Mitogen and co‐mitogen stimulation of lymphocytes inhibited by three Ca⁺⁺ antagonists

The Ca⁺⁺ requirement for in vitro lymphocyte stimulation by lectins is well known and can be demonstrated by the use of Ca⁺⁺ chelators. In this study, three Ca⁺⁺ antagonists were examined for their effects on lymphocyte proliferation. [³H]‐thymidine incorporation was employed to measure DNA synthesis in several systems. Stimulation and proliferation were achieved by the addition of one of the following: the mitogenic lectin concanavalin A (ConA); the combination of two co‐mitogens, the calcium ionophore A23187 and the phorbol ester, 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA), neither of which is mitogenic alone; or the non‐mitogenic lectin, wheat germ agglutinin (WGA) with TPA. These mitogenic systems were tested for their sensitivity to the Ca⁺⁺ channel blockers verapamil and nicardipine and the intracellular Ca⁺⁺ antagonist TMB‐8. We found that the ConA and WGA plus TPA treated cells were inhibited approximately 50% by 10 μM verapamil, nicardipine or TMB‐8. The stimulation caused by A23187 and TPA was only inhibited by TMB‐8 and nicardipine. The inhibitory effects caused by the Ca⁺⁺ antagonists could not be reversed by the addition of exogenous Ca⁺⁺ (0.1–1.5 mM), but were reversed by repeated washings in antagonist free media. Using TMB‐8 we saw an apparent intracellular Ca⁺⁺ dependence throughout the G1 phase. Previous studies using Ca⁺⁺ chelators or Ca⁺⁺ antagonists suggested an endpoint at about halfway through this period.