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• W2022690810 abstract "We recently reported that the rate of efflux of cholesterol from cells to high density lipoprotein (HDL) was related to the expression level of scavenger receptor class B type I (SR-BI). Moreover, the expression of this receptor in atheromatous arteries raises the possibility that SR-BI mediates cholesterol efflux in the arterial wall (Ji, Y., Jian, B., Wang, N., Sun, Y., de la Llera Moya, M., Phillips, M. C., Rothblat, G. H., Swaney, J. B., and Tall, A. R. (1997) J. Biol. Chem. 272, 20982–20985). In this paper we describe studies that suggest that the presence of phospholipid on acceptor particles plays an important role in modulating interaction with the SR-BI. Specifically, enrichment of serum with phospholipid resulted in marked stimulation of cholesterol efflux from cells that had higher levels of SR-BI expression, like Fu5AH or Y1-BS1 cells, and little or no stimulation in cells with low SR-BI levels, such as Y-1 cells. Stimulation of efflux by phospholipid enrichment was also a function of SR-BI levels in Chinese hamster ovary cells transfected with the SR-BI gene. Efflux to protein-free vesicles prepared with 1-palmitoyl-2-oleoylphosphatidyl-choline also correlated with SR-BI levels, suggesting that phospholipid, as well as protein, influences the interaction that results in cholesterol efflux. By contrast, cholesterol efflux from a non-cell donor showed no stimulation consequent to phospholipid enrichment of the serum acceptor. These results may help to explain observations in the literature that document an increased risk of atherosclerosis in patients with depressed levels of HDL phospholipid even in the face of normal HDL cholesterol levels. We recently reported that the rate of efflux of cholesterol from cells to high density lipoprotein (HDL) was related to the expression level of scavenger receptor class B type I (SR-BI). Moreover, the expression of this receptor in atheromatous arteries raises the possibility that SR-BI mediates cholesterol efflux in the arterial wall (Ji, Y., Jian, B., Wang, N., Sun, Y., de la Llera Moya, M., Phillips, M. C., Rothblat, G. H., Swaney, J. B., and Tall, A. R. (1997) J. Biol. Chem. 272, 20982–20985). In this paper we describe studies that suggest that the presence of phospholipid on acceptor particles plays an important role in modulating interaction with the SR-BI. Specifically, enrichment of serum with phospholipid resulted in marked stimulation of cholesterol efflux from cells that had higher levels of SR-BI expression, like Fu5AH or Y1-BS1 cells, and little or no stimulation in cells with low SR-BI levels, such as Y-1 cells. Stimulation of efflux by phospholipid enrichment was also a function of SR-BI levels in Chinese hamster ovary cells transfected with the SR-BI gene. Efflux to protein-free vesicles prepared with 1-palmitoyl-2-oleoylphosphatidyl-choline also correlated with SR-BI levels, suggesting that phospholipid, as well as protein, influences the interaction that results in cholesterol efflux. By contrast, cholesterol efflux from a non-cell donor showed no stimulation consequent to phospholipid enrichment of the serum acceptor. These results may help to explain observations in the literature that document an increased risk of atherosclerosis in patients with depressed levels of HDL phospholipid even in the face of normal HDL cholesterol levels. The deposition of lipids in vessel walls and the development of atheromas are thought to be opposed by removal of the cholesterol and transport back to the liver for secretion, a process known as reverse cholesterol transport (1Fielding C.J. Curr. Opin. Lipidol. 1991; 2: 376-378Crossref Scopus (30) Google Scholar). Efflux of cellular free cholesterol from peripheral cells to the acceptor particles is the first step of this process. Although the mechanisms of cellular cholesterol efflux remain to be clarified, certain classes of high density lipoproteins (HDL) 1The abbreviations used are: HDL, high density lipoprotein; LDL, low density lipoprotein; SR-BI, scavenger receptor class B type I; CHO, Chinese hamster ovary; CHO-high, high SR-BI expression CHO cells; DMPC, dimyristoylphosphatidylcholine; FBS, fetal bovine serum; MβCD, methyl-β-cyclodextrin; MLV, multilamellar vesicles; PL, phospholipid; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; SUV, small unilamellar vesicles; CMV, cytomegalovirus. are believed to play a key role in this process (2Oram J.F. Albers J.J. Cheung M.C. Bierman E.L. J. Biol. Chem. 1981; 256: 8348-8356Abstract Full Text PDF PubMed Google Scholar, 3Fielding C.J. Fielding P.E. J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar). Two general models have been proposed regarding cholesterol removal from cells: a nonspecific, aqueous diffusion pathway and a specific pathway that may involve a cell surface receptor (3Fielding C.J. Fielding P.E. J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar, 4Johnson W.J. Mahlberg F.H. Rothblat G.H. Phillips M.C. Biochim. Biophys. Acta. 1991; 1085: 273-298Crossref PubMed Scopus (389) Google Scholar, 5Rothblat G.H. Mahlberg F.H. Johnson W.J. Phillips M.C. J. Lipid Res. 1992; 33: 1091-1098Abstract Full Text PDF PubMed Google Scholar, 6Oram J.F. Yokoyama S. J. Lipid Res. 1996; 37: 2473-2491Abstract Full Text PDF PubMed Google Scholar). In the aqueous diffusion model, cholesterol molecules first desorb from the cell membrane and are then incorporated into acceptor particles after traversing the intervening aqueous layer by diffusion. The theoretical aspects of this mechanism have been extensively reviewed (4Johnson W.J. Mahlberg F.H. Rothblat G.H. Phillips M.C. Biochim. Biophys. Acta. 1991; 1085: 273-298Crossref PubMed Scopus (389) Google Scholar, 7Phillips M.C. Johnson W.J. Rothblat G.H. Biochim. Biophys. Acta. 1987; 906: 223-276Crossref PubMed Scopus (424) Google Scholar). Although this model was derived from the studies of free cholesterol transport between model membranes (unilamellar vesicles) (8Phillips M.C. McLean L.R. Stoudt G.W. Rothblat G.H. Atherosclerosis. 1980; 36: 409-422Abstract Full Text PDF Scopus (79) Google Scholar), the kinetics of cholesterol transfer from living cells to phospholipid-containing acceptors can be adequately described by this model (9Rothblat G.H. Phillips M.C. J. Biol. Chem. 1982; 257: 4775-4782Abstract Full Text PDF PubMed Google Scholar). In this process, free cholesterol transfers between donor and acceptors bidirectionally and, for net cholesterol removal to occur, a concentration gradient must be established between the cell surface and the acceptors. This gradient depends upon many properties of both the cell membrane and cholesterol acceptors, such as the cholesterol and phospholipid content, the arrangement of the cholesterol domains within the cell membrane, and the sizes or numbers of acceptors present in the aqueous solution (4Johnson W.J. Mahlberg F.H. Rothblat G.H. Phillips M.C. Biochim. Biophys. Acta. 1991; 1085: 273-298Crossref PubMed Scopus (389) Google Scholar, 10Johnson W.J. Mahlberg F.H. Chacko G.K. Phillips M.C. Rothblat G.H. J. Biol. Chem. 1988; 263: 14099-14106Abstract Full Text PDF PubMed Google Scholar, 11Mahlberg F.H. Glick J.M. Lund-Katz S. Rothblat G.H. J. Biol. Chem. 1991; 266: 19930-19937Abstract Full Text PDF PubMed Google Scholar, 12Mahlberg F.H. Rothblat G.H. J. Biol. Chem. 1992; 267: 4541-4550Abstract Full Text PDF PubMed Google Scholar, 13Davidson W.S. Lund-Katz S. Johnson W.J. Anantharamaiah G.M. Palgunachari N. Sergrest J.P. Rothblat G.H. Phillips M.C. J. Biol. Chem. 1994; 269: 22975-22982Abstract Full Text PDF PubMed Google Scholar, 14Davidson W.S. Gillotte K.L. Lund-Katz S. Johnson W.J. Rothblat G.H. Phillips M.C. J. Biol. Chem. 1995; 270: 5882-5890Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 15Davidson W.S. Rodrigueza W.V. Lund-Katz S. Johnson W.J. Rothblat G.H. Phillips M.C. J. Biol. Chem. 1995; 270: 17106-17113Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). When an acceptor particle has a large capacity to absorb cholesterol (a cholesterol “sink”) and is present in excess, the rate-limiting step for cholesterol removal is desorption of cholesterol from the plasma membrane (4Johnson W.J. Mahlberg F.H. Rothblat G.H. Phillips M.C. Biochim. Biophys. Acta. 1991; 1085: 273-298Crossref PubMed Scopus (389) Google Scholar). The second model involves a pathway by which an acceptor promotes cholesterol efflux by interaction with cell surface sites (3Fielding C.J. Fielding P.E. J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar, 6Oram J.F. Yokoyama S. J. Lipid Res. 1996; 37: 2473-2491Abstract Full Text PDF PubMed Google Scholar, 16Oram J.F. Johnson C.J. Brown T.A. J. Biol. Chem. 1987; 262: 2405-2410Abstract Full Text PDF PubMed Google Scholar,17Hara H. Yokoyama S. J. Biol. Chem. 1991; 266: 3080-3086Abstract Full Text PDF PubMed Google Scholar). Lipid-free apoA-I or lipid-poor pre-β-HDL particles are believed to operate by this mechanism. Through reversible interactions with the plasma membrane, apoA-I not only solubilizes cholesterol and phospholipid directly from the plasma membrane but also stimulates mobilization of pools of cholesterol that are readily accessible to esterification by acyl-CoA:cholesterol acyltransferase (16Oram J.F. Johnson C.J. Brown T.A. J. Biol. Chem. 1987; 262: 2405-2410Abstract Full Text PDF PubMed Google Scholar, 18Oram J.F. Mendez A.J. Slotte J.P. Johnson T.F. Arterioscler. Thromb. 1991; 11: 403-414Crossref PubMed Scopus (140) Google Scholar, 19Oram J.F. Arteriosclerosis. 1983; 3: 420-432Crossref PubMed Google Scholar, 20Mendez A.J. Oram J.F. Bierman E.L. J. Biol. Chem. 1991; 266: 10104-10111Abstract Full Text PDF PubMed Google Scholar), an enzyme localized to the rough endoplasmic reticulum. Both lipid-free apoA-I and lipid-poor pre-β-HDL particles presumably function effectively as natural shuttles to transport cholesterol between cells and larger α-HDL, since they have a relatively lower cholesterol capacity compared with α-HDL (1Fielding C.J. Curr. Opin. Lipidol. 1991; 2: 376-378Crossref Scopus (30) Google Scholar, 21Atger V.M. de la Llera Moya M. Stoudt G.W. Rodrigueza W.V. Phillips M.C. Rothblat G.H. J. Clin. Invest. 1997; 99: 773-780Crossref PubMed Scopus (161) Google Scholar). Receptor-mediated endocytosis plays an important role in lipoprotein metabolism. Although the LDL receptor is well characterized, the metabolism of HDL by any receptor-mediated pathway is still unclear. Several cell membrane proteins have been described that bind to HDL (for reviews, see Refs. 4Johnson W.J. Mahlberg F.H. Rothblat G.H. Phillips M.C. Biochim. Biophys. Acta. 1991; 1085: 273-298Crossref PubMed Scopus (389) Google Scholar and 6Oram J.F. Yokoyama S. J. Lipid Res. 1996; 37: 2473-2491Abstract Full Text PDF PubMed Google Scholar), but the physiological implications are obscure. Recently, a member of the scavenger receptor family, the class B scavenger receptor SR-BI, was shown to bind HDL with high affinity (22Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2010) Google Scholar). This receptor can also bind to many other ligands, such as native LDL, modified proteins (acetylated LDL, oxidized LDL, maleylated bovine serum albumin), and anionic phospholipids (23Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar, 24Rigotti A. Acton S. Krieger M. J. Biol. Chem. 1995; 270: 16221-16224Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). However, unlike other scavenger receptors such as the LDL scavenger receptor, which binds to its ligand and internalizes the whole particle, SR-BI in certain cells can bind HDL reversibly and mediate selective cholesteryl ester uptake, leaving the HDL particles largely intact (22Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2010) Google Scholar). SR-BI has highest expression in the adrenal gland, ovary, testis, and liver, where the selective uptake is important to the function of these tissues (25Glass C. Pittman R.C. Weinstein D.B. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5435-5439Crossref PubMed Scopus (423) Google Scholar, 26Glass C. Pittman R.C. Civen M. Steinberg D. J. Biol. Chem. 1985; 260: 744-750Abstract Full Text PDF PubMed Google Scholar). It has also been demonstrated that in vivo the level of SR-BI is under feedback regulation in response to changes of cholesterol stores (27Wang N. Weng W. Breslow J.L. Tall A.R. J. Biol. Chem. 1996; 271: 21001-21004Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Recently, work in our laboratories showed that SR-BI overexpression can markedly enhance the bi-directional cholesterol flux in transfected CHO cells. A strong correlation between the rate of efflux to HDL or serum and the expression levels of SR-BI was also observed in several other cell lines (28Ji Y. Jian B. Wang N. Sun Y. de la Llera Moya M. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. J. Biol. Chem. 1997; 272: 20982-20985Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar). Furthermore, we found that SR-BI mRNA is expressed in the thickened intima of atheromatous aorta, suggesting a potential role of SR-BI in cholesterol flux in the arterial wall. Since SR-BI binds to a variety of lipoproteins that contain different apolipoprotein components and binds to anionic phospholipids (23Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar, 24Rigotti A. Acton S. Krieger M. J. Biol. Chem. 1995; 270: 16221-16224Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar), it is possible that phospholipids on the surface of lipoprotein particles play an important role in mediating the binding. Therefore, in this paper, we correlated the ability of PL to potentiate efflux, which was either incorporated into the lipoprotein fractions of serum or into small unilamellar vesicles, with the expression levels of SR-BI in various cell types. Pooled human serum was obtained from normolipemic volunteers. The HDL3 fraction was isolated from the 1.125–1.21 g/ml fraction of serum by sequential ultracentrifugation (29Hatch F.T. Lees R.S. Adv. Lipid Res. 1968; 6: 1-68Crossref PubMed Google Scholar) and dialyzed against phosphate-buffered saline. Aliquots of HDL3 stock solution (10 mg of protein/ml; 4.8 mg of PL/ml) were stored at −70 °C and thawed before use. Dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) were purchased from Avanti Polar Lipids, Inc. 5,5′-dithiobis(2-nitrobenzoic acid) was purchased from Pierce. [1,2-3H]cholesterol was purchased from NEN Life Science Products. Leupeptin, aprotinin, pepstatin A, phenylmethylsulfonylfluoride, and gentamicin were purchased from Sigma. Methyl-β-cyclodextrin (MβCD) was either purchased from Sigma or was a gift from Cerestar USA, Inc. (Hammond, IN). Eagle's minimal essential medium, Dulbecco's modified Eagle's medium, Ham's F-12, Ham's F-10, RPMI 1640, fetal bovine serum (FBS), calf serum, horse serum, and geneticin were purchased from Life Technologies, Inc. The stock concentration of MβCD was 5 mm in culture medium and was diluted to a final concentration of 0.05 mm. Sandoz compound 58035 was a gift from Dr. John Heider. Tissue culture flasks and plates were obtained from Corning Glass Works (Corning, NY). DMPC was dissolved in methanol, and the solvent was evaporated under a stream of N2 at room temperature; any remaining solvent was removed by vacuum overnight. Buffer (150 mm NaCl, 10 mmTris, pH 7.4) was added, and the sample was warmed to 40–50 °C. The lipid was then vortexed vigorously for 1 min, placed in a low power sonication bath for 10 min, and vortexed again for 30 s to obtain turbid dispersions of multilamellar vesicles. POPC-SUV were made using the sonication technique as described previously (9Rothblat G.H. Phillips M.C. J. Biol. Chem. 1982; 257: 4775-4782Abstract Full Text PDF PubMed Google Scholar). POPC in chloroform was dried under a stream of N2 to form a thin film on a test tube wall and then placed in a vacuum for 2 h to remove any remaining solvent. Buffer (150 mm NaCl, 10 mm Tris, 1 mmEDTA, pH 7.4) was added to bring the concentration of phospholipid to 10 mg/ml and vortexed to generate MLV. SUV were prepared from MLV by sonicating at 0 °C for 15 min followed by a 1-min cooling period for several cycles until the dispersion was almost clear. After sonication, the vesicle dispersion was centrifuged for 2 h at 40,000 rpm (Beckman 50 Ti fixed angle rotor) to remove any titanium particles and large multilamellar vesicles. Serum was heated at 56 °C for 30 min in the presence of 2 mm5,5′-dithiobis(2-nitrobenzoic acid) to inactivate complement, which is toxic to some cell types, such as L cells (30Fedoroff S. Doerr J. J. Natl. Cancer Inst. 1962; 29: 331-353PubMed Google Scholar) and to inhibit lecithin:cholesterol acyltransferase. The serum was then incubated with DMPC-MLV at 24 °C for 2 h at a ratio of 4 mg of DMPC/ml of serum. After incubation, aliquots were removed and frozen at −70 °C. The phospholipid concentration for serum and DMPC-modified serum were 1.5 and 4.8 mg/ml, respectively. Since our preliminary studies showed that freezing and heat treatment did not influence the ability of serum or DMPC-modified serum to remove cholesterol from Fu5AH cells, a large amount of serum or DMPC-modified serum were prepared and used throughout this study to minimize the experimental variability. The murine SR-BI cDNA (28Ji Y. Jian B. Wang N. Sun Y. de la Llera Moya M. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. J. Biol. Chem. 1997; 272: 20982-20985Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar) was subcloned into a mammalian expression vector pRc/CMV (Invitrogen) and transfected into Chinese hamster ovary (CHO) cells by electroporation as described previously (28Ji Y. Jian B. Wang N. Sun Y. de la Llera Moya M. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. J. Biol. Chem. 1997; 272: 20982-20985Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar). Stable transformants with moderate SR-BI overexpression (CHO-low) were selected with 0.8 mg/ml geneticin and maintained with 0.3 mg/ml in Ham's F-12 containing 5% FBS. The high expression cells (CHO-high) were obtained by further screening the original geneticin-resistant pool with fluorescence-activated cell sorting using HDL labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyaninepercholate (Molecular Probes, Inc.). Rat hepatoma cells (Fu5AH) were grown in Eagle's minimal essential medium supplemented with 5% calf serum. Mouse L cell fibroblasts were grown in Eagle's minimal essential medium supplemented with 10% FBS. Two lines of mouse adrenal tumor cells, Y1 and Y1-BS1, which differ in their expression levels of SR-BI (28Ji Y. Jian B. Wang N. Sun Y. de la Llera Moya M. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. J. Biol. Chem. 1997; 272: 20982-20985Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar) were grown in Ham's F-10 with 12.5% horse serum and 5% FBS. Chinese hamster ovary cells (CHO-K1) were grown in Ham's F-12 with 7.5% FBS, and three transfected CHO cells were grown in Ham's F-12 with 5% FBS. Mouse J774 macrophages were grown in RPMI 1640 with 10% FBS. Mouse RAW 2.1 macrophages were grown in Dulbecco's modified Eagle's medium with 10% FBS. All media were supplemented with 50 mg/ml gentamicin except for the transfected CHO cells, which were supplemented with geneticin at a concentration of 0.3 mg/ml. Cells were plated in 24-well plates 3 days before the experiment and labeled for 2 days with 1–2 μCi of [3H]cholesterol in 1 ml of growth medium to obtain confluent radiolabeled cell monolayers. Before the experiments, the labeling medium was replaced with equilibrating medium for 18 h to allow the equilibration of labeled cholesterol among cellular pools. Sandoz 58035, an inhibitor of acyl-CoA:cholesterol acyltransferase, was present at a concentration of 1 μg/ml during the labeling, equilibration, and efflux periods in all experiments to prevent sequestration of the label in an intracellular cholesteryl ester pool. More than 95% total cholesterol was in free cholesterol form in all cell types after the experiments. Y1-BS1 cells were provided by Dr. David L. Williams. The cholesterol efflux ability of the serum, DMPC-modified serum, MβCD-supplemented serum, HDL3, and POPC-SUV were evaluated by methods described previously (31de la Llera Moya M. Atger V. Paul J.L. Fournier N. Moatti N. Giral P. Friday K.E. Rothblat G.H. Arterioscler. Thromb. 1994; 14: 1056-1065Crossref PubMed Google Scholar). Cholesterol acceptors included serum or DMPC-modified serum, which were customarily diluted in efflux medium to 5% that of the original concentration; SUV was diluted to 1 mg/ml and HDL3 was diluted to 75 μg/ml with medium before the experiment. In some samples, MβCD was added to the 5% serum before the experiment at a concentration of 0.05 mm. The different cholesterol acceptors were incubated with [3H]cholesterol-labeled cells for various time periods at 37 °C, and 100-μl aliquots were taken to monitor efflux at indicated time points. These aliquots were filtered through 0.45-μm Multiscreen (96 screens) filtration plates (Millipore), and 75-μl aliquots were then counted to determine the release of labeled cholesterol from the cells. The total [3H]cholesterol in the cells was extracted with isopropanol from dried cell monolayers that had been washed with phosphate-buffered saline. In some experiments, cholesterol-impregnated glass fiber filters (Corning) were used as an inert cholesterol donor. Filters were cut to fit multiwell plates, 50 μl of an ethanol cholesterol solution was applied to each filter, and the filters were dried overnight. Each filter contained 0.5 μCi of [3H]cholesterol and 5 μg of cholesterol mass, which is similar to the average cholesterol content of the cell monolayers used in the efflux assays. The filters were then exposed to 2 ml of culture medium containing the various acceptors tested with the cells. The amount of radiolabeled cholesterol released to the medium was expressed as the fraction of the total radioactive cholesterol present initially in the cells or filters; culture medium without added acceptors was used as a control for all experiments. The net movement of cholesterol mass between cells and acceptors was not monitored in this study. Each data point was the result of triplicate determinations from one or two different experiments. Cells grown in monolayers were homogenized by a N2 cavitation technique using N2 (300 p.s.i) for 30 min before the release of pressure. Protease inhibitors (0.5 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin A, 0.2 mm phenylmethylsulfonyl fluoride and 1 mm EDTA) were present in the homogenizing buffer (100 mm Tris). Cell membranes were collected by ultracentrifugation (50 Ti rotor, 32,000 rpm for 1 h), and proteins were resolved by SDS-PAGE. Immunoblotting was performed as described previously (28Ji Y. Jian B. Wang N. Sun Y. de la Llera Moya M. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. J. Biol. Chem. 1997; 272: 20982-20985Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar) using anti-serum to rodent SR-BI (27Wang N. Weng W. Breslow J.L. Tall A.R. J. Biol. Chem. 1996; 271: 21001-21004Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar) followed by chemiluminescence detection (Amersham Pharmacia Biotech) and densitometric quantitation. The efflux data were analyzed as originally proposed by Johnson et al. (32Johnson W.J. Bamberger M.J. Latta R.A. Rapp P.E. Phillips M.C. Rothblat G.H. J. Biol. Chem. 1986; 261: 5766-5776Abstract Full Text PDF PubMed Google Scholar) and as described in detail for this system by Davidson et al. (13Davidson W.S. Lund-Katz S. Johnson W.J. Anantharamaiah G.M. Palgunachari N. Sergrest J.P. Rothblat G.H. Phillips M.C. J. Biol. Chem. 1994; 269: 22975-22982Abstract Full Text PDF PubMed Google Scholar). Briefly, the kinetic analysis assumed a closed system where unesterified, free cholesterol exists in one of two kinetic pools, the cellular or the acceptor pool. The equilibration of cholesterol between these two pools was fitted to the single exponential equation: Y =H1 e −gt +H2. Y represents the fraction of radiolabeled cholesterol remaining in the cells, t is the incubation time, H1 is a pre-exponential term that reflects the fraction of cellular free cholesterol that is lost to the medium at steady state, g is the sum of the rate constants for efflux (ke) and influx (ki), and H2 is a constant that represents the fraction of labeled cell cholesterol that remains associated with the cells at equilibrium due to a constant retrograde flux of free cholesterol from the extracellular acceptor back into the cells. H1, g, and H2 are variables that were fitted to the experimental data by nonlinear regression. The half-times of efflux were calculated as t1/2 = ln 2/ke, where ke = g ×H1. Vmax and EC50 values for the efflux of cholesterol from CHO cells (Fig. 7) were calculated using the equation y =Vmax × x /[EC50 +x]. To compare the ability of different cells to donate cholesterol to various acceptors, human serum, HDL3and POPC-SUV were incubated with [3H]cholesterol-labeled cell monolayers for 8 h. Fig. 1shows the time course of cholesterol efflux from two cell types that represent two extreme patterns of efflux. It can be seen that after 8 h of incubation 40% of free cholesterol was released from Fu5AH cells to serum, whereas only about 20% was released from Y1 cells. Efflux to HDL3 and SUV is also faster in Fu5AH cells compared with Y1 cells. POPC-SUV, at a concentration of 1 mg of PL/ml of efflux medium, was able to promote cholesterol efflux, comparable to HDL3 at 75 μg of protein/ml. These data agree with previous work from this laboratory that demonstrated that the Fu5AH cells release free cholesterol much faster than other cells (33DeLamatre J. Wolfbauer G. Phillips M.C. Rothblat G.H. Biochim. Biophys. Acta. 1986; 875: 419-428Crossref PubMed Scopus (44) Google Scholar). The results obtained from Fu5AH and Y1 cells suggested that similar efflux patterns were obtained whether the acceptors were serum, HDL3, or POPC-SUV. To examine this further, a variety of cell types were studied that spanned a range of efflux from fast to slow. Serum (5%), HDL3 (75 μg of protein/ml) or POPC-SUV (1 mg/ml) were incubated with cholesterol-labeled cells for 8 h at 37 °C, and aliquots were removed at various time points. Rate constants (k) for efflux to different cholesterol acceptors were obtained from the single exponential curve fit to the time course data. As shown in Fig. 2, there is a very strong linear correlation (r 2 = 0.98) between efflux to serum and to HDL3 in all the cell types examined. Surprisingly, a strong correlation was also observed between serum and POPC-SUV with a correlation coefficient of 0.94, which suggests that a common cellular component may be involved in mediating the cholesterol efflux to serum and POPC-SUV. This finding is consistent with our previous data that HDL-PL is the component of HDL that best reflects the efflux efficiency of the serum (34Fournier N. Paul J. Atger V. de la Llera Moya M. Rothblat G.H. Moatti N. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2685-2691Crossref PubMed Scopus (139) Google Scholar, 35Fournier N. de la Llera Moya M. Burkey B. Swaney J.B. Paterniti J.J. Moatti N. Atger V. Rothblat G.H. J. Lipid Res. 1996; 37: 1704-1711Abstract Full Text PDF PubMed Google Scholar). Recently, the scavenger receptor SR-BI was found to bind HDL with high affinity (22Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2010) Google Scholar). Previously, data from our laboratories have suggested that this receptor might contribute to cholesterol efflux by promoting bi-directional flux (28Ji Y. Jian B. Wang N. Sun Y. de la Llera Moya M. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. J. Biol. Chem. 1997; 272: 20982-20985Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar). Having characterized the cholesterol efflux properties of a variety of cells, the SR-BI expression level was measured by preparing cell membrane proteins and total RNA for Western blot and RNase protection assays, followed by densitometry of chemiluminescence images; mouse liver membranes were used as a control in all assays. As shown in Fig. 3, the expression level of SR-BI was found to vary significantly among different cell types and appears to be correlated with the efflux of cell cholesterol to both serum and POPC-SUV. Panel A shows a combination of data previously reported for serum as acceptor (28Ji Y. Jian B. Wang N. Sun Y. de la Llera Moya M. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. J. Biol. Chem. 1997; 272: 20982-20985Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar), to which additional analyses and cell types have been added; the plot shows the saturation effect at high levels of SR-BI (>1,000 units), whereas the inset emphasizes the linear relationship at low levels of SR-BI; similar results were found for POPC-SUV (panel B). Note that Y1 cells, although adrenal-derived, were found to have slow efflux in response to serum, HDL, or PL vesicles (Fig. 1) and very low SR-BI expression, whereas Fu5AH cells have the highest rate of efflux and SR-BI levels among various cell types studied (Fig. 3). Another line of the adrenal-derived Y1 cells (Y1-BS1) had a moderate SR-BI expression level and intermediate efflux values (Fig. 3). Previously we reporte" @default.
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