Peroxynitrite induces haem oxygenase-1 in vascular endothelial cells: a link to apoptosis

Peroxynitrite (ONOO^-) is a potent oxidizing agent generated by the interaction of nitric oxide (NO) and the superoxide anion. In physiological solution, ONOO^- rapidly decomposes to a hydroxyl radical, one of the most reactive free radicals, and nitrogen dioxide, another species able to cause oxidative damage. In the present study we investigated the effect of ONOO^- on the expression of haem oxygenase-1 (HO-1), an inducible protein that is highly up-regulated by oxidative stress. Exposure of bovine aortic endothelial cells to ONOO^- (250-1000 μM) produced a concentration-dependent increase in haem oxygenase activity and HO-1 protein expression. This effect was completely abolished by the ONOO^- scavengers uric acid and N-acetylcysteine, and partly attenuated by 1,3-dimethyl-2-thiourea, a scavenger of hydroxyl radicals. ONOO^- also produced a concentration-dependent increase in apoptosis and cytotoxicity, which were considerably decreased by uric acid and N-acetylcysteine. A 70% decrease in apoptosis was observed when cells were exposed to ONOO^- in the presence of 10 μM tin protoporphyrin IX (SnPPIX), an inhibitor of haem oxygenase activity. When SnPPIX was added 5 min after ONOO^-, apoptosis decreased by only 40%, which suggests that an interaction between ONOO^- and the protoporphyrin occurs in our system. Increased haem oxygenase activity by pretreatment of cells with haemin resulted in elevated bilirubin production and was associated with a substantial decrease (35%) in ONOO^--mediated apoptosis. These results indicate the ability of ONOO^- to modulate the expression of the stress protein HO-1 and suggest that the haem oxygenase pathway contributes to protection against the cytotoxic action of ONOO^-.