The plasma clearance of human α2-macroglobulin-trypsin complex in the rat is mainly accounted for by uptake into hepatocytes

Rats were given intravenous injections of ¹²⁵I-labelled human α₂-macroglobulin·trypsin. The half-time of disappearance of radioactivity from arterial blood was 2 min. External counting showed that radioactivity in the liver was maximal by 10 min and then decreased slowly. 87% of the injected dose was recovered in the liver by 10 min. Light- and electron microscopic autoradiography carried out on samples of liver fixed with glutaraldehyde 3 min or 30 min after the injection showed that 85–90% of the grains were over the hepatocytes and 4–9% were over the Kupffer cells. Thus, uptake into hepatocytes, and not into Kupffer cells as believed previously, appears to account for the major part of the uptake of α₂-macroglobulin·trypsin by the liver and thereby for its rapid removal from the blood.     

Binding and receptor-mediated endocytosis of pregnancy zone protein-proteinase complex in rat macrophages

125I-labelled pregnancy zone protein complex was injected intravenously in rats and after 6 min uptake into cells of the liver and spleen was determined by electron microscopic autoradiography. The liver took up 68% of the injected radioactivity; 61% was in the hepatocytes and 7% was in the liver macrophages (Kupffer cells). The spleen took up 3-4% and nearly all the radioactivity was in the macrophages of the red pulp. The uptake per cell volume was several times higher in the macrophage than in the hepatocyte. The radioactivity associated with macrophages was largely in endocytotic vacuoles and lysosomes. Binding of labelled pregnancy zone protein complex to peritoneal macrophages at 4 degrees C was 2-3 times higher than binding of the homologous alpha 2-macroglobulin complex. The two ligands competed for binding to the same receptors and the difference was due to a higher affinity of the pregnancy zone protein complex (Kd approx. 60 pM). After binding to the receptor, this ligand was internalised within 2-3 min at 37 degrees C and radioactivity inside the cells largely represented intact pregnancy zone protein complex. Radioactivity was released from the cell as iodotyrosine after a lag time of about 10 min. It is concluded that pregnancy zone protein complex is bound with a high affinity to the alpha 2-macroglobulin receptors in rat macrophages followed by receptor-mediated endocytosis and degradation of the ligand in the lysosomes.     

Lactate, alanine and glutamine metabolism in isolated canine pup liver cells

125I-Labelled alpha 2-macroglobulin-trypsin complex (125I-labelled alpha 2-macroglobulin X trypsin) was associated to isolated rat adipocytes and hepatocytes with a half-time of about 60 min at 37 degrees C. The association of 0.5 micrograms/ml 125I-labelled alpha 2-macroglobulin X trypsin was inhibited by unlabelled alpha 2-macroglobulin X trypsin with a half-inhibition constant of about 8 micrograms/ml (11 nM). 125I-Labelled alpha 2-macroglobulin became cell-associated to a smaller extent (10-40% of that of alpha 2-macroglobulin X trypsin) and the half-inhibition constant was about 35 micrograms/ml in adipocytes. The cell association of 125I-labelled alpha 2-macroglobulin X trypsin was markedly inhibited by dansylcadaverine, bacitracin, omission of Ca2+ from the medium or pretreatment of the cells with trypsin. After incubation for 180 min more than 60% of the cell-associated 125I-labelled alpha 2-macroglobulin X trypsin was not removed by treatment of the cells with trypsin-EDTA and represented probably internalized material. 125I-Labelled alpha 2-macroglobulin X trypsin was degraded to trichloroacetic acid-soluble fragments by suspensions of both cell types but only to a negligible extent by incubation media preincubated with these cells. The rate of degradation of 0.5 micrograms/ml 125I-labelled alpha 2-macroglobulin was approx. 40% of that of 125I-labelled alpha 2-macroglobulin X trypsin. Degradation of 125I-labelled alpha 2-macroglobulin X trypsin was abolished by a high concentration (0.5 mg/ml) of alpha 2-macroglobulin X trypsin. It is concluded that alpha 2-macroglobulin X trypsin by a specific and saturable mechanism is bound to, internalized and degraded by isolated rat adipocytes and hepatocytes.     

Association of alpha 2-macroglobulin-thrombin and alpha 2-macroglobulin-plasmin complexes with isolated hepatocytes

125I-labelled alpha 2-macroglobulin complexed with thrombin or plasmin bound to hepatocytes in a concentration- and time-dependent manner. The apparent Kd values calculated from displacement experiments were 7.9 X 10(-8) M for alpha 2-macroglobulin-thrombin and 8.5 X 10(-8) M for alpha 2-macroglobulin-plasmin. Association of these complexes was only partially reversible; after a 180 min incubation period, 50-60% of the bound radioactivity was internalized by the cells. alpha 2-Macroglobulin itself bound also to hepatocytes, but the affinity of the alpha 2-macroglobulin complexes was higher than that of the inhibitor alone, and alpha 2-macroglobulin was not internalized, either. 125I-labelled thrombin or plasmin bound to hepatocytes as well. These bindings were also concentration-dependent and could be decreased with an excess of unlabelled ligands. Binding rates and amounts of the bound proteinases were higher than those of their alpha 2-macroglobulin complexes. The alpha 2-macroglobulin-thrombin complex competed with the alpha 2-macroglobulin-plasmin complex in binding to hepatocytes, whereas there was no competition between these complexes and the antithrombin III-thrombin complex. These results suggest that the binding sites of hepatocytes for alpha 2-macroglobulin-proteinase and antithrombin III-proteinase complexes are different.     

Clearance of acetyl low density lipoprotein by rat liver endothelial cells. Implications for hepatic cholesterol metabolism

We have studied the hepatic uptake of human [14C] cholesteryl oleate labeled acetyl low density lipoprotein (LDL). Acetyl-LDL injected intravenously into rats was cleared from the blood with a half-life of about 10 min. About 80% of the injected acetyl-LDL was recovered in the liver after 1 h. Initially, most of the [14C]cholesterol was recovered in liver endothelial cells (about 60%). Some radioactivity (about 15%) was also recovered in the hepatocytes, while the Kupffer cells and stellate cells contained only small amounts of the label (less than 5%). About 1 h after injection, radioactivity started to disappear from endothelial cells and appeared instead in hepatocytes. Radioactivity subsequently declined in hepatocytes as well. After a lag phase of 4 h, significant amounts of radioactivity were recovered in bile. The in vitro uptake and hydrolysis of [14C]cholesteryl oleate-labeled acetyl-LDL were saturable in isolated rat liver endothelial cells. Native LDL does neither affect the uptake nor the hydrolysis of acetyl-LDL. Ammonia and monensin reduced the hydrolysis of acetyl-LDL in isolated liver endothelial cells. Furthermore, monensin at concentrations above 10 microM completely blocked the binding of acetyl-LDL to the liver endothelial cells, suggesting that the receptor for acetyl-LDL is trapped inside the cells. The liver endothelial cells may be involved in the protection against atherogenic lipoproteins, e.g. liver endothelial cells may mediate uptake of cholesterol from plasma and transfer of cholesterol to the hepatocytes for further secretion into the bile.     

A solid-phase immunosorbent assay to determine the proteinase binding capacity of alpha 2-macroglobulin using 125I-trypsin as indicator proteinase

Hyperimmune sera against human alpha 2 macroglobulin were raised in rabbits following immunization with 's' alpha 2-macroglobulin over half a year. Immunoglobulins were prepared by DEAE-Sephacel anion exchange chromatography. The immunoglobulin preparations showed a remarkably high and equal titer for 's' and 'f' alpha 2-macroglobulin (plasma alpha 2-macroglobulin fully saturated with pig pancreas trypsin), which amounted to 6.4 X 10(-6) as revealed by passive hemagglutination. Immunoimmobilization experiments revealed that at equilibrium, 's' alpha 2-macroglobulin and both 'f' alpha 2-macroglobulins (27 and 82% saturation of 's' alpha 2-macroglobulin with trypsin) had been bound to the same degree from the fluid phase to the monospecific antibodies that had been adsorbed to polystyrene tubes. Comparison of quantitative gel scans for disappearance of the intact alpha 2-macroglobulin subunit (Mr 182000) with 125I-labeled trypsin binding capacity of immunoimmobilized alpha 2-macroglobulin-trypsin complexes showed conspicuous agreement. Rocket immunoelectrophoresis did not give significant differences between 's' alpha 2-macroglobulin and 'f' alpha 2-macroglobulin. In the fluid phase, a binding ratio of 2.4 mol trypsin/mol alpha 2-macroglobulin was observed. Saturation of solid phase immunoimmobilized 's' alpha 2-macroglobulin with trypsin could be accomplished by incubation with a 100-200-fold molar excess of enzyme for 10 min. The solid-phase experiments showed a binding ratio of 2.0 mol trypsin/mol alpha 2-macroglobulin. The high molar excess of trypsin needed to saturate solid-phase immunoimmobilized alpha 2-macroglobulin, which binds 20% less trypsin than in the liquid phase, is partially explained by an enhancement of the negative cooperativity of trypsin binding to alpha 2-macroglobulin found in the liquid-phase system. Assessment of the trypsin-binding capacity of alpha 2-macroglobulin immunoadsorbed from synovial fluids (n = 19) of patients with seropositive rheumatoid arthritis yielded an inactive alpha 2-macroglobulin of 0-53% when compared to the trypsin-binding capacity of normal plasma alpha 2-macroglobulin.     

Uptake and degradation of insulin and alpha 2-macroglobulin-trypsin complex in rat adipocytes. Evidence for different pathways

The cell association and degradation of insulin and alpha 2-macroglobulin-trypsin complex were measured in rat adipocytes with or without various inhibitors in the attempt to clarify whether the two ligands were taken up by the same or by different pathways. Several inhibitors, and particularly those of membrane traffic, lysosomal function and transglutaminase activity, affected the two ligands differently. Thus, chloroquine (100 microM) reduced both the uptake of alpha 2-macroglobulin X trypsin and its receptor-mediated degradation by about 70%. In contrast, the uptake of insulin was increased 2-3-times and the receptor-mediated degradation was only slightly reduced. Methylamine (10 mM) and ammonium chloride (10 mM) reduced degradation of alpha 2-macroglobulin X trypsin markedly without affecting that of insulin. Leupeptin (100 microM) increased uptake and reduced degradation of alpha 2-macroglobulin X trypsin without affecting insulin. Dansylcadaverine (500 microM) almost abolished uptake and degradation of alpha 2-macroglobulin X trypsin but had little effect on insulin. Moreover, uptake and degradation of alpha 2-macroglobulin X trypsin was much more sensitive than insulin to the action of metabolic inhibitors such as dinitrophenol and cyanide. The results show that the two ligands are taken up by functionally different systems. In addition, they support the hypothesis that lysosomes play a relatively minor role in the receptor-mediated degradation of insulin.     

Characterization, size estimation and solubilization of alpha-macroglobulin complex receptors in liver membranes

Receptors for alpha 2-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of 125I-labelled alpha 2-macroglobulin.trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8-9.0. The half-time for association was about 5 min at 37 degrees C in contrast to about 5 h at 4 degrees C. The half-saturation constant was about 100 pM at 4 degrees C and 1 nM at 37 degrees C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 +/- 71 kDa (S.D., n = 7) for alpha 2-macroglobulin.trypsin binding activity. Affinity cross-linking to rat and human membranes of 125I-labelled rat alpha 1-inhibitor-3.chymotrypsin, a 210 kDa analogue which binds to the alpha 2-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55-60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked alpha 2-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound 125I-alpha 1-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]propane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400-500 kDa alpha 2-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

Uridine catabolism in Kupffer cells, endothelial cells, and hepatocytes

Kupffer cells, endothelial cells, and hepatocytes were separated by centrifugal elutriation. The rate of uracil formation from [2-14C]uridine, the first step in uridine catabolism, was monitored in suspensions of the three different liver cell types. Kupffer cells demonstrated the highest rate of uridine phosphorolysis. 15 min after the addition of the nucleoside the label in uracil amounted to 51%, 13%, and 19% of total radioactivity in the medium of Kupffer cells, endothelial cells, and hepatocytes, respectively. If corrected for Kupffer cell contamination, hepatocyte suspensions demonstrated similar activities as endothelial cells. In contrast to non-parenchymal cells, hepatocytes continuously cleared uracil from the incubation medium. The lack of uracil consumption by Kupffer cells and endothelial cells points to uracil as the end-product of uridine catabolism in these cells. Kupffer cells and endothelial cells did not produce radioactive CO2 upon incubation in the presence of [2-14C]uridine. Hepatocytes, however, were able to degrade uridine into CO2, beta-alanine, and ammonia as demonstrated by active formation of volatile radioactivity from the labeled nucleoside. There was almost no detectable formation of thymine from thymidine or of cytosine, uracil, or uridine from cytidine by any of the different cell types tested. These results are in line with low thymidine phosphorolysis and cytidine deamination in rat liver. Our studies suggest a co-operation of Kupffer cells, endothelial cells, and hepatocytes in the breakdown of uridine from portal vein blood with uridine phosphorolysis predominantly occurring in Kupffer cells and with uracil catabolism restricted to parenchymal liver cells.     

Complexes of rat alpha 1-macroglobulin and subtilisin are endocytosed by parenchymal liver cells.

Rat alpha 1-macroglobulin was isolated from plasma. Gel electrophoresis of the denatured and reduced protein showed two bands, with Mr values of 163 000 and 37 000. The large subunit contained an autolytic site. This subunit was also split after reaction of the macroglobulin with trypsin. Electron microscopy showed that the macroglobulin changed towards a more compact conformation after reaction with this proteinase. Subtilisin, or alpha 1-macroglobulin, was labelled with a sucrose-containing radio-iodinated group that stays in lysosomes after endocytosis and breakdown of the tagged protein. After intravenous injection into rats, alpha 1-macroglobulin was cleared from plasma with first-order kinetics, showing a half-life of about 9 h, whereas complexes of alpha 1-macroglobulin and subtilisin were cleared with half-lives of only 3 min. Liver contained about 60% of the label at 30 min after injection of complexes. About 90% of the liver radioactivity was found in parenchymal cells isolated after perfusion of the liver with a collagenase solution. Subcellular fractionation indicated a lysosomal localization of the complexes. We conclude that endocytosis by parenchymal liver cells is the major cause of the rapid clearance of alpha 1-macroglobulin-proteinase complexes from plasma.     

Binding and receptor-mediated endocytosis of pregnancy zone protein-proteinase complex in rat macrophages

¹²⁵I-labelled pregnancy zone protein complex was injected intravenously in rats and after 6 min uptake into cells of the liver and spleen was determined by electron microscopic autoradiography. The liver took up 68% of the injected radioactivity; 61% was in the hepatocytes and 7% was in the liver macrophages (Kupffer cells). The spleen took up 3–4% and nearly all the radioactivity was in the macrophages of the red pulp. The uptake per cell volume was several times higher in the macrophage than in the hepatocyte. The radioactivity associated with macrophages was largely in endocytotic vacuoles and lysosomes. Binding of labelled pregnancy zone protein complex to peritoneal macrophages at 4°C was 2–3-times higher than binding of the homologous α₂-macroglobulin complex. The two ligands competed for binding to the same receptors and the difference was due to a higher affinity of the pregnancy zone protein complex (K_d approx. 60 pM). After binding to the receptor, this ligand was internalised within 2–3 min at 37°C and radioactivity inside the cells largely represented intact pregnancy zone protein complex. Radioactivity was released from the cell as iodotyrosine after a lag time of about 10 min. It is concluded that pregnancy zone protein complex is bound with a high affinity to the α₂-macroglobulin receptors in rat macrophages followed by receptor-mediated endocytosis and degradation of the ligand in the lysosomes.     

The acute-phase induction of alpha 2-macroglobulin in rat hepatocyte primary cultures: action of a hepatocyte-stimulating factor, triiodothyronine and dexamethasone

During inflammation a number of liver-derived plasma proteins increases in concentration. In the rat these so-called acute-phase proteins are mainly proteinase inhibitors, such as alpha 1-proteinase inhibitor, alpha 1-acute-phase globulin and alpha 2-macroglobulin. At present, the mechanisms responsible for the enhanced synthesis of acute-phase proteins are poorly understood. Therefore, we have studied the induction of alpha 2-macroglobulin synthesis in rat hepatocyte primary cultures. Adrenaline, triiodothyronine, estradiol and progesterone were tested for their ability to stimulate alpha 2-macroglobulin synthesis. Only triiodothyronine induced alpha 2-macroglobulin synthesis markedly. However, the presence of dexamethasone was a prerequisite for alpha 2-macroglobulin induction indicating a permissive action of glucocorticoids. Besides glucocorticoids and triiodothyronine a non-dialyzable factor (HSF) derived from rat Kupffer cells or human peripheral blood monocytes was found to be able to stimulate alpha 2-macroglobulin synthesis in hepatocytes. Equal amounts of HSF activity were found in conditioned media from lipopolysaccharide-stimulated and unstimulated rat Kupffer cells as well as in human monocytes. Since the supernatants of unstimulated rat Kupffer cells or human monocytes did not exhibit interleukin 1 activity, HSF activity distinct from interleukin 1 must exist. No HSF activity was found in media conditioned by rat Kupffer cells which had been treated with dexamethasone. Hepatocyte primary cultures were incubated with [35S]methionine-labeled proteins secreted by rat Kupffer cells. A 30 kDa polypeptide was found to be bound to or internalized by rat hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelial binding of transferrin in fractionated liver cell suspensions

Several studies using crude liver cell suspensions incubated with labeled transferrin have led to a conclusion that hepatocytes have transferrin receptors. When a visual probe, which permits evaluation of transferrin binding to individual cells, was used, the binding was unexpectedly found to be limited to endothelial cells in liver cell suspensions. Neither hepatocytes nor Kupffer cells contained transferrin receptors. In the present study, we fractionated liver cell suspensions using metrizamide gradients and centrifugal elutriation to obtain hepatocytes, Kupffer cell and endothelial cell fractions of high purity. Incubation of these fractions with 125I- or 59Fe-labeled transferrin led to exclusive binding to endothelial cells but not hepatocytes nor Kupffer cells. Kinetic analysis demonstrated Kd of 1.9 X 10(-7) M, Bmax of 3.1 pmol/10(6) cells per min, corresponding to 2.1 X 10(5) molecules/cell per min. At 4 degrees C, the binding reached a steady-state plateau within 5 min. Comparison of our data with those of previous investigators demonstrates a consistency if we consider that crude liver cell suspensions are contaminated with 2-3% endothelial cells. Thus, the previously reported findings may be entirely due to the contamination of crude liver cell suspensions with a small number of endothelial cells.     

Induction of rat alpha 2-macroglobulin in vivo and in hepatocyte primary cultures: synergistic action of glucocorticoids and a Kupffer cell-derived factor

Turpentine injection into rats elicits enhanced secretion of acute phase proteins including alpha 2-macroglobulin (alpha 2M). Hypophysectomized rats, however, do not respond in this way unless dexamethasone is given together with turpentine. On the other hand, dexamethasone injection alone did not result in an induction of alpha 2M synthesis. When a medium of Kupffer cell cultures was added to hepatocytes, a dose-dependent stimulation of alpha 2M synthesis of up to 4-fold after 10-12 h was observed. However, the presence of low concentrations (10(-9)M) of dexamethasone was essential for the stimulatory effect. We conclude that the acute phase induction of alpha 2M in hepatocytes requires the synergistic action of glucocorticoids and a non-dialysable factor secreted by Kupffer cells.     

Uptake of rat and human alpha 2-macroglobulin-trypsin complexes into rat and human cells

Uptake of rat and human alpha 2-macroglobulin-trypsin complexes was measured in rat hepatocytes, rat and human adipocytes and human fibroblasts. Uptake and degradation of 125I-labelled rat complex were about one-third of that of the human complex in the various isolated cell types. In rat hepatocytes, the apparent Km for cell association of the rat complex was about 16 nM as compared to about 6 nM for the human complex. The Vmax values were similar, about 1 X 10(4) molecules X cell-1 X min-1. Thus, rat alpha 2-macroglobulin (an acute-phase protein) complexed with trypsin follows the same pathways of uptake as the human homologue, although with a somewhat lower affinity for the uptake system.     

Distinct properties of the recognition sites for beta-very low density lipoprotein (remnant receptor) and alpha 2-macroglobulin (low density lipoprotein receptor-related protein) on rat parenchymal cells.

The properties of the recognition sites for alpha 2-macroglobulin (alpha 2-macroglobulin receptor; low density lipoprotein receptor-related protein) and beta-migrating very low density lipoprotein (beta-VLDL) (remnant receptor) on rat parenchymal cells were directly compared to analyze whether both substrates are recognized and internalized by the same receptor system. In cholesterol-fed rats, the large circulating pool of beta-VLDL is unable to diminish the liver uptake of 125I-labeled alpha 2-macroglobulin, while liver uptake of 125I-labeled beta-VLDL in these rats is reduced by 87.3% at 10 min after injection. In vitro competition studies with isolated parenchymal liver cells demonstrate that the binding of 125I-labeled alpha 2-macroglobulin to rat parenchymal cells is not effectively competed for by beta-VLDL, whether this lipoprotein is additionally enriched in apolipoprotein E or not. Binding of alpha 2-macroglobulin to parenchymal cells requires the presence of calcium, while binding of beta-VLDL does not. Incubation of parenchymal cells for 1 h with proteinase K reduced the subsequent binding of alpha 2-macroglobulin by 90.1%, while the binding of beta-VLDL was reduced by only 20.2%. In the presence of monensin, the association of alpha 2-macroglobulin to parenchymal cells at 2 h of incubation was reduced by 64.7%, while the association of beta-VLDL was not affected. Preincubation of parenchymal cells with monensin for 60 min at 37 degrees C reduced the subsequent binding of alpha 2-macroglobulin by 54.5%, while binding of beta-VLDL was only reduced by 14.6%. The results indicate that the recognition sites for alpha 2-macroglobulin and beta-VLDL on rat parenchymal cells do exert different properties and are therefore likely to reside on different molecules.     

Different fate in vivo of oxidatively modified low density lipoprotein and acetylated low density lipoprotein in rats. Recognition by various scavenger receptors on Kupffer and endothelial liver cells

Human low density lipoprotein was oxidized (Ox-LDL) by exposure to 5 microM Cu2+ and its fate in vivo was compared to acetylated low density lipoprotein (Ac-LDL). Ox-LDL, when injected into rats, is rapidly removed from the blood circulation by the liver, similarly as Ac-LDL. A separation of rat liver cells into parenchymal, endothelial, and Kupffer cells at 10 min after injection of Ox-LDL or Ac-LDL indicated that the Kupffer cell uptake of Ox-LDL is 6.8-fold higher than for Ac-LDL, leading to Kupffer cells as the main liver site for Ox-LDL uptake. In vitro studies with isolated liver cells indicated that saturable high affinity sites for Ox-LDL were present on both endothelial and Kupffer cells, whereby the capacity of Kupffer cells to degrade Ox-LDL is 6-fold higher than for endothelial cells. Competition studies showed that unlabeled Ox-LDL competed as efficiently (90%) as unlabeled Ac-LDL with the cell association and degradation of 125I-labeled Ac-LDL by endothelial and Kupffer cells. However, unlabeled Ac-LDL competed only partially (20-30%) with the cell association and degradation of 125I-labeled Ox-LDL by Kupffer cells, while unlabeled Ox-LDL or polyinosinic acid competed for 70-80%. It is concluded that the liver contains, in addition to the scavenger (Ac-LDL) receptor which interacts efficiently with both Ac-LDL and Ox-LDL and which is concentrated on endothelial cells, an additional specific Ox-LDL receptor which is highly concentrated on Kupffer cells. In vivo the specific Ox-LDL recognition site on Kupffer cells will form the major protection system against the occurrence of the atherogenic Ox-LDL particles in the blood.     

Uptake of lactosylated low-density lipoprotein by galactose-specific receptors in rat liver.

The liver contains two types of galactose receptors, specific for Kupffer and parenchymal cells respectively. These receptors are only expressed in the liver, and therefore are attractive targets for the specific delivery of drugs. We provided low-density lipoprotein (LDL), a particle with a diameter of 23 nm in which a variety of drugs can be incorporated, with terminal galactose residues by lactosylation. Radioiodinated LDL, lactosylated to various extents (60-400 mol of lactose/ mol of LDL), was injected into rats. The plasma clearance and hepatic uptake of radioactivity were correlated with the extent of lactosylation. Highly lactosylated LDL (greater than 300 lactose/LDL) is completely cleared from the blood by liver within 10 min. Pre-injection with N-acetylgalactosamine blocks liver uptake, which indicates that the hepatic recognition sites are galactose-specific. The hepatic uptake occurs mainly by parenchymal and Kupffer cells. At a low degree of lactosylation, approx. 60 lactose/LDL, the specific uptake (ng/mg of cell protein) is 28 times higher in Kupffer cells than in parenchymal cells. However, because of their much larger mass, parenchymal cells are the main site of uptake. At high degrees of lactosylation (greater than 300 lactose/LDL), the specific uptake in Kupffer cells is 70-95 times that in parenchymal cells. Under these conditions, Kupffer cells are, despite their much smaller mass, the main site of uptake. Thus not only the size but also the surface density of galactose on lactosylated LDL is important for the balance of uptake between Kupffer and parenchymal cells. This knowledge should allow us to design particulate galactose-bearing carriers for the rapid transport of various drugs to either parenchymal cells or Kupffer cells.     

Involvement of various organs in the initial plasma clearance of differently glycosylated rat liver secretory proteins

The initial plasma clearance and organ distribution of alpha 1-acid glycoprotein and alpha 2-macroglobulin carrying different types of oligosaccharide, side chains was studied in rats. The differently glycosylated proteins were synthesized by rat hepatocytes in culture in the presence of tunicamycin (unglycosylated form), swainsonine (hybrid type), or 1-deoxymannojirimycin (high-mannose type). Deglycosylated glycoproteins (Asn-GlcNAc) were obtained by endoglucosaminidase H treatment of high-mannose-type glycoproteins. Ten minutes after intravenous injection 3% of complex type, 26% of hybrid type, 84% of high-mannose type. 64% of unglycosylated and 80% of deglycosylated alpha 1-acid glycoprotein disappeared from the plasma. The respective values for alpha 2-macroglobulin were 26%, 42%, 59% and 67%. When the clearance of total hepatic secretory proteins was examined, major differences between glycosylated and unglycosylated (glyco)proteins were found, particularly in the case of low-molecular-mass polypeptides. Whereas complex-type alpha 1-acid glycoprotein and alpha 2-macroglobulin showed no accumulation in various organs, hybrid-type alpha 1-acid glycoprotein and alpha 2-macroglobulin were present in spleen and liver. High-mannose-type alpha 1-acid glycoprotein and alpha 2-macroglobulin also accumulated mainly in spleen and liver. Spleen had the highest specific activity; liver, due to its larger organ mass, represented the major organ for the uptake of high-mannose-type glycoproteins. Competition experiments with mannan and GlcNAc-bovine-serum-albumin showed a mannose/GlcNAc receptor-mediated removal. Whereas unglycosylated alpha 1-acid glycoprotein was taken up by the kidney, unglycosylated alpha 2-macroglobulin was found in the spleen. Deglycosylated glycoproteins (Asn-GlcNAc) were removed from the plasma via two different mechanisms: firstly, clearance by the kidney similar to the unglycosylated glycoproteins; secondly, clearance by a mannose/GlcNAc receptor-mediated uptake mainly into the spleen. We conclude that N-linked oligosaccharide side chains are important for the plasma survival of hepatic secretory glycoproteins and that unphysiologically glycosylated forms are cleared by different mechanisms.     

Endocytosis of lactate dehydrogenase isoenzyme M4 in rats in vivo. Experiments with enzyme labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose.

1. Pig lactate dehydrogenase isoenzyme M4 was labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose and injected intravenously into rats. Previous work has shown that this label does not influence the clearance of the enzyme (half-life about 26 min) and that it is retained within the lysosomes for several hours after endocytosis and breakdown of the protein [De Jong, Bouma & Gruber (1981) Biochem. J. 198, 45--51]. 2. The distribution of the radioactivity over a large number of tissues was determined 2 h after injection. A high percentage of the injected dose was found in liver (41%), spleen (10%) and bone including marrow (21%). 3. Autoradiography indicated uptake of the enzyme mainly by Kupffer cells of the liver, by spleen macrophages and by bone marrow macrophages. 4. Liver cells were isolated 1 h after injection of the enzyme. Kupffer cells, endothelial cells and parenchymal cells were found to endocytose the enzyme at rates corresponding to 4230, 35 and 25 ml of plasma/day per g of cell protein, respectively. 5. Previous injection of carbon particles greatly reduced the uptake of the enzyme by liver and spleen, but the uptake by bone marrow was not significantly changed.