title: Prostaglandin E₂regulation of cystic fibrosis transmembrane conductance regulator activity and airway surface liquid volume requires gap junctional communication.
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.
authors.affiliation: Laboratory of Clinical Investigation III, Department of Pediatrics, Geneva University Hospitals, 4 Gabrielle-Perret-Gentil, Geneva 4, Switzerland.
authors.forename: K E Ludwig
authors.initials: KE
authors.lastname: Scheckenbach
authors.forename: Davide
authors.initials: D
authors.lastname: Losa
authors.forename: Tecla
authors.initials: T
authors.lastname: Dudez
authors.forename: Marc
authors.initials: M
authors.lastname: Bacchetta
authors.forename: Scott
authors.initials: S
authors.lastname: O'Grady
authors.forename: Sophie
authors.initials: S
authors.lastname: Crespin
authors.forename: Marc
authors.initials: M
authors.lastname: Chanson
mesh_terms: D015720:5'-Nucleotidase / Q000378:metabolism; D000241:Adenosine / Q000378:metabolism; D015153:Blotting, Western; D002450:Cell Communication / Q000187:drug effects; D002460:Cell Line; D016764:Cell Polarity; D002712:Chlorides / Q000378:metabolism; D017630:Connexins / Q000378:metabolism; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D015232:Dinoprostone / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D058851:GPI-Linked Proteins / Q000378:metabolism; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D006706:Homeostasis; D006801:Humans; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D009079:Mucociliary Clearance / Q000187:drug effects; D009093:Mucus / Q000378:metabolism*; D064801:Phospholipase A2 Inhibitors; D054467:Phospholipases A2 / Q000378:metabolism; D034622:RNA Interference; D018078:Receptors, Prostaglandin E / Q000378:metabolism; D018047:Receptors, Purinergic P1 / Q000378:metabolism; D020545:Respiratory Mucosa / Q000187:drug effects / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D013499:Surface Properties; D013997:Time Factors
chemical_list: C505032:CFTR protein, human; D002712:Chlorides; D017630:Connexins; D016861:Cyclooxygenase Inhibitors; D058851:GPI-Linked Proteins; D064801:Phospholipase A2 Inhibitors; D018078:Receptors, Prostaglandin E; D018047:Receptors, Purinergic P1; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D054467:Phospholipases A2; D015720:5'-Nucleotidase; C548241:NT5E protein, human; D000241:Adenosine; D015232:Dinoprostone