Hypoxia potentiates nitric oxide-mediated apoptosis in endothelial cells via peroxynitrite-induced activation of mitochondria-dependent and -independent pathways

J Biol Chem. 2004 Feb 6;279(6):4425-32. doi: 10.1074/jbc.M310582200. Epub 2003 Nov 3.


Nitric oxide (NO*) at low concentrations is cytoprotective for endothelial cells; however, elevated concentrations of NO* (> or =1 micromol/liter), as may be achieved during inflammatory states, can induce apoptosis and cell death. Hypoxia is associated with tissue inflammation and ischemia and, therefore, may modulate the effects of NO* on endothelial function. To examine the influence of hypoxia on NO*-mediated apoptosis, we exposed bovine aortic endothelial cells (BAEC) to (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (diethylenetriamine NONOate, DETA-NO) (1 mmol/liter) under normoxic or hypoxic conditions (pO2 = 35 mm of Hg) and measured the indices of apoptotic cell death. BAEC treated with DETA-NO under normoxic conditions demonstrated increased levels of histone-associated DNA fragments, which was confirmed by terminal dUTP nick-end labeling assay, and hypoxic conditions augmented this response. To determine whether mitochondrial dysfunction was one mechanism by which NO* initiated apoptosis under hypoxic conditions, we evaluated mitochondrial membrane potential in (Psim). Exposure to DETA-NO resulted in a decrease in Psim and concomitant release of cytochrome c and caspase-9 activation, which were enhanced by hypoxia. By utilizing Rho0 BAEC (Rho0-EC), which lack functional mitochondria, we demonstrated that dissipation of Psim was associated with increased reactive oxygen species generation and peroxynitrite formation. Moreover, in Rho0-EC we identified activation of caspase-8 as part of the mitochondrial-independent pathway of apoptosis. To establish that peroxynitrite mediated mitochondrial damage and apoptosis, we treated BAEC and Rho0-EC with the peroxynitrite scavenger uric acid and found that the indices of apoptosis were decreased significantly. These findings confirm that high flux of NO* under hypoxic conditions promotes cell death via mitochondrial damage and mitochondrial-independent mechanisms by peroxynitrite.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Apoptosis / physiology*
  • Cattle
  • Cell Hypoxia / physiology*
  • Cells, Cultured
  • Endothelium, Vascular / cytology*
  • Endothelium, Vascular / drug effects
  • Endothelium, Vascular / metabolism*
  • Membrane Potentials / drug effects
  • Mitochondria / metabolism
  • Nitric Oxide / metabolism*
  • Nitric Oxide Donors / pharmacology
  • Oxidative Stress
  • Peroxynitrous Acid / metabolism
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • Triazenes / pharmacology
  • bcl-2-Associated X Protein


  • 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene
  • Nitric Oxide Donors
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • Triazenes
  • bcl-2-Associated X Protein
  • Peroxynitrous Acid
  • Nitric Oxide