Properties of CFTR activated by the xanthine derivative X-33 in human airway Calu-3 cells

2000 ◽  
Vol 279 (6) ◽  
pp. C1925-C1937 ◽  
Author(s):  
Laurence Bulteau ◽  
Renaud Dérand ◽  
Yvette Mettey ◽  
Thierry Métayé ◽  
M. Rachel Morris ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Cystic Fibrosis Gene ◽  
Intracellular Camp ◽  
Airway Epithelial ◽  
Whole Cell ◽  
Xanthine Derivative ◽  
Human Airway ◽  
High Level

The pharmacological activation of the cystic fibrosis gene protein cystic fibrosis transmembrane conductance regulator (CFTR) was studied in human airway epithelial Calu-3 cells, which express a high level of CFTR protein as assessed by Western blot and in vitro phosphorylation. Immunolocalization shows that CFTR is located in the apical membrane. We performed iodide efflux, whole cell patch-clamp, and short-circuit recordings to demonstrate that the novel synthesized xanthine derivative 3,7-dimethyl-1-isobutylxanthine (X-33) is an activator of the CFTR channel in Calu-3 cells. Whole cell current activated by X-33 or IBMX is linear, inhibited by glibenclamide and diphenylamine-2-carboxylate but not by DIDS or TS-TM calix[4]arene. Intracellular cAMP was not affected by X-33. An outwardly rectifying Cl− current was recorded in the absence of cAMP and X-33 stimulation, inhibited by DIDS and TS-TM calix[4]arene. With the use of short-circuit recordings, X-33 and IBMX were able to stimulate a large concentration-dependent CFTR transport that was blocked by glibenclamide but not by DIDS. Our results show that manipulating the chemical structure of xanthine derivatives offers an opportunity to identify further specific activators of CFTR in airway cells.

scholarly journals Short-term consequences of F508del-CFTR thermal instability on CFTR-dependent transepithelial currents in human airway epithelial cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Lionel Froux ◽  
Christelle Coraux ◽  
Edouard Sage ◽  
Frédéric Becq
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Short Circuit ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Loss Of Function ◽  
Membrane Expression ◽  
Human Airway ◽  
Function Loss ◽  
Human Airway Epithelial Cells

Abstract Loss-of-function mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) channel in human airway epithelial cells are responsible for Cystic Fibrosis. A deleterious impact of physiological temperature on CFTR plasma membrane expression, residence and channel activity is characteristic of the most common and severe CF mutation, F508del. Using primary human F508del-airway epithelial cells and CF bronchial epithelial CFBE41o- cell lines expressing F508del- or WT-CFTR, we examined the effects of temperature (29 °C-39 °C) on the amplitude and stability of short-circuit CFTR-dependent currents over time and the efficiency of pharmacological strategies to stably restore F508del-CFTR function. We show that F508del-CFTR functional instability at 37 °C is not prevented by low temperature or VX-809 correction, genistein and VX-770 potentiators, nor by the combination VX-809/VX-770. Moreover, F508del-CFTR-dependent currents 30 minutes after CFTR activation at 37 °C did not significantly differ whether a potentiator was used or not. We demonstrate that F508del-CFTR function loss is aggravated at temperatures above 37 °C while limited by a small decrease of temperature and show that the more F508del-CFTR is stimulated, the faster the current loss happens. Our study highlights the existence of a temperature-dependent process inhibiting the function of F508del-CFTR, possibly explaining the low efficacy of pharmacological drugs in clinic.

scholarly journals Human Nasal Epithelial Organoids for Therapeutic Development in Cystic Fibrosis

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 603
Author(s):  
Zhongyu Liu ◽  
Justin D. Anderson ◽  
Lily Deng ◽  
Stephen Mackay ◽  
Johnathan Bailey ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Ex Vivo ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Genotypic Diversity ◽  
Mutant Gene ◽  
Airway Epithelial ◽  
Predictive Capacity ◽  
Apical Side

We describe a human nasal epithelial (HNE) organoid model derived directly from patient samples that is well-differentiated and recapitulates the airway epithelium, including the expression of cilia, mucins, tight junctions, the cystic fibrosis transmembrane conductance regulator (CFTR), and ionocytes. This model requires few cells compared to airway epithelial monolayer cultures, with multiple outcome measurements depending on the application. A novel feature of the model is the predictive capacity of lumen formation, a marker of baseline CFTR function that correlates with short-circuit current activation of CFTR in monolayers and discriminates the cystic fibrosis (CF) phenotype from non-CF. Our HNE organoid model is amenable to automated measurements of forskolin-induced swelling (FIS), which distinguishes levels of CFTR activity. While the apical side is not easily accessible, RNA- and DNA-based therapies intended for systemic administration could be evaluated in vitro, or it could be used as an ex vivo biomarker of successful repair of a mutant gene. In conclusion, this highly differentiated airway epithelial model could serve as a surrogate biomarker to assess correction of the mutant gene in CF or other diseases, recapitulating the phenotypic and genotypic diversity of the population.

Murine colonic mucosa hyperproliferation. I. Elevated CFTR expression and enhanced cAMP-dependent Cl−secretion

2000 ◽  
Vol 278 (5) ◽  
pp. G753-G764 ◽  
Author(s):  
Shahid Umar ◽  
Jason Scott ◽  
Joseph H. Sellin ◽  
William P. Dubinsky ◽  
Andrew P. Morris
Keyword(s):  
Cystic Fibrosis ◽  
Fluid Transport ◽  
Cellular Proliferation ◽  
Neck Region ◽  
Anion Channel ◽  
Cellular Level ◽  
Cystic Fibrosis Gene ◽  
Mrna And Protein Expression

Fluid transport in the large intestine is mediated by the cystic fibrosis gene product and cAMP-dependent anion channel cystic fibrosis transmembrane conductance regulator (CFTR). cAMP-mediated Cl−secretion by gastrointestinal cell lines in vitro has been positively correlated with the insertion of CFTR into the apical membrane of differentiated senescent colonocytes and negatively correlated with the failure of CFTR to insert into the plasma membrane of their undifferentiated proliferating counterparts. In native tissues, this relationship remains unresolved. We demonstrate, in a transmissible murine colonic hyperplasia (TMCH) model, that (8-fold) colonocyte proliferation was accompanied by increased cellular CFTR mRNA and protein expression (8.3- and 2.4-fold, respectively) and enhanced mucosal cAMP-dependent Cl−secretion (2.3-fold). By immunofluorescence microscopy, cellular CFTR expression was restricted to the apical pole of cells at the base of the epithelial crypt. In contrast, increased cellular proliferation in vivo led to increases in both the cellular level and the total number of cells expressing this anion channel, with cellular CFTR staining extending into the crypt neck region. Hyperproliferating colonocytes accumulated large amounts of CFTR in apically oriented subcellular perinuclear compartments. This novel mode of CFTR regulation may explain why high endogenous levels of cellular CFTR mRNA and protein within the TMCH epithelium were not matched with larger increases in transmucosal CFTR Cl−current.

G protein G alpha i-2 inhibits outwardly rectifying chloride channels in human airway epithelial cells

1995 ◽  
Vol 269 (2) ◽  
pp. C451-C456 ◽  
Author(s):  
E. M. Schwiebert ◽  
D. C. Gruenert ◽  
W. B. Guggino ◽  
B. A. Stanton
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
G Protein ◽  
G Proteins ◽  
Airway Epithelial Cells ◽  
Protein G ◽  
Airway Epithelial ◽  
Human Airway ◽  
Cl Channels ◽  
Human Airway Epithelial Cells

Previously we demonstrated that the heterotrimeric G protein, G alpha i-2, inhibits cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels in human airway epithelial cells (E. M. Schwiebert, F. Gesek, L. Ercolani, C. Wjasow, D. C. Gruenert, and B. A. Stanton. Am. J. Physiol. 267 (Cell Physiol. 36): C272-C281, 1994, and E. M. Schwiebert, N. L. Kizer, D. C. Gruenert, and B. A. Stanton. Proc. Natl. Acad. Sci. USA 89: 10623-10627, 1992). The goal of the present study was to determine if G proteins also regulate outwardly rectifying Cl- channels (ORCC), a distinct class of Cl- channels regulated defectively by protein kinase A (PKA) in cystic fibrosis (CF). To this end, we used the patch-clamp technique to study ORCC in a normal human airway epithelial cell line (9HTEo-) that expresses CFTR and ORCC. Stimulation of G proteins with GTP and GTP gamma S decreased the single-channel open probability (Po) of ORCC, whereas inhibition of G proteins by GDP beta S increased the Po. Moreover, pertussis toxin (PTX), an uncoupler of Gi and G(o) subclasses of heterotrimeric G proteins, also increased the Po. Purified G alpha i-2 decreased the Po. In contrast, other PTX-sensitive G proteins, G alpha i-1, G alpha i-3, and G alpha o, had no effect on Po. We propose that G alpha i-2 couples to a receptor whose agonist negatively regulates ORCC in human airway epithelial cells.

Ozone-induced release of cytokines and fibronectin by alveolar macrophages and airway epithelial cells

1994 ◽  
Vol 266 (6) ◽  
pp. L612-L619 ◽  
Author(s):  
R. B. Devlin ◽  
K. P. McKinnon ◽  
T. Noah ◽  
S. Becker ◽  
H. S. Koren
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Alveolar Macrophages ◽  
Airway Epithelial Cells ◽  
Airway Hyperreactivity ◽  
Ozone Exposure ◽  
Airway Epithelial ◽  
Human Airway ◽  
Human Airway Epithelial Cells

Acute exposure of animals and humans to ozone results in decrements in lung function, development of airway hyperreactivity, inflammation, edema, damage to pulmonary cells, and production of several compounds with tissue damaging, fibrinogenic or fibrotic potential. The contribution of airway epithelial cells and alveolar macrophages to these processes is unclear. In this study we have directly exposed human alveolar macrophages and human airway epithelial cells to ozone in vitro and measured the cytotoxic effects of ozone, as well as the production of the inflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), and fibronectin, all of which are substantially elevated in the bronchoalveolar lavage fluid of humans exposed to ozone. Cells were grown on rigid, collagen-impregnated filter supports, and the interaction of cells with ozone facilitated by exposing them to the gas with medium below the support but no medium on top of the cells. The results show that, although macrophages are much more sensitive to ozone than epithelial cells, they do not produce increased amounts of IL-6, IL-8, or fibronectin following ozone exposure. In contrast, epithelial cells produce substantially more of all three proteins following ozone exposure, and both IL-6 and fibronectin are secreted vectorially. An immortalized human airway epithelial cell line (BEAS 2B) was used in these experiments since human airway epithelial cells are infrequently available for in vitro studies. Data from this study extend previous findings which suggest that the BEAS cell line is a useful model to study the interaction between airway epithelial cells and environmental toxicants.

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