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CARDIOVASCULAR

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

Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences (K.H., N.N., M.H., F.M., A.H., H.K.) and 21st Century Center of Excellence Program (H.K.), Kyushu University, Fukuoka, Japan

Proteinase-activated receptors 1 and 4 (PAR₁ and PAR₄) 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 G_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 PAR₁-mediated Ca²⁺ signal. PAR₄-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 G_i/o and induced NO production in a manner mostly independent of Ca²⁺ but dependent on the PI3K/Akt pathway, whereas PAR₁ activates both the Ca²⁺-dependent and -independent mechanisms.

Address correspondence to: Hideo Kanaide, Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. E-mail: kanaide{at}molcar.med.kyushu-u.ac.jp

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