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 E 2 (PGE 2 ) 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 PGE 2 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 N G ‐nitro‐ L ‐arginine‐methyl‐ester ( L ‐NAME). IL‐1β alone (1–50 u ml −1 ) induced PGE 2 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 PGE 2 (3.61±0.55 to 0.51±0.04 pg/10 4 cells; P <0.001) and nitrite (34.33±8.07 to 0 pmol/10 4 cells; P <0.001) production. Restoration of the PGE 2 response (0.187±0.053 to 15.46±2.59 pg/10 4 cells; P <0.001) was observed after treating cells with the same cytokine stimulus and L ‐NAME 10 −6 M , but with the addition of the NOS substrate L ‐arginine (0 to 10 −5 M ). Cells incubated with IL‐1β 3 u ml −1 for 6 h, either alone or with addition of the NO donor S‐nitroso‐acetyl‐penicillamine (SNAP) (0 to 10 −4 M ), demonstrated increased PGE 2 formation (1.23±0.03 to 2.92±0.19 pg/10 4 cells; P < 0.05). No increase in PGE 2 formation was seen when the experiment was repeated in the presence of the guanylate cyclase inhibitor methylene blue (50 μ M ). Cells treated with SNAP alone did not demonstrate an increased PGE 2 formation. Cells incubated with IL‐1β 3 u ml −1 for 6 h in the presence of dibutyryl cyclic guanylate monophosphate (0 to 10 −3 M ) also demonstrated an increased PGE 2 response (2.56±0.21 to 4.53±0.64 pg/10 4 cells; P <0.05). These data demonstrate that in a human airway epithelial cell culture system, both exogenous and endogenous NO increase the activity of the COX‐2 pathway in the setting of inflammatory cytokine stimulation, and that this effect is likely to be mediated by guanylate cyclase. This suggests a role for NO in the regulation of human airway inflammation.