Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, blocks lysosomal cholesterol trafficking in macrophages.
Certain steroids having an oxo group at the C-17 or C-20 position such as pregnenolone and dehydroisoandrosterone inhibit the cholesterol transport from lysosomes to other cellular sites. Taking advantage of the fact that the inhibition is reversed upon removal of the steroids, we studied the factors that control the cholesterol transport from lysosomes to other cellular sites in macrophages. Macrophages that accumulated unesterified cholesterol in their lysosomes were prepared by incubating cells with liposomes containing cholesterol and phosphatidylserine in the presence of a steroid inhibitor. These cells were chased by means of steroid washout, and then the effects of various pharmacological agents on the subsequent metabolism of cholesterol were examined. When the cells were chased in the absence of the agents, some of the cholesterol was converted to cholesteryl esters in the cells, and others were desorbed into the medium as unesterified forms, suggesting recovery of lysosomal cholesterol trafficking. Among the agents tested, bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, completely blocked both cholesterol esterification and cholesterol desorption at 10 nM. Moreover, agents that neutralize the lysosomal proton gradient, such as ammonium chloride and chloroquine, also reduced both of the processes. Fluorescent microscopic examination of bafilomycin A1-treated cells revealed extensive filipin-cholesterol staining of perinuclear lysosomes. From these data, we conclude that acidic pH is required for the efflux of cholesterol from lysosomes to other cellular sites.
Furuchi
T
T
Department of Health Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Aikawa
K
K
Arai
H
H
Inoue
K
K
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001692:Biological Transport; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism*; D003078:Colchicine / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism; D018942:Macrolides; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D011284:Pregnenolone / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors*; D014617:Vacuoles / Q000201:enzymology*
D000900:Anti-Bacterial Agents; D018942:Macrolides; D003571:Cytochalasin B; D011284:Pregnenolone; C040929:bafilomycin A1; D002784:Cholesterol; D006180:Proton-Translocating ATPases; D003078:Colchicine