Measurements of airway surface liquid height and mucus transport by fluorescence microscopy, and of ion composition by X-ray microanalysis

Roomans, Godfried M.; Kozlova, Inna; Nilsson, Hans C.; Vanthanouvong, Viengphet; Button, Brian; Tarran, Robert

doi:10.1016/j.jcf.2004.05.029

Cited by 23 publications

(9 citation statements)

References 18 publications

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“…The cells were washed with PBS, followed by apical addition of Texas Red-labeled 70-kDa dextran/PBS (2.5 mg/ml; Invitrogen) to measure AvSL height. For studies of basal AvSL regulation, 25 l of Texas Red-labeled 70-kDa dextran, dextran, PBS, and perflurocarbon (50 l) were added, and the cells were imaged immediately and serially for 24 h at five predetermined sites, as previously described (32). Studies of forskolin or UTP were performed by incubating 25 l of Texas Red-labeled 70-kDa dextran, PBS fluorophore on the cell surface for 30 min at 37°C, followed by removal of excess dextran to initiate studies under steady state "thin film" conditions.…”

Section: Atp Concentration and Releasementioning

confidence: 99%

Human Alveolar Type II Cells Secrete and Absorb Liquid in Response to Local Nucleotide Signaling

Bove

Grubb

Okada

et al. 2010

Journal of Biological Chemistry

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A balance sheet describing the integrated homeostasis of secretion, absorption, and surface movement of liquids on pulmonary surfaces has remained elusive. It remains unclear whether the alveolus exhibits an intra-alveolar ion/liquid transport physiology or whether it secretes ions/liquid that may communicate with airway surfaces. Studies employing isolated human alveolar type II (AT2) cells were utilized to investigate this question. Human AT2 cells exhibited both epithelial Na

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Section: Atp Concentration and Releasementioning

confidence: 99%

Human Alveolar Type II Cells Secrete and Absorb Liquid in Response to Local Nucleotide Signaling

Bove

Grubb

Okada

et al. 2010

Journal of Biological Chemistry

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“…1 ). Inhaled bacteria adhere to the mucin and are continuously displaced upward by coordinated cilia beating activity [ 17 , 18 ]. Comparative analysis of the bioelectric properties of airway epithelia from normal donors and CF patients revealed that PCL height is regulated by a delicate balance between the activities of the epithelial sodium channel (ENAC) and the cystic fibrosis transmembrane regulator (CFTR) [ 19 ].…”

Section: Nucleotide-mediated Bacterial Clearancementioning

confidence: 99%

E-NTPDases in human airways: Regulation and relevance for chronic lung diseases

Burch

Picher

2006

Purinergic Signalling

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Chronic obstructive lung diseases are characterized by the inability to prevent bacterial infection and a gradual loss of lung function caused by recurrent inflammatory responses. In the past decade, numerous studies have demonstrated the importance of nucleotide-mediated bacterial clearance. Their interaction with P2 receptors on airway epithelia provides a rapid Fon-and-off_ signal stimulating mucus secretion, cilia beating activity and surface hydration. On the other hand, abnormally high ATP levels resulting from damaged epithelia and bacterial lysis may cause lung edema and exacerbate inflammatory responses. Airway ATP concentrations are regulated by ecto nucleoside triphosphate diphosphohydrolases (E-NTPDases) which are expressed on the mucosal surface and catalyze the sequential dephosphorylation of nucleoside triphosphates to nucleoside monophosphates (ATP Y ADP Y AMP). The common bacterial product, Pseudomonas aeruginosa lipopolysaccharide (LPS), induces an acute reduction in azide-sensitive ENTPDase activities, followed by a sustained increase in activity as well as NTPDase 1 and NTPDase 3 expression. Accordingly, chronic lung diseases, including cystic fibrosis (CF) and primary ciliary dyskinesia, are characterized by higher rates of nucleotide elimination, azide-sensitive E-NTPDase activities and expression. This review integrates the biphasic regulation of airway E-NTPDases with the function of purine signaling in lung diseases. During acute insults, a transient reduction in E-NTPDase activities may be beneficial to stimulate ATP-mediated bacterial clearance. In chronic lung diseases, elevating E-NTPDase activities may represent an attempt to prevent P2 receptor desensitization and nucleotide-mediated lung damage.

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“…Typically 10-12 beads were analyzed from each sample. For quantitative analysis, the data were compared to the results obtained on beads soaked in salt solutions of different concentrations (50mM-250mM) [21,22,24].…”

Section: Frozen-hydrated Samplesmentioning

confidence: 99%

X-ray Microanalysis of Apical Fluid in Cystic Fibrosis Airway Epithelial Cell Lines

Kozlova¹,

Nilsson²,

Henriksnäs³

et al. 2006

Cell Physiol Biochem

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The ionic composition of the fluid lining the airways (airway surface liquid, ASL) in healthy subjects and patients with cystic fibrosis (CF) has been a matter of controversy. It has been attempted to resolve conflicting theories by using cell cultures, but published results show a wide variety of values for the ionic concentrations in the apical fluid in these cultures. To investigate CFTR-mediated HCO 3 -conductance and the role of HCO 3 - in regulating ASL pH we determined the pH of the fluid covering the apical surface of airway epithelial cells. A normal (16HBE14o - ) and a CF (CFBE41o - ) bronchial epithelial cell line were grown on membrane inserts in both a liquid-liquid interface culture system for 7 days, and in an air-liquid interface culture system for one month. The elemental composition of the fluid covering the apical surface was determined by X-ray microanalysis of frozen-hydrated specimens, or by X-ray microanalysis of Sephadex beads that had been equilibrated with the apical fluid. Analysis showed that the apical fluid had a Na + and Cl -concentration of about 80-100 mM and thus was slightly hypotonic. The ionic concentrations were somewhat higher in air-liquid interface than in liquid-liquid interface cultures. The apical fluid in CF cells had significantly higher concentrations of Na and Cl than that in control cultures. In control cultures, the concentrations of Na and Cl in the apical fluid increased if glibenclamide, an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR) was added to the apical medium. Exposing the cells to the metabolic inhibitor NaCN also resulted in a significant increase of the Na and Cl concentrations in the apical fluid. The results agree with the notion that these cell cultures are mainly absorptive cells, and that ion absorption by the CF cells is reduced compared to that in normal cells. The pH measurements of the fluid covering the apical part of cell cultures support the notion that bicarbonate ions may be transported by CFTR, and that this can be inhibited by specific CFTR inhibitors.

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Measurements of airway surface liquid height and mucus transport by fluorescence microscopy, and of ion composition by X-ray microanalysis

Cited by 23 publications

References 18 publications

Human Alveolar Type II Cells Secrete and Absorb Liquid in Response to Local Nucleotide Signaling

Human Alveolar Type II Cells Secrete and Absorb Liquid in Response to Local Nucleotide Signaling

E-NTPDases in human airways: Regulation and relevance for chronic lung diseases

X-ray Microanalysis of Apical Fluid in Cystic Fibrosis Airway Epithelial Cell Lines

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