Abstract
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.
Highlights
Nitric oxide (NO1⁄7), an endogenous signaling molecule that modulates vessel wall function, has both cytoprotective and cytotoxic effects on the vascular endothelium [1]
Hypoxia Enhances NO1⁄7-mediated Cell Death—To examine the effect of hypoxia on cell death in endothelial cells exposed to high concentrations of NO1⁄7, bovine aortic endothelial cells (BAEC) were treated with the NO1⁄7 donor diethylenetriamine NONOate (DETA-NO), which yields 3 mol/liter NO1⁄7 flux, comparable with amounts produced in vivo by the inducible isoform of nitric oxide synthase [18]
When peroxynitrite was scavenged by uric acid in BAEC, mitochondrial membrane potential was maintained after exposure to DETA-NO under either normoxic (88 Ϯ 3 versus Ϯ 2% control, p Ͻ 0.05) or hypoxic conditions (73 Ϯ 2 versus Ϯ 4% control, p Ͻ 0.01), suggesting that peroxynitrite initiated apoptosis by inducing mitochondrial damage
Summary
Nitric oxide (NO1⁄7), an endogenous signaling molecule that modulates vessel wall function, has both cytoprotective and cytotoxic effects on the vascular endothelium [1]. Prolonged exposure to elevated levels of NO1⁄7 suppresses mitochondrial respiration by inhibition of cytochrome oxidase (complex IV), resulting in a decrease in inner mitochondrial membrane potential (m) and induction of mitochondrial permeability transition to effect release of cytochrome c into the cell cytosol. These events activate caspase-9, which in turn activates downstream executioner caspases to commence intracellular proteolysis, internucleosomal DNA fragmentation, and eventual cell death (9 –11). Hypoxia, which is associated with tissue inflammation and ischemia, has been shown to increase mitochondrial ROS generation in endothelial cells [16] and to potentiate NO1⁄7-mediated. To date the combined influence of hypoxia and NO1⁄7 on mitochondria-dependent apoptosis in endothelial cells has not been determined
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