Effect of calmodulin antagonists on lysosomal enzyme secretion and phospholipid metabolism in guinea-pig macrophages

Abstract

The effects of calmodulin antagonists on the secretion of lysosomal enzyme and lipid metabolism in guinea-pig peritoneal macrophages were studied. Calmodulin antagonists, such as trifluoperazine, dibucaine and quinacrine, inhibited the secretion of N-acetyl-β-d-glucosaminidase from cytochalasin B-treated macrophages when the macrophages were stimulated by the chemotactic peptide, formylmethionyl-leucyl-phenylalanine (f Met-Leu-Phe) or the Ca2+ ionophore A23187. The effect of calmodulin antagonists on the incorporation of [32P]Pi or [3H]glycerol into glycerolipids as well as on the redistribution of [14C]glycerol or [3H]arachidonic acid in [14C]glycerol- or [3H]arachidonic acid-prelabelled lipids were examined. Trifluoperazine, dibucaine or quinacrine stimulated [32P]Pi incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) without significant effect on the labelling of phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), lysophosphatidylcholine (lyso-PtdCho) and lysophosphatidylethanolamine (lyso-PtdEtn). The incorporation of [32P]Pi into phosphatidylcholine (PtdCho) was, on the contrary, inhibited. When calmodulin antagonists were added to macrophages stimulated by fMet-Leu-Phe, [32P]Pi incorporation into PtdIns and PtdA was synergistically increased compared with that induced only by calmodulin antagonists. Trifluoperazine inhibited the incorporation of [3H]glycerol into PtdCho, triacylglycerol and PtdEtn. Also in this case, the incorporation of [3H]glycerol into PtdA and PtdIns was greatly enhanced. But [3H]glycerol incorporation into PtdSer, lyso-PtdEtn and lyso-PtdCho was not affected by the drug. On the other hand, diacylglycerol labelling with [3H]glycerol was maximally activated by 10μm-trifluoperazine and levelled off with the increasing concentration. When the effect of calmodulin antagonists on the redistribution of [14C]glycerol among lipids was examined in pulse-chase experiments, no significant effect on [14C]glycerol redistribution in PtdEtn, PtdCho, PtdIns, PtdSer, PtdA and tri- and di-acylglycerol could be detected. When macrophages prelabelled with [3H]arachidonic acid were treated with trifluoperazine, dibucaine or quinacrine, the [3H]arachidonic acid moiety in PtdEtn and PtdCho was decreased and that in PtdA was increased. The formation of [arachidonate-3H]diacylglycerol and non-esterified [3H]-arachidonic acid was also enhanced, but the increase in [3H]arachidonic acid was only observed at concentrations between 1 and 50μm. [Arachidonate-3H]PtdIns was not significantly affected. The activated formation of [arachidonate-3H]PtdA, diacylglycerol and non-esterified arachidonic acid by these drugs was synergistically enhanced in the presence of fMet-Leu-Phe.