Modulation of particulate nitric oxide synthase activity and peroxynitrite synthesis in cholesterol enriched endothelial cell membranes

Abstract

Endothelium-derived relaxing factor/nitric oxide (EDRF/NO) is produced by the vascular wall and is a key modulator of vascular tone and blood pressure. Since reduced EDRF/NO release from the endothelium is a major key event in the development of atherosclerosis, we investigated the effect of cholesterol on endothelial cell particulate (membrane-bound) NO synthase activity. Low concentrations (up to 0.2 mM) of liposomal cholesterol progressively activated plasma membrane-bound NO synthase. Increasing cholesterol concentration above that which maximally stimulated enzyme activity produced a progressive inhibition with respect to the control value. In time course experiments using endothelial cell plasma membranes enriched with cholesterol, changes in NO production were followed by analogous changes in soluble guanylate cyclase activity (sGC). N-Monomethyl-L-arginine (L-NMMA) (1 mM) inhibited particulate NO synthase activity at all cholesterol concentrations used with subsequent decreases in cGMP production. Egg lecithin liposomes (free of cholesterol) had no effect on NO synthase activity. A three-fold increase in Superoxide (O 2 −) and a 2.5 fold increase in NO formation followed by an eight-fold increase in peroxynitrite (ONOO −) production by cholesterol-treated microsomes isolated from endothelial cells was observed, one which rose further up to eight-fold in the presence of Superoxide dismutase (SOD) (10 U/mL). Cholesterol had no effect on Lubrol-PX solubilized membrane-bound NO synthase or on cytosolic (soluble) NO synthase activities of endothelial cells. Cholesterol modulated lipid fluidity of plasma membranes labelled with 1,6-diphenyl-1, 3,5-hexatriene (DPH) as indicated by the steady state fluorescence anisotropy [( r o r ) − 1] − 1 . Arrhenius plots of [( r o r ) − 1] −1 indicated that the lipid phase separation of the membranes at 26.2 ± 1.5 ° was elevated to 34.4 ± 1.9 ° in cholesterol-enriched membranes, consistent with a general decrease in membrane fluidity. Cholesterol-enriched plasma membranes treated with egg lecithin liposomes showed a lipid phase separation at 27.5 ± 1.6 °, indicating the reversible effect of cholesterol on membrane lipid fluidity. Arrhenius plots of NO synthase activity exhibited break point at 26.9 ± 1.8 ° which rose to 35.6 ± 2.1 ° in 0.5 mM cholesterol-treated plasma membranes and decreased to 21.5 ± 1.4 ° in plasma membranes treated with 0.2 mM cholesterol. The allosteric properties of plasma membrane-bound NO synthase inhibited by Mn 2+ (as reflected by changes in the Hill coefficient) were changed by cholesterol, consistent with modulations of the fluidity of the lipid microenvironment of the enzyme. Our findings suggest that incorporation of high concentrations of cholesterol into endothelial cell membranes causes down-regulation of NO synthase by producing an increased packing of bulk lipids. In contrast, cholesterol incorporation at low concentrations up-regulates NO synthase by increasing the fluidity of the lipid microenvironment of the enzyme. The present studies concerning the behaviour of particulate NO synthase and rate of NO release with respect to the structure and function of the biomembranes provide important new clues as to the role of this fascinating molecule in atherosclerosis.