Abstract
Membrane potential changes accompanying Ca2+ influx stimulated by release of Ca2+ from intracellular stores (store-regulated Ca2+ uptake) were monitored in BAPTA-loaded rat thymic lymphocytes using the fluorescent indicator bis(1,3-diethylthiobarbituric acid)trimethine oxonol. Depletion of [Ca2+] i stores by the application of thapsigargin, ionomycin or cyclopiazonic acid induced a depolarization which was (i) dependent upon BAPTA-loading, (ii) dependent upon extracellular Ca2+, (iii) independent of extracellular Na+ and (iv) abolished by 5 mm extracellular Ni2+. This depolarization was followed by a charybdotoxin-sensitive repolarization and subsequent hyperpolarization to values approximating the K+ equilibrium potential, consistent with secondary activation of a K+ conductance. These membrane potential changes temporally correlated with Ca2+ influx from the extracellular medium as measured fluorimetrically with indo-1. The divalent cation permeability sequence was investigated by monitoring the magnitude of the depolarization observed following the addition of 4 mm Ca2+, Mn2+, Ba2+ or Sr2+ to cells pretreated with doses of thapsigargin or ionomycin known to activate the store-regulated calcium uptake pathway. On the basis of these experiments, we conclude that the store-regulated Ca2+ uptake pathway has the following permeability sequence: Ca2+ > Mn2+ ≫ Ba2+, Sr2+ with Mn2+ displaying significant permeability relative to Ca2+. This pathway is distinguishable from other divalent cation uptake pathways reported in other cells types on the basis of its activation by thapsigargin and its high Mn2+ permeability.
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Alvarez, J., Montero, M., Garcia-Sancho, J. 1991. Cytochrome P-450 may link intracellular Ca2+ stores with plasma membrane Ca2+ influx. Biochem J. 274:193–197.
Bijsterbosch, M.K., Rigley, K.P., Klaus, G.G.B. 1986. Cross-linking of surface immunoglobulins on B lymphocytes induces both intracellular Ca2+ release and Ca2+ influx: analysis with indo-1. Biochem. Biophys. Res. Commun. 137:500–506
Chow, S.C., Jondal, M. 1990. Ca2+ entry in T cells is activated by emptying the inositol 1,4,5-trisphosphate sensitive Ca2+ pool. Cell Calcium 11:641–646
Densmore, J.J., Szabo, G., Gray, L. 1991. A voltage-gated calcium channel is linked to the antigen receptor in Jurkat T lymphocytes. FEBS Lett. 312:161–164
Dupuis, G., Heroux, J., Payet, M.D. 1989. Characterization of Ca2+ and K+ currents in the human Jurkat T-cell line: Effects of phytohaemagglutinin. J. Physiol. 412:135–154
Freedman, M., Raff, C.M., Gomperts, B. 1975. Induction of increased calcium uptake in mouse T lymphocytes by concanavalin A and its modulation by cyclic nucleotides. Nature 255:378–382
Gelfand, E.W., Cheung, R.K., Grinstein, S. 1984. Role of membrane potential in the regulation of lectin-induced calcium uptake. J. Cell Physiol. 121:533–539
Gouy, H., Cefai, D., Brogger Christensen, S., Debre, P., Bismuth, G. 1990. Ca2+ influx in human T lymphocytes is induced independently of inositol phosphate production by mobilization of intracellular Ca2+ stores. A study with the Ca2+ endoplasmic reticulum-ATPase inhibitor thapsigargin. Eur. J. Immunol. 20:2269–2275
Grinstein, S., Dixon, S.J. 1989. Ion transport, membrane potential, and cytoplasmic pH in lymphocytes: Changes during activation. Physiol. Rev. 69:417–481
Grinstein, S., Cohen, S., Rothstein, A. 1984. Cytoplasmic pH regulation in thymic lymphocytes by an amiloride-sensitive Na+/H+ antiport. J. Gen. Physiol. 83:341–370
Grinstein, S., Goetz, J.D., Rothstein, A. 1984. 22Na+ fluxes through thymic lymphocytes: I. Na+/Na+ and Na+/H+ exchange through an amiloride-sensitive pathway. J. Gen. Physiol. 84:565–584
Grinstein, S., Smith, J.D. 1989. Ca2+ induces charybdotoxin-sensitive membrane potential changes in rat lymphocytes. Am. J. Physiol. 257:C197-C206
Grynkiewicz, G., Poenie, M., Tsien, R.Y. 1985. A new generation of Ca2+ indicators with greatly improved fluorescent properties. J. Biol. Chem. 260:3440–3450
Hoth, M., Penner, R. 1991. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature 355:353–355
Khan, A.A., Steiner, J.P., Snyder, S.H. 1992. Plasma membrane inositol 1,4,5-trisphosphate receptor of lymphocytes: Selective enrichment in sialic acid and unique binding specificity. Proc. Natl. Acad. Sci. USA 89:2849–2853
Kuno, M., Gardner, P. 1987. Ion channels activated by inositol 1,4,5-trisphosphate in plasma membrane of human T-lymphocytes. Nature 326:301–304
Kuno, M., Goronzy, J., Weyand, C.M., Gardner, P. 1986. Singlechannel and whole cell recordings of mitogen-regulated inward currents in human cloned helper T lymphocytes. Nature 323:269–273
Lewis, R.S., Cabalan, M.D. 1989. Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells. Cell Regul. 1:99–112
MacDougal, S., Grinstein, S., Gelfand, E.W. 1988. Activation of Ca2+-dependent K+ channels in human B-lymphocytes by antiimmunoglobulin. J. Clin. Invest. 81:449–454
Mahaut-Smith, M.P., Schlicter, L.C. 1989. Ca2+-activated K+ channels in lymphocytes. Pfluegers Arch. 414(Suppl. 1):S164
Mahaut-Smith, M.P., Mason M.J. 1991. Single channel recordings of Ca2+-activated K+ channels in rat lymphocytes: Activation by concanavalin A. J. Physiol. 439:513–528
Mason, M.J., Garcia-Rodriguez, C., Grinstein, S. 1991. Effect of endomembrane Ca2+-ATPase inhibitors on the Ca2+-permeability of the plasma membrane: Comparison of the effects of thapsigargin, 2,5-di(tert-butyl)1,4-hydroquinone and cyclopiazonic acid in rat thymic lymphocytes. J. Biol Chem. 266:20856–20862
Mason, M.J., Grinstein, S. 1990. Effect of cytoplasmic acidification on the membrane potential of T-lymphocytes: Role of trace metals. J. Membrane Biol 116:139–148
Mason, M.J., Mahaut-Smith, M.P., Grinstein, S. 1991. Role of intracellular Ca2+ in the regulation of the plasma membrane Ca2+ permeability of rat thymic lymphocytes. J. Biol. Chem. 266:10872–10879
Mason, M.J., Mayer, B., Hymel, L.J. 1993. Inhibition of Ca2+ transport pathways in rat thymic lymphocytes by econazole, miconazole and SKF 96365. Am. J. Physiol 33:C654-C662
McDonald, T.V., Premack, B.A., Gardner, P. 1993. Flash photolysis of caged inositol 1,4,5-trisphosphate activates plasma membrane calcium current in human T-cells. J. Biol. Chem. 268:3889–3896
Montero, M., Alvarez, J., Garcia-Sancho, J. 1990. Uptake of Ca2+ and refilling of intracellular Ca2+ stores in Ehrlich-ascites tumour cells and in rat thymocytes. Biochem. J. 271:535–540
Ng, J., Fredholm, B.B., Jondal, M., Andersson, T. 1990. Regulation of receptor-mediated calcium influx across the plasma membrane in a human leukemic T-cell line: Evidence of its dependence on an initial calcium mobilization from intracellular stores. Biochim. Biophys. Acta 971:207–214
Ng, J., Gustavsson, J., Jondal, M., Andersson, T. 1988. Regulation of calcium influx across the plasma membrane of the Tleukemic cell line JURKAT: Dependence on a rise in cytosolic free calcium can be dissociated from formation of inositol phosphate. Biochim. Biophys. Acta 1053:97–105
Oettgen, H.C., Terhorst, C., Cantley, L.C., Rossoff, P.M. 1985. Stimulation of the T3-T cell receptor complex induces a membrane-potential-sensitive calcium influx. Cell 40:583–590
Pecht, I., Corcia, A., Trisorio Liuzzi, M.P., Alcover, A., Reinherz, E.L. 1987. Ion channels activated by specific Ti or T3 antibodies in plasma membranes of human T cells. EMBO J. 6:1935–1939
Rink, T.J., Montecucco, C., Hesketh, T.R., Tsien, R.Y. 1980. Lymphocyte membrane potential assessed with fluorescent probes. Biochim. Biophys. Acta 595:65–70
Scharff, O., Foder, B., Thastrup, O., Hofmann, B., Moller, J., Ryder, L.P., Jacobsen, K.D., Langhoff, E., Dickmeiss, E., Christensen, S.B., Skinhoj, P., Svejgaard, A. 1988. Effect of thapsigargin on cytoplasmic Ca2+ and proliferation of human lymphocytes in relation to AIDS. Biochim. Biophys. Acta 972:257–264
Taylor, M.J., Metcalfe, J.C., Hesketh, T.R., Moore, J.P. 1984. Mitogens increase phosphorylation of phosphoinositides in thymocytes. Nature 312:462–465
Tsien, R.Y., Pozzan, T., Rink, T.J. 1982. T-cell mitogens cause early changes in cytoplasmic free Ca2+ and membrane potential in lymphocytes. Nature 295:68–70
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This work is supported by grants from the American Heart Association, Louisiana Affiliate (LA-92-6-28), Louisiana Education Quality Support Fund (LEQSF(1993-96)-RD-A-31) and Tulane University Graduate Program in Molecular and Cellular Biology.
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Wilson, O.I., Marriott, I., Mahaut-Smith, M.P. et al. Isolation and characterization of membrane potential changes associated with release of calcium from intracellular stores in rat thymic lymphocytes. J. Membarin Biol. 137, 159–168 (1994). https://doi.org/10.1007/BF00233485
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DOI: https://doi.org/10.1007/BF00233485