title: Distinct roles of protease-activated receptors in signal transduction regulation of endothelial nitric oxide synthase.
abstract: Protease-activated receptors (PARs), such as PAR1 and PAR2, have been implicated in the regulation of endothelial NO production. We hypothesized that PAR1 and PAR2 distinctly regulate the activity of endothelial NO synthase through the selective phosphorylation of a positive regulatory site, Ser(1179), and a negative regulatory site, Thr(497), in bovine aortic endothelial cells. A selective PAR1 ligand, TFLLR, stimulated the phosphorylation of endothelial NO synthase at Thr(497). It had a minimal effect on Ser(1179) phosphorylation. In contrast, a selective PAR2 ligand, SLIGRL, stimulated the phosphorylation of Ser(1179) with no noticeable effect on Thr(497). Thrombin has been shown to transactivate PAR2 through PAR1. Thus, thrombin, as well as a peptide mimicking the PAR1 tethered ligand, TRAP, stimulated phosphorylation of both sites. Also, thrombin and SLIGRL, but not TFLLR, stimulated cGMP production. A G(q) inhibitor blocked thrombin- and SLIGRL-induced Ser(1179) phosphorylation, whereas it enhanced thrombin-induced Thr(497) phosphorylation. In contrast, a G(12/13) inhibitor blocked thrombin- and TFLLR-induced Thr(497) phosphorylation, whereas it enhanced the Ser(1179) phosphorylation. Although a Rho-kinase inhibitor, Y27632, blocked the Thr(497) phosphorylation, other inhibitors that targeted Rho-kinase failed to block TFLLR-induced Thr(497) phosphorylation. These data suggest that PAR1 and PAR2 distinctly regulate endothelial NO synthase phosphorylation and activity through G(12/13) and G(q), respectively, delineating the novel signaling pathways by which the proteases act on protease-activated receptors to potentially modulate endothelial functions.
authors.affiliation: Cardiovascular Research Center and Department of Physiology, Temple University School of Medicine, 3420 N Broad St, Philadelphia, PA 19140, USA.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Suzuki
authors.forename: Evangeline D
authors.initials: ED
authors.lastname: Motley
authors.forename: Kunie
authors.initials: K
authors.lastname: Eguchi
authors.forename: Akinari
authors.initials: A
authors.lastname: Hinoki
authors.forename: Heigoro
authors.initials: H
authors.lastname: Shirai
authors.forename: Vabren
authors.initials: V
authors.lastname: Watts
authors.forename: Laura N
authors.initials: LN
authors.lastname: Stemmle
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Fields
authors.forename: Satoru
authors.initials: S
authors.lastname: Eguchi
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000378:metabolism; D019204:GTP-Binding Proteins; D006801:Humans; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation; D044463:Receptor, PAR-1 / Q000819:agonists / Q000378:metabolism*; D044464:Receptor, PAR-2 / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013917:Thrombin / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000378:metabolism
chemical_list: D009842:Oligopeptides; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; C099570:seryl-leucyl-isoleucyl-glycyl-arginyl-leucine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D054460:rho-Associated Kinases; D013917:Thrombin; D019204:GTP-Binding Proteins; D044365:GTP-Binding Protein alpha Subunits, G12-G13