Activation of NAD(P)H oxidases by thromboxane A2 receptor uncouples endothelial nitric oxide synthase.
OBJECTIVE
The thromboxane receptor (TPr) and multiple TPr ligands, including thromboxane A(2) (TxA(2)) and prostaglandin H(2), 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.


RESULTS
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 TxA(2) mimetics, for 24 hours markedly increased the release of superoxide anions (O(2)(·-)) 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 O(2)(·-) were accompanied by the membrane translocation of the p67(phox) subunit of NAD(P)H oxidase. Pharmacological or genetic inhibition of either NAD(P)H oxidase or TPr abolished IBOP-induced O(2)(·-) 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 p67(phox) and O(2)(·-) production. Finally, exposure of isolated mouse aortae to IBOP markedly increased O(2)(·-) in wild-type but not in those from gp91(phox) knockout mice.


CONCLUSIONS
We conclude that TPr activation via PKC-ζ-mediated NAD(P)H oxidase activation increases both O(2)(·-) and ONOO(-), resulting in eNOS uncoupling in endothelial cells.
Department of Medicine, University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA.
Miao
M
Zhang
Ping
P
Song
Jian
J
Xu
Ming-Hui
MH
Zou
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology*; D019746:Intramolecular Oxidoreductases / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D030421:Peroxynitrous Acid / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D034622:RNA Interference; D011971:Receptors, Immunologic / Q000172:deficiency / Q000235:genetics; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000819:agonists* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D000099221:Protein Kinase C zeta
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D001708:Biopterins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D006152:Cyclic GMP; D003577:Cytochrome P-450 Enzyme System; D004791:Enzyme Inhibitors; D005231:Fatty Acids, Unsaturated; D019746:Intramolecular Oxidoreductases; D019255:NADPH Oxidases; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D030421:Peroxynitrous Acid; D010750:Phosphoproteins; D011493:Protein Kinase C; D011971:Receptors, Immunologic; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D013481:Superoxides; D014443:Tyrosine; D000099221:Protein Kinase C zeta; C105949:Pirb protein, mouse; C079789:neutrophil cytosol factor 67K; C061750:7-(3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo(2.2.1)heptan-2-yl)-5-heptenoic acid; C002744:3-nitrotyrosine; C496315:Nos3 protein, mouse; C022461:prostacyclin synthetase; C003402:sapropterin