Search tips
Search criteria 


Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
J Cell Biol. 1982 August 1; 94(2): 325–334.
PMCID: PMC2112871

Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator


A new, fluorescent, highly selective Ca2+ indicator , "quin2", has been trapped inside intact mouse and pig lymphocytes, to measure and manipulate cytoplasmic free Ca2+ concentrations, [Ca2+]i. Quin2 is a tetracarboxylic acid which binds Ca2+ with 1:1 stoichiometry and an effective dissociation constant of 115 nM in a cationic background mimicking cytoplasm. Its fluorescence signal (excitation 339 nm, emission 492 nm) increases about fivefold going from Ca-free to CA- saturated forms. Cells are loaded with quin2 by incubation with its acetoxymethyl ester, which readily permeates the membrane and is hydrolyzed in the cytoplasm, thus trapping the impermeant quin2 there. The intracellular quin2 appears to be free in cytoplasm, not bound to membranes and not sequestered inside organelles. The fluorescence signal from resting cells indicates a [Ca2+]i of near 120 nM. The millimolar loadings of quin2 needed for accurately calibrated signals do not seem to perturb steady-state [Ca2+]i, but do somewhat slow or blunt [Ca2+]i transients. Loadings of up to 2mM are without serious toxic effects, though above this level some lowering of cellular ATP is observed. [Ca2+]i was well stabilized in the face of large changes in external Ca2+. Alterations of Na+ gradients, membrane potential, or intracellular pH had little effect. Mitochondrial poisons produced a small increase in [Ca2+]i, probably due mostly to the effects of severe ATP depletion on the plasma membrane. Thus intracellulary trapped chelators like quin2 offer a method to measure or buffer [Ca2+]i in hitherto intractable cell types.

Full Text

The Full Text of this article is available as a PDF (1.0M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Alvarez-Leefmans FJ, Rink TJ, Tsien RY. Free calcium ions in neurones of Helix aspersa measured with ion-selective micro-electrodes. J Physiol. 1981 Jun;315:531–548. [PubMed]
  • Baker PF, Knight DE. Gaining access to the site of exocytosis in bovine adrenal medullary cells. J Physiol (Paris) 1980 Sep;76(5):497–504. [PubMed]
  • Brinley FJ., Jr Calcium buffering in squid axons. Annu Rev Biophys Bioeng. 1978;7:363–392. [PubMed]
  • Denton RM, McCormack JG, Edgell NJ. Role of calcium ions in the regulation of intramitochondrial metabolism. Effects of Na+, Mg2+ and ruthenium red on the Ca2+-stimulated oxidation of oxoglutarate and on pyruvate dehydrogenase activity in intact rat heart mitochondria. Biochem J. 1980 Jul 15;190(1):107–117. [PubMed]
  • Ferreira HG, Lew VL. Proceedings: Ca transport and Ca pump reversal in human red blood cells. J Physiol. 1975 Nov;252(2):86P–87P. [PubMed]
  • Hesketh TR, Smith GA, Houslay MD, Warren GB, Metcalfe JC. Is an early calcium flux necessary to stimulate lymphocytes? Nature. 1977 Jun 9;267(5611):490–494. [PubMed]
  • Lichtman AH, Segel GB, Lichtman MA. Calcium transport and calcium-ATPase activity in human lymphocyte plasma membrane vesicles. J Biol Chem. 1981 Jun 25;256(12):6148–6154. [PubMed]
  • Lyall RM, Dubois JH, Crumpton MJ. Ionomycin stimulates T-lymphocytes to grow. Biochem Soc Trans. 1980 Dec;8(6):720–721. [PubMed]
  • Montecucco C, Pozzan T, Rink T. Dicarbocyanine fluorescent probes of membrane potential block lymphocyte capping, deplete cellular ATP and inhibit respiration of isolated mitochondria. Biochim Biophys Acta. 1979 Apr 19;552(3):552–557. [PubMed]
  • Montecucco C, Rink TJ, Pozzan T, Metcalfe JC. Triggering of lymphocyte capping appears not to require changes in potential or ion fluxes across the plasma membrane. Biochim Biophys Acta. 1980;595(1):65–70. [PubMed]
  • Murphy E, Coll K, Rich TL, Williamson JR. Hormonal effects on calcium homeostasis in isolated hepatocytes. J Biol Chem. 1980 Jul 25;255(14):6600–6608. [PubMed]
  • Palmer LG, Civan MM. Distribution of Na+, K+ and Cl- between nucleus and cytoplasm in Chironomus salivary gland cells. J Membr Biol. 1977 May 6;33(1-2):41–61. [PubMed]
  • Pozzan T, Corps AN, Montecucco C, Hesketh TR, Metcalfe JC. Cap formation by various ligands on lymphocytes shows the same dependence on high cellular ATP levels. Biochim Biophys Acta. 1980 Nov 18;602(3):558–566. [PubMed]
  • Rink TJ. The influence of sodium on calcium movements and catecholamine release in thin slices of bovine adrenal medulla. J Physiol. 1977 Apr;266(2):297–325. [PubMed]
  • Rink TJ, Montecucco C, Hesketh TR, Tsien RY. Lymphocyte membrane potential assessed with fluorescent probes. Biochim Biophys Acta. 1980;595(1):15–30. [PubMed]
  • Scott ID, Akerman KE, Nicholls DG. Calcium-ion transport by intact synaptosomes. Intrasynaptosomal compartmentation and the role of the mitochondrial membrane potential. Biochem J. 1980 Dec 15;192(3):873–880. [PubMed]
  • Siebert G. The limited contribution of the nuclear envelope to metabolic compartmentation. Biochem Soc Trans. 1978;6(1):5–9. [PubMed]
  • Tsien RY. New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry. 1980 May 27;19(11):2396–2404. [PubMed]
  • Tsien RY. A non-disruptive technique for loading calcium buffers and indicators into cells. Nature. 1981 Apr 9;290(5806):527–528. [PubMed]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press