Mechanisms and pathways of excess cholesterol removal from intracellular sites of accumulation to extracellular cholesterol acceptors remain poorly defined. To gain further insights, compounds known to affect cellular protein transport pathways were tested for their effects on high density lipoprotein (HDL)-mediated cholesterol efflux from cultured cells enriched with cholesterol. Monensin, nigericin, and brefeldin A inhibited the ability of HDL to decrease cellular cholesterol esterification, stimulate sterol biosynthesis, and promote the efflux of labeled cholesterol and cholesterol mass from fibroblasts and smooth muscle cells. HDL-mediated decrease in cell cholesterol esterification was inhibited up to 80% by these compounds compared with control incubations over an HDL concentration of 5-100 µg/ml and up to 18 h of incubation. Up-regulation of sterol biosynthesis after depletion of cell cholesterol by HDL increased over 10-fold; however, inclusion of monensin or brefeldin A during the incubation completely prevented the increase of sterol biosynthesis by HDL. Efflux of [H]cholesterol to HDL from prelabeled cells was inhibited up to 40% by these compounds, and this effect persisted when cholesterol esterification was blocked. Similarly, monensin and brefeldin A inhibited up to 50% of HDL-mediated cholesterol mass efflux relative to controls. Treatment of cells with cholesterol oxidase demonstrated an increase of intracellular cholesterol after exposure to monensin or nigericin and to a lesser extent with brefeldin A. These data show that monensin, nigericin, and brefeldin A sequester cholesterol from sites normally available for efflux by HDL. Since these compounds act by disruption of Golgi complex structure and function, a role for this intracellular organelle in transport of cholesterol between intracellular sites and the plasma membrane for eventual removal by extracellular acceptors such as HDL is suggested.

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