Reactive oxygen nitrogen species decrease cystic fibrosis transmembrane conductance regulator expression and cAMP-mediated Cl- secretion in airway epithelia.
We investigated putative mechanisms by which nitric oxide modulates cystic fibrosis transmembrane conductance regulator (CFTR) expression and function in epithelial cells. Immunoprecipitation followed by Western blotting, as well as immunocytochemical and cell surface biotinylation measurements, showed that incubation of both stably transduced (HeLa) and endogenous CFTR expressing (16HBE14o-, Calu-3, and mouse tracheal epithelial) cells with 100 microm diethylenetriamine NONOate (DETA NONOate) for 24-96 h decreased both intracellular and apical CFTR levels. Calu-3 and mouse tracheal epithelial cells, incubated with DETA NONOate but not with 100 microm 8-bromo-cGMP for 96 h, exhibited reduced cAMP-activated short circuit currents when mounted in Ussing chambers. Exposure of Calu-3 cells to nitric oxide donors resulted in the nitration of a number of proteins including CFTR. Nitration was augmented by proteasome inhibition, suggesting a role for the proteasome in the degradation of nitrated proteins. Our studies demonstrate that levels of nitric oxide that are likely to be encountered in the vicinity of airway cells during inflammation may nitrate CFTR resulting in enhanced degradation and decreased function. Decreased levels and function of normal CFTR may account for some of the cystic fibrosis-like symptoms that occur in chronic inflammatory lung diseases associated with increased NO production.
Department of Medicine, and The Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama, Birmingham, Alabama 35233, USA.
Zsuzsa
Z
Bebok
Karoly
K
Varga
James K
JK
Hicks
Charles J
CJ
Venglarik
Timea
T
Kovacs
Lan
L
Chen
Karin M
KM
Hardiman
James F
JF
Collawn
Eric J
EJ
Sorscher
Sadis
S
Matalon
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002712:Chlorides / Q000378:metabolism*; D000242:Cyclic AMP / Q000502:physiology*; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000187:drug effects / Q000235:genetics*; D004594:Electrophysiology / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009603:Nitroso Compounds / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D026361:Reactive Nitrogen Species / Q000502:physiology*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000502:physiology*; D014132:Trachea; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
C505032:CFTR protein, human; D002712:Chlorides; D015853:Cysteine Proteinase Inhibitors; D009097:Multienzyme Complexes; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C094210:2,2'-(hydroxynitrosohydrazono)bis-ethanamine; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D000242:Cyclic AMP; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D006152:Cyclic GMP