title: Distinct Ca2+ requirement for NO production between proteinase-activated receptor 1 and 4 (PAR1 and PAR4) in vascular endothelial cells.
abstract: Proteinase-activated receptors 1 and 4 (PAR(1) and PAR(4)) are the major receptors mediating thrombin-induced NO production in endothelial cells. The intracellular signaling following their activation still remains to be elucidated. The present study provides the first evidence for the distinct Ca(2+) requirement for the NO production between PAR(1) and PAR(4). The activation of PAR(1) by the activating peptide (PAR(1)-AP) elevated cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and activated NO production in porcine aortic and human umbilical vein endothelial cells, whereas it had little effect on bovine aortic endothelial cells. PAR(4) activation by PAR(4)-AP consistently induced NO production without an appreciable [Ca(2+)](i) elevation in three types of endothelial cells. The PAR(1)-mediated NO production was significantly inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), whereas the PAR(4)-mediated NO production was resistant. NO production following the PAR(1) and PAR(4) activation was significantly inhibited by pertussis toxin, but it was resistant to a Galpha(q/11) inhibitor, YM254890 [(1R)-1-[(3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-[(1R)-1-methoxyethyl]-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl]-2-methylpropyl rel-(2S,3R)-2-acetamido-3-hydroxy-4-methylpentanoate]. However, YM254890 abrogated the PAR(1)-mediated Ca(2+) signal. PAR(4)-mediated NO production was substantially inhibited by the inhibitors of phosphotidylinositol-3 kinase (PI3K) and Akt, as well as by the dominant negative mutant of Akt. The PAR(1)-mediated NO production was relatively resistant to inhibitors of PI3K. An immunoblot analysis revealed a transient increase in the phosphorylation of Akt and endothelial NO synthase following the PAR(4) stimulation. In conclusion, PAR(1) and PAR(4) engage distinct signal transduction mechanisms to activate NO production in vascular endothelial cells. PAR(4) preferably activates Galpha(i/o) and induced NO production in a manner mostly independent of Ca(2+) but dependent on the PI3K/Akt pathway, whereas PAR(1) activates both the Ca(2+)-dependent and -independent mechanisms.
authors.affiliation: Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Katsuya
authors.initials: K
authors.lastname: Hirano
authors.forename: Namie
authors.initials: N
authors.lastname: Nomoto
authors.forename: Mayumi
authors.initials: M
authors.lastname: Hirano
authors.forename: Fumi
authors.initials: F
authors.lastname: Momota
authors.forename: Akiko
authors.initials: A
authors.lastname: Hanada
authors.forename: Hideo
authors.initials: H
authors.lastname: Kanaide
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044385:GTP-Binding Protein alpha Subunits / Q000037:antagonists & inhibitors; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D037342:Pertussis Toxin / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D044463:Receptor, PAR-1 / Q000378:metabolism*; D018179:Receptors, Thrombin / Q000378:metabolism*; D013552:Swine; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D002118:Calcium; D002867:Chromones; D004533:Egtazic Acid; D004791:Enzyme Inhibitors; D044385:GTP-Binding Protein alpha Subunits; D009025:Morpholines; D019331:NG-Nitroarginine Methyl Ester; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D009842:Oligopeptides; D010456:Peptides, Cyclic; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D000077191:Wortmannin; C482003:AYPGKG-NH(2); C462999:PAR-1-activating peptide; C475455:YM-254890; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C112908:protease-activated receptor 4