The effect of NO-donors on chloride efflux, intracellular Ca2+ concentration and mRNA expression of CFTR and ENaC in cystic fibrosis airway epithelial cells

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 (Delta F508/Delta 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 Ca2+ 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 C2+ 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 alpha-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 Ca2+-activated Cl- channels. (c) 2013 Published by Elsevier Inc.