Platelet high affinity low density lipoprotein binding and import of lipoprotein derived phospholipids

The binding of low density lipoprotein (LDL) to the platelet cell membrane could facilitate the transfer of phospholipids from LDL to the platelets. A polyclonal antibody against the platelet glycoproteins IIb/IIIa inhibited the high affinity binding of 125I-LDL by up to 80%. The transfer of pyrene (py)-labeled sphingomyelin (SM), phosphatidylcholine and phosphatidylethanolamine from LDL to the platelets was unaffected by the antibody. The lectin wheat germ agglutinin (WGA) reduced the binding of 125I-LDL to the platelets by approximately 80%. In contrast, the lectin stimulated the transfer of SM from LDL into the platelets by about three-fold. WGA also specifically augmented the transfer of py-SM between lipid vesicles and the platelets, the stimulation being abolished in the presence of N-acetylglucosamine. Dextran sulfate (DS) increased the specific binding of 125I-LDL to the platelets by up to 2.8-fold. On the other hand, the import of LDL-derived py-phospholipids was unaffected by DS. Together, the results indicate that the phospholipid transfer from LDL to the platelets is independent of the high affinity LDL binding to the platelets and is specifically stimulated by WGA. Thus, the interactions of platelets with LDL phospholipids differ markedly from those with the apoprotein components of the lipoproteins.     

Virulent Escherichia coli strains for chicks bind fibronectin and type II collagen

125I-fibronectin and 125I-collagen (type II) binding was detected in Escherichia coli strains isolated from chickens and poults. High fibronectin binding-strains also bind the 29 kD aminoterminal fragment of fibronectin. Binding properties in strain CK28 were partially characterized. The highest binding of 125I-fibronectin and 125I-collagen for strain CK28 was obtained with bacteria grown at 33 degrees C. Binding of 125I-fibronectin, its 125I-29 kD fragment, and 125I-collagen, was very rapid, reaching a maximum in 5 min. Binding of 125I-fibronectin and 125I-collagen was considerably inhibited by preincubation of bacteria with unlabelled fibronectin and unlabelled type I collagen respectively, but not inhibited with human immunoglobulin G or bovine serum albumin. Inhibition experiments showed that the reversibility of 125I-fibronectin binding was estimated at approximately 50%, while reversibility for 125I-collagen binding was higher than 90%. Receptors for fibronectin, its 29 kD fragment, and collagen were released from the bacterial surface by treatment at different temperatures, and surface material released at 100 degrees C inhibited binding. There was cross-inhibition for both fibronectin and collagen binding when unlabelled fibronectin and unlabelled collagen were used as inhibitors, suggesting that binding receptors for both proteins may be closely located.     

Native and modified low-density-lipoprotein interaction with human platelets in normal and homozygous familial-hypercholesterolaemic subjects.

The binding of low-density lipoproteins (LDL) as well as LDL modified by cyclohexanedione (CHD-LDL) to gel-filtered platelets (GFP) and its effect on platelet function were studied in normal and in homozygous familial hypercholesterolaemic (HFH) subjects. Only normal-derived LDL could significantly compete with normal 125I-labelled LDL for binding to normal platelets. When GFP from normal subjects were incubated with normal LDL at concentrations of 25-200 micrograms of protein/ml, platelet aggregation in the presence of thrombin (0.5 i.u./ml) was increased by 65-186%. CHD-LDL, at similar concentrations, caused the opposite effect and decreased platelet aggregation by 26-47%. Both LDL and CHD-LDL (100 micrograms/ml) from HFH patients, when incubated with normal GFP, caused a significant reduction in platelet aggregation (33 and 50% respectively). When HFH-derived platelets were used, both patient LDL and CHD-LDL (but not the normal lipoprotein) could markedly compete with the patient 125I-labelled LDL for binding to the platelets. LDL and CHD-LDL (100 micrograms/ml) from normal subjects decreased aggregation of HFH-platelets by 52 and 85% respectively, while corresponding concentrations of LDL derived from HFH subjects (HFH-LDL) and CHD-LDL derived from HFH subjects (CHD-HFH-LDL) increased platelet aggregation by 165 and 65% respectively. The present results support the following conclusions: platelet activation by LDL in normal subjects is through the arginine-rich apoprotein-binding site; more than one binding site for LDL exists on platelets; under certain circumstances, LDL binding can cause a reduction in platelet activity; specificity for LDL binding to the platelets resides in different regions of the lipoprotein in HFH and in normal subjects. We have thus suggested a model for LDL-platelet interaction in normal and in HFH subjects.     

Characterisation of heterologous and homologous low-density lipoprotein binding to apolipoprotein B,E receptors on porcine adrenal cortex membranes: enhanced binding of trypsin-modified human low-density lipoprotein

The characteristics of the binding of homologous and heterologous (human) LDL to membrane preparations from porcine adrenal cortex have been determined. The membranes displayed a single class of high-affinity, saturable binding site for both 125I-labelled porcine and human LDL, which was dependent on divalent cations, in addition to a low-affinity, non-saturable component(s). Porcine LDL displaced both 125I-labelled porcine and 125I-labelled human LDLs from the high-affinity binding site more effectively than human LDL, reflecting the lower Kd, (13.2 micrograms/ml) for porcine than human (Kd 19.2 micrograms/ml) LDL. These values are comparable to those obtained for half-maximal binding of human and bovine LDLs in a bovine adrenocortical membrane system (Kovanen, P.T., Basu, S.K., Goldstein, J.L. and Brown, M.S. (1979) Endocrinology 104, 610-616). Tryptic modification of porcine LDL (T-LDL) diminished its ability to compete with 125I-labelled native LDL for the high-affinity binding site; in contrast, 125I-labelled porcine T-LDL showed an elevated receptor affinity (Kd 9.7 micrograms/ml) and was more efficiently displaced by its unlabelled counterpart than by native porcine LDL. Tryptic treatment of human LDL similarly increased its binding affinity (Kd 8.3 micrograms/ml), although in this case, the unlabelled T-LDL displaced not only 125I-labelled human T-LDL but also 125I-labelled human LDL from the high-affinity site more effectively than native LDL. We conclude that (i) porcine adrenocortical membranes possess binding sites specific for LDL and resembling the apolipoprotein B,E receptors already demonstrated in murine, bovine and human adrenal cortex; (ii) tryptic modification of porcine LDL may remove or destroy segments of apolipoprotein B100 which contribute to receptor recognition sites on the surface of the particle; (iii) trypsinised porcine LDL may interact with the membrane binding site by a mechanism differing from that by which native LDL binds, and (iv) trypsinisation of human LDL may cleave or remove species-specific segments of the B100 protein at or close to the receptor recognition site(s) on the particle, thus decreasing structural differences between porcine and human LDL, and thereby enhancing its binding affinity for the porcine receptor.     

High-density lipoprotein binding to bovine adrenal cortex membranes

Binding studies were performed with bovine adrenal cortex membranes, human 125I-labelled high-density lipoprotein (HDL) and modified photoactivable derivatives of 125I-labelled HDL, namely 125I-labelled HDL-amidinophenylazide and 125I-labelled HDL-amidopropionyldithiophenylazide. The purity of the apolipoprotein composition of the 125I-labelled HDL and photoactivable 125I-labelled HDL used in the binding studies was determined by Coomassie blue and silver staining, and by measuring 125I-labelled cpm after SDS-polyacrylamide gel electrophoresis. About 45% of the 125I-labelled HDL binding to the membranes occurred in the presence of excess EDTA and only unlabelled HDL competed for the binding site. The 125I-labelled interaction with this binding site on the membranes did not require calcium. In addition, 40% of the 125I-labelled HDL binding was to an EDTA-sensitive site, and unlabelled HDL and low-density lipoprotein (LDL) competed for the binding site. Consequently, adrenal cortex membranes have binding sites which show cross reactivity for both HDL and LDL. Modification of 58% of the apolipoprotein lysine residues of 125I-labelled HDL with methylazidophenylimidate, a reagent which maintains the positive charge at lysine residues, had little affect on binding to EDTA-sensitive and insensitive sites. In contrast, modification of 35% of apolipoprotein lysine residues of 125I-labelled HDL with N-succinimidyl(4-azidophenyldithio)propionate, a reagent which converts charged amino lysines to amide bonds, showed binding properties which were almost totally inhibited by EDTA.     

Specific saturable binding of rat high-density lipoproteins to rat kidney membranes

The binding of rat 125I-labelled high-density lipoprotein (HDL) to rat kidney membranes was studied using HDL fractions varying in their apolipoprotein E content. The apolipoprotein E/apolipoprotein A-I ratio (g/g) in the HDL fractions ranged from essentially 0 to 1.5. All these HDL preparations showed the same binding characteristics. The saturation curves, measured at 0 degrees C in the presence of 2% bovine serum albumin, consisted of two components: low-affinity non-saturable binding and high-affinity binding (Kd about 40 micrograms of HDL protein/ml). Scatchard analyses of the high-affinity binding suggest a single class of non-interacting binding sites. These sites could be purified together with the plasma membrane marker enzyme 5'-nucleotidase. The binding of rat HDL to rat kidney membranes was not sensitive to high concentrations of EDTA, relatively insensitive to pronase treatment and influenced by temperature. The specific binding of rat HDL was highest at acid pH and showed an additional optimum at pH 7.5. On a total protein basis unlabelled rat VLDL competed as effectively as unlabelled rat HDL for binding of 125I-labelled rat HDL to partially purified kidney membranes. Rat LDL, purified by chromatography on concanavalin A columns and human LDL did not compete. Unlabelled human HDL was a much weaker competitor than unlabelled rat HDL and the maximal specific binding of 125I-labelled human HDL was only 10% of the value for 125I-labelled rat HDL.     

Characterization of the high-affinity receptors on Swiss 3T3 cells which mediate the binding, internalization and degradation of the mitogenic peptide bombesin.

Bombesin and bombesin-related peptides such as gastrin-releasing peptide (GRP) stimulate DNA synthesis and proliferation of Swiss 3T3 cells in culture. We have used 125I-labelled [Tyr4]bombesin and 125I-labelled GRP to characterize and identify the receptors for these peptides on Swiss 3T3 cells. The binding of 125I-[Tyr4]bombesin, which retained full biological activity, was maximal between 20 and 30 min incubation at 37 degrees C, after which continued incubation led to a decline in cell-associated radioactivity. This decline was markedly slowed by the presence of lysosomal enzyme inhibitors. Specificity of the binding site was indicated by the competitive inhibition of binding by bombesin-related peptides, but not by unrelated peptides and growth factors. Scatchard analysis of binding data indicated a single class of high-affinity receptors. The calculated value for the dissociation constant (Kd) was 2.1 nM and each cell possesses approx. 240,000 receptors. Because [Tyr4]bombesin has no free amino group, 125I-GRP was used in chemical cross-linking studies. When disuccinimidyl suberate was used to covalently couple 125I-GRP to the cells, two major radiolabelled complexes were detected with molecular masses of approx. 80,000-85,000 and 140,000. The binding of 125I-[Tyr4]bombesin to the cells was pH-dependent with maximal binding at pH 6.5-7.5 and effectively no specific binding at pH values below 4.5. At 37 degrees C, cell-associated 125I-[Tyr4]bombesin quickly became resistant to removal by acidic buffers, suggesting its rapid transfer to an intracellular compartment. However, pre-incubation with unlabelled [Tyr4]bombesin did not induce down-regulation of bombesin receptors as measured by the subsequent binding of 125I-[Tyr4]bombesin. In contrast with the Swiss 3T3 cells, specific binding of 125I-[Tyr4]bombesin was not detectable in two cell lines which are biologically unresponsive to bombesin-related peptides.     

Low density lipoprotein binding, internalization, and degradation in human adipose cells.

Human adipose tissue derives its cholesterol primarily from circulating lipoproteins. To study fat cell-lipoprotein interactions, low density lipoprotein (LDL) uptake and metabolism were examined using isolated human adipocytes. The 125I-labelled LDL (d = 1.025-1.045) was bound and incorporated by human fat cells in a dose-dependent manner with an apparent Km of 6.9 + 0.9 microgram LDL protein/mL and a Vmax of 15-80 microgram LDL protein/mg lipid per 2 h. In time-course studies, LDL uptake was characterized by rapid initial binding followed by a linear accumulation for at least 4 h. The 125I-labelled LDL degradation products (trichloroacetic acid soluble iodopeptides) accumulated in the incubation medium in a progressive manner with time. Azide and F- inhibited LDL internalization and degradation, suggesting that these processes are energy dependent. Binding and cellular internalization of 125I-labelled LDL lacked lipoprotein class specificity in that excess (25-fold) unlabelled very low density lipoprotein (VLDL) (d less than 1.006) and high density lipoprotein (HDL) (d = 1.075-1.21) inhibited binding and internalization of 125I-labelled LDL. On an equivalent protein basis HDL was the most potent. The 125I-labelled LDL binding to an adipocyte plasma membrane preparation was a saturable process and almost completely abolished by a three- to four-fold greater concentration of HDL. The binding, internalization, and degradation of LDL by human adipocytes resembled that reported by other mesenchymal cells and could account for a significant proportion of in vivo LDL catabolism. It is further suggested that adipose tissue is an important site of LDL and HDL interactions.     

Low density lipoprotein receptors and catabolism in primary cultures of rabbit hepatocytes.

Rabbit 125I-labelled low density lipoproteins (LDL) were incubated with primary monolayer cultures of rabbit hepatocytes in studies designed to assess the role of liver in LDL catabolism at the cellular level. After hepatocytes were preincubated for 20 h in lipoprotein-free medium, they exhibited time- and concentration-dependent interaction with 125I-labelled DLD at concentrations to 1 mg LDL protein/ml and times to 24 h. After a 3 h (37 degrees C) incubation with 50 microgram LDL protein/ml, hepatocytes bound 400 ng (LDL protein)/mg (cell protein), internalized 280 ng/mg, and degraded 660 ng/mg. Internalization and degradation may be greater than indicated by these values since pulse studies suggested the presence of a deiodinase which attacks cell associated 125I-labelled LDL. The amounts of LDL bound to hepatocytes after 3 h (37 degrees C) were similar to amounts for fibroblasts, but DLD internalization and degradation were considerably less. Rabbit hyperlipidemic 125I-labelled DLD showed the same amount of binding but 1.39 times more internalization and degradation than normolipidemic 125I-labelled LDL. Binding of both control and hyperlipidemic LDL was 3-fold greater at 24 and 42 h than at O or 3 h but addition of a 50-fold molar excess of high density lipoproteins (HDL) prevented increased LDL binding with time. Induction of specific high affinity receptors for binding LDL was shown to occur by preincubation of hepatocytes for increasing periods in lipoprotein-free medium and then measuring 125I-labelled LDL binding at 4 degrees C in the presence and absence of excess unlabelled LDL. Finally, hepatocytes took up 40 times more LDL than sucrose or dextran over a 24-h period, an indication that the uptake of LDL occurs via some mechanism other than simple bulk fluid endocytosis.     

The binding of low density lipoprotein by liver membranes in the pig.

Porcine liver membranes are capable of high affinity binding of homologous low density lipoproteins (LDL). Binding is time and temperature dependant and substrate saturable. High affinity binding sites are half saturated at 11 μg/ml lipoprotein-protein. The binding of ¹²⁵I-LDL is inhibited by unlabelled homologous LDL, very low density lipoproteins (VLDL) and high density lipoproteins (HDL) and also be human LDL and HDL, but not by unrelated proteins tested. The binding and displacement patterns with membranes from several other porcine tissues are similar to those of liver membranes. These results suggest the presence of “lipoprotein binding sites” in liver membranes which recognize structural features common to the lipoproteins and further indicate that liver membranes are not unique in their ability to bind LDL.     

Lipoprotein-associated paf (LA-paf) was found in washed human platelets and monocyte/macrophage-like U937 cells

Beyond cholesterol, inflammatory ether phospholipids such as platelet-activating factor (paf) may play a role in atherogenesis. (1) We detected a paf-like compound (‘LA-paf’) associated with human serum lipoproteins, mainly in LDL but not with the lipoprotein-poor fraction. (2) LA-paf was also found in washed human platelets, from where it was partially released during platelet aggregation in response to paf (50 nM) or thrombin (1 U). In addition, resident monocyte/macrophage-like U937 cells carried huge amounts of LA-paf (41 ng per 10⁷ cells) and metabolized added [³H]paf to a labelled compound co-eluting with the retention time of LA-paf in standard HPLC. (3) Functionally, LA-paf had a comparable potency to synthetic paf, because LA-paf aggregated washed aspirin-treated platelets in a concentration-dependent manner. The specific paf receptor antagonist WEB2086 inhibited the platelet aggregation induced by three distinct LA-paf preparations as compared with synthetic paf with similar inhibitory concentrations (IC₅₀: 35.6 ± 12.8, 24.0 ± 4.0, 38.0 ± 15.8 nM for LA-paf, and 43.6 ± 6.5 nM for synthetic paf), indicating that LA-paf interacted with paf receptors. (4) However, LA-paf had a distinct retention time using high-pressure liquid chromatography (HPLC) as compared with synthetic paf. LA-paf eluted at 9–15 min and synthetic paf at 21–24 min. In addition, total and non-specific [³H]paf binding to intact washed human platelets was affected differently by the two unlabelled agonists: while LA-paf increased total and non-specific (but not specific) binding in a significant manner (P < 0.002 and P < 0.007) as LDL did (P < 0.006 and P < 0.03), synthetic paf decreased total binding (P < 0.03). Similarly, low-density lipoproteins (LDL) increased significantly the total [³H]paf binding. In contrast, paf did not affect specific [¹²⁵I]LDL binding to human fibroblasts. Our results show the presence of LA-paf in lipoproteins,     

Enhanced binding of phospholipase-A2-modified low density lipoprotein by human adipocytes

Recognition of low density lipoprotein (LDL) by human adipocytes is not dependent on the classical LDL (apoprotein B-E) receptor. To assess whether LDL phospholipids have a role in adipocyte-LDL interactions, binding studies were carried out with human LDL modified with cobra venom phospholipase A2 (PLA2) and freshly isolated adipocytes and purified adipocyte plasma membranes prepared from surgical biopsies. LDL incubated with PLA2 showed increased monoacylphospholipid content, decreased diacylphospholipid content, and increased anodic migration on agarose gel electrophoresis. LDL cholesterol, triglyceride, and protein content remained unchanged. Typically, modification of 16 and 47% of LDL phospholipids enhanced specific binding of 125I-labelled LDL to plasma membranes progressively from 3.1 micrograms LDL bound/mg membrane protein (control) to 5.8 and 28.2 micrograms LDL bound/mg membrane protein, respectively. Nonspecific binding was not altered significantly. Excess unlabelled native LDL and high density lipoprotein (HDL3) effectively inhibited binding of PLA2-modified LDL. Freshly isolated adipocytes also showed enhanced binding and uptake of PLA2-modified LDL (0.1 vs. 0.9 micrograms LDL/10(6) cells x 2 h), control vs. modified). The results demonstrate that alterations of LDL phospholipids significantly enhance LDL binding and suggest a regulatory role for phospholipids in lipoprotein-cell interaction. Furthermore, the results support the view that human adipose tissue may be involved in the metabolism of modified lipoproteins, in vivo.     

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.     

Processing and characterization of the low density lipoprotein receptor in the human colonic carcinoma cell subclone HT29-18: A potential pathway for delivering therapeutic drugs and genes

Low density lipoprotein (LDL) processing has been investigated in the subcloned human colonic carcinoma cell line HT29-18. LDL binding at 4°C was a saturable process in relation to time and LDL concentration. The Kd for LDL binding was 11 g/ml. ApoE-free HDL₃ or acetylated LDL did not significantly compete with¹²⁵I-LDL binding, up to 500 g/ml.¹²⁵I-LDL binding was decreased by 70% in HT29-18 cells preincubated for 24 hours in culture medium containing 100 g/ml unlabelled LDL. Ligand blotting studies performed on HT29-18 homogenates using colloidal gold labelled LDL indicated the presence of one autoradiographic band corresponding to an apparent molecular weight of 130 kDa, which is consistent with the previously reported molecular weight of the LDL receptor in human fibroblasts. At 37°C,¹²⁵I-LDL was actively internalized by HT29-18 cells and lysosomal degradation occurred as demonstrated by the inhibitory effect of chloroquine. LDL uptake and degradation by HT29-18 cells also resulted in a marked decrease in endogenous sterol synthesis. These data demonstrate that the HT29-18 human cancerous intestinal cells are able to specifically bind and internalize LDL, and that LDL processing results in down-regulation of sterol biosynthesis. Thus, intestinal epithelial cells possess specific LDL receptors that can be exploited to accomplish drug delivery and gene transfer via the receptor-mediated endocytosis pathway.Abbreviations HDL, HCL₃ high density lipoprotein - LDL low density lipoprotein     

Binding of fibrinogen molecules to pig platelets and their membranes

Following addition of ADP, 125I-labelled fibrinogen binds specifically to pig platelets. This binding is completely inhibited by the unlabelled fibrinogen. Quantitative analysis indicates the presence of 12,400-25,000 molecules of fibrinogen which can be bound with an association constant of 5 . 10(8) M-1 to platelets. Fibrinogen receptors were found to be active in the isolated platelet membranes as well. Quantitative analysis of the saturable binding of fibrinogen to the platelet membranes showed that these receptors react with the same affinity with fibrinogen molecules. In contrast to the intact platelets, the platelet membranes can specifically bind fibrinogen in the absence of ADP. We conclude that a specific receptor for fibrinogen is exposed on the surface as a result of cell damage which is the first step of the platelet membrane isolation.     

Uptake and degradation of human very-low-density lipoproteins by rat liver endothelial cells in culture

Rat liver endothelial cells in primary cultures take up and degrade 125I-labelled human very-low-density lipoproteins (VLDL) in a saturable fashion at physiological triacylglycerol concentrations. The iodinated VLDL are readily taken up by the freshly isolated endothelial cells and degradation products appear in the medium about 30 min after the addition of VLDL to the cultures. Uptake and degradation at 37 degrees C are effectively inhibited by unlabelled human VLDL, low-density lipoproteins (LDL), high-density lipoproteins and lymph chylomicrons, but only modestly by acetylated LDL. Purified apolipoproteins E and C-III:1 also compete with the uptake of iodinated VLDL, but when degradation was studied for longer periods of time, such a competition could not be demonstrated. This may be due to the fact that the added apolipoproteins become associated with the lipoproteins. In binding experiments at 7 degrees C, iodinated apolipoprotein C III:1 bound to the liver endothelial cells in a manner characteristic of receptor binding with a dissociation constant of 0.5 microM. This binding could not only be inhibited by unlabelled apolipoprotein C-III:1 but also by unlabelled apolipoprotein E. The results indicate that rat liver endothelial cells carry receptors for VLDL and that these recognize the apolipoproteins E, C-III and B on the lipoprotein surface. Considering the large endothelial surface and high blood flow through the liver, significant quantities of lipoproteins can be taken up and degraded, thus influencing the levels of circulating lipoproteins in the in vivo situation.     

Effect of the anti-receptor ligand-blocking 225 monoclonal antibody on EGF receptor endocytosis and sorting

The anti-receptor antibody, 225 mAb, is known to block binding of ligand to the epidermal growth factor receptor (EGFR). However, the effect of this neutralizing antibody on EGFR endocytosis, trafficking and degradation remains unclear. Here, we demonstrate that endocytosis of (125)I-225 mAb occurs, albeit with a slower rate than that of EGF. Using pulse chase assays, we show that internalized (125)I-225 mAb is recycled to the surface much more efficiently than internalized (125)I-EGF. Also, we found that internalization of (125)I-225 mAb, in contrast to that of EGF, is independent of receptor tyrosine kinase activity, as evidenced by its insensitivity to AG1478, a specific EGFR tyrosine kinase inhibitor. Analysis of the levels of cell surface and total EGFR showed that treatment with 225 mAb results in a 30-40% decrease in surface EGFR and a relatively slow downregulation of total EGFR. Taken together, these data indicate that 225 mAb induces internalization and downregulation of EGFR via a mechanism distinct from that underlying EGF-induced EGFR internalization and downregulation.     

Effect of prostaglandin E1 on low density lipoprotein apo-B-receptor binding in human, rat and swine liver in vitro.

The effect of PGE1 on low density lipoprotein (LDL) apo-B-receptor binding was examined in human, rat and swine liver. Autologous LDL (for humans and swines) and homologous LDL (for rats) were isolated by ultracentrifugation and labelled with 123I using Iodogen followed by purification with dialysis. LDL-concentrations of 0.1-6 micrograms protein/ml were used for direct binding assays investigating the specific binding of labelled LDL in presence of increasing PGE1-concentrations (100 pM to 100 microM). In separate experiments the effect of PGE1 on displacement of specifically bound 123I-LDL by unlabelled ones was studied. The binding capacities estimated by Scatchard analysis were similar for human and rat liver LDL-apo-B-receptor binding, however, swine liver exhibited a significantly (p less than 0.001) lower binding capacity for 123I-LDL. PGE1 significantly (p less than 0.01-0.001) increased the amount of 123I-LDL specifically bound to the liver apo-B-receptors and the binding affinity in all liver preparations of the 3 species in a dose-dependent manner. PGE1 also significantly increased competition of unlabelled LDL for 123I-LDL bound to its specific apo-B-receptors in a dose-dependent manner (p less than 0.01-0.001) with an ED50 of 123 +/- 64 nM for human liver, 901 +/- 102 nM for rat liver obtained during anaesthesia, 74 +/- 23 nM for rat liver obtained after decapitation and 941 +/- 121 nM for swine liver. In human liver iloprost (ED50 = 876 +/- 53 nM) and PGI2 (ED50 = 52 +/- 12 microM) were less effective than PGE1, PGE2 had no effect on LDL-induced competition. It is concluded that PGE1 renders LDL more sensitive for apo-B-receptor binding suggesting a potential hypolipidemic action of PGE1.     

Impaired binding of low density lipoprotein to hepatic membranes from uremic guinea pigs.

Chronic renal failure is associated with abnormalities in lipoprotein metabolism that may contribute to premature atherosclerosis and early mortality in patients on dialysis. In previous studies, we found that plasma clearance of radiolabelled low density lipoprotein (LDL) was retarded in nephrectomized guinea pigs left with one-sixth of normal functioning renal mass. To elucidate potential mechanisms of delayed LDL clearance, we compared binding of LDL to hepatic membranes from both normal and uremic guinea pigs. One hundred micrograms of the 8000-100,000 X g hepatic microsomal protein was incubated with 125I-labelled normal guinea pig LDL (10-150 micrograms/mL) for 1 h at 37 degrees C, and the membrane washed and pelleted by centrifugation in a Beckman Ti 42.2 rotor. Parallel incubations with excess unlabelled LDL were done to determine specific binding. LDL specific binding to uremic hepatic membranes was significantly impaired compared with normal ones. The major abnormality, as determined by Scatchard transformation of the binding data, was a reduction of the apparent maximal binding of LDL to uremic membranes, with an average Bmax of 4.1 micrograms/mg protein compared with 6.6 micrograms/mg protein for normal hepatic microsomes. The affinity of LDL for uremic liver membranes was only slightly diminished with a mean apparent Kd of 35.2 micrograms/mL in comparison with 21.8 micrograms/mL for normal liver membranes. These results provide a biochemical explanation for the diminished LDL clearance in uremia and may account for the dyslipidemia of renal failure.     

Characterization of the LDL receptor in rat promegakaryoblasts in culture

Rat promegakaryoblasts (RPM, a precursor platelet cell line) in culture exhibited a capacity to bind, take up and degrade¹²⁵I-LDL. The low density lipoprotein (LDL) binding showed the following characteristics: (a) high affinity, (b) saturability, (c) specificity, (d) down-regulation, after exposure to 25 hydroxycholesterol. Furthermore the proteolytic degradation of¹²⁵I-LDL by RPMs was inhibited by chloroquine which interferes with the lysosomal degradation processes. These findings show LDL receptor cell biology of RPM to be of the classical type and to differ from that of platelets.