Regulation of high density lipoprotein receptors in cultured macrophages: role of acyl-CoA:cholesterol acyltransferase

The interaction of human serum high density lipoproteins (HDL) with mouse peritoneal macrophages and human blood monocytes was studied. Saturation curves for binding of apolipoprotein E-free [125I]HDL3 showed at least two components: non-specific binding and specific binding that saturated at approximately 40 micrograms HDL protein/ml. Scatchard analysis of specific binding of apo E-free [125I]-HDL3 to cultured macrophages yielded linear plots indicative of a single class of specific binding sites. Pretreatment of [125I]HDL3 with various apolipoprotein antibodies (anti apo A-I, anti apo A-II, anti apo C-II, anti apo C-III and anti apo E) and preincubation of the cells with anti-idiotype antibodies against apo A-I and apo A-II prior to the HDL binding studies revealed apolipoprotein A-I as the ligand involved in specific binding of HDL. Cellular cholesterol accumulation via incubation with acetylated LDL led to an increase in HDL binding sites as well as an increase in the activity of the cytoplasmic cholesterol esterifying enzyme acyl-CoA:cholesterol acyltransferase (ACAT). Incubation of the cholesterol-loaded cells in the presence of various ACAT inhibitors (Sandoz 58.035, Octimibate-Nattermann, progesterone) revealed a time- and dose-dependent amplification in HDL binding and HDL-mediated cholesterol efflux. It is concluded that the homeostasis of cellular cholesterol in macrophages is regulated in part by the number of HDL binding sites and that ACAT inhibitors enhance HDL-mediated cholesterol efflux from peripheral cells.     

Specific binding and uptake of apolipoprotein E-free high density lipoproteins by cultured liver parenchymal cells of copper-deficient rats

The influence of copper deficiency on the binding and uptake of apolipoprotein E-free high density lipoprotein (apo E-free HDL) in cultured rat hepatic parenchymal cells was examined in this study. Male weanling Sprague-Dawley rats were randomly divided into two treatments, a Cu-adequate (7.33 mg Cu/kg diet) or a Cu-deficient (1.04 mg Cu/kg diet) group. After 7 weeks, plasma apo E-free HDL were isolated by a combination of ultracentrifugation, gel filtration, and heparin-Sepharose affinity chromatography. Parenchymal cells were isolated from collagenase perfused liver of Cu-deficient and adequate rats and cultured for 16 hours at 37 degrees C prior to incubation with iodinated apo E-free HDL from the same treatment group. Cells were incubated with 5 microg/ml(125) I-apo E-free HDL for 2, 6, or 12 hours in the presence or absence of 200 microg/ml (40-fold) excess unlabeled apo E-free HDL. Increases in specific binding at 4 degrees C and specific cell-associated uptake at 37 degrees C as a function of time were observed with cells and HDL from Cu-deficient rats. Cells were also incubated for 6 hours with 8 concentrations of (125)I-apo E-free HDL in the presence or absence of excess unlabeled HDL. Although no significant increase in specific binding was detected at 4 degrees C as a function of ligand concentration, the response tended to be higher at 5 to 15 microg HDL/ml for the Cu-deficient treatment. However, at 37 degrees C the specific cell-associated uptake was increased markedly with cells and HDL from Cu-deficient rats. The observed increases in HDL binding and uptake indicate that these processes may be enhanced in Cu-deficient rats. These data are also consistent with recent in vivo results which indicate that plasma clearance and tissue uptake of HDL are increased in Cu-deficient rats.     

Uptake of rat plasma HDL subfractions labeled with [3H]cholesteryl linoleyl ether or with 125I by cultured rat hepatocytes and adrenal cells

Rat plasma low- and high-density lipoproteins were labeled with [3H]cholesteryl linoleyl ether and isolated by rate-zonal ultracentrifugation into apolipoprotein B-containing LDL, apolipoprotein E-containing HDL1 and apolipoprotein E-poor HDL2. These fractions were incubated with cultured rat hepatocytes and comparable amounts of all lipoproteins were taken up by the cells. Rat HDL was isolated at d 1.085-1.21 g/ml and apolipoprotein E-free HDL was prepared by heparin Sepharose chromatography. The original HDL and the apolipoprotein E-free HDL were labeled with 125I or with [3H]cholesteryl linoleyl ether and incubated with rat hepatocytes or adrenal cells in culture. The uptake of apolipoprotein E-free [3H]cholesterol linoleyl ether HDL by the cultured hepatocytes was 20-40% more than that of the original HDL. Comparison of uptake of cholesteryl ester moiety (represented by uptake of [3H]cholesteryl linoleyl ether) and of protein moiety (represented by metabolism of 125I-labeled protein) was carried out using both original and apolipoprotein E-free HDL. In experiments in which low concentrations of HDL were used, the ratio of 3H/125I exceeded 1.0. In cultured adrenal cells, the uptake of [3H]cholesteryl linoleyl ether-labeled HDL was stimulated 3-6-fold by 1 X 10(-7) M ACTH, while the uptake of 125I-labeled HDL increased about 2-fold. The ratio of 3H/125I representing cellular uptake was 2-3 and increased to 5 in ACTH-treated cells. The present results indicate that in cultured rat hepatocytes the uptake of homologous HDL does not depend on the presence of apolipoprotein E. Evidence was also presented for an uptake of cholesteryl ester independent of protein uptake in cultured rat adrenal cells and to a lesser extent in rat hepatocytes.     

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.     

Specific binding of iodothyronines with apolipoprotein A-1 in high density lipoprotein particle isolated from human serum by affinity chromatography on thyroxine-sepharose

The kinetic and equilibrium characteristics of interaction of thyroxine (T4) and its structural analogs with a high density lipoprotein (HDL) fraction isolated from human serum by T4-Sepharose affinity chromatography and containing apolipoprotein A-I (apo A-I) as a sole protein component, were studied. The binding of [125I]T4 to apo A-I-HDL reached a maximum after 40 min and did not change during the next 80 min of incubation at 0 degrees--22 degrees C. Dissociation of [125I]T4 induced by the addition of excess unlabeled T4 to the complex solution proceeded more intensely on a time scale at 0--2 degrees C than at 22 degrees C. Incubation of apo A-I-HDL with increasing concentrations of T4 showed that the binding is saturable. The data analysis using different computer programs revealed the presence in apo A-I-HDL of a single class of binding sites with K alpha = (4.0 +/- 2.1).10(-7) M- and Bmax = 1.7 +/- 0.8 nmol T4/mg of protein. Naturally occurring iodothyronines, their analogs and D-isomers of thyroid hormones competed with [125I]T4 for the binding sites on apo A-I-HDL with the following inhibitory potencies: L-T4 = D-T4 greater than or equal to 3,3',5-triiodo-L-thyronine = 3,3',5-triiodo-D-thyronine greater than 3,5-diiodo-L-thyronine = 3,3',5- triiodothyroacetic acid greater than 3,3',5-triiodothyropropionic acid greater than or equal to 3,5-diiodo-L-thyrosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of human low and high density lipoproteins on the binding of rat intermediate density lipoproteins to rat liver membranes

Upon incubation with rat liver membranes, radioiodinated rat intermediate density lipoproteins (IDL) interacted with at least two binding sites having a low and a high affinity as demonstrated by the curvilinear Scatchard plots obtained from the specific binding data. The purpose of our work was to identify the nature of these binding sites. Human low density lipoproteins (LDL), contain apolipoprotein B only, and human high density lipoproteins (HDL3), containing neither apolipoprotein B nor E, were both capable of decreasing the specific binding of rat 125I-IDL. The Scatchard analysis clearly revealed that only the low affinity component was affected by the addition of these human lipoproteins. In fact, the low affinity binding component gradually decreased as the amount of human LDL or HDL3 increased in the binding assay. At a 200-fold excess of human LDL or HDL3, the low affinity binding was totally masked, and the Scatchard plot of the specific 125I-IDL binding became linear. Only the high affinity binding component was left, enabling a precise measurement of its binding parameters. In a series of competitive displacement experiments in which the binding assay contained a 200-fold excess of human LDL or HDL3, only unlabeled rat IDL effectively displaced the binding of rat 125I-IDL. We conclude that the low affinity binding of rat IDL to rat liver membranes is due to weak interactions with unspecified lipoprotein binding sites. The camouflage of these sites by human lipoproteins makes possible the study of IDL binding to the high affinity component which likely represents the combined effect of IDL binding to both the remnant and the LDL receptors.     

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.     

Apoprotein A-1 is a cofactor independent substrate of protein kinase C

Apoprotein A-1 (apo A-1), the predominant protein constituent of high density lipoproteins (HDL), was phosphorylated by protein kinase C (PKC). Optimal phosphorylation of lipid-free apo A-1 occurs in the absence of calcium, phosphatidyl serine (PS), and diolein (DO). However, HDL-bound apo A-1 was not phosphorylated by PKC. Furthermore, addition of either native or reconstituted HDL particles to lipid-free apo A-1 resulted in a concentration-dependent inhibition of phosphorylation. It appears that the phosphorylatable sites on apo A-1 are involved in hydrophobic interaction with the lipids of HDL. Apo A-1 is a novel substrate of PKC because it does not require calcium and lipid cofactors for optimal phosphorylation.     

5-Iodo-A-85380 binds to alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors (nAChRs) as well as alpha4beta2* subtypes

Recent work suggests that 5-iodo-A-85380, a radioiodinated analog of the 3-pyridyl ether A-85380, represents a promising imaging agent for non-invasive, in vivo studies of alphaAbeta2* nicotinic acetylcholine receptors (nAChRs; *denotes receptors containing the indicated subunits), because of its low non-specific binding, low in vivo toxicity and high selectivity for alpha4beta2* nAChRs. As an approach to elucidate nAChR subtypes expressed in striatum, we carried out competitive autoradiography in monkey and rat brain using 5-[125I]iodo-A-85380 ([125I]A-85380) and [125I]alpha-conotoxin MII, a ligand that binds with high affinity to alpha6* and alpha3* nAChRs, but not to alpha4beta2* nAChRs. Although A-85380 is reported to be selective for alpha4beta2* nAChRs, we observed that A-85380 completely inhibited [125I]alpha-conotoxin MII binding in rat striatum and that A-85380 blocked >90% of [125I] alpha-conotoxin MII sites in monkey caudate and putamen. These results suggest that A-85380 binds to non-alpha4beta2* nAChRs, including putative alpha6* nAChRs. Experiments to determine the percentage of [125I]A-85380 sites that contain alpha-conotoxin MII-sensitive (alpha6beta2*) nAChRs indicate that they represent about 10% of [125I]A-85380 sites in rodent striatum and about 30% of sites in monkey caudate and putamen. These data are important for identifying alterations in nicotinic receptor subtypes in Parkinson's disease and other basal ganglia disorders both in in vitro and in in vivo imaging studies.     

Interaction in vivo and in vitro of apolipoprotein E-free high-density lipoprotein with parenchymal, endothelial and Kupffer cells from rat liver.

The interaction of apolipoprotein (apo) E-free high-density lipoprotein (HDL) with parenchymal, endothelial and Kupffer cells from liver was characterized. At 10 min after injection of radiolabelled HDL into rats, 1.0 +/- 0.1% of the radioactivity was associated with the liver. Subfractionation of the liver into parenchymal, endothelial and Kupffer cells, by a low-temperature cell-isolation procedure, indicated that 77.8 +/- 2.4% of the total liver-associated radioactivity was recovered with parenchymal cells, 10.8 +/- 0.8% with endothelial cells and 11.3 +/- 1.7% with Kupffer cells. It can be concluded that inside the liver a substantial part of HDL becomes associated with endothelial and Kupffer cells in addition to parenchymal cells. With freshly isolated parenchymal, endothelial and Kupffer cells the binding properties for apo E-free HDL were determined. For parenchymal, endothelial and Kupffer cells, evidence was obtained for a saturable, specific, high-affinity binding site with Kd and Bmax. values respectively in the ranges 10-20 micrograms of HDL/ml and 25-50 ng of HDL/mg of cell protein. In all three cell types nitrosylated HDL and low-density lipoproteins did not compete for the binding of native HDL, indicating that lipids and apo B are not involved in specific apo E-free HDL binding. Very-low-density lipoproteins (VLDL), however, did compete for HDL binding. The competition of VLDL with apo E-free HDL could not be explained by label exchange or by transfer of radioactive lipids or apolipoproteins between HDL and VLDL, and it is therefore suggested that competition is exerted by the presence of apo Cs in VLDL. The results presented here provide evidence for a high-affinity recognition site for HDL on parenchymal, liver endothelial and Kupffer cells, with identical recognition properties on the three cell types. HDL is expected to deliver cholesterol from peripheral cells, including endothelial and Kupffer cells, to the liver hepatocytes, where cholesterol can be converted into bile acids and thereby irreversibly removed from the circulation. The observed identical recognition properties of the HDL high-affinity site on liver parenchymal, endothelial and Kupffer cells suggest that one receptor may mediate both cholesterol efflux and cholesterol influx, and that the regulation of this bidirectional cholesterol (ester) flux lies beyond the initial binding of HDL to the receptor.     

Characterization of high-density lipoprotein binding and cholesterol efflux in cultured mouse adipose cells

Binding of high-density lipoproteins to cultured mouse Ob1771 adipose cells was studied, using labeled human HDL3, mouse HDL and apolipoprotein AI- or AII-containing liposomes. In each case, saturation curves were obtained, yielding linear Scatchard plots. The Kd values were found to be respectively 18, 42, 30 and 3.4 micrograms/ml, whereas the maximal binding capacities were found to be 160, 100, 90 and 21 ng/mg of cell protein. Apoprotein AI not inserted into liposomes did not bind. The binding of 125I-HDL3 was competitively inhibited by apolipoprotein AI-containing liposomes greater than mouse HDL greater than HDL3. The binding of 125I-labeled apolipoprotein AI- and 125I-labeled apolipoprotein AII-containing liposomes was competitively inhibited by HDL3, apolipoprotein AI- and apolipoprotein AII-containing liposomes. Dimyristoylphosphatidylcholine liposomes containing or not cholesterol did not interfere with the binding of labeled HDL3 or apolipoprotein-containing liposomes. Binding studies on crude membranes of Ob1771 adipose cells revealed the presence of intracellular binding sites for LDL and HDL3. Thus, adipose cells have specific binding sites for apolipoprotein E-free HDL and apolipoprotein AI (or AII) is the ligand for these binding sites. Long-term exposure of adipose cells to LDL cholesterol as a function of LDL concentration led to an accumulation of cellular unesterified cholesterol. This process was saturable and reversible as a function of time and concentration by exposure to HDL3 or apolipoprotein AI-containing liposomes, whereas apolipoprotein AII-containing liposomes did not promote any cholesterol efflux. Since long-term exposure of adipose cells to LDL and HDL3 did not affect the number of apolipoprotein B,E receptors and apolipoprotein E-free binding sites, respectively, it appears that adipose cells do not show efficient cholesterol homeostasis and thus could accumulate or mobilize unesterified cholesterol.     

Binding of lipoproteins and regulation of cholesterol synthesis in cultured mouse adipose cells

Binding of human lipoproteins to cultured mouse Ob17 preadipose and adipose cells was studied, using labeled VLDL, LDL and apoprotein E-free HDL. In each case, saturation curves were obtained, yielding linear Scatchard plots. The Kd values were found to be respectively 6.4, 31 and 24 micrograms/ml for VLDL, LDL and apoprotein E-free HDL, whereas the maximal numbers of binding sites per cell were 4.2 X 10(4), 1.5 X 10(4) and 2.5 X 10(5). The binding of 125I-LDL was competitively inhibited by LDL greater than VLDL greater than total HDL; human LDL and mouse LDL were equipotent in competition assays. Methylated LDL and apoprotein E-free HDL were not competitors. In contrast, the binding of 125I-apoprotein E-free HDL was competitively inhibited by apoprotein E-free HDL greater than total HDL and the binding of 125I-HDL3 by mouse HDL. Thus, mouse adipose cells possess distinct apoprotein B, E and apoprotein E-free HDL binding sites which can recognize heterologous or homologous lipoproteins. The cell surface receptor of LDL in mouse preadipose cells shows similarities with that described for human fibroblasts, since: (1) the LDL binding initiated the process of internalization and degradation of the apoprotein B and apoprotein E-containing lipoproteins; (2) receptor-mediated uptake of cholesterol LDL led to a parallel but incomplete decrease in the [14C]acetate incorporation into cholesterol and in the activity of HMG-CoA reductase. Growing (undifferentiated) or growth-arrested cells (differentiated or not) showed no significant changes in the Kd values for lipoprotein binding. In contrast, the maximal number of binding sites correlated with the proliferative state of the cells and was independent of cell differentiation. The results are discussed with respect to cholesterol accumulation in adipose cells.     

Binding of apolipoprotein A-I and A-II after recombination with phospholipid vesicles to the high density lipoprotein receptor of luteinized rat ovary

To determine the apolipoprotein specificity of high density lipoprotein (HDL) receptor, apolipoprotein A-I (apo-AI) and apolipoprotein A-II (apo-AII) purified from high density lipoprotein3 (HDL3) were reconstituted into dimyristoyl phosphatidylcholine vesicles (DMPC) and their ability to bind to luteinized rat ovarian membranes was examined. Both 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC were shown to bind to ovarian membranes with Kd = 2.87 and 5.70 micrograms of protein/ml, respectively. The binding of both 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC was inhibited by unlabeled HDL3, apo-A-I.DMPC, apo-A-II.DMPC, apo-C-I.DMPC, apo-C-II.DMPC, apo-C-III1.DMPC, and apo-C-III2.DMPC, but not by DMPC vesicles, bovine serum albumin.DMPC or low density lipoprotein. Since the binding labeled apo-A-I.DMPC and apo-A-II.DMPC was inhibited by the DMPC complexes of apo-C groups, the direct binding of 125I-apo-C-III1.DMPC was also demonstrated with Kd = 9.6 micrograms of protein/ml. In addition, unlabeled apo-A-I.DMPC, and apo-A-II.DMPC, as well as apo-C.DMPC, inhibited 125I-HDL3 binding. 125I-apo-A-I, 125I-apo-A-II, and 125I-apo-C-III1 in the absence of DMPC also bind to the membranes. These results suggest that HDL receptor recognizes apolipoprotein AI, AII, and the C group and that the binding specificity of the reconstituted lipoproteins is conferred by their apolipoprotein moiety rather than the lipid environment. In vivo pretreatment of rats with human chorionic gonadotropin resulted in an increase of 125I-apo-A-I.DMPC, 125I-apo-A-II.DMPC, and 125I-apo-C-III1.DMPC binding activities. However, no induction of binding activity was observed when the apolipoprotein was not included in DMPC vesicles. An examination of the equilibrium dissociation constant and binding capacity for 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC after human chorionic gonadotropin treatment revealed that the increase in binding activity was due to an increase in the number of binding sites rather than a change in the binding affinity. These results further support our contention that apo-A-I, apo-A-II, and the apo-C group bind to HDL receptor. In conclusion, the HDL receptor of luteinized rat ovary recognizes apolipoproteins A-I, A-II, and the C group but not low density lipoprotein, and the binding is induced by human chorionic gonadotropin in vivo.     

Uptake of lipoprotein-associated alpha-tocopherol by primary porcine brain capillary endothelial cells

From the severe neurological syndromes resulting from vitamin E deficiency, it is evident that an adequate supply of the brain with alpha-tocopherol (alphaTocH), the biologically most active member of the vitamin E family, is of utmost importance. However, uptake mechanisms of alphaTocH in cells constituting the blood-brain barrier are obscure. Therefore, we studied the interaction of low (LDL) and high (HDL) density lipoproteins (the major carriers of alphaTocH in the circulation) with monolayers of primary porcine brain capillary endothelial cells (pBCECs) and compared the ability of these two lipoprotein classes to transfer lipoprotein-associated alphaTocH to pBCECs. With regard to potential binding proteins, we could identify the presence of the LDL receptor and a putative HDL3 binding protein with an apparent molecular mass of 100 kDa. At 4 degrees C, pBCECs bound LDL with high affinity (K(D) = 6 nM) and apolipoprotein E-free HDL3 with low affinity (98 nM). The binding capacity was 20,000 (LDL) and 200,000 (HDL3) lipoprotein particles per cell. alphaTocH uptake was approximately threefold higher from HDL3 than from LDL when [14C]alphaTocH-labeled lipoprotein preparations were used. The majority of HDL3-associated alphaTocH was taken up in a lipoprotein particle-independent manner, exceeding HDL3 holoparticle uptake 8- to 20-fold. This uptake route is less important for LDL-associated alphaTocH (alphaTocH uptake approximately 1.5-fold higher than holoparticle uptake). In line with tracer experiments, mass transfer studies with unlabeled lipoproteins revealed that alphaTocH uptake from HDL3 was almost fivefold more efficient than from LDL. Biodiscrimination studies indicated that uptake efficacy for the eight different stereoisomers of synthetic alphaTocH is nearly identical. Our findings indicate that HDL could play a major role in supplying the central nervous system with alphaTocH in vivo.     

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.     

Modification of human high density lipoprotein (HDL3) with tetranitromethane and the effect on its binding to isolated rat liver plasma membranes

Apolipoprotein E-free high density lipoproteins (HDL) bind to various cells and cell membrane preparations, with properties typical of ligand-receptor interactions. In order to further characterize the binding sites and to investigate the functional role of binding, a chemically modified HDL without the specific binding properties would be highly desirable. We have reacted human HDL3 with tetranitromethane, a relatively specific nitrating reagent for tyrosine residues, in 50 mM Tris HCL buffer, pH 8.0, and at a reagent concentration 10 times the molar excess of tyrosine residues. The resulting nitrated HDL3 completely lost its ability to bind to high affinity saturable binding sites of rat liver plasma membranes, as determined by competitive binding with 125I-labeled HDL3, and also by direct binding assays using 125I-labeled nitrated HDL3. Although nitrated HDL3 did not bind to the high affinity saturable binding sites, it bound to the membranes, but the binding was not saturable, and was not competed for by unlabeled nitrated HDL3. On agarose gel electrophoresis, pH 8.6, the nitrated HDL3 moved ahead of the control HDL3, indicating an increase in negative charges in the molecule. No difference in size was noted in the nitrated HDL3 when analyzed either by negative stain electron microscopy or by gel filtration chromatography. Spectroscopic analysis of the nitrated HDL3 at pH 8.0 revealed a prominent absorption with maximum at around 360 nm, but none in the region expected for nitrotyrosine residues. At pH 10.0, however, the nitrated HDL3 showed an absorption band with a maximum at around 440 nm, possibly related to nitrotyrosine residues. Nitrotyrosine was detected in the nitrated HDL3 on amino acid analysis. Comparison of the amino acid analysis of the nitrated HDL3 and control HDL3 showed no difference in composition of any of the amino acids except tyrosine; tyrosine content was reduced more than 90% in the nitrated HDL3. SDS-polyacrylamide gel electrophoresis analysis of apoproteins of nitrated HDL3 revealed changes in apolipoprotein profile. Bands corresponding to the apolipoproteins of the starting HDL3 almost disappeared and a series of new bands appeared at the high molecular weight region of the gel, indicating extensive cross-linking of apolipoproteins during the reaction. In addition, a substantial amount of phospholipids and cholesteryl esters, but not unesterified cholesterol, was found covalently linked, possibly through the unsaturated centers of the fatty acid chains, to apolipoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Apolipoprotein E-rich HDL binding to liver plasma membranes in copper-deficient rats

Copper deficiency in rats raises plasma cholesterol concentration while reducing live cholesterol concentration. One consequence of this cholesterol redistribution is the accumulation of a large high-density lipoprotein (HDL) particle rich in apolipoprotein E (apo E). The purpose of this study was to determine, using an in vitro binding assay, if the interaction of apo E-rich HDL with hepatic lipoprotein binding sites may be affected by copper deficiency. Male Sprague-Dawley rats were divided into two dietary treatments (copper-deficient and -adequate) and placed on a dietary regimen for 8 weeks. Subsequent to exsanguination, hepatic plasma membranes were prepared and apo E-rich HDL was isolated from rats of each treatment by ultracentrifugation, agarose column chromatography, and heparin-Sepharose affinity chromatography. Total binding and experimentally derived specific binding of 125I-apo E-rich HDl to hepatic plasma membranes indicated greater binding when lipoproteins and membranes from copper-deficient animals were used in the assay compared to controls. Scatchard analysis of specific binding data indicated that equilibrium binding affinity (Kd) was also affected by copper deficiency. The hepatic binding sites recognizing apo E-rich HDL were not affected by EDTA or pronase, of relatively high capacity, and recognized a variety of other rat lipoproteins.     

A quantitative assay for the non-covalent association between apolipoprotein[a] and apolipoprotein B: an alternative measure of Lp[a] assembly

Increasing evidence suggests that the assembly of lipoprotein[a] (Lp[a]) proceeds in two steps. In the first step, non-covalent interactions between apolipoprotein[a] (apo[a]) and apolipoprotein B (apoB) of low density lipoprotein (LDL) form a dissociable apo[a]:LDL complex. In the second step, a covalent disulfide linkage forms the stable Lp[a] particle. Several methods are currently used to study the assembly of Lp[a], however, these methods are laborious, time-consuming, and not suitable for a high throughput screening. We report here the development of a rapid and simple assay based on the binding of labeled LDL to a Lp[a]/apo[a] substrate which is immobilized on the surface of a microtiter plate. Quantification of bound LDL provides a measure of the extent of complex formation. Labeled LDL bound to both Lp[a] and apo[a] substrates with similar affinity. Plasma lipoproteins containing apoB as well as free apo[a] were capable of competing with LDL binding. The binding of LDL to Lp[a]/apo[a] was inhibited by L-proline and lysine analogs, which are known to inhibit the non-covalent association between apo[a] and apoB. Using this method we have found that nicotinic acid and captopril are able to inhibit the association of apo[a] with apoB. This method is compatible with automation and can be applied to a high throughput screening of inhibitors of Lp[a] formation.     

Site-directed mutagenesis of an apolipoprotein E mutant, apo E5(Glu3----Lys) and its binding to low density lipoprotein receptors.

Apo E5(Glu3----Lys) is a naturally occurring apolipoprotein E (apo E) mutant found in patients with hyperlipoproteinemia and atherosclerosis. It has been shown to have a high affinity for low density lipoprotein (LDL) receptors. In this study, mutant apo E5 was produced by Chinese hamster ovary cells by means of an in vitro site-directed mutagenesis technique, and its LDL receptor binding activity was assessed. The apo E5 obtained from gene expression bound more readily to the LDL receptor than did plasma apo E3. The concentrations required for 50% competitive binding of 125I-labeled LDL to the LDL receptors were 58.9 ng/ml for plasma apo E3 and 25.7 ng/ml for the expressed apo E5. The expressed apo E5 displayed 229% normal binding. This result is highly consistent with that obtained with plasma apo E5, which showed 217% normal binding. Although the experimental apo E isoproteins contained more sialic acid than plasma apo E, the extent of sialylation had no effect on the receptor binding of apo E.     

Plasma factors controlling atrial natriuretic peptide (ANP) aggregation: role of lipoproteins

We have previously shown that human plasma atrial alpha-natriuretic peptide (alpha-hANP) sequestering is a protective phenomenon against amyloid aggregation. In the present work, the possible role of lipoproteins as alpha-hANP binding factors has been investigated in vitro using an experimental model, developed in our laboratory, that allows to work at physiological concentrations. This approach consists of gel filtration on Sephacryl S-300 HR of big alpha-[(125)I]hANP generated in phosphate buffered saline or in human normal plasma supplemented or not with lipoproteins. The results of these experiments indicate that high density lipoproteins (HDL) are responsible for the ANP binding phenomenon observed in vitro, while low density lipoproteins and very low density lipoproteins do not directly interact with ANP. Moreover, the HDL remodeling process occurring in vitro has been analyzed during plasma incubation by monitoring the redistribution of lipids and apolipoproteins among the HDL subclasses. The changes in HDL size and composition observed in incubated plasma were compared with the redistribution of endogenous and labeled big ANP. The obtained results revealed that both tend to follow the molecular rearrangement in plasma of apolipoprotein A-I containing particles and suggested that, among HDL species, the small particles are mainly involved in the ANP binding phenomenon. This hypothesis was further demonstrated by ligand blotting experiments that confirmed the existence of differences in the ability of HDL particles to bind alpha-[(125)I]hANP.