NADPH-oxidase and a hydrogen peroxide-sensitive K+ channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines

Pulmonary neuroepithelial bodies (NEB) are widely distributed throughout the airway mucosa of human and animal lungs. Based on the observation that NEB cells have a candidate oxygen sensor enzyme complex (NADPH oxidase) and an oxygen-sensitive K+ current, it has been suggested that NEB may function as airway chemoreceptors. Here we report that mRNAs for both the hydrogen peroxide sensitive voltage gated potassium channel subunit (KH2O2) KV3.3a and membrane components of NADPH oxidase (gp91phox and p22phox) are coexpressed in the NEB cells of fetal rabbit and neonatal human lungs. Using a microfluorometry and dihydrorhodamine 123 as a probe to assess H2O2 generation, NEB cells exhibited oxidase activity under basal conditions. The oxidase in NEB cells was significantly stimulated by exposure to phorbol esther (0.1 μM) and inhibited by diphenyliodonium (5 μM). Studies using whole-cell voltage clamp showed that the K+ current of cultured fetal rabbit NEB cells exhibited inactivating properties similar to KV3.3a transcripts expressed in Xenopus oocyte model. Exposure of NEB cells to hydrogen peroxide (H2O2, the dismuted by-product of the oxidase) under normoxia resulted in an increase of the outward K+ current indicating that H2O2 could be the transmitter modulating the O2-sensitive K+ channel. Expressed mRNAs or orresponding protein products for the NADPH oxidase membrane cytochrome b as well as mRNA encoding KV3.3a were identified in small cell lung carcinoma cell lines. The studies presented here provide strong evidence for an oxidase-O2 sensitive potassium channel molecular complex operating as an O2 sensor in NEB cells, which function as chemoreceptors in airways and in NEB related tumors. Such a complex may represent an evolutionary conserved biochemical link for a membrane bound O2-signaling mechanism proposed for other cells and life forms.

Production of β-defensins by human airway epithelia

Human β-defensins (HBDs) are antimicrobial peptides that may play a role in mucosal defense. Diminished activity of these peptides has been implicated in the pathogenesis of cystic fibrosis (CF) lung disease. We show that HBD-1 and HBD-2 mRNAs are expressed in excised surface and submucosal gland epithelia from non-CF and CF patients. The pro-inflammatory cytokine interleukin-1β stimulated the expression of HBD-2 but not HBD-1 mRNA and peptide in primary cultures of airway epithelia. HBD-1 was found in bronchoalveolar lavage (BAL) fluid from normal volunteers, CF patients, and patients with inflammatory lung diseases, whereas HBD-2 was detected in BAL fluid from patients with CF or inflammatory lung diseases, but not in normal volunteers. Both HBD-1 and HBD-2 were found in BAL fluid in concentrations of several ng/ml, and both recombinant peptides showed salt-sensitive bactericidal activity. These data suggest that in the lung HBD-2 expression is induced by inflammation, whereas HBD-1 may serve as a defense in the absence of inflammation.

Emergency airway management by intensive care unit nurses with the intubating laryngeal mask airway and the laryngeal tube

When using the laryngeal tube and the intubating laryngeal mask airway (ILMA), the medium-size (maximum volume 1100 ml) versus adult (maximum volume 1500 ml) self-inflating bags resulted in significantly lower lung tidal volumes. No gastric inflation occurred when using both devices with either ventilation bag. The newly developed medium-size self-inflating bag may be an option to further reduce the risk of gastric inflation while maintaining sufficient lung ventilation. Both the ILMA and laryngeal tube proved to be valid alternatives for emergency airway management in the experimental model used.

NO chemical events in the human airway during the immediate and late antigen-induced asthmatic response

A wealth of evidence supports increased NO (NO⋅) in asthma, but its roles are unknown. To investigate how NO participates in inflammatory airway events in asthma, we measured NO⋅ and NO⋅ chemical reaction products [nitrite, nitrate, S-nitrosothiols (SNO), and nitrotyrosine] before, immediately and 48 h after bronchoscopic antigen (Ag) challenge of the peripheral airways in atopic asthmatic individuals and nonatopic healthy controls. Strikingly, NO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} was the only NO⋅ derivative to increase during the immediate Ag-induced asthmatic response and continued to increase over 2-fold at 48 h after Ag challenge in contrast to controls [P < 0.05]. NO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document} was not affected by Ag challenge at 10 min or 48 h after Ag challenge. Although SNO was not detectable in asthmatic airways at baseline or immediately after Ag, SNO increased during the late response to levels found in healthy controls. A model of NO⋅ dynamics derived from the current findings predicts that NO⋅ may have harmful effects through formation of peroxynitrite, but also subserves an antioxidant role by consuming reactive oxygen species during the immediate asthmatic response, whereas nitrosylation during the late asthmatic response generates SNO, safe reservoirs for removal of toxic NO⋅ derivatives.

Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma

Airway hyperresponsiveness (AHR), goblet cell metaplasia, and mucus overproduction are important features of bronchial asthma. To elucidate the molecular mechanisms behind these pulmonary pathologies, we examined for genes preferentially expressed in the lungs of a murine model of allergic asthma by using suppression subtractive hybridization (SSH). We identified a gene called gob-5 that had a selective expression pattern in the airway epithelium with AHR. Here, we show that gob-5, a member of the calcium-activated chloride channel family, is a key molecule in the induction of murine asthma. Intratracheal administration of adenovirus-expressing antisense gob-5 RNA into AHR-model mice efficiently suppressed the asthma phenotype, including AHR and mucus overproduction. In contrast, overexpression of gob-5 in airway epithelia by using an adenoviral vector exacerbated the asthma phenotype. Introduction of either gob-5 or hCLCA1, the human counterpart of gob-5, into the human mucoepidermoid cell line NCI-H292 induced mucus production as well as MUC5AC expression. Our results indicated that gob-5 may play a critical role in murine asthma, and its human counterpart hCLCA1 is therefore a potential target for asthma therapy.

Defective regulatory volume decrease in human cystic fibrosis tracheal cells because of altered regulation of intermediate conductance Ca2+-dependent potassium channels

The cystic fibrosis transmembrane conductance regulator (CFTR) protein has the ability to function as both a chloride channel and a channel regulator. The loss of these functions explains many of the manifestations of the cystic fibrosis disease (CF), including lung and pancreatic failure, meconium ileus, and male infertility. CFTR has previously been implicated in the cell regulatory volume decrease (RVD) response after hypotonic shocks in murine small intestine crypts, an effect associated to the dysfunction of an unknown swelling-activated potassium conductance. In the present study, we investigated the RVD response in human tracheal CF epithelium and the nature of the volume-sensitive potassium channel affected. Neither the human tracheal cell line CFT1, expressing the mutant CFTR-ΔF508 gene, nor the isogenic vector control line CFT1-LC3, engineered to express the βgal gene, showed RVD. On the other hand, the cell line CFT1-LCFSN, engineered to express the wild-type CFTR gene, presented a full RVD. Patch-clamp studies of swelling-activated potassium currents in the three cell lines revealed that all of them possess a potassium current with the biophysical and pharmacological fingerprints of the intermediate conductance Ca2+-dependent potassium channel (IK, also known as KCNN4). However, only CFT1-LCFSN cells showed an increase in IK currents in response to hypotonic challenges. Although the identification of the molecular mechanism relating CFTR to the hIK channel remains to be solved, these data offer new evidence on the complex integration of CFTR in the cells where it is expressed.

Phenotypic and physiologic characterization of transgenic mice expressing interleukin 4 in the lung: lymphocytic and eosinophilic inflammation without airway hyperreactivity.

To investigate the contribution of interleukin-4 (IL-4) to airway inflammation in vivo and to explore directly its relationship to airway reactivity, we created transgenic mice in which the murine cDNA for IL-4 was regulated by the rat Clara cell 10 protein promoter. Expression was detected only in the lung and not in thymus, heart, liver, spleen, kidney, or uterus. The expression of IL-4 elicited hypertrophy of epithelial cells of the trachea, bronchi, and bronchioles. Hypertrophy is due, at least in part, to the accumulation of mucus glycoprotein. Histologic examination of parenchyma revealed multinucleated macrophages and occasional islands of cells consisting largely of eosinophils or lymphocytes. Analysis of lung lavage fluid revealed the presence of a leukocytic infiltrate consisting of lymphocytes, neutrophils and eosinophils. Mice expressing IL-4 had greater baseline airway resistance but did not demonstrate hyperreactivity to methacholine. Thus, the expression of IL-4 selectively within the lung elicits an inflammatory response characterized by epithelial cell hypertrophy, and the accumulation of macrophages, lymphocytes, eosinophils, and neutrophils without resulting in an alteration in airway reactivity to inhaled methacholine.

Inducible expression of an antibiotic peptide gene in lipopolysaccharide-challenged tracheal epithelial cells.

Mammals continually confront microbes at mucosal surfaces. A current model suggests that epithelial cells contribute to defense at these sites, in part through the production of broad-spectrum antibiotic peptides. Previous studies have shown that invertebrates can mount a host defense response characterized by the induction in epithelia] cells of a variety of antibiotic proteins and peptides when they are challenged with microorganisms, bacterial cell wall/membrane components, or traumatic injury [Boman, H.G. & Hultmark, D. (1987) Annu. Rev. Microbiol. 41, 103-126J. However, factors that govern the expression of similar defense molecules in mammalian epithelial cells are poorly understood. Here, a 13-fold induction of the endogenous gene encoding tracheal antimicrobial peptide was found to characterize a host response of tracheal epithelia] cells (TECs) exposed to bacterial lipopolysaccharide (LPS). Northern blot data indicated that TECs express CD14, a well-characterized LPS-binding protein known to mediate many LPS responses. A monoclonal antibody to CD14 blocked the observed tracheal antimicrobial peptide induction by LPS under serum-free conditions. Together the data support that CD14 of epithelial cell origin mediates the LPS induction of an antibiotic peptide gene in TECs, providing evidence for the active participation of epithelial cells in the host's local defense response to bacteria. Furthermore, the data allude to a conservation of this host response in evolution and suggest that a similar inducible pathway of host defense is prevalent at mucosal surfaces of mammals.

Asbestos induces nuclear factor kappa B (NF-kappa B) DNA-binding activity and NF-kappa B-dependent gene expression in tracheal epithelial cells.

Nuclear factor kappa B (NF-kappa B) is a transcription factor regulating expression of genes intrinsic to inflammation and cell proliferation--features of asbestos-associated diseases. In studies here, crocidolite asbestos caused protracted and dose-responsive increases in proteins binding to nuclear NF-kappa B-binding DNA elements in hamster tracheal epithelial (HTE) cells. This binding was modulated by cellular glutathione levels. Antibodies recognizing p65 and p50 protein members of the NF-kappa B family revealed these proteins in two of the DNA complexes. Transient transfection assays with a construct containing six NF-kappa B-binding DNA consensus sites linked to a luciferase reporter gene indicated that asbestos induced transcriptional activation of NF-kappa B-dependent genes, an observation that was confirmed by northern blot analyses for c-myc mRNA levels in HTE cells. Studies suggest that NF-kappa B induction by asbestos is a key event in regulation of multiple genes involved in the pathogenesis of asbestos-related lung cancers.

Continuous nitric oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo.

Nitric oxide (NO) is an important mediator of inflammatory responses in the lung and a key regulator of bronchomotor tone. An airway NO synthase (NOS; EC 1.14.13.39) has been proposed as a source of endogenous NO in the lung but has not been clearly defined. Through molecular cloning, we conclusively demonstrate that NO synthesis in normal human airways is due to the continuous expression of the inducible NOS (iNOS) isoform in airway epithelial cells. Although iNOS mRNA expression is abundant in airway epithelial cells, expression is not detected in other pulmonary cell types, indicating that airway epithelial cells are unique in the continuous pattern of iNOS expression in the lung. In situ analysis reveals all airway epithelial cell types express iNOS. However, removal of epithelial cells from the in vivo airway environment leads to rapid loss of iNOS expression, which suggests expression is dependent upon conditions and/or factors present in the airway. Quantitation of NOS activity in epithelial cell lysates indicates nanomolar levels of NO synthesis occur in vivo. Remarkably, the high-level iNOS expression is constant in airway epithelium of normal individuals over time. However, expression is strikingly decreased by inhaled corticosteroids and beta-adrenergic agonists, medications commonly used in treatment of inflammatory airway diseases. Based upon these findings, we propose that respiratory epithelial cells are key inflammatory cells in the airway, functioning in host defense and potentially playing a role in airway inflammation.