In vivo analysis of fluid transport in cystic fibrosis airway epithelia of bronchial xenografts

1996 ◽  
Vol 270 (5) ◽  
pp. C1326-C1335 ◽  
Author(s):  
Y. Zhang ◽  
J. Yankaskas ◽  
J. Wilson ◽  
J. F. Engelhardt
Keyword(s):  
Cystic Fibrosis ◽  
Fluid Transport ◽  
Xenograft Model ◽  
Na Channel ◽  
Transmembrane Conductance Regulator ◽  
Fluid Absorption ◽  
Cystic Fibrosis Airway ◽  
In Vivo Analysis ◽  
Cf Transmembrane Conductance Regulator

An in vivo human bronchial xenograft model system was used to simultaneously analyze electrolyte and fluid transport defects in fully differentiated human cystic fibrosis (CF) and non-CF proximal airways. CF airways demonstrated three discernible defects when compared with non-CF, including 1) a lack of adenosine 3',5'-cylic monophosphate (cAMP)-inducible Cl- secretion, 2) a fourfold higher basal fluid absorption rate, and 3) an altered regulation of fluid absorption in response to amiloride-stimulated changes in Na+ transport. A unique finding in this study demonstrated that treatment of epithelia with amiloride led to a greater than threefold decrease in the rate of fluid absorption in CF tissues as contrasted to a greater than threefold increase in the rate of fluid absorption in non-CF tissues. The removal of apical Na+ from amiloride-treated non-CF xenografts was capable of ablating this amiloride-induced increase in fluid absorption. In light of the recent interactions demonstrated between CF transmembrane conductance regulator (CFTR) and the rat epithelial, amiloride-sensitive Na+ channel, these findings implicate additional complexities between the Na+ conductance pathways and fluid transport in normal and CF proximal airways. Such findings suggest that CFTR may also regulate amiloride-insensitive Na+ channels.

Ileal smooth muscle dysfunction and remodeling in cystic fibrosis

2012 ◽  
Vol 303 (1) ◽  
pp. G1-G8 ◽  
Author(s):  
P.-A. Risse ◽  
L. Kachmar ◽  
O. S. Matusovsky ◽  
M. Novali ◽  
F. R. Gil ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Smooth Muscle ◽  
Electrical Field Stimulation ◽  
Mouse Strains ◽  
Maximal Velocity ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Velocity Of Shortening ◽  
Cf Transmembrane Conductance Regulator

Patients with cystic fibrosis (CF) often suffer from gastrointestinal cramps and intestinal obstruction. The CF transmembrane conductance regulator (CFTR) channel has been shown to be expressed in vascular and airway smooth muscle (SM). We hypothesized that the absence of CFTR expression alters the gastrointestinal SM function and that these alterations may show strain-related differences in the mouse. The aim of this study was to measure the contractile properties of the ileal SM in two CF mouse models. CFTR−/− and CFTR+/+ mice were studied on BALB/cJ and C57BL/6J backgrounds. Responsiveness of ileal strips to electrical field stimulation (EFS), methacholine (MCh), and isoproterenol was measured. The mass and the cell density of SM layers were measured morphometrically. Finally, the maximal velocity of shortening (Vmax) and the expression of the fast (+)insert myosin isoform were measured in the C57BL/6J ileum. Ileal hyperreactivity was observed in response to EFS and MCh in CFTR−/− compared with CFTR+/+ mice in C57BL/6J background. This latter observation was not reproduced by acute inhibition of CFTR with CFTRinh172. BALB/cJ CFTR−/− mice exhibited a significant increase of SM mass with a lower density of cells compared with CFTR+/+, whereas no difference was observed in the C57BL/6J background. In addition, in this latter strain, ileal strips from CFTR−/− exhibited a significant increase in Vmax compared with control and expressed a greater proportion of the fast (+)insert SM myosin isoform with respect to total myosin. BALB/cJ CFTR−/− ilium had a greater relaxation to isoproterenol than the CFTR+/+ mice when precontracted with EFS, but no difference was observed in response to exogeneous MCh. In vivo, the lack of CFTR expression induces a different SM ileal phenotype in different mouse strains, supporting the importance of modifier genes in determining intestinal SM properties.

Enhanced colonic Na+ absorption in cystic fibrosis mice versus normal mice

1997 ◽  
Vol 272 (2) ◽  
pp. G393-G400 ◽  
Author(s):  
B. R. Grubb ◽  
R. C. Boucher
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Proximal Colon ◽  
Na Channel ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Constant Dose ◽  
Cf Transmembrane Conductance Regulator

Because there are reports that electrogenic Na+ absorption is increased in colonic epithelia of cystic fibrosis (CF) subjects, we tested whether amiloride-sensitive Na+ absorption was increased in the colonic epithelia of CF mice compared with normal mice on high- or low-Na+ diets. When mice consumed a diet high in Na+, none of the colonic regions (distal colon, proximal colon, or cecum) from either group of mice exhibited an amiloride-sensitive short-circuit current (Isc). However, when mice were placed on a low-Na+ diet for 2 wk, all three intestinal regions from the CF mice exhibited a significant response to amiloride (P < or = 0.05). In contrast, normal mice on the low-Na+ diet exhibited an amiloride-sensitive Isc that was smaller and only significant in the cecum and distal colon. Measurement of plasma aldosterone levels revealed that the CF mice on the low-Na+ diet had significantly greater aldosterone levels than similarly treated controls [8,906 +/- 1,039 (n = 14) vs. 5,243 +/- 1,410 pg/ml (n = 14), respectively]. When mice were infused with a constant dose of aldosterone (1 microg x g(-1) x day(-1)) for 7 days, the distal colon of the CF mice still had a significantly greater amiloride-sensitive Isc than did the normal distal colon. If the presence of CF transmembrane conductance regulator (CFTR) down-regulates Na+ absorption in the colonic tissue from normal mice, our data suggest that at least some CFTR may be colocalized with the Na+ channel. Alternatively, other factors may be involved.

scholarly journals Airway surface fluid volume and Cl content in cystic fibrosis and normal bronchial xenografts

1999 ◽  
Vol 276 (2) ◽  
pp. C469-C476 ◽  
Author(s):  
Yulong Zhang ◽  
John F. Engelhardt
Keyword(s):  
Cystic Fibrosis ◽  
Solid State ◽  
Xenograft Model ◽  
Fluid Volume ◽  
Molar Ratio ◽  
Free Solution ◽  
Fluid Absorption

We describe the use of an in vivo human bronchial xenograft model of cystic fibrosis (CF) and non-CF airways to investigate pathophysiological alterations in airway surface fluid (ASF) volume (Vs) and Cl content. Vs was calculated based on the dilution of an impermeable marker, [3H]inulin, during harvesting of ASF from xenografts with an isosmotic Cl-free solution. These calculations demonstrated that Vs in CF xenographs (28 ± 3.0 μl/cm2; n = 17) was significantly less than that of non-CF xenografts (35 ± 2.4 μl/cm2; n = 30). The Cl concentration of ASF ([Cl]s) was determined using a solid-state AgCl electrode and adjusted for dilution during harvesting using the impermeable [3H]inulin marker. Cumulative results demonstrate small but significant elevations ( P < 0.045) in [Cl]s in CF (125 ± 4 mM; n = 27) compared with non-CF (114 ± 4 mM; n = 48) xenografts. To investigate potential mechanisms by which CF airways may facilitate a higher level of fluid absorption yet retain slightly elevated levels of Cl, we sought to evaluate the capacity of CF and non-CF airways to absorb both 22Na and36Cl. Two consistent findings were evident from these studies. First, in both CF and non-CF xenografts,22Na and36Cl were always absorbed in an equal molar ratio. Second, CF xenografts hyperabsorbed (∼1.5-fold higher) both 22Na and36Cl compared with non-CF xenografts. These results substantiate previously documented findings of elevated Na absorption in CF airways and also suggest that the slightly elevated [Cl]s found in this study of CF xenograft epithelia does not occur through a mechanism of decreased apical permeability to Cl.

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.

scholarly journals Chlorzoxazone or 1-EBIO increases Na+absorption across cystic fibrosis airway epithelial cells

2001 ◽  
Vol 281 (5) ◽  
pp. L1123-L1129 ◽  
Author(s):  
Lin Gao ◽  
James R. Yankaskas ◽  
Catherine M. Fuller ◽  
Eric J. Sorscher ◽  
Sadis Matalon ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Fold Increase ◽  
Short Circuit Current ◽  
Airway Disease ◽  
Airway Epithelial Cells ◽  
Transmembrane Conductance Regulator ◽  
Airway Epithelial ◽  
Cystic Fibrosis Airway ◽  
Cf Transmembrane Conductance Regulator

Previous studies demonstrated that chlorzoxazone or 1-ethyl-2-benzimidazolinone (1-EBIO) enhances transepithelial Cl− secretion by increasing basolateral K+ conductance ( G K) (Singh AK, Devor DC, Gerlach AC, Gondor M, Pilewski JM, and Bridges RJ. J Pharmacol Exp Ther 292: 778–787, 2000). Hence these compounds may be useful to treat cystic fibrosis (CF) airway disease. The goal of the present study was to determine whether chlorzoxazone or 1-EBIO altered ion transport across ΔF508-CF transmembrane conductance regulator homozygous CFT1 airway cells. CFT1 monolayers exhibited a basal short-circuit current that was abolished by apical amiloride (inhibition constant 320 nM) as expected for Na+ absorption. The addition of chlorzoxazone (400 μM) or 1-EBIO (2 mM) increased the amiloride-sensitive I sc ∼2.5-fold. This overlapping specificity may preclude use of these compounds as CF therapeutics. Assaying for changes in the basolateral G K with a K+ gradient plus the pore-forming antibiotic amphotericin B revealed that chlorzoxazone or 1-EBIO evoked an ∼10-fold increase in clotrimazole-sensitive G K. In contrast, chlorzoxazone did not alter epithelial Na+ channel-mediated currents across basolateral-permeabilized monolayers or in Xenopus oocytes. These data further suggest that alterations in basolateral G K alone can modulate epithelial Na+ transport.

scholarly journals Abnormal glutathione transport in cystic fibrosis airway epithelia

1999 ◽  
Vol 277 (1) ◽  
pp. L113-L118 ◽  
Author(s):  
Lin Gao ◽  
Kwang Jin Kim ◽  
James R. Yankaskas ◽  
Henry Jay Forman
Keyword(s):  
Cystic Fibrosis ◽  
Antioxidant Defense ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Airway Epithelia ◽  
Cystic Fibrosis Airway ◽  
Lung Lining Fluid ◽  
Cf Transmembrane Conductance Regulator ◽  
Glutamyl Transpeptidase ◽  
Glutathione Transport

Glutathione (GSH) is a potentially important component of antioxidant defense in the epithelial lung lining fluid. Cystic fibrosis (CF) patients have chronic inflammation in which oxidative stress can be a factor. To examine the hypothesis that the transport of GSH content was defective in CF patients, intracellular and extracellular GSH were measured by HPLC. Four cell lines were used: CFT1 cells [with defective CF transmembrane conductance regulator (CFTR), ΔF508 homozygous, two clones] and one of the CFT1 clones transfected with either normal CFTR (CFTR repleted) or β-galactosidase. GSH content in the apical fluid was 55% lower in CFTR-deficient cultures than in CFTR-repleted cells ( P < 0.001). In contrast, intracellular GSH content was similar in CFT1 cells and CFTR-repleted cells. γ-Glutamyl transpeptidase activity, which degrades extracellular GSH, did not account for differences in apical GSH. Rather, GSH efflux of CFTR-deficient cells was lower than that of CFTR-repleted cells. These studies suggested that decreased GSH content in the apical fluid in CF resulted from abnormal GSH transport associated with a defective CFTR.

scholarly journals Synthesis and Therapeutic Applications of Iminosugars in Cystic Fibrosis

2020 ◽  
Vol 21 (9) ◽  
pp. 3353 ◽  
Author(s):  
Anna Esposito ◽  
Daniele D’Alonzo ◽  
Maria De Fenza ◽  
Eliana De Gregorio ◽  
Anna Tamanini ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Biological Activities ◽  
Transmembrane Conductance Regulator ◽  
Medical Interventions ◽  
Processing Enzymes ◽  
Wide Range ◽  
Pharmacological Potential ◽  
Cf Transmembrane Conductance Regulator

Iminosugars are sugar analogues endowed with a high pharmacological potential. The wide range of biological activities exhibited by these glycomimetics associated with their excellent drug profile make them attractive therapeutic candidates for several medical interventions. The ability of iminosugars to act as inhibitors or enhancers of carbohydrate-processing enzymes suggests their potential use as therapeutics for the treatment of cystic fibrosis (CF). Herein we review the most relevant advances in the field, paying attention to both the chemical synthesis of the iminosugars and their biological evaluations, resulting from in vitro and in vivo assays. Starting from the example of the marketed drug NBDNJ (N-butyl deoxynojirimycin), a variety of iminosugars have exhibited the capacity to rescue the trafficking of F508del-CFTR (deletion of F508 residue in the CF transmembrane conductance regulator), either alone or in combination with other correctors. Interesting results have also been obtained when iminosugars were considered as anti-inflammatory agents in CF lung disease. The data herein reported demonstrate that iminosugars hold considerable potential to be applied for both therapeutic purposes.

Involvement of αENaC and Nedd4-2 in the conversion from lung fluid secretion to fluid absorption at birth in the rat as assayed by RNA interference analysis

2007 ◽  
Vol 293 (4) ◽  
pp. L1069-L1078 ◽  
Author(s):  
Tianbo Li ◽  
Shyny Koshy ◽  
Hans G. Folkesson
Keyword(s):  
Extravascular Lung Water ◽  
Fluid Secretion ◽  
Alveolar Epithelial Cells ◽  
Na Channel ◽  
Fluid Absorption ◽  
Lung Water ◽  
Fourfold Increase ◽  
Lung Fluid ◽  
Near Term

To explore interactions between the epithelial Na channel (ENaC) and neural precursor expressed, developmentally downregulated protein 4-2 (Nedd4-2) at the conversion of the rat lung from fluid secretion to absorption at birth, we used small-interfering RNA (siRNA) against αENaC and Nedd4-2. siRNA-generating plasmid DNA (pDNA) was administered via trans-thoracic intrapulmonary (ttip) injection 24 h before ENaC and Nedd4-2 expression, extravascular lung water, and mortality were measured. αENaC mRNA and protein were specifically reduced by ∼65% after pSi-4 injection. Nedd4-2 mRNA and protein were reduced by ∼60% after pSi-N1 injection. Interestingly, αENaC and βENaC mRNA and protein expression were increased after Nedd4-2 silencing. Extravascular lung water was significantly increased after αENaC silencing and reduced after Nedd4-2 silencing. αENaC silencing resulted in a fourfold increase in newborn mortality, whereas silencing Nedd4-2 did not affect mortality. We also isolated distal lung epithelial (DLE) cells after in vivo αENaC or Nedd4-2 silencing and measured αENaC or Nedd4-2 expression in freshly isolated DLE cells. In these DLE cells, there were attenuated αENaC or Nedd4-2 mRNA and protein, thus demonstrating that αENaC and Nedd4-2 silencing occurred in alveolar epithelial cells after ttip injection. We also looked for pDNA by PCR to determine pDNA presence in the lungs and found strong evidence for pDNA presence in both lungs. Thus we provide evidence that ENaC and Nedd4-2 are involved in the transition from lung fluid secretion to fluid absorption near term and at birth.

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