Previous in vivo studies have indicated that prolonged exposure to NO + HYP(but not NO alone) causes significant lung injury in neonatal piglets. Peroxynitrate (ONOO), the product of NO and superoxide, can cause protein nitration and oxidant tissue injury and is thought to be a causative agent in the injury process. Nitrotyrosine (NT), a stable byproduct of ONOO, has been used as a specific indicator of ONOO formation. To determine whether NT formation occurs in vitro, cultured human alveolar epithelial cells (A549) were grown in room air (RA), 95% oxygen (HYP), 2mM SNAP (an NO donor) or HYP + SNAP. Western blot analysis, using a polyclonal antibody against NT, revealed NT-containing proteins at 25 and 35 kD from cells exposed to SNAP or SNAP + HYP. In vivo studies were then performed with 1-3 d old piglets, mechanically ventilated with RA (n=2), 100% oxygen (n=3), 100 PPM NO (n=4) or 100 PPM NO and 90% oxygen (n=4) for 48 h. Animals were sacrificed, the lungs removed and Western blot analysis performed for NT. Results demonstrated one major 35 kD protein in lung tissue from animals exposed to NO or NO + 90% oxygen, but not in animals exposed to RA or 100% oxygen. Control blots with preabsorbed antibody showed no specific staining. These results indicate that while NT may be a specific marker of ONOO formation, the presence of NT alone may not be a specific indicator of lung tissue injury. In addition, these data suggest that sufficient amounts of superoxide may be present both in tissue culture and in vivo to yield ONOO in the absence of an additional superoxide donor. Further studies are necessary to more clearly elucidate the role of protein nitration in lung injury.
Supported by a grant from the American Lung Association.