Regulation of the inducible cyclo‐oxygenase pathway in human cultured airway epithelial (A549) cells by nitric oxide

In airway epithelium, nitric oxide (NO) is synthesized in the setting of inflammation by inducible nitric oxide synthase (iNOS). Although the role of epithelial derived NO in the regulation of human airways is unknown, prostaglandin E2 (PGE2) is recognised as an important inhibitory mediator in human airways. Cyclo‐oxygenase (COX) is the rate limiting enzyme in the production of prostanoids and since inflammatory pathways enhance the expression of an inducible COX (COX‐2), both COX‐2 and iNOS may be co‐expressed in response to an inflammatory stimulus. Although regulation of the COX‐2 pathway by NO has been demonstrated in animal models, its potential importance in human airway epithelium has not been investigated. The effect of endogenous and exogenous NO on the COX‐2 pathway was investigated in the A549 human airway epithelial cell culture model. Activity of the COX‐2 pathway was assessed by PGE2 EIA, and iNOS pathway activity by nitrite assay. A combination cytokine stimulus of interferon gamma (IFNγ) 100 u ml−1, interleukin‐1β (IL‐1β) 1 u ml−1 and lipopolysaccharide (LPS) 10 μg ml−1 induced nitrite formation which could be inhibited by the competitive NOS inhibitor NG‐nitro‐L‐arginine‐methyl‐ester (L‐NAME). IL‐1β alone (1–50 u ml−1) induced PGE2 formation without significant nitrite formation, a response which was inhibited by the COX‐2 specific inhibitor nimesulide. Submaximal stimuli used for further experiments were IFNγ 100 u ml−1, IL‐1β 1 u ml−1 and LPS 10 μg ml−1 to induce both the iNOS and COX‐2 pathways, and IL‐1β 3 u ml−1 to induce COX‐2 without iNOS activity. Cells treated with IFNγ 100 u ml−1, IL‐1β 1 u ml−1 and LPS 10 μg ml−1 for 48 h either alone, or with the addition of L‐NAME (0 to 10−2 M), demonstrated inhibition by L‐NAME of PGE2 (3.61±0.55 to 0.51±0.04 pg/104 cells; P