Experiments are proposed to study intracellular processing of lipoproteins by monocyte-derived macrophages cultured from White Carneau pigeons. The hypothesis is that modified LDLs and high capacity low affinity binding of betaVLDL stimulates macropinocytosis. Uptake via the stimulated macropinocytosis rather than internalization by receptor-mediated endocytosis is associated with foam cell formation. Modified LDL stimulated macropinocytosis also causes uptake of co-internalized lipoproteins via fluid-phase, or high capacity, low-affinity binding, or nonspecific binding to the membrane resulting in foam cell formation. These studies will be done by a combination of ultrastructural and biochemical approaches. Ultrastructurally receptor-mediated coated-pit endocytosis will be differentiated from macropinocytosis by localization of colloidal gold conjugated ligands in pits and vesicles (diameter less than or equal to 0.05) and endosomes (diameter less than or equal to 0.2microm) for receptor-mediated endocytosis versus entry into macropinosomes (diameter greater than or equal to 0.5 microm under membrane ruffled surface) for macropinocytosis. Phase-contrast light microscopy will identify the stimulation of membrane ruffling and phase-bright macropinosomes (greater than or equal to 0.5microm). Fluorescent microscopy with co-localization with fluid-phase markers or endocytic markers will be used to study trafficking of lipoproteins in either macropinosomes or endosomes, respectively. Stimulation of the fluid-phase uptake will be measured with 14C-sucrose, lucifer yellow (LY), horseradish peroxidase (HRP), and dextrans Additionally macropinocytosis and endocytosis will be functionally differentiated by wortmannin, nocodazole, hypertonic medium, and cytoplasmic acidification. The mechanism of stimulated uptake and degradation of LDL in the presence of modified LDLs will be examined using the biochemical parameters of binding, degradation, acyl-CoA; cholesterol acyltransferase (ACAT) stimulation and cholesterol loading. The role of receptors and non-specific binding will be determined by competition studies. The proposed studies will provide potential mechanism(s) for native LDL to cause in vitro foam cell formation.
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