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-l (HO-1), an inducible protein that is highly up-regulated by oxidative stress. Exposure of bovine aortic endothelial cells to ONOO- (250-1000 mu M) 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-acetyl-cysteine, 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 mu M 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-.