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Prostaglandin E₂regulation of cystic fibrosis transmembrane conductance regulator activity and airway surface liquid volume requires gap junctional communication

Scheckenbach, K E Ludwig
O'Grady, Scott
Published in American Journal of Respiratory Cell and Molecular Biology. 2011, vol. 44, no. 1, p. 74-82
Abstract Stimulation of the cystic fibrosis transmembrane conductance regulator (CFTR) by protease-activated receptors (PARs) at the basolateral membranes and by adenosine receptors (ADO-Rs) at the apical membrane maintain airway surface liquid (ASL) volume, which is required to ensure hydrated and clearable mucus. Both pathways involve the release of prostaglandin E₂ (PGE₂) and the stimulation of their basolateral receptors (EP-Rs). We sought to determine whether gap junctions contribute to the coordination of these pathways for modulating CFTR activity and mucus hydration. We used RT-PCR and Western blotting to determine connexin (Cx), CD73, and EP-R expression in a Calu-3 airway epithelial cell line grown on Transwell (Corning Costar, Cambridge, MA) inserts. We used dye coupling to evaluate gap junctional intercellular communication (GJIC). We used Ussing chamber studies and X-Z confocal microscopy to monitor Cl(-) secretion and ASL volume regulation. We found that connexin 43 (Cx43)-mediated GJIC was increased either by endogenous ADO after the hydrolysis of purine nucleotides by CD73 or by the direct activation of ADO-Rs. Inhibition of phospholipase A2 and cyclooxygenase prevented ADO-dependent increases in GJIC, suggesting the involvement of PGE₂. PGE₂ was found to increase GJIC markedly by stimulating EP4-Rs. The modulation of ADO signaling also affected the PAR-dependent activation of CFTR. The reduction of GJIC by CD73 or Cx43 inhibition prevented PAR-evoked CFTR currents in Ussing chambers. The inhibition of GJIC resulted in a failure of PGE₂ to increase ASL volume in Calu-3 cells and in primary cultures of well-differentiated human airway epithelial cells. Thus, gap junctions coordinate a signaling network comprising CFTR, ADO-Rs, PARs, and EP-Rs, and are required for ASL volume homeostasis.
Keywords 5'-Nucleotidase/metabolismAdenosine/metabolismBlotting, WesternCell Communication/drug effectsCell LineCell PolarityChlorides/metabolismConnexins/metabolismCyclooxygenase Inhibitors/pharmacologyCystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolismDinoprostone/metabolismEpithelial Cells/drug effects/metabolismGPI-Linked Proteins/metabolismGap Junctions/drug effects/metabolismHomeostasisHumansMembrane PotentialsMicroscopy, ConfocalMucociliary Clearance/drug effectsMucus/metabolismPhospholipases A2/antagonists & inhibitors/metabolismRNA InterferenceReceptors, Prostaglandin E/metabolismReceptors, Purinergic P1/metabolismRespiratory Mucosa/drug effects/metabolismReverse Transcriptase Polymerase Chain ReactionSignal Transduction/drug effectsSurface PropertiesTime Factors
PMID: 20167933
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Research group Mucoviscidose et jonctions GAP (229)
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SCHECKENBACH, K E Ludwig et al. Prostaglandin E₂regulation of cystic fibrosis transmembrane conductance regulator activity and airway surface liquid volume requires gap junctional communication. In: American Journal of Respiratory Cell and Molecular Biology, 2011, vol. 44, n° 1, p. 74-82. https://archive-ouverte.unige.ch/unige:21573

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Deposited on : 2012-06-13

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