Methylamine-treated low density lipoproteins elicit different responses in HepG2 cells and macrophages |
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Authors: | Eugen Koren Nassrin Dashti Paul R. Wilson Diana M. Lee |
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Affiliation: | (1) Lipoprotein and Atherosclerosis Research Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, 73104 Oklahoma City, OK, USA;(2) Department of Medicine, University of Oklahoma Health Sciences Center, 73104 Oklahoma City, OK, USA;(3) Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 73104 Oklahoma City, OK, USA;(4) Present address: Arthritis and Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma, USA;(5) Department of Nutrition Science, Division of Pediatric Gastroenterology and Nutrition, The University of Alabama at Birmingham, 35233 Birmingham, AL;(6) Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, 73104 Oklahoma City, OK, USA |
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Abstract: | Recent results from this laboratory have demonstrated the existence of labile thiolester bonds in apolipoprotein B (ApoB). Thiolester bonds can be cleaved with nucleophiles such as methylamine, resulting in conformational change. The purpose of this study was to explore whether the cellular interactions would be altered after methylamine treatment of low density lipoproteins (LDL). Human hepatoma cells, HepG2, and human monocyte derived macrophages were used for these studies. Fresh LDL were incubated with methylamine under mild alkaline conditions under N2 and with preservatives for 24 h. The methylamine-treated LDL showed particle size and net charge identical to fresh native LDL. In addition, no oxidative modification of LDL occurred under the experimental conditions. The methylamine-treated LDL were indistinguishable from native LDL in HepG2 cells as judged by binding, degradation, cholesterol accumulation andde novo sterol synthesis. However, methylamine-treated LDL caused an increased accumulation of cholesteryl esters in macrophages which was comparable to the accumulation caused by acetylated LDL. Dual color digital imaging fluorescence microscopy revealed no competition between acetylated and methylamine-treated LDL, suggesting that the excessive uptake of methylamine-treated LDL was not mediated by the scavenger receptor. The increased accumulation of cholesteryl ester in macrophages also did not appear to stem from the classical LDL receptor. These results suggest that a new receptor binding domain is exposed due to the conformational change upon treatment of LDL with methylamine. (Mol Cell Biochem124: 67–79, 1993)Abbreviations LDL low density lipoproteins (d 1.032–1.043 g/ml for this study) - ApoB apolipoprotein B - MA methylamine - TBAR thiobarbituric acid reactive - HepG2 human hepatoma cell line - HMG-CoA reductase, -hydroxy--methylglutaryl CoA reductase - DIFM digital imaging fluorescence microscopy - FITC fluorescence isothiocyanate - 2M 2M-macroglobulin - BSA bovine serum albumin - PBS phosphate buffered saline - ACA -amino caproic acid - SDS-PAGE polyacrylamide gel electrophoresis containing SDS - TCA trichloroacetic acid - LRP lipoprotein receptor-related protein |
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Keywords: | apolipoprotein B intramolecular thiolester conformational change cholesteryl ester accumulation receptor |
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