Distinct Ca2+ requirement for NO production between proteinase-activated receptor 1 and 4 (PAR1 and PAR4) in vascular endothelial cells

Katsuya Hirano, Namie Nomoto, Mayumi Hirano, Fumi Momota, Akiko Hanada, Hideo Kanaide

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26 Citations (Scopus)


Proteinase-activated receptors 1 and 4 (PAR1 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 Ca2+ requirement for the NO production between PAR1 and PAR4. The activation of PAR1 by the activating peptide (PAR1-AP) elevated cytosolic Ca2+ concentrations ([Ca2+]i) and activated NO production in porcine aortic and human umbilical vein endothelial cells, whereas it had little effect on bovine aortic endothelial cells. PAR4 activation by PAR4-AP consistently induced NO production without an appreciable [Ca2+]i elevation in three types of endothelial cells. The PAR1-mediated NO production was significantly inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), whereas the PAR4-mediated NO production was resistant. NO production following the PAR1 and PAR4 activation was significantly inhibited by pertussis toxin, but it was resistant to a Gαq/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 PAR1-mediated Ca2+ 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 PAR1-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 PAR4 stimulation. In conclusion, PAR1 and PAR4 engage distinct signal transduction mechanisms to activate NO production in vascular endothelial cells. PAR4 preferably activates Gαi/o and induced NO production in a manner mostly independent of Ca2+ but dependent on the PI3K/Akt pathway, whereas PAR 1 activates both the Ca2+-dependent and -independent mechanisms.

Original languageEnglish
Pages (from-to)668-677
Number of pages10
JournalJournal of Pharmacology and Experimental Therapeutics
Issue number2
Publication statusPublished - Aug 2007
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology


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