Summary
Jurkat and MOLT-4 cultured T lymphoblasts were loaded with low concentrations (30–50 μm) of indo-1 and with high concentrations (3.5–4.5mm) of quin-2, respectively, in order to follow the activation of calcium transport pathways after stimulation of the cells by a monoclonal antibody against the T cell antigen receptor (aCD3), or after the addition of thapsigargin, a presumed inhibitor of endoplasmic reticulum calcium pump. In the indo-1 loaded cells the dynamics of the intracellular calcium release and the calcium influx could be studied, while in the quin-2 overloaded cells the changes in cytoplasmic free calcium concentration ([Ca2+] i ) were strongly buffered and the rate of calcium influx could be quantitatively determined. We found that in Jurkat lymphoblasts, in the absence of external calcium, both aCD3 and thapsigargin induced a rapid calcium release from internal stores, while upon the readdition of external calcium an increased rate of calcium influx could be observed in both cases, aCD3 and thapsigargin released calcium from the same intracellular pools. The calcium influx induced by either agent was of similar magnitude and had a nonadditive character if the two agents were applied simultaneously. As demonstrated in quin-2 overloaded cells, a significant initial rise in [Ca2+] i or a pronounced depletion of internal calcium pools was not required to obtain a rapid calcium influx. The activation of protein kinase C by phorbol ester abolished the internal calcium release and the calcium influx induced by aCD3, while having only a small effect on these phenomena when evoked by thapsigargin. Membrane depolarization by gramicidin inhibited the rapid calcium influx in both aCD3- and thapsigargin-treated cells, although it did not affect the internal calcium release produced by either agent. In MOLT-4 cells, which have no functioning antigen receptors, aCD3 was ineffective in inducing a calcium signal, while thapsigargin produced similar internal calcium release and external calcium influx to those observed in Jurkat cells.
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References
Aboolian, A., Van der Molen, M., Nord, E.P. 1989. Differential effects of phorbol esters on PGE2 and bradykinin-induced elevation of [Ca2+] i in MDCK cells.Am. J. Physiol. 256:F1135-F1143
Alcover, A., Ramarli, D., Richardson, N.E., Chang, H.-C., Reinherz, E.L. 1987. Functional and molecular aspects of human T lymphocyte activation via T3-Ti and T11 pathways.Immunol. Rev. 95:5–36
Brayden, D.J., Hanley, M.R., Thastrup, O., Cuthbert, A.W. 1989. Thapsigargin, a new calcium-dependent epithelial anion secretagogue.Br. J. Pharmacol. 98:809–816
Cantrell, D.A., Davies, A.A., Crumpton, M.J. 1985. Activators of protein kinase C down-regulate and phosphorylate the T3/T-cell antigen receptor complex of human T-lymphocytes.Proc. Natl. Acad. Sci. USA 82:8158–8162
Crofts, J.N., Barritt, G.J. 1989. The measurement of Ca2+ inflow across the liver plasma membrane by using quin2 and studies of the roles of Na+ and extracellular Ca2+ in the mechanism of Ca2+ inflow.Biochem. J. 264:61–70
Crumpton, J.M., Cantrell, D.A., Davies, A.A., Verbi, W. 1987. The effect and role of protein kinase C activation in the stimulation of human T lymphocytes.In: Leukocyte Typing III. White Cell Differentiation Antigens. A.J. McMichael, editor. pp. 918–922, Oxford University Press, Oxford
DiVirgilio, F., Lew, P.D., Andersson, T., Pozzan, T. 1987. Plasma membrane potential modulates chemotactic peptidestimulated cytosolic free Ca2+ changes in human neutrophils.J. Biol. Chem. 262:4574–4579
Fewtrell, C., Sherman, E. 1987. IgE receptor-activated calcium permeability pathway in rat basophilic leukemia cells: Measurement of the unidirectional influx of calcium using quin-2-buffered cells.Biochemistry 26;6995–7003
Foder, B., Scharff, O., Thastrup, O. 1989. Ca2+ transients and Mn2+ entry in human neutrophils induced by thapsigargin.Cell Calcium 10:477–490
Gardner, P. 1989. Calcium and T lymphocyte activation.Cell 59:15–20
Gelfand, E.W., Cheung, R.K., Mills, G.B., Grinstein, S. 1987a. Role of membrane potential in the response of human T lymphocytes to phytohemagglutinin.J. Immunol. 138:527–531
Gelfand, E.W., Mills, G.B., Cheung, R.K., Lee, J.W.W., Grinstein, S. 1987b. Transmembrane ion fluxes during activation of human T lymphocytes: Role of Ca2+, Na+/H+ exchange and phospholipid turnover.Immunol. Rev. 95:59–87
Gray, L.S., Gnarra, J.R., Russell, J.H., Engelhard, V.H. 1987. The role of K+ in the regulation of the increase in intracellular Ca2+ mediated by the T lymphocyte antigen receptor.Cell 50:119–127
Grinstein, S., Klip, A. 1989. Calcium homeostasis and the activation of calcium channels in cells of the immune system.Bull. N. Y. Acad. Sci. 65:69–79
Grynkiewicz, G., Poenie, M., Tsien, R.Y. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties.J. Biol. Chem. 260:3440–3450
Gupta, S. 1989. Mechanism of transmembrane signalling in human T cell activation.Mol. Cell. Biochem. 91:45–50
Harnett, M.M., Klaus, G.G.B. 1988. Protein kinase-C activators inhibit the antigen receptor-coupled polyphosphoinositide phosphodiesterase in murine lymphocytes-B.FEBS Lett. 239:281–284
Hesketh, T.R., Smith, G.A., Moore, J.P., Taylor, M.V., Metcalfe, J.C. 1983. Free cytoplasmic calcium concentration and mitogenic stimulation of lymphocytes.J. Biol. Chem. 258:4876–4882
Imboden, J.B., Weiss, A. 1987. The T-cell antigen receptor regulates sustained increase in cytoplasmic free Ca2+ through external Ca2+ influx and ongoing intracellular Ca2+ mobilization.Biochem. J. 247:685–700
Imboden, J.B., Weiss, A., Stobo, J.D. 1985. The antigen receptor on a human T cell line initiates activation by increasing cytoplasmic free calcium.J. Immunol. 134:663–665
Isakov, N., Mally, M., Scholz, W., Altman, A. 1987. T-lymphocyte activation: The role of protein kinase C and the bifurcating inositol phospholipid signal transduction pathway.Immunol. Rev. 95:89–111
Jackson, T.R., Patterson, S.I., Thastrup, O., Hanley, M.R. 1988. A novel tumour promoter, thapsigargin, transiently increases cytoplasmic free Ca2+ without generation of inositol phosphates in NG115-401L neuronal cells.Biochem. J. 253:81–86
Johansson, J.S., Haynes, D.H. 1988. Deliberate quin2 overload as a method forin situ characterization of active calcium extrusion system and cytoplasmic calcium binding: Application to human platelet.J. Membrane Biol. 104:147–163
Kovács, T., Tordai, A., Szász, I., Sarkadi, B., Gárdos, G. 1990. Membrane depolarization inhibits thrombin-induced calcium influx and aggregation in human platelets.FEBS Lett. 266:171–174
Kuno, M., Goronzy, J., Weyand, C.M., Gardner, P. 1986. Single-channel and whole-cell recordings of mitogen-regulated inward currents in human cloned helper T lymphocytes.Nature 323:269–273
Lagast, H., Pozzan, T., Waldvogel, F.A., Lew, P.D. 1984. Phorbol myristate acetate stimulates ATP-dependent calcium transport by the plasma membrane of neutrophils.J. Clin. Invest. 73:878–883
Laskey, R.E., Adams, D.J., Johns, A., Rubanyi, G.M., van Breemen, C. 1990. Membrane potential and Na+−K+ pump activity modulate resting and bradykinin-stimulated changes in cytosolic free calcium in cultured endothelial cells from bovine atria.J. Biol. Chem. 265:2613–2619
Law, G.J., Pachter, J.A., Thastrup, O., Hanley, M., Dannies, P.S. 1990. Thapsigargin, but not caffeine, blocks the ability of thyrotropin-releasing hormone to release Ca2+ from intracellular store in GH4C1 pituitary cells.Biochem. J. 267:359–364
Lewis, R.S., Cahalan, M.D. 1989. Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells.Cell Reg. 1:99–112
MacIntyre, D.E., McNicol, A., Drummond, A.H. 1985. Tumourpromoting phorbol esters inhibit agonist-induced phosphatidate formation and calcium flux in human platelets.FEBS Lett. 180:160–164
Magócsi, M., Sarkadi, B., Kovács, T., Gárdos, G. 1989. Thrombin-induced activation of calcium transport pathways and their role in platelet functions.Biochim. Biophys. Acta 984:88–96
Meldolesi, J., Pozzan, T. 1987. Pathways of Ca2+ influx at the plasma membrane: Voltage-, receptor-, and second messenger-operated channels.Exp. Cell. Res. 171:271–283
Mellors, A., Stalmach, M.E., Cohen, A. 1985. Co-mitogenic tumor promoters suppress the phosphatidylinositol response in lymphocytes during early mitogenesis.Biochim. Biophys. Acta 833:181–191
Merritt, J.E., Rink, T.J. 1987. Regulation of cytosolic free calcium in fura-2 loaded rat parotid acinar cells.J. Biol. Chem. 262:17362–17369
Mohr, F.Ch., Fewtrell, C. 1987. The relative contributions of extracellular and intracellular calcium to secretion from tumor mast cells.J. Biol. Chem. 262:10638–10643
Naccache, P.H., Molski, T.F.P., Borgeat, P., White, J.R., Sha'afi, R.I. 1985. Phorbol esters inhibit the fMet-Leu-Phe-and leukotriene B4-stimulated calcium mobilization and enzyme secretion in rabbit neutrophils.J. Biol. Chem. 260:2125–2131
Ng, J., Fredholm, B.B., Jondal, M., Andersson, T. 1988. 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
Nishizuka, Y. 1986. Studies and perspectives of protein kinase C.Science 233:305–312
Nishizuka, Y. 1988. The molecular heterogeneity of protein kinase C and its implications for cellular regulation.Nature 334:661–665
Owen, C.S. 1988. Quantitation of lymphocyte intracellular free calcium signals using indo-1.Cell Calcium 9:141–147
Pecht, M., Corcia, A., Liuzzi, M.P.T., Alcover, A., Reinherz, E.L. 1987. Ion channels activated by specific Ti and T3 antibodies in plasma membranes of human T cells.EMBO J. 6:1935–1939
Penner, R., Matthews, G., Neher, E. 1988. Regulation of calcium influx by second messengers in rat mast cells.Nature 334:499–504
Pollock, W.K., Sage, S.O., Rink, T.J. 1987. Stimulation of calcium efflux from fura-2-loaded platelets activated by thrombin or phorbol myristate acetate.FEBS Lett. 210:132–136
Putney, J.W., Jr. 1986. A model for receptor-regulated calcium entry.Cell Calcium 7:1–12
Putney, J.W., Jr., Takemura, H., Hughues, A.R., Horstman, D.A., Thastrup, O. 1989. How do inositol phosphates regulate calcium signalling?FASEB J. 3:1899–1905
Rasmussen, U., Christensen, S.B., Sandberg, F. 1978. Thapsigargin and thapsigargicin, two new histamine liberators fromThapsia garganica.Acta Pharmaceut. Suec. 15:133–140
Sage, S.O., Reast, R., Rink, T.J. 1990. ADP evokes biphasic Ca2+ influx in fura-2-loaded human platelets. Evidence for Ca2+ entry regulated by the intracellular calcium store.Biochem. J. 265:675–680
Sarkadi, B., Tordai, A., Gárdos, G. 1990a. Membrane depolarization selectively inhibits receptor-operated calcium channels in human T (Jurkat) lymphoblasts.Biochim. Biophys. Acta 1027:130–140
Sarkadi, B., Tordai, A., Müller, M., Gárdos, G. 1990b. Regulation of stimulus-induced calcium transport pathways in human T (Jurkat) lymphoblasts.Mol. Immunol. 27:1297–1306
Savage, A.L., Biffen, M., Martin, B.R. 1989. Vasopressin-stimulated Ca2+ influx in rat hepatocytes is inhibited in high-K medium.Biochem. J. 260:821–827
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
Smallwood, J.I., Gugi, B., Rasmussen, H. 1988. Regulation of erythrocyte Ca2+ pump activity by protein kinase C.J. Biol. Chem. 263:2195–2202
Takemura, H., Hughes, A.R., Thastrup, O., Putney, J.W., Jr. 1989. Activation of calcium entry by the tumor promoter thapsigargin in parotid acinar cells. Evidence that an intracellular calcium pool and not an inositol phosphate, regulates calcium fluxes at the plasma membrane.J. Biol. Chem. 264:12266–12271
Takemura, H., Putney, J.W., Jr. 1989. Capacitative calcium entry in parotid acinar cells.Biochem. J. 258:409–412
Takemura, H., Thastrup, O., Putney, J.W., Jr. 1990. Calcium efflux across the plasma membrane of rat parotid acinar cells is unaffected by receptor activation or by the microsomal calcium ATPase inhibitor, thapsigargin.Cell Calcium 11:11–17
Thastrup, O., Cullen, P.I., Drobak, K., Hanley, M.K., Dawson, A.P. 1990. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase.Proc. Natl. Acad. Sci. USA 87:2466–2470
Thastrup, O., Dawson, A.P., Scharff, O., Foder, B., Cullen, P.J., Drobak, B.K., Bjerrum, P.J., Christensen, S.B., Hanley, M.R. 1989. Thapsigargin, a novel molecular probe for studying intracellular calcium release and storage.Agents & Actions 27:17–23
Thastrup, O., Foder, B., Scharff, O. 1987. The calcium mobilizing and tumor promoting agent, thapsigargin elevates the platelet cytoplasmic free calcium concentration to a higher steady state level. A possible mechanism of action for the tumor promotion.Biochem. Biophys. Res. Commun. 142:654–660
Tordai, A., Sarkadi, B., Görög, Gy., Gárdos, G. 1989. Inhibition of the CD3-mediated calcium signal by protein kinase C activators in human T (Jurkat) lymphoblastoid cells.Immunol. Lett. 20:47–52
Tsien, R.Y. 1983. Intracellular measurements of ion activities.Annu. Rev. Biophys. Bioeng. 12:91–116
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 295:69–71
Willems, P.H.G.M., Van Den Broek, B.A.M., Van Os, C.H., De Pont, J.J.H.H.M. 1989. Inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ release in permeabilized pancreatic acinar cells by hormonal and phorbol ester pretreatment.J. Biol. Chem. 264:9762–9767
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Sarkadi, B., Tordai, A., Homolya, L. et al. Calcium influx and intracellular calcium release in anti-CD3 antibody-stimulated and thapsigargin-treated human T lymphoblasts. J. Membrain Biol. 123, 9–21 (1991). https://doi.org/10.1007/BF01993958
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DOI: https://doi.org/10.1007/BF01993958