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.

Wortmannin-sensitive trafficking steps in the endocytic pathway in rat liver endothelial cells

2001 ◽  
Vol 357 (2) ◽  
pp. 497-503 ◽  
Author(s):  
Rune KJEKEN ◽  
Seyed A. MOUSAVI ◽  
Andreas BRECH ◽  
Gareth GRIFFITHS ◽  
Trond BERG
Keyword(s):  
Endothelial Cells ◽  
Rat Liver ◽  
Intracellular Transport ◽  
Mannose Receptor ◽  
Endocytic Pathway ◽  
Receptor Ligand ◽  
Surface Receptors ◽  
Phosphatidylinositol Kinases ◽  
Early Endosomes ◽  
Liver Endothelial Cells

Liver endothelial cells (LECs) play an important homoeostatic role by removing potentially harmful macromolecules from blood. The extremely efficient endocytosis in LECs makes these cells an interesting model for the study of the involvement of phosphoinositides in the different steps of the endocytic process. In the present investigation we have studied the effect of wortmannin, an inhibitor of phosphatidylinositol kinases, on uptake, recycling and intracellular transport of 125I-labelled ovalbumin, which is taken up in LECs via mannose-receptor-mediated endocytosis. Wortmannin was found to inhibit both uptake and degradation of ovalbumin. Further studies indicated that the reduced uptake via the mannose receptor was due both to a reduction of the number of surface receptors and a reduction in the rate of receptor–ligand internalization. Transport of ligand from endosomes to lysosomes was prevented, leading to increased recycling of internalized ligand. Wortmannin treatment released the Rab5 effector EEA1 from the endosomes and caused reduced size of early endosomes.

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 Studies in vitro on the uptake and degradation of sodium hyaluronate in rat liver endothelial cells

1984 ◽  
Vol 223 (3) ◽  
pp. 617-626 ◽  
Author(s):  
B Smedsrød ◽  
H Pertoft ◽  
S Eriksson ◽  
J R E Fraser ◽  
T C Laurent
Keyword(s):  
Endothelial Cells ◽  
Rat Liver ◽  
Degradation Products ◽  
Binding Capacity ◽  
Primary Cultures ◽  
Sodium Hyaluronate ◽  
Physiological Concentration ◽  
Liver Endothelial Cells

Rat liver endothelial cells in primary cultures at 7 degrees C bind radioactively labelled sodium hyaluronate (HA; Mr 400 000) specifically and with high affinity (Kd = 6 × 10(-11) M). Maximal binding capacity is approx. 10(4) molecules per cell. Inhibition experiments with unlabelled HA and oligosaccharides from HA indicate that each molecule is bound by several receptors acting co-operatively and that the single receptor recognizes a tetra- or hexa-saccharide sequence of the polysaccharide. At 37 degrees C the liver endothelial cells endocytose the HA. The process combines the features of a receptor-mediated and a fluid-phase endocytosis. The rate of internalization does not show any saturation with increasing HA concentration, but is approximately proportional to the polysaccharide concentration at and above the physiological concentration. At 50 micrograms of free HA/l each liver endothelial cell accumulates 0.1 fg of the polysaccharide/min. Fluorescent HA accumulates in perinuclear granules, presumably lysosomes. Degradation products from HA appear in the medium about 30 min after addition of the polysaccharide to the cultures. The radioactivity from HA containing N-[3H]acetyl groups or 14C in the sugar rings is recovered mainly as [3H]acetate and [14C]acetate respectively. Estimations of the capacity of liver endothelial cells to internalize and degrade HA in vitro indicate that these cells may be primarily responsible for the clearance of HA from human blood in vivo.

Tissue Plasminogen Activator is Endocytosed by Mannose and Galactose Receptors of Rat Liver Cells

1988 ◽  
Vol 59 (03) ◽  
pp. 480-484 ◽  
Author(s):  
Bård Smedsrød ◽  
Monica Einarsson ◽  
Håkan Pertoft
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Rat Liver ◽  
Side Chains ◽  
Diisopropyl Fluorophosphate ◽  
Tissue Plasminogen ◽  
Rat Liver Cells ◽  
Liver Endothelial Cells ◽  
Parenchymal Cells

SummaryExperiments were carried out to charact erize the specificity of uptake of tPA in rat liver cells. Endocytosis in liver endothelial cells of the native carbohydrate variants of tissue plasminogen activator (tPA), and tPA inactivated by diisopropyl fluorophosphate was found to be competitive, suggesting that the determinant being recognized by these cells is different from the active site. Fibronectin and urokinase, which show partial homology with tPA, did not compete with tPA for uptake in liver endothelial cells. Hyaluronic acid, collagen, or IgG, which are endocytosed by specific receptors in liver endothelial cells, did not interfere with the uptake.Reduced endocytosis by liver endothelial cells was observed with tPA modified in the carbohydrate side chains, suggesting that these structures are important for uptake. Ovalbumin, mannan, mannose, fructose, and EDTA, but not galactose, effectively inhibited uptake in liver endothelial cells of both native and diisopropyl fluorophosphate-inhibited tPA, but had very little effect on the uptake of tPA modified in the carbohydrate side chains.Endocytosis of native tPA by parenchymal cells could be inhibited by galactose, ovalbumin, and EDTA, but not by mannose.These results suggest that endocytosis of tPA by liver endothelial cells and parenchymal cells is mediated by the mannose and galactose receptors, respectively.

Uptake and Degradation of Tissue Plasminogen Activator in Rat Liver

1988 ◽  
Vol 59 (03) ◽  
pp. 474-479 ◽  
Author(s):  
Monica Einarsson ◽  
Bård Smedsrød ◽  
Håkan Pertoft
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Rat Liver ◽  
Liver Cells ◽  
Terminal Phase ◽  
Tissue Plasminogen ◽  
Liver Endothelial Cells ◽  
Parenchymal Cells

SummaryThe mechanism of uptake of tissue plasminogen activator (tPA) in rat liver was studied. Radio-iodinated tPA was removed from the circulation after intravenous administration in a biphasic mode. The initial half life, t1/2(α), and the terminal phase, t1/2(β), were determined to be 0.5 min and 7.5 min, resp. Separation of the liver cells by collagenase perfusion and density centrifugation, revealed that the uptake per cell was two to three times higher in the non-parenchymal cells than in the parenchymal cells.Endocytosis of fluorescein isothiocyanate-labelled or 125I-labelled tPA was studied in pure cultures of liver cells in vitro. Liver endothelial cells and parenchymal cells took up and degraded tPA. Endocytosis was more efficient in liver endothelial cells than in parenchymal cells, and was almost absent in Kupffer cells.Competitivb inhibition experiments showing that excess unlabelled tPA could compete with the uptake and degradation of 125I-tPA, suggested that liver endothelial cells and parenchymal cells interact with the activator in a specific manner. Endocytosis of trace amounts of 125I-tPA in cultures of liver endothelial cells and parenchymal cells was inhibited by 50% in the presence of 19 nM unlabelled tPA. Agents that interfere with one or several steps of the endocytic machinery inhibited uptake and degradation of 125I-tPA in both cell types.These findings suggest that 1) liver endothelial cells and parenchymal cells are responsible for the rapid hepatic clearance of intravenously administered tPA; 2) the activator is taken up in these cells by specific endocytosis, and 3) endocytosed tPA is transported to the lysosomes where it is degraded.

Nitric Oxide Down-Regulates Endocytosis in Rat Liver Endothelial Cells

1996 ◽  
Vol 222 (3) ◽  
pp. 688-693 ◽  
Author(s):  
Iñigo Martinez ◽  
Baldur Sveinbjørnsson ◽  
Bård Smedsrød
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Rat Liver ◽  
Liver Endothelial Cells
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