Abstract
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-1β, and interferon-γ for 12 h. Cytokines significantly increased the expression and activity of iNOS, and reduced generation of cGMP in response to stimulation with NO donorS-nitroso-glutathione (GSNO). Patch-clamp studies showed that 100 μM 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 μM 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−.
Footnotes
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Send reprint requests to: Dr. M. Duszyk, Department of Physiology, University of Alberta, 7-46 Medical Sciences Bldg., Edmonton, Alberta T6G 2H7 Canada. E-mail: marek.duszyk{at}ualberta.ca
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↵1 This work was supported by the Canadian Cystic Fibrosis Foundation and the Medical Research Council of Canada. B.K. was supported by a fellowship award from the Canadian Cystic Fibrosis Foundation. M.W.R is a Medical Research Council scientist, and M.D. is an Alberta Heritage Foundation for Medical Research (AHFMR) Senior Scholar.
- Abbreviations:
- IFN-γ
- interferon-γ
- TNF
- tumor necrosis factor
- iNOS
- inducible nitric-oxide synthase
- IL-β
- interleukin-1β
- NO
- nitric oxide
- eNOS
- endothelial nitric-oxide synthase
- IBMX
- 3-isobutyl-1-methylxanthine
- GSNO
- S-nitroso-glutathione
- ODQ
- 1H-[1,2,4]oxadiazole [4,3-α]quinoxalin-1-one
- DCF-DA
- 2,7-dihydrodichlorofluorescein diacetate
- DCF-H
- 2,7-dihydrodichlorofluorescein
- 1400W
- N-(3-(aminomethyl)benzyl)acetamidine
- SOD
- superoxide dismutase
- LDH
- lactate dehydrogenase
- 8-Br-cGMP
- 8-bromo-cGMP
- Received April 14, 2000.
- Accepted July 10, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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