ser1177 phosphorylation through Gq and a calcium and protein kinase C (PKC)-delta sensitive, but phosphatidylinositol 3-kinase (PI3K)/Akt-independent pathway. However, the mechanism for eNOS-Thr495 phosphorylation by PAR agonists is unknown. 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). Using Western blot analysis and the Griess Reagent assay, we found that both agonists phosphorylated Thr495 in a time- and dose-dependent manner and significantly decreased nitrite production, respectively. Pretreatment of cells with the PAR-1 inhibitor, SCH-79797, resulted in a significant decrease in thrombin- and TFLLR-induced phosphorylation of eNOS-Thr495 and an increase in nitrite production. We further demonstrated that inhibition of Rho with C3 exoenzyme or dominant negative (dn) RhoA, and inhibition of Rho-Kinase (ROCK) with Y-27632 caused a significant decrease in thrombin and TFLLR-induced Thr495 phosphorylation. Blockade of the Rho/ROCK pathway also caused an increase in nitrite production. This suggests that PAR-1 regulates eNOS activity via phosphorylation of eNOS-Thr495, which is dependent upon activation of the Rho/ROCK pathway. These findings will be beneficial in further understanding the signaling pathways that regulate eNOS-induced NO production, which plays an important role in endothelial dysfunction associated with cardiovascular disease.
authors.affiliation: Department of Cardiovascular Biology, Meharry Medical College, 1005 D. B. Todd Blvd., Nashville, TN 37208, USA.
authors.forename: Vabren L
authors.initials: VL
authors.lastname: Watts
authors.forename: Evangeline D
authors.initials: ED
authors.lastname: Motley
mesh_terms: D036002:ADP Ribose Transferases / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D001905:Botulinum Toxins / Q000494:pharmacology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D005799:Genes, Dominant; D006801:Humans; D008954:Models, Biological; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D044463:Receptor, PAR-1 / Q000378:metabolism*; D044464:Receptor, PAR-2 / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D010975:Platelet Aggregation Inhibitors
chemical_list: D000577:Amides; C415424:N3-cyclopropyl-7-((4-(1-methylethyl)phenyl)methyl)-7H-pyrrolo(3, 2-f)quinazoline-1,3-diamine; D009573:Nitrites; D009842:Oligopeptides; C462999:PAR-1-activating peptide; D011725:Pyridines; D011758:Pyrroles; D011799:Quinazolines; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D010769:Phosphothreonine; C108830:Y 27632; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D054460:rho-Associated Kinases; D013917:Thrombin; D001905:Botulinum Toxins; D020742:rhoA GTP-Binding Protein; D010975:Platelet Aggregation Inhibitors