title: The effect of NO-donors on chloride efflux, intracellular Ca(2+) concentration and mRNA expression of CFTR and ENaC in cystic fibrosis airway epithelial cells.
abstract: Since previous studies showed that the endogenous bronchodilator, S-nitrosglutathione (GSNO), caused a marked increase in CFTR-mediated chloride (Cl(-)) efflux and improved the trafficking of CFTR to the plasma membrane, and that also the nitric oxide (NO)-donor GEA3162 had a similar, but smaller, effect on Cl(-) efflux, it was investigated whether the NO-donor properties of GSNO were relevant for its effect on Cl(-) efflux from airway epithelial cells. Hence, the effect of a number of other NO-donors, sodium nitroprusside (SNP), S-nitroso-N-acetyl-DL-penicillamine (SNAP), diethylenetriamine/nitric oxide adduct (DETA-NO), and diethylenetriamine/nitric oxide adduct (DEA-NONOate) on Cl(-) efflux from CFBE (∆F508/∆F508-CFTR) airway epithelial cells was tested. Cl(-) efflux was determined using the fluorescent N-(ethoxycarbonylmethyl)-6-methoxyquinoliniu bromide (MQAE)-technique. Possible changes in the intracellular Ca(2+) concentration were tested by the fluorescent fluo-4 method in a confocal microscope system. Like previously with GSNO, after 4 h incubation with the NO-donor, an increased Cl(-) efflux was found (in the order SNAP>DETA-NO>SNP). The effect of DEA-NONOate on Cl(-) efflux was not significant, and the compound may have (unspecific) deleterious effects on the cells. Again, as with GSNO, after a short (5 min) incubation, SNP had no significant effect on Cl(-) efflux. None of the NO-donors that had a significant effect on Cl(-) efflux caused significant changes in the intracellular Ca(2+) concentration. After 4 h preincubation, SNP caused a significant increase in the mRNA expression of CFTR. SNAP and DEA-NONOate decreased the mRNA expression of all ENaC subunits significantly. DETA-NO caused a significant decrease only in α-ENaC expression. After a short preincubation, none of the NO-donors had a significant effect, neither on the expression of CFTR, nor on that of the ENaC subunits in the presence and absence of L-cysteine. It can be concluded that the effect of GSNO on Cl(-) efflux is, at least in part, due to its properties as an NO-donor, and the effect is likely to be mediated by CFTR, not by Ca(2+)-activated Cl(-) channels.
authors.affiliation: School of Health and Medical Sciences, University of Örebro, Örebro University Hospital, Örebro, Sweden.
authors.forename: Igor
authors.initials: I
authors.lastname: Oliynyk
authors.forename: Rashida
authors.initials: R
authors.lastname: Hussain
authors.forename: Ahmad
authors.initials: A
authors.lastname: Amin
authors.forename: Marie
authors.initials: M
authors.lastname: Johannesson
authors.forename: Godfried M
authors.initials: GM
authors.lastname: Roomans
mesh_terms: D001980:Bronchi / Q000473:pathology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D053503:Epithelial Sodium Channels / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D014230:Triazoles / Q000494:pharmacology
chemical_list: D002712:Chlorides; D053503:Epithelial Sodium Channels; D020030:Nitric Oxide Donors; D012333:RNA, Messenger; D014230:Triazoles; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C082874:GEA 3162; D002118:Calcium