Phosphatidylethanol stimulates calcium-dependent cytosolic phospholipase A(2) activity of a macrophage cell line (RAW 264.7)

The synthesis of inflammation mediators produced from arachidonic acid is regulated primarily by the cellular concentration of free arachidonic acid. Since intracellular arachidonic acid is almost totally present as phospholipid esters, the concentration of intracellular arachidonic acid is primarily dependent on the balance between the release of arachidonic acid from membrane phospholipids and the uptake of arachidonic acid into membrane phospholipids. Cytosolic phospholipase A(2) is a calcium-dependent enzyme that catalyzes the stimulus-coupled hydrolysis of arachidonic acid from membrane phospholipids. Following exposure of macrophages to various foreign or endogenous stimulants, cytosolic phospholipase A(2) is activated. Treatment with these compounds may also stimulate phospholipase D activity, and, in the presence of ethanol, phospholipase D catalyzes the syn thesis of phosphatidylethanol. A cell-free system was used to evaluate the effect of phosphatidylethanol on cytosolic phospholipase A(2) activity. Phosphatidylethanol (0.5 mu M) added to 1-stearoyl-2-[H-3]-arachidonoyl-sn-gly-cero-3-phosphocholine vesicles stimulated cytosolic phospholipase A(2) activity. However, high concentrations (20-100 mu M) of phosphatidylethanol inhibited cytosolic phospholipase Az activity. Phosphatidic acid, the normal phospholipase D product, also stimulated cytosolic phospholipase Ap activity at 0.5 CIM, but had an inhibitory effect on cytosolic phospholipase Ap activity at concentrations of 50 and 100 mu M Ethanol (20-200 mM), the precursor of phosphatidylethanol, added directly to the assay did not alter cytosolic phospholipase A(2) activity. These results suggest that phosphatidylethanol alters the physical properties of the substrate, and at lower concentrations of anionic phospholipids the substrate is more susceptible to hydrolysis. However, at high concentrations, phosphatidylethanol either reverses the alterations in physical properties of the substrate or phosphatidylethanol may be competing as the substrate. Both interactions may result in lower cytosolic phospholipase A(2) activity. Copyright (C) 2000 National Science Council, ROC and S. Karger AG. Basel.