Absence of CFTR is associated with pleiotropic effects on mucins in mouse gallbladder epithelial cells

2006 ◽  
Vol 291 (6) ◽  
pp. G1148-G1154 ◽  
Author(s):  
Rahul Kuver ◽  
Thomas Wong ◽  
Johanne Henriette Klinkspoor ◽  
Sum P. Lee
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Pleiotropic Effects ◽  
Mrna Levels ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Mucous Granule ◽  
Organ Systems ◽  
Gene And Protein Expression ◽  
Quantitative Immunoblotting

Mucus of cystic fibrosis patients exhibits altered biochemical composition and biophysical behavior, but the causal relationships between altered cystic fibrosis transmembrane conductance regulator (CFTR) function and the abnormal mucus seen in various organ systems remain unclear. We used cultured gallbladder epithelial cells (GBEC) from wild-type and Cftr(−/−) mice to investigate mucin gene and protein expression, kinetics of postexocytotic mucous granule content expansion, and biochemical and ionic compositions of secreted mucins. Muc1, Muc3, Muc4, Muc5ac, and Muc5b mRNA levels were significantly lower in Cftr(−/−) GBEC compared with wild-type cells, whereas Muc2 mRNA levels were higher in Cftr(−/−) cells. Quantitative immunoblotting demonstrated a trend toward lower MUC1, MUC2, MUC3, MUC5AC, and MUC5B mucin levels in Cftr(−/−) cells compared with cells from wild-type mice. In contrast, the levels of secreted MUC1, MUC3, MUC5B, and MUC6 mucins were significantly higher from Cftr(−/−) cells; a trend toward higher levels of secreted MUC2 and MUC5AC was also noted from Cftr(−/−) cells. Cftr(−/−) cells demonstrated slower postexocytotic mucous granule content expansion. Calcium concentration was significantly elevated in the mucous gel secreted by Cftr(−/−) cells compared with wild-type cells. Secreted mucins from Cftr(−/−) cells contained higher sulfate concentrations. Thus absence of CFTR is associated with pleiotropic effects on mucins in murine GBEC.

scholarly journals Enhancement of alveolar epithelial sodium channel activity with decreased cystic fibrosis transmembrane conductance regulator expression in mouse lung

2011 ◽  
Vol 301 (4) ◽  
pp. L557-L567 ◽  
Author(s):  
Ahmed Lazrak ◽  
Asta Jurkuvenaite ◽  
Lan Chen ◽  
Kim M. Keeling ◽  
James F. Collawn ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Alveolar Epithelial ◽  
Cystic Fibrosis Transmembrane

We sought to establish whether the cystic fibrosis transmembrane conductance regulator (CFTR) regulates the activity of amiloride-sensitive sodium channels (ENaC) in alveolar epithelial cells of wild-type, heterozygous ( Cftr +/−), knockout ( Cftr −/−), and ΔF508-expressing mice in situ. RT-PCR studies confirmed the presence of CFTR message in freshly isolated alveolar type II (ATII) cells from wild-type mice. We patched alveolar type I (ATI) and ATII cells in freshly prepared lung slices from these mice and demonstrated the presence of 4-pS ENaC channels with the following basal open probabilities (Po): wild-type=0.21 ± 0.015: Cftr +/−=0.4 ± 0.03; ΔF508=0.55 ± 0.01; and Cftr −/−=and 0.81 ± 0.016 (means ± SE; n ≥ 9). Forskolin (5 μM) or trypsin (2 μM), applied in the pipette solution, increased the Po and number of channels in ATII cells of wild-type, Cftr +/−, and ΔF508, but not in Cftr −/− mice, suggesting that the latter were maximally activated. Western blot analysis showed that lungs of all groups of mice had similar levels of α-ENaC; however, lungs of Cftr +/− and Cftr −/− mice had significantly higher levels of an α-ENaC proteolytic fragment (65 kDa) that is associated with active ENaC channels. Our results indicate that ENaC activity is inversely correlated to predicted CFTR levels and that CFTR heterozygous and homozygous mice have higher levels of proteolytically processed ENaC fragments in their lungs. This is the first demonstration of functional ENaC-CFTR interactions in alveolar epithelial cells in situ.

Endoplasmic reticulum stress and the unfolded protein response regulate genomic cystic fibrosis transmembrane conductance regulator expression

2007 ◽  
Vol 292 (2) ◽  
pp. C756-C766 ◽  
Author(s):  
András Rab ◽  
Rafal Bartoszewski ◽  
Asta Jurkuvenaite ◽  
John Wakefield ◽  
James F. Collawn ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Er Stress ◽  
Mrna Levels ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Cystic Fibrosis Transmembrane

The unfolded protein response (UPR) is a cellular recovery mechanism activated by endoplasmic reticulum (ER) stress. The UPR is coordinated with the ER-associated degradation (ERAD) to regulate the protein load at the ER. In the present study, we tested how membrane protein biogenesis is regulated through the UPR in epithelia, using the cystic fibrosis transmembrane conductance regulator (CFTR) as a model. Pharmacological methods such as proteasome inhibition and treatment with brefeldin A and tunicamycin were used to induce ER stress and activate the UPR as monitored by increased levels of spliced XBP1 and BiP mRNA. The results indicate that activation of the UPR is followed by a significant decrease in genomic CFTR mRNA levels without significant changes in the mRNA levels of another membrane protein, the transferrin receptor. We also tested whether overexpression of a wild-type CFTR transgene in epithelia expressing endogenous wild-type CFTR activated the UPR. Although CFTR maturation is inefficient in this setting, the UPR was not activated. However, pharmacological induction of ER stress in these cells also led to decreased endogenous CFTR mRNA levels without affecting recombinant CFTR message levels. These results demonstrate that under ER stress conditions, endogenous CFTR biogenesis is regulated by the UPR through alterations in mRNA levels and posttranslationally by ERAD, whereas recombinant CFTR expression is regulated only by ERAD.

Basal chloride currents in murine airway epithelial cells: modulation by CFTR

1998 ◽  
Vol 274 (4) ◽  
pp. C904-C913 ◽  
Author(s):  
R. Tarran ◽  
M. A. Gray ◽  
M. J. Evans ◽  
W. H. Colledge ◽  
R. Ratcliff ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Airway Epithelial Cells ◽  
Transmembrane Conductance Regulator ◽  
Airway Epithelial ◽  
Wild Type ◽  
Patch Clamp Technique ◽  
Chloride Currents ◽  
Cation Permeability ◽  
Current Voltage ◽  
Respiratory Cells

We have isolated ciliated respiratory cells from the nasal epithelium of wild-type and cystic fibrosis (CF) null mice and used the patch-clamp technique to investigate their basal conductances. Current-clamp experiments on unstimulated cells indicated the presence of K+ and Cl− conductances and, under certain conditions, a small Na+conductance. Voltage-clamp experiments revealed three distinct Cl− conductances. I tv-indep was time and voltage independent with a linear current-voltage ( I- V) plot; I v-actexhibited activation at potentials greater than ±50 mV, giving an S-shaped I- Vplot; and I hyp-act was activated by hyperpolarizing potentials and had an inwardly rectified I- Vplot. The current density sequence was I hyp-act = I v-act ≫ I tv-indep. These conductances had Cl−-to- N-methyl-d-glucamine cation permeability ratios of between 2.8 and 10.3 and were unaffected by tamoxifen, flufenamate, glibenclamide, DIDS, and 5-nitro-2-(3-phenylpropylamino) benzoic acid but were inhibited by Zn2+ and Gd3+. I tv-indep and I v-act were present in wild-type and CF cells at equal density and frequency. However, I hyp-actwas detected in only 3% of CF cells compared with 26% of wild-type cells, suggesting that this conductance may be modulated by cystic fibrosis transmembrane conductance regulator (CFTR).

Sign in / Sign up

Export Citation Format

Share Document