Measurement of Intracellular Free Calcium Ion Concentration in Cell Populations Using Fura-2

  • Philip A Iredale
  • John M. Dickenson
Part of the Methods in Molecular Biology™ book series (MIMB, volume 41)

Absrtact

The early methods for calcium measurement involved microinjection of calcium-sensitive proteins, such as aequorin or obelin, into large cells (1, 2) or the use of microelectrodes (3). Both techniques are still employed, however, with much improved sensitivity allowing investigation of a greater range of cell types. In the early 1980s, the “Null Point method” was introduced (4), which involved addition of a metallochromic calcium indicator (Arsenazo III) to cells permeabilized with digitonin. Using this technique, the accumulation and release of calcium from intracellular stores could be recorded. A major advance in calcium measurement was made when Tsien and his colleagues (5, 6) introduced fluorescent calcium indicators. The first to be used was quin-2: its structure was based on the novel calcium chelator 1,2-bis-(O-aminophenoxyl-ethane-N, N,N′,N′-tetraacetic acid (BAPTA) (7, 8), a double aromatic analog of EGTA. The major problem of inducing a hydrophilic polycarboxylate anion to cross the plasma membrane was overcome by the addition of an acetoxymethyl ester group (AM), thus producing a lipophilic, membrane-permeant molecule (quin-2 AM) that, once within the cytoplasm, was subject to attack by intracellular enzymes, which cleaved the ester bond and left the calcium-sensitive free acid trapped within the cell (5). A number of improved calcium indicators have since been developed (e.g., fura-2, indo-1, and fluo-3), but the basic principles of dye loading and continuous calcium reporting remain the same.

References

  1. 1.
    Ridgway, E. B. and Ashley, C. C. (1967) Calcium transients in single muscle fibres. Biochem. Biophys. Res. Commun. 29, 229–234.PubMedCrossRefGoogle Scholar
  2. 2.
    Campbell, A. K., Lea, T. J., and Ashley, C. C. (1979) Coelenterate photoproteins, in Detection and Measurement of Free Ca 2+ in Cells (Ashley, C. C and Campbell, A. K., eds.), Elsevier, North-Holland, Amsterdam, pp. 13–72.Google Scholar
  3. 3.
    Amman, D, Meier, P. C, and Simon, W. (1979) Ca2+ measurements using microelectrodes, in Detection and Measurement of Free Ca 2+ in Cells (Ashley, C. C. and Campbell, A K, eds), Elsevier, North-Holland, Amsterdam, pp. 117–129Google Scholar
  4. 4.
    Murphey, E., Coll, K., Rich, T. L., and Williamson, J. R. (1980) Hormonal effects on calcium homeostasis in isolated hepatocytes. J. Biol. Chem. 255, 6600–6608Google Scholar
  5. 5.
    Tsien, R. Y. (1981) A non-disruptive technique for loading buffers and indicators into cells. Nature 290, 527,528.CrossRefGoogle Scholar
  6. 6.
    Tsien, R. Y., Pozzan, T., and Rink T J. (1982) Calcium homoeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J. Cell Biol. 94, 325–334.PubMedCrossRefGoogle Scholar
  7. 7.
    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, 2396–2404PubMedCrossRefGoogle Scholar
  8. 8.
    Cobbold, P. H. and Rink, T. J (1987) Fluorescence and bioluminescence measure-ment of cytoplasmic free calcium. Biochem J. 248, 313–328.PubMedGoogle Scholar
  9. 9.
    Tsien, R. Y., Rink, T. J., and Poenie, M. (1985) Measurement of cytosolic free Ca2+ in individual cells using fluorescence microscopy with dual excitation wavelengths. Cell Calcium 6, 145–157.PubMedCrossRefGoogle Scholar
  10. 10.
    Gynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biochem. Sci. 260, 3440–3450.Google Scholar
  11. 11.
    Iredale, P. A., Martin, K. F., Hill, S. J., and Kendall, D. A. (1992) Agonist-induced changes in [Ca2+], in N1E-115 cells: differential effects of bradykinin and carbachol. Eur. J. Pharmacol. 26, 163–168.Google Scholar
  12. 12.
    Iredale, P. A., Martin, K F, Alexander, S. P. H., Hill, S. J., and Kendall, D. A. (1992) Inositol 1,4,5 trisphosphate generation and calcium mobilisation via activation of an atypical P2-receptor in the neuronal cell line N1E-115. Br. J. Pharmacol 107, 1083–1087.PubMedGoogle Scholar
  13. 13.
    Iredale, P. A, Martin, K. F, Alexander, S. P. H., Hill, S J, and Kendall, D. A. (1992) Qualitative differences in [Ca2+], and InsP3 generation following stimula-tion of N1E-115 cells with micromolar and millimolar ATP. Biochem Pharmacol. 44, 1479–1487PubMedCrossRefGoogle Scholar
  14. 14.
    Iredale, P. A., Martin, K. F., Hill, S. J., and Kendall, D. A. (1993) The effects of B-phorbol-12,13 dibutyrate on agonist-induced InsP3 generation and [Ca2+], increases in N1E-115 cells: differential modulation of responses to angiotensin II and bradykinin. Biochem. Pharmacol. 45, 611–617.PubMedCrossRefGoogle Scholar
  15. 15.
    Dickenson, J. M. and Hill, S. J. (1991) Histamine stimulated increases in intracellular calcium in the smooth muscle cell line, DDT1MF-2. Biochem. Pharmacol. 42, 1545–1550.PubMedCrossRefGoogle Scholar
  16. 16.
    Dickenson, J. M. and Hill, S. J. (1992) Histamine H1-receptor-mediated calcium influx in DDT,MF-2 cells Biochem. J. 284, 425–431PubMedGoogle Scholar
  17. 17.
    Dickenson, J M. and Hill, S. J. (1993) Adenosine Arreceptor stimulated increases in intracellular calcium in the smooth muscle cell line, DDTiMF-2. Br. J. Pharmacol. 108, 85–92.PubMedGoogle Scholar
  18. 18.
    Dickenson, J. M. and Hill, S. J. (1993) Intracellular cross talk between receptors coupled to phospholipase C via pertussis toxin-sensitive and insensitive G proteins in DD MF-2 cells. Br J. Pharmacol. 109, 719–724.PubMedGoogle Scholar
  19. 19.
    Dickenson, J. M., White, T. E., and Hill, S. J. (1993) The effects of elevated cyclic AMP levels on histamine H1-receptor-stimulated inositol phospholipid hydrolysis and calcium mobilization in the smooth muscle cell line, DDT1MF-2. Biochem. J. 292, 409–417PubMedGoogle Scholar
  20. 11.
    Walsh, J. P. and Bell, R. M. (1986) sn-l,2-Diacylglycerol kinase of Eschericia coh. Mixed micellar analysis of the phospholipid cofactor requirement and divalent cation dependence. J. Biol. Chem. 261, 6239–6247.PubMedGoogle Scholar
  21. 12.
    Wright, T. M., Ranagan, L. A., Shin, H. S., and Raben, D M (1988) Kinetic analysis of 1,2-diacylglycerol mass levels in cultured fibroblasts. J. Biol. Chem. 263, 9374–9380PubMedGoogle Scholar
  22. 13.
    Leach, K. L., Ruff, V. A., Wright, T. M, Pessin, M. S., and Raben D M (1991) Dissociation of protein kinase C activation and sn-1,2-diacylglycerol formation: comparison of phosphatidylinositol and phosphatidylcholine derived diglycerides in alpha thrombin stimulated fibroblasts. J Biol. Chem. 266, 3215–3221.PubMedGoogle Scholar
  23. 14.
    Jamal, Z., Martin, A., Gomez-Munoz, A., and Brindley D. N. (1991) Plasma membrane fractions from rat liver contain a pbospatidate phosphohydrolase distinct from that in endoplasmic reticulum and cytosol. J. Biol. Chem. 266, 2988–2996.PubMedGoogle Scholar
  24. 15.
    Boarder, M. R. and Purkiss, J. R. (1993) Assay of phospholipase D as a neuronal receptor effector mechanism, in Neuroprotocols, vol 3 (Bleasedale, J. E. and Fisher, S. K., eds.), Academic, New York, pp. 157–164.Google Scholar
  25. 16.
    Purkiss, J. R., Murrin, R. A. J., Owen, P. J., and Boarder M. R. (1991) Lack of phospholipase D activity in chromaffin cells: bradykinin stimulated phosphatidic acid formation involves phospholipase C in chromaffin cells but phospholipase D in PC12 cells. J. Neurochem. 57, 1084–1087.PubMedCrossRefGoogle Scholar
  26. 17.
    Challiss, R. J. A., Wilkes, L. C, Patel, V., Purkiss, J. R., and Boarder M. R. (1993) Phospholipase D activation regulates endothelin-1 stimulation of phosphoinositidespecific phospholipase C in SK-N-MC cells. FEBS Lett. 327, 157–160.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc , Totowa, NJ 1995

Authors and Affiliations

  • Philip A Iredale
    • 1
  • John M. Dickenson
    • 1
  1. 1.Department of Physiology and PharmacologyQueen’s Medical CentreNottinghamUK

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