Residual chloride secretion in intestinal tissue of ΔF508 homozygous twins and siblings with cystic fibrosis

doi:10.1053/gast.2000.8524

Gastroenterology

Volume 119, Issue 1, July 2000, Pages 32-40

https://doi.org/10.1053/gast.2000.8524 Get rights and content

Abstract

Background & Aims: Cholinergic stimulation of chloride secretion is impaired in the intestines of patients with cystic fibrosis (CF). However, intestinal chloride secretion has been observed in patients with mild CF mutations. The aim of this study was to investigate residual Cl⁻ secretion in the intestine of ΔF508 homozygous CF patients, and examine the contribution of cystic fibrosis transmembrane conductance regulator (CFTR) and alternative Cl⁻ conductances. Twins and siblings with identical CFTR genotypes were investigated to determine the impact of factors other than CFTR on chloride secretion. Methods: Chloride secretion in rectal tissue was investigated by applying Ca²⁺ and adenosine 3',5'-cyclic monophosphate (cAMP)-linked agonists before and after the inhibition of alternative Cl⁻ conductances with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Results: cAMP-mediated Cl⁻ secretion was observed in 73% of patients, and 20% showed DIDS-sensitive Ca²⁺-activated Cl⁻ secretion. This DIDS-sensitive alternative chloride conductance was seen only in CF patients who also responded to cAMP agonists. Chloride secretion was more concordant within monozygous twins than within dizygous pairs. Conclusions: These results suggest the presence of CFTR-mediated Cl⁻ secretion in a subgroup of patients, implying that a portion of ΔF508 CFTR can be processed in vivo and function as a chloride channel in the apical membrane of intestinal cells. Moreover, a considerable number of ΔF508 homozygous patients express chloride conductances other than CFTR in their intestinal epithelia.

GASTROENTEROLOGY 2000;119:32-40

Section snippets

Subjects

Twin and sibling pairs with CF with different genotypes were enrolled for the European Cystic Fibrosis Twin and Sibling Study. For the study described here, only subjects homozygous for the CFTR gene mutation ΔF508 were selected. We invited 72 patients belonging to dizygous twin or sibling pairs and 26 patients belonging to monozygous twin pairs. Patients were investigated in or near their home countries at selected CF core centers in Hannover, Innsbruck, London, Rotterdam, and Verona.

Chloride-secretory response in CF intestine

Of the participating patients belonging to a sibling or dizygous twin pair, 49 responses to 8-bromo-cAMP + forskolin, 56 to carbachol, and 41 to histamine could be determined. Eighteen 8-bromo-cAMP + forskolin, 20 carbachol, and 14 histamine responses from participating patients of monozygous twin pairs were collected. The mean basal transepithelial resistance of the rectal biopsy specimens of our group of ΔF508 homozygous CF patients was 27 Ω·cm². The mean basal Isc was 20 μA/cm².

Patients for

Discussion

This study investigated the presence and frequency of chloride-secretory responses in the rectal tissue of ΔF508 homozygous CF twins and siblings upon stimulation with cAMP- and Ca²⁺-linked agonists, before and after incubation of the tissue with DIDS. Forty (73%) of the ΔF508 homozygotes expressed a cAMP-stimulated chloride-secretory response in their intestinal tissue. Because cAMP-regulated chloride conductance is indicative of CFTR,2, 3, 27 this finding suggests the presence of some active

Acknowledgements

The authors thank the collaborating patients, parents, physicians and scientists for their cooperation, particularly H. Ellemunter (Innsbruck), G. Mastella (Verona), S. Thomas (London), J. Versloot, H. Otten, and R. Samlal-Soedhoe (The Netherlands).

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The clinical pictures of the disease of two Russian patients with cystic fibrosis with a rare nonsense variant c.831G>A (p.Trp277*) are described. The first case is a patient with the genotype comprising variant c.54-5940_273+10250del21kb (CFTRdele2,3), and the genotype of the second case included variant c.1521_1523delCTT (F508del). Patient 1, whose genotype had two class I genetic variants, revealed severe violations of CFTR synthesis based on the intestinal current measurements (ICM) and results obtained in the intestinal organoids. In both cases of patients with genetic variant c.831G>A, a severe course of cystic fibrosis was observed.
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CFTR function measurements in intestinal organoids may help to better characterise individual disease expression in F508del homozygous people. Our objective was to study correlations between CFTR function as measured with forskolin-induced swelling in rectal organoids with clinical parameters in adult patients with homozygous F508del mutations.
Multicentre observational study. Thirty-four adults underwent rectal biopsy, pulmonary function tests (FEV₁ and FVC), chest X-ray and chest CT. Body-mass index (BMI) was assessed at study visit and exacerbation rate was determined during five years prior to study visit. Organoids were cultured and measured after stimulation with 5 µm forskolin for three hours to quantitate CFTR residual function.
FIS was positively correlated with FEV₁ (r = 0.36, 95% CI 0.02–0.62, p = 0.04) and BMI (r = 0.42, 95% CI 0.09–0.66, p = 0.015). FIS was negatively correlated with PRAGMA-CF CT score for% of disease (r = −0.37, 95% CI -0.62- -0.03, p = 0.049). We found no significant correlation between FIS and chest radiography score for CF (r = −0.16, 95% CI -0.48–0.20, p = 0.44). We observed a trend between higher FIS and a lower mean number of exacerbations over the last 5 years of observation, but this was not statistically significant (Poisson regression, p = 0.089).
FIS of intestinal organoids varied between subjects with homozygous F508del and correlated with pulmonary and nutritional parameters. These findings suggest that differences at low CFTR residual function may contribute to clinical heterogeneity in F508del homozygous patients and small changes in CFTR residual function might impact long-term disease expression.
Inhibition of calpain 1 restores plasma membrane stability to pharmacologically rescued Phe508del-CFTR variant
2019, Journal of Biological Chemistry
Citation Excerpt :
This mutation is mainly characterized by protein misfolding and premature degradation by the endoplasmic reticulum (ER) quality control, preventing the mutant protein from trafficking to the cell surface (5–7). Although Phe508del ER retention is not complete, the very small fraction of channels that reach the cell surface possesses only partial activity because of a gating defect (5, 8, 9) and show a severely decreased plasma membrane (PM) half-life, because of accelerated endocytosis and lysosomal degradation (10, 11). At present, there are two approved corrector drugs that target the molecular defects in Phe508del (12–14).
Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del-Na⁺/H⁺-exchange regulatory factor-1 complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR– or rPhe508del-containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 down-regulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an appealing target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function.
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Cystic fibrosis (CF) is one of the most common autosomal recessive disorders associated with decreased longevity in the Caucasian population. The disease is classically described as a triad; chronic obstructive pulmonary disease, exocrine pancreatic insufficiency, and elevation of chloride concentration in sweat. The underlying pathophysiology involves abnormal ion transport due to dysfunction of the CF transmembrane conductance regulator (CFTR). Altered salt and water movement across epithelia interferes with the hydration and ionic composition of mucus secretions. Abnormal mucus impairs clearance and defense systems, leading to inflammation and destruction of the lungs, the pancreas, and the developing vas deferens in males. The development of bioavailable small molecules that augment CFTR function is revolutionizing the treatment of CF.
Ion Channels of the Gastrointestinal Epithelial Cells
2018, Physiology of the Gastrointestinal Tract, Sixth Edition
This chapter summarizes the role of epithelial ion (K⁺, Na⁺, and Cl⁻) channels in the gastrointestinal (GI) tract. Both cAMP- and Ca^2 +-activated K⁺ channels are localized on both apical and basolateral membranes of the entire GI tract. Apical K⁺ channels regulate H⁺,K⁺-ATPase-mediated acid (i.e., H⁺) secretion is stomach, while it regulates active K⁺ secretion in colon. Basolateral K⁺ channels regulate active Cl⁻ secretion by maintaining cell membrane potential. Epithelial Na⁺ channels (ENaC), which are present in the apical membrane of colon, mediates active Na⁺ absorption. Cystic fibrosis transmembrane regulator (CFTR) Cl⁻ channels that regulate electrogenic Cl⁻ secretion are present on the apical membranes of the entire intestine, while Slc26a9 Cl⁻ channels are strongly expressed in the upper GI tract. TMEM16 Ca^2 +-activated Cl⁻ channels are important for the increase in intracellular Ca^2 + concentration during purinergic and cholinergic signaling.

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^☆: Address requests for reprints to: Inez Bronsveld, Laboratory of Pediatrics, rm Ee-1500, Erasmus University Rotterdam, P.O. Box 1738, 3000 Rotterdam, The Netherlands. e-mail: [email protected]; fax: (31) 20-8706751.

^☆☆: This work was executed as part of the European Cystic Fibrosis Twin and Sibling Study and supported by the BIOMED II program of the EU, the Deutsche Forschungsgemeinschaft, the Deutsche Fördergesellschaft für die Mukoviszidoseforschung eV, and the Mukoviszidose eV.

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Alimentary TractResidual chloride secretion in intestinal tissue of ΔF508 homozygous twins and siblings with cystic fibrosis☆,☆☆

Abstract

Section snippets

Subjects

Chloride-secretory response in CF intestine

Discussion

Acknowledgements

Higher bioelectric potentials due to decreased chloride absorption in the sweat glands of patients with cystic fibrosis

N Engl J Med

Cyclic AMP–activated chloride channels in CFTR-transfected cystic fibrosis pancreatic epithelial cells

Am J Physiol

Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR)

Cell

Cystic fibrosis

Increased bioelectrical potential difference across respiratory epithelia in cystic fibrosis

N Engl J Med

Failure of cholinergic stimulation to induce a secretory response from the rectal mucosa in cystic fibrosis

Gut

Ion transport abnormalities in rectal suction biopsies from children with cystic fibrosis

Gastroenterology

Determinants of mild symptoms in cystic fibrosis patients: residual chloride secretion measured in rectal biopsies in relation to the genotype

J Clin Invest

Modulation of disease severity in cystic fibrosis transmembrane conductance regulator deficient mice by a secondary genetic factor

Nat Genet

Relationship of a non-cystic fibrosis transmembrane conductance regulator-mediated chloride conductance to organ-level disease in Cftr(−/−) mice

Proc Natl Acad Sci U S A

The murine calcium-sensitive chloride channel (mCaCC) is widely expressed in secretory epithelia and in other select tissues

Histochem Cell Biol

Genomic cloning, molecular characterization, and functional analysis of human CLCA1, the first human member of the family of Ca2+-activated Cl− channel proteins

Genomics

Expression of delta F508 cystic fibrosis transmembrane conductance regulator protein and related chloride transport properties in the gallbladder epithelium from cystic fibrosis patients

Hepatology

Molecular cloning and transmembrane structure of hCLCA2 from human lung, trachea, and mammary gland

Am J Physiol

A mouse model for the cystic fibrosis ΔF508 mutation

EMBO J

Efficiency of two different nine-loci short tandem repeat systems for DNA typing purposes

Clin Chem

On the potential of simple repetitive DNA for fingerprinting in clinical, forensic, and evolutionary dynamic studies

Clin Invest

Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits

Nature

Arachidonic acid metabolites and chloride secretion in rabbit distal colonic mucosa

Am J Physiol

Alimentary Tract
Residual chloride secretion in intestinal tissue of ΔF508 homozygous twins and siblings with cystic fibrosis☆,☆☆

Genomic cloning, molecular characterization, and functional analysis of human CLCA1, the first human member of the family of Ca²⁺-activated Cl⁻ channel proteins

Amiloride-sensitive epithelial Na⁺ channel is made of three homologous subunits