Skip to main content
SpringerLink
Log in

Fluorescence and Laser Photon Counting: Measurements of Epithelial [Ca2+]i or [Na+]i with Ciliary Beat Frequency

  • Published:
Annals of Biomedical Engineering Aims and scope Submit manuscript

Abstract

We describe a system we developed that enabled simultaneous measurements of either epithelial calcium ion concentration ([Ca2+]i) or sodium ion concentration [Na+]i with the ciliary beat frequency (CBF) in native ciliated epithelia using either Fura-2 (AM) or SBFI (AM) ratiometric fluorescence photon counting along with nonstationary laser light scattering. Studies were performed using native epithelial tissues obtained from ovine tracheae. The dynamic range of the laser light-scattering system was determined by a simulated light “beating” experiment. The nonstationary CBF was demonstrated by the time-frequency analysis of the raw photon count sequences of backscattered heterodyne photons from cultured and native epithelia. Calibrations of calcium and sodium ion concentrations were performed using the respective Fura-2 and SBFI impermanent salts as well as in native epithelia. The cumulative responses of 10−6, 10−5, and 10−4} M nifedipine on [Ca2+]i together with the CBF as well as the cumulative responses of 10−5, 10−4, and 10−3 M amiloride on [Na+]i together with the CBF were also determined. Nifedipine decreased [Ca2+]i but had no effect on CBF. Amiloride decreased [Na+]i and CBF. Stimulation of CBF corresponded with either an increase of [Na+]i or an increase of [Ca2+]i. Decreases of [Na+]i or substantial decreases of [Ca2+]i were associated with decreases in the CBF. These data demonstrate the utility of this system for investigating the regulatory mechanisms of intracellular ions dynamics and the CBF in native epithelia. © 1998 Biomedical Engineering Society.

PAC98: 8780+s, 8722-q, 4262Be

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

REFERENCES

  1. Aiello, E., J. Kennedy, and C. Hernandez. Stimulation of frog ciliated cells in culture by acetylcholine and substance P. Comp. Biochem. Physiol.99C:497-506, 1991.

    Google Scholar 

  2. Al-Bazzaz, F., and T. Jayaram. Ion transport by canine tracheal mucosa: Effect of elevation of cellular calcium. Exp. Lung Res.2:121-130, 1981.

    Google Scholar 

  3. BenShimol, Y., I. Dinstein, A. Meisels, and Z. Priel. Ciliary motion features from digitized video photography. J. Comput.-Assist. Microsc.3:103-116, 1991.

    Google Scholar 

  4. Boitano, S., M. L. Woodruff, and E. R. Dirksen. Evidence for voltage-sensitive, calcium-conducting channels in airway epithelial cells. Am. J. Physiol.269(Cell Physiol.38):C1547-C1556, 1995.

    Google Scholar 

  5. Borzak, S., R. A. Kelly, B. K. Kramer, Y. Matoba, J. D. Marsh, and M. Reers. In situcalibration of Fura-2 and BCECF fluorescence in adult rat ventricular myocytes. Am. J. Physiol.259(Heart Circ. Physiol):H973-H981, 1990.

    Google Scholar 

  6. Busa, W. B. Spectral characterization of the effect of viscosity on Fura-2 fluorescence: Excitation wavelength optimization abolishes the viscosity artifact. Cell Calcium13:313- 319.

  7. Chandra T., D. B. Yeates, I. F. Miller, and L. B. Wong. Stationary and nonstationary correlation-frequency analysis of heterodyne mode laser light scattering: Magnitude and periodicity of canine tracheal ciliary beat frequency in vivo. Biophys. J.66:878-890, 1994.

    Google Scholar 

  8. Di Benedetto, G., C. J. Magnus, P. T. A. Gray, and A. Mehta. Calcium regulation of ciliary beat frequency in human respiratory epithelium in vitro. J. Physiol. (London)439:103-113, 1991.

    Google Scholar 

  9. Dirksen, E. R., and M. J. Sanderson. Regulation of ciliary activity in the mammalian respiratory tract. Biorheology27:533-545, 1990.

    Google Scholar 

  10. Gilon, P., and J. C. Henquin. Activation of muscarinic receptors increases the concentration of free Na+ in mouse pancreatic B-cells. Fed. Euro. Biochem. Soc. 315:353-356, 1993.

    Google Scholar 

  11. Grynkiewicz, G., M. Poenie, and R. Y. Tsien. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem.260:3440-3450, 1985.

    Google Scholar 

  12. Harris, R. A., and J. W. Hanrahan. Effects of EGTA on calcium signaling in airway epithelial cells. Am. J. Physiol.267 (Cell Physiol. 36):C1426-C1434, 1994.

    Google Scholar 

  13. Kao, J. P. Y. Practical aspects of measuring [Ca2+]iwith fluorescent indicators. In: Methods in Cell Biology: A practical guide to the study of calcium in living cells, Vol. 40, edited by R. Nuccitelli. San Diego, CA: Academic, 1994, pp. 155-181.

    Google Scholar 

  14. Korngreen, A., and Z. Priel. Simultaneous measurement of ciliary beating and intracellular calcium. Biophys. J.67:377- 380, 1994.

    Google Scholar 

  15. Korngreen, A., and Z. Priel. Purinergic stimulation of rabbit ciliated airway epithelia: Control by multiple calcium stores. J. Physiol. (London)497.1:53-66, 1996.

    Google Scholar 

  16. Lansley, A. B., M. J. Sanderson, and E. R. Dirksen. Control of the beat cycle of respiratory tract cilia by Ca2+ and CAMP. Am. J. Physiol.263(Lung Cell Mol. Physiol. 7):L232-L242, 1992.

    Google Scholar 

  17. Mao, H., and L. B. Wong. Depolarization of cell membrane is associated with an increase in ciliary beat frequency (CBF). Biochem. Biophys. Res. Commun.215:1014-1021, 1995.

    Google Scholar 

  18. Mason, S. J., A. M. Paeadiso, and R. C. Boucher. Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airways epithelium. Br. J. Pharmacol. 103:1649-1656, 1991.

    Google Scholar 

  19. Minta, A., and R. Y. Tsien. Fluorescent indicators for cytosolic sodium. J. Biol. Chem.264:19,449-19,457, 1989.

    Google Scholar 

  20. Moore, E. D. W., P. L. Beckerm, K. E. Fogarty, D. A. Williams, and F. S. Fay. Ca2+ imaging in single living cells: Theoretical and practical issues. Cell Calcium11:157-179, 1990.

    Google Scholar 

  21. Negulescu, P. A., and T. E. Machen. Intracellular ion activities and membrane transport in parietal cells measured with fluorescent dyes. Methods Enzymol. 192:38-81, 1990.

    Google Scholar 

  22. Nilius, B., G. Schwartz, and G. Droogmans. Control of intracellular calcium by membrane potential in human melanoma cells. Am. J. Physiol.(Cell Physiol.) 34:C1501-C1510, 1993.

    Google Scholar 

  23. Romet, S., D. Schoevaert, and F. Marano. Dynamic image analysis applied to the study of ciliary beat on cultured ciliated epithelial cells from rabbit trachea. Cell Biol. 71:183- 190, 1991.

    Google Scholar 

  24. Tornquist, K., and E. Ekokoski. Intracellular free sodium concentrations in GH4Cl cells. J. Cell Physiol.154:608-614, 1993.

    Google Scholar 

  25. Uematsu, D., J. H. Greenberg, M. Reivich, S. Kobayashi, and A. Karp. In vivofluormetric measurement of changes in cytosolic free calcium from the cat cortex during anoxia. J. Cerebral Blood Flow Metabol. 8:367-374, 1988.

    Google Scholar 

  26. Uto, A., H. Arai, and Y. Ogawa. Reassessment of Fura-2 and the ratio method for determination of intracellular Ca2+concentrations. Cell Calcium12:29-37, 1991.

    Google Scholar 

  27. Villalon, M., T. R. Hinds, and P. Verdugo. Stimulus-response coupling in mammalian ciliated cells: Demonstration of two mechanisms of control for cytosolic [Ca2+]i. Biophys. J.56:1255-1258, 1989.

    Google Scholar 

  28. Wong, L. B., I. F. Miller, and D. B. Yeates. Stimulation of ciliary beat frequency by autonomic agonists: In vivo. J. Appl. Physiol. 65:971-981, 1988.

    Google Scholar 

  29. Wong, L. B., and D. B. Yeates. Dynamics of the regulation of ciliated epithelial function. Comments Theoretical Biol. V4:183-208, 1997.

    Google Scholar 

  30. Yeates, D. B., G. Besseries, and L. B. Wong. Physiochemical properties of mucus and its propulsion. In: The Lung, Scientific Foundations, 2nd ed., Chap. 33, edited by R. G. Crystal, J. B. West, P. Barnes, N. S. Chernick, and E. R. Weibel. Philadelphia: Lippincott-Raven, 1996, pp. 487-503.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Pulmonary Biophysics and Bioengineering Research Laboratory, Department of Medicine and Bioengineering Program, University of Illinois at Chicago, Chicago, IL

    Hua Mao & Lid B. Wong

Authors
  1. Hua Mao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lid B. Wong
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mao, H., Wong, L.B. Fluorescence and Laser Photon Counting: Measurements of Epithelial [Ca2+]i or [Na+]i with Ciliary Beat Frequency. Annals of Biomedical Engineering 26, 666–678 (1998). https://doi.org/10.1114/1.124

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1114/1.124

Search

Navigation