Remodeling and Shuttling

Abstract

In normal physiology, cells are exposed to cholesterol acceptors of different sizes simultaneously. The current study examined the possible interactions between two different classes of acceptors, one large (large unilamellar phospholipid vesicles, LUVs) and one small (HDL or other small acceptors), added separately or in combination to Fu5AH rat hepatoma cells. During a 24-hour incubation, LUVs of palmitoyl-oleoyl phosphatidylcholine at 1 mg phospholipid (PL) per milliliter extracted ≈20% of cellular unesterified cholesterol (UC) label and mass in a slow, continuous fashion (half-time [t½] for UC efflux was ≈50 hours) and human HDL 3 at 25 μg PL per milliliter extracted ≈15% cellular UC label with no change in cellular cholesterol mass (t½ of ≈8 hours). In contrast, the combination of LUVs and HDL 3 extracted over 90% of UC label (t½ of ≈4 hours) and ≈50% of the UC mass, indicating synergy. To explain this synergy, specific particle interactions were examined, namely, remodeling, in which the two acceptors alter each other's composition and thus the ability to mobilize cellular cholesterol, and shuttling, in which the small acceptor ferries cholesterol from cells to the large acceptor. To examine remodeling, LUVs and HDL were coincubated and reisolated before application to cells. This HDL became UC depleted, PL enriched, and lost a small amount of apolipoprotein A-I. Compared with equivalent numbers of control HDL particles, remodeled HDL caused faster efflux (t½ ≈4 hours) and exhibited a greater capacity to sequester cellular cholesterol over 24 hours (≈38% versus ≈15% for control HDL), consistent with their enrichment in PL. Remodeled LUVs still extracted ≈20% of cellular UC. Thus, remodeling accounted for some but not all of the synergy between LUVs and HDL. To examine shuttling, several approaches were used. First, reisolation of particles after an 8-hour exposure to cells revealed that HDL contained very little of the cellular UC label. The label was found almost entirely with the LUVs, suggesting that LUVs continuously stripped the HDL of cellular UC. Second, bidirectional flux studies demonstrated that LUVs blocked the influx of HDL UC label into cells, while the rate of efflux of cellular UC was maintained. These kinetic effects explained the massive net loss of cellular UC to LUVs with HDL. Third, cyclodextrin, an artificial small acceptor that does not acquire PL and hence does not become remodeled, exhibitedCorrespondence to Dr Michael C. Phillips, Department of Biochemistry, Medical College of Pennsylvania and Hahnemann University, 2900 Queen Ln, Philadelphia, PA 19129. Portions of this work were published in abstract form in Abstracts Submitted to the Council on Arteriosclerosis for the 68th Scientific Sesssions of the American Heart Association. 1995:54. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript substantial synergy with LUVs, supporting shuttling. Thus, the presence of large and small acceptors together can overcome intrinsic deficiencies in...

Mechanism of ATP-binding Cassette Transporter A1-mediated Cellular Lipid Efflux to Apolipoprotein A-I and Formation of High Density Lipoprotein Particles

“…Approximately 20-nm-diameter SUV were prepared by sonication (24), and 100-or 200-nm-diameter LUV were prepared by extrusion through polycarbonate filters (25). Enthalpies of apo A-I binding to unilamellar egg PC vesicles were measured by isothermal titration calorimetry as described (20,24).…”

Clustering Induces Redistribution of Syndecan-4 Core Protein into Raft Membrane Domains

“…Plasma membrane cholesterol depletion was accomplished by pretreatment of cultured cells with ␤-cyclodextrin (10 M), which removes unesterified cholesterol from the cell membrane (19) for 30 min at 37°C immediately followed by chilling the cells to 4°C.…”

Cyclodextrins as catalysts for the removal of cholesterol from macrophage foam cells.

“…In addition, small unilamellar vesicles could be substituted for the LUV. These effects are not specific to macrophages because in studies with Fu5AH hepatoma cells, HDL particles or cyclodextrin were found to be able to shuttle cell cholesterol to LUV (29). mulate cholesterol in both the free and ester pools.…”