Binding and degradation of human high-density lipoproteins by human hepatoma cell line HepG2

The catabolism of human HDL was studied in human hepatoma cell line HepG2. The binding of 125I-labeled HDL at 4 degrees C was time-dependent and reached completion within 2 h. The observed rates of binding of 125I-labeled HDL at 4 degrees C and uptake and degradation at 37 degrees C indicated the presence of both high-affinity and low-affinity binding sites for this lipoprotein density class. The specific binding of 125I-labeled HDL accounted for 55% of the total binding capacity. The lysosomal degradation of 125I-labeled HDL was inhibited 25 and 60% by chloroquine at 50 and 100 microM, respectively. Depolymerization of microtubules by colchicine (1 microM) inhibited the degradation of 125I-labeled HDL by 36%. Incubation of cells with HDL caused no significant change in the cellular cholesterol content or in the de novo sterol synthesis and cholesterol esterification. Binding and degradation of 125I-labeled HDL was not affected by prior incubation of cells with HDL. When added at the same protein concentration, unlabeled VLDL, LDL and HDL had similar inhibitory effects on the degradation of 125I-labeled HDL, irrespective of a short or prolonged incubation time. Reductive methylation of unlabeled HDL had no significant effect on its capacity to inhibit the 125I-labeled HDL degradation. The competition study indicated no correlation between the concentrations of apolipoproteins A-I, A-II, B, C-II, C-III, E and F in VLDL, LDL and HDL and the inhibitory effect of these lipoprotein density classes on the degradation of 125I-labeled HDL. There was, however, some association between the inhibitory effect and the levels of apolipoprotein D and C-I.     

Interactions of high-density lipoprotein 3 with brain capillary endothelial cells

High-density lipoprotein 3 (HDL3) binds to capillary endothelial cells when their lumen surfaces are exposed to 125I-HDL3 by post-mortem perfusion of whole brain. Kinetic studies of binding of HDL3 to isolated membranes show that HDL3 binds only to endothelial membranes with high affinity (Kd = 7 micrograms/ml). Trypsin treatment of membranes abolishes HDL3 binding. High-affinity binding sites for HDL3 were recovered when endothelial cells from bovine brain capillaries were maintained in culture (Kd = 13 micrograms/ml HDL3 protein). The characteristics of the binding were preserved up to the 6th passage. Competition experiments using isolated luminal membranes or cultured endothelial cells indicate that only HDL3 and not LDL or methylated LDL, are able to compete binding of 125I-HDL3. Furthermore, the inhibition of 125I-HDL3 binding by lipoprotein A-I and lipoprotein A-I:A-II strongly suggests that apolipoprotein A-I is implicated in the formation of HDL3-receptor complexes. The binding is increased by loading cells with free cholesterol or LDL cholesterol. In addition, surface-bound 125I-HDL3 remains sensitive to mild trypsin treatment after subsequent incubation of BBCE at 37 degrees C. HDL3 bound to the cell surface is not endocytosed, but rather rapidly released into the medium after binding (t1/2 = 5 min).     

Binding of partially reassembled high-density lipoprotein to isolated human small intestine epithelial cells. Effect of lipid composition

The binding of human high-density lipoprotein (HDL3), apolipoprotein A-I (apoA-I) and recombinants of apoA-I with cholesterol and/or dimyristoylphosphatidylcholine (DMPC) to the HDL receptor on isolated human small intestine epithelial cells was studied. ApoA-I competed for 125I-labelled HDL3 binding sites less effectively than HDL3, and a lower amount of 125I-labelled apoA-I than 125I-HDL3 was bound to cells. The apoA-I/DMPC recombinant competed for 125I-HDL3 binding sites nearly as well as HDL3, and 125I-apoA-I/DMPC recombinant bound to cells with at least the same efficiency as 125I-HDL3. The apoA-I/DMPC/cholesterol recombinant failed to compete for 125I-HDL3 binding sites, and the 125I-apoA-I/DMPC/cholesterol complex binding to cells was several-fold lower than that of other particles. All particles bound to cells with similar dissociation constants. Tetranitromethane-modified HDL3 failed to bind to high-affinity specific binding sites and compete with 125I-HDL3 for binding. The results obtained make it possible to assume that, while apoA-I may be a determinant of the HDL receptor, the lipid composition of the lipoprotein may affect its interaction with the receptor.     

The amphipathic alpha-helical repeats of apolipoprotein A-I are responsible for binding of high density lipoproteins to HepG2 cells.

Nine monoclonal antibodies (mAbs) against apoA-I reacting with distinct but overlapping epitopes covering more than 90% of the sequence have been used to block the interaction of 125I-labeled high density lipoprotein (125I-HDL) with HepG2 cells in order to delineate the cell binding domain of apolipoprotein A-I (apoA-I). While 2 mAbs reacting with epitopes exclusively localized in the N-terminal region (residues 1 to 86) enhanced slightly association of 125I-HDL, all other mAbs, which react with epitopes localized in the regions of amphipathic alpha-helical repeats, inhibited that association by 9 to 15%. Although this inhibition is not significant compared to the effect of an irrelevant mAb, combination of these mAbs could significantly inhibit the association of 125I-HDL (32 to 43%) as could polyclonal antibodies (up to 95%). These results are compatible with the concept of HDL binding to these cells via the nonexclusive interaction of each of the amphipathic alpha-helical repeats of apoA-I. When the same approach was applied to block the association of 3H-cholesteryl ether (CE)-labeled HDL to HepG2 cells, each anti-apoA-I could inhibit by 15 to 25% the cellular association of cholesteryl ether while mAbs in combination or polyclonal antibodies could inhibit this association up to 45% or 60%, respectively. The cholesteryl ether radioactivity that remained associated with the cells (40%) in the presence of polyclonal antibodies could be effectively blocked by addition of an antibody against the receptor binding domain of apoE (1D7). Therefore, the differential cellular association of cholesteryl ether compared to apolipoprotein can be explained by the presence of apoE secreted by HepG2 and apoE or apoB/E receptors. Thus, we conclude that the optimum uptake of both cholesteryl ether and apoA-I of HDL by cells requires the accessibility of the entire apoA-I and the cooperative binding of the amphipathic alpha-helical repeats to HepG2 cell membranes. This type of interaction would explain the competitive binding observed for apoA-I, -A-II, and -A-IV by others.     

Studies on the proteins involved in the interaction of high-density lipoprotein with isolated human small intestine epithelial cells.

Treatment of 125I-labelled high-density lipoprotein ([125I]HDL3) with monospecific polyclonal antibodies against apolipoproteins A-I and A-II resulted in a dose-dependent inhibition of the [125I]HDL3 binding to isolated human small intestine epithelial cells by 25% and 50%, respectively. Both antibodies also inhibited intracellular degradation of [125I]HDL3 by 80%. Treatment of enterocytes with polyclonal antibody against apolipoprotein A-I binding protein, a putative HDL receptor, inhibited both binding and degradation of [125I]HDL3 by these cells by 50%. Antibodies to apolipoprotein A-I, A-II and apo A-I-binding protein also inhibited [125I]HDL3 binding to cholesterol-loaded cells.     

Comparison of the metabolic behavior of rat apolipoproteins A-I and A-IV, isolated from both lymph chylomicrons and serum high density lipoproteins

Rat apolipoprotein (apo) A-I and A-IV, isolated from both lymph chylomicrons and serum high density lipoproteins (HDL) were analyzed by isoelectric focusing. Lymph chylomicron apo A-I consisted for 81 +/- 2% of the pro form and for 19 +/- 2% of the mature form, while apo A-I isolated from serum HDL was present for 36 +/- 4% in the pro form and for 64 +/- 4% in the mature form. Apo A-IV also showed two major protein bands after analysis by isoelectric focusing. The most prominent component is the more basic protein that amounts to 80 +/- 2% in apo A-IV isolated from lymph chylomicrons and to 60 +/- 3% in apo A-IV isolated from serum HDL. Apo A-I (or apo A-IV), isolated from both sources (lymph chylomicrons or serum HDL), was iodinated and the radioactive apolipoproteins were incorporated into rat serum lipoproteins. The resulting labeled HDL was isolated from serum by molecular sieve chromatography on 6% agarose columns and injected intravenously into rats. No difference in the fractional turnover rate or the tissue uptake of the two labeled HDL preparations was observed, neither for apo A-I nor for apo A-IV. It is concluded that the physiological significance of the extracellular pro apo A-I conversion or the post-translational modification of apo A-IV is not related to the fractional turnover rate in serum or to the rate of catabolism in liver and kidneys.     

Interaction of high density lipoprotein with its receptor on cultured fibroblasts and macrophages. Evidence for reversible binding at the cell surface without internalization

Cultured extrahepatic cells possess a specific high affinity receptor for high density lipoprotein (HDL) that is induced by cholesterol delivery to cells. Current results suggest that HDL receptors on cultured human fibroblasts and mouse peritoneal macrophages promote reversible binding of HDL to the cell surface without internalization of lipoprotein particles. When 125I-HDL3 was bound to cultured cells at 0 degrees C and then warmed to 37 degrees C after removal of unbound lipoprotein, most of the cell surface-bound HDL was released rapidly (t1/2 = 3 min) into the medium without entering a cellular pool that was inaccessible to digestion by trypsin at 0 degrees C. This lack of internalization of HDL was evident under conditions where internalization of 125I-low density lipoprotein and 125I-transferrin were readily detected. When cells were exposed to 125I-HDL3 at 37 degrees C, only a trace amount of iodinated apoprotein remained associated with cells after treatment of cells with trypsin. Fibroblasts treated with medium containing increasing concentrations of cholesterol exhibited a dose-dependent increase in reversible, trypsin-sensitive binding of 125I-HDL3 at 37 degrees C without an attendant increase in trypsin-resistant binding. These results suggest that reversible binding of HDL to its cell-surface receptor without subsequent endocytosis of receptor-HDL complexes is the mechanism by which HDL receptors facilitate cholesterol transport from cells.     

Human high density lipoprotein (HDL3) binding to rat liver plasma membranes

The binding of human 125I-labeled HDL3 to purified rat liver plasma membranes was studied. 125I-labeled HDL3 bound to the membranes with a dissociation constant of 10.5 micrograms protein/ml and a maximum binding of 3.45 micrograms protein/mg membrane protein. The 125I-labeled HDL3-binding activity was primarily associated with the plasma membrane fraction of the rat liver membranes. The amount of 125I-labeled HDL3 bound to the membranes was dependent on the temperature of incubation. The binding of 125I-labeled HDL3 to the rat liver plasma membranes was competitively inhibited by unlabeled human HDL3, rat HDL, HDL from nephrotic rats enriched in apolipoprotein A-I and phosphatidylcholine complexes of human apolipoprotein A-I, but not by human or rat LDL, free human apolipoprotein A-I or phosphatidylcholine vesicles. Human 125I-labeled apolipoprotein A-I complexed with egg phosphatidylcholine bound to rat liver plasma membranes with high affinity and saturability, and the binding constants were similar to those of human 125I-labeled HDL3. The 125I-labeled HDL3-binding activity of the membranes was not sensitive to pronase or phospholipase A2; however, prior treatment of the membranes with phospholipase A2 followed by pronase digestion resulted in loss of the binding activity. Heating the membranes at 100 degrees C for 30 min also resulted in an almost complete loss of the 125I-labeled HDL3-binding activity.     

Characterization of the specific binding of rat apolipoprotein E-deficient HDL to rat hepatic plasma membranes

We have used a preparation of rat liver plasma membranes to study the binding of rat apolipoprotein E-deficient HDL to rat liver. The membranes were found to bind HDL by a saturable process that was competed for by excess unlabeled HDL. The binding was temperature-dependent and was 85% receptor-mediated when incubated at 4, 22 and 37 degrees C. The affinity of the binding site for the HDL was consistent at all temperatures, while the maximum binding capacity increased at higher temperatures. The specific binding of HDL to the membranes did not require calcium and was independent of the concentration of NaCl in the media. The effect of varying the pH of the media on HDL binding was small, being 30% higher at pH 6.5 than at pH 9.0. Both rat HDL and human HDL3 were found to compete for the binding of rat HDL to the membranes, whereas rat VLDL remnants and human LDL did not compete. At 4 degrees C, complexes of dimyristoylphosphatidylcholine (DMPC) and apolipoproteins A-I, A-IV and the C apolipoproteins, but not apolipoprotein E, competed for HDL binding to the membranes. At 22 and 37 degrees C, all DMPC-apolipoprotein complexes competed to a similar extent, DMPC vesicles that contained no protein did not compete for the binding of HDL. These results suggest that the rat liver possesses a specific receptor for apolipoprotein E-deficient HDL that recognizes apolipoproteins A-I, A-IV and the C apolipoproteins as ligands.     

The role of apolipoproteins of HDL in the selective uptake of cholesteryl linoleyl ether by cultured rat and bovine adrenal cells

Rat adrenal cells in culture were used to study the uptake of cholesteryl linoleyl ether [( 3H]cholesteryl linoleyl ether), a nonhydrolyzable analog of cholesteryl ester. When [3H]cholesteryl linoleyl ether was added in the form of liposomes, its uptake was enhanced by adrenocorticotropin (ACTH) and by addition of milk lipoprotein lipase and interfered by heparin. When the adrenal cells were incubated with homologous [3H]cholesteryl linoleyl ether-HDL, ACTH treatment also resulted in an increase in [3H]cholesteryl linoleyl ether uptake. The uptake of [3H]cholesteryl linoleyl ether was in excess of the uptake and metabolism of 125I-labeled HDL protein and was not sensitive to heparin. Unlabeled HDL or delipidated HDL reduced very markedly the uptake of [3H]cholesteryl linoleyl ether, while addition of phosphatidylcholine liposomes had little effect. Attempts were made to deplete and enrich the adrenal cells in cholesterol and, while depletion resulted in a decrease in [3H]cholesteryl linoleyl ether-HDL uptake, enrichment of cells with cholesterol had no effect. Among the individual apolipoproteins tested, apolipoprotein A-I and the C apolipoproteins reduced [3H]cholesteryl linoleyl ether uptake, while apolipoprotein E was not effective. Since the labeled ligand studied was a lipid, these effects could not be due to an exchange of apolipoproteins, but indicated competition for binding sites. Preferential uptake of human [3H]cholesteryl linoleyl ether-HDL3 by bovine adrenal cells was found when compared to the uptake and metabolism of 125I-labeled HDL. The present results suggest that the preferential uptake of HDL cholesteryl ester (as studied with [3H]cholesteryl linoleyl ether) requires an interaction between the apolipoproteins of HDL and cell surface components.     

Studies on the binding and degradation of human very-low-density lipoproteins by human hepatoma cell line HepG2

The regulation of the hepatic catabolism of normal human very-low-density lipoproteins (VLDL) was studied in human-derived hepatoma cell line HepG2. Concentration-dependent binding, uptake and degradation of 125I-labeled VLDL demonstrated that the hepatic removal of these particles proceeds through both the saturable and non-saturable processes. In the presence of excess unlabeled VLDL, the specific binding of 125-labeled VLDL accounted for 72% of the total binding. The preincubation of cells with unlabeled VLDL had little effect on the expression of receptors, but reductive methylation of VLDL particles reduced their binding capacity. Chloroquine and colchicine inhibited the degradation of 125I-labeled VLDL and increased their accumulation in the cell, indicating the involvement of lysosomes and microtubuli in this process. Receptor-mediated degradation was associated with a slight (13%) reduction in de novo sterol synthesis and had no significant effect on the cellular cholesterol esterification. Competition studies demonstrated the ability of unlabeled VLDL, low-density lipoproteins (LDL) and high-density lipoproteins (HDL) to effectively compete with 125I-labeled VLDL for binding to cells. No correlation was observed between the concentrations of apolipoproteins A-I, A-II, C-I, C-II and C-III of unlabeled lipoproteins and their inhibitory effect on 125I-labeled VLDL binding. When unlabeled VLDL, LDL and HDL were added at equal contents of either apolipoprotein B or apolipoprotein E, their inhibitory effect on the binding and uptake of 125I-labeled VLDL only correlated with apolipoprotein E. Under similar conditions, the ability of unlabeled VLDL, LDL and HDL to compete with 125I-labeled LDL for binding was a direct function of only their apolipoprotein B. These results demonstrate that in HepG2 cells, apolipoprotein E is the main recognition signal for receptor-mediated binding and degradation of VLDL particles, while apolipoprotein B functions as the sole recognition signal for the catabolism of LDL. Furthermore, the lack of any substantial regulation of beta-hydroxy-beta-methylglutaryl-CoA reductase and acyl-CoA:cholesterol acyltransferase activities subsequent to VLDL degradation, in contrast to that observed for LDL catabolism, suggests that, in HepG2 cells, the receptor-mediated removal of VLDL proceeds through processes independent of those involved in LDL catabolism.     

Distribution of apolipoproteins A-I and A-IV among lipoprotein classes in rat mesenteric lymph, fractionated by molecular sieve chromatography

The distribution of apolipoproteins A-I and A-IV among lymph lipoprotein fractions was studied after separation by molecular sieve chromatography, avoiding any ultracentrifugation. Lymph was obtained from rats infused either with a glucose solution or with a triacylglycerol emulsion. Relative to glucose infusion, triacylglycerol infusion caused a 20-fold increase in the output of triacylglycerol, coupled with a 4-fold increase in output of apolipoprotein A-IV. The output of apolipoprotein A-I was only elevated 2-fold. Chromatography on 6% agarose showed that lymph apolipoproteins A-I and A-IV are present on triacylglycerol-rich particles and on particles of the size of HDL. In addition, apolipoprotein A-IV is also present as 'free' apolipoprotein A-IV. The increase in apolipoprotein A-I output is caused by a higher output of A-I associated with large chylomicrons only, while the increase in apolipoprotein A-IV output is reflected by an increased output in all lymph lipoprotein fractions, including lymph HDL and 'free' apolipoprotein A-IV. The increased level of 'free' A-IV, seen in fatty lymph, may contribute to, and at least partly explain, the high concentrations of 'free' apolipoprotein A-IV present in serum obtained from fed animals.     

Characterization of Na+-dependent phosphate uptake in cultured kidney cells (JTC-12) from monkey.

O-(4-Diazo-3-[125I]iodobenzoyl)sucrose ([125I]DIBS), a novel labelling compound specifically designed to study the catabolic sites of serum proteins [De Jong, Bouma, & Gruber (1981) Biochem. J. 198, 45-51], was applied to study the tissue sites of degradation of serum lipoproteins. [125I]DIBS-labelled apolipoproteins (apo) E and A-I, added in tracer amounts to rat serum, associate with high-density lipoproteins (HDL) just like conventionally iodinated apo E and A-I. No difference is observed between the serum decays of chromatographically isolated [125I]DIBS-labelled and conventionally iodinated HDL labelled specifically in either apo E or apo A-I. When these specifically labelled HDLs are injected into fasted rats, a substantial [125I]DIBS-dependent 125I accumulation occurs in the kidneys and in the liver. No [125I]DIBS-dependent accumulation is observed in the kidneys after injection of labelled asialofetuin or human low-density lipoprotein. It is concluded that the kidneys and the liver are important sites of catabolism of rat HDL apo E and A-I.     

Characteristics of the binding of high-density lipoprotein3 by intact cells and membrane preparations of rat intestinal mucosa

We have investigated the binding of high-density lipoprotein (HDL3, d = 1.12-1.21 g/ml), and apolipoprotein E-deficient human and rat HDL, obtained by heparin-Sepharose affinity chromatography, to intact cells and membrane preparations of rat intestinal mucosal cells. Binding of 125I-labeled HDL3 to the basolateral plasma membranes was characterised by a saturable, specific process (Kd = 21 micrograms of HDL3 protein/ml, Bmax = 660 ng HDL3 protein/mg membrane protein) and E-deficient human HDL demonstrated a similar affinity for the binding site. The basolateral plasma membranes isolated from proximal and distal portion of rat small intestine showed similar binding affinities for HDL3, whereas the interaction of HDL with brush-border membranes was characterised by mainly nonspecific and nonsaturable binding. The binding of 125I-labeled HDL3 to basolateral plasma membranes was competitively inhibited by unlabeled HDL3 but less efficiently by unlabeled human LDL. The putative HDL receptor was not dependent on the presence of divalent cations but was markedly influenced by temperature and sensitive to pronase treatment. We have also demonstrated, using whole intestinal mucosal cells, that lysine and arginine-modified HDL3 inhibited binding of normal 125I-labeled HDL3 to the same extent as normal excess HDL3. These data suggest that basolateral plasma membranes of rat intestinal mucosal cells possess a specific receptor for HDL3 which contains mainly apolipoprotein A-I and A-II, and the mechanisms of recognition of HDL3 differ from those involved in binding to the B/E receptor.     

Binding of human apolipoprotein A-IV to human hepatocellular plasma membranes

We have investigated the binding of human apolipoprotein A-IV (apo A-IV) to human hepatocellular plasma membranes. Addition of increasing concentrations of radiolabeled apo A-IV to hepatic plasma membranes, in the presence and absence of a 25-fold excess of unlabeled apo A-IV, revealed saturation binding to the membranes with a KD of 154 nM and a binding maximum of 1.6 ng/microgram of membrane protein. The binding was temperature-insensitive, partially calcium-dependent, abolished when apo A-IV was denatured by guanidine hydrochloride or when the membranes were treated with Pronase and decreased when apo A-IV was incorporated into phospholipid/cholesterol proteoliposomes. In displacement studies using purified apolipoproteins and isolated lipoproteins, only unlabeled apo A-IV, apo A-I and high-density lipoproteins effectively competed with radiolabeled apo A-IV for membrane binding sites. We conclude that human apo A-IV exhibits high-affinity binding to isolated human hepatocellular plasma membranes which is saturable, reversible and specific.     

Effects of apolipoproteins A-IV and A-I on the uptake of phospholipid liposomes by hepatocytes

We examined the effects of apolipoproteins A-IV and A-I on the catabolism of whole particles by hepatoma G2 cells and cultured primary hepatocytes. For this type of experiment, high density lipoprotein is unsuitable, because all of its lipid and protein components independently dissociate and exchange and hence poorly trace whole particle catabolism. We therefore used phosphatidylcholine liposomes with radioactive tracers entrapped within their aqueous cores. Apolipoproteins A-IV, A-I, or E added to liposomes became liposome-associated and produced no detectable release of encapsulated label. As a positive control, apolipoprotein E doubled the uptake of labeled liposomes by hepatoma cells, compared to apolipoprotein-free controls, and this increase could be blocked by the addition of excess unlabeled low density lipoprotein. Degradation of labeled liposomes by hepatoma cells was increased 6-fold by the addition of apolipoprotein E. In contrast, neither apolipoprotein A-IV nor A-I increased cellular uptake or degradation of the particles. Similar results were obtained with primary hepatocytes. In studies using apolipoprotein combinations, apolipoproteins A-IV and A-I were each able to displace apolipoprotein E from liposomes and thereby reduce cellular uptake. Our data indicate that apolipoproteins A-IV and A-I do not facilitate uptake or degradation of whole particles by liver-derived cells in vitro. However, these apolipoproteins may modulate receptor-mediated uptake of particles by reducing the amount of particle-bound apolipoprotein E.     

Human apolipoprotein A-IV binds to apolipoprotein A-I/A-II receptor sites and promotes cholesterol efflux from adipose cells

Cholesterol efflux was studied in cultured mouse adipose cells after preloading with low density lipoprotein cholesterol. Exposure to complexes containing human apolipoprotein A-IV and L-alpha-dimyristoylphosphatidylcholine (DMPC) as well as to human lipoprotein particles containing apolipoprotein A-IV but not apolipoprotein A-I and particles containing apolipoproteins A-IV and A-I showed that both artificial and native apolipoprotein A-IV-containing particles were able to promote cholesterol efflux at 37 degrees C as a function of time and concentration. The half-maximal concentration was found to be 0.3 X 10(-6) M for apolipoprotein A-IV.DMPC complexes. Binding experiments performed in intact cells at 4 degrees C with labeled apolipoprotein A-IV.DMPC complexes showed the existence of specific binding sites, with a Kd value of 0.32 x 10(-6) M and a maximal binding capacity of 223,000 sites/cell. By cross-competition experiments with labeled and unlabeled complexes containing apolipoprotein A-IV, A-I, or A-II, it appeared that all three apolipoproteins bind to the same cell-surface recognition sites. It is suggested that apolipoprotein A-IV, which is present in the interstitial fluid surrounding adipose cells in vivo at concentrations similar to those required in vitro for the promotion of cholesterol efflux, plays a critical role in cholesterol removal from peripheral cells.     

The role of apolipoprotein A-IV in reverse cholesterol transport studied with cultured cells and liposomes derived from an ether analog of phosphatidylcholine

Cholesterol efflux was studied in a model system in culture using apolipoproteins and phospholipids added in the form of liposomes at concentrations expected to be present in the extracellular fluid. Fibroblasts were seeded in medium containing [3H]cholesterol-labeled serum, grown till confluent, and the [3H]cholesterol efflux was studied in serum-free medium. Addition of delipidated HDL apolipoprotein resulted in a very low release of [3H]cholesterol, which did not increase with time of exposure or concentration of apolipoproteins. Addition of increasing amounts of HDL apolipoprotein to liposomes prepared from either dioleoylphosphatidylcholine (PC) or its nonhydrolysable ether analog, dioleylphosphatidylcholine (DOEPC) resulted in a 3-5-fold increase of [3H]cholesterol efflux, over that achieved with liposomes alone. This model system permitted the test of the putative role of apolipoprotein A-IV in cholesterol removal from cells. The ability of apolipoprotein A-IV to enhance [3H]cholesterol efflux from cells by DOEPC liposomes was compared to that of apolipoproteins A-I, E and C, which were added at equimolar concentrations. At nM concentrations, apolipoproteins A-IV, A-I and E were equally able to enhance cholesterol efflux, while C apolipoproteins were less effective at these low concentrations. Mixtures prepared from apolipoprotein A-IV, A-I and E and PC or DOEPC liposomes were equally effective in cholesterol removal, while phosphatidylethanolamine liposome apolipoprotein mixtures had a much lower capacity. The present study provides the first evidence that apolipoprotein A-IV can play a role in reverse cholesterol transport as was suggested on the basis of high concentrations of this apolipoprotein in nonlipoprotein form in plasma and extracellular fluid. The efficacy of DOEPC liposomes to serve as cholesterol acceptors might be of potential value for enhancement of reverse cholesterol transport in vivo.     

Immune-regulation of the apolipoprotein A-I/C-III/A-IV gene cluster in experimental inflammation

Apolipoprotein A-IV is a member of the apo A-I/C-III/A-IV gene cluster. In order to investigate its hypothetical coordinated regulation, an acute phase was induced in pigs by turpentine oil injection. The hepatic expression of the gene cluster as well as the plasma levels of apolipoproteins were monitored at different time periods. Furthermore, the involvement of the inflammatory mediators' interleukins 1 and 6 and tumor necrosis factor in the regulation of this gene cluster was tested in cultured pig hepatocytes, incubated with those mediators and apo A-I/C-III/A-IV gene cluster expression at the mRNA level was measured. In response to turpentine oil-induced inflammation, a decreased hepatic apo A-IV mRNA expression was observed (independent of apo A-I and apo C-III mRNA) not correlating with the plasma protein levels. The distribution of plasma apo A-IV experienced a shift from HDL to larger particles. In contrast, the changes in apo A-I and apo C-III mRNA were reflected in their corresponding plasma levels. Addition of cytokines to cultured pig hepatocytes also decreased apo A-IV and apo A-I mRNA levels. All these results show that the down-regulation of apolipoprotein A-I and A-IV messages in the liver may be mediated by interleukin 6 and TNF-alpha. The well-known HDL decrease found in many different acute-phase responses also appears in the pig due to the decreased expression of apolipoprotein A-I and the enlargement of the apolipoprotein A-IV-containing HDL.     

Effects of Iodination on Cytoagglutination by and Toxicity of Ricinus communis Lectins

Iodinations of two Ricinus communis lectins, ricin D and hemagglutinin (CBH), with potassium iodide at pH 7.0 and 0°C led to inactivation of the cytoagglutinating activity on sarcoma 180 ascites tumor cells as well as the toxicity to HeLa cells of ricin D, whereas the cytoagglutinating activity of CBH was affected slightly. In the presence of lactose, which binds to ricin D, one tyrosyl residue in the B-chain of ricin D was protected from iodination and 40% of the cytoagglutinating activity was retained. This protection against iodination was not observed in the presence of glucose, which does not bind to ricin D. This suggested that the protected tyrosyl residue in the B-chain of ricin D may be situated at or near the saccharide binding site and directly involved in the binding to the saccharide moieties of the cellular receptors.

Adsorption of the iodinated ricin D to Sepharose 4B indicated that one of the two saccharide binding sites in ricin D is still intact and participates in the binding to saccharide: ricin D was altered from divalent to monovalent by the iodination.

We found from binding experiments with ¹²⁵I-labeled iodinated ricin D to HeLa cells, that the low toxicity of the iodinated ricin D may be attributed mainly to its decreased internalization into the cells and that the divalent binding of ricin D to the cellular receptors is important for this internalization.