Abstract
cADP-ribose (cADPr) has recently been shown to release Ca2+ from an intracellular store of permeabilized T lymphocyte cell lines (Guse, A. H., da Silva, C. P., Emmrich, F., Ashamu, G. A., Potter, B. V. L., and Mayr, G. W. (1995) J. Immunol. 155, 3353-3359). Using permeabilized Jurkat and HPB. ALL T lymphocytes, the effects of varying concentrations of inorganic phosphate and Mg2+ on cADPr-induced Ca2+ release were investigated. cADPr-induced Ca2+ release was dependent on the concentration of inorganic phosphate, showing very low Ca2+ release activity between 0.5 and 2 mM inorganic phosphate. At 4 to 5 mM inorganic phosphate, the cADPr-induced Ca2+ release was much more pronounced, reaching maximal values at 10 mM inorganic phosphate. The underlying mechanism for this stimulatory effect was an increased loading of the cADPr-sensitive Ca2+ store, which was demonstrated by enhanced resequestration of Ca2+ selectively into the cADPr-sensitive Ca2+ store. The free Mg2+ concentration also influenced cADPr-induced Ca2+ release in permeabilized cells: at 0 and 8.58 mM the release was nearly completely abolished, whereas at 1.06 mM maximal Ca2+ release by cADPr was observed. High performance liquid chromatographic analysis of exogenously added cADPr revealed that the catabolism of cADPr at varying Mg2+ and Pi concentrations had only minor relevance for the modulatory effects observed. To correlate the effects of inorganic phosphate and Mg2+ on cADPr-induced Ca2+ release observed in the permeabilized cell preparations, measurements of these ions in intact Jurkat T lymphocytes were carried out. Intact Jurkat T cells stimulated via the T cell receptor middle dotCD3 complex did not respond with significant elevation of the free intracellular Mg2+ concentration. In contrast, stimulation via the T cell receptor middle dotCD3 complex resulted in an increase in the intracellular inorganic phosphate concentration. These data indicate a role for the intracellular inorganic phosphate concentration in the regulation of cADPr-mediated Ca2+ release in T lymphocytes.
Highlights
To correlate the effects of inorganic phosphate and Mg2؉ on cADPr-induced Ca2؉ release observed in the permeabilized cell preparations, measurements of these ions in intact Jurkat T lymphocytes were carried out
Effect of Varying [Pi] and [Mg2ϩ] on the Total Intracellular Ca2ϩ Pool Content in Permeabilized T Cells—During the first experiments using an altered composition of the intracellular buffer, it became clear that changing the parameters [Pi] and [Mg2ϩ]free would influence the Ca2ϩ pool size of the permeabilized cell preparation (Table I)
In this report we demonstrate that: (i) the Ca2ϩ pool content of permeabilized T lymphocytes was significantly altered by changing the standard composition of the intracellular buffer used; (ii) [Pi] and [Mg2ϩ] both can modulate cADPr-induced Ca2ϩ release in permeabilized T lymphocyte cell lines; (iii) the stimulatory effect of increasing [Pi] is likely due to an increased loading of the cADPr-sensitive intracellular Ca2ϩ pool; and (iv) [Pi]i increased on stimulation of TCR1⁄7CD3, whereas [Mg2ϩ]i did not
Summary
Ins(1,4,5)P3, D-myo-inositol-1,4,5trisphosphate; cADPr, cADP-ribose; HPLC, high performance liquid chromatography; [Mg2ϩ]free, free Mg2ϩ concentration; [Mg2ϩ]i, free intracellular Mg2ϩ concentration; [Pi], inorganic phosphate concentration; [Pi]i, intracellular inorganic phosphate concentration; RyR, ryanodine receptor; TCR1⁄7CD3, T cell receptor1⁄7CD3 complex. We report here that: (i) cADPr-induced Ca2ϩ release in permeabilized Jurkat T lymphocytes is modulated by the concentration of Pi and Mg2ϩ; and (ii) the stimulatory effect of high [Pi] on cADPr-induced Ca2ϩ release was most likely due to enhanced loading of the cADPr-sensitive intracellular Ca2ϩ store. The intracellular free Mg2ϩ ([Mg2ϩ]i) was not altered by stimulation of intact mag-Fura2-loaded Jurkat T cells, whereas stimulation of the TCR1⁄7CD3 resulted in a marked increase in the intracellular Pi concentration
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