Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulatordependent bicarbonate secretion (original) (raw)
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Science (New York, N.Y.), 2014
Lung disease in people with cystic fibrosis (CF) is initiated by defective host defense that predisposes airways to bacterial infection. Advanced CF is characterized by a deficit in mucociliary transport (MCT), a process that traps and propels bacteria out of the lungs, but whether this deficit occurs first or is secondary to airway remodeling has been unclear. To assess MCT, we tracked movement of radiodense microdisks in airways of newborn piglets with CF. Cholinergic stimulation, which elicits mucus secretion, substantially reduced microdisk movement. Impaired MCT was not due to periciliary liquid depletion; rather, CF submucosal glands secreted mucus strands that remained tethered to gland ducts. Inhibiting anion secretion in non-CF airways replicated CF abnormalities. Thus, impaired MCT is a primary defect in CF, suggesting that submucosal glands and tethered mucus may be targets for early CF treatment.
American Journal of Respiratory Cell and Molecular Biology, 2013
Cystic fibrosis (CF) is caused by mutations in the tightly regulated anion channel cystic fibrosis transmembrane conductance regulator (CFTR), yet much of the pathology in this disease results from mucus obstruction of the small airways and other organs. Mucus stasis has been attributed to the abnormal luminal environment of CF airways, which results from dehydration of the mucus gel or low bicarbonate concentration. We show here that CFTR and MUC5AC are present in single mucin-containing granules isolated from a human airway epithelial cell line and from highly differentiated airway primary cell cultures. CFTR was not detected in MUC5AC granules from CFTR knockdown cells or CF primary cells. The results suggest a direct link between CFTR and the mucus defect.
CFTR and bicarbonate secretion to epithelial cells
News in physiological sciences, 2003
T he cystic fibrosis transmembrane conductance regulator (CFTR) plays a crucial role in maintaining fluid secretion of epithelial cells of the airways and the intestine. Defective CFTR leads to an imbalance between fluid absorption and secretion in the lungs of cystic fibrosis (CF) patients, resulting in a relatively dehydrated mucus layer on the airways. However, the onset of clear symptoms of impaired lung function remains highly variable. A striking contrast can be found when one examines the exocrine pancreas. Among all CF patients, 7090% are born with pancreatic insufficiency, which means that >98% of the pancreatic capacity is already lost (17). Even in the seemingly pancreatic-sufficient patients, the ratio between alkaline fluid and secreted digestive enzymes is significantly decreased (8). Clinicians have been using the amount of residual pancreatic function to classify CF patients into severe and mild cases. Under physiological conditions, the secreted HCO 3-rich fluid and electrolytes serve to flush the digestive enzymes from the acini and ducts of the pancreas. Thus impaired HCO 3 secretion results in poor clearance of the digestive enzymes, and their premature activation eventually causes the destruction of the pancreas in CF. During the past few years, it has been shown that a similar defect in HCO 3 secretion can also be found in the small and large intestine (16) and, surprisingly, also in the airway mucosa (18). We would assert that a similar sequela as in the pancreas follows from impaired HCO 3 secretion in the submucosal glands and airways of CF patients. Analogous to the pancreas, the submucosal glands secrete mucins, protease inhibitors, antibiotic peptides, and enzymes that must be flushed from the glands onto the airway surface epithelium. Moreover, the physical properties of mucus are intrinsically dependent on the electrolyte composition of the fluid. Thus the results of recent studies on submucosal gland serous cells should provide important new insights into the mechanism of HCO 3 secretion in airway epithelial cells and have significant implications in the future treatment of CF.
MUCLIN expression in the cystic fibrosis transmembrane conductance regulator knockout mouse
Gastroenterology, 1997
Background & Aims: Cystic fibrosis is characterized hydrate composition. These changes are believed to conby increased secretion of glycoconjugates with altered tribute to inspissation of the lumina of these organs, carbohydrate composition, but no specific gene prodwhich can lead to destruction of the exocrine pancreas ucts that show these changes have been identified. and obstruction of the intestine (meconium ileus) in a The aim of this study was to use a recently described significant proportion of affected individuals. 2 Despite sulfated mucin-like glycoprotein (MUCLIN; formerly intense investigation, it remains unclear how the loss of called gp300) as a model glycoconjugate to study such functional CFTR results in the pleiotropic effects seen changes in the gastrointestinal system in the cystic in this disease, especially the increase in glycoconjugate fibrosis transmembrane conductance regulator (CFTR) secretion and altered carbohydrate composition of these knockout mouse (cftr m1Unc). Methods: Western and secretions. Northern blots were used to determine the tissue levels Changes in the carbohydrate composition in CF inof MUCLIN and its messenger RNA (mRNA) in normal clude increased levels of sulfate and fucose and decreased and CFTR knockout mice. Immunocytochemistry was levels of sialic acid. 6,7,9-11 Because altered composition used to determine the localization of MUCLIN. Results: of mucus will affect their hydrodynamic properties, they MUCLIN is expressed in the normal mouse intestinal tract, pancreas, and gallbladder. In CFTR knockout can contribute to clogging of airways and exocrine gland mice, MUCLIN shows increased expression at both ductular systems. 2,12 The nature of the changes in carbo-mRNA and protein levels in pancreas and duodenum, hydrate composition is controversial. Some investigators but not in the gallbladder. In the duodenum, MUCLIN provide evidence that posttranslational processing of prowas localized intracellularly in crypt enterocytes and teins is altered in CF, whereas others propose that the on the luminal surface, and luminal surface labeling expression of proteins to which carbohydrates are added was dramatically increased in the CFTR knockout is altered. 13 For example, Wesley et al. 8 compared normal mouse. In the CFTR knockout mouse duodenum and and CF intestinal mucous compositions and found ingallbladder, MUCLIN showed retarded electrophoretic creased total carbohydrate content but no change in migration indicating altered posttranslational proamino acid profiles, indicating altered posttranslational cessing. Conclusions: MUCLIN shows increased exmodification. On the other hand, Chace et al. 12 compared pression and possibly altered posttranslational prothe composition of airway mucus from asthmatic patients cessing in the CFTR knockout mouse and will serve as with that of CF patients and found that in addition to a good model for understanding changes in the compoincreased sulfate levels, the amino acid composition of sition of mucous secretions in patients with this disease.
Journal of Biological Chemistry, 2011
In most HCO 3 ؊-secreting epithelial tissues, SLC26 Cl ؊ /HCO 3 ؊ transporters work in concert with the cystic fibrosis transmembrane conductance regulator (CFTR) to regulate the magnitude and composition of the secreted fluid, a process that is vital for normal tissue function. By contrast, CFTR is regarded as the only exit pathway for HCO 3 ؊ in the airways. Here we show that Cl ؊ /HCO 3 ؊ anion exchange makes a major contribution to transcellular HCO 3 ؊ transport in airway serous cells. Real-time measurement of intracellular pH from polarized cultures of human Calu-3 cells demonstrated cAMP/PKA-activated Cl ؊dependent HCO 3 ؊ transport across the luminal membrane via CFTR-dependent coupled Cl ؊ /HCO 3 ؊ anion exchange. The
The Journal of Physiology, 2007
Submucosal glands line the cartilaginous airways and produce most of the antimicrobial mucus that keeps the airways sterile. The glands are defective in cystic fibrosis (CF), but how this impacts airway health remains uncertain. Although most CF mouse strains exhibit mild airway defects, those with the C57Bl/6 genetic background have increased airway pathology and susceptibility to Pseudomonas. Thus, they offer the possibility of studying whether, and if so how, abnormal submucosal gland function contributes to CF airway disease. We used optical methods to study fluid secretion by individual glands in tracheas from normal, wild-type (WT) mice and from cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice (Cftr m1UNC /Cftr m1UNC ; CF mice). Glands from WT mice qualitatively resembled those in humans by responding to carbachol and vasoactive intestinal peptide (VIP), although the relative rates of VIP-and forskolin-stimulated secretion were much lower in mice than in large mammals. The pharmacology of mouse gland secretion was also similar to that in humans; adding bumetanide or replacement of HCO 3 − by Hepes reduced the carbachol response by ∼50%, and this inhibition increased to 80% when both manoeuvres were performed simultaneously. It is important to note that glands from CFTR knockout mice responded to carbachol but did not secrete when exposed to VIP or forskolin, as has been shown previously for glands from CF patients. Tracheal glands from WT and CF mice both had robust secretory responses to electrical field stimulation that were blocked by tetrodotoxin. It is interesting that local irritation of the mucosa using chili pepper oil elicited secretion from WT glands but did not stimulate glands from CF mice. These results clarify the mechanisms of murine submucosal gland secretion and reveal a novel defect in local regulation of glands lacking CFTR which may also compromise airway defence in CF patients.
The Journal of Physiology, 2012
The mechanisms of anion and fluid transport by airway submucosal glands are not well understood and may differ from those in surface epithelium. • The Calu-3 cell line is often used as a model for submucosal gland serous cells and has cAMP-stimulated fluid secretion; however, it does not actively transport chloride under short-circuit conditions. • In this study we show that fluid secretion requires chloride, bicarbonate and sodium, that chloride is the predominant anion in Calu-3 secretions, and that a large fraction of the basolateral chloride loading during cAMP stimulation occurs by Cl − /HCO 3 − exchange. • The results suggest a novel cellular model for anion and fluid secretion by Calu-3 and submucosal gland acinar cells Abstract Anion and fluid secretion are both defective in cystic fibrosis (CF); however, the transport mechanisms are not well understood. In this study, Cl − and HCO 3 − secretion was measured using genetically matched CF transmembrane conductance regulator (CFTR)-deficient and CFTR-expressing cell lines derived from the human airway epithelial cell line Calu-3. Forskolin stimulated the short-circuit current (I sc) across voltage-clamped monolayers, and also increased the equivalent short-circuit current (I eq) calculated under open-circuit conditions. I sc was equivalent to the HCO 3 − net flux measured using the pH-stat technique, whereas I eq was the sum of the Cl − and HCO 3 − net fluxes. I eq and HCO 3 − fluxes were increased by bafilomycin and ZnCl 2 , suggesting that some secreted HCO 3 − is neutralized by parallel electrogenic H + secretion. I eq and fluid secretion were dependent on the presence of both Na + and HCO 3 −. The carbonic anhydrase inhibitor acetazolamide abolished forskolin stimulation of I eq and HCO 3 − secretion, suggesting that HCO 3 − transport under these conditions requires catalysed synthesis of carbonic acid. Cl − was the predominant anion in secretions under all conditions studied and thus drives most of the fluid transport. Nevertheless, 50-70% of Cl − and fluid transport was bumetanide-insensitive, suggesting basolateral Cl − loading by a sodium-potassium-chloride cotransporter 1 (NKCC1)-independent mechanism. Imposing a transepithelial HCO 3 − gradient across basolaterally permeabilized Calu-3 cells sustained a forskolin-stimulated current, which was sensitive to CFTR inhibitors and drastically reduced in CFTR-deficient cells. Net HCO 3 − secretion was increased by bilateral Cl − removal and therefore did not require apical Cl − /HCO 3 − exchange. The results suggest a model in which most HCO 3 − is recycled basolaterally by exchange
Impaired pancreatic ductal bicarbonate secretion in cystic fibrosis
JOP : Journal of the pancreas, 2001
Patients with cystic fibrosis demonstrate a defect in HCO(3)(-) secretion by their pancreatic duct cells. However, attempts toward understanding or correcting this defect have been hampered by a lack of knowledge regarding the cellular and molecular mechanisms mediating HCO(3)(-) transport in these cells. Recent functional and molecular studies indicate a major role for a basolateral electrogenically-driven Na(+):HCO(3)(-) cotransporter (NBC1) in mediating the transport of HCO(3)(-) into the duct cells. The HCO(3)(-) exits at the lumen predominantly via two recently discovered apical HCO(3)(-) transporters. cAMP, which mediates the stimulatory effect of secretin on pancreatic ductal HCO(3)(-) secretion, potentiates the basolateral Na(+):HCO(3)(-) cotransporter due to generation of a favorable electrogenic gradient as a result of membrane depolarization by Cl(-)-secreting cystic fibrosis transmembrane conductance regulator (CFTR). Two apical HCO(3)(-) transporters drive the secretion...
JOP : Journal of the pancreas, 2001
While cystic fibrosis transmembrane conductance regulator (CFTR) is well known to function as a Cl(-) channel, some mutations in the channel protein causing cystic fibrosis (CF) disrupt another vital physiological function, HCO(3)(-) transport. Pathological implications of derailed HCO(3)(-) transport are clearly demonstrated by the pancreatic destruction that accompany certain mutations in CF. Despite the crucial role of HCO(3)(-) in buffering pH, little is known about the relationship between cause of CF pathology and the molecular defects arising from specific mutations. Using electrophysiological techniques on basolaterally permeabilized preparations of microperfused native sweat ducts, we investigated whether: a) CFTR can act as a HCO(3)(-) conductive channel, b) different conditions for stimulating CFTR can alter its selectivity to HCO(3)(-) and, c) pancreatic insufficiency correlate with HCO(3)(-) conductance in different CFTR mutations. We show that under some conditions sti...