NNOS goes nuclear: nNOS‐ and NADPH oxidase‐derived reactive oxygen/nitrogen species promote oxidative nuclear damage in alveolar epithelial cells.

Abstract

Rationale: Although emerging evidence implicates a role for angiotensin II (Ang II)-stimulated reactive oxygen and nitrogen species (ROS/RNS) formation in acute lung injury (ALI), details of the mechanism are lacking. We hypothesized that compartmentalized generation of superoxide (O2-) and nitric oxide (·NO) may be key events in the Ang II-stimulated progression of ALI. Methods: Human alveolar epithelial (A549) cells were treated with Ang II +/− the specific nNOS inhibitor NPA, or the NADPH oxidase inhibitor, apocynin, then observed for ROS/RNS generation and localization of nNOS and Nox4. A549 cells were further analyzed via western blot, or confocal microscopy, for poly (ADP-ribose) (PAR) polymer formation, an indicator of oxidative nuclear damage. Results: Compared to unstimulated controls, Ang II increased ROS/RNS production 7.4 fold, an effect blocked by NPA or apocynin. Ang II causes Nox4 and nNOS to transiently co-immunoprecipitate, and co-localize at the peri-nuclear region within 15 minutes. Subsequently, nNOS translocates to the nucleus, suggesting that nNOS may regulate nuclear signaling directly. Furthermore, PAR polymers, which are undetectable in resting conditions, were generated following Ang II stimulation, an effect blocked with apocynin or NPA. Conclusions: These data suggest that nNOS/Nox mediate Ang II-induced nuclear oxidative damage in lung epithelial cells.