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J. Biol. Chem. 286 (30): 26277-26286

© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Rho Signaling Regulates Pannexin 1-mediated ATP Release from Airway Epithelia*Formula

Lucia Seminario-Vidal{ddagger}§1, Seiko F. Okada{ddagger}1, Juliana I. Sesma{ddagger}, Silvia M. Kreda{ddagger}, Catharina A. van Heusden{ddagger}, Yunxiang Zhu{ddagger}, Lisa C. Jones{ddagger}, Wanda K. O'Neal{ddagger}, Silvia Penuela, Dale W. Laird, Richard C. Boucher{ddagger}, , and Eduardo R. Lazarowski{ddagger}2

From the {ddagger}Cystic Fibrosis/Pulmonary Research and Treatment Center and
§Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, North Carolina 27599 and
the Department of Anatomy and Cell Biology, University of Western Ontario, London N6A 5C1, Ontario, Canada

ABSTRACT Back to Top

Abstract: ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia.

Key Words: ATP • Epithelial Cell • Purinergic Receptor • Rho • TRP Channels • ATP Release • Airway Epithelia • Pannexin 1

Received for publication May 12, 2011.

FOOTNOTES Back to Top

1 Both authors contributed equally to this work.

2 To whom correspondence should be addressed: Cystic Fibrosis/Pulmonary Research and Treatment Center, 7017 Thurston-Bowles Building, CB 7248, University of North Carolina, Chapel Hill, NC 27599-7248. Tel.: 919-966-0991; Fax: 919-966-5178; E-mail: eduardo_lazarowski{at}med.unc.edu.

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