The long term objective of this proposal is to investigate the physiological role of sphingomyeIin (SPH) in plasma, where it is the most abundant phospholipid next to phosphatidylcholine (PC). In contrast to its functions in membrane cholesterol metabolism and signal transduction, the role of SPH in the metabolism of plasma lipoproteins has received little attention, although its concentration is significantly increased in atherosclerosis and aging. Based on preliminary data, the PI proposes that SPH modulates plasma lipolytic activities, reverse cholesterol transport pathways, and lipid peroxidation reactions, thereby preventing excessive turnover and premature degradation of PC and cholesterol.The PI will test the hypothesis that SPH, by virtue of its structural similarities to PC, competitively inhibits not only LCAT, as he previously showed, but also other lipolytic enzymes that hydrolyze PC, such as hepatic lipase and secretory phospholipases A2. He will also investigate the mechanism(s) involved in the inhibition of these activities, by employing monolayer techniques, enzyme kinetics, and structural modifications of the SPH molecule. The role of SPH in the exchange of free cholesterol and cholesteryl ester (CE) between lipoproteins, and in the selective uptake of HDL CE by various cells in culture will be studied by manipulating the SPH concentration of native lipoproteins and recombinant HDL (rHDL) particles. Since the surface concentration of CE determines its transfer rate, the effect of SPH on the partitioning of CE into surface lipids of rHDL will be investigated by [13C] NMR. The possible role of membrane SPH in the function of SR-B1 receptor will also be investigated.The PI proposes to test the novel hypothesis that SPH inhibits lipid peroxidation by retarding the propagation of lipid peroxy radicals and that the increased oxidative susceptibility of small dense LDL, compared to buoyant LDL, is due to the low SPH/PC ratio in the former. He will correlate the oxidizability of various LDL subfractions and synthetic liposomes with their SPH/PC ratios. The mechanism by which SPH inhibits lipid peroxidation will be investigated by using structural analogs of SPH, by oxidizing fluorescent probes which are specifically localized in the surface or core of lipoproteins, and by studying the effects of SPH on membrane fluidity and lateral diffusion rates.The results from these studies should provide new insights into the physiological functions of SPH in lipoproteins, and its possible relevance to atherosclerosis and inflammation.
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