Measuring Cytoplasmic Calcium in Single Living Cells Using Fluorescent Probes

  • Ron Jacob
  • Christopher D. Benham
Chapter
Part of the Topics in Molecular and Structural Biology book series (TMSB)

Abstract

Calcium is an important intracellular second messenger in that it often links events occurring at the cell surface, such as a membrane potential depolarization or the binding of a hormone to a receptor, with events occurring within the cell, such as activation of the contractile apparatus or secretion. The importance of calcium was recognized long before it was possible to measure it at the very low concentrations that occur intracellularly, but an improved understanding of its role developed when its direct measurement became feasible. However, the earlier techniques for measuring cytoplasmic free calcium ion concentration ([Ca2+]j) not only were technically difficult, but also were confined to use with large cells (e.g. squid giant axon or large muscle cells) because of the need to introduce some form of micropipette into the cell, either to microinject the indicator or as part of a Ca2+-sensitive microelectrode (but see Campbell et al., 1985, for a review of alternative strategies for introducing photoproteins into cells). The introduction of the fluorescent indicators has overcome these technical difficulties, resulting in their widespread use. A very good comprehensive review of optical indicators of [Ca2+] is Blinks et al (1982), although it mentions only the first of the fluorescent indicators (quin2).

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References

  1. Almers, W. and Neher, E. (1985). The Ca signal from fura-2 loaded mast cells depends strongly on the method of dye-loading. FEBS Lett., 192, 13–18CrossRefPubMedGoogle Scholar
  2. Barcenas-Ruiz, L. and Weir, W.G. (1987). Voltage dependence of intracellular [Ca2+]; transients in guinea-pig ventricular myocytes. Circ. Res., 61, 148–154CrossRefPubMedGoogle Scholar
  3. Baylor, S.M. and Hollingworth, S. (1988). Fura-2 calcium transients in frog skeletal muscle fibres. J. Physiol, 403, 151–192PubMedCentralCrossRefPubMedGoogle Scholar
  4. Becker, P.L. and Fay, F.S. (1987). Photobleaching of fura-2 and its effect on determination of calcium concentrations. Am. J. Physiol., 253, C613–C618Google Scholar
  5. Benham, CD. (1989). Voltage-gated and agonist mediated rises in intracellular Ca in rat clonal pituitary cells (GH3) held under voltage clamp. J. Physiol, 415, 145–158CrossRefGoogle Scholar
  6. Bioprobes (1988). Ajournai published by Molecular Probes Inc., P.O. Box 22010, Eugene, OR 97402, USAGoogle Scholar
  7. Blinks, J.R., Wier, W.G., Hess, P. and Prendergast, F.G. (1982). Measurement of Ca2+ concentrations in living cells. Prog. Biophys. Molec. Biol, 40, 1–114CrossRefGoogle Scholar
  8. Bright, G.R., Fisher, G.W., Rogowska, J. and Taylor, D.L. (1987). Fluorescence ratio imaging microscopy: Temporal and spatial measurement of cytoplasmic pH. J. Cell Biol, 104, 1019– 1033Google Scholar
  9. Campbell, A.K., Dormer, R.L. and Halle, M.B. (1985), Coelenterate photoproteins as indicators of cytoplasmic free Ca2+ in small cells. Cell Calcium, 6, 69–82Google Scholar
  10. Cannell, M.B., Berlin, J.R. and Lederer, W.J. (1987). Effect of membrane potential changes on the calcium transient in single rat cardiac muscle cells. Science, N.Y., 238, 1419–1423CrossRefGoogle Scholar
  11. Cobbold, P.H. and Bourne, P.K. (1984). Aequorin measurements of free calcium in single heart cells. Nature, 312, 444–446CrossRefPubMedGoogle Scholar
  12. Cobbold, P.H. and Rink, T.J. (1987). Fluorescence and bioluminescence measurement of cytoplasmic free calcium. Biochem. J., 248, 313–328PubMedCentralCrossRefPubMedGoogle Scholar
  13. DiVirgilio, F., Steinberg, T.H., Swanson, J.A. and Silverstein, S.A. (1988). Fura-2 secretion and sequestration in macrophages. J. Immunol., 140, 915–920Google Scholar
  14. Foskett, J.K. (1988). Simultaneous Nomarski and fluorescence imaging during video microscopy of cells. Am. J. Physiol, 255, C566–C571Google Scholar
  15. Gray, P.T.A. (1988). Oscillations of free cytosolic calcium evoked by cholinergic and catecholaminergic agonists in rat parotid acinar cells. J. Physiol, 406, 35–53PubMedCentralCrossRefPubMedGoogle Scholar
  16. Grynkiewicz, G, Poenie, M. and Tsien, R.Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem., 260, 3440–3450PubMedGoogle Scholar
  17. Gunter, T.E., Restrepo, D. and Gunter, K.K. (1988). Conversion of esterified fura-2 and indo-1 to Ca2+-sensitive forms by mitochondria. Am. J. Physiol, 255, C304–C310Google Scholar
  18. Gurney, A.M. and Lester, H.A. (1987). Light flash physiology with synthetic photosensitive compounds. Physiol. Rev., 67, 583–617PubMedGoogle Scholar
  19. Hallam, T.J., Pearson, J.D. and Needham, L.A. (1988). Thrombin-stimulated elevation of human endothelial-cell cytoplasmic free calcium concentration causes prostacyclin production. Biochem. J., 251, 243–249PubMedCentralCrossRefPubMedGoogle Scholar
  20. Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J. (1981). Improved patchclamp techniques for high resolution current recording from cells and cell free membrane patches. Pflügers Arch., 381, 85–100CrossRefGoogle Scholar
  21. Harootunian, A.T., Kao, J.P.Y. and Tsien, R.Y. (1988). Agonist-induced calcium oscillations in depolarized fibroblasts and their manipulation by photoreleased Ins(l,4,5)P3, Ca2+, and Ca2+ buffer. Cold Spring Harbor Symp., 53 (in press)Google Scholar
  22. Illsley, N.P. and Verkman, A.S. (1987). Membrane chloride transplant measured using a chloride-sensitive fluorescent probe. Biochemistry, 26, 1215–1219CrossRefPubMedGoogle Scholar
  23. Jackson, A.P., Timmerman, M.P., Bagshaw, C.R. and Ashley, C.C. (1987). The kinetics of calcium binding to fura-2 and indo-1. FEBS Lett., 216, 35–39CrossRefPubMedGoogle Scholar
  24. Jacob, R. (1990). Imaging cytoplasmic free calcium in histamine stimulated endothelial cells and in fMet-Leu-Phe stimulated neutrophils. Cell Calcium, 11, 241–249CrossRefPubMedGoogle Scholar
  25. Jacob, R., Merritt, J.E., Hallam, T.J. and Rink, T.J. (1988). Repetitive spikes in cytoplasmic calcium evoked by histamine in human endothelial cells. Nature, 335, 40–45CrossRefPubMedGoogle Scholar
  26. Kao, J.P.Y. and Tsien, R.Y. (1988). Ca2+-binding kinetics of fura-2 and azo-1 from temperature-jump relaxation measurements. Biophys. J., 53, 635–640PubMedCentralCrossRefPubMedGoogle Scholar
  27. Kassotis, J., Steinberg, S.F., Ross, S., Bilezikian, J.P. and Robinson, R.B. (1987). An inexpensive dual-excitation apparatus for fluorescence microscopy. Pflügers Arch., 409, 47–51CrossRefPubMedGoogle Scholar
  28. Keating, S. M, Wensel, T.G., Meyer, T. and Stryer, L. (1989). Nanosecond fluorescence and emission anisotropy kinetics of fura-2 in single cells. Biophys. J., 55, 518aGoogle Scholar
  29. Klein, M.G., Simon, B.J., Szucs, G. and Schneider, M.F. (1988). Simultaneous recording of calcium transients in skeletal muscle using high-and low-affinity calcium indicators. Biophys. J., 53, 971–988PubMedCentralCrossRefPubMedGoogle Scholar
  30. Konishi, M., Olson, A., Hollingworth, S. and Baylor, S.M. (1988). Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements. Biophys. J., 54, 1089–1104PubMedCentralCrossRefPubMedGoogle Scholar
  31. Kurtz, I. and Balaban, R.S. (1985). Fluorescence emission spectroscopy of 1,4-dihydroxyphtha-lonitrile. A method for determining intracellular pH in cultured cells. Biophys. J., 48, 499–508PubMedCentralCrossRefPubMedGoogle Scholar
  32. Kurtz, I. and Katzka, P. (1988), Rapid scanning excitation microfluorometry using an acoustooptic tunable filter. Biophys. J., 53, 197aGoogle Scholar
  33. Lemasters, J.L., DiGuseppi, J., Nieminen, A.-L. and Herman, B. (1987). Blebbing, free Ca2+ and mitochondrial membrane potential preceding cell death in hepatocytes. Nature, 325, 78– 81Google Scholar
  34. Lindau, M. and Neher, E. (1988). Patch-clamp techniques for time resolved capacitanceGoogle Scholar
  35. measurements in single cells. Pflügers Arch., 411, 137–146Google Scholar
  36. Lückhoff, A. (1986). Measuring cytosolic free calcium concentration in endothelial cells with indo-1: the pitfall of using the ratio of two fluorescence intensities recorded at different wavelengths. Cell Calcium, 7, 233–248CrossRefPubMedGoogle Scholar
  37. Lukacs, G.L. and Kapus, A. (1987). Measurement of the matrix free Ca2+ concentration in heart mitochondria by entrapped fura-2 and quin2. Biochem. J., 248, 609–613PubMedCentralCrossRefPubMedGoogle Scholar
  38. Malgaroli, A., Milani, D., MeIdolesi, J. and Pozzan, T. (1987). Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells. J. Cell Biol, 105, 2145–2155CrossRefPubMedGoogle Scholar
  39. Minta, A., Harootunian, A.T., Kao, J.P.Y. and Tsien, R.Y. (1987). New fluorescent indicators for intracellular sodium and calcium. J. Cell Biol., 105, 89aGoogle Scholar
  40. Moore, E.D.W., Minta, A., Tsien, R.Y. and Fay, F.S. (1989). Measurement of intracellular [Na+], with SBFI, and Na+ regulation in smooth muscle cells. Biophys. J., 55, 471aGoogle Scholar
  41. Murphy, E., Freudenrich, C. C, Levy, L.A., London, R.E. and Lieberman, M. (1989). Monitoring cytosolic free magnesium in cultured chick heart cells using a new fluorescent indicator, FURAPTRA. Proc. Natl Acad. Sei. USA, 86, 2981–2984CrossRefGoogle Scholar
  42. Murphy, E., Jacob, R. and Lieberman, M. (1985). Cytosolic free calcium in chick heart cells. Its role in cell injury. J. Molec. Cell Cardiol, 17, 221–231CrossRefGoogle Scholar
  43. Negulescu, P.A., Harootunian, A., Minta, A., Tsien, R.Y. and Machen, T.E. (1988). Intracellular sodium regulation in rabbit gastric glands determined using a fluorescent sodium indicator. J. Gen. Physiol, 92, 26aGoogle Scholar
  44. Paradiso, A.M., Tsien, R.Y. and Machen, T.E. (1987). Digital image processing of intracellular pH in gastric oxyntic and chief cells. Nature, 325, 447–450CrossRefPubMedGoogle Scholar
  45. Peeters, G.A., Hlady, V., Bridge, J.H.B. and Barry, W.H. (1987). Simultaneous measurement of calcium transients and motion in cultured heart cells. Am. J. Physiol, 253, H1400–H1408Google Scholar
  46. Penner, R. (1988). Multiple signalling pathways control stimulus-secretion coupling in rat peritoneal mast cells. Proc. Natl Acad. Sei. USA, 85, 9856–9860Google Scholar
  47. Poenie, M., Tsien, R.Y. and Schmitt-Verhulst, A.-M. (1987). Sequential activation and lethal hit measured by [Ca2+l in individual cytolytic T cells and targets. EMBO J., 6, 2223–2232PubMedCentralPubMedGoogle Scholar
  48. Popov, E.G., Gavrilov, I.Y., Pozin, E.Y. and Gabbbasov, Z.A. (1988). Multiwavelength method for measuring concentration of free cytosolic calcium using the fluorescent probe indo-1. Arch. Biochem. Biophys., 261, 91–96CrossRefPubMedGoogle Scholar
  49. Raju, B., Murphy, E., Levy, L.A., Hall, R.D. and London, R.E. (1989). A fluorescent indicator for measuring cytosolic free magnesium. Am. J. Physiol., 256, C54O–C548Google Scholar
  50. Rapp, G. and Guth, K. (1988). A low cost high intensity flash device for photolysis experiments. Pflügers Arch., 411, 200–203CrossRefPubMedGoogle Scholar
  51. Rink, T.J., Tsien, R.Y. and Pozzan, T. (1982). Cytoplasmic pH and free Mg2+ in lymphocytes. J. Cell Biol, 95, 189–196CrossRefPubMedGoogle Scholar
  52. Scanlon, M., Williams, D.A. and Fay, F.S. (1987). A Ca2+-insensitive form of fura-2 associated with polymorphonuclear leukocytes. J. Biol. Chem., 262, 6308–6312PubMedGoogle Scholar
  53. Simpson, A.W.M. and Rink, T.J. (1987). Elevation of pH; is not an essential step in calcium mobilisation in fura-2 loaded human platelets. FEBS Lett., 222, 144–148CrossRefPubMedGoogle Scholar
  54. Smith, G.A., Hesketh, T.R. and Metcalfe, J.C. (1988). Design and properties of a fluorescent indicator of intracellular free Na+ concentration. Biochem. J., 250, 227–232PubMedCentralCrossRefPubMedGoogle Scholar
  55. Spring, K.R. and Lowy, R.J. (1989). Characteristics of low light level television cameras. Meth. Cell Biol, 29, 269–289CrossRefGoogle Scholar
  56. Spring, K.R. and Smith, P.D. (1987). Illumination and detection systems for quantitative fluorescence microscopy. J. Microsc, >147, 265–278Google Scholar
  57. Steinberg, S.F., Bilezikian, J.P. and Al-Awqati, Q. (1987). Fura-2 fluorescence is localized to mitochondria in endothelial cells. Am. J. Physiol, 253, C744–C747Google Scholar
  58. Tank, D.W., Sugimori, M., Connor, J.A. and Llinas, R.R. (1988). Spatially resolved calcium dynamics of mammalian Purkinje cells in cerebellar slice. Science, N.Y., 242, 773–777CrossRefGoogle Scholar
  59. Thayer, S.A., Sturek, M. and Miller, R.J. (1988). Measurement of neuronal Ca transients using simultaneous microfluorimetry and electrophysiology. Pflügers Arch., 412, 216–223CrossRefPubMedGoogle Scholar
  60. Thomas, J.A., Buchsbaum, R.N., Zimniak, A. and Racker, E. (1979). Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopie probes generated in situ. Biochemistry, 18, 2210–2218CrossRefPubMedGoogle Scholar
  61. Timmerman, M.P. and Ashley, C.C. (1986). Fura-2 diffusion and its use as an indicator of transient free calcium changes in single striated muscle cells. FEBS Lett., 209, 1–8CrossRefPubMedGoogle Scholar
  62. Tsien, R.Y. (1980). New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry, 19, 2396– 2404Google Scholar
  63. Tsien, R.Y. (1981). A non-disruptive technique for loading calcium buffers and indicators into cells. Nature, 290, 527–528CrossRefPubMedGoogle Scholar
  64. Tsien, R.Y. (1989). Fluorescent probes of cell signalling. Ann. Rev. Neurosci, 12, 227–253CrossRefPubMedGoogle Scholar
  65. Tsien, R.Y. and Poenie, M. (1986). Fluorescence ratio imaging: a new window into intracellular ionic signalling. Trends Biochem. Sei., 11, 450–455CrossRefGoogle Scholar
  66. Tsien, R.Y., Pozzan, T. and Rink, T.J. (1982). Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J. Cell Biol, 94, 325–334CrossRefPubMedGoogle Scholar
  67. Tsien, R.Y., Rink, T.J. and Poenie, M. (1985). Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths. Cell Calcium, 6, 145–157CrossRefPubMedGoogle Scholar
  68. White, J.G., Amos, W.B. and Fordham, M. (1987). An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. J. Cell Biol., 105, 41–8CrossRefPubMedGoogle Scholar
  69. Wier, G., Cannell, M.B., Berlin, J.R., Marban, E. and Lederer, W.J. (1987). Cellular and subcellular heterogeneity of [Ca2+]j in single heart cells revealed by fura-2. Science, N.Y., 235, 325–328CrossRefGoogle Scholar
  70. Williams, D.A., Fogarty, K.E., Tsien, R.Y. and Fay, F.S. (1985). Calcium gradients in single muscle cells revealed by the digital imaging microscope using fura-2. Nature, 318, 558–561CrossRefPubMedGoogle Scholar

Copyright information

© The Macmillan Press Ltd 1991

Authors and Affiliations

  • Ron Jacob
  • Christopher D. Benham

There are no affiliations available

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