INHIBITION OF INDUCIBLE NITRIC-OXIDE SYNTHASE IN MACROPHAGES BY OXIDIZED LOW-DENSITY LIPOPROTEINS

Uptake of oxidized low-density lipoprotein (LDL) by monocyte/macrophag es to form ''foam'' cells has been implicated in atherogenesis. Activa ted monocyte/macrophages synthesize nitric oxide (NO) from L-arginine. NO is cytotoxic, antiproliferative, and a vasodilator that inhibits p latelet and monocyte adhesion. NO synthase mRNA, protein, and enzyme a ctivity were induced in J774.A1 macrophages activated with lipopolysac charide and gamma interferon. When macrophages were incubated with oxi dized LDL for 24 hours and activated, there was a dose- and time-depen dent inhibition of NO synthesis, assessed as nitrite accumulation in t he media. When activated cells were incubated with nontoxic doses of l ipoprotein (25 mu g/mL), neither native LDL nor acetyl LDL inhibited N O production, whereas oxidized LDL produced 50% inhibition. Levels of enzyme protein were unchanged by Western blot. Inhibition was a functi on of the degree of oxidation of LDL but was independent of cholestery l esterification by the cells. Incubations of oxidized LDL with cells that had been pretreated with dextran sulfate or cytochalasin B yielde d no evidence that inhibition was dependent on the scavenger receptor or directed endocytosis. Kinetic studies of inducible NO synthase from J774.A1 cells that were incubated with increasing doses of oxidized L DL indicated a pattern of noncompetitive inhibition. Inhibition of the enzyme was produced by lipids extracted from oxidized LDL but not by lipids extracted from native LDL. Because phosphatidylcholine (PC) is converted to lysophosphatidylcholine (LPC) during the oxidation of LDL , the effects of LPC were investigated. PC vesicles containing LPC did not inhibit enzyme activity but produced modest reductions in nitrite accumulation from cells. In contrast, PC vesicles had no significant effect. The data indicate that oxidized LDL lipid inhibits the activit y of inducible NO synthase in activated macrophages. NO production by this enzyme and its inhibition by oxidized LDL lipid may influence cel l-to-cell interactions and vasomotor tone in atherosclerotic blood ves sels.