Internalization of high density lipoproteins into an human hepatoma cell line (HepG2); a new approach.
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Apolipoproteins↗
Biomedical subjects
X Collet
Publications and source records attributed to X Collet.
Site-specific detection and structural characterization of the glycosylation of human plasma proteins lecithin:cholesterol acyltransferase and apolipoprotein D using HPLC/electrospray mass spectrometry and sequential glycosidase digestion.
Site-specific structural characterization of the glycosylation of human lecithin:cholesterol acyltransferase (LCAT) was carried out using microbore reversed-phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESIMS). A recently described mass spectrometric technique involving monitoring of carbohydrate-specific fragment ions during HPLC/ESIMS was employed to locate eight different groups of glycopeptides in a digest of a human LCAT protein preparation. In addition to the four expected N-linked glycopeptides of LCAT, a di-O-linked glycopeptide was detected, as well as three additional glycopeptides. Structural information on the oligosaccharides from all eight glycopeptides was obtained by sequential glycosidase digestion of the glycopeptides followed by HPLC/ESIMS. All four potential N-linked glycosylation sites (Asn20, Asn84, Asn272, and Asn384) of LCAT were determined to contain sialylated triantennary and/or biantennary complex structures. Two unanticipated O-linked glycosylation sites were identified at Thr407 and Ser409 of the LCAT O-linked glycopeptide, each of which contain sialylated galactose beta 1-->3N-acetylgalactosamine structures. The three additional glycopeptides were determined to be from a copurifying protein, apolipoprotein D, which contains potential N-linked glycosylation sites at Asn45 and Asn78. These glycopeptides were determined to bear sialylated triantennary oligosaccharides or fucosylated sialylated biantennary oligosaccharides. Previous studies of LCAT indicated that removal of the glycosylation site at Asn272 converts this protein to a phospholipase (Francone OL, Evangelista L, Fielding CJ, 1993, Biochim Biophys Acta 1166:301-304). Our results indicate that the carbohydrate structures themselves are not the source of this functional discrimination; rather, it must be mediated by the structural environment around Asn272.
Amino Acid Sequence↗
Purification and characterization of recombinant human apolipoprotein A-II expressed in Escherichia coli.
We have expressed recombinant human apolipoprotein A-II (apoA-II) in Escherichia coli, as a fusion protein with Schistosoma japonicum glutathione-S-transferase (GST). The GST-AII fusion protein was recovered by affinity chromatography using glutathione as a ligand. After thrombin cleavage and removal of the GST carrier, recombinant apoA-II was obtained in a highly purified form and was exclusively composed of dimeric apoA-II. Kinetics of association to dimyristoylglycerophosphocholine (Myr2GroPCho) vesicles showed that recombinant apoA-II exhibited the same pattern of association as human plasma apoA-II. Electron microscopic analysis of the complexes showed a typical pattern of rouleaux, characteristic of stacked discs, with a diameter similar to that determined by gradient-gel electrophoresis. Circular dichroism measurements showed that the alpha-helical content of both plasma and recombinant apoA-II increased similarly when the proteins associated with Myr2GroPCho vesicles, at the expense of a random-coil structure. Lipid-bound apoA-II consisted of 70-72% alpha helices, suggesting the presence of three 18-residue alpha helices/apoA-II monomer. Cross-linking experiments indicated that Myr2GroPCho complexes contained two molecules dimeric apoA-II/vesicle. Recombinant apoA-II was as efficient as plasma apoA-II in associating with HDL subclasses, and in displacing apoA-I from dipalmitoylglycerophosphocholine/cholesterol/apoA-I complexes, most likely due to its highly ordered secondary structure when associated with Myr2GroPCho vesicles. These findings demonstrate that recombinant apoA-II exhibits the same structural and functional properties as human plasma apoA-II. Thus, the expression system utilized is appropriate to produce mutagenized forms to further structure/function analysis.
Amino Acid Sequence↗
Hepatic lipase induces the formation of pre-beta 1 high density lipoprotein (HDL) from triacylglycerol-rich HDL2. A study comparing liver perfusion to in vitro incubation with lipases.
High density lipoprotein subfractions with a pre-beta migration play a key role in the reverse cholesterol transport. The origin of these particles is not yet clearly defined. We propose to verify a possible origin of these particles during the catabolism of high density lipoprotein2 (HDL2) by hepatic lipase using two different models. A rat liver perfusion of native human HDL2 in the presence of heparin induced, after 30 min, the formation of the pre-beta 1 HDL subspecies. Human HDL2 enriched with triacylglycerols, perfused in the same conditions, led after 15 min to an enhanced production of this pre-beta 1 HDL population, as compared with the results obtained with native HDL2. A reduction of the alpha-HDL2 fraction was also evident. After perfusion, a similar formation of pre-beta 1 HDL from triacylglycerol-rich HDL2 was observed in absence of heparin. When these HDL2 were incubated in vitro for 120 min at 37 degrees C in the presence of partially purified rat hepatic lipase, the appearance of pre-beta 1 HDL was again found and associated with a decrease in size of the remaining alpha-HDL subfractions as compared with original HDL2. On the contrary, the incubation of the same HDL2 with snake venom phospholipase A2 produced no pre-beta HDL. These results evidence the role of the triacylglycerol lipase activity of hepatic lipase in the formation of pre-beta 1 HDL from triacylglycerol-rich HDL2.
Animals↗
Specific binding of free apolipoprotein A-I to a high-affinity binding site on HepG2 cells: characterization of two high-density lipoprotein sites.
In this paper, we present the first evidence that free apoA-I, without association with lipids, binds only to a high-affinity binding site (Kd = 1.8 microgram/mL, Bmax = 63.12 ng/mL). This is a new binding site of higher affinity (80-100 times) but of lower capacity than the binding sites already described for HDL. This is also the first evidence on HepG2 cells both of a high-affinity site (Kd = 0.685 microgram/mL, Bmax = 39.86 ng/mL) and of a low-affinity site (Kd = 55.65 micrograms/mL, Bmax = 665.45 ng/mL) for HDL. ApoA-I-DMPC complexes also present two binding components comparable to the HDL3 binding sites. This free apoA-I binding is specific, as shown by competition experiments, and allowed us to specifically study this high-affinity site, without interference of the low-affinity one. Kinetic rates of association/dissociation for the high-affinity site were faster than for the low-affinity site (10 and 20 min versus 40 and 30 min, respectively). The kinetic Kd values, derived from association and dissociation rate constants (Kd = 55.14 and 2.91 micrograms/mL), were of similar magnitude as the Kd values calculated by Scatchard analysis. These data confirm that HDL3 binding sites characterized by saturation experiments follow the law of mass action, indicative of ligand-receptor interaction. In summary, HepG2 cells present high HDL3 binding sites which are able to bind free apoA-I in contrast with the low-affinity HDL3 binding sites.
Apolipoprotein A-I↗
Hepatic lipase promotes the uptake of HDL esterified cholesterol by the perfused rat liver: a study using reconstituted HDL particles of defined phospholipid composition.
The role of hepatic triacylglycerol lipase (H-TGL) in promoting the liver uptake of high density lipoprotein (HDL) free and esterified cholesterol was studied in a recirculating rat liver perfusion, a situation where the enzyme is physiologically expressed and is active at the vascular bed. For this purpose, reconstituted HDL of defined phospholipid composition were prepared, containing either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, a substrate for H-TGL, or 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphocholine, a non-hydrolyzable analog. Reconstituted HDL were then used in the perfused rat liver system. The main results are the following. 1) Reconstituted HDL were obtained by sonication of lipids and apolipoproteins and isolated by ultracentrifugation in the 1.07-1.21 g/ml density interval. Reconstituted HDL containing either diacylphosphatidylcholine or alkyl-acyl-phosphatidylcholine were similar in terms of chemical composition, apparent size, and apolipoprotein A-I immunoreactivity, and were comparable to native HDL3. 2) Reconstituted HDL were labeled with free [14C]cholesterol and [3H]cholesteryl ether, a non-hydrolyzable tracer of esterified cholesterol, and were perfused through the rat liver. Liver uptake of [3H]cholesteryl ether was 2.5-fold higher from reconstituted HDL containing diacylphospholipid than from HDL reconstituted with alkyl-acyl-phospholipids. Liver uptake of free [14C]cholesterol was identical in both cases. 3) H-TGL-depleted rat livers were obtained by a 12-min preperfusion in the presence of heparin, displacing 90% of the enzymatic activity. The residual activity in the perfusate was inhibited by a specific antibody directed against rat H-TGL. Liver uptake of [3H]cholesteryl ether from reconstituted HDL containing diacylphospholipid was reduced by 35% in hepatic lipase-depleted livers compared to controls. On the other hand, hepatic lipase depletion had no effect on the liver uptake of esterified cholesterol from HDL reconstituted with alkyl-acyl-phospholipids. The above findings support a role for the phospholipase A1 activity of H-TGL in stimulating the delivery of HDL esterified cholesterol to liver cells.
Animals↗
Site-directed mutagenesis and structure-function analysis of the human apolipoprotein A-I. Relation between lecithin-cholesterol acyltransferase activation and lipid binding.
We have mutagenized the human apoA-I gene and have generated cell lines which express normal and mutant apoA-I forms. Point mutations were introduced which changed Gln-1, Gln-2 to Arg,Arg, Pro99 to His, and Pro121 to His. In addition, the following amino acid deletions (delta) were generated: delta 113-124, delta 148-186, delta 212-233, and delta 213-243. The apoA-I form isolated from the culture medium of C127 cells was analyzed for its ability to activate lecithin-cholesterol acyltransferase (LCAT) and to bind to phospholipid vesicles and high density lipoprotein (HDL). Compared with the wild type (WT) apoA-I, the relative activation of LCAT achieved by the point mutations Gln-1, Gln-2----Arg,Arg, Pro99----His, and Pro121----His were 106 +/- 7, 92 +/- 6, and 77 +/- 9%, respectively. Kinetic analysis of one mutant apoA-I form showed similar Vmax but a 15-fold increase in the Km of the mutant apoA-I form. Furthermore, the activation achieved by the internal deletion mutants delta 113-124, delta 148-186, delta 212-233, and delta 213-243 was 47 +/- 3, 0.5 +/- 0.4, 28 +/- 4 and 13 +/- 5%, respectively. Mutants deficient in their ability to activate LCAT displayed alterations in liposome and HDL binding, compared with WT as determined by density gradient ultracentrifugation analysis of the culture medium. Thus, the peak recovery (approximately 50%) of apoA-I bound to HDL was at density 1.14 g/ml for the WT apoA-I, at 1.18 g/ml for the mutants delta 113-124 and delta 148-186, and at d greater than 1.21 g/ml for the delta 212-233 and delta 213-243. Electron microscopy of the proteoliposome LCAT substrate generated by WT and mutant apoA-I forms showed that the carboxyl-terminal deletion mutants which displayed aberrant binding to HDL also displayed reduced ability to convert the spherical lecithin-cholesterol vesicles into discs compared with WT. The findings suggest that (a) the importance of the carboxyl terminus of apoA-I for LCAT activation is related to its ability to bind to lipid and/or to form discoidal substrate for LCAT, and (b) the interaction of several domains of apoA-I are required for the activation of LCAT.
Algorithms↗
Differential effects of lecithin and cholesterol on the immunoreactivity and conformation of apolipoprotein A-I in high density lipoproteins.
Recently identified epitopes in apoA-I define a distinct N-terminal region with a complex tertiary structure, characterized by multiple discontinuous epitopes. Other epitopes are constituted of short domains centered either on beta-turns or random coils or on the 22-mer amphipathic alpha-helices (Marcel, Y. L., Provost, P. R., Koa, H., Raffaï, E., Vu Dac, N., Fruchart, J.-C., and Rassart, E. (1991) J. Biol. Chem. 266, 3644-3653). The compared immunoreactivity of seven epitopes studies here in response first to delipidation of high density lipoprotein (HDL) apoA-I by detergents, and second to modifications of HDL lipid composition by phospholipase A2 or by enrichment in surface lipids demonstrates that apoA-I has a flexible conformation which is readily responsive to the nature and concentration of bound lipids and that the structure of lipid-free apoA-I is significantly different from that of HDL-bound apoA-I, possibly representing a condensed molecule with several masked domains. In HDL apoA-I, these epitopes define five distinct domains which are characterized by particular responses to lipid modifications. However, two domains, each starting at the N-terminal beta-turn of an amphipathic alpha-helical repeat (residues 99-121 and 186-209, respectively) have almost identical immunoreactivity whether after detergent treatment or after changes in cholesterol and phospholipid levels, a property which probably reflects the known periodicity of apoA-I structural 22-mers. The immunoreactivity of a discontinuous epitope, representative of the N-terminal domain, is inversely related to the concentration of phospholipids, a unique characteristic among the epitopes tested here which indicates that the complex N-terminal region interacts with phospholipids, either directly or indirectly. These studies demonstrate that the conformation of multiple domains of HDL apoA-I is dependent on lipid phase composition and differentially affected by cholesterol and phospholipids.
Apolipoprotein A-I↗
Effects of inhibitors of N-linked oligosaccharide processing on the secretion, stability, and activity of lecithin:cholesterol acyltransferase.
The structure and function of the carbohydrate moiety of human lecithin:cholesterol acyltransferase (LCAT) were determined by using several glycosidases in reaction with the isolated plasma protein or by using specific inhibitors of glycoprotein assembly with cultured cells secreting LCAT activity. Analysis of the plasma enzyme indicated that almost all of the large carbohydrate moiety of LCAT (approximately 25% w/w) was N-linked with part of the high-mannose and part of the complex type. This analysis was confirmed with metabolic inhibitors of carbohydrate processing by using CHO cells stably transfected with the human LCAT gene. Inhibitors of the subsequent processing of the N-linked high-mannose chains formed by glucosidase activity were without effect on either the secretion rate or the catalytic activity of LCAT. The inhibition of catalytic activity by glucosidase inhibitors applied to both the phospholipase and the acyltransferase activities of LCAT. The reduction of the LCAT catalytic rate by terminal glycosidase inhibitors was without effect on apparent Km and did not affect enzyme stability. These data indicate an unusual specific role for high-mannose carbohydrates in the catalytic mechanism of LCAT.
Alkaloids↗
Hepatic lipase-mediated hydrolysis versus liver uptake of HDL phospholipids and triacylglycerols by the perfused rat liver.
Hepatic triacylglycerol-lipase-mediated hydrolysis and liver uptake of high-density lipoprotein (HDL) lipid components were studied in a recirculating rat liver perfusion, a situation where the enzyme is physiologically expressed and active at the vascular bed. Human native HDL were labelled with tri-[3H]oleoylglycerol, [N-methyl-3H]dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl,2-[14C]linoleoylphosphatidylcholine (PLPC), 1-palmitoyl,2-[14C]linoleoylphosphatidyl-ethanolamine (PLPE) and 1-palmitoyl,2-[14C]palmitoylphosphatidylethanolamine (DPPE). (1) Relative degradation rates of phosphatidylethanolamine molecular species were 2- to 10-fold higher than those of phosphatidylcholine. Considering [14C] PLPC and [14C] PLPE as representative of HDL phosphatidylcholine and phosphatidylethanolamine, respectively, the amounts of lysophosphatidylcholine and lysophosphatidylethanolamine generated after a 60 min perfusion were comparable. The enzyme showed a clear preference for the molecular species bearing an unsaturated fatty acid at the 2 position of glycerol; this was the most pronounced in the case of phosphatidylethanolamine molecular species. (2) Relative liver uptake of HDL-phosphatidylethanolamine was 4- to 5-fold higher than that of HDL-phosphatidylcholine, irrespective of the constitutive fatty acids. Nevertheless, mass estimation indicated that 3 times more molecules of phosphatidylcholine than of phosphatidylethanolamine were transferred. No correlation could be found between the relative degradation rates of phospholipids and their relative liver uptake, indicating a dissociation between the two processes. (3) Perfusate decay and relative liver uptake of labelled HDL-triacylglycerol were higher than that of any phospholipid class. No circulating radiolabelled free fatty acids accumulated in the perfusate, but they were found acylated into liver cell phospholipids and triacylglycerols. (4) A prior 10-12-min washout of the liver vascular bed with heparin removed over 80% of the hepatic lipase activity, as assessed by specific immunoinhibition. Hepatic lipase-depleted liver displayed impaired phospholipid hydrolysis and triacyglycerol uptake, whereas the transfer of HDL phospholipids to liver tissue was unaffected.
Animals↗
Behaviour of phospholipase modified-HDL towards cultured hepatocytes. II. Increased cell cholesterol storage and bile acid synthesis.
Human total HDL (hydrated density 1.070-1.210), HDL2 (1.070-1.125), HDL3 (1.125-1.210) or HDL separated by heparin affinity chromatography were treated with or without purified phospholipase A2 from Crotalus adamanteus. Control and treated HDL were reisolated and were then incubated with cultured hepatocytes. 1. Mass measurements evidenced a time-dependent cholesterol enrichment in hepatocytes cultured in the absence of lipoproteins. Addition of HDL2 still enhanced by 25% the cell cholesterol content and down-regulated endogenous sterol synthesis in similar proportions. Conversely, HDL3 slightly decreased the amount of free cholesterol in hepatocytes (-12%). 2. Incubations with phospholipase A2-treated HDL resulted in a 35%-50% increase of both the cellular cholesterol esterification and the cholesterylester accumulation, when compared to cells cultured in the presence of control-HDL. This effect was observed with HDL2, HDL3 and combining the data with all subfractions. 3. Cultured hepatocytes secreted cholic and beta-muricholic acids as major bile acids and HDL2 showed a tendency to stimulate their secretion. Phospholipase treatment of HDL again induced an increased production by hepatocytes of those two bile acids. Thus, whereas HDL2 and HDL3 display different behaviours with respect to cell cholesterol content, neosynthesis and bile acid secretion, their modifications by phospholipases always orientate the cell sterol metabolism in the same direction: increased cholesterylester accumulation and bile acid production.
Animals↗
Phospholipase activity of lecithin-cholesterol acyltransferase.
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Apolipoprotein A-I↗
Behaviour of phospholipase-modified HDL towards cultured hepatocytes. I. Enhanced transfers of HDL sterols and apoproteins.
Human HDL subfractions (HDL2, HDL3, or HDL separated by heparin affinity chromatography) were labelled either on their apolipoprotein moiety with 125I or on their sterols: unesterified [14C]cholesterol and [3H]cholesteryl linoleyl ether, a non-hydrolysable analog of esterified cholesterol. HDL subfractions were then treated with or without phospholipase A2 from Crotalus adamanteus in presence of albumin leading to a 72-82% phosphatidylcholine degradation. Control and treated HDL were reisolated and then addressed to cultured rat hepatocytes. (A) During incubations, unesterified [14C]cholesterol from HDL3 readily appeared in hepatocytes. The specific uptake of HDL esterified cholesterol calculated from [3H]cholesteryl ether was 2-4-times less important. Uptake of HDL cholesterol tended to saturate at 150-200 micrograms/ml HDL protein. A prior phospholipase treatment of HDL3 stimulated by 2-5-fold the uptake of [3H]cholesteryl ether, whereas the transfer of free [14C]cholesterol was minimally increased. The uptake of 3H/14C-labelled sterols from HDL2 was 2-3-times higher than from HDL3. (B) Parallel experiments were conducted with 125I-labelled HDL subfractions. At 37 degrees C, the specific uptake and degradation of HDL3 125I-apolipoprotein were about 2-fold enhanced following treatment of HDL3 with phospholipase A2. Uptakes of apolipoprotein and of esterified cholesterol were compared, indicating a preferential delivery of the sterol over apoprotein (X5). The dissociation was still more pronounced with phospholipase-treated HDL3. Competition experiments showed that 12-times more unlabelled HDL3 were required to half reduce the uptake of HDL3 [3H]cholesteryl ether than to impede similarly the HDL 125I-apolipoprotein recovered in cells. Uptake of 125I-labelled apolipoprotein from HDL2 was quantitatively comparable to that from HDL3. (C) Binding of 125I-HDL subfractions was followed at 4 degrees C. A specific binding was observed for HDL2 and HDL3, although kinetic parameters were quite different (KD of 9 and 25 micrograms/ml, respectively). Following phospholipolysis, both the specific and non-specific contributions to total binding were increased. Hence, hepatocytes take up more 125I-labelled apolipoprotein and 3H/14C-labelled sterols from lipolysed HDL than from unmodified particles. This is associated to changes in the binding characteristics.
Animals↗
Phosphatidylcholine and triacylglycerol hydrolysis in HDL as induced by hepatic lipase: modulation of the phospholipase activity by changes in the particle surface or in the lipid core.
(1) Human HDL2 (d 1.063-1.125) and HDL3 (d 1.125-1.210), labelled with 2-[14C]oleoylphosphatidylcholine (PC), and with/without tri[3H]oleoylglycerol, were incubated with a partially purified human hepatic triacylglycerol lipase, at pH 8.5. PC hydrolysis was linear up to 90-120 min incubation and within a range of lipase activities, from 50 to 500 mIU/ml. At low degrees of lipolysis, the hydrolysis of triacylglycerol was linearly related to that of PC, but the relative degradation rate was 10-fold higher for the former, which was thus very rapidly consumed. HDL subfractions were then differentiated in terms of PC hydrolysis. Km values were 0.32 and 0.43 mM for HDL2 PC and HDL3 PC, respectively. The corresponding Vmax values expressed for 200 mIU/ml hepatic lipase activity were 41.0 nmol PC hydrolysed/ml per h (HDL2) and 28.6 nmol PC/ml per h (HDL3). (2) HDL3 were modified in the presence of VLDL by inducing triacylglycerol lipolysis in VLDL with a semi-purified human plasma or bovine milk lipoprotein lipase (LPL). Lipolysis-modified HDL3 (LIP-HDL3) were mostly enriched in free cholesterol (+80%, P less than 0.05) and to a lesser extent in triacylglycerol (+33%). As a consequence, 45% of the LIP-HDL3 was reisolated in the HDL2-density interval, and is referred to as light LIP-HDL3. LIP-HDL3 displayed a 65% increase in its reactivity towards hepatic lipase compared to control HDL3. The light LIP-HDL3 showed the lowest Km (0.19 mM PC) and the highest Vmax (69 nmol/ml per h) of all HDL tested. Coincubation of HDL3 with VLDL and albumin did not alter the further reactivity of HDL3 towards hepatic lipase. Cholesterol loading of HDL3 by celite-cholesterol dispersions also led to an enhanced reactivity, though less important than with the lipolysis modification. (3) HDL3 were also modified by coincubation with VLDL and the lecithin-cholesterol acyltransferase-inhibited plasma fraction of d greater than 1.21 g/ml, thus allowing the cholesteryl ester transfer reaction to occur. The modified HDL3 (CET-HDL3) were depleted in esterified cholesterol (-25%, P less than 0.05) and enriched in triacylglycerol (+70%, P less than 0.05). However, these particles behaved like control HDL3 in their reactivity towards hepatic triacylglycerol lipase. Thus, the hydrolysis of HDL PC mediated by hepatic triacylglycerol lipase appears to be influenced by changes occurring in the particle's surface rather than in the lipid core.
Humans↗
Uptake of HDL unesterified and esterified cholesterol by human endothelial cells. Modulation by HDL phospholipolysis and cell cholesterol content.
Human HDL (1.070-1.210), doubly labelled with 3H/14C-labelled unesterified cholesterol and 3H-labelled esterified cholesterol were incubated for 1-5 h with monolayer cultures of human endothelial cells. HDL were preincubated for 60-120 min the presence of albumin and with/without purified phospholipase A2 (control HDL, phospholipase A2 HDL) before dilution in the cell culture medium. Average phosphatidylcholine (PC) degradation was 62.10% +/- 2.57% (range 45-80%). A purified lipase/phospholipase A1 from guinea pig pancreas was used in some experiments (range of PC hydrolysis: 16-70%). (1) 3H/14C-labelled unesterified cholesterol and 3H-labelled esterified cholesterol appeared in cells during 0-5 h incubations. Trypsin treatment allowed a simple adsorption of HDL onto the cell surface to be avoided, and most of the 3H-labelled esterified cholesterol transferred to cells was hydrolysed. Cell uptake of radioactive cholesterol increased as a function of HDL concentration but no saturation was achieved at the highest lipoprotein concentration used (200 micrograms cholesterol/ml). Flux of 3H/14C-labelled unesterified cholesterol was related to the cell cholesterol content, suggesting that it might partly represent an exchange process. The cell cholesterol content was slightly increased after 5 h incubation with HDL (+16%). (2) Pretreatment of HDL with purified phospholipase A2 doubled on average the amount of cell recovered 3H-labelled esterified cholesterol, while the flux of 3H/14C-labelled unesterified cholesterol was enhanced by 15-25%. Both transfer and cell hydrolysis of 3H-labelled esterified cholesterol were increased. A stimulation was also observed using purified lipase/phospholipase A1, provided that a threshold phospholipid degradation was achieved (between 27 and 45%). (3) Endothelial cells were conditioned in different media so as to modulate their charge in cholesterol. The uptake of 3H-labelled esterified cholesterol was found to be significantly higher in cholesterol-enriched cells compared to the sterol-depleted state. Finally, movements of 3H-labelled esterified cholesterol from HDL to endothelial cells were essentially unaffected by cell density or by the presence of partially purified cholesterol ester transfer protein. The possible roles of the transfer of HDL esterified cholesterol to endothelial cells and its modulation by phospholipases are discussed.
Biological Transport↗
Disposal of HDL-cholesterol to cultured granulosa cells, endothelial cells and hepatocytes: effects of HDL-phospholipolysis.
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Animals↗