Nitric oxide (NO) has been discovered to be a potent vasodilator. Endogenous NO is produced by many cell types and is important in regulating basal vasomotor tone. Exogenous NO has recently been used very successfully to treat pulmonary hypertension in infants and children. In most cases, these patients receive simultaneous oxygen therapy. Unfortunately, there are few reports on the combined toxicity of NO and hyperoxia, either of which can be toxic alone. Together, the potential for the formation of peroxynitrite(ONOO˙) - an extremely reactive and toxic free radical - is increased. To directly test this potential toxicity, cultured human alveolar epithelial(A549) cells were grown in room air (control), hyperoxia (95% O2), 2 mM SNAP (S-nitroso-N-acetyl penicillamine - an NO donor) and hyperoxia + SNAP. Live cells were counted in each of the groups above daily for the next six days, and media and gasses were refreshed each day. Cells began to die in hyperoxia after 4 to 5 d, but cell counts were relatively unchanged in NO. However, cells exposed to both NO and hyperoxia began to die on day 2, and died rapidly thereafter. This cytotoxic effect was clearly synergistic, the death rate far exceeding any additive effect. Some cells were grown on cover slips for later immunocytochemistry for nitrotyrosine, which is a stable byproduct of peroxynitrite (which is too reactive to detect directly). Immunoreactive nitrotyrosine was most abundant only in cells exposed to NO + hyperoxia. Western blots indicate that a variety of proteins become nitrated under various conditions. However, a unique band appeared on blots only after culture in NO + hyperoxia. These experiments indicate that NO and hyperoxia have a synergistic cytotoxic effect on alveolar cells in culture, which is probably mediated by production of peroxynitrite.(Funded by grants from the NIH-NHLBI, ALA and Winthrop-University Hospital)