Lung inflammation is associated with enhanced expression of proinflammatory cytokines and increased production of nitric oxide (NO) by inducible NO synthase (iNOS). To investigate the possible relationship between cytokine-induced expression of iNOS and epithelial ion channel function, we measured whole-cell current in A549 cells treated with a mixture of cytokines: tumor necrosis factor, interleukin-1 beta, and interferon-gamma for 12 h. Cytokines significantly increased the expression and activity of iNOS, and reduced generation of cGMP in response to stimulation with NO donor S-nitroso-glutathione (GSNO). Patch-clamp studies showed that 100 microM GSNO increased the whole-cell current from 11.2 +/- 1.8 to 19.6 +/- 2.7 pA/pF (n = 16) in control cells, but had no effect in cytokine-treated cells (n = 9). N-(3-(Aminomethyl)benzyl)acetamidine (1400W), a selective inhibitor of iNOS, restored activation of the current by GSNO in cytokine-treated cells, indicating a crucial role for iNOS in this process. Cells treated with cytokines showed increased levels of peroxynitrite (ONOO(-)), compared with the control, or cells that were treated with the cytokines and 1400W or superoxide dismutase/catalase. Treatment of cells with 100 microM ONOO(-) had no effect on the whole-cell current, but in contrast to untreated cells, subsequent application of GSNO did not activate the current. In conclusion, cytokine-induced expression of iNOS affects activation of the whole-cell current via NO/cGMP pathway, likely by increasing the generation of ONOO(-).