CHOLESTEROL TRANSPORT FROM PLASMA-MEMBRANES TO INTRACELLULAR MEMBRANES IS INHIBITED BY 3-BETA-[2-(DIETHYLAMINO)ETHOXY]ANDROST-5-EN-17-ONE

The compound U18666A 3beta-[2-(diethylamino)ethoxylandrost-5-en-17-one ) has been shown to inhibit the cellular transfer of low-density lipop rotein-derived cholesterol from lysosomes to plasma membranes (Liscum and Faust (1989) J. Biol. Chem. 264, 11796-806). We have in this study examined the effects of U18666A on cholesterol translocation from pla sma membranes to intracellular membranes. Translocation of plasma memb rane cholesterol was induced by degradation of plasma membrane sphingo myelin. The sphingomyelinase-induced activation of the acyl-CoA choles terol acyl transferase (ACAT) reaction was completely inhibited in a d ose-dependent manner by U18666A, both in cultured human skin fibroblas ts and baby hamster kidney cells. Half-maximal inhibition (within 60 m in) was obtained with 0.5-1 mug/ml of U18666A. A time-course study ind icated that the onset of inhibition was rapid (within 10-15 min), and reversible if U18666A was removed from the incubation mixture. Using a cholesterol oxidase assay, we observed that the extent of plasma memb rane cholesterol translocation in sphingomyelinase-treated HSF cells w as significantly lowered in the presence of U18666A (at 3 mug/ml). The effect of U18666A on cholesterol translocation was also fully reversi ble when the drug was withdrawn. In mouse Leydig tumor cells, labeled to constant specific activity with [H-3]cholesterol, the compound U186 66A inhibited in a dose-dependent manner the cyclic AMP-stimulated sec retion of [H-3]steroid hormones. The effects seen with compound U18666 A appeared to be specific for this molecule, since another hydrophobic amine, imipramine, did not in our experiments affect cholesterol tran slocation or ACAT activation. Since different cell types display sensi tivity to U18666A in various intracellular cholesterol transfer proces ses, they appear to have a common U18666A-sensitive regulatory mechani sm.