Distinct Ca2+ Requirement for NO Production between Proteinase-Activated Receptor 1 and 4 (PAR1 and PAR4) in Vascular Endothelial Cells

• Published in: The Journal of pharmacology and experimental therapeutics Vol. 322; no. 2; pp. 668 - 677
• Main Authors: Hirano, Katsuya, Nomoto, Namie, Hirano, Mayumi, Momota, Fumi, Hanada, Akiko, Kanaide, Hideo
• Format: Journal Article
• Language: English
• Published: United States American Society for Pharmacology and Experimental Therapeutics 01.08.2007
• Subjects: Androstadienes - pharmacology; Animals; Calcium - metabolism; Cattle; Cell Line; Cells, Cultured; Chromones - pharmacology; Dose-Response Relationship, Drug; Egtazic Acid - analogs & derivatives; Egtazic Acid - pharmacology; Endothelial Cells - drug effects; Endothelial Cells - metabolism; Enzyme Inhibitors - pharmacology; GTP-Binding Protein alpha Subunits - antagonists & inhibitors; Humans; Morpholines - pharmacology; NG-Nitroarginine Methyl Ester - pharmacology; Nitric Oxide - metabolism; Nitric Oxide Synthase Type III - metabolism; Oligopeptides - pharmacology; Peptides, Cyclic - pharmacology; Pertussis Toxin - pharmacology; Phosphatidylinositol 3-Kinases - antagonists & inhibitors; Phosphatidylinositol 3-Kinases - metabolism; Phosphorylation - drug effects; Receptor, PAR-1 - metabolism; Receptors, Thrombin - metabolism; Swine
• ISSN: 0022-3565; 1521-0103
• DOI: 10.1124/jpet.107.121038

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 Gα q/11 inhibitor, YM254890 [(1 R )-1-{(3 S ,6 S ,9 S ,12 S ,18 R ,21 S ,22 R )-21-acetamido-18-benzyl-3-[(1 R )-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-(2 S ,3 R )-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 Gα 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.