[The functions of receptors on the liver cells].
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Animals↗
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[Receptors and molecular heterogeneity of lipoprotein particles containing apolipoprotein A-I].
High density lipoproteins (HDL) are heterogeneous, with respect to their hydrated density (HDL2, HDL3), and to their apolipoprotein composition (Lp A-I : A-II contains both apolipoprotein A-I and A-II, Lp A-I contains apolipoprotein A-I but not apolipoprotein A-II). Lp A-I and Lp A-I and Lp A-I : A-II particles have different metabolic functions. Only Lp A-I particles seem to be involved in the antiatherogenic role of HDL. Alcohol consumption raises Lp A-I : A-II level but not Lp A-I. Different tissue possess specific binding sites for HDL: steroidogenic tissue, hepatocytes peripheral cells. Apolipoprotein A-I and/or A-II are possible ligands. HLD, after binding to the receptor, can provide the cells with cholesterol, or promote an efflux of cholesterol from the cells and the "reverse cholesterol transport" from the peripheral cells to the liver. The HDL subfractions possess different metabolic roles: binding of Lp A-I to mouse adipose cell receptors promotes cholesterol efflux. Apo A-II are antagonists for this effect.
Animals↗
Identification of a rat hepatic high density lipoprotein-binding protein.
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Animals↗
The influence of cellular and lipoprotein cholesterol contents on the flux of cholesterol between fibroblasts and high density lipoprotein.
Previous studies indicate that free cholesterol moves passively between high density lipoprotein (HDL) and cell plasma membranes by uncatalyzed diffusion of cholesterol molecules in the extracellular aqueous phase. By this mechanism, the rate constants for free cholesterol influx (Cli) and efflux (ke) should not be very sensitive to the free cholesterol content of cells or HDL. Thus, at a given HDL concentration, the unidirectional influx and efflux of cholesterol mass (Fi, Fe) should be proportional to the cholesterol content of HDL and cells, respectively, and net efflux of cholesterol mass (Fe-Fi greater than 0) should occur when either cells are enriched with cholesterol or HDL is depleted of cholesterol. We have examined the influence of cell and HDL free cholesterol contents on the bidirectional flux of free cholesterol between HDL and human fibroblasts and also attempted to detect some dependence of flux on the binding of HDL to the cells. In the range of HDL concentrations from 1 to 1000 micrograms of protein/ml, ke for cell free cholesterol approximately doubled for every 10-fold increase in HDL concentration, reaching 0.04 h-1 at 1000 micrograms of HDL/ml. ke and Cli were not influenced by the doubling of fibroblast free cholesterol content (from 31 +/- 5 to 62 +/- 13 micrograms of cholesterol/mg of protein). There was an approximate exchange of cholesterol between HDL and the unenriched fibroblasts (e.g. at [HDL] = 100 micrograms/ml, Fe and Fi = 3.2 and 3.0 micrograms of cholesterol/[4 h.mg of cell protein], respectively). In contrast, there was substantial net efflux from the enriched cells (at [HDL] = 100 micrograms/ml, Fe and Fi = 5.5 and 3.1 micrograms of cholesterol/[4 h.mg of cell protein], respectively). The rate constants for cholesterol flux were not influenced by changing the free cholesterol content of HDL, so that there was net efflux of cell cholesterol in the presence of cholesterol-depleted HDL and net influx from cholesterol-rich HDL. The Kd of HDL binding to fibroblasts was reduced from 1.7 to 0.9 micrograms/ml by the enrichment of the cells with free cholesterol; this increase in affinity for HDL was not reflected in enhanced rate constants for cholesterol flux. The inhibition of specific HDL binding by treatment of the lipoprotein with dimethyl suberimidate did not affect cholesterol flux using either control or cholesterol-rich cells at any HDL concentration in the range 1-1000 micrograms/ml. The above results are consistent with the concept that net movement of free cholesterol between cells and HDL occurs by passive, mass-action effects.(ABSTRACT TRUNCATED AT 400 WORDS)
Biological Transport↗
Rotational diffusion of human lipoproteins and their receptors as determined by time-resolved phosphorescence anisotropy.
Time-resolved phosphorescence anisotropy has been used to assess the rotational dynamics of human serum lipoproteins labeled with phosphorescent probes of high triplet yield. Labeling the lipid phase of low density, very low density, and high density lipoproteins with an eosinyl fatty acid revealed the existence of two motions. The shorter time constant was attributed to motion of the chromophore within the lipoprotein particle, while the longer time constant represented the global tumbling of the particles in solution. The measured correlation times for this global motion were about twice those predicted from the Stokes-Einstein relationship. Covalent labeling of the apolipoproteins of the low and high density lipoproteins with erythrosin revealed the existence of segmental motion of labeled domains of the apolipoprotein within their respective particles. The correlation times for this motion were within the range 10-50 microseconds. The binding of low density lipoproteins to receptors on membranes isolated from the adrenal cortex resulted in a freezing of the global motion, but maintenance of the faster segmental motion of the labeled domains of the apolipoprotein. The experiments imply that in these membranes there is no global motion of the low density lipoprotein-receptor complex on the phosphorescence time scale. Similar results were found for the binding of high density lipoproteins to liver plasma membranes. The contributions of nonspecific binding of the labeled lipoproteins to the measured phosphorescence anisotropy were carefully assessed.
Adrenal Cortex↗
Receptor-mediated internalization of high density lipoprotein by rat sinusoidal liver cells: identification of a nonlysosomal endocytic pathway by fluorescence-labeled ligand.
Rat sinusoidal liver cells possess the surface receptor for high density lipoprotein (HDL) (Murakami, M., S. Horiuchi, K. Takata, and Y. Morino. 1987. J. Biochem. (Tokyo) 101: 729-741). The present study was undertaken to determine whether cell surface-bound HDL underwent subsequent endocytic internalization by using 125I-labeled HDL and fluorescein isothiocyanate-labeled HDL (FITC-HDL). The cell-associated radioactivity obtained by a 40-min incubation with 125I-labeled HDL at 37 degrees C was released into the medium as acid-precipitable forms upon further incubation at 37 degrees C. When further incubated at 0 degree C instead of 37 degrees C, however, this release was significantly reduced. A similar phenomenon was observed after the cell-associated ligands had been treated with trypsin. The cell-associated ligands obtained after a 1-hr incubation with 125I-labeled HDL at 0 degree C were largely counted for by those bound to the outer surface of the cells, thus suggesting that HDL is internalized into cells at 37 degrees C but not at 0 degree C. Moreover, when cells were incubated with FITC-HDL at 0 degree C, the cell-associated ligands were found in a pH 7.2 +/- 0.1 compartment, whereas when incubated at 37 degrees C, its microenvironmental pH became much more acidic, exhibiting pH 6.2 +/- 0.1. Furthermore, this value returned to 7.1 +/- 0.1 upon treatment with carbonylcyanide m-chlorophenylhydrazone known to dissipate the total protonomotive force. These results suggest, therefore, that the internalization process does follow receptor-mediated binding of HDL in rat sinusoidal liver cells. This notion was also supported by fluorescence microscopic observations.
Animals↗
Cell-surface binding sites for high density lipoproteins do not mediate efflux of cholesterol from human fibroblasts in tissue culture.
The present investigation was designed to test the hypothesis that binding sites for high density lipoproteins (HDL3) on cell surfaces of peripheral tissues mediate cholesterol efflux from these cells. This hypothesis had been formulated to explain two observations: 1) HDL3 binding to peripheral cells and HDL3-mediated cholesterol efflux from these cells had both been found to saturate at similar unbound (free) HDL3 concentrations; and 2) both of these processes had been found to be similarly "up-regulated" by loading the cells with cholesterol. In the present study, however, we found that the "specific" binding of HDL3 to cholesterol-loaded human fibroblasts was saturated at a free HDL3 concentration of approximately 20 micrograms protein/ml, whereas efflux of cholesterol from these cells to HDL3 did not "saturate" even at a free HDL3 concentration of 2000 micrograms protein/ml. In addition, we found that the increase in cholesterol efflux caused by loading the fibroblasts with cholesterol was no greater when the acceptor particles were HDL3 than when albumin or phospholipid vesicles served as acceptors, despite a marked increase in HDL3 binding to these cells. Because HDL3 binding to these cells and HDL3-mediated cholesterol efflux from these cells do not saturate at similar free HDL3 concentrations, and because the cholesterol-induced increase in HDL3 binding is not accompanied by a similar increase in cholesterol efflux that is specific for HDL3, we conclude that the described HDL3 binding sites on human fibroblasts do not mediate cholesterol efflux.
Carrier Proteins↗
Up-regulation of high density lipoprotein receptor activity by gamma-interferon associated with inhibition of cell proliferation.
High density lipoprotein (HDL) binds to cell surface receptors and promotes selective removal of excess cholesterol from intracellular pools. The activity of this receptor is up-regulated when cells become loaded with cholesterol, but the relative degree of up-regulation depends on the growth state of the cells. The current study demonstrates that treatment of proliferating fibroblasts with gamma-interferon (IFN) increases the activity of the HDL receptor in association with a decrease in the rate of cell proliferation. Addition of IFN during the growth phase reduced the number of cells but had little effect on total cell protein, indicating that IFN inhibited cell proliferation but produced larger cells. IFN treatment increased the number of high affinity receptors for HDL on the surface of cholesterol-loaded fibroblasts, whether receptor binding was expressed per cell or per unit of cell surface area, cell protein, or cell cholesterol. IFN treatment also appeared to increase the amount of 110-kDa HDL binding protein in fibroblast membranes that has been postulated to represent the HDL receptor molecule. The IFN-induced increase in HDL receptor activity was associated with an enhanced ability of HDL3 to remove cholesterol from intracellular pools. These results are consistent with the hypothesis that inhibition of cell proliferation increases HDL receptor-mediated transport of excess cholesterol from cells, possibly to rid cells of cholesterol that accumulates in response to a reduced rate of membrane synthesis.
Carrier Proteins↗
The influence of the isolation technique of influenza virus nucleoprotein on its antigenic properties.
ELISA has been used to study the antigenic properties 1. of influenza virus nucleoprotein (NP-1) isolated from virions with the help of preparative polyacrylamide gel electrophoresis (PAGE); 2. of virion ribonucleoprotein (NP-2), and 3. of NP structures prepared by dissociation of ribonucleoprotein into RNA and protein in sucrose gradient containing NaCl (NP-3). The investigation of immunologic cross-reactivity has shown complete identity of NP-2 and NP-3 and their striking difference from NP-1. In contrast to NP-2, NP-3 was not contaminated by other virus antigens, it was a good immunogen and could be used for preparation of monospecific antisera of high titre. NP-1 did not induce a high antibody response,however, like NP-2 and NP-3, it retained its capacity to react with antisera to native virus. Owing to its simple production and high yield, this protein can be used in serodiagnosis for testing the antibody level against NP-protein in convalescent sera.
Animals↗
Identification of apolipoproteins involved in the interaction of human high density lipoprotein3 with receptors on cultured cells.
Human high density lipoprotein (HDL), devoid of apolipoproteins E or B, binds with high affinity and specificity to cultured cells derived from several tissues. In order to investigate the ligand specificity of the putative receptor, we have performed competitive inhibition studies to identify the components of high density lipoprotein that bind to cell surfaces of rat adrenal cortical cells and human skin fibroblasts. Radiolabeled HDL3 was displaced with unlabeled apolipoprotein-dimyristoylphosphatidylcholine recombinant particles containing AI, AII, CIII-1, and E apolipoproteins, but not by dimyristoylphosphatidylcholine complexed to albumin or by low density lipoprotein. Because exchange may readily occur between apolipoproteins in HDL and in recombinants this observation may not be truly representative of ligand competition. Further experiments using Fab fragments prepared from pure IgG to each apolipoprotein showed that binding of radioiodinated HDL to cells was suppressed following preincubation of HDL with Fab fragments raised against apolipoproteins AI or AII but not against apolipoproteins E or CIII-1 or albumin. In additional studies with apolipoprotein recombinants specific saturable binding was demonstrated between apo-AI or -AII recombinants and adrenocortical cells whereas binding of apo-CIII-2 was characterized by a large nonsaturable component which almost equaled the specific binding. The data, therefore, provide evidence for the involvement of the two major apolipoproteins (AI and AII) in HDL recognition by cellular receptors.
Adrenal Cortex↗
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.
Animals↗
Stimulation of rat ovarian cell steroidogenesis by high density lipoproteins modified with tetranitromethane.
Human high density lipoprotein (devoid of apo-E) was modified by nitration of tyrosine residues with tetranitromethane. As a result of extensive cross-linking, monomeric apo-A-I was markedly depleted, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the modified HDL did not effectively bind to high-affinity sites present on dispersed rat ovarian cells and isolated rat ovarian membranes. Nonetheless, the modified HDL retained the ability to stimulate steroidogenesis by both dispersed rat ovarian cells and cultured rat granulosa cells to a degree at least equal to that of native HDL. Modified HDL stimulated luteal steroidogenesis under basal conditions and when cells were stimulated with luteinizing hormone or 8-bromo-cAMP. Although modified HDL did not effectively bind to high-affinity sites, it exhibited substantial "nonspecific" or "low-affinity" binding which was not displaceable by native HDL. These data suggest that high-affinity binding is not an essential event in the "HDL pathway" and that HDL can deliver its sterols through low-affinity cellular associations.
Animals↗
[Protein phosphatases].
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Carrier Proteins↗
Specific binding of high density lipoprotein (HDL3) is not related to sterol synthesis in rat intestinal mucosa.
There is good evidence that high density lipoprotein (HDL) is involved in the flux of cholesterol into the cells of some organs and out of the cells of other tissues. Because we have previously found that HDL is bound specifically by mucosal cells of the small intestine, we have examined the possibility that this was associated with regulation of cholesterol flux. We have, therefore, compared the specific binding of 125I-labeled HDL3 with cholesterol synthesis in mucosal cells obtained from rats that had been treated to alter intestinal cholesterol metabolism. The rate of sterol synthesis measured in tissue slices, by the incorporation of [3H]water into sterols, was altered up to fivefold by treatment with cholestyramine (to induce bile salt loss), by surformer treatment (to reduce absorption of cholesterol), and by biliary diversion. Yet the capacity of mucosal cells to bind, internalize, and degrade 125I-labeled HDL3 was unchanged. Cholesterol feeding influenced neither the interaction of 125I-labeled HDL3 with cells nor the rate of sterol synthesis. Furthermore, the interactions of 125I-labeled HDL3 with mucosal cells isolated from the proximal and distal halves of the intestine or between the upper and lower villus cells were similar, despite differences in sterol synthesis. These data suggest that, in rat intestine, the specific binding of HDL is not related to sterol synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
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)
Animals↗
Obligatory role of cholesterol and apolipoprotein E in the formation of large cholesterol-enriched and receptor-active high density lipoproteins.
The formation of large cholesterol-enriched high density lipoproteins (HDL1/HDLc) from typical HDL3 requires lecithin:cholesterol acyltransferase activity, additional cholesterol, and a source of apolipoprotein (apo-) E. The present study explores the role of apo-E in promoting HDL1/HDLc formation and in imparting to these lipoprotein particles the ability to interact with the apo-B,E(low density lipoprotein (LDL] receptor. Incubation of normal canine serum with cholesterol-loaded mouse peritoneal macrophages resulted in the formation of HDL1/HDLc that competed with 125I-LDL for binding to the apo-B,E(LDL) receptors on cultured human fibroblasts. Cholesterol efflux from macrophages was necessary because incubation of normal canine serum with nonloaded macrophages did not cause HDL1/HDLc formation. However, cholesterol delivery to the serum was not sufficient to result in HDL1/HDLc formation. Apolipoprotein E had to be available. Incubation of apo-E-depleted canine serum with cholesterol-loaded J774 cells, a macrophage cell line that does not synthesize apo-E, demonstrated that no HDL1/HDLc formation was detected even in the presence of significant cholesterol efflux. However, addition of exogenous apo-E to the serum during the incubation with cholesterol-loaded J744 cells promoted the formation of large receptor-active HDL1/HDLc. The receptor binding activity of these particles produced in vitro correlated with the amount of apo-E incorporated into the HDL1/HDLc. Apolipoproteins A-I and C-III were ineffective in promoting HDL1/HDLc formation; thus, apo-E was unique in allowing HDL1/HDLc formation. These results demonstrate that when lecithin:cholesterol acyltransferase activity, cholesterol, and apo-E are present in serum, typical HDL can be transformed in vitro into large cholesterol-rich HDL1/HDLc that are capable of binding to lipoprotein receptors.
Animals↗
Human lipoprotein metabolism and the liver.
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Apolipoproteins↗