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

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 Gα_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. 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 Gα_i/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.