Regulation of Nitric Oxide by Cigarette Smoke in Airway Cells

Background and Objectives: Exhaled nitric oxide (NO) is decreased by smoking while oxides of nitrogen such as nitrites/nitrates (NOx) are increased. It was hypothesised that in vitro cigarette smoke extract (CSE) would either inhibit NO generation by increasing the NO synthase inhibitor, NG, NG-dimethyl-L-arginine (ADMA) or increase NOx levels via an oxidation pathway, which in turn could be inhibited by the antioxidant N-acetylcysteine NAC. Methods: Transformed airway cells (A549) were cultured with control medium, 1.0% CSE in culture medium, or 0.8 mM NAC with 1.0% CSE. Baseline L-arginine, NOx and ADMA levels were measured in the media. Conditioned media were then sampled at 1hour, 6 hours, 24 hours, 48 hours and 72 hours after incubation. Results: CSE induced significantly higher NOx levels (mean (SD) peak increase of 135.8 (126.6)% after incubation for 6 hours (p < 0.0005)). NAC pre-treatment partially reversed this effect to 35.6 (21.4)% at 6 hours (p = 0.009). ADMA levels were significantly higher in the CSE conditioned media compared with control media (p = 0.02) while NAC pre-treatment did not affect ADMA levels. Conclusions: CSE increased NOx which was partially reversed by NAC pre-treatment. ADMA levels were also increased after CSE exposure, suggesting that it activates the NO pathway via oxidative-stress while inhibition probably occurs via both ADMA and NOS.