Effects of Enrichment of Fibroblasts with Unesterified Cholesterol on the Efflux of Cellular Lipids to Apolipoprotein A-I
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m108268200
ISSN1083-351X
AutoresKristin Gillotte-Taylor, Margaret Nickel, William J. Johnson, Omar L. Francone, Paul Holvoet, Sissel Lund‐Katz, George H. Rothblat, Michael C. Phillips,
Tópico(s)Lipid metabolism and biosynthesis
ResumoThis study elucidates the factors underlying the enhancement in efflux of human fibroblast unesterified cholesterol and phospholipid (PL) by lipid-free apolipoprotein (apo) A-I that is induced by cholesterol enrichment of the cells. Doubling the unesterified cholesterol content of the plasma membrane by incubation for 24 h with low density lipoprotein and lipid/cholesterol dispersions increases the pools of PL and cholesterol available for removal by apoA-I from about 0.8–5%; the initial rates of mass release of cholesterol and PL are both increased about 6-fold. Expression of the ATP binding cassette transporter A1 (ABCA1) is critical for this increased efflux of lipids, and cholesterol loading of the fibroblasts over 24 h increases ABCA1 mRNA about 12-fold. The presence of more ABCA1 and cholesterol in the plasma membrane results in a 2-fold increase in the level of specific binding of apoA-I to the cells with no change in binding affinity. Characterization of the species released from either control or cholesterol-enriched cells indicates that the plasma membrane domains from which lipids are removed are cholesterol-enriched with respect to the average plasma membrane composition. Cholesterol enrichment of fibroblasts also affects PL synthesis, and this leads to enhanced release of phosphatidylcholine (PC) relative to sphingomyelin (SM); the ratios of PC to SM solubilized from control and cholesterol-enriched fibroblasts are ∼2/1 and 5/1, respectively. Biosynthesis of PC is critical for this preferential release of PC and the enhanced cholesterol efflux because inhibition of PC synthesis by choline depletion reduces cholesterol efflux from cholesterol-enriched cells. Overall, it is clear that enrichment of fibroblasts with unesterified cholesterol enhances efflux of cholesterol and PL to apoA-I because of three effects, 1) increased PC biosynthesis, 2) increased PC transport via ABCA1, and 3) increased cholesterol in the plasma membrane. This study elucidates the factors underlying the enhancement in efflux of human fibroblast unesterified cholesterol and phospholipid (PL) by lipid-free apolipoprotein (apo) A-I that is induced by cholesterol enrichment of the cells. Doubling the unesterified cholesterol content of the plasma membrane by incubation for 24 h with low density lipoprotein and lipid/cholesterol dispersions increases the pools of PL and cholesterol available for removal by apoA-I from about 0.8–5%; the initial rates of mass release of cholesterol and PL are both increased about 6-fold. Expression of the ATP binding cassette transporter A1 (ABCA1) is critical for this increased efflux of lipids, and cholesterol loading of the fibroblasts over 24 h increases ABCA1 mRNA about 12-fold. The presence of more ABCA1 and cholesterol in the plasma membrane results in a 2-fold increase in the level of specific binding of apoA-I to the cells with no change in binding affinity. Characterization of the species released from either control or cholesterol-enriched cells indicates that the plasma membrane domains from which lipids are removed are cholesterol-enriched with respect to the average plasma membrane composition. Cholesterol enrichment of fibroblasts also affects PL synthesis, and this leads to enhanced release of phosphatidylcholine (PC) relative to sphingomyelin (SM); the ratios of PC to SM solubilized from control and cholesterol-enriched fibroblasts are ∼2/1 and 5/1, respectively. Biosynthesis of PC is critical for this preferential release of PC and the enhanced cholesterol efflux because inhibition of PC synthesis by choline depletion reduces cholesterol efflux from cholesterol-enriched cells. Overall, it is clear that enrichment of fibroblasts with unesterified cholesterol enhances efflux of cholesterol and PL to apoA-I because of three effects, 1) increased PC biosynthesis, 2) increased PC transport via ABCA1, and 3) increased cholesterol in the plasma membrane. Plasma high density lipoprotein (HDL) 1The abbreviations used are: HDLhigh density lipoproteinrHDLreconstituted HDLLDLlow density lipoproteinABCA1ATP binding cassette transporter A1apoapolipoproteinCDcyclodextrinFCfree (unesterified) cholesterolMEMminimal essential mediumPBSphosphate-buffered salt solutionPCphosphatidylcholinePLphospholipidSMsphingomyelin levels are inversely correlated with the onset of coronary artery disease, and this protective effect is due at least in part to the role of HDL in mediating cholesterol transport from peripheral cells to sites of reutilization and degradation in the reverse cholesterol transport process (for a review, see Ref. 1.Barter P.J. Rye K.-A. Atherosclerosis. 1996; 121: 1-12Abstract Full Text PDF PubMed Scopus (342) Google Scholar). HDL and its main protein moiety, apolipoprotein (apo) A-I, drive the initial unesterified (free) cholesterol (FC) efflux from the periphery as well as the subsequent esterification of the cholesterol by lecithin-cholesterol acyltransferase (2.Fielding C.J. Fielding P.E. J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar, 3.Oram J.F. Yokoyama S. J. Lipid Res. 1996; 37: 2473-2491Abstract Full Text PDF PubMed Google Scholar) and selective uptake of the cholesteryl ester in the liver by scavenger receptor class B type 1 (SR-B1) (4.Rigotti A. Trigatti B.L. Penman M. Rayburn H. Herz J. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12610-12615Crossref PubMed Scopus (761) Google Scholar). HDL comprises a very heterogeneous class of particles, particularly with respect to the particle size, density, protein content, and surface charge; the structure of the particles affects their efficiency in participating in the various steps of the reverse cholesterol transport pathway (1.Barter P.J. Rye K.-A. Atherosclerosis. 1996; 121: 1-12Abstract Full Text PDF PubMed Scopus (342) Google Scholar). high density lipoprotein reconstituted HDL low density lipoprotein ATP binding cassette transporter A1 apolipoprotein cyclodextrin free (unesterified) cholesterol minimal essential medium phosphate-buffered salt solution phosphatidylcholine phospholipid sphingomyelin The efflux of cellular FC is initiated by one of two distinct mechanisms, depending on the degree of lipidation of the apoA-I (5.Phillips M.C. Gillotte K.L. Haynes M.P. Johnson W.J. Lund-Katz S. Rothblat G.H. Atherosclerosis. 1998; 137: 13-17Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 6.Gillotte K.L. Davidson W.S. Lund-Katz S. Rothblat G.H. Phillips M.C. J. Lipid Res. 1998; 39: 1918-1928Abstract Full Text Full Text PDF PubMed Google Scholar). Fully lipidated apoA-I or mature HDL species have been established to mediate this process through passive diffusion. In this aspect of FC efflux, FC molecules spontaneously desorb from the plasma membrane, diffuse through the aqueous phase surrounding the cell, and incorporate into an HDL particle upon collision. This so-called aqueous diffusion mechanism has been extensively described in the literature (for a review, see Ref. 7.Phillips M.C. Johnson W.J. Rothblat G.H. Biochim. Biophys. Acta. 1987; 906: 223-276Crossref PubMed Scopus (424) Google Scholar). This simple diffusion process can be facilitated by the presence of scavenger receptor class B, type I (SR-B1) in the plasma membrane of the cell (8.Rothblat G.H. de la Llera-Moya M. Atger V. Kellner-Weibel G. Williams D.L. Phillips M.C. J. Lipid Res. 1999; 40: 781-796Abstract Full Text Full Text PDF PubMed Google Scholar). ApoA-I molecules that have dissociated from HDL particles and exist in a lipid-free (poor) state (9.Barter P.J. Rye K. Curr. Opin. Lipidol. 1996; 7: 82-87Crossref PubMed Scopus (171) Google Scholar) mediate cellular phospholipid (PL) and FC efflux by an active mechanism involving interaction of apoA-I with the cell surface (3.Oram J.F. Yokoyama S. J. Lipid Res. 1996; 37: 2473-2491Abstract Full Text PDF PubMed Google Scholar, 10.Yokoyama S. Biochim. Biophys. Acta. 1998; 1392: 1-15Crossref PubMed Scopus (108) Google Scholar). The resulting protein-lipid complexes enlarge in size through the further incorporation of lipids and interaction with lecithin-cholesterol acyltransferase as they progress through the reverse cholesterol transport pathway (2.Fielding C.J. Fielding P.E. J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar). The efflux of cellular lipids to incompletely lipidated apoA-I molecules is a critical pathway for supply of PL to the HDL pool. Recent studies of Tangier disease have shown that mutations that inactivate the ATP binding cassette transporter 1 (ABCA1) are the cause of the low HDL levels in this disease (for a review, see Ref. 11.Oram J.F. Lawn R.M. J. Lipid Res. 2001; 42: 1173-1179Abstract Full Text Full Text PDF PubMed Google Scholar). ABCA1 is critical for efflux of cellular PL and FC to apoA-I because FC-loaded Tangier disease fibroblasts are defective in this regard (12.Oram J.F. Vaughan A.M. Curr. Opin. Lipidol. 2000; 11: 253-260Crossref PubMed Scopus (243) Google Scholar). This behavior is in striking contrast to that of normal human fibroblasts, which exhibit significant stimulation of FC and PL efflux when FC-loaded (13.Bielicki J.K. Johnson W.J. Weinberg R.B. Glick J.M. Rothblat G.H. J. Lipid Res. 1992; 33: 1699-1709Abstract Full Text PDF PubMed Google Scholar, 14.Remaley A.T. Farsi B.D. Shirali A.C. Hoeg J.M. Brewer H.J. J. Lipid Res. 1998; 39: 1231-1238Abstract Full Text Full Text PDF PubMed Google Scholar, 15.Lawn R.M. Wade D.P. Garvin M.R. Wang X. Schwartz K. Porter J.G. Seilhamer J.J. Vaughan A.M. Oram J.F. J. Clin. Invest. 1999; 104: 25-31Crossref PubMed Scopus (658) Google Scholar). The above observations are consistent with a key role for ABCA1 in PL and FC efflux because expression of ABCA1 in fibroblasts is up-regulated by cholesterol loading (15.Lawn R.M. Wade D.P. Garvin M.R. Wang X. Schwartz K. Porter J.G. Seilhamer J.J. Vaughan A.M. Oram J.F. J. Clin. Invest. 1999; 104: 25-31Crossref PubMed Scopus (658) Google Scholar, 16.Fielding P.E. Nagao K. Hakamata H. Chimini G. Fielding C.J. Biochemistry. 2000; 39: 14113-14120Crossref PubMed Scopus (183) Google Scholar). Expression of ABCA1 can be regulated by ligands for the nuclear receptor LXR (17.Venkateswaran A. Laffitte B.A. Joseph S.B. Mak P.A. Wilpitz D.C. Edwards P.A. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12097-12102Crossref PubMed Scopus (848) Google Scholar) and by cAMP (18.Oram J.F. Lawn R.M. Garvin M.R. Wade D.P. J. Biol. Chem. 2000; 275: 34508-34511Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). The appearance of the transporter at the cell surface enhances apoA-I binding and efflux of cellular PL and FC (18.Oram J.F. Lawn R.M. Garvin M.R. Wade D.P. J. Biol. Chem. 2000; 275: 34508-34511Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar, 19.Wang N. Silver D.L. Costet P. Tall A.R. J. Biol. Chem. 2000; 275: 33053-33058Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 20.Abe-Dohmae S. Suzuki S. Wada Y. Aburatani H. Vance D.E. Yokoyama S. Biochemistry. 2000; 39: 11092-11099Crossref PubMed Scopus (101) Google Scholar). Comparison of different cell types indicates that there is a general correlation between expression of ABCA1 and lipid efflux to apoA-I (21.Bortnick A.E. Rothblat G.H. Stoudt G. Hoppe K.L. Royer L.J. McNeish J. Francone O.L. J. Biol. Chem. 2000; 275: 28634-28640Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). ABCA1 is likely to be involved in PL and FC transport to the cell surface because related transporters translocate PL molecules across membranes (22.Smit J.J. Schinkel A.H. Oude E. Groen A.K. Wagenaar E. van Deemter L. Mol C.A. Ottenhoff R. van der Lugt N.M. van Roon M.A. Cell. 1993; 75: 451-462Abstract Full Text PDF PubMed Scopus (1336) Google Scholar). However, FC loading of cells up-regulates expression of other proteins likely to modulate lipid flux within cells. Thus, caveolin mRNA levels are up-regulated by FC loading of fibroblasts, and the rate of FC efflux is proportional to the caveolin mRNA levels (23.Fielding C.J. Bist A. Fielding P.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3753-3758Crossref PubMed Scopus (219) Google Scholar). Likewise, FC loading of macrophages and fibroblasts stimulates phosphatidylcholine (PC) biosynthesis and up-regulation of CTP:phosphocholine cytidyltransferase (24.Tabas I. Marathe S. Keesler G.A. Beatini N. Shiratori Y. J. Biol. Chem. 1996; 271: 22773-22781Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 25.Leppimaki P. Mattinen J. Slotte J.P. Eur. J. Biochem. 2000; 267: 6385-6394Crossref PubMed Scopus (26) Google Scholar). Active trafficking of lipid vesicles between the plasma membrane and the cell interior is critical because disruption of Golgi membranes by agents such as brefeldin (26.Mendez A.J. J. Lipid Res. 1997; 38: 1807-1821Abstract Full Text PDF PubMed Google Scholar, 27.Remaley A.T. Schumacher U.K. Stonik J.A. Farsi B.D. Nazih H. Brewer H.J. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1813-1821Crossref PubMed Scopus (191) Google Scholar) and of calcium-dependent retroendocytosis (28.Takahashi Y. Smith J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11358-11363Crossref PubMed Scopus (207) Google Scholar) inhibits apoA-I-mediated FC and PL efflux. In sum, it seems clear that a continuing supply of PL and FC to the exofacial leaflet of the plasma membrane is required for efficient efflux to apoA-I. In prior studies (6.Gillotte K.L. Davidson W.S. Lund-Katz S. Rothblat G.H. Phillips M.C. J. Lipid Res. 1998; 39: 1918-1928Abstract Full Text Full Text PDF PubMed Google Scholar) using human skin fibroblasts containing normal levels of FC, we examined the kinetics of efflux of cellular FC and PL to lipid-free apoA-I (pre-β-HDL). It was shown that in a 10-min incubation FC and PL are released together and that a finite pool of about 1% of plasma membrane cholesterol is available. This process was termed membrane microsolubilization and defined as follows. "This process involves apolipoprotein-mediated solubilization into the extracellular medium of a small amount of plasma membrane PL and FC; the mechanism is facilitated by interaction of apolipoprotein with the plasma membrane and occurs without obvious cellular damage" (6.Gillotte K.L. Davidson W.S. Lund-Katz S. Rothblat G.H. Phillips M.C. J. Lipid Res. 1998; 39: 1918-1928Abstract Full Text Full Text PDF PubMed Google Scholar). Subsequently, the features of the apolipoprotein required for membrane microsolubilization were investigated (29.Gillotte K.L. Zaiou M. Lund-Katz S. Anantharamaiah G.M. Holvoet P. Dhoest A. Palgunachari M.N. Segrest J.P. Weisgraber K.H. Rothblat G.H. Phillips M.C. J. Biol. Chem. 1999; 274: 2021-2028Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). It was concluded that the mechanism of apoA-I-mediated FC efflux involves two essential features, 1) the membrane lipids must be organized in a manner that allows insertion of α-helices among the lipids, and 2) the α-helices must exhibit sufficient binding affinity to induce the apolipoprotein-lipid association. It now seems apparent that ABCA1 activity is critical for the first feature, but the sizes and composition of the particles released by apoA-I remain to be established. FC loading of fibroblasts can have direct effects on plasma membrane composition and structure by altering the FC/PL ratio and indirect effects via up-regulation of ABCA1 (15.Lawn R.M. Wade D.P. Garvin M.R. Wang X. Schwartz K. Porter J.G. Seilhamer J.J. Vaughan A.M. Oram J.F. J. Clin. Invest. 1999; 104: 25-31Crossref PubMed Scopus (658) Google Scholar, 16.Fielding P.E. Nagao K. Hakamata H. Chimini G. Fielding C.J. Biochemistry. 2000; 39: 14113-14120Crossref PubMed Scopus (183) Google Scholar). To better define these contributions, we have examined how FC enrichment of fibroblasts affects FC and PL efflux to apoA-I. Since FC loading of fibroblasts has opposite effects on PC and sphingomyelin (SM) biosynthesis (25.Leppimaki P. Mattinen J. Slotte J.P. Eur. J. Biochem. 2000; 267: 6385-6394Crossref PubMed Scopus (26) Google Scholar, 30.Leppimaki P. Kronqvist R. Slotte J.P. Biochem. J. 1998; 335: 285-291Crossref PubMed Scopus (40) Google Scholar), the contribution of PL synthesis to ABCA1-mediated release of lipids to apoA-I was investigated. Enrichment of the cells with FC enhances efflux of FC because of increases in both the amount of FC in the plasma membrane and the expression of ABCA1; the latter effect combined with increased PC biosynthesis stimulates release of PC. 1-Palmitoyl-2-oleoylphosphatidylcholine and bovine brain sphingomyelin were purchased from Avanti Polar Lipids (Birmingham, AL) (+99% grade). [1,2-3H]cholesterol (43.5 Ci/mmol), [4-14C]cholesterol (51.3 mCi/mmol), and [methyl-3H]choline chloride (81 Ci/mmol) were obtained from PerkinElmer Life Sciences. Minimal essential medium and phosphate-buffered saline (PBS) were purchased from BioWhittaker (Walkersville, MD). Bovine calf and fetal serum were supplied by Invitrogen. Methyl-β-cyclodextrin and 2-hydroxyl-propyl-β-cyclodextrin were generous gifts from Cerestar (Hammond, IN). All other reagents were purchased from Sigma. HDL3 and LDL were isolated from fresh plasma obtained from normolipidemic donors by sequential ultracentrifugation, and apoA-I was obtained from HDL as described previously (6.Gillotte K.L. Davidson W.S. Lund-Katz S. Rothblat G.H. Phillips M.C. J. Lipid Res. 1998; 39: 1918-1928Abstract Full Text Full Text PDF PubMed Google Scholar, 29.Gillotte K.L. Zaiou M. Lund-Katz S. Anantharamaiah G.M. Holvoet P. Dhoest A. Palgunachari M.N. Segrest J.P. Weisgraber K.H. Rothblat G.H. Phillips M.C. J. Biol. Chem. 1999; 274: 2021-2028Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). A previously described (29.Gillotte K.L. Zaiou M. Lund-Katz S. Anantharamaiah G.M. Holvoet P. Dhoest A. Palgunachari M.N. Segrest J.P. Weisgraber K.H. Rothblat G.H. Phillips M.C. J. Biol. Chem. 1999; 274: 2021-2028Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) apoA-I deletion mutant (Δ190–243) in which a C-terminal segment was removed was used in selected experiments. Reconstituted HDL (rHDL) particles containing 100:1 (mol:mol) 1-palmitoyl-2-oleoylphosphatidylcholine/apoA-I) were prepared and purified using the cholate dispersion/Bio-Bead removal technique as described in detail previously (31.Gillotte K.L. Davidson W.S. Lund-Katz S. Rothblat G.H. Phillips M.C. J. Biol. Chem. 1996; 271: 23792-23798Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Normal human skin GM3468A fibroblasts (passages 2:1, mol:mol), 50 μg of protein/ml human LDL, 2 mg/ml fatty acid-free bovine serum albumin, and either 10–20 μCi/ml [3H]cholesterol or 20 μCi/ml [3H]choline chloride. After this preparation, the monolayers (35–50 μg of cell protein/well) were washed 4× with MEM containing HEPES (50 mm) (MEM-HEPES), and 1 ml of an acceptor preparation was applied for FC or PL efflux measurements. The cellular content of ABCA1 mRNA was determined by Northern analysis as described previously (21.Bortnick A.E. Rothblat G.H. Stoudt G. Hoppe K.L. Royer L.J. McNeish J. Francone O.L. J. Biol. Chem. 2000; 275: 28634-28640Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). MEM devoid of choline was prepared for experiments designed to understand the importance of PL synthesis in apoA-I-mediated FC efflux. In these studies all manipulations were carried out in parallel plates with media either containing or lacking choline. Control and FC-enriched fibroblasts were grown in the choline-deficient media for 3–6 days; this treatment resulted in decreases of cellular PL phosphorus relative to protein of about one-third. This result indicated that incubation of the fibroblasts in choline-free medium inhibited PC synthesis as expected (24.Tabas I. Marathe S. Keesler G.A. Beatini N. Shiratori Y. J. Biol. Chem. 1996; 271: 22773-22781Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In some experiments, methyl-β-cyclodextrin (CD)·FC complexes at a final CD concentration of 5 mm (CD:cholesterol (mol:mol) = 12:1) were utilized to rapidly enrich the monolayers 2–3-fold with FC (32.Christian A.E. Haynes M.P. Phillips M.C. Rothblat G.H. J. Lipid Res. 1997; 38: 2264-2272Abstract Full Text PDF PubMed Google Scholar). In one experiment 2-hydroxy-propyl-β-CD was utilized to ensure that effects were not specific for methyl-β-cyclodextrin; in this case the CD solution was prepared at 25 mm with a CD:cholesterol molar ratio of 40:1. The results with 2-hydroxypropyl-β-CD were identical to those obtained with methyl-β-cyclodextrin treatments. FC and PL efflux measurements were conducted as described before (6.Gillotte K.L. Davidson W.S. Lund-Katz S. Rothblat G.H. Phillips M.C. J. Lipid Res. 1998; 39: 1918-1928Abstract Full Text Full Text PDF PubMed Google Scholar, 29.Gillotte K.L. Zaiou M. Lund-Katz S. Anantharamaiah G.M. Holvoet P. Dhoest A. Palgunachari M.N. Segrest J.P. Weisgraber K.H. Rothblat G.H. Phillips M.C. J. Biol. Chem. 1999; 274: 2021-2028Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 33.Davidson W.S. Lund-Katz S. Johnson W.J. Anantharamaiah G.M. Palgunachari M.N. Segrest J.P. Rothblat G.H. Phillips M.C. J. Biol. Chem. 1994; 269: 22975-22982Abstract Full Text PDF PubMed Google Scholar). The kinetic models used to analyze the measured time courses of FC and PL efflux from fibroblasts are presented under "." As described before for this system (33.Davidson W.S. Lund-Katz S. Johnson W.J. Anantharamaiah G.M. Palgunachari M.N. Segrest J.P. Rothblat G.H. Phillips M.C. J. Biol. Chem. 1994; 269: 22975-22982Abstract Full Text PDF PubMed Google Scholar, 34.Johnson 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), Model 1, in which all the FC is available for exchange and located in either the cellular pool or the acceptor pool, was used to analyze FC efflux to rHDL particles. The efflux time courses were fitted to Equation 1 by nonlinear regression, and the rate constants for FC flux were derived from the fitting variables using Equation 2, Equation 3, Equation 4, Equation 5, Equation 6. The apparent rate constant for efflux (ke) was used to calculate the halftime (t½) of efflux from the relationship t½ = ln 2/ke. Model 3, in which there are accessible and inaccessible pools of cellular FC and PL, was applied to the efflux induced by the presence of lipid-free apoA-I in the extracellular medium. Equation 16 was used to estimate the initial distribution of cellular FC between accessible and isolated pools. Fibroblasts grown to confluence and labeled with [3H]cholesterol as described above were incubated with 0.2% w/v trypsin at 0 °C for 10 min. The trypsin was removed, and the monolayers were washed 2× with MEM-HEPES containing 0.1% (w/v) soybean trypsin inhibitor and 2× with MEM-HEPES, warming the cells to 37 °C. Cholesterol efflux to apoA-I was performed as described above. The effectiveness of the trypsin treatment in removing cell surface proteins was assessed by using cells in which the surface proteins were radioiodinated (35.Shyng S.L. Huber M.T. Harris D.A. J. Biol. Chem. 1993; 268: 15922-15928Abstract Full Text PDF PubMed Google Scholar). Briefly, confluent cells in 35-mm dishes were washed on ice with PBS then incubated for 20 min with 1 ml of PBS containing 400 μg of glucose, 40 μg of lactoperoxidase, 20 μg of glucose oxidase, and 100 μCi of Na[125I]. The reaction was quenched by rinsing cells 2× with PBS containing 1 mm tyrosine and 10 mm sodium metabisulfite followed by 2 additional washes with PBS. The labeled cell monolayers were then treated with trypsin as described above, and the radioactivity released into the media was compared with the total125I incorporated into the monolayers. Approximately 50% of the total 125I was released upon trypsin treatment; this value was corrected for the release of counts measured in the absence of trypsin. The effects of cAMP on cellular cholesterol efflux were examined because this treatment has been shown in macrophages and fibroblasts to promote expression of ABCA1 (15.Lawn R.M. Wade D.P. Garvin M.R. Wang X. Schwartz K. Porter J.G. Seilhamer J.J. Vaughan A.M. Oram J.F. J. Clin. Invest. 1999; 104: 25-31Crossref PubMed Scopus (658) Google Scholar, 18.Oram J.F. Lawn R.M. Garvin M.R. Wade D.P. J. Biol. Chem. 2000; 275: 34508-34511Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar, 20.Abe-Dohmae S. Suzuki S. Wada Y. Aburatani H. Vance D.E. Yokoyama S. Biochemistry. 2000; 39: 11092-11099Crossref PubMed Scopus (101) Google Scholar, 21.Bortnick A.E. Rothblat G.H. Stoudt G. Hoppe K.L. Royer L.J. McNeish J. Francone O.L. J. Biol. Chem. 2000; 275: 28634-28640Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Chlorophenylthio-cAMP was incubated with radiolabeled control or FC-enriched fibroblasts at 0.3 mm in MEM-HEPES at 37 °C for 18 h before the initiation of cholesterol efflux measurements. For comparison, MEM-HEPES alone was incubated for 18 h before the efflux experiment. As described previously (36.Sakr S.W. Williams D.L. Stoudt G.W. Phillips M.C. Rothblat G.H. Biochim. Biophys. Acta. 1999; 1438: 85-98Crossref PubMed Scopus (93) Google Scholar), radioiodinated apoA-I was incubated with the fibroblast monolayers at concentrations ranging from 0.1 to 100 μg/ml for 1 h at 4 °C to measure the total binding of apoA-I to the cell surface. The nonspecific binding was determined by measuring the same concentration dependence of binding with a 40-fold excess of unlabeled apoA-I present at each concentration. From the difference between total and nonspecific binding, the specific binding of apoA-I was calculated and fitted by nonlinear regression to the one-site binding equation, Y = Bmax ×X/(Kd + X). From this equation, the maximal binding (Bmax) and concentration of apoA-I required to reach half-maximal binding (Kd), were determined for both the control and FC-enriched fibroblasts. Cells labeled with [14C]cholesterol and [3H]choline chloride were washed 4× with PBS, and the monolayers were exposed to 50 mm formaldehyde and 2 mm dithiothreitol in PBS for 90 min at 37 °C (37.Scott R.E. Perkins R.G. Zschunke M.A. Hoerl B.J. Maercklein P.B. J. Cell Sci. 1979; 35: 229-243Crossref PubMed Scopus (47) Google Scholar, 38.Scott R.E. Maercklein P.B. J. Cell Sci. 1979; 35: 245-252Crossref PubMed Google Scholar). The incubation buffer was removed and discarded, and the monolayers were washed with hypertonic PBS (0.44m NaCl, pH 7.6). This treatment resulted in the release of a large number of plasma membrane vesicles (0.5–5.0-μm diameter (39.Bellini F. Phillips M.C. Pickell C. Rothblat G.H. Biochim. Biophys. Acta. 1984; 777: 209-215Crossref PubMed Scopus (26) Google Scholar)); the buffer was removed and collected for analysis. Aliquots were extensively extracted as described above to remove any free [3H]choline present, and radioactivity was determined by liquid scintillation counting. The composition of the plasma membrane vesicles, which is assumed to be approximately representative of the plasma membrane as a whole, was computed from the specific activities of the FC and PL in the cell at t = 0. Protein concentration was determined by a modification of the Lowry assay (40.Markwell M.A. Haas S.M. Bieber L.L. Tolbert N.E. Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5347) Google Scholar). Total and FC contents of the cellular lipid extracts were quantified by gas-liquid chromatography using cholesteryl methyl ether as an internal standard. PL mass was determined as inorganic phosphorus (41.Bartlett G.R. J. Biol. Chem. 1959; 234: 466-468Abstract Full Text PDF PubMed Google Scholar), and the mass of choline-containing PL was determined by an enzymatic assay (Wako). The composition of the choline-containing PL appearing in the medium upon stimulation of PL efflux from the cell monolayers was determined by a one-dimensional TLC separation procedure. Samples were collected and extracted from free [3H]choline as described above. Further isolation of PL and separation into the various PL subclasses was achieved through two sequential developments in chloroform:methanol:ammonium hydroxide (75:25:4 (v:v:v)) (Silica Gel G). Migration distances of lysophosphatidylcholine, sphingomyelin, and phosphatidylcholine were determined by iodine staining of lanes containing purified standards of these specie
Referência(s)