Abstract
The effects of the lectins concanavalin A, succinylated concanavalin A, and wheat germ agglutinin on the free cytoplasmic Ca2+ concentration in mouse thymocytes were measured using the fluorescent Ca2+ indicator "quin 2" (Tsien, R. Y. (1980) Biochemistry 19, 2396-2404) and compared with the metabolic and mitogenic effects of the lectins on the cells. Within 1 min of adding each ligand, there is a dose-dependent increase in the free cytoplasmic Ca2+ concentration reported by quin 2. This response is selective for Ca2+, but it does not coincide closely with the subsequent mitogenic stimulation at 48 h by concanavalin A or succinyl concanavalin A. The nonmitogenic lectin wheat germ agglutinin also causes an increase in free cytoplasmic Ca2+ concentration and early metabolic stimulation of the cells, but stimulation is self-aborted before DNA synthesis occurs. At the intracellular concentrations of quin 2 required for measurement of the free Ca2+ concentration, the chelator causes early metabolic stimulation of the cells very similar to that produced by concanavalin A and the mitogenic Ca2+ ionophore A23187. Thus, phosphatidylinositol metabolism and lactate production are stimulated in mouse thymocytes and pig lymphocytes within 1 h of loading with quin 2 and significant increases in RNA synthesis occur after 8 h. Quin 2 causes mitogenic stimulation of pig lymphocytes measured as increased [3H]thymidine uptake at 48 h, that is variable but substantial in most experiments (up to 100% of the stimulation by A23187). The chelator itself has no significant activity as a Ca2+ ionophore, but the apparent free Ca2+ concentration in the cells increases both with the concentration of intracellular quin 2 and with the extracellular Ca2+ concentration. These data leave open the possibility that quin 2 itself affects the concentration of free Ca2+ or other cations in the cells.
Highlights
The effedts of the lectins concanavalin A, succiny- cated that mitogenic lectins suchas concanavalin Acan cause lated concanavalin A, and wheat germ agglutinin on substantial increases in 45Ca2+ associatedwith the cells, but the free cytoplasmic Ca2+concentration in mouse thy- the magnitude and durationof the effect and itsrelevance to mocytes were measured using the fluorescent Ca2+in- mitogenic stimulation arehighly controversial [5,6,7]
M constitutestheprimary mitogeniscignal fotrhe transition out of the resting state (Go)into the cell cycle and that this signal must persist for about 20 h to commit the maximum proportion of cells to DNA synthesis [1].If [Cali is mitogenic stimulation at 48 h by concanavalin A or lowered back into theGo range
The nonmitogenic lectin mitogen from thecells) before commitment to DNA synthesis wheat germ agglutinin causes an increase in free has occurred, the cells will return to the resting (Go) cytoplasmic Ca2+concentration andearly metabolic state
Summary
[Ca], in Thymocytes Loaded with quin 2-The estimation of the per cent saturation of quin 2 by Ca2' is illustrated in. The ion selectivity of the effect of ConA is consistent with ConA causes maximal mitogenic stimulation over a wider an increase in [Cali as a specific response of thymocytes to range of concentrations thanConA and at 10 pg/ml caused a the ligand, rather than a general increase in ion permeability similar increase in [Calito optimal ConA. It should be noted, of the plasma membrane as a result of the cross-linking of that the dose-responsecurvesfor the increase in proteins by ConA.
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