QUANTITATION OF THE POOL OF CHOLESTEROL ASSOCIATED WITH ACYL-COA - CHOLESTEROL ACYLTRANSFERASE IN HUMAN FIBROBLASTS

The esterification of cholesterol in homogenates of human fibroblasts was explored as a means of estimating the size of the pool of choleste rol associated with the endoplasmic reticulum (ER) in vivo. The ration ale was that the acyl-coenzyme A:cholesterol acyltransferase (ACAT) in homogenates should have access only to cholesterol associated with th e (rough) ER membrane fragments in which it resides. Reacting whole ho mogenates to completion with an excess of [C-14]oleoyl-CoA converted s imilar to 0.1-2% of total cell free cholesterol to [C-14]cholesteryl e sters. Control studies indicated that membranes not associated with AC AT did not contribute cholesterol to this reaction. The extent of in v itro cholesterol esterification varied with pretreatment of the cells. Exposing intact cells to serum lipoproteins, oxysterols, or sphingomy elinase increased cholesterol esterification in homogenates severalfol d; exposing the cells to mevinolin or cholesterol oxidase had the oppo site effect. The variation in cholesterol esterification did not corre late with either the total cellular cholesterol or the intrinsic activ ity of ACAT, neither of which was changed significantly by the pretrea tments. Rather, the total amount of cholesterol esterified in homogena tes paralleled the rate of cholesterol esterification in the correspon ding intact cells. The pool of cholesterol esterified in vitro therefo re ap pears to reflect that associated with the ER in vivo. Since seve ral of the mechanisms keeping cell cholesterol under tight feedback co ntrol are themselves located in the ER, this pool might not only be re gulated physiologically, but could, in turn, help to regulate homeosta tic effector pathways.