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Pannexin 1 Contributes to ATP Release in Airway Epithelia

¹ Division of Pulmonary and Critical Care, ² Department of Cell Biology and Anatomy, and ³ Department of Physiology and Biophysics, University of Miami, Miami, Florida

Correspondence and requests for reprints should be addressed to Matthias Salathe, M.D., 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. E-mail: msalathe{at}med.miami.edu

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) µM, recalculated to a normal airway surface liquid volume. Using date- and culture-matched cells (each n 16 from 4 different lungs), the pannexon inhibitors carbenoxolone (10 µM) and probenecid (1 mM), but not the connexon inhibitor flufenamic acid (100 µM), 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.

Key Words: pannexin • ATP release • airway epithelia

CLINICAL RELEVANCE Pannexin 1 contributes to release of ATP, an important paracrine regulator of mucociliary function, in airway epithelia. Given the regulation of pannexins, the findings described in this article may have important implications for the availability of ATP in the airway surface liquid in airway homeostasis and disease.