Pflügers Archiv - European Journal of Physiology

, Volume 464, Issue 6, pp 671–680 | Cite as

Methods to measure and analyze ciliary beat activity: Ca2+ influx-mediated cilia mechanosensitivity

  • Wen-Er Li
  • Weiwei Chen
  • Yun-Fei Ma
  • Qing-Rong Tuo
  • Xiao-Jing Luo
  • Ting Zhang
  • Wen-Bo Sai
  • Jing Liu
  • Jinhua Shen
  • Zhi-Gang Liu
  • Yun-Min Zheng
  • Yong-Xiao Wang
  • Guangju Ji
  • Qing-Hua Liu
Signaling and Cell Physiology

Abstract

Airway ciliary beat activity (CBA) plays a pivotal role in protecting the body by removing mucus and pathogens from the respiratory tract. Since CBA is complicated and cannot be characterized by merely frequency, we recorded CBA using laser confocal line scanning and defined six parameters for describing CBA. The values of these parameters were all above 0 when measured in beating ciliated cells from mouse tracheae. We subsequently used 10 μM adenosine-5′-triphosphate (ATP) to stimulate ciliated cells and simultaneously recorded intracellular Ca2+ levels and CBA. We found that intracellular Ca2+ levels first increased, followed by an increase in CBA. Among the six parameters, frequency, amplitude, and integrated area significantly increased, whereas rise time, decay time, and full duration at half maximum markedly decreased. The results suggest that these six parameters are appropriate for assessing CBA and that increased intracellular Ca2+ levels might enhance CBA. We next used our established methods to observe changes in mechanically stimulated cilia tips. We found that mechanical stimulation-induced changes in both intracellular Ca2+ levels and CBA were not only similar to those induced by ATP, but were also blocked by treatment with a Ca2+ chelator, BAPTA-AM, (10 μM) for 10 min. Moreover, while the same blockage was observed under Ca2+-free conditions, addition of 2 mM Ca2+ into the chamber restored increases in both intracellular Ca2+ levels and CBA. Taken together, we have provided a novel method for real-time measurement and complete analysis of CBA as well as demonstrated that mechanical stimulation of cilia tips resulted in Ca2+ influx that led to increased intracellular Ca2+ levels, which in turn triggered CBA enhancement.

Keywords

Ciliated cells Ciliary beat activity Intracellular Ca2+ Mechanical stimulation 

References

  1. 1.
    Dalhamn T (1955) A method for determination in vivo of the rate of ciliary beat and mucous flow in the trachea. Acta Physiol Scand 33:1–5PubMedCrossRefGoogle Scholar
  2. 2.
    Dalhamn T (1960) The determination in vivo of the rate of ciliary beat in the trachea. Acta Physiol Scand 49:242–250PubMedCrossRefGoogle Scholar
  3. 3.
    Doyle RT, Moninger T, Debavalya N, Hsu WH (2006) Use of confocal linescan to document ciliary beat frequency. J Microsc 223:159–164PubMedCrossRefGoogle Scholar
  4. 4.
    Evans JH, Sanderson MJ (1999) Intracellular calcium oscillations regulate ciliary beat frequency of airway epithelial cells. Cell Calcium 26:103–110PubMedCrossRefGoogle Scholar
  5. 5.
    Geary CA, Davis CW, Paradiso AM, Boucher RC (1995) Role of CNP in human airways: cGMP-mediated stimulation of ciliary beat frequency. Am J Physiol 268:L1021–L1028PubMedGoogle Scholar
  6. 6.
    Kakuta Y, Kanno T, Sasaki H, Takishima T (1985) Effect of Ca2+ on the ciliary beat frequency of skinned dog tracheal epithelium. Respir Physiol 60:9–19PubMedCrossRefGoogle Scholar
  7. 7.
    Korngreen A, Priel Z (1996) Purinergic stimulation of rabbit ciliated airway epithelia: control by multiple calcium sources. J Physiol 497(Pt 1):53–66PubMedGoogle Scholar
  8. 8.
    Lansley AB, Sanderson MJ (1999) Regulation of airway ciliary activity by Ca2+: simultaneous measurement of beat frequency and intracellular Ca2+. Biophys J 77:629–638PubMedCrossRefGoogle Scholar
  9. 9.
    Lorenzo IM, Liedtke W, Sanderson MJ, Valverde MA (2008) TRPV4 channel participates in receptor-operated calcium entry and ciliary beat frequency regulation in mouse airway epithelial cells. Proc Natl Acad Sci USA 105:12611–12616PubMedCrossRefGoogle Scholar
  10. 10.
    Ma W, Silberberg SD, Priel Z (2002) Distinct axonemal processes underlie spontaneous and stimulated airway ciliary activity. J Gen Physiol 120:875–885PubMedCrossRefGoogle Scholar
  11. 11.
    Ma W, Korngreen A, Uzlaner N, Priel Z, Silberberg SD (1999) Extracellular sodium regulates airway ciliary motility by inhibiting a P2X receptor. Nature 400:894–897PubMedCrossRefGoogle Scholar
  12. 12.
    Moore WD, Engelmann TW (1869) On ciliary motion. J Anat Physiol 3:420–435PubMedGoogle Scholar
  13. 13.
    Nlend MC, Bookman RJ, Conner GE, Salathe M (2002) Regulator of G-protein signaling protein 2 modulates purinergic calcium and ciliary beat frequency responses in airway epithelia. Am J Respir Cell Mol Biol 27:436–445PubMedGoogle Scholar
  14. 14.
    Porter ME, Sale WS (2000) The 9 + 2 axoneme anchors multiple inner arm dyneins and a network of kinases and phosphatases that control motility. J Cell Biol 151:F37–F42PubMedCrossRefGoogle Scholar
  15. 15.
    Salathe M, Bookman RJ (1999) Mode of Ca2+ action on ciliary beat frequency in single ovine airway epithelial cells. J Physiol 520(Pt 3):851–865PubMedCrossRefGoogle Scholar
  16. 16.
    Sanderson MJ, Dirksen ER (1986) Mechanosensitivity of cultured ciliated cells from the mammalian respiratory tract: implications for the regulation of mucociliary transport. Proc Natl Acad Sci USA 83:7302–7306PubMedCrossRefGoogle Scholar
  17. 17.
    Sanderson MJ, Lansley AB, Dirksen ER (1992) Regulation of ciliary beat frequency in respiratory tract cells. Chest 101:69S–71SPubMedGoogle Scholar
  18. 18.
    Verdugo P (1980) Ca2+-dependent hormonal stimulation of ciliary activity. Nature 283:764–765PubMedCrossRefGoogle Scholar
  19. 19.
    Wirschell M, Yamamoto R, Alford L, Gokhale A, Gaillard A et al (2011) Regulation of ciliary motility: conserved protein kinases and phosphatases are targeted and anchored in the ciliary axoneme. Arch Biochem Biophys 510:93–100PubMedCrossRefGoogle Scholar
  20. 20.
    Zhang L, Sanderson MJ (2003) Oscillations in ciliary beat frequency and intracellular calcium concentration in rabbit tracheal epithelial cells induced by ATP. J Physiol 546:733–749PubMedCrossRefGoogle Scholar
  21. 21.
    Zhang L, Sanderson MJ (2003) The role of cGMP in the regulation of rabbit airway ciliary beat frequency. J Physiol 551:765–776PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Wen-Er Li
    • 1
  • Weiwei Chen
    • 1
  • Yun-Fei Ma
    • 1
  • Qing-Rong Tuo
    • 1
  • Xiao-Jing Luo
    • 1
  • Ting Zhang
    • 1
  • Wen-Bo Sai
    • 1
  • Jing Liu
    • 1
  • Jinhua Shen
    • 1
  • Zhi-Gang Liu
    • 2
  • Yun-Min Zheng
    • 3
  • Yong-Xiao Wang
    • 3
  • Guangju Ji
    • 4
  • Qing-Hua Liu
    • 1
  1. 1.Institute for Medical Biology, College of Life SciencesSouth-Central University for NationalitiesWuhanChina
  2. 2.School of MedicineShenzhen UniversityShenzhenChina
  3. 3.Center for Cardiovascular SciencesAlbany Medical CollegeAlbanyUSA
  4. 4.National Laboratory of Biomacromolecules, Institute of BiophysicsChinese Academy of SciencesBeijingChina

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