Role of inducible nitric-oxide synthase in regulation of whole-cell current in lung epithelial cells.
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(-).
Kamosinska
B
B
Department of Physiology, University of Alberta, Edmonton, Alberta, Canada.
Radomski
A
A
Man
S F
SF
Radomski
M W
MW
Duszyk
M
M
D006152:Cyclic GMP / Q000378:metabolism / Q000494:pharmacology; D016207:Cytokines / Q000494:pharmacology*; D004789:Enzyme Activation; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D007473:Ion Channels / Q000378:metabolism; D008168:Lung / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009566:Nitrates / Q000378:metabolism / Q000502:physiology; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D009603:Nitroso Compounds / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011994:Recombinant Proteins; D026422:S-Nitrosoglutathione; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016207:Cytokines; D007375:Interleukin-1; D007473:Ion Channels; D009566:Nitrates; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C039642:peroxynitric acid; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D007371:Interferon-gamma; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D005978:Glutathione; D006152:Cyclic GMP