cGMP stimulates sodium and chloride currents in rat tracheal airway epithelia

1997 ◽  
Vol 272 (3) ◽  
pp. C911-C922 ◽  
Author(s):  
E. M. Schwiebert ◽  
E. D. Potter ◽  
T. H. Hwang ◽  
J. S. Woo ◽  
C. Ding ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Epithelial Cells ◽  
Ion Transport ◽  
Cation Channels ◽  
Enzyme Analysis ◽  
Airway Epithelial ◽  
Cyclic Monophosphate ◽  
Tracheal Epithelial Cells ◽  
Tracheal Epithelial

To test the hypothesis that guanosine 3',5'-cyclic monophosphate (cGMP) regulates ion transport in airway epithelial cells, we measured short-circuit current (I(sc)) and (22)Na+ fluxes in primary cultured rat tracheal epithelial cells. In Cl- -containing Ringer solution, I(sc) was increased by approximately 17 microA/cm2 after application of 1 mM 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), whereas, in Cl- -free solutions, the Na+ -mediated component was approximately 5 microA/cm2, suggesting a cGMP stimulation of Cl-secretory current and a smaller Na+ absorptive current. Inward and net mucosal-to-serosal (22)Na+ flux was doubled in the presence of 2 mM 8-BrcGMP. To determine whether nucleotide-gated channels play a role in this transepithelial Na+ absorption, blockers of nucleotide-gated cation channels were used to inhibit I(sc). The cGMP-stimulated Na+-mediated I(sc) was blocked by as little as 500 nM dichlorobenzamil or 50 microM L-cis-diltiazem, which are known blockers for cyclic nucleotide-gated cation channels. These agents also blocked the basal (non-cGMP-stimulated) current when measured in the presence of 10 microM amiloride, which blocks current through 5-pS amiloride-sensitive Na+ channels. To document whether the distribution of nucleotide-gated nonselective cation channels was consistent with a role in airway epithelial transport, in situ hybridization was performed. In situ hybridization of mRNA encoding for nucleotide-gated cation channels was found in epithelial cell layers of rat trachea, bronchi, bronchioles, and alveolar cells but not in smooth muscle layers or tracheal cartilage. Reverse transcriptase-polymerase chain reaction, restriction enzyme analysis, and sequencing of the cDNA transcribed from mRNA of whole lung and tracheal epithelial cells indicate that a channel highly homologous to the retinal nucleotide-gated nonselective cation channel (CNG1) is present. Thus these data, along with evidence supporting the existence of signal transduction pathways elevating intracellular levels of cGMP, indicate that cGMP regulates transepithelial ion transport in lung epithelial tissues.

Paraoxonase-2 deficiency enhancesPseudomonas aeruginosaquorum sensing in murine tracheal epithelia

2007 ◽  
Vol 292 (4) ◽  
pp. L852-L860 ◽  
Author(s):  
David A. Stoltz ◽  
Egon A. Ozer ◽  
Carey J. Ng ◽  
Janet M. Yu ◽  
Srinivasa T. Reddy ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Quorum Sensing ◽  
Airway Epithelial Cell ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Airway Epithelia ◽  
Tracheal Epithelial Cells ◽  
Human Airway ◽  
Tracheal Epithelial

Pseudomonas aeruginosa is an important cause of nosocomial infections and is frequently present in the airways of cystic fibrosis patients. Quorum sensing mediates P. aeruginosa's virulence and biofilm formation through density-dependent interbacterial signaling with autoinducers. N-3-oxododecanoyl homoserine lactone (3OC12-HSL) is the major autoinducer in P. aeruginosa. We have previously shown that human airway epithelia and paraoxonases (PONs) degrade 3OC12-HSL. This study investigated the role of PON1, PON2, and PON3 in airway epithelial cell inactivation of 3OC12-HSL. All three PONs were present in murine tracheal epithelial cells, with PON2 and PON3 expressed at the highest levels. Lysates of tracheal epithelial cells from PON2, but not PON1 or PON3, knockout mice had impaired 3OC12-HSL inactivation compared with wild-type mice. In contrast, PON1-, PON2-, or PON3-targeted deletions did not affect 3OC12-HSL degradation by intact epithelia. Overexpression of PON2 enhanced 3OC12-HSL degradation by human airway epithelial cell lysates but not by intact epithelia. Finally, using a quorum-sensing reporter strain of P. aeruginosa, we found that quorum sensing was enhanced in PON2-deficient airway epithelia. In summary, these results show that loss of PON2 impairs 3OC12-HSL degradation by airway epithelial cells and suggests that diffusion of 3OC12-HSL into the airway cells can be the rate-limiting step for degradation of the molecule.

Erythromycin increases bactericidal activity of surface liquid in human airway epithelial cells

2005 ◽  
Vol 289 (4) ◽  
pp. L565-L573 ◽  
Author(s):  
Kota Ishizawa ◽  
Tomoko Suzuki ◽  
Mutsuo Yamaya ◽  
Yu Xia Jia ◽  
Seiichi Kobayashi ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Bactericidal Activity ◽  
Culture Medium ◽  
Airway Epithelial Cells ◽  
Airway Surface Liquid ◽  
Airway Epithelial ◽  
Tracheal Epithelial Cells ◽  
Tracheal Epithelial ◽  
Surface Liquid ◽  
Number Of Bacteria

Macrolide antibiotics have clinical benefits in patients with diffuse panbronchiolitis and in patients with cystic fibrosis. Although many mechanisms have been proposed, the precise mechanisms are still uncertain. We examined the effects of erythromycin on bactericidal activity of airway surface liquid secreted by cultured human tracheal epithelial cells. Airway surface liquid was collected by washing the surface of human tracheal epithelial cells with a sodium solution (40 meq/l). Methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa were incubated with airway surface liquid, and the number of surviving bacteria was examined. The number of bacteria in airway surface liquid from the cells cultured in medium alone was significantly lower than that in the sodium solution. Furthermore, the number of bacteria in airway surface liquid from the cells treated with erythromycin was significantly lower than that in airway surface liquid from the cells treated with solvent alone. The production of mRNA and protein of human β-defensin-1 and human β-defensin-2 was significantly increased by erythromycin. Bactericidal activity of airway surface liquid was observed at low concentrations (40 meq/l) of sodium but not at higher concentrations (≥80 meq/l). Airway surface liquid did not contain significant amounts of antibiotics supplemented in the culture medium. Erythromycin at the levels in airway surface liquid and in culture medium did not inhibit bacterial growth. These results suggest that erythromycin may increase bactericidal activity of airway surface liquid in human airway epithelial cells through human β-defensins production and reduce susceptibility of the airway to bacterial infection.

Adenosine 3:5′ Cyclic Monophosphate Synthesis by Human Tracheal Epithelial Cells

1990 ◽  
Vol 2 (1) ◽  
pp. 33-39 ◽  
Author(s):  
Carolyn M. Kercsmar ◽  
Michael D. Infeld ◽  
Catherine L. Silski ◽  
Pamela B. Davis
Keyword(s):  
Epithelial Cells ◽  
Cyclic Monophosphate ◽  
Tracheal Epithelial Cells ◽  
Tracheal Epithelial

Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population

2002 ◽  
Vol 283 (6) ◽  
pp. L1315-L1321 ◽  
Author(s):  
Yingjian You ◽  
Edward J. Richer ◽  
Tao Huang ◽  
Steven L. Brody
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Airway Epithelial Cell ◽  
Airway Epithelial ◽  
Tracheal Epithelial Cells ◽  
Proliferation And Differentiation ◽  
Tracheal Epithelial

Highly regulated programs for airway epithelial cell proliferation and differentiation during development and repair are often disrupted in disease. These processes have been studied in mouse models; however, it is difficult to isolate and identify epithelial cell-specific responses in vivo. To investigate these processes in vitro, we characterized a model for primary culture of mouse tracheal epithelial cells. Small numbers of cells seeded at low density (7.5 × 104 cells/cm2) rapidly proliferated and became polarized. Subsequently, supplemented media and air-liquid interface conditions resulted in development of highly differentiated epithelia composed of ciliated and nonciliated cells with gene expression characteristic of native airways. Genetically altered or injured mouse tracheal epithelial cells also reflected in vivo patterns of airway epithelial cell gene expression. Passage of cells resulted in continued proliferation but limited differentiation after the first passage, suggesting that transit-amplifying cell populations were present but with independent programs for proliferation and differentiation. This approach provides a high-fidelity in vitro model for evaluation of gene regulation and expression in mouse airway epithelial cells.

A subset of mouse tracheal epithelial basal cells generates large colonies in vitro

2004 ◽  
Vol 286 (4) ◽  
pp. L631-L642 ◽  
Author(s):  
Kelly G. Schoch ◽  
Adriana Lori ◽  
Kimberlie A. Burns ◽  
Tracy Eldred ◽  
John C. Olsen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Epithelial Cells ◽  
The Body ◽  
Growth Potential ◽  
Small Subset ◽  
Basal Cells ◽  
Airway Epithelial ◽  
Epithelial Stem Cells ◽  
Tracheal Epithelial Cells ◽  
Tracheal Epithelial

Airway epithelial stem cells are not well characterized. To examine clonal growth potential, we diluted single, viable B6.129S7-Gtrosa26 (Rosa26) mouse tracheal epithelial cells that constitutively express β-galactosidase into non-Rosa26 cells in an air-liquid interface cell culture model; 1.7% of the cells formed colonies of varying size, and, on average, 0.1% of the cells formed large colonies. Thus only a small subset of cells displayed progenitorial capacity suggestive of stem or early transient amplifying cells. Prior studies identified cells with high keratin 5 (K5) promoter activity in specific niches in the mouse trachea and these cells corresponded to the location of bromodeoxyuridine label-retaining cells, thought to be stem cells (Borthwick DW, Shahbazian M, Todd KQ, Dorin JR, and Randell SH, Am J Respir Cell Mol Biol: 24: 662–670, 2001). To explore the hypothesis that stem cells were present in the K5-expressing compartment, we created transgenic mice in which enhanced green fluorescent protein (EGFP) was driven by the K5 promoter. These mice expressed EGFP in most basal cells of the body including a subset of tracheal basal cells apparently located in positions similar to previously identified stem cell niches. Flow cytometrically purified EGFP-positive cells had an overall colony-forming efficiency 4.5-fold greater than EGFP-negative cells, but the ability to generate large colonies was 12-fold greater. Thus adult mouse tracheal epithelial cells with progenitorial capacity sufficient to generate large colonies reside in the basal cell compartment. These studies are a first step toward purification and characterization of airway epithelial stem cells.

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.

scholarly journals Regulation of guanosine 3′:5′-cyclic monophosphate in ovine tracheal epithelial cells

1997 ◽  
Vol 120 (7) ◽  
pp. 1249-1254 ◽  
Author(s):  
Simon P. Range ◽  
Elaine D. Holland ◽  
Graham P. Basten ◽  
Alan J. Knox
Keyword(s):  
Epithelial Cells ◽  
Cyclic Monophosphate ◽  
Tracheal Epithelial Cells ◽  
Tracheal Epithelial

scholarly journals Connexin mimetic peptides reversibly inhibit Ca2+signaling through gap junctions in airway cells

2000 ◽  
Vol 279 (4) ◽  
pp. L623-L630 ◽  
Author(s):  
Scott Boitano ◽  
W. Howard Evans
Keyword(s):  
Epithelial Cells ◽  
Gap Junctions ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Gap Junctional Communication ◽  
Tracheal Epithelial Cells ◽  
Junctional Communication ◽  
Mimetic Peptides ◽  
Intracellular Ca ◽  
Tracheal Epithelial

The effect of peptides with sequences derived from connexins, the constituent proteins of gap junctions, on mechanically stimulated intercellular Ca2+signaling in tracheal airway epithelial cells was studied. Three peptides with sequences corresponding to connexin extracellular loop regions reversibly restricted propagation of Ca2+ waves to neighboring cells. Recovery of communication began within 10 min of removal of the peptides, with inhibition totally reversed by 20–40 min. The peptides were shown to be more effective in inhibiting Ca2+ waves than glycyrrhetinic acid or oleamide. Inhibition of intercellular Ca2+ waves by connexin mimetic peptides did not affect the Ca2+ response to extracellular ATP. Although the intracellular Ca2+ response of tracheal epithelial cells to ATP was greatly reduced by either pretreatment with high doses of ATP or application of apyrase, mechanically stimulated intercellular Ca2+ signaling was not affected by these agents. We conclude that connexin mimetic peptides are effective and reversible inhibitors of gap junctional communication of physiologically significant molecules that underlie Ca2+wave propagation in tracheal epithelial cells and propose a potential mechanism for the mode of action of mimetic peptides.

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