Activation of NAD(P)H oxidases by thromboxane A2 receptor uncouples endothelial nitric oxide synthase

Arterioscler Thromb Vasc Biol. 2011 Jan;31(1):125-32. doi: 10.1161/ATVBAHA.110.207712. Epub 2010 Oct 14.


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.

Methods and 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / pharmacology*
  • Animals
  • Apoptosis / drug effects
  • Biopterin / analogs & derivatives
  • Biopterin / metabolism
  • Bridged Bicyclo Compounds, Heterocyclic / pharmacology*
  • Cattle
  • Cell Survival / drug effects
  • Cells, Cultured
  • Cyclic GMP / metabolism
  • Cytochrome P-450 Enzyme System / metabolism
  • Endothelial Cells / drug effects*
  • Endothelial Cells / enzymology
  • Endothelial Cells / pathology
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Fatty Acids, Unsaturated / pharmacology*
  • Intramolecular Oxidoreductases / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • NADPH Oxidases / antagonists & inhibitors
  • NADPH Oxidases / genetics
  • NADPH Oxidases / metabolism*
  • Nitric Oxide / metabolism
  • Nitric Oxide Synthase Type III / metabolism*
  • Oxidative Stress / drug effects
  • Peroxynitrous Acid / metabolism
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / metabolism
  • Protein Processing, Post-Translational
  • Protein Transport
  • RNA Interference
  • Receptors, Immunologic / deficiency
  • Receptors, Immunologic / genetics
  • Receptors, Thromboxane A2, Prostaglandin H2 / agonists*
  • Receptors, Thromboxane A2, Prostaglandin H2 / metabolism
  • Signal Transduction / drug effects*
  • Superoxides / metabolism
  • Time Factors
  • Tyrosine / analogs & derivatives
  • Tyrosine / metabolism


  • Bridged Bicyclo Compounds, Heterocyclic
  • Enzyme Inhibitors
  • Fatty Acids, Unsaturated
  • Phosphoproteins
  • Pirb protein, mouse
  • Receptors, Immunologic
  • Receptors, Thromboxane A2, Prostaglandin H2
  • neutrophil cytosol factor 67K
  • Superoxides
  • 7-(3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo(2.2.1)heptan-2-yl)-5-heptenoic acid
  • Peroxynitrous Acid
  • Biopterin
  • Nitric Oxide
  • 3-nitrotyrosine
  • Tyrosine
  • 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid
  • Cytochrome P-450 Enzyme System
  • Nitric Oxide Synthase Type III
  • Nos3 protein, mouse
  • NADPH Oxidases
  • protein kinase C zeta
  • Protein Kinase C
  • Intramolecular Oxidoreductases
  • prostacyclin synthetase
  • sapropterin
  • Cyclic GMP