Abstract
BackgroundIt has been suggested that low μM 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.MethodsThe 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.ResultsTreatment with 60 μM 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 μM 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.ConclusionPrevious 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.
Highlights
It has been suggested that low μM concentrations of S-nitrosoglutathione (GSNO), an endogenous bronchodilator, may promote maturation of the defective cystic fibrosis (CF) transmembrane conductance regulator (CFTR)
GSNO increases delF508 CFTR function in primary nasal epithelial cells from CF patients In the nasal tall columnar ciliated (TCC) epithelial cells from CF patients, GSNO treatment increased the relative cAMP-dependent efflux rate (JCFTR) from -0.19 ± 0.06 to 0.11 ± 0.08 percent/s, reaching the efflux level observed in healthy nasal columnar cells
Chloride efflux in nasal cells from healthy persons was not affected by GSNO treatment (Fig. 1b, 1c)
Summary
It has been suggested that low μM concentrations of S-nitrosoglutathione (GSNO), an endogenous bronchodilator, may promote maturation of the defective cystic fibrosis (CF) transmembrane conductance regulator (CFTR). The disease is caused by mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel present in the apical membrane of epithelia in many organs, including airways [2]. We show that GSNO increases maturation and function of delF508 CFTR using both airway epithelial cell lines and primary nasal epithelial cells from CF patients. CFTR is synthesized in the endoplasmic reticulum (ER) and transported to the Golgi complex where, after N-glycosylation, it becomes a mature protein that via the secretory pathway reaches the plasma membrane. Yoo et al [7] reported an alternative pathway of CFTR trafficking between ER and Golgi complex that may affect CFTR expression in the cell membrane
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