Chloride conductance of CFTR facilitates basal Cl−/HCO3−exchange in the villous epithelium of intact murine duodenum

2005 ◽  
Vol 288 (6) ◽  
pp. G1241-G1251 ◽  
Author(s):  
Janet E. Simpson ◽  
Lara R. Gawenis ◽  
Nancy M. Walker ◽  
Kathryn T. Boyle ◽  
Lane L. Clarke
Keyword(s):  
Exchange Rate ◽  
Apical Membrane ◽  
Anion Channel ◽  
Duodenal Mucosa ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Anion Exchanger 1 ◽  
Direct Exposure ◽  
Mucus Barrier ◽  
Cf Transmembrane Conductance Regulator

Villi of the proximal duodenum are situated for direct exposure to gastric acid chyme. However, little is known about active bicarbonate secretion across villi that maintains the protective alkaline mucus barrier, a process that may be compromised in cystic fibrosis (CF), i.e., in the absence of a functional CF transmembrane conductance regulator (CFTR) anion channel. We investigated Cl−/HCO3−exchange activity across the apical membrane of epithelial cells located at the midregion of villi in intact duodenal mucosa from wild-type (WT) and CF mice using the pH-sensitive dye BCECF. Under basal conditions, the Cl−/HCO3−exchange rate was reduced by ∼35% in CF compared with WT villous epithelium. Cl−/HCO3−exchange in WT and CF villi responded similarly to inhibitors of anion exchange, and membrane depolarization enhanced rates of Cl−out/HCO3−inexchange in both epithelia. In anion substitution studies, anionin/HCO3−outexchange rates were greater in WT epithelium using Cl−or NO3−, but decreased to the level of the CF epithelium using the CFTR-impermeant anion, SO42−. Similarly, treatment of WT epithelium with the CFTR-selective blocker glybenclamide decreased the Cl−/HCO3−exchange rate to the level of CF epithelium. The mRNA expression of Slc26a3 (downregulated in adenoma) and Slc26a6 (putative anion exchanger-1) was similar between WT and CF duodena. From these studies of murine duodenum, we conclude 1) characteristics of Cl−/HCO3−exchange in the villous epithelium are most consistent with Slc26a6 activity, and 2) Cl−channel activity of CFTR facilitates apical membrane Cl−in/HCO3−outexchange by providing a Cl−“leak” under basal conditions.

Liquid secretion inhibitors reduce mucociliary transport in glandular airways

2002 ◽  
Vol 283 (2) ◽  
pp. L329-L335 ◽  
Author(s):  
Stephen T. Ballard ◽  
Laura Trout ◽  
Anil Mehta ◽  
Sarah K. Inglis
Keyword(s):  
Beat Frequency ◽  
Anion Channel ◽  
Ciliary Beat Frequency ◽  
Mucociliary Transport ◽  
Transmembrane Conductance Regulator ◽  
Inhibitory Effects ◽  
Transmembrane Conductance ◽  
Regulator Protein ◽  
Liquid Absorption ◽  
Cf Transmembrane Conductance Regulator

Because of its possible importance in cystic fibrosis (CF) pulmonary pathogenesis, the effect of anion and liquid secretion inhibitors on airway mucociliary transport was examined. When excised porcine tracheas were treated with ACh to induce gland liquid secretion, the rate of mucociliary transport was increased nearly threefold from 2.5 ± 0.5 to 6.8 ± 0.8 mm/min. Pretreatment with both bumetanide and dimethylamiloride (DMA), to respectively inhibit Cl− and HCO[Formula: see text]secretion, significantly reduced mucociliary transport in the presence of ACh by 92%. Pretreatment with the anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid similarly reduced mucociliary transport in ACh-treated airways by 97%. These agents did not, however, reduce ciliary beat frequency. Luminal application of benzamil to block liquid absorption significantly attenuated the inhibitory effects of bumetanide and DMA on mucociliary transport. We conclude that anion and liquid secretion is essential for normal mucociliary transport in glandular airways. Because the CF transmembrane conductance regulator protein likely mediates Cl−, HCO[Formula: see text], and liquid secretion in normal glands, we speculate that impairment of gland liquid secretion significantly contributes to defective mucociliary transport in CF.

scholarly journals Rab11b Regulates the Apical Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator in Polarized Intestinal Epithelial Cells

2009 ◽  
Vol 20 (8) ◽  
pp. 2337-2350 ◽  
Author(s):  
Mark R. Silvis ◽  
Carol A. Bertrand ◽  
Nadia Ameen ◽  
Franca Golin-Bisello ◽  
Michael B. Butterworth ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Apical Membrane ◽  
Expression System ◽  
Anion Channel ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
T84 Cells ◽  
Anion Secretion ◽  
Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, ΔF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.

scholarly journals Postnatal airway growth in cystic fibrosis piglets

2017 ◽  
Vol 123 (3) ◽  
pp. 526-533 ◽  
Author(s):  
Ryan J. Adam ◽  
Mahmoud H. Abou Alaiwa ◽  
Drake C. Bouzek ◽  
Daniel P. Cook ◽  
Nicholas D. Gansemer ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Lung Disease ◽  
Lung Volume ◽  
Growth And Development ◽  
Anion Channel ◽  
Postnatal Period ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Disease Pathogenesis ◽  
Cf Transmembrane Conductance Regulator

Mutations in the gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) anion channel cause CF. The leading cause of death in the CF population is lung disease. Increasing evidence suggests that in utero airway development is CFTR-dependent and that developmental abnormalities may contribute to CF lung disease. However, relatively little is known about postnatal CF airway growth, largely because such studies are limited in humans. Therefore, we examined airway growth and lung volume in a porcine model of CF. We hypothesized that CF pigs would have abnormal postnatal airway growth. To test this hypothesis, we performed CT-based airway and lung volume measurements in 3-wk-old non-CF and CF pigs. We found that 3-wk-old CF pigs had tracheas of reduced caliber and irregular shape. Their bronchial lumens were reduced in size proximally but not distally, were irregularly shaped, and had reduced distensibility. Our data suggest that lack of CFTR results in aberrant postnatal airway growth and development, which could contribute to CF lung disease pathogenesis. NEW & NOTEWORTHY This CT scan-based study of airway morphometry in the cystic fibrosis (CF) postnatal period is unique, as analogous studies in humans are greatly limited for ethical and technical reasons. Findings such as reduced airway lumen area and irregular caliber suggest that airway growth and development are CF transmembrane conductance regulator-dependent and that airway growth defects may contribute to CF lung disease pathogenesis.

Activation of Ca2+- and cAMP-sensitive K+ channels in murine colonic epithelia by 1-ethyl-2-benzimidazolone

1999 ◽  
Vol 277 (1) ◽  
pp. C111-C120 ◽  
Author(s):  
A. W. Cuthbert ◽  
M. E. Hickman ◽  
P. Thorn ◽  
L. J. MacVinish
Keyword(s):  
Channel Blocker ◽  
Apical Membrane ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Camp Content ◽  
Colonic Crypts ◽  
Electrical Gradient ◽  
Intracellular Ca ◽  
Cf Transmembrane Conductance Regulator ◽  
Sustained Increase

1-Ethyl-2-benzimidazolone (EBIO) caused a sustained increase in electrogenic Cl− secretion in isolated mouse colon mucosae, an effect reduced by blocking basolateral K+ channels. The Ca2+-sensitive K+ channel blocker charybdotoxin (ChTX) and the cAMP-sensitive K+channel blocker 293B were more effective when the other had been added first, suggesting that both types of K+ channel were activated. EBIO did not cause Cl− secretion in cystic fibrosis (CF) colonic epithelia. In apically permeabilized colonic mucosae, EBIO increased the K+ current when a concentration gradient was imposed, an effect that was completely sensitive to ChTX. No current sensitive to trans-6-cyano-4-( N-ethylsulfonyl- N-methylamino)-3-hydroxy-2,2-dimethylchromane (293B) was found in this condition. However, the presence of basolateral cAMP-sensitive K+channels was demonstrated by the development of a 293B-sensitive K+ current after cAMP application in permeabilized mucosae. In isolated colonic crypts EBIO increased cAMP content but had no effect on intracellular Ca2+. It is concluded that EBIO stimulates Cl−secretion by activating Ca2+-sensitive and cAMP-sensitive K+ channels, thereby hyperpolarizing the apical membrane, which increases the electrical gradient for Cl− efflux through the CF transmembrane conductance regulator (CFTR). CFTR is also activated by the accumulation of cAMP as well as by direct activation.

scholarly journals Rescuing ΔF508 CFTR with trimethylangelicin, a dual-acting corrector and potentiator

2014 ◽  
Vol 307 (6) ◽  
pp. L431-L434 ◽  
Author(s):  
James F. Collawn ◽  
Lianwu Fu ◽  
Rafal Bartoszewski ◽  
Sadis Matalon
Keyword(s):  
Anion Channel ◽  
Transmembrane Conductance Regulator ◽  
Nucleotide Binding Domain ◽  
Transmembrane Conductance ◽  
Surface Stability ◽  
Molecular Defects ◽  
Δf508 Cftr ◽  
Cftr Protein ◽  
Cf Transmembrane Conductance Regulator ◽  
Phenylalanine Residue

Since the discovery of the cystic fibrosis (CF) gene that encodes the CF transmembrane conductance regulator (CFTR) in 1989, there has been considerable progress in understanding the molecular defects associated with different mutations in the CFTR protein. Small molecule “potentiators” have led the way as a drug therapeutic approach for correcting channel gating mutations such as the G551D mutation. Therapies for correcting the most common folding mutation in CFTR, ΔF508, however, have proven to be much more challenging. The protein-folding problem appears to be associated with both nucleotide binding domain (NBD) instability and domain interface interactions that are caused by the loss of the phenylalanine residue in NBD 1. Given the inherent complexity in the sequential folding pathway for this very large multidomain protein, it has been suggested that correcting the proper folding, anion channel function, and cell surface stability of the ΔF508 CFTR protein will require a multidrug approach to fix each of these compounding problems. Here we discuss a recent publication (Favia M, Mancini MT, Bezzerri V, Guerra L, Laselva O, Abbattiscianni AC, Debellis L, Reshkin SJ, Gambari R, Cabrini G, Casavola V. Am J Physiol Lung Cell Mol Physiol 307: L48–L61, 2014), however, that offers hope that single drug therapies are still possible.

scholarly journals Metaproteomics to Decipher CF Host-Microbiota Interactions: Overview, Challenges and Future Perspectives

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 892
Author(s):  
Pauline Hardouin ◽  
Raphael Chiron ◽  
Hélène Marchandin ◽  
Jean Armengaud ◽  
Lucia Grenga
Keyword(s):  
Cystic Fibrosis ◽  
Treatment Outcome ◽  
Chronic Infection ◽  
Phenotypic Variability ◽  
Anion Channel ◽  
Hereditary Disease ◽  
Transmembrane Conductance Regulator ◽  
Future Perspectives ◽  
Transmembrane Conductance ◽  
Cf Transmembrane Conductance Regulator

Cystic fibrosis (CF) is a hereditary disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, triggering dysfunction of the anion channel in several organs including the lung and gut. The main cause of morbidity and mortality is chronic infection. The microbiota is now included among the additional factors that could contribute to the exacerbation of patient symptoms, to treatment outcome, and more generally to the phenotypic variability observed in CF patients. In recent years, various omics tools have started to shed new light on microbial communities associated with CF and host–microbiota interactions. In this context, proteomics targets the key effectors of the responses from organisms, and thus their phenotypes. Recent advances are promising in terms of gaining insights into the CF microbiota and its relation with the host. This review provides an overview of the contributions made by proteomics and metaproteomics to our knowledge of the complex host–microbiota partnership in CF. Considering the strengths and weaknesses of proteomics-based approaches in profiling the microbiota in the context of other diseases, we illustrate their potential and discuss possible strategies to overcome their limitations in monitoring both the respiratory and intestinal microbiota in sample from patients with CF.

scholarly journals Outcomes of early repeat sweat testing in infants with cystic fibrosis transmembrane conductance regulator-related metabolic syndrome (CRMS)/CF screen-positive, inconclusive diagnosis (CFSPID)

2021 ◽  
Author(s):  
vito terlizzi ◽  
Laura Claut ◽  
Carla Colombo ◽  
Antonella Tosco ◽  
Alice Castaldo ◽  
...  
Keyword(s):  
Metabolic Syndrome ◽  
Definitive Diagnosis ◽  
First Year ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Sweat Chloride ◽  
Sweat Testing ◽  
Cf Transmembrane Conductance Regulator ◽  
First Year Of Life

Background: Reaching early and definitive diagnosis in infants with cystic fibrosis (CF) transmembrane conductance regulator-related metabolic syndrome (CRMS)/CF screen-positive, inconclusive diagnosis (CFSPID) is a priority of all CF newborn screening programs. Currently, sweat testing is the gold standard for CF diagnosis or exclusion. We assessed outcomes in a cohort of Italian CRMS/CFSPID infants who underwent repeat sweat testing in the first year of life. Methods: This multicentre, prospective study analysed clinical data and outcomes in CRMS/CFSPID infants born between September 1, 2018 and December 31, 2019, and followed until June 30, 2020. All subjects underwent CF transmembrane conductance regulator (CFTR) gene sequencing and the search for CFTR macrodeletions/macroduplications, and repeat sweat testing in the first year of life. Results: Fifty subjects (median age at end of follow-up, 16 months [range, 7–21 months]) were enrolled. Forty-one (82%) had the first sweat chloride in the intermediate range. During follow up, 150 sweat tests were performed (range, 1–7/infant). After a median follow-up of 8.5 months (range 1–16.2 months), 11 (22%) subjects were definitively diagnosed as follows: CF (n=2 [4%]) at 2 and 5 months, respectively; healthy carrier (n=8 [16%]), at a median age of 4 months (range 2–8 months); and healthy (n=1 [2%]) at 2 months of age. Inconclusive diagnosis remained in 39 (78%) infants. Conclusions: Early repeat sweat testing in the first year of life can shorten the time to definitive diagnosis in screening positive subjects with initial sweat chloride levels in the intermediate range.

Bumetanide blocks CFTRG Cl in the native sweat duct

1999 ◽  
Vol 276 (1) ◽  
pp. C231-C237 ◽  
Author(s):  
M. M. Reddy ◽  
P. M. Quinton
Keyword(s):  
Basolateral Membrane ◽  
Electrical Conductance ◽  
Anion Channel ◽  
Membrane Resistance ◽  
Activation Process ◽  
Transmembrane Conductance Regulator ◽  
Sweat Duct ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane ◽  
Human Sweat Duct

Bumetanide is well known for its ability to inhibit the nonconductive Na+-K+-2Cl−cotransporter. We were surprised in preliminary studies to find that bumetanide in the contraluminal bath also inhibited NaCl absorption in the human sweat duct, which is apparently poor in cotransporter activity. Inhibition was accompanied by a marked decrease in the transepithelial electrical conductance. Because the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is richly expressed in the sweat duct, we asked whether bumetanide acts by blocking this anion channel. We found that bumetanide 1) significantly increased whole cell input impedance, 2) hyperpolarized transepithelial and basolateral membrane potentials, 3) depolarized apical membrane potential, 4) increased the ratio of apical-to-basolateral membrane resistance, and 5) decreased transepithelial Cl− conductance ( G Cl). These results indicate that bumetanide inhibits CFTR G Clin both cell membranes of this epithelium. We excluded bumetanide interference with the protein kinase A phosphorylation activation process by “irreversibly” phosphorylating CFTR [by using adenosine 5′- O-(3-thiotriphosphate) in the presence of a phosphatase inhibition cocktail] before bumetanide application. We then activated CFTR G Clby adding 5 mM ATP. Bumetanide in the cytoplasmic bath (10−3 M) inhibited ∼71% of this ATP-activated CFTR G Cl, indicating possible direct inhibition of CFTR G Cl. We conclude that bumetanide inhibits CFTR G Clin apical and basolateral membranes independent of phosphorylation. The results also suggest that >10−5 M bumetanide cannot be used to specifically block the Na+-K+-2Cl−cotransporter.

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