Evidence for prolonged cell-surface contact of acetyl-LDL before entry into macrophages.
Acetyl-LDL stimulates acyl-CoA:cholesterol acyltransferase (ACAT) much more effectively than LDL in mouse peritoneal macrophages. Previous work with another potent ACAT stimulator, beta-VLDL, suggested that atherogenic lipoproteins may use internalization pathways distinct from that of LDL. Brief incubation of fluorescently labeled acetyl-LDL and LDL followed by a short chase period without lipoproteins was used to compare endocytic pathways. LDL was delivered rapidly to perinuclear vesicles, corresponding to late endosomes and lysosomes. A substantial fraction (> 40%) of acetyl-LDL was initially retained in the cell periphery, while the rest was rapidly delivered to late endosomes that also contained LDL. Fluorescence of peripheral 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI)-acetyl-LDL could be quenched by TNBS, indicating accessibility of the peripheral acetyl-LDL to the extracellular space. Quantification of fluorescence intensities demonstrated that > 40% of the cell-associated DiI-acetyl-LDL but only about 10% of DiI-LDL fluorescence was quenchable by TNBS after a 3-minute chase. Fucoidin can efficiently displace DiI-acetyl-LDL bound to cells at 0 degree C. DiI-acetyl-LDL in the TNBS-quenchable peripheral compartments, however, was resistant to fucoidin. Electron microscopy of colloidal gold-acetyl-LDL showed that acetyl-LDL on the cell surface was often associated with microvilli or ruffles. After clearance from the surface, the peripheral acetyl-LDL was also delivered to the late endosomes and lysosomes. These results indicate that a substantial portion of acetyl-LDL enters macrophages through a pathway that initially differs from that of LDL. This pathway involves a prolonged retention of acetyl-LDL on the plasma membrane. This surface retention may affect ACAT activation in macrophages.
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Department of Pathology, Columbia University, College of Physicians and Surgeons, New York, NY, USA.
Tabas
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Leopold
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Jones
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Maxfield
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D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008079:Lipoproteins, VLDL / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000378:metabolism*; D008565:Membrane Proteins; D051379:Mice; D008854:Microscopy, Electron; D011134:Polysaccharides / Q000378:metabolism; D011971:Receptors, Immunologic / Q000378:metabolism; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; D051122:Scavenger Receptors, Class B; D013499:Surface Properties; D013997:Time Factors; D000511:alpha-Macroglobulins / Q000378:metabolism
D008077:Lipoproteins, LDL; D008079:Lipoproteins, VLDL; D008565:Membrane Proteins; D011134:Polysaccharides; D011971:Receptors, Immunologic; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; C454739:Scarb1 protein, mouse; D051122:Scavenger Receptors, Class B; C032629:acetyl-LDL; D000511:alpha-Macroglobulins; C007789:fucoidan