The possible mechanism by which a divalent cation ionophore, A 23 187, initiated the activation of lymphocytes was investigated. Rabbit lymphocytes were cultured in the presence of the ionophore, the T cell mitogen Con A, or the B cell mitogen GARIG serum. The proliferative response, assessed by incorporation of 3H-thymidine into DNS, as well as the increase in incorporation of 3H-uridine induced by the ionophore was comparable to that caused by the receptor-binding B mitogen in T-B mixtures. Thymocytes responded weakly and inconsistently to the ionophore, whereas purified B lymphocytes proved to be highly reactive, suggesting that A 23 187 is primarily a B mitogen in rabbits. Removal of macrophages did not impair the activating capacity of the ionophore. To initiate cell activation the ionophore had to be present for at least some hours.
At optimal concentrations of Ca++ (1 mM), increasing doses of Mg++, which has the same binding affinity to the ionophore but does not support activation, did not suppress the response to the ionophore. Similarly, Mn++ which binds to the ionophore with a 100-fold higher affinity than Ca++ was not able to interfere with the activation of lymphocytes. Both ions, however, inhibited the ionophore-dependent Ca++ influx at concentrations not affecting activation. Within 1 hr of contact the ionophore increased the turnover of the phospholipid fatty acids. The dose response curve for the activation of the membrane phospholipid metabolism and that for the stimulation of RNA or DNA synthesis were identical.
These data suggest that the ionophore A 23 187 interacts directly with rabbit (B) lymphocytes to induce cell activation. The activating capacity does not depend on the translocation of Ca++. We propose that the ionophore intercalates into the lipid phase of the plasma membrane, initiating the same changes in the membrane itself that are induced by mitogens that bind to surface receptors, i.e., increasing the turnover of membrane phospholipids.