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T Berg

Publications and source records attributed to T Berg.

At least 217 records · Page 12Linked to original sources
PubMed1990

The effect of vanadate on receptor-mediated endocytosis of asialoorosomucoid in rat liver parenchymal cells.

Vanadate is a phosphate analogue that inhibits enzymes involved in phosphate release and transfer reactions (Simons, T. J. B. (1979) Nature 281, 337-338). Since such reactions may play important roles in endocytosis, we studied the effects of vanadate on various steps in receptor-mediated endocytosis of asialoorosomucoid labeled with 125I-tyramine-cellobiose (125I-TC-AOM). The labeled degradation products formed from 125I-TC-AOM are trapped in the lysosomes and may therefore serve as lysosomal markers in subcellular fractionation studies. Vanadate reduced the amount of active surface asialoglycoprotein receptors approximately 70%, but had no effect on the rate of internalization and retroendocytosis of ligand. The amount of surface asialoglycoprotein receptors can be reduced by lowering the incubation temperature gradually from 37 to 15 degrees C (Weigel, P. H., and Oka, J. A. (1983) J. Biol. Chem. 258, 5089-5094); vanadate affected only the temperature--sensitive receptors. Vanadate inhibited degradation of 125I-TC-AOM 70-80%. Degradation was much more sensitive to vanadate than binding; half-maximal effects were seen at approximately 1 mM vanadate for binding and approximately 0.1 mM vanadate for degradation. By subcellular fractionation in sucrose and Nycodenz gradients, it was shown that vanadate completely prevented the transfer of 125I-TC-AOM from endosomes to lysosomes. Therefore, the inhibition of degradation by vanadate was indirect; in the presence of vanadate, ligand did not gain access to the lysosomes. The limited degradation in the presence of vanadate took place in a prelysosomal compartment. Vanadate did not affect cell viability and ATP content.

Adenosine Triphosphate↗
PubMed1990

Exocrine and endocrine release of kallikrein after reflex-induced salivary secretion.

Exocrine and endocrine release of rat submandibular gland kallikrein has been shown to be low after parasympathetic and beta-adrenergic stimulation but greatly increased after alpha-adrenergic stimulation. In the present study, release of glandular kallikrein was investigated under conditions known to give a reflex-induced salivary gland response. Heat stress induced a rich flow of saliva originating in the submandibular glands. Salivary kallikrein secretory rate was higher than after parasympathetic stimulation but lower than after sympathetic stimulation (P less than 0.005). Only heat stress increased circulating glandular kallikrein (12.7 +/- 0.8 ng ml-1 before heat exposure and 53.3 +/- 14.1 ng ml-1 40 min afterwards, P less than 0.005). There were no indications that the endocrine release of kallikrein was due to non-specific leakage. Atropine abolished heat-induced salivation and endocrine kallikrein secretion, possibly through interference with central pathways (P less than 0.05). However, phentolamine did not, which may indicate as an yet unidentified mediator of endogenous kallikrein release. The salivary gland response to acid and ether was comparable to that observed after parasympathetic nerve stimulation and was abolished by atropine (P less than 0.005). Stimuli known to influence other salivary gland ductal cells, such as aggression and starvation followed by drinking, also did not increase the plasma concentration of glandular kallikrein. The fact that various conditions which induce salivation did not increase circulating glandular kallikrein, coupled with the fact that kallikrein concentration was the highest in animals that died from heat stress, may suggest that the increase in circulating glandular kallikrein seen after heat stress may be pathological and could contribute to the development of heat shock.

Animals↗
PubMed1990

Endocytosis mediated by the mannose receptor in liver endothelial cells. An immunocytochemical study.

Immunocytochemical labeling of ultrathin cryosections from rat liver showed that mannose-terminated glycoproteins are removed rapidly from the blood stream mainly by the sinusoidal endothelial cells. The mannose-terminated glycoprotein ovalbumin was injected intravenously into rats 1 min, 6 min, and 24 min before perfusion fixation of the liver. Several minor and at least three major subcellular compartments were shown to be involved in the endocytic process. One minute after injection, ovalbumin was found at the cell surface, in coated pits, in coated vesicles, in tubular structures, and bound to the membrane of large early endosomes of which some showed a cisternal structure. After 6 min, ovalbumin was found in the lumen of large electron-lucent late endosomes and after 24 min in electron-dense structures, presumably lysosomes. The early endosomes have an ultrastructure which, together with the labeling pattern, indicates that this compartment has the same function as the CURL identified in parenchymal liver cells. The results are in accordance with recent biochemical findings indicating that ovalbumin endocytosed by endothelial cells is found sequentially in three different subcellular fractions depending on the time between injection and cooling for fractionation (G. M. Kindberg, T. Berg: Intracellular transport of endocytosed mannose terminated glycoproteins in rat liver endothelial cells. In: E. Wisse, D. L. Knook, K. Decker (eds.): Cells of the Hepatic Sinusoid. Vol. 2. pp. 120-124. Kupffer Cell Foundation. Rijswijk The Netherlands 1989).

Animals↗
PubMed1990

Internalization of retinol-binding protein in parenchymal and stellate cells of rat liver.

We have studied uptake of retinol-binding protein (RBP) by rat liver cells. First, we compared the in vivo uptake in different liver cells of 125I-labeled RBP with that of other well-known ligands. We found that the ligands studied were recognized differently by the various cell types in the liver, and that RBP was most efficiently taken up by parenchymal and stellate cells. We then studied the in vivo uptake of RBP in liver cells by immunocytochemistry at the electron microscopic level using ultrathin cryosections. Ten min after injection, RBP was localized to parenchymal cells and stellate cells. In these cells, RBP was detected on the cell surface and in vesicles near the cell surface. RBP was observed mainly in association with the membrane in these vesicles. Two hours after injection, RBP was localized not only on the cell surface and in vesicles close to the cell surface, but also in larger vesicles located deeper in the cytoplasm of these cells. RBP in larger vesicles was observed at a distance from the vesicular membrane. Finally, we compared the distribution of endocytosed RBP in liver parenchymal cells with that of asialo-orosomucoid, a ligand known to be internalized by receptor-mediated endocytosis. We detected both ligands on the cell surface and in small vesicles located close to the cell surface and in larger vesicles located deeper in the cytoplasm. Asialo-orosomucoid and RBP were seldom observed in the same small vesicles, but the larger vesicles contained both ligands. These data suggest that RBP is internalized in parenchymal and stellate cells of the liver by receptor-mediated endocytosis.

Animals↗
PubMed1989

A multicompartmental model of fluid-phase endocytosis in rabbit liver parenchymal cells.

Fluid-phase endocytosis was studied in isolated rabbit liver parenchymal cells by using 125I-poly(vinylpyrrolidone) (PVP) as a marker. First, uptake of 125I-PVP by cells was determined. Also, cells were loaded with 125I-PVP for 20, 60 and 120 min, and release of marker was monitored for 120-220 min. Then we used the Simulation, Analysis and Modeling (SAAM) computer program and the technique of model-based compartmental analysis to develop a mechanistic model for fluid-phase endocytosis in these cells. To fit all data simultaneously, a model with three cellular compartments and one extracellular compartment was required. The three kinetically distinct cellular compartments are interpreted to represent (1) early endosomes, (2) a prelysosomal compartment equivalent to the compartment for uncoupling of receptor and ligand (CURL) and/or multivesicular bodies (MVB), and (3) lysosomes. The model predicts that approx. 80% of the internalized 125I-PVP was recycled to the medium from the early-endosome compartment. The apparent first-order rate constant for this recycling was 0.094 min-1, thus indicating that an average 125I-PVP molecule is recycled in 11 min. The model also predicts that recycling to the medium occurs from all three intracellular compartments. From the prelysosomal compartment, 40% of the 125I-PVP molecules are predicted to recycle to the medium and 60% are transferred to the lysosomal compartment. The average time for recycling from the prelysosomal compartment to the medium was estimated to be 66 min. For 125I-PVP in the lysosomal compartment, 0.3%/min was transferred back to the medium. These results, and the model developed to interpret the data, predict that there is extensive recycling of material endocytosed by fluid-phase endocytosis to the extracellular environment in rabbit liver parenchymal cells.

Animals↗
PubMed1989

Low-density-lipoprotein receptors in different rabbit liver cells.

Receptor-dependent uptake mechanisms for low-density lipoprotein (LDL) were studied in rabbit liver parenchymal and non-parenchymal cells. Hybridization studies with a cDNA probe revealed that mRNA for the apo (apolipoprotein) B,E receptor was present in endothelial and Kupffer cells as well as in parenchymal cells. By ligand-blotting experiments we showed that apo B,E-receptor protein was present in both parenchymal and non-parenchymal cells. Studies of binding of homologous LDL in cultured rabbit parenchymal cells suggested that about 63% of the specific LDL binding was mediated via the apo B,E receptor. Approx. 47% of the specific LDL binding was dependent on Ca2+, suggesting that specific Ca2+-dependent as well as Ca2+-independent LDL-binding sites exist in liver parenchymal cells. Methylated LDL bound to the parenchymal cells in a saturable manner. Taken together, our results showed that apo B,E receptors are present in rabbit liver endothelial and Kupffer cells as well as in the parenchymal cells, and that an additional saturable binding activity for LDL may exist on rabbit liver parenchymal cells. This binding activity was not inhibited by EGTA or reductive methylation of lysine residues in apo B. LDL degradation in parenchymal cells was mainly mediated via the apo B,E receptor.

Animals↗
PubMed1989

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

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.

Animals↗
PubMed1989

Extremely rapid endocytosis mediated by the mannose receptor of sinusoidal endothelial rat liver cells.

Isolated sinusoidal endothelial rat liver cells (EC) in suspension bound and internalized ovalbumin, a mannose-terminated glycoprotein, in a saturable manner. The binding and uptake were Ca2+-dependent and were effectively inhibited by alpha-methyl mannoside and yeast mannan, but not by galactose or asialoglycoproteins. This corresponds to the binding specificity described for the mannose receptor of macrophages and non-parenchymal liver cells. Binding studies indicated a surface pool of 20,000-25,000 mannose receptors per cell, with a dissociation constant of 6 x 10(-8) M. Uptake and degradation of ovalbumin by isolated EC were inhibited by weak bases and ionophores which inhibit acidification of endocytic vesicles and dissociation of receptor-ligand complexes. Cycloheximide had no effect on uptake or degradation. Degradation, but not uptake, was inhibited by leupeptin. We conclude that ovalbumin dissociates from the mannose receptors in the endosomal compartment and the receptors are recycled to the cell surface, while the ovalbumin is directed to the lysosomes for degradation. A fraction of the internalized ovalbumin was recycled intact to the cell surface and escaped degradation (retroendocytosis). The rate of internalization of ovalbumin by isolated EC was very fast, with a Ke (endocytotic rate constant) of 4.12 min-1, which corresponds to a half-life of 10 s for the surface pool of receptor-ligand complexes. To our knowledge, this is the highest Ke reported for a receptor-mediated endocytosis system.

Animals↗
PubMed1989

Heterogeneity of degradation of B-cell endocytosed monoclonal antibodies reacting with different sIgM epitopes.

The degradation of a panel of monoclonal antibodies (MoAb) bound to surface IgM (sIgM) was studied in three human Burkitt's lymphoma cell lines. The panel included MoAb that recognize several distinct epitopes associated with the F(c mu)5 domain, the c mu 2 domain and kappa or lambda light chains. The amount of degraded MoAb and the rate of their degradation varied considerably between the various antibodies. Properties of MoAb such as avidity or ability to cross-link sIgM did not significantly influence their degradation. The most consistent correlation between rate of degradation and MoAb used was the location of the epitope recognized by the individual MoAb. Thus, 7 out of 8 anti-light chain MoAb were degraded at a higher rate than 5 out of 5 anti-F(c mu)5 MoAb. One anti-c mu 2 MoAb was degraded at a rate similar to the majority of anti-light chain MoAb. The intracellular transport of an anti-kappa light chain MoAb and an anti-F(c mu)5 MoAb was studied in detail by subcellular fractionation in sucrose gradients. We found that the anti-kappa light chain MoAb was transported more rapidly to lysosomes than the anti-F(c mu)5 MoAb, showing that they were sorted differently intracellularly.

Animals↗
PubMed1989

Role of kinin in regulation of rat submandibular gland blood flow.

In tissues rich in kallikrein, vasodilator kinins, acting as paracrine hormones, may play a role in the local regulation of blood flow. We studied the role of kinins in the regulation of blood flow in the rat submandibular gland using a kinin analogue with antagonistic properties, [DArg0]Hyp3-Thi5-8[DPhe7]bradykinin. When infused into the carotid artery (20 micrograms/min/rat), this antagonist blocked the effect of bradykinin (25-250 ng/kg, intracarotid injection) on glandular blood flow. In nephrectomized rats, the antagonist also blocked the increase in glandular blood flow caused by enalaprilat, a kininase II converting enzyme inhibitor. At a dose of 20 micrograms/min/rat, the antagonist produced no detectable change in basal glandular blood flow; however, at a higher dose (100 micrograms/min/rat), it caused a significant decrease (p less than 0.001). In eight of 10 rats, blood flow decreased by 75% or more; this effect was not blocked by the alpha-adrenergic receptor antagonist phentolamine. After antagonist infusion was stopped, blood flow returned toward normal. Sympathetic nerve stimulation of the gland induced vasoconstriction followed by poststimulatory vasodilatation. In rats displaying severe vasoconstriction after the antagonist, postsympathetic vasodilatation was abolished even when stimulation was performed after the antagonist infusion had been stopped and blood flow returned toward normal. Although a direct vasoconstrictor effect of the kinin antagonist cannot be completely ruled out, these data suggest that, in the rat submandibular gland, kinins may play a role in regulation of basal blood flow and vasodilatation after converting enzyme inhibitor or sympathetic stimulation.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1989

Intracellular transport and degradation of 125I-tyramine cellobiose-labelled low-density lipoprotein endocytosed in vivo in rat liver cells studied by means of subcellular fractionation.

The intracellular transport and degradation of in vivo endocytosed 125I-tyramine cellobiose-labelled low density lipoprotein (125I-TC-LDL) in rat liver cells were studied by means of subcellular fractionation in Nycodenz, sucrose and Percoll density gradients, as well as by means of analytical differential centrifugation. Initially, labelled LDL was located in endocytic vesicles of low densities. Subsequently, acid-soluble and acid-precipitable radioactivities were found in organelles with buoyant densities distinctly lower than that of the main peaks of the lysosomal marker enzymes acid phosphatase and N-acetyl-beta-glucosaminidase. These prelysosomal organelles may represent multivesicular bodies (MVBs). Finally, 6 h after injection and onwards, the acid-soluble radioactivity cosegregated completely with the two lysosomal marker enzymes, suggesting that the degradation products were in secondary lysosomes. The rate of intracellular processing of LDL was very slow compared to that of asialoglycoproteins, suggesting that LDL followed a unique intracellular pathway, that may be specific for this type of ligand.

Animals↗
PubMed1989

Kinin antagonist does not protect against the hypotensive response to endotoxin, anaphylaxis or acute pancreatitis.

The vasodilator bradykinin (Bk) has long been though to participate in shock induced by endotoxemia, anaphylaxis and acute pancreatitis. Recently developed kinin antagonists have made it possible to test this hypothesis. We studied the effect of two of them. DArg0Hyp3-Thi5.8-DPhe7-Bk (45 and 220 micrograms/kg/min) and Lys-Lys-Hyp2-Thi5.8-DPhe7-Bk (100 micrograms/kg/min) on the early hypotensive response to Escherichia coli lipopolysaccharide (LPS). Rats infused with the antagonist vehicle were used as controls. At 45 micrograms/kg/min, DArg0-Hyp3-Thi5.8-dPhe7-Bk prevented the hypotensive response to high doses of Bk; however, neither antagonist prevented the hypotensive response to LPS. Circulating kinins measured 3 min after injecting LPS or vehicle were similar (16.3 +/- 1.4 vs. 26.0 +/- 7.2 pg/ml; P greater than .23). In allergically sensitized rats, 500 micrograms/kg/min DArg0-Hyp3-Thi5.8-DPhe-7-Bk did not alter the hypotensive (anaphylactic) response to antigen challenge (P greater than .38). Similarly, hypotension caused by development of acute pancreatitis in rats was not prevented by infusion of DArg0-Hyp3-Thi5.8-DPhe7-Bk at 200 micrograms/kg/min, 10 min) (P greater than .69). These results indicate that in the rate formation of kinins is not a major contributor to the hypotensive response observed in early endotoxemia, anaphylaxis and acute pancreatitis.

Acute Disease↗
PubMed1988

Degradation of a monoclonal anti-mu chain antibody in a human surface IgM-positive B cell line starts in prelysosomal vesicle.

The surface IgM-mediated endocytosis and intracellular transport of an anti-F(c mu)5 mAb was studied by using subcellular fractionation in sucrose gradients. The results of such experiments showed that antibody was initially endocytosed in vesicles of low density, and later transferred to a presumably lysosomal compartment of higher density. SDS-PAGE analysis of gradient fractions showed that high Mr degradation fragments of the endocytosed antibody were formed in the low density vesicles before terminal degradation could be recorded. The partial degradation of the antibody was not blocked by low temperature or enzyme inhibitors, such as leupeptin and benzyloxycarbonyl-phenylalanylalanine-diazomyethyl-ketone, all of which severely retarded terminal degradation. The data also suggested that the recycling of partially degraded antibody to the cell surface employed a pool of such low density prelysosomal vesicles.

Animals↗
PubMed1988

Uptake of LDL in parenchymal and non-parenchymal rabbit liver cells in vivo. LDL uptake is increased in endothelial cells in cholesterol-fed rabbits.

1. Hepatic uptake of low-density lipoprotein (LDL) in parenchymal cells and non-parenchymal cells was studied in control-fed and cholesterol-fed rabbits after intravenous injection of radioiodinated native LDL (125I-TC-LDL) and methylated LDL (131I-TC-MetLDL). 2. LDL was taken up by rabbit liver parenchymal cells, as well as by endothelial and Kupffer cells. Parenchymal cells, however, were responsible for 92% of the hepatic LDL uptake. 3. Of LDL in the hepatocytes, 89% was taken up via the B,E receptor, whereas 16% and 32% of the uptake of LDL in liver endothelial cells and Kupffer cells, respectively, was B,E receptor-dependent. 4. Cholesterol feeding markedly reduced B,E receptor-mediated uptake of LDL in parenchymal liver cells and in Kupffer cells, to 19% and 29% of controls, respectively. Total uptake of LDL in liver endothelial cells was increased about 2-fold. This increased uptake is probably mediated via the scavenger receptor. The B,E receptor-independent association of LDL with parenchymal cells was not affected by the cholesterol feeding. 5. It is concluded that the B,E receptor is located in parenchymal as well as in the non-parenchymal rabbit liver cells, and that this receptor is down-regulated by cholesterol feeding. Parenchymal cells are the main site of hepatic uptake of LDL, both under normal conditions and when the number of B,E receptors is down-regulated by cholesterol feeding. In addition, LDL is taken up by B,E receptor-independent mechanism(s) in rabbit liver parenchymal, endothelial and Kupffer cells. The non-parenchymal liver cells may play a quantitatively important role when the concentration of circulating LDL is maintained at a high level in plasma, being responsible for 26% of hepatic uptake of LDL in cholesterol-fed rabbits as compared with 8% in control-fed rabbits. The proportion of hepatic LDL uptake in endothelial cells was greater than 5-fold higher in the diet-induced hypercholesterolaemic rabbits than in controls.

Animals↗
PubMed1988

High sensitivity towards monensin of receptor-mediated endocytosis of formaldehyde treated albumin by liver endothelial cells.

Endocytosis of formaldehyde-treated serum albumin (f-albumin) in isolated liver sinusoidal endothelial cells was studied. Uptake occurs via the scavenger receptor and was found to be very sensitive to the ionophore monensin. Binding at 4 degrees C of f-albumin was reduced to 50% of control values by preincubation for 2 min with 2 microM monensin. Both uptake and degradation of f-albumin were more sensitive to monensin. No lag-phase in the inhibitory effect on uptake and degradation was detected. A concentration of 0.1 microM monensin reduced uptake of f-albumin by 50%. Degradation of internalized f-albumin was reduced by 50% in the presence of 0.2 microM monensin. Since uptake and degradation of f-albumin were very sensitive to monensin, the effect of introducing the drug during endocytosis of the ligand was tested. All processing of f-albumin stopped instantly upon addition of monensin; hence, there seems to be no step in the endocytic process beyond which monensin is ineffective. The data suggest that the scavenger receptor of liver endothelial cells is internalized and recycled very rapidly.

Animals↗