Miao Zhang, Ping Song, Jian Xu, Ming-Hui Zou
The thromboxane receptor (TPr) and multiple TPr ligands, including thromboxane A2 (TxA2) and prostaglandin H2, are elevated during vascular and atherothrombotic diseases. How TPr stimulation causes vascular injury remains poorly defined. This study was conducted to investigate the mechanism by which TPr stimulation leads to vascular injury. Exposure of bovine aortic endothelial cells to either [1S-(1α,2β(5Z),3α(1E,3R),4α]-7-[3-(3-hydroxy-4-(d′-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1] heptan-2-yl]-5′-heptenoic acid (IBOP) or U46619, 2 structurally related TxA2 mimetics, for 24 hours markedly increased the release of superoxide anions (O2·−) and peroxynitrite (ONOO−) but reduced cyclic GMP, an index of nitric oxide bioactivity. IBOP also significantly suppressed activity of endothelial nitric oxide synthase (eNOS), increased enzyme-inactive eNOS monomers, and reduced levels of tetrahydrobiopterin, an essential eNOS cofactor. IBOP- and U46619-induced increases in O2·− were accompanied by the membrane translocation of the p67phox subunit of NAD(P)H oxidase. Pharmacological or genetic inhibition of either NAD(P)H oxidase or TPr abolished IBOP-induced O2·− formation. Furthermore, TPr activation significantly increased protein kinase C-ζ (PKC-ζ) in membrane fractions and PKC-ζ phosphorylation at Thr410. Consistently, PKC-ζ inhibition abolished TPr activation-induced membrane translocation of p67phox and O2·− production. Finally, exposure of isolated mouse aortae to IBOP markedly increased O2·− in wild-type but not in those from gp91phox knockout mice. We conclude that TPr activation via PKC-ζ-mediated NAD(P)H oxidase activation increases both O2·− and ONOO−, resulting in eNOS uncoupling in endothelial cells.