Abstract
Human airway epithelial cells express pannexin 1 (Panx1) channels to release ATP, which regulates mucociliary clearance. Airway inflammation causes mucociliary dysfunction. Exposure of primary human airway epithelial cell cultures to IFN-γ for 48 h did not alter Panx1 protein expression but significantly decreased ATP release in response to hypotonic stress. The IFN-γ-induced functional down-regulation of Panx1 was due to the up-regulation of dual oxidase 2 (Duox2). Duox2 suppression by siRNA led to an increase in ATP release in control cells and restoration of ATP release in cells treated with IFN-γ. Both effects were reduced by the pannexin inhibitor probenecid. Duox2 up-regulation stoichiometrically increases H2O2 and proton production. H2O2 inhibited Panx1 function temporarily by formation of disulfide bonds at the thiol group of its terminal cysteine. Long-term exposure to H2O2, however, had no inhibitory effect. To assess the role of cellular acidification upon IFN-γ treatment, fully differentiated airway epithelial cells were exposed to ammonium chloride to alkalinize the cytosol. This led to a 2-fold increase in ATP release in cells treated with IFN-γ that was also inhibited by probenecid. Duox2 knockdown also partially corrected IFN-γ-mediated acidification. The direct correlation between intracellular pH and Panx1 open probability was shown in oocytes. Therefore, airway epithelial cells release less ATP in response to hypotonic stress in an inflammatory environment (IFN-γ exposure). Decreased Panx1 function is a response to cell acidification mediated by IFN-γ-induced up-regulation of Duox2, representing a novel mechanism for mucociliary dysfunction in inflammatory airway diseases.
Highlights
ATP is a key regulator of the innate pulmonary host defense by activating purinergic P2Y2 receptors, which promote chloride secretion by calcium-activated ClϪ channels, inhibit Naϩ absorption by epithelial Naϩ channels, increase ciliary beat fre
Pannexin proteins form pannexons, which are channels that open at resting membrane potential because of mechanical stress and in response to extracellular ATP when co-expressed with P2Y2 receptors [8, 9]
We showed the electrophysiological presence and activity of pannexin 1 (Panx1) in primary normal human bronchial epithelial cells
Summary
Chemicals and Solutions—Unless stated otherwise, all materials were purchased from Sigma-Aldrich The culture was allowed to equilibrate for 100 s to re-establish basal conditions, and (time 0), 15 l of a hypotonic solution (H2O with 1 mM CaCl2 and 1 mM MgCl2) was added to the apical compartment, causing hypotonic stress for an additional 200 s, as described previously [5, 7]. B, left panel, average traces (n ϭ 5 lung donors, 3 replicates each) of ATP release of NHBE cells Ϯ 48 h of preincubation with IFN-␥ (100 ng/ml). E, representative Western blotting for the quantification shown in D for Duox and Duox, pannexin 1 (Panx1), and -actin. MRNA levels were assayed by real-time PCR using TaqMan kits as described previously [15], and Western blotting was done as above. Statistics—Statistical analysis was performed by Student’s t test and analysis of variance or Kruskal Wallis test with appropriate post tests for at least three independent experiments, with significance accepted at p Ͻ 0.05
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