scholarly journals Intracellular transport of formaldehyde-treated serum albumin in liver endothelial cells after uptake via scavenger receptors

1989 ◽  
Vol 258 (2) ◽  
pp. 511-520 ◽  
Author(s):  
W Eskild ◽  
G M Kindberg ◽  
B Smedsrød ◽  
R Blomhoff ◽  
K R Norum ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Serum Albumin ◽  
Intravenous Injection ◽  
Intracellular Transport ◽  
Liver Homogenate ◽  
Scavenger Receptors ◽  
Coated Pits ◽  
Mitochondrial Fractions ◽  
Liver Endothelial Cells

Endocytosis of formaldehyde-treated serum albumin (FSA) mediated by the scavenger receptor was studied in rat liver endothelial cells. Suspended cells had about 8000 receptors/cell, whereas cultured cells had about 19,000 receptors/cell. Kd was 10(-8) M in both systems. Cell-surface scavenger receptors were found exclusively in coated pits by electron microscopy, by using ligand labelled with colloidal gold. Cell-surface-bound FSA could be released by decreasing the pH to 6.0; it was therefore possible to assess the rate of internalization of surface-bound ligand. This rate was very high: t1/2 for internalization of ligand prebound at 4 degrees C was 24 s. The endocytic rate constant at 37 degrees C, Ke, measured as described by Wiley & Cunningham [(1982) J. Biol. Chem. 257, 4222-4229], was 2.44 min-1, corresponding to t1/2 = 12 s. Uptake of FSA at 37 degrees C after destruction of one cell-surface pool of receptors by Pronase was decreased to 60%. This finding is compatible with a relatively large intracellular pool of receptors. The intracellular handling of 125I-tyramine-cellobiose-labelled FSA (125I-TC-FSA) was studied by subcellular fractionation in sucrose gradients, Nycodenz gradients or by differential centrifugation. The density distributions of degraded and undegraded 125I-TC-FSA after fractionation of isolated non-parenchymal cells and whole liver were similar, when studied in Nycodenz and sucrose gradients, suggesting that the subcellular distribution of the ligand was not influenced by the huge excess of non-endothelial material in a whole liver homogenate. Fractionation in sucrose gradients showed that the ligand was sequentially associated with organelles banding at 1.14, 1.17 and 1.21 g/ml. At 9-12 min after intravenous injection the ligand was in a degradative compartment, as indicated by the accumulation of acid-soluble radioactivity at 1.21 g/ml. A rapid transfer of ligand to the lysosomes was also indicated by the finding that a substantial proportion of the ligand could be degraded by incubating mitochondrial fractions prepared 12 min after intravenous injection of the ligand. The results indicate that FSA is very rapidly internalized and transferred through an endosomal compartment to the lysosomes. The endosomes are gradually converted into lysosomes between 9 and 12 min after injection of FSA. The rate-limiting step in the intracellular handling of 125I-TC-FSA is the degradation in the lysosomes.

scholarly journals Intracellular transport of endocytosed proteins in rat liver endothelial cells

1990 ◽  
Vol 270 (1) ◽  
pp. 205-211 ◽  
Author(s):  
G M Kindberg ◽  
E Stang ◽  
K J Andersen ◽  
N Roos ◽  
T Berg
Keyword(s):  
Endothelial Cells ◽  
Rat Liver ◽  
Intracellular Transport ◽  
Degradation Products ◽  
Liver Homogenate ◽  
Subcellular Fractionation ◽  
Early Endosomes ◽  
Liver Endothelial Cells ◽  
Ultrathin Cryosections ◽  
Two Populations

1. Receptor-mediated endocytosis of mannose-terminated glycoproteins in rat liver endothelial cells has been followed by means of subcellular fractionation and by immunocytochemical labelling of ultrathin cryosections after intravenous injection of ovalbumin. For subcellular-fractionation studies the ligand was labelled with 125-tyramine-cellobiose adduct, which leads to labelled degradation products being trapped intracellularly in the organelle where the degradation takes place. 2. Isopycnic centrifugation in sucrose gradients of a whole liver homogenate showed that the ligand is sequentially associated with three organelles with increasing buoyant densities. The ligand was, 1 min after injection, recovered in a light, slowly sedimenting vesicle and subsequently (6 min) in larger endosomes. After 24 min the ligand was recovered in dense organelles, where also acid-soluble degradation products accumulated. 3. Immunocytochemical labelling of ultrathin cryosections showed that the ligand appeared rapidly after internalization in coated vesicles and subsequently in two larger types of endosomes. In the ‘early’ endosomes (1 min after injection) the labelling was seen closely associated with the membrane of the vesicle; after 6 min the ligand was evenly distributed in the lumen. At 24 min after injection the ligand was found in the lysosomes. 4. A bimodal distribution of endothelial cell lysosomes with different buoyant densities was revealed by centrifugation in iso-osmotic Nycodenz gradients, suggesting that two types of lysosomes are involved in the degradation of mannose-terminated glycoproteins in liver endothelial cells. Two populations of lysosomes were also revealed by sucrose-density-gradient centrifugation after injection of large amounts of yeast invertase. 5. In conclusion, ovalbumin is transferred rapidly through three endosomal compartments before delivering to the lysosomes. The degradation seems to take place in two populations of lysosomes.

scholarly journals Glucagon receptors in endothelial and Kupffer cells of mouse liver.

1988 ◽  
Vol 36 (9) ◽  
pp. 1081-1089 ◽  
Author(s):  
J Watanabe ◽  
K Kanai ◽  
S Kanamura
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Kupffer Cells ◽  
Mouse Liver ◽  
Intracellular Transport ◽  
Coated Pits ◽  
Sinusoidal Cells ◽  
Nonparenchymal Cells ◽  
Glucagon Receptors

To determine whether hepatic sinusoidal cells contain glucagon receptors and, if so, to study the significance of the receptors in the cells, binding of [125I]-glucagon to nonparenchymal cells (mainly endothelial cells and Kupffer cells) isolated from mouse liver was examined by quantitative autoradiography and biochemical methods. Furthermore, the pathway of intracellular transport of colloidal gold-labeled glucagon (AuG) was examined in vivo. Autoradiographic and biochemical results demonstrated many glucagon receptors in both endothelial cells and Kupffer cells, and more receptors being present in endothelial cells than in Kupffer cells. In vivo, endothelial cells internalized AuG particles into coated vesicles via coated pits and transported the particles to endosomes, lysosomes, and abluminal plasma membrane. Therefore, receptor-mediated transcytosis of AuG occurs in endothelial cells. The number of particles present on the abluminal plasma membrane was constant if the amount of injected AuG increased. Therefore, the magnitude of receptor-mediated transcytosis of AuG appears to be regulated by endothelial cells. Kupffer cells internalized the ligand into cytoplasmic tubular structures via plasma membrane invaginations and transported the ligand exclusively to endosomes and lysosomes, suggesting that the ligand is degraded by Kupffer cells.

scholarly journals Binding of hyaluronate and chondroitin sulphate to liver endothelial cells

1986 ◽  
Vol 234 (3) ◽  
pp. 653-658 ◽  
Author(s):  
T C Laurent ◽  
J R E Fraser ◽  
H Pertoft ◽  
B Smedsrød
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Chondroitin Sulphate ◽  
Mutual Exclusion ◽  
Degree Of Polymerization ◽  
Multiple Site ◽  
Large Molecules ◽  
Liver Endothelial Cell ◽  
Liver Endothelial Cells ◽  
Inhibition Studies

Hyaluronate is taken up and metabolized in liver endothelial cells by means of a receptor. To characterize the interaction with the receptor, two preparations of 3H-labelled hyaluronate, of Mr 4 × 10(5) and 6.4 × 10(6), and a series of hyaluronate oligosaccharides were bound to cultured liver endothelial cells at 7 degrees C. The dissociation constant varied between 4.6 × 10(-6) M for an octasaccharide and 9 × 10(-12) M for the largest polymer. The Mr-dependence for the series of oligosaccharides was explained by the increased probability of binding due to the repetitive sequence along the chain. The high affinity of high-Mr hyaluronate for the receptor could also be mainly ascribed to this effect, which rules out any major contribution of co-operative multiple-site attachment to the cell surface. Each liver endothelial cell can bind 10(5) oligosaccharides, about 10(4) molecules with Mr 4 × 10(5) and about 10(3) molecules with Mr 6.4 × 10(6). This is explained by mutual exclusion of large molecules from the cell surface. Chondroitin sulphate is also bound to liver endothelial cells. Inhibition studies showed that it binds to the same receptor as hyaluronate and with an affinity that is about 3-fold higher than that of hyaluronate of the same degree of polymerization.

scholarly journals Intracellular transport of transferrin- and asialoorosomucoid-colloidal gold conjugates to lysosomes after receptor-mediated endocytosis.

1985 ◽  
Vol 33 (11) ◽  
pp. 1134-1144 ◽  
Author(s):  
M R Neutra ◽  
A Ciechanover ◽  
L S Owen ◽  
H F Lodish
Keyword(s):  
Acid Phosphatase ◽  
Cell Surface ◽  
Colloidal Gold ◽  
Intracellular Transport ◽  
Ruthenium Red ◽  
Endocytic Pathway ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Gold Probe ◽  
20 Nm

Proteins coupled to colloidal gold particles have been widely used to visualize the uptake and intracellular transport of specific ligands by receptor-mediated endocytosis. The intracellular route of lysosome-directed ligands such as asialoglycoproteins (ASGP) are apparently unaltered by conjugation to gold, but the pathway of transferrin, a ligand that normally recycles to the cell surface, was reported to be altered by conjugation to 15-20 nm gold. In this study, we sought to determine whether a smaller transferrin-gold probe would recycle, and whether it might enter the same endosomal and lysosomal compartments as does a larger, lysosome-directed ASGP gold probe by visualizing their simultaneous uptake in human hepatoma (HepG2) cells. In the same cells, endocytosis of fluid-phase protein was followed using the soluble tracer native ferritin; lysosomal compartments were identified by acid phosphatase cytochemistry; and cell surfaces were labeled with ruthenium red or cationized ferritin. During the first 10 min of uptake at 37 degrees C, specific receptor-bound ferrotransferrin (FeTf)-8 nm gold and asialoorosomucoid (ASOR)-20 nm gold were clustered together in coated pits and entered the same coated vesicles, smooth vesicles, and tubules in the peripheral cytoplasm. At later times, however, transferrin-gold did not return to the cell surface; unlike native transferrin, this gold probe accompanied ASOR-gold into multivesicular bodies (MVB). The MVBs that contained probes were at first devoid of acid phosphatase activity, but at 30 min, enzyme activity was detected in a few MVBs. Native ferritin was present, along with gold probes, in all compartments of the endocytic pathway. We conclude that the normal intracellular pathway of transferrin is altered by its association with a colloidal gold particle.

scholarly journals Signal-dependent distribution of cell surface P-selectin in clathrin-coated pits affects leukocyte rolling under flow

2003 ◽  
Vol 163 (6) ◽  
pp. 1385-1395 ◽  
Author(s):  
Hendra Setiadi ◽  
Rodger P. McEver
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Secretory Granules ◽  
Rho Kinase ◽  
Cytoplasmic Domain ◽  
Endocytic Pathway ◽  
Leukocyte Rolling ◽  
Coated Pits ◽  
Transfected Cells

Flowing leukocytes roll on P-selectin that is mobilized from secretory granules to the surfaces of endothelial cells after stimulation with histamine or thrombin. Before it is internalized, P-selectin clusters in clathrin-coated pits, which enhances its ability to support leukocyte rolling. We found that thrombin and histamine induced comparable exocytosis of P-selectin on endothelial cells. However, compared with histamine, thrombin decreased the recruitment of P-selectin into clathrin-coated pits, slowed the internalization of P-selectin, and reduced the number and stability of neutrophils rolling on P-selectin. Significantly more RhoA was activated in thrombin- than in histamine-stimulated endothelial cells. Inhibitors of RhoA or its effector, Rho kinase, reversed thrombin's ability to inhibit the internalization and adhesive function of P-selectin in endothelial cells. Experiments with transfected cells confirmed that the inhibitory actions of thrombin and Rho kinase on P-selectin required its cytoplasmic domain. Thus, a signaling event affects both the function and clearance of a protein that enters the constitutive clathrin-mediated endocytic pathway.

scholarly journals New Scavenger Receptor-Like Receptors for the Binding of Lipopolysaccharide to Liver Endothelial and Kupffer Cells

1998 ◽  
Vol 66 (11) ◽  
pp. 5107-5112 ◽  
Author(s):  
Marijke van Oosten ◽  
Erika van de Bilt ◽  
Theo J. C. van Berkel ◽  
Johan Kuiper
Keyword(s):  
Endothelial Cells ◽  
Kupffer Cells ◽  
Binding Sites ◽  
Oxidized Ldl ◽  
Scavenger Receptor ◽  
Scavenger Receptors ◽  
Liver Endothelial Cells ◽  
Parenchymal Cells

ABSTRACT Lipopolysaccharide (LPS) is cleared from the blood mainly by the liver. The Kupffer cells are primarily responsible for this clearance; liver endothelial and parenchymal cells contribute to a lesser extent. Although several binding sites have been described, only CD14 is known to be involved in LPS signalling. Among the other LPS binding sites that have been identified are scavenger receptors. Scavenger receptor class A (SR-A) types I and II are expressed in the liver on endothelial cells and Kupffer cells, and a 95-kDa receptor, identified as macrosialin, is expressed on Kupffer cells. In this study, we examined the role of scavenger receptors in the binding of LPS by the liver in vivo and in vitro. Fucoidin, a scavenger receptor ligand, significantly reduced the clearance of 125I-LPS from the serum and decreased the liver uptake of 125I-LPS about 40%. Within the liver, the in vivo binding of 125I-LPS to Kupffer and liver endothelial cells was decreased 72 and 71%, respectively, while the binding of 125I-LPS to liver parenchymal cells increased 34% upon fucoidin preinjection. Poly(I) inhibited the binding of 125I-LPS to Kupffer and endothelial cells in vitro 73 and 78%, respectively, while poly(A) had no effect. LPS inhibited the binding of acetylated low-density lipoprotein (acLDL) to Kupffer and liver endothelial cells 40 and 55%, respectively, and the binding of oxidized LDL (oxLDL) to Kupffer and liver endothelial cells 65 and 61%, respectively. oxLDL and acLDL did not significantly inhibit the binding of LPS to these cells. We conclude that on both endothelial cells and Kupffer cells, LPS binds mainly to scavenger receptors, but SR-A and macrosialin contribute to a limited extent to the binding of LPS.

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