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

Andreas Schmid, Ge Bai, Nathalie Schmid, Manuela Zaccolo, Lawrence E. Ostrowski, Gregory E. Conner, Nevis Fregien, Matthias Salathe

Research output: Contribution to journalArticle

55 Scopus citations

Abstract

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 (I 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.

Original languageEnglish (US)
Pages (from-to)4176-4186
Number of pages11
JournalJournal of Cell Science
Volume119
Issue number20
DOIs
StatePublished - Oct 15 2006

Keywords

  • Air-liquid interface
  • cAMP
  • Ciliary beating
  • Kinetics relationship
  • Protein kinase A

ASJC Scopus subject areas

  • Cell Biology

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