Dual Ca2+ requirement for optimal lipid peroxidation of low density lipoprotein by activated human monocytes.

Abstract

IntroductionThe oxidative modification of LDL seems a key event in atherogenesis and may participate in inflammatory tissue injury. Our previous studies suggested that the process of LDL oxidation by activated human monocytes/macrophages required O-and activity of intracellular lipoxygenase. Herein, we studied the mechanisms involved in this oxidative modification of LDL. In this study, we used the human monocytoid cell line U937 to examine the role of Ca2+ in U937 cell-mediated lipid peroxidation of LDL. U937 cells were activated by opsonized zymosan.Removal of Ca2+ from cell culture medium by EGTA inhibited U937 cell-mediated peroxidation of LDL lipids. Therefore, Ca 2+ influx and mobilization were examined for their influence on U937 cell-mediated LDL lipid peroxidation. Ca2+ channel blockers nifedipine and verapamil blocked both Ca2+ influx and LDL lipid peroxidation by activated U937 cells. The inhibitory effects of nifedipine and verapamil were dose dependent. TMB-8 and ryanodine, agents known to prevent Ca2' release from intracellular stores, also caused a dose-dependent inhibition of LDL lipid peroxidation by activated U937 cells while exhibiting no effect on Ca2+ influx. Thus, both Ca2+ influx through functional calcium channels and Ca2+ mobilization from intracellular stores participate in the oxidative modification of LDL by activated U937 cells. 45Ca2+ uptake experiments revealed profound Ca2+ influx during the early stages of U937 cell activation, however, the Ca2+ ionophore 4-bromo A23187 was un- Oxidized LDL has been shown to be toxic to proliferating target cells in vitro, such as fibroblasts and endothelial cells (1,4,5). If activated monocytes/macrophages were able to oxidize LDL in vivo, the resultant toxic lipoproteins could mediate tissue injury in a variety of inflammatory responses. This may likely occur in the developing atherosclerotic lesion where lipid-laden foam cells of monocyte origin are surrounded by LDL accumulating in high concentration in the vascular interstitial space (5,6). Thus, it appears that phagocyte-derived free radicals, both superoxide anion and lipid peroxyl radicals, oxidize LDL and that the products oflipid peroxidation carried by oxidized LDL can effect more extensive or more distant tissue injury than that directly mediated by monocyte-derived free radicals.Although both the release ofreactive oxygen species by activated human monocytes/macrophages and the requirement for cellular lipoxygenases have been correlated with oxidation of LDL lipids, the precise mechanisms involved in the oxidation are not entirely understood. In particular, little is known regarding the intracellular signaling required for activated monocytes to mediate oxidation of LDL. Studies on the required intracellular signaling can help elucidate the specific mechanisms of activated monocyte-mediated oxidation of LDL and conversion of LDL to cytotoxin as well as suggest optimal means for intervening and preventing this process. tors, complement receptors, and as a stimulator of phagoc...

Role of endogenous ceruloplasmin in low density lipoprotein oxidation by human U937 monocytic cells.

“…The addition of Chelex-100 to zymosan-activated U937 cells suppressed LDL oxidation in a concentrationdependent manner with a maximal inhibition of 80% found at 4 mg/ml of the solid-phase chelator (not shown). This result is consistent with an oxidant role for ceruloplasmin, and in fact for the specific oxidant ceruloplasmin copper, and is also consistent with studies by others showing that chelators of divalent cations block LDL oxidation by other monocytic and leukocytic cells (10,40,41).…”

“…This finding distinguishes these cells from endothelial cells and smooth muscle cells that require exogenous copper or iron salts in the medium to achieve optimal oxidation rates (8,9). One explanation is that monocytic cells may provide their own transition metal ions to maintain high oxidation rates and the inhibition of monocytic cell lipid oxidation by divalent cation chelators is consistent with this idea (10). Alternatively, macrophage-derived hypochlorous acid or nitric oxide may be precursors of highly reactive hydroxyl radicals by metal ion-independent mechanisms (11).…”

Myosin Light Chain Kinase Regulates Capacitative Ca 2+ Entry in Human Monocytes/Macrophages

“…For example, leukocytes adhering to endothelial cells demonstrate multiple Ca 2ϩ transients 4 ; the binding of oxidatively damaged red blood cells by macrophages is almost totally Ca 2ϩ dependent 5 ; and oxidized LDL-induced activation of protein kinase C (PKC) leading to macrophage growth also involves a rise in [Ca 2ϩ ] i . 6 In addition, optimal oxidative modification of LDL by monocytes/macrophages requires Ca 2ϩ release from intracellular stores and Ca 2ϩ entry, 7 and LDL receptor-mediated lipoprotein degradation is also Ca 2ϩ dependent. 8 In many cells, intracellular Ca 2ϩ stores are mobilized after either the production of inositol trisphosphate or blockade of the endoplasmic reticulum (ER) Ca 2ϩ pump, and it has been postulated that Ca 2ϩ store depletion can trigger transmembranous Ca 2ϩ entry, the capacitative Ca 2ϩ entry (CCE) model.…”

Protein Kinase Cα Regulates Human Monocyte O·̄2Production and Low Density Lipoprotein Lipid Oxidation

“…During the preparation procedure, LDL was protected from exposure to light and oxidation (6,7). LDL was filter sterilized and stored in 0.5 mM EDTA.…”