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

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  • 2010-02-20
Published in:
  • American journal of respiratory cell and molecular biology. - 2011
5'-Nucleotidase
Adenosine
Blotting, Western
Cell Communication
Cell Line
Cell Polarity
Chlorides
Connexins
Cyclooxygenase Inhibitors
Cystic Fibrosis Transmembrane Conductance Regulator
Dinoprostone
Epithelial Cells
GPI-Linked Proteins
Gap Junctions
Homeostasis
Humans
Membrane Potentials
Microscopy, Confocal
Mucociliary Clearance
Mucus
Phospholipase A2 Inhibitors
Phospholipases A2
RNA Interference
Receptors, Prostaglandin E
Receptors, Purinergic P1
Respiratory Mucosa
Reverse Transcriptase Polymerase Chain Reaction
Signal Transduction
Surface Properties
Time Factors
English 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.
Language
  • English
Open access status
green
Identifiers
Persistent URL
https://sonar.ch/global/documents/205977
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