Protease-activated receptors (PARs), such as
PAR1 and PAR2, have been implicated in the regulation of
endothelial NO production. We hypothesized that
PAR1 and PAR2 distinctly regulate the activity of
endothelial NO synthase through the
selective phosphorylation of a positive
regulatory site, Ser(1179), and a negative
regulatory site, Thr(497), in bovine
aortic endothelial cells. A
selective PAR1 ligand, TFLLR, stimulated the
phosphorylation of
endothelial NO synthase at Thr(497). It had a minimal effect on Ser(1179)
phosphorylation. In contrast, a
selective PAR2 ligand, SLIGRL, stimulated the
phosphorylation of Ser(1179) with no noticeable effect on Thr(497).
Thrombin has been shown to transactivate PAR2 through
PAR1. Thus,
thrombin, as well as a
peptide mimicking the
PAR1 tethered ligand, TRAP, stimulated
phosphorylation of both sites. Also,
thrombin and SLIGRL, but not TFLLR, stimulated cGMP production. A G(q) inhibitor blocked thrombin- and SLIGRL-induced Ser(1179)
phosphorylation, whereas it enhanced thrombin-induced Thr(497)
phosphorylation. In contrast, a G(12/13) inhibitor blocked thrombin- and TFLLR-induced Thr(497)
phosphorylation, whereas it enhanced the Ser(1179)
phosphorylation. Although a
Rho-kinase inhibitor, Y27632, blocked the Thr(497)
phosphorylation, other inhibitors that targeted
Rho-kinase failed to block TFLLR-induced Thr(497)
phosphorylation. These data suggest that
PAR1 and PAR2 distinctly regulate
endothelial NO synthase phosphorylation and activity through G(12/13) and G(q), respectively, delineating the novel
signaling pathways by which the
proteases act on
protease-activated receptors to potentially modulate
endothelial functions.
