NSP4 elicits age-dependent diarrhea and Ca2+mediated I−influx into intestinal crypts of CF mice

1999 ◽  
Vol 277 (2) ◽  
pp. G431-G444 ◽  
Author(s):  
Andrew P. Morris ◽  
Jason K. Scott ◽  
Judith M. Ball ◽  
Carl Q.-Y. Zeng ◽  
Wanda K. O’Neal ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intestinal Epithelial ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Gene Encoding ◽  
Gastrointestinal Complications ◽  
Age Dependent ◽  
Intracellular Ca ◽  
Cf Transmembrane Conductance Regulator ◽  
Mouse Pup

Homologous disruption of the murine gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to the loss of cAMP-mediated ion transport. Mice carrying this gene defect exhibit meconium ileus at birth and gastrointestinal plugging during the neonatal period, both contributing to high rates of mortality. We investigated whether infectious mammalian rotavirus, the recently characterized rotaviral enterotoxin protein NSP4, or its active NSP4114–135peptide, can overcome these gastrointestinal complications in CF (CFTRm3Baynull mutation) mice. All three agents elicited diarrhea when administered to wild-type (CFTR+/+), heterozygous (CFTR+/−), or homozygous (CFTR−/−) 7- to 14-day-old mouse pups but were ineffective when given to older mice. The diarrheal response was accompanied by non-age-dependent intracellular Ca2+mobilization within both small and large intestinal crypt epithelia. Significantly, NSP4 elicited cellular I−influx into intestinal epithelial cells from all three genotypes, whereas both carbachol and the cAMP-mobilizing agonist forskolin failed to evoke influx in the CFTR−/−background. This unique plasma membrane halide permeability pathway was age dependent, being observed only in mouse pup crypts, and was abolished by either the removal of bath Ca2+or the transport inhibitor DIDS. These findings indicate that NSP4 or its active peptide may induce diarrhea in neonatal mice through the activation of an age- and Ca2+-dependent plasma membrane anion permeability distinct from CFTR. Furthermore, these results highlight the potential for developing synthetic analogs of NSP4114–135to counteract chronic constipation/obstructive bowel syndrome in CF patients.

scholarly journals Cooh-Terminal Truncations Promote Proteasome-Dependent Degradation of Mature Cystic Fibrosis Transmembrane Conductance Regulator from Post-Golgi Compartments

2001 ◽  
Vol 153 (5) ◽  
pp. 957-970 ◽  
Author(s):  
Mohamed Benharouga ◽  
Martin Haardt ◽  
Norbert Kartner ◽  
Gergely L. Lukacs
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cell Surface Biotinylation ◽  
Partially Unfolded ◽  
Cf Transmembrane Conductance Regulator

Impaired biosynthetic processing of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, constitutes the most common cause of CF. Recently, we have identified a distinct category of mutation, caused by premature stop codons and frameshift mutations, which manifests in diminished expression of COOH-terminally truncated CFTR at the cell surface. Although the biosynthetic processing and plasma membrane targeting of truncated CFTRs are preserved, the turnover of the complex-glycosylated mutant is sixfold faster than its wild-type (wt) counterpart. Destabilization of the truncated CFTR coincides with its enhanced susceptibility to proteasome-dependent degradation from post-Golgi compartments globally, and the plasma membrane specifically, determined by pulse–chase analysis in conjunction with cell surface biotinylation. Proteolytic cleavage of the full-length complex-glycosylated wt and degradation intermediates derived from both T70 and wt CFTR requires endolysosomal proteases. The enhanced protease sensitivity in vitro and the decreased thermostability of the complex-glycosylated T70 CFTR in vivo suggest that structural destabilization may account for the increased proteasome susceptibility and the short residence time at the cell surface. These in turn are responsible, at least in part, for the phenotypic manifestation of CF. We propose that the proteasome-ubiquitin pathway may be involved in the peripheral quality control of other, partially unfolded membrane proteins as well.

Applicability of Different Antibodies for the Immunohistochemical Localization of CFTR In Respiratory and Intestinal Tissues of Human and Murine Origin

2003 ◽  
Vol 51 (9) ◽  
pp. 1191-1199 ◽  
Author(s):  
Laurent Doucet ◽  
Filipa Mendes ◽  
Tristan Montier ◽  
Pascal Delépine ◽  
Deborah Penque ◽  
...  
Keyword(s):  
Immunohistochemical Localization ◽  
Tissue Preservation ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Immunofluorescence Method ◽  
Colon Tissue ◽  
Gene Encoding ◽  
Cftr Protein ◽  
Cf Transmembrane Conductance Regulator ◽  
Apical Localization

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, which has a major role as a chloride (Cl−) channel. Although perhaps all functions of CFTR are still not fully characterized, localization studies are necessary to understand the consequences of the more than 1000 mutations thus far identified. Our aim was to determine the histological localization of CFTR on respiratory and colon epithelia of human and murine origin with a panel of several antibodies produced against different CFTR epitopes, using an indirect immunofluorescence method. Our results on human tissues confirm the apical localization of CFTR in ciliated cells of the respiratory mucosa and show that in colon tissue CFTR is observed in both apical and basolateral membranes of epithelial cells from colon crypts. However, poor tissue preservation of colon biopsies after immunohistochemistry (IHC) raises doubts about the latter localization. Contrary to human, mouse colon epithelium (not biopsed) presents good tissue preservation and evidences many cylindrical surface cells with high apical expression of CFTR. For the antibodies's sensitivity, we demonstrate that MATG1061, 24-1, M3A7, and MPCT-1 give good results, allowing the histological localization of CFTR protein of both human and murine origin.

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.

PAR-2-activated secretion by airway gland serous cells: role for CFTR and inhibition by Pseudomonas aeruginosa

2021 ◽  
Vol 320 (5) ◽  
pp. L845-L879
Author(s):  
Derek B. McMahon ◽  
Ryan M. Carey ◽  
Michael A. Kohanski ◽  
Nithin D. Adappa ◽  
James N. Palmer ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluid Secretion ◽  
Transmembrane Conductance Regulator ◽  
Contrast Imaging ◽  
Transmembrane Conductance ◽  
Intracellular Ca ◽  
Influx Pathways ◽  
Cf Transmembrane Conductance Regulator ◽  
Protease Activated Receptor 2 ◽  
Conducting Airways

Airway submucosal gland serous cells are important sites of fluid secretion in conducting airways. Serous cells also express the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). Protease-activated receptor 2 (PAR-2) is a G protein-coupled receptor that activates secretion from intact airway glands. We tested if and how human nasal serous cells secrete fluid in response to PAR-2 stimulation using Ca2+ imaging and simultaneous differential interference contrast imaging to track isosmotic cell shrinking and swelling reflecting activation of solute efflux and influx pathways, respectively. During stimulation of PAR-2, serous cells exhibited dose-dependent increases in intracellular Ca2+. At stimulation levels >EC50 for Ca2+, serous cells simultaneously shrank ∼20% over ∼90 s due to KCl efflux reflecting Ca2+-activated Cl− channel (CaCC, likely TMEM16A)-dependent secretion. At lower levels of PAR-2 stimulation (<EC50 for Ca2+), shrinkage was not evident due to failure to activate CaCC. Low levels of cAMP-elevating VIP receptor (VIPR) stimulation, also insufficient to activate secretion alone, synergized with low-level PAR-2 stimulation to elicit fluid secretion dependent on both cAMP and Ca2+ to activate CFTR and K+ channels, respectively. Polarized cultures of primary serous cells also exhibited synergistic fluid secretion. Pre-exposure to Pseudomonas aeruginosa conditioned media inhibited PAR-2 activation by proteases but not peptide agonists in primary nasal serous cells, Calu-3 bronchial cells, and primary nasal ciliated cells. Disruption of synergistic CFTR-dependent PAR-2/VIPR secretion may contribute to reduced airway surface liquid in CF. Further disruption of the CFTR-independent component of PAR-2-activated secretion by P. aeruginosa may also be important to CF pathophysiology.

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.

Role of membrane trafficking in plasma membrane solute transport

1994 ◽  
Vol 267 (1) ◽  
pp. C1-C24 ◽  
Author(s):  
N. A. Bradbury ◽  
R. J. Bridges
Keyword(s):  
Plasma Membrane ◽  
Solute Transport ◽  
Membrane Trafficking ◽  
Molecular Mechanisms ◽  
Glucose Transporter ◽  
Water Channel ◽  
Transport Proteins ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Regulated Trafficking

Cells can rapidly and reversibly alter solute transport rates by changing the kinetics of transport proteins resident within the plasma membrane. Most notably, this can be brought about by reversible phosphorylation of the transporter. An additional mechanism for acute regulation of plasma membrane transport rates is by the regulated exocytic insertion of transport proteins from intracellular vesicles into the plasma membrane and their subsequent regulated endocytic retrieval. Over the past few years, the number of transporters undergoing this regulated trafficking has increased dramatically, such that what was once an interesting translocation of a few transporters has now become a widespread modality for regulating plasma membrane solute permeabilities. The aim of this article is to review the models proposed for the regulated trafficking of transport proteins and what lines of evidence should be obtained to document regulated exocytic insertion and endocytic retrieval of transport proteins. We highlight four transporters, the insulin-responsive glucose transporter, the antidiuretic hormone-responsive water channel, the urinary bladder H(+)-ATPase, and the cystic fibrosis transmembrane conductance regulator Cl- channel, and discuss the various approaches taken to document their regulated trafficking. Finally, we discuss areas of uncertainty that remain to be investigated concerning the molecular mechanisms involved in regulating the trafficking of proteins.

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.

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