u-18666a and cholesteryl-oleate

The objective of this work was to develop a cell-free system for studying the transfer of cholesterol from lysosomes to membrane acceptor particles. The methods involved: 1) loading of CHO cells at 15 degrees C with [3H]cholesteryl oleate-reconstituted LDL, such that it accumulated undegraded in endosomes; 2) homogenization of cells, followed by preparation of an endosome-lysosome donor fraction; 3) incubation of the donor fraction at 37 degrees C in a defined cytosol-like medium containing acceptor particles of egg phosphatidylcholine small unilamellar vesicles (PC-SUV); and 4) measurement of cholesteryl oleate (CO) hydrolysis and transfer of the resulting free cholesterol (FC) to vesicles. During cell-free incubation, LDL-loaded endosomes fused with lysosomes leading to the lysosomal hydrolysis of LDL cholesteryl ester. Maximal hydrolysis of approximately 50% was achieved in 4-8 h. This hydrolysis was inhibited by lysosomotropic agents, proton ionophores, or removal of ATP and GTP from the medium, indicating that it took place in sealed lysosomes. In the absence of PC-SUV, the release of LDL-derived FC from lysosomes was "< or =" 10%/8 h. This was increased to a maximum of 25-30%/8 h at 3 mg/ml of PC-SUV. In contrast, the release of undegraded CO was 5-15%/8 h and not stimulated by PC-SUV, suggesting that the transfer of FC to PC-SUV was selective and not due to the uncontrolled release of lysosomal contents. In comparisons between CHO-K1 cells and sterol transport-defective CHO(2-2) cells, lysosomes from the latter cell were 35% less efficient as donors of cholesterol for transfer to egg phosphatidylcholine small unilamellar vesicles, indicating that these methods reproduce an important aspect of sterol trafficking in cells. In addition, this result suggests that the mutation in CHO(2-2) has a direct effect on the lysosomes of these cells.

Topics: Acetylglucosaminidase; Androstenes; Animals; Anticholesteremic Agents; Biological Transport; Calcium; Cell-Free System; CHO Cells; Cholesterol; Cholesterol Esters; Cricetinae; Hydrolysis; Imipramine; Lipoproteins, LDL; Liposomes; Lysosomes; Nucleotides; Phosphatidylcholines; Subcellular Fractions

Lysosomal accumulation of unesterified (free) cholesterol, following the phagocytic incorporation of cholesteryl oleate lipid droplets, was quantitatively characterized in a murine J774 macrophage foam cell model. The induction of phagocytic incorporation by the macrophages, using an inverted culture technique, allowed the rapid delivery of large amounts of cholesteryl ester droplets to the lysosomes, leading to the subsequent generation of free cholesterol. The lysosomally generated free cholesterol was differentiated from the membrane cholesterol by a double radiolabeling procedure. Free cholesterol accumulation was quantitated in a population of low density lipid-filled lysosomes prepared by ultracentrifugal isolation of a floating lipid fraction from a homogenate of the cholesteryl ester-loaded cells. About 10% of the total N-acetyl-beta-glucosaminidase activity, a lysosomal marker, was recovered in the lipid fraction. Negligible amounts of alkaline phosphodiesterase-1, a plasma membrane marker, or membrane cholesterol were present in this fraction. Electron microscopic and cytochemical analysis of the isolated lipid fraction revealed the presence of lysosomes in the fraction with a diameter ranging from 1.5 to 4 microns. Continued hydrolysis of incorporated cholesteryl ester over a 24-h incubation resulted in approximately 30% of the generated free cholesterol in lipid-filled lysosomes. The accumulation of free cholesterol occurred whether or not the cholesterol esterifying enzyme, acyl-CoA: cholesterol acyltransferase, was inhibited. In addition, substantial amounts of free cholesterol accumulated even in the presence of efficient cholesterol acceptor particles, apolipoprotein high density lipoprotein-phosphatidylcholine complexes which stimulate cholesterol efflux. Also, increased accumulation of free cholesterol in the lipid fraction was observed when cholesteryl ester-loaded cells were treated with the compound U-18666A which blocks the movement of lysosomal cholesterol. The data demonstrate that the phagocytic incorporation and hydrolysis of cholesteryl ester lipid droplets by macrophage foam cells lead to a substantial accumulation of free cholesterol in the lipid-filled lysosomes. This process could result in a build-up of lysosomal free cholesterol in macrophage foam cells during the progression of atherosclerotic plaque.

Topics: Androstenes; Animals; Anticholesteremic Agents; Carbon Radioisotopes; Cell Line; Cell Membrane; Cholesterol; Cholesterol Esters; Dimethyl Sulfoxide; Foam Cells; Kinetics; Lysosomes; Membrane Lipids; Mice; Microscopy, Electron; Phagocytosis; Tritium

Fluorescent microscopic examination of fibroblasts cultured with low density lipoprotein (LDL) and progesterone (10 micrograms/ml) for 24 h revealed extensive filipin-cholesterol staining of perinuclear lysosomes. Levels of unesterified cholesterol were 2-fold greater than in fibroblasts cultured with LDL alone. Progesterone strongly blocked cholesteryl ester synthesis. When cellular uptake of LDL was monitored in the presence of 58035, a specific inhibitor of acyl-CoA:cholesterol acyltransferase, excess unesterified cholesterol was not stored in lysosomes. Discontinuation of LDL uptake in conjunction with progesterone washout markedly reversed the filipin-cholesterol staining of lysosomes. Reversal of the lysosomal cholesterol lipidosis was associated with a rapid burst of cholesteryl ester synthesis and a normalization of the cellular levels of free and esterified cholesterol. In contrast to normal cells, progesterone removal from Niemann-Pick C fibroblasts did not reverse the lysosomal cholesterol accumulation of these mutant cultures. The metabolic precursor of progesterone, pregnenolone, also induced extensive accumulation of cholesterol in lysosomes. Other steroids induced less vacuolar cholesterol accumulation in the following decreasing order: corticosterone and testosterone, promegestone, RU 486. The relative inhibition of cellular cholesterol esterification by the steroids paralleled their respective abilities to sequester cholesterol in lysosomes rather than their inhibition of acyl-CoA:cholesterol acyltransferase activity in cell-free extracts. The progesterone-related inhibition and restoration of lysosomal cholesterol trafficking is a useful experimental means of studying intracellular cholesterol transport. A particularly important feature of its utility is the facile reversibility of the steroid-induced block. The lysosomal cholesterol lipidosis established with a hydrophobic amine, U18666A, was not as readily reversed.

Topics: Androstenes; Anticholesteremic Agents; Cells, Cultured; Cholesterol; Cholesterol Esters; Corticosterone; Fibroblasts; Humans; Lipoproteins, LDL; Lysosomes; Microscopy, Fluorescence; Mifepristone; Niemann-Pick Diseases; Oleic Acid; Oleic Acids; Progesterone; Reference Values; Testosterone