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 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.

[1]  T. Lieb,et al.  Prolonged increase in ciliary beat frequency after short‐term purinergic stimulation in human airway epithelial cells , 2002, The Journal of physiology.

[2]  M. Sanderson,et al.  Control of the beat cycle of respiratory tract cilia by Ca2+ and cAMP. , 1992, The American journal of physiology.

[3]  G. Mellgren,et al.  Novel (Rp)-cAMPS Analogs as Tools for Inhibition of cAMP-kinase in Cell Culture , 1995, The Journal of Biological Chemistry.

[4]  M. Sanderson,et al.  Oscillations in ciliary beat frequency and intracellular calcium concentration in rabbit tracheal epithelial cells induced by ATP , 2003, The Journal of physiology.

[5]  T. Hamasaki,et al.  cAMP-stimulated phosphorylation of an axonemal polypeptide that copurifies with the 22S dynein arm regulates microtubule translocation velocity and swimming speed in Paramecium. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[6]  M. Kondo,et al.  Adenosine-mediated cyclic AMP-dependent inhibition of ciliary activity in rabbit tracheal epithelium. , 1989, The American review of respiratory disease.

[7]  Susan S. Taylor,et al.  Fluorescence ratio imaging of cyclic AMP in single cells , 1991, Nature.

[8]  P. Verdugo,et al.  beta-Adrenergic stimulation of respiratory ciliary activity. , 1980, Journal of applied physiology: respiratory, environmental and exercise physiology.

[9]  T. Wyatt,et al.  Both cAMP and cGMP are required for maximal ciliary beat stimulation in a cell-free model of bovine ciliary axonemes. , 2005, American journal of physiology. Lung cellular and molecular physiology.

[10]  O. Danos,et al.  Regeneration of a well‐differentiated human airway surface epithelium by spheroid and lentivirus vector‐transduced airway cells , 2004, The journal of gene medicine.

[11]  J. W. Karpen,et al.  Review Article: Cyclic AMP Sensors in Living Cells: What Signals Can They Actually Measure? , 2002, Annals of Biomedical Engineering.

[12]  G. Conner,et al.  Regulation of human airway ciliary beat frequency by intracellular pH , 2004, The Journal of physiology.

[13]  J. Schaack,et al.  In Vivo Assessment of Local Phosphodiesterase Activity Using Tailored Cyclic Nucleotide–Gated Channels as Camp Sensors , 2001, The Journal of general physiology.

[14]  G. Di Benedetto,et al.  Effect of cyclic AMP on ciliary activity of human respiratory epithelium. , 1991, The European respiratory journal.

[15]  L. Naldini,et al.  Transduction of a gene expression cassette using advanced generation lentiviral vectors. , 2002, Methods in enzymology.

[16]  M J Sanderson,et al.  Mechanosensitive and beta-adrenergic control of the ciliary beat frequency of mammalian respiratory tract cells in culture. , 1989, The American review of respiratory disease.

[17]  S. Randell,et al.  Mucin gene expression during differentiation of human airway epithelia in vitro. Muc4 and muc5b are strongly induced. , 1999, American journal of respiratory cell and molecular biology.

[18]  Tullio Pozzan,et al.  Discrete Microdomains with High Concentration of cAMP in Stimulated Rat Neonatal Cardiac Myocytes , 2002, Science.

[19]  Zvi Priel,et al.  Distinct Axonemal Processes Underlie Spontaneous and Stimulated Airway Ciliary Activity , 2002, The Journal of general physiology.

[20]  Scott H Randell,et al.  Transcellular thiocyanate transport by human airway epithelia , 2004, The Journal of physiology.

[21]  Xiaodong Cheng,et al.  Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[22]  T. Mccaffrey,et al.  Dual signal transduction mechanisms modulate ciliary beat frequency in upper airway epithelium. , 1996, The American journal of physiology.

[23]  R. Pickles.  Physical and biological barriers to viral vector-mediated delivery of genes to the airway epithelium. , 2004, Proceedings of the American Thoracic Society.

[24]  Intracellular Ca2+ Regulates the Phosphorylation and the Dephosphorylation of Ciliary Proteins Via the NO Pathway , 2004, The Journal of general physiology.

[25]  M. Salathe,et al.  Mode of Ca2+ action on ciliary beat frequency in single ovine airway epithelial cells , 1999, The Journal of physiology.

[26]  M J Sanderson,et al.  Regulation of airway ciliary activity by Ca2+: simultaneous measurement of beat frequency and intracellular Ca2+. , 1999, Biophysical journal.

[27]  A. Mehta,et al.  Asymmetric interactions between phosphorylation pathways regulating ciliary beat frequency in human nasal respiratory epithelium in vitro. , 1996, The Journal of physiology.

[28]  M. Kondo,et al.  Effect of cAMP on ciliary function in rabbit tracheal epithelial cells. , 1989, Journal of applied physiology.

[29]  Martin J. Lohse,et al.  Novel Single Chain cAMP Sensors for Receptor-induced Signal Propagation*♦ , 2004, Journal of Biological Chemistry.

[30]  S. Brody,et al.  Role of f-box factor foxj1 in differentiation of ciliated airway epithelial cells. , 2004, American journal of physiology. Lung cellular and molecular physiology.

[31]  Lawrence E Ostrowski,et al.  Characterization of an A-kinase anchoring protein in human ciliary axonemes. , 2002, Molecular biology of the cell.

[32]  T. Wyatt,et al.  Regulation of ciliary beat frequency by both PKA and PKG in bovine airway epithelial cells. , 1998, American journal of physiology. Lung cellular and molecular physiology.

[33]  G. Conner,et al.  Regulator of G-protein signaling protein 2 modulates purinergic calcium and ciliary beat frequency responses in airway epithelia. , 2002, American journal of respiratory cell and molecular biology.

[34]  Susan S. Taylor,et al.  A genetically encoded, fluorescent indicator for cyclic AMP in living cells , 1999, Nature Cell Biology.

[35]  M. Lohse,et al.  Real-time Monitoring of the PDE2 Activity of Live Cells , 2005, Journal of Biological Chemistry.

[36]  M. Salathe,et al.  Effects of albuterol enantiomers on ciliary beat frequency in ovine tracheal epithelial cells. , 2002, Journal of applied physiology.

[37]  C. W. Davis,et al.  Differential effects of UTP, ATP, and adenosine on ciliary activity of human nasal epithelial cells. , 2001, American journal of physiology. Cell physiology.

[38]  A. Wanner,et al.  Cyclic AMP-dependent phosphorylation of a 26 kD axonemal protein in ovine cilia isolated from small tissue pieces. , 1993, American journal of respiratory cell and molecular biology.

[39]  T. Hamasaki,et al.  In vitro phosphorylation of Paramecium axonemes and permeabilized cells. , 1989, Cell motility and the cytoskeleton.

[40]  P. Cohen,et al.  Specificity and mechanism of action of some commonly used protein kinase inhibitors. , 2000, The Biochemical journal.

[41]  L. Ostrowski,et al.  Targeting expression of a transgene to the airway surface epithelium using a ciliated cell-specific promoter. , 2003, Molecular therapy : the journal of the American Society of Gene Therapy.