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

• Published in: Journal of cell science Vol. 119; no. 20; pp. 4176 - 4186
• Main Authors: Schmid, Andreas, Bai, Ge, Schmid, Nathalie, Zaccolo, Manuela, Ostrowski, Lawrence E, Conner, Gregory E, Fregien, Nevis, Salathe, Matthias
• Format: Journal Article
• Language: English
• Published: England The Company of Biologists Limited 15.10.2006
• Subjects: 8-Bromo Cyclic Adenosine Monophosphate - analogs & derivatives; 8-Bromo Cyclic Adenosine Monophosphate - pharmacology; Cell Line, Tumor; Cell Movement - drug effects; Cilia - drug effects; Cilia - metabolism; Cilia - physiology; Colforsin - pharmacology; Cyclic AMP - metabolism; Cyclic AMP - pharmacology; Cyclic AMP-Dependent Protein Kinases - antagonists & inhibitors; Cyclic AMP-Dependent Protein Kinases - genetics; Cyclic AMP-Dependent Protein Kinases - metabolism; Dose-Response Relationship, Drug; Epithelial Cells - drug effects; Epithelial Cells - metabolism; Epithelial Cells - physiology; Fluorescence Resonance Energy Transfer; Forkhead Transcription Factors - genetics; Humans; Immunohistochemistry; Isoquinolines - pharmacology; Kinetics; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Lung - metabolism; Lung - pathology; Phosphorylation - drug effects; Promoter Regions, Genetic - genetics; Protein Binding - drug effects; Protein Subunits - antagonists & inhibitors; Protein Subunits - genetics; Protein Subunits - metabolism; Recombinant Fusion Proteins - metabolism; Sulfonamides - pharmacology; Thionucleotides - pharmacology; Time Factors
• ISSN: 0021-9533; 1477-9137
• DOI: 10.1242/jcs.03181

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.