Involvement of calcium in the thimerosal-stimulated formation of leukotriene by fMLF in human polymorphonuclear leukocytes

Abstract

Only small amounts of leukotrienes could be detected by reverse-phase HPLC analysis after stimulation of human polymorphonuclear leukocytes (PMN) by the receptor agonist N-formymethionyl-leucyl-phenylalanine (fMLP). Preincubation of the cells with the organomercury compound thimerosal prior to fMLP-addition, however, resulted in the formation of significant amounts of 5-lipoxygenase derived metabolites. This effect was dose-dependent with respect both to fMLP and thimerosal. Thimerosal alone did neither lead to the formation of HPLC-detectable leukotrienes nor to the release of arachidonic acid in [1− 14C]arachidonic acid prelabelled cells. The formation of leukotrienes by fMLP/thimerosal required extracellular Ca 2+. Measurements of intracellular Ca 2+-levels revealed that (i) thimerosal alone is able to release Ca 2+ from internal stores and (ii) thimerosal causes a persistent accumulation of Ca 2+ within the cells after stimulation by fMLP. We conclude that by the synergistic action of fMLP and thimerosal the Ca 2+-levels exceed the threshold for phospholipase A 2 activation resulting in the liberation of arachidonic acid and subsequently in the formation of 5-lipoxygenase products. Our results suggest that thimerosal may provide a model for leukotriene formation under pathophysiological conditions when SH-group oxidation leads to increased intracellular Ca 2+-levels. k]abr|PMN, polymorphonuclear leukocytes; fMLP, N-formyl-methionyl-leucyl-phenylalanine; DMSO, dimethylsulfoxide; HO-Δ 4-Ach, hydroxyicosatetraenoic acid; HPLC, high performance liquid chromatography; LT, leukotriene. Enzymes: Acyl-CoA:lysophosphatidylacyltransferase (EC 2.3.1.23); Ca 2+-ATPase (EC 3.6.1.8); lactate dehydrogenase (EC 1.1.1.27); 5-lipoxygenase (EC 1.13.11.34); phospholipase A 2 (EC 3.1.1.4); phospholipase C (EC 3.1.4.3); proteinkinase C (EC 2.7.1.37).