Journal
JOURNAL OF CELL SCIENCE
Volume 119, Issue 20, Pages 4176-4186Publisher
COMPANY BIOLOGISTS LTD
DOI: 10.1242/jcs.03181
Keywords
cAMP; ciliary beating; protein kinase A; kinetics relationship; air-liquid interface
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Funding
- NHLBI NIH HHS [HL-070199, R01 HL060644, HL-60644, HL-67206] Funding Source: Medline
- Telethon [TCP00089] Funding Source: Medline
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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 mu M, 10 mu M, 20 mu M) and, in permeabilized cells, basolateral cAMP (20 mu M, 50 mu M, 100 mu M) 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.
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