Cellular cholesterol efflux mediated by cyclodextrins. Demonstration Of kinetic pools and mechanism of efflux.
The efflux of cholesterol from cells in culture to cyclodextrin acceptors has been reported to be substantially more rapid than efflux induced by other known acceptors of cholesterol (Kilsdonk, E. P. C., Yancey, P., Stoudt, G., Bangerter, F. W., Johnson, W. J., Phillips, M. C., and Rothblat, G. H. (1995) J. Biol. Chem. 270, 17250-17256). In this study, we compared the kinetics of cholesterol efflux from cells with 2-hydroxypropyl-beta-cyclodextrins and with discoidal high density lipoprotein (HDL) particles to probe the mechanisms governing the remarkably rapid rates of cyclodextrin-mediated efflux. The rate of cholesterol efflux was enhanced by shaking cells growing in a monolayer and further enhanced by placing cells in suspension to achieve maximal efflux rates. The extent of efflux was dependent on cyclodextrin concentration, 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 of cholesterol: a fast pool (half-time (t1/2) approximately 19-23 s) and a slow pool with t1/2 of 15-30 min. Two distinct kinetic pools of cholesterol 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 incubating cells with solutions of cyclodextrins complexed with increasing 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 from cells and model membranes was compared; the activation energies were 7 kcal/mol and 2 kcal/mol, respectively. The equivalent activation energy observed with apo-HDL-phospholipid acceptor particles was 20 kcal/mol. It seems that cyclodextrin molecules are substantially more efficient than phospholipid acceptors, because cholesterol molecules desorbing from a membrane surface can diffuse directly into the hydrophobic core of a cyclodextrin molecule without having to desorb completely into the aqueous phase before being sequestered by the acceptor.
Yancey
P G
PG
Department of Biochemistry, Medical College of Pennsylvania and Hahnemann University, Philadelphia, Pennsylvania 19129, USA.
Rodrigueza
W V
WV
Kilsdonk
E P
EP
Stoudt
G W
GW
Johnson
W J
WJ
Phillips
M C
MC
Rothblat
G H
GH
D000818:Animals; D002250:Carbon Radioisotopes; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D003505:Cyclodextrins / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D007739:L Cells; D008075:Lipoproteins, HDL / Q000302:isolation & purification / Q000378:metabolism; D053440:Lipoproteins, HDL3; D051379:Mice; D008954:Models, Biological; D011865:Radioisotope Dilution Technique; D012867:Skin; D014316:Tritium
D002250:Carbon Radioisotopes; D003505:Cyclodextrins; D008075:Lipoproteins, HDL; D053440:Lipoproteins, HDL3; D014316:Tritium; D002784:Cholesterol