CELLULAR CHOLESTEROL EFFECT MEDIATED BY CYCLODEXTRINS - DEMONSTRATIONOF KINETIC POOLS AND MECHANISM OF EFFLUX

In this study, we compared the kinetics of cholesterol efflux from cel ls with 2-hydroxypropyl-beta-cyclodextrins and with discoidal high den sity lipoprotein (HDL) particles to probe the mechanisms governing the remarkably rapid rates of cyclodextrin-mediated efflux. The rate of c holesterol efflux was enhanced by shaking cells growing in a monolayer and further enhanced by placing cells in suspension to achieve maxima l efflux rates, The extent of efflux was dependent on cyclodextrin con centration, and maximal efflux was observed at concentrations >50 mM. For several cell types, biexponential kinetics of cellular cholesterol efflux were observed, indicating the existence of two kinetic pools o f cholesterol: a fast pool (half-time (t(1/2)) similar to 19-23 s) and a slow pool with t(1/2) of 15-30 min. Two distinct kinetic pools of c holesterol were also observed with model membranes (large unilamellar cholesterol-containing vesicles), implying that the cellular pools are in the plasma membrane, Cellular cholesterol content was altered by i ncubating cells with solutions of cyclodextrins complexed with increas ing levels of cholesterol. The number of kinetic pools was unaffected by raising the cellular cholesterol content, but the size of the fast pool increased, After depleting cells of the fast pool of cholesterol, this pool was completely restored after a 40-min recovery period. The temperature dependence of cyclodextrin-mediated cholesterol efflux fr om cells and model membranes was compared; the activation energies wer e 7 kcal/mol and 2 kcal/mol, respectively. The equivalent activation e nergy observed with apo-HDL-phospholipid acceptor particles was 20 kca l/mol, It seems that cyclodextrin molecules are substantially more eff icient than phospholipid accepters, because cholesterol molecules deso rbing from a membrane surface can diffuse directly into the hydrophobi c core of a cyclodextrin molecule without having to desorb completely into the aqueous phase before being sequestered by the acceptor.