valinomycin and indo-1

CD3 mAb induced calcium movements are unaffected by hyperpolarization of the membrane in Jurkat T cells treated with valinomycin. By contrast, the CD3 induced Ca2+ influx was impaired by depolarization of the membrane with either gramicidin or by equimolar substitution of KCl for NaCl in the medium. In depolarized cells, the synthesis of phosphatidylserine was strongly diminished as a result of impaired transport of the [3H]serine substrate. In depolarized cells, the CD3-induced release of Ca2+ from intracellular stores (endoplasmic reticulum) was unaffected. Emptying of the Ca2+ stores by CD3 was shown by the lack of effect of additional treatment of the cells with the Ca2+ ionophore, ionomycin. The empty status of the calcium stores was also confirmed by measurements of phosphatyidylserine synthesis through the Ca2+ -dependent base exchange enzyme system that was found to be significantly decreased despite the low amount synthesized in the presence of a defective [3H]serine transport in depolarized cells.

Topics: Antibodies, Monoclonal; Barbiturates; Calcium; CD3 Complex; Fluorescent Dyes; Gramicidin; Humans; Indoles; Ionomycin; Ionophores; Isoxazoles; Jurkat Cells; Membrane Potentials; Phosphatidylserines; T-Lymphocytes; Valinomycin

Jurkat and MOLT-4 cultured T lymphoblasts were loaded with low concentrations (30-50 microM) of indo-1 and with high concentrations (3.5-4.5 mM) 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.

Topics: Aminoquinolines; Antibodies, Monoclonal; Antigens, Differentiation, T-Lymphocyte; Biological Transport, Active; Calcium; CD3 Complex; Cell Line; Cells, Cultured; Endoplasmic Reticulum; Fluorescent Antibody Technique; Gramicidin; Humans; Indoles; Receptors, Antigen, T-Cell; T-Lymphocytes; Terpenes; Thapsigargin; Valinomycin