Inhaled nitric oxide (NO) is efficacious and in increasing clinical use in the therapy of persistent pulmonary hypertension of the newborn. The potential adverse effects of inhaled NO are unknown and represent the focus of the present studies. Treatment of mice with inhaled NO at doses of 20 to 100 ppm(5 h) was found to prime alveolar macrophages (AM) to release increased amounts of reactive oxygen and nitrogen intermediates in response to inflammatory mediators. These effects were dose and time dependent and were not observed at doses of NO ≤ 10 ppm. Increased NO production by AM was correlated with increased iNOS protein expression, as determined by Western blotting and immunofluorescence. We also found that inhalation of nitric oxide resulted in increased peroxynitrite production by AM, which was associated with increased nitrotyrosine expression in lung sections. Overproduction of reactive oxygen and nitrogen intermediates has been implicated in oxidant-induced tissue damage. In this regard, we found increased protein in bronchoalveolar lavage (BAL) fluid, suggesting that inhaled NO induces alveolar epithelial damage. To evaluate the role of endogenous NO in tissue injury, we used knockout mice lacking the iNOS gene. Stimulated AM from these animals did not produce detectable quantities of nitrite or nitrate. In contrast to AM from control mice, AM from knockout mice were not primed to produce increased superoxide anion or peroxynitrite. Moreover, BAL fluid protein was not increased in the knockout strain following inhalation of NO doses up to 100 ppm. These data suggest that reactive nitrogen intermediates produced by primed lung macrophages play a role in the tissue damage induced by inhaled NO.
