u-18666a and 7-ketocholesterol

Macrophages are well recognized as key pathophysiologic agents in many chronic inflammatory diseases, especially atherosclerosis. During atherogenesis process, low density lipoproteins (LDL) undergo oxidation (oxLDL) and become highly atherogenic as they induce a strong accumulation of cholesterol in subendothelial macrophages leading to the formation of foam cells, the major cellular component of fatty streaks. OxLDL are enriched in oxidation products of cholesterol called oxysterols involved in the regulation of cholesterol homeostasis, by their ability to induce cellular oxidative stress and cytotoxicity. Little is known about intracellular oxysterol production in macrophages. Using both radiochemical and mass analyzes, we showed that THP1 macrophages promote the intracellular oxidation of LDL derived-cholesterol as well as intracellular cholesterol, this later mechanism being enhanced by exposure with native or oxLDL. We demonstrated that in both THP1 and Raw 267.4 cells cholesterol oxidation occurs in the late endosomal compartment. Most oxysterols were produced by non-enzymatic routes (7-ketocholesterol and 7α/β-hydroxycholesterol) but enzymatically formed 7α-, 27-hydroxycholesterol were also quantified. Incubation of THP1 macrophages with nLDL or oxLDL, induced a 2- and 100-fold increase in oxysterol production, respectively. Both oxysterols derived from LDL cholesterol and cellular cholesterol were readily exported to HDL whereas apoA1 was inefficient, showing that HDL plays a major role in the removal of excess oxysterols in THP1 macrophages.

Topics: Androstenes; Animals; Apolipoprotein A-I; Cells, Cultured; Cholesterol; Endosomes; Humans; Intracellular Space; Ketocholesterols; Lipoproteins, HDL; Lipoproteins, LDL; Macrophages; Mice; Oxidation-Reduction; RAW 264.7 Cells

The hypocholesterolemic agent 3 beta-(2-diethylaminoethoxy)androst-5-en-17-one . HCl (U18666A) is known to induce experimental epilepsy. The possibility that this drug interferes with cholesteryl ester formation in glioblastoma cells was examined. The incorporation of radioactive oleic acid into cellular cholesteryl ester was drastically and specifically inhibited by U18666A. The inhibitory effect of U18666A persisted in different oleic acid concentrations. Kinetic studies revealed the rapidity of U18666A action. U18666A was found to be ineffective in inhibiting acyl-CoA:cholesterol acyltransferase activity when it was added directly to the cell homogenates. In contrast, the acyltransferase activity was greatly diminished in homogenates derived from U18666A-treated cells. Thus, U18666A appeared to block cellular cholesteryl ester biosynthesis by indirectly inactivating acyl-CoA:cholesterol acyltransferase activity in a cell-dependent manner. The potent inhibition of cholesteryl ester formation by U18666A represents one unique aspect of the drug which might contribute to its ability to induce chronic epileptiform activity.

Topics: Androstenes; Animals; Anticholesteremic Agents; Cell Line; Cholesterol Esters; Desmosterol; Glioma; Ketocholesterols; Neuroglia; Oleic Acid; Oleic Acids; Rats; Squalene; Sterol O-Acyltransferase; Time Factors