A method for measuring Cl efflux from dispersed cells of airway epithelium

1995 ◽  
Vol 78 (3) ◽  
pp. 1197-1202 ◽  
Author(s):  
T. Ohrui ◽  
B. Q. Shen ◽  
R. J. Mrsny ◽  
J. H. Widdicombe
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Room Temperature ◽  
Primary Cultures ◽  
Cell Types ◽  
Airway Epithelial Cells ◽  
Isolated Cells ◽  
T84 Cells ◽  
Tracheal Epithelial Cells ◽  
3T3 Fibroblasts

This paper describes a method for measuring the increase in halide permeability of isolated airway epithelial cells induced by adenosine 3′,5′-cyclic monophosphate (cAMP). Suspensions of isolated cells, known to contain the cystic fibrosis transmembrane conductance regulator (CFTR), were placed in the upper part of a Swinnex filter holder containing a filter with pores of 0.65 micron diameter. Medium was perfused over the cells at room temperature and collected at minute intervals following its passage through the filter. Experiments were performed on Calu-3 and T84 cells (human lung and colonic epithelial cell lines), primary cultures of dog and human tracheal epithelium, and Swiss 3T3 fibroblasts stably transfected with CFTR. In all cell types, addition of agents that elevate cAMP led to increases in the rates of loss of 36Cl and 125I. However, in human tracheal epithelial cells, warming the medium from room temperature to 37 degrees C was a more effective way of stimulating tracer efflux. Increases in efflux in response to either temperature or cAMP-elevating agents were inhibited by diphenylamine-2-carboxylate, a blocker of CFTR. Reproducible increases in tracer efflux were seen with as few as 10(6) cells. Cells that had been trypsinized off their culture dishes responded better than cells that had been scraped off, although treatment of scraped cells with trypsin enhanced their responsiveness to cAMP-elevating agents. Cystic fibrosis is characterized by the lack of a cAMP-activated Cl conductance in the apical membrane of airway epithlia.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Transcriptomic profile of cystic fibrosis airway epithelial cells undergoing repair

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Alice Zoso ◽  
Aderonke Sofoluwe ◽  
Marc Bacchetta ◽  
Marc Chanson
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Airway Epithelium ◽  
Primary Cultures ◽  
Cell Types ◽  
Airway Epithelial Cells ◽  
Molecular Signature ◽  
Airway Epithelial ◽  
Transcriptomic Profile ◽  
Different Cell Types

Abstract Pathological remodeling of the airway epithelium is commonly observed in Cystic Fibrosis (CF). The different cell types that constitute the airway epithelium are regenerated upon injury to restore integrity and maintenance of the epithelium barrier function. The molecular signature of tissue repair in CF airway epithelial cells has, however, not well been investigated in primary cultures. We therefore collected RNA-seq data from well-differentiated primary cultures of bronchial human airway epithelial cells (HAECs) of CF (F508del/F508del) and non-CF (NCF) origins before and after mechanical wounding, exposed or not to flagellin. We identified the expression changes with time of repair of genes, the products of which are markers of the different cell types that constitute the airway epithelium (basal, suprabasal, intermediate, secretory, goblet and ciliated cells as well as ionocytes). Researchers in the CF field may benefit from this transcriptomic profile, which covers the initial steps of wound repair and revealed differences in this process between CF and NCF cultures.

Differential expression of ORCC and CFTR induced by low temperature in CF airway epithelial cells

1995 ◽  
Vol 268 (1) ◽  
pp. C243-C251 ◽  
Author(s):  
M. E. Egan ◽  
E. M. Schwiebert ◽  
W. B. Guggino
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Incubation Temperature ◽  
Cell Types ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Channel Activity ◽  
Patch Clamp Recording ◽  
Cl Channel ◽  
Channel Expression

When nonepithelial cell types expressing the delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) mutation are grown at reduced temperatures, the mutant protein can be properly processed. The effect of low temperatures on Cl- channel activity in airway epithelial cells that endogenously express the delta F508-CFTR mutation has not been investigated. Therefore, we examined the effect of incubation temperature on both CFTR and outwardly rectifying Cl- channel (ORCC) activity in normal, in cystic fibrosis (CF)-affected, and in wild-type CFTR-complemented CF airway epithelia with use of a combination of inside-out and whole cell patch-clamp recording, 36Cl- efflux assays, and immunocytochemistry. We report that incubation of CF-affected airway epithelial cells at 25-27 degrees C is associated with the appearance of a protein kinase A-stimulated CFTR-like Cl- conductance. In addition to the appearance of CFTR Cl- channel activity, there is, however, a decrease in the number of active ORCC when cells are grown at 25-27 degrees C, suggesting that the decrease in incubation temperature may be associated with multiple alterations in ion channel expression and/or regulation in airway epithelial cells.

Na-K-Cl cotransport in nystatin-treated tracheal cells: regulation by isoproterenol, apical UTP, and [Cl]i

1994 ◽  
Vol 266 (5) ◽  
pp. C1440-C1452 ◽  
Author(s):  
M. Haas ◽  
D. G. McBrayer
Keyword(s):  
Epithelial Cells ◽  
Plasma Membranes ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Chloride Secretion ◽  
Airway Epithelial Cells ◽  
Tracheal Epithelial Cells ◽  
Cl Transport ◽  
Monovalent Ions ◽  
Stimulation Of

Chloride secretion in mammalian airway epithelia is stimulated by beta-adrenergic agonists via an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent mechanism and by apical triphosphate nucleotides (ATP, UTP) via a cAMP-independent mechanism. Both types of secretagogues are known to stimulate apical Cl channels in airway cells; however, to maintain a stimulated rate of secretion, basolateral Cl influx via Na-K-Cl cotransport must be upregulated in parallel with apical Cl efflux. To examine the regulation of basolateral cotransport activity and its relationship to apical Cl efflux, we examined Cl transport in confluent primary cultures of dog tracheal epithelial cells treated with nystatin, an antibiotic that increases the permeability of plasma membranes to small monovalent ions, including Cl. By applying nystatin to the apical membrane of these cultures, apical Cl permeability could be increased to the point where transepithelial Cl transport is limited by transport across the basolateral membrane, which reflects primarily the activity of the cotransporter. In cultures of tracheal cells not treated with nystatin, transepithelial (basolateral-to-apical) 36Cl flux was increased two- to threefold by exposure to isoproterenol (5 microM, basolateral) or apical UTP (10 microM). Apical application of nystatin (400 units/ml) increased the basal level of transepithelial 36Cl flux approximately 1.5-fold and eliminated UTP stimulation of this flux, although an approximately twofold stimulation by isoproterenol persisted. Nystatin treatment also abolished UTP stimulation of saturable, basolateral [3H]bumetanide binding, a measure of functioning Na-K-Cl cotransporters in these cells; isoproterenol stimulation of binding was only mildly inhibited by nystatin treatment. Lowering intracellular Cl concentration ([Cl]i) by incubating cultures with apical media containing nystatin and reduced [Cl] (NO3 replacement) increased both basolateral-to-apical 36Cl flux and [3H]bumetanide binding in the absence of secretagogues or cell shrinkage. The results support our previous suggestion, based entirely on [3H]bumetanide binding [M. Haas, D. G. McBrayer, and J. R. Yankaskas. Am. J. Physiol. 264 (Cell. Physiol. 32): C189-C200, 1993], that UTP stimulation of basolateral Na-K-Cl cotransport in airway epithelial cells is entirely secondary to, and requires, an increase in apical Cl efflux, and further suggest that a decrease in [Cl]i may be a signal for cotransport activation in response to UTP. In addition, a cAMP-dependent cascade initiated by isoproterenol appears to directly stimulate the cotransporter.

Glucocorticoids induce neutral endopeptidase in transformed human tracheal epithelial cells

1991 ◽  
Vol 260 (2) ◽  
pp. L83-L89 ◽  
Author(s):  
D. B. Borson ◽  
D. C. Gruenert
Keyword(s):  
Epithelial Cells ◽  
Cell Types ◽  
Neutral Endopeptidase ◽  
Airway Epithelial Cells ◽  
Biologically Active ◽  
Airway Epithelial ◽  
Tracheal Epithelial Cells ◽  
Endopeptidase Activity ◽  
Airway Responses

Neutral endopeptidase (NEP, also known as enkephalinase, CALLA, or EC 3.4.24.11) is a membrane-bound peptidase present in many different cell types. Previous studies have shown that it modulates the actions of a variety of biologically active peptides on several airway responses. More recent studies have demonstrated that reductions in neutral endopeptidase activity in animal airways is associated with increased responses to exogenously applied and endogenously released peptides. To study the regulation of NEP expression, we used human airway epithelial cells transformed in vitro with an origin-defective SV40 plasmid. Enzymatic activity, measured using [3H-Tyr,D-Ala2]leucine enkephalin, increased with cell density (1.4 ng/10(6) cells at 530 cells/cm2 and 21 ng/10(6) cells at confluence, 400 X 10(3) cells/cm2). In both confluent and nonconfluent cultures, the glucocorticoid budesonide increased neutral endopeptidase activity in time- and concentration-dependent fashions. Maximal increases of 10 ng/10(6) cells greater than control were observed after 6 days of incubation at 10(-7) M budesonide. Dexamethasone also increased NEP, suggesting that the effect is due to glucocorticoid receptor effects. Transcription, as assessed by Northern blot analysis of total cellular RNA, showed that NEP-specific RNAs also increased with increasing concentration of glucocorticoid. We conclude that neutral endopeptidase can be increased by cell growth or density and by glucocorticoids and that the effects of glucocorticoids are mediated by increased NEP gene expression.

NF-κB activation and sustained IL-8 gene expression in primary cultures of cystic fibrosis airway epithelial cells stimulated with Pseudomonas aeruginosa

2005 ◽  
Vol 288 (3) ◽  
pp. L471-L479 ◽  
Author(s):  
Theresa Joseph ◽  
Dwight Look ◽  
Thomas Ferkol
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Inflammatory Response ◽  
Cell Cultures ◽  
Primary Cultures ◽  
Airway Epithelial Cells ◽  
Mrna Levels ◽  
Airway Epithelial

The progression of lung disease in cystic fibrosis (CF) is characterized by an exuberant inflammatory response mounted by the respiratory epithelium that is further exacerbated by bacterial infection. Recent studies have demonstrated upregulation of nuclear factor-κB (NF-κB) in response to infection in genetically modified cell culture models, which is associated with expression of interleukin (IL)-8. Using human airway epithelial cells grown in primary culture, we examined in vitro activation of NF-κB in cells isolated from five CF (ΔF508/ΔF508) and three non-CF (NCF) patients in response to Pseudomonas aeruginosa. Immunofluorescence, gel-shift, and immunoblot assays demonstrated a rapid translocation of NF-κB subunits (p50 and p65) to the nucleus in both CF and NCF cell cultures. However, nuclear extracts from CF cells both before and following P. aeruginosa stimulation revealed elevated NF-κB activation compared with NCF cells. Additionally, elevated nuclear levels of the NF-κB inhibitor IκBα were detected in nuclei of CF cells after P. aeruginosa stimulation, but this increase was transient. There was no difference in IL-8 mRNA levels between CF and NCF cells early after stimulation, whereas expression was higher and sustained in CF cells at later times. Our results also demonstrated increased baseline translocation of NF-κB to nuclei of primary CF epithelial cell cultures, but intranuclear IκBα may initially block its effects following P. aeruginosa stimulation. Thus, IL-8 mRNA expression was prolonged after P. aeruginosa stimulation in CF epithelial cells, and this sustained IL-8 expression may contribute to the excessive inflammatory response in CF.

scholarly journals Acid ceramidase rescues cystic fibrosis mice from pulmonary infections

2020 ◽  
Author(s):  
Katrin Anne Becker ◽  
Rabea Verhaegh ◽  
Hedda-Luise Verhasselt ◽  
Simone Keitsch ◽  
Matthias Soddemann ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Pathogenic Bacteria ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Pulmonary Infections ◽  
Acid Ceramidase ◽  
Tracheal Epithelial Cells ◽  
Infection Susceptibility ◽  
Tracheal Epithelial

Previous studies have shown that sphingosine kills a variety of pathogenic bacteria, including Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus. Sphingosine concentrations are decreased in airway epithelial cells of cystic fibrosis (CF) mice and this defect has been linked to the infection susceptibility of these mice. Here, we tested whether genetic overexpression of the acid ceramidase rescues cystic fibrosis mice from pulmonary infections with P. aeruginosa. We demonstrate that transgenic overexpression of the acid ceramidase in CF mice corresponds to an overexpression of the acid ceramidase in bronchial and tracheal epithelial cells and normalizes ceramide and sphingosine levels in bronchial and tracheal epithelial cells. In addition, expression of β1-integrin, which is ectopically expressed on the luminal surface of airway epithelial cells in cystic fibrosis mice - an alteration that is very important for mediating pulmonary P. aeruginosa infections of cystic fibrosis, is normalized in cystic fibrosis airways upon overexpression of acid ceramidase. Most importantly, overexpression of acid ceramidase protects cystic fibrosis mice from pulmonary P. aeruginosa infections. Infection of CF mice or CF mice that were inhaled with sphingosine with P. aeruginosa or a P. aeruginosa mutant that is resistant to sphingosine indicate that sphingosine and not a metabolite kills P. aeruginosa upon pulmonary infection. These studies further support the use of acid ceramidase and its metabolite sphingosine as a potential treatment of cystic fibrosis.

Morphology and locomotion of individual epithelial cells in culture

1985 ◽  
Vol 78 (1) ◽  
pp. 105-115 ◽  
Author(s):  
R.M. Brown ◽  
C.A. Middleton
Keyword(s):  
Epithelial Cells ◽  
Primary Cultures ◽  
Time Lapse ◽  
Cell Types ◽  
Epidermal Cells ◽  
Cell Type ◽  
Isolated Cells ◽  
Cell Behaviour ◽  
Significant Movement ◽  
Different Cell Types

The behaviour in culture of epithelial cells derived from chick embryo pigmented retina epithelium (PRE), corneal epithelium (CE) and epidermis has been studied using time-lapse cinemicrography. The analysis concentrated on the morphology and movement of individual isolated cells, lacking contacts with other cells, during a 24h period starting 1–3 h after the cells were plated out in primary cultures. Isolated cells from all three sources could change morphology and reversibly exhibited either a poorly spread or a well-spread morphology. While poorly spread, the different cell types all appeared similar and all blebbed vigorously. In contrast, while well spread, the cells did not bleb significantly but there were other differences between them. Well-spread CE cells were always polarized by the presence of a dominant leading lamella but well-spread PRE cells were always unpolarized. Well-spread epidermal cells exhibited both a polarized and an unpolarized morphology. The tendency of individual isolated cells to change morphology varied with cell type. PRE cells were the most stable. Nearly 80% of them retained the same morphology throughout the period of analysis and only 1% of them showed three or more changes in morphology during this period. In contrast, 22% of CE cells and 37% of epidermal cells showed three or more changes in morphology during the period of observation. Isolated cells of all three types spent a greater proportion of the time exhibiting a poorly spread morphology than they spent exhibiting any alternative well-spread morphology. The analysis revealed a relationship between the morphology of isolated cells and the speed of their locomotion. Only cells with a well-spread polarized morphology showed significant movement. CE and epidermal cells with this morphology moved three to four times faster than their counter-parts with a poorly spread morphology or, in the case of epidermal cells, with a well-spread but unpolarized morphology. Actively moving PRE cells were not seen and this correlates with the absence of cells with a well-spread polarized morphology from cultures of this type. These findings are discussed in the light of similar investigations of cell behaviour in other epithelial cell types and fibroblasts.

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