IP-10 Induction by LPS Stimulation Is Compromised in Cystic Fibrosis Bronchial Epithelial Cells.

Chronic bronchitis, a disease mainly of cigarette smokers, shares many clinical features with cystic fibrosis, a disease of altered ion transport, suggesting that the negative effects of cigarette smoke on mucociliary clearance may be mediated through alterations in ion transport. We tested the hypothesis that cigarette smoke extract would inhibit chloride secretion in human bronchial epithelial cells. In agreement with studies in canine trachea, cigarette smoke extract inhibited net chloride secretion without affecting sodium transport. We performed microelectrode impalements and impedance analysis studies to investigate the physiological mechanisms of this inhibition. These data demonstrated that cigarette smoke extract caused an acute increase in membrane resistances in conjunction with apical membrane hyperpolarization, an effect consistent with inhibition of an apical membrane anion conductance. After this acute phase, both membrane resistances decreased while membrane potentials continued to hyperpolarize, indicating that cigarette smoke extract also inhibited the basolateral entry of chloride into the cell. Furthermore, cigarette smoke extract caused an increase in mucin secretion. Therefore, the ion transport phenotype of human bronchial epithelial cells exposed to cigarette smoke extract is similar to that of cystic fibrosis epithelia in which there is sodium absorption out of proportion to chloride secretion in the setting of increased mucus secretion.

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Activity and inhibition of prostasin and matriptase on apical and basolateral surfaces of human airway epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00303.2011 ◽

2012 ◽

Vol 303 (2) ◽

pp. L97-L106 ◽

Cited By ~ 11

Author(s):

Shilpa Nimishakavi ◽

Marina Besprozvannaya ◽

Wilfred W. Raymond ◽

Charles S. Craik ◽

Dieter C. Gruenert ◽

...

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Cell Surface ◽

Selective Inhibition ◽

Airway Epithelial Cells ◽

Bronchial Epithelial Cells ◽

Apical Surface ◽

Airway Epithelial ◽

Wild Type ◽

Bronchial Epithelial

Prostasin is a membrane-anchored protease expressed in airway epithelium, where it stimulates salt and water uptake by cleaving the epithelial Na+ channel (ENaC). Prostasin is activated by another transmembrane tryptic protease, matriptase. Because ENaC-mediated dehydration contributes to cystic fibrosis (CF), prostasin and matriptase are potential therapeutic targets, but their catalytic competence on airway epithelial surfaces has been unclear. Seeking tools for exploring sites and modulation of activity, we used recombinant prostasin and matriptase to identify substrate t-butyloxycarbonyl-l-Gln-Ala-Arg-4-nitroanilide (QAR-4NA), which allowed direct assay of proteases in living cells. Comparisons of bronchial epithelial cells (CFBE41o−) with and without functioning cystic fibrosis transmembrane conductance regulator (CFTR) revealed similar levels of apical and basolateral aprotinin-inhibitable activity. Although recombinant matriptase was more active than prostasin in hydrolyzing QAR-4NA, cell surface activity resisted matriptase-selective inhibition, suggesting that prostasin dominates. Surface biotinylation revealed similar expression of matriptase and prostasin in epithelial cells expressing wild-type vs. ΔF508-mutated CFTR. However, the ratio of mature to inactive proprostasin suggested surface enrichment of active enzyme. Although small amounts of matriptase and prostasin were shed spontaneously, prostasin anchored to the cell surface by glycosylphosphatidylinositol was the major contributor to observed QAR-4NA-hydrolyzing activity. For example, the apical surface of wild-type CFBE41o− epithelial cells express 22% of total, extractable, aprotinin-inhibitable, QAR-4NA-hydrolyzing activity and 16% of prostasin immunoreactivity. In conclusion, prostasin is present, mature and active on the apical surface of wild-type and CF bronchial epithelial cells, where it can be targeted for inhibition via the airway lumen.

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