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

Abstract

Background: It has been suggested that low microM 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 microM 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 microM 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.

Figure 1

The effect of GSNO treatment (60 μM for 4 hours) on the Cl- relative efflux rate measured in nasal epithelial cells with the MQAE fluorescence method. Individual data from (a) CF patients and (b) healthy subjects; horizontal line indicates the mean. (c) Data are expressed as mean ± SEM and the number of subjects in each group is shown in the parentheses beside the legend. Significant difference from the control (paired Student's t-test) is indicated by asterisks (** p < 0.01).

Figure 2

Immunoblot analysis on homozygous ΔF508 CFBE cultured cells treated with various concentrations of GSNO. Western blot analysis of CFTR was performed on whole cell extracts from CFBE cells using the monoclonal antibody 596, in the absence or presence of GSNO at different concentrations for 4 hours. Lane 1-control; lane 2- 0.5 μM; lane 3- 5 μM; and lane 4- 10 μM of GSNO. 50 μg of protein was loaded to each line.

Figure 3

The effect of GSNO and GEA in CFBE (A) and CFSME (B) cells on the Cl^-efflux rate. Cells were treated with 60 μM GSNO for 4 hours or 25 μM GEA for the indicated time, then the Cl^- efflux rate was measured with the fluorescent indicator MQAE. "J basal": the basal efflux rate (recorded during exposure to Cl^--free SR without agonists). "J cAMP": the cAMP-stimulated efflux rate (recorded during exposure to Cl^--free SR containing 20 μM forskolin and 100 μM IBMX). For some experiments, during the basal efflux 400 μM DIDS was used to block Cl^- channels (different from CFTR), while the CFTR specific inhibitor CFTR_inh-172 (40 μM) was used during the cAMP-stimulated efflux. Data are expressed as mean ± SEM and the number of experiments in each group is shown in each column. Significant difference from the control (ANOVA) is indicated by asterisks (* p < 0.05, ** p < 0.01).

Figure 4

The effect of GSNO on intracellular ion composition in CFSME cells assessed by X-ray microanalysis method. "Control": Cells kept 2 min in Cl^--free SR. "GSNO": cells treated with 60 μM GSNO for 4 hours, then exposed for 3 min to Cl^--free SR. fors+IBMX = 3 min exposure to Cl^--free SR containing 20 μM forskolin and 100 μM IBMX. Data are expressed as mean ± SEM. Significant difference from the control (ANOVA) is indicated by asterisks (* p < 0.05, ** p < 0.01).

Figure 5

Plot of the intracellular calcium concentration in the CFBE cells. In the control experiment (continuous line) the cells were exposed only to ATP (the duration is indicated by the horizontal line). In another experiment (dotted line) the cells were exposed first to GSNO, then, after a 5 minutes washing step, to ATP. The two results were overlapped. Plots represent the averaged results of 3–6 experiments.