Inhibition of T3 Mediated T‐Cell Proliferation by Ca2+‐Channel Blockers and Inhibitors of Ca2+/Phospholipid‐Dependent Kinase

The potential roles of Ca2+ ions in the response of T lymphocytes to stimulation with monoclonal antisera to the T3 antigen were investigated by means of pharmacological agents that predominantly inhibit the flux of C a2+ ions into cells (verapamil, nifedipine) or the activity of C a2+-dependent kinases (trifluoperazine, polymyxin B). As assessed by uptake of [3H]thymidine. proliferation induced with anti T3-recombinant IL-2 at 72 h was inhibited by >80% in the presence of nifedipine at 50 μM, and almost completely arrested (>95% inhibition) with the other agents at the same concentration. Further quantitative assays of the effects of polymyxin B and trifluoperazine on C-kinase labelling of exogenous substrate showed a major reduction with both agents, but inhibition was substantially greater with polymyxin B that with trifluoperazine (IC50= 14 and 70 μM respectively). These results were confirmed by qualitative assessment of C a2+/phospholipid-dependent phosphorylation of endogenous substrates, which demonstrated major phosphoproteins of MW 56,000. 52.000, 43,000. and 20,000, and dose-dependent reduction in labelling in the presence of polymyxin B. Similar results were obtained under more physiological conditions in intact cells labelled with 32P orthophosphate. These findings indicate several possible roles for C a2+ in T cell activation, and several possible levels of activity, including modulation of calmodulin-dependent kinases and effects on C a2+/phospholipid-dependent kinases and C a2+ channels.