Received for publication February 7, 2007.
Revised May 3, 2007.
Accepted for publication May 8, 2007.

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

Abstract

Proteinase-activated receptor 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 [Ca²⁺]_i and activated NO production in porcine aortic and human umbilical vein endothelial cells, while 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 BAPTA, while 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. However, YM254890 abrogated the PAR₁-mediated Ca²⁺ signal. PAR₄-mediated NO production was substantially inhibited by the inhibitors of phosphotidylinositol-3 kinase and Akt, and also by the dominant negative mutant of Akt. The PAR₁-mediated NO production was relatively resistant to inhibitors of phosphotidylinositol-3 kinase. An immunoblot analysis revealed a transient increase in the phosphorylation of Akt and eNOS 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 phosphotidylinositol-3 kinase-Akt pathway, while PAR₁ activates both the Ca²⁺-dependent and independent mechanisms.

Key words: calcium, endothelial cells, nitric oxide, receptor, signal transduction, thrombin