Activation of chloride transport in CF airway epithelial cell lines and primary CF nasal epithelial cells by S-nitrosoglutathione

• Background: It has been suggested that low mu M concentrations of S-nitrosoglutathione (GSNO), an endogenous bronchodilator, may promote maturation of the defective cystic fibrosis (CF) transmembrane conductance regulator ( CFTR). Because nitric oxide ( NO) and GSNO levels appear to be low in the CF airway, there is an interest in the possibility that GSNO replacement could be of therapeutic benefit in CF.Methods: The effect of GSNO on chloride (Cl-) transport was investigated in primary nasal epithelial cells obtained from CF patients homozygous for the delF508 mutation, as well as in two CF cell lines (CFBE and CFSME), using both a fluorescent Cl- indicator and X-ray microanalysis. Maturation of delF508 CFTR was determined by immunoblotting.Results: Treatment with 60 mu M GSNO for 4 hours increased cAMP-induced chloride efflux in nasal epithelial cells from 18 out of 21 CF patients, but did not significantly affect Cl- efflux in cells from healthy controls. This Cl- efflux was confirmed by measurements with a fluorescent Cl- indicator in the CFBE and CFSME cell lines. The effect of GSNO on Cl- efflux in CFBE cells could be inhibited both by a specific thiazolidinone CFTR inhibitor (CFTRinh-172) and by 4,4'-diisothiocyanatodihydrostilbene- 2,2'-disulfonic acid (H2DIDS). X-ray microanalysis showed that, following 4 hours incubation with 60 mu M GSNO, cAMP agonists caused a decrease in the cellular Cl- concentration in CFBE cells, corresponding to Cl- efflux. GSNO exposure resulted in an increase in the protein expression and maturation, as shown by immunoblot analysis. GSNO did not increase the cytosolic Ca2+ concentration in cultured airway epithelial cells.Conclusion: Previous studies have suggested that treatment with GSNO promotes maturation of delF508-CFTR, consistent with our results in this study. Here we show that GSNO increases chloride efflux, both in the two CF cell lines and in primary nasal epithelial cells from delF508-CF patients. This effect is at least in part mediated by CFTR. GSNO may be a candidate for pharmacological treatment of the defective chloride transport in CF epithelial cells.

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