Real-time analysis of cAMP-mediated regulation of ciliary motility in single primary human airway epithelial cells.
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 microM, 10 microM, 20 microM) and, in permeabilized cells, basolateral cAMP (20 microM, 50 microM, 100 microM) 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.
Schmid
Andreas
A
Division of Pulmonary and Critical Care Medicine, University of Miami School of Medicine, 1600 NW 10th Avenue, RMSB 7063, Miami, FL 33136, USA.
Bai
Ge
G
Schmid
Nathalie
N
Zaccolo
Manuela
M
Ostrowski
Lawrence E
LE
Conner
Gregory E
GE
Fregien
Nevis
N
Salathe
Matthias
M
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects*; D002923:Cilia / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer; D051858:Forkhead Transcription Factors / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008168:Lung / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors
C105593:8-bromoadenosine-3',5'-cyclic monophosphorothioate; C106349:FOXJ1 protein, human; D051858:Forkhead Transcription Factors; D007546:Isoquinolines; D008164:Luminescent Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D013449:Sulfonamides; D013873:Thionucleotides; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide