Pannexin 1 contributes to ATP release in airway epithelia.
ATP is a paracrine regulator of critical airway epithelial cell functions, but the mechanism of its release is poorly understood. Pannexin (Panx) proteins, related to invertebrate innexins, form channels (called pannexons) that are able to release ATP from several cell types. Thus, ATP release via pannexons was examined in airway epithelial cells. Quantitative RT-PCR showed Panx1 expression in normal human airway epithelial cells during redifferentiation at the air-liquid interface (ALI), at a level comparable to that of alveolar macrophages; Panx3 was not expressed. Immunohistochemistry showed Panx1 expression at the apical pole of airway epithelia. ALI cultures exposed to hypotonic stress released ATP to an estimated maximum of 255 (+/-64) nM within 1 minute after challenge (n = 6 cultures from three different lungs) or to approximately 1.5 (+/-0.4) microM, recalculated to a normal airway surface liquid volume. Using date- and culture-matched cells (each n > or = 16 from 4 different lungs), the pannexon inhibitors carbenoxolone (10 microM) and probenecid (1 mM), but not the connexon inhibitor flufenamic acid (100 microM), inhibited ATP release by approximately 60%. The drugs affected Panx1 currents in Xenopus oocytes expressing exogenous Panx1 correspondingly. In addition, suppression of Panx1 expression using lentivirus-mediated production of shRNA in differentiated airway epithelial cells inhibited ATP release upon hypotonic stress by approximately 60% as well. These data not only show that Panx1 is expressed apically in differentiated airway epithelial cells but also that it contributes to ATP release in these cells.
Ransford
George A
GA
Division of Pulmonary and Critical Care Medicine (R-47), University of Miami Miller School of Medicine, 1600 NW 10th Ave., RMSB 7058, Miami, FL 33136, USA.
Fregien
Nevis
N
Qiu
Feng
F
Dahl
Gerhard
G
Conner
Gregory E
GE
Salathe
Matthias
M
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002229:Carbenoxolone / Q000494:pharmacology; D054337:Cell Dedifferentiation; D002478:Cells, Cultured; D017630:Connexins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005439:Flufenamic Acid / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D007038:Hypotonic Solutions; D016676:Macrophages, Alveolar / Q000378:metabolism; D051379:Mice; D009079:Mucociliary Clearance; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009997:Osmotic Pressure; D019899:Paracrine Communication; D011339:Probenecid / Q000494:pharmacology; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D020545:Respiratory Mucosa / Q000187:drug effects / Q000378:metabolism*; D013312:Stress, Physiological; D013997:Time Factors; D014162:Transfection; D014981:Xenopus
D017630:Connexins; D007038:Hypotonic Solutions; D009419:Nerve Tissue Proteins; C492764:PANX1 protein, human; C503631:Panx1 protein, mouse; D012333:RNA, Messenger; D005439:Flufenamic Acid; D000255:Adenosine Triphosphate; D002229:Carbenoxolone; D011339:Probenecid