Peroxynitrite (ONOO-), an anion and a potent oxidant, generated by the interaction of nitric oxide (NO) and superoxide is able to induce apoptosis in HL-60 human leukemia cells in a time- and concentration-dependent manner. Characteristic morphology of apoptosis can be observed 3 h after HL-60 cells are exposed to 10 microM ONOO-. Treatment of HL-60 cells with increasing concentrations of ONOO- from 1 to 100 microM confirms the concentration dependence of apoptosis as evidenced by: 1) degradation of nuclear DNA of these cells into integer multiples of approximately 200 base pairs; 2) colorimetric DNA fragmentation assay; and 3) evidence of condensation of chromatin and nuclear fragmentation shown by propidium iodide staining. Under the same conditions, peroxynitrite causes apoptosis in another transformed cell line, U-937 cells, but is ineffective at inducing apoptosis in normal endothelial cells derived from human umbilical cord and normal human peripheral blood mononuclear cells. This direct evidence of peroxynitrite inducing apoptosis implicated a new function of this potent oxidant.

In the present study, the contribution of nitric oxide (NO), superoxide, and peroxynitrite to the inflammatory response induced by myocardial ischemia-reperfusion (MI/R) was investigated. Male Sprague-Dawley rats were anesthetized, and the left main coronary artery was ligated for 20 min and reperfused for 5 h. MI/R induced severe arrhythmias, indicated by a significantly elevated arrhythmia score in the MI/R group compared with that in the sham control group. Creatine kinase activity in the left ventricular free wall of the MI/R group was significantly reduced by 38%. In contrast, myeloperoxidase activity in the left ventricular free wall of the MI/R group was increased by 140%. Similarly, superoxide and tissue NO levels in the ischemic region of the heurt were increased by 140 and 90%, respectively. Superoxide and NO values in the nonischemic regions were similar to the sham control group. Total NO synthase (NOS) activity was elevated by 212%; moreover, inducible NOS (iNOS) activity increased 6.7-fold in the ischemic vs. nonischemic regions. MI/R also induced both systemic and remote organ (lung) inflammatory responses. Circulating neutrophils and plasma NO levels were increased by 163 and 138%, respectively, in MI/R rats compared with sham control animals. NO levels and superoxide generation were increased by 90 and 176%, respectively, in the lung tissues. The expression of iNOS and peroxynitrite generation were demonstrated by immunohistochemical staining with polyclonal anti-iNOS and monoclonal anti-nitrotyrosine antibodies, respectively. Sections of both the ischemic area of the ventricular wall and the lung tissue of MI/R animals exhibited a marked immunoreactivity with anti-iNOS and anti-nitrotyrosine antibodies, indicating the presence of iNOS and nitrotyrosine. Our data indicate that NO, superoxide, and peroxynitrite formation are elevated after reperfusion of the ischemic heart, suggesting that these inflammatory mediators may be involved in MI/R injury.

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