Effect of calmodulin and calmodulin antagonists on the Ca²⁺ uptake by the intracellular Ca²⁺-accumulating system of guinea pig peritoneal macrophages treated with saponin

Guinea pig peritoneal macrophages were treated with saponin, and the effects of calmodulin and 'calmodulin antagonists' on the Ca²⁺ uptake of saponin-treated macrophages were examined. With this intracellular Ca²⁺-accumulating system we found that intact macrophages contained 672 ng/4 X 10⁶ cells (2874 ng/mg of protein), which was reduced to 64 ng/4 X 10⁶ cells (799 ng/mg of protein) by treatment of the macrophages with saponin. Exogeneously added calmodulin affected neither the maximal capacity of the Ca²⁺ uptake nor the apparent affinity of Ca²⁺ of the saponin-treated macrophages. All of the calmodulin antagonists examined [chlorpromazine, trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, and N-(6-aminohexyl)-1-naphthalenesulfonamide] inhibited the Ca²⁺ uptake of saponin-treated macrophages. However, the concentrations of these drugs required for half-maximal inhibition of the Ca²⁺ uptake were much higher than those described for the calmodulin stimulation of phosphodiesterase. Troponin I, which inhibited calmodulin-stimulated phosphodiesterase activity, did not inhibit Ca²⁺ uptake. Inhibition of Ca²⁺ uptake by chlorpromazine and trifluoperazine was not reduced by the addition of excess calmodulin and was not altered by changes in concentration of both Mg-ATP and free Ca²⁺. The Ca²⁺ accumulated in saponin-treated macrophages was released by the addition of chlorpromazine and trifluoperazine, and, after removal of the drugs, Ca²⁺ accumulation was restored. This release of Ca²⁺ by chlorpromazine and trifluoperazine was concentration-dependent, and the concentration required for half-maximal release of Ca²⁺ was similar to that for half-maximal inhibition of Ca²⁺ uptake. From these findings, we conclude that the intracellular Ca²⁺-accumulating system in guinea pig peritoneal macrophages was not stimulated by calmodulin. Although calmodulin antagonists inhibited Ca²⁺ uptake and released accumulated Ca²⁺ in saponin-treated macrophages, these effects may be unrelated to the specific effect of the drugs on calmodulin.