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First published online 19 September 2006
doi: 10.1242/jcs.03181

Journal of Cell Science 119, 4176-4186 (2006)
Published by The Company of Biologists 2006
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Research Article

Real-time analysis of cAMP-mediated regulation of ciliary motility in single primary human airway epithelial cells

Andreas Schmid1, Ge Bai1, Nathalie Schmid1, Manuela Zaccolo2, Lawrence E. Ostrowski3, Gregory E. Conner1,4, Nevis Fregien1,4 and Matthias Salathe1,*

1 Division of Pulmonary and Critical Care Medicine, University of Miami School of Medicine, 1600 NW 10th Avenue, RMSB 7063, Miami, FL 33136, USA
2 Dulbecco Telethon Institute at the Venetian Institute of Molecular Medicine, Padova, Italy
3 Cystic Fibrosis Center, University of North Carolina, Chapel Hill, NC, USA
4 Department of Cell Biology and Anatomy, University of Miami, Miami, FL, USA

* Author for correspondence (e-mail: msalathe{at}miami.edu)

Accepted 25 July 2006

Airway ciliary beat frequency regulation is complex but in part influenced by cyclic adenosine monophosphate (cAMP)-mediated changes in cAMP-dependent kinase activity, yet the cAMP concentration required for increases in ciliary beat frequency and the temporal relationship between ciliary beat frequency and cAMP changes are unknown. A lentiviral gene transfer system was developed to express a fluorescence resonance energy transfer (FRET)-based cAMP sensor in ciliated cells. Expression of fluorescently tagged cAMP-dependent kinase subunits from the ciliated-cell-specific foxj1 promoter enhanced expression in fully differentiated ciliated human airway epithelial cells, and permitted simultaneous measurements of ciliary beat frequency and cAMP (represented by the FRET ratio). Apical application of forskolin (1 µM, 10 µM, 20 µM) and, in permeabilized cells, basolateral cAMP (20 µM, 50 µM, 100 µM) caused dose-dependent, albeit similar and simultaneous–increases in cAMP and ciliary beat frequency. However, decreases in cAMP preceded decreases in ciliary beat frequency, suggesting that either cellular cAMP decreases before ciliary cAMP or the dephosphorylation of target proteins by phosphatases occur at a rate slower than the rate of cAMP hydrolysis.

Key words: cAMP, Ciliary beating, Protein kinase A, Kinetics relationship, Air-liquid interface




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