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

Nitric oxide (NO.) at low concentrations is cytoprotective for endothelial cells; however, elevated concentrations of NO. (greater than or equal to1 mumol/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 (pO(2) = 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 (psi(m)). Exposure to DETA-NO resulted in a decrease in psi(m) and concomitant release of cytochrome c and caspase-9 activation, which were enhanced by hypoxia. By utilizing Rho(0) BAEC (Rho(0)-EC), which lack functional mitochondria, we demonstrated that dissipation of psi(m) was associated with increased reactive oxygen species generation and peroxynitrite formation. Moreover, in Rho(0)-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 Rho(0)-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.