Effects of membrane lipids and -proteins and cytoskeletal proteins on the kinetics of cholesterol exchange between high density lipoprotein and human red blood cells, ghosts and microvesicles

To better understand the effects of plasma membrane lipids and proteins and the cytoskeleton on the kinetics of cellular cholesterol efflux, the effects of (1), selectively depleting either sphingomyelin (SM) or phosphatidylcholine (PC); (2), cross-linking the cytoskeleton, and (3), removing certain cytoskeletal and integral membrane proteins on radiolabelled cholesterol efflux from red blood cells (RBC) have been studied. When RBC were treated with either phospholipase A2 or sphingomyelinase C to hydrolyze either 30-40% of the PC or 40-50% of the SM, respectively, the halftimes (t1/2) for cholesterol efflux to excess HDL3 were not significantly altered, with the values being 4.4 +/- 0.8 h or 3.7 +/- 0.4 h, respectively, compared to 4.6 +/- 0.6 h for control RBC. To investigate the effects of the cytoskeleton on the rate of free cholesterol (FC) desorption from the plasma membrane, the cytoskeletal proteins were cross-linked by either heat-treatment or exposure to diamide and cholesterol efflux from ghosts of these cells was measured. Cross-linking the cytoskeletal proteins by diamide treatment resulted in no significant change in t1/2 for treated (3.6 +/- 0.6 h) compared to control (4.2 +/- 0.4 h) ghosts: this suggests that the cytoskeleton does not play a large role in modulating cholesterol efflux. To investigate the effects of membrane proteins on cholesterol efflux, RBC microvesicles, containing mainly band 3 and 4 proteins and little of the cytoskeletal proteins, such as spectrin (bands 1,2) or actin (band 5), were obtained by incubation with the ionophore A23187. With excess HDL3 present, microvesicles exhibited a t1/2 of 4.2 +/- 1.9 h (compared to the t1/2 of 4.2 +/- 0.4 h for control ghosts). The results described in this paper suggest that neither changing the SM/PC ratio in the membrane nor cross-linking the cytoskeletal proteins nor removing the cytoskeleton changes the t1/2 for cholesterol efflux to excess HDL3. Presumably, the cholesterol-phospholipid interactions are insensitive to these perturbations in membrane structure.