Biomedical subjects

R Blomhoff

Publications and source records attributed to R Blomhoff.

At least 109 records · Page 6Linked to original sources

PubMed1985

Intracellular transport of endocytosed chylomicron [3H]retinyl ester in rat liver parenchymal cells. Evidence for translocation of a [3H]retinoid from endosomes to endoplasmic reticulum.

The intracellular transport of chylomicron remnants labeled with [3H]retinyl ester was studied in rat liver parenchymal cells by means of subcellular fractionation in Nycodenz and sucrose density gradients. The data presented indicate that endocytosed chylomicron remnant [3H]retinyl ester initially is located in low density endosomes. Radioactivity is subsequently transferred to a denser vesicle. Equilibrium as well as rate zonal centrifugation suggest that this denser [3H] retinoid-containing vesicle may represent endoplasmic reticulum. We have compared the intracellular transport of chylomicron remnant [3H]retinyl ester and 125I-asialofetuin. The receptor-mediated endocytosis of asialoglycoproteins in rat liver parenchymal cells is a thoroughly studied system. Our results suggest that the [3H] retinoid and 125I-asialofetuin follow the same path initially to the endosomes. After transit in endosomes, the intracellular transport differs. While asialofetuin is transported to the lysosomes, the retinoid is probably transferred to the endoplasmic reticulum.

Animals↗

PubMed1985

Hepatic retinol metabolism. Distribution of retinoids, enzymes, and binding proteins in isolated rat liver cells.

The main retinoids and some binding proteins and enzymes involved in retinol metabolism have been quantified in different types of rat liver cells. Hepatic perisinusoidal stellate cells contained 28-34 nmol of retinoids/10(6) cells, and parenchymal liver cells contained 0.5-0.8 nmol of retinoids/10(6) cells, suggesting that as much as 80% of more of total liver retinoids might be stored in stellate cells with the rest stored in parenchymal cells. Isolated endothelial cells and Kupffer cells contained very low levels of retinoids. More than 98% of the retinoids recovered in stellate cells were retinyl esters. Isolated parenchymal and stellate cell preparations both contained considerable retinyl palmitate hydrolase and acyl-CoA:retinol acyltransferase activities. Parenchymal cells accounted for about 75-80% of the total hepatic content of these two enzyme activities, with the rest located in stellate cells. On a cell protein basis, the concentrations of both of these activities were much greater in stellate cells than in parenchymal cells. In contrast, cholesteryl oleate and triolein hydrolase activities were fairly evenly distributed in all types of liver cells. Large amounts of cellular retinol binding proteins were also found in parenchymal and stellate cells. Although parenchymal cells accounted for more than 90% of hepatic cellular retinol binding protein, the concentration of the protein in stellate cells (per unit protein) was 22 X greater than that in parenchymal cells. Stellate cells were also enriched in cellular retinoic acid binding protein. Thus, both parenchymal and stellate cells contain substantial amounts of retinoids and of the enzymes and intracellular binding proteins involved in retinol metabolism. Stellate cells are particularly enriched in these several components.

Acyltransferases↗

PubMed1985

Hepatic uptake of [3H]retinol bound to the serum retinol binding protein involves both parenchymal and perisinusoidal stellate cells.

We have studied the hepatic uptake of retinol bound to the circulating retinol binding protein-transthyretin complex. Labeled complex was obtained from the plasma of donor rats that were fed radioactive retinol. When labeled retinol-retinol binding protein-transthyretin complex was injected intravenously into control rats, about 45% of the administered dose was recovered in liver after 56 h. Parenchymal liver cells were responsible for an initial rapid uptake. Perisinusoidal stellate cells initially accumulated radioactivity more slowly than did the parenchymal cells, but after 16 h, these cells contained more radioactivity than the parenchymal cells. After 56 h, about 70% of the radioactivity recovered in liver was present in stellate cells. For the first 2 h after injection, most of the radioactivity in parenchymal cells was recovered as unesterified retinol. The radioactivity in the retinyl ester fraction increased after a lag period of about 2 h, and after 5 h more than 60% of the radioactivity was recovered as retinyl esters. In stellate cells, radioactivity was mostly present as retinyl esters at all time points examined. Uptake of retinol in both parenchymal cells and stellate cells was reduced considerably in vitamin A-deficient rats. Less than 5% of the injected dose of radioactivity was found in liver after 5-6 h (as compared to 25% in control rats), and the radioactivity recovered in liver from these animals was mostly in the unesterified retinol fraction. Studies with separated cells in vitro suggested that both parenchymal and stellate cells isolated from control rats were able to take up retinol from the retinol-retinol binding protein-transthyretin complex. This uptake was temperature dependent.

Animals↗

PubMed1985

Retinol esterification in cultured rat liver cells.

Retinol esterification was examined in cultured hepatocytes and stellate cells from the rat. Esterification of [3H]retinol was linear for 2 h in both cell types. By increasing the concentration of retinol in the medium, there was a marked increase in retinol esterification in both cell types. The capacity for esterification of retinol was in the same order of magnitude in the two cell types at 3.5 microM-retinol in the medium. This represents a rate of retinol esterification which far exceeds that required to esterify the amount of retinol absorbed in the intestine. It was demonstrated in particulate homogenates from cultured hepatocytes that the esterification of retinol was dependent on acyl-CoA. Addition of 25-hydroxycholesterol or mevalonolactone promoted an increase in cholesterol esterification, whereas retinol esterification was unaffected, suggesting that cholesterol and retinol are esterified by two different enzymes. Some 80% of vitamin A in cultured hepatocytes is retinyl esters, mostly retinyl palmitate. By adding 87 microM-retinol in the medium the cells accumulated 100-fold free retinol and 2.5-3.0-fold retinyl esters within 1 h. When retinol-loaded cells were incubated without retinol, there was a marked decrease especially in free but also in esterified retinol. In the presence of 1 mM-oleic acid in the medium the amount of retinyl oleate was twice that in control cells.

Acyltransferases↗

PubMed1985

Intracellular transport of asialoglycoproteins in rat hepatocytes. Evidence for two subpopulations of lysosomes.

The intracellular transport and degradation of asialoorosomucoid (AOM) in isolated rat hepatocytes was studied by means of subcellular fractionation in Nycodenz gradients. The asialoglycoprotein was labelled by covalent attachment of a radioiodinated tyramine-cellobiose adduct ( [125I]TC) which leads to labelled degradation products being trapped intracellularly and thus serving as markers for the degradative organelles. The ligand was initially (1 min) in a slowly sedimenting (small) vesicle and subsequently in larger endosomes. Acid-soluble, radioactive degradation products were first found in a relatively light lysosome whose distribution coincided in the gradient with that of the larger endosome. Later (30 min) degradation products were found in denser lysosomes which banded in the same region of the gradient as the lysosomal enzyme, beta-acetylglucosaminidase. Colchicine, monensin and leupeptin all inhibited degradation of [125I]tyramine-cellobiose asialoorosomucoid ( [125I]TC-AOM) and reduced the formation of degradation products in both the light and the dense lysosomes. In presence of monensin and colchicine no undegraded ligand was seen in the dense lysosome, suggesting that uptake in these vesicles was inhibited. Leupeptin allowed accumulation of undegraded ligand in the dense lysosome. Therefore, transfer from light to dense lysosomes is not dependent on degradation as such. In the presence of monensin two peaks of undegraded ligand were found in the gradients. It seems possible that in the monensin-sensitive endosomes, dissociation of the ligand-receptor complex is inhibited, allowing ligand to recycle with the receptors in small vesicles.

Animals↗

PubMed1985

Intracellular degradation of asialoglycoproteins in hepatocytes starts in a subgroup of lysosomes.

Isolated rat hepatocytes take up and degrade [125I]tyramine-cellobiose-labelled asialofetuin [( 125I]TC-AF). The labelled degradation products are trapped at the site of degradation. The intracellular transport of [125I]TC-AF was studied by means of cell fractionation in Nycodenz gradients. The labelled ligand was kept in a small, slowly sedimenting vesicle during the first minutes after uptake in the cells, and was then transferred to a larger endosome. Labelled degradation products first appeared in an organelle with the same density distribution as the larger endosome and then in a denser organelle. These observations suggest that two types of lysosome, 'light' lysosomes and 'dense', are sequentially involved in the degradation of the asialoglycoprotein. The bulk of the lysosomal enzymes is associated with the dense lysosome.

Acetylglucosaminidase↗

PubMed1985

Uptake and degradation of bovine testes beta-galactosidase by parenchymal and nonparenchymal rat liver cells.

The plasma half-life of beta-galactosidase in rat was about 1.5 min. Ten minutes after in vivo injection, 45% of the enzyme was recovered in liver, with hepatocytes and endothelial cells as the predominant cell types responsible for uptake. In vitro uptake of beta-galactosidase in hepatocytes and nonparenchymal liver cells was saturable, Ca2+-dependent and it could be partly inhibited by mannose or alpha-methyl-mannoside.

Animals↗

PubMed1985

Hepatic uptake of circulating IgG immune complexes.

IgG antibodies were found to increase the uptake of circulating dinintrophenylated human serum albumin (DNP-HSA) preparations by the nonparenchymal liver cells in rats. Highly DNP-conjugated HSA was taken up by the Kupffer cells both when given alone and when complexed by IgG. More lightly DNP-conjugated HSA was taken up mainly by the liver endothelial cells. Here, IgG promoted the antigen uptake both by the Kupffer cells and by the endothelial cells. Uptake of IgG immune complexes (IgG-ICs) by the sinusoidal endothelial cells of the liver is a new aspect on the function of these cells. Whether or not this phenomenon is Fc receptor-mediated is discussed. A heat-labile serum factor was found to direct the ICs to the Kupffer cells. This implies that serum complement and hepatic C3 receptors are essential for the physiological clearance of circulating immune complexes.

Animals↗

PubMed1985

Retinol and retinyl esters in parenchymal and nonparenchymal rat liver cell fractions after long-term administration of ethanol.

Chronic ethanol consumption reduces the liver retinoid store in man and rat. We have studied the effect of ethanol on some aspects of retinoid metabolism in parenchymal and nonparenchymal liver cells. Rats fed 36% of total energy intake as ethanol for 5-6 weeks had the liver retinoid concentration reduced to about one-third, as compared to pair-fed controls. The reduction in liver retinoid affected both the parenchymal and the nonparenchymal cell fractions. Plasma retinol level was normal. Liver uptake of injected chylomicron [3H]retinyl ester was similar in the experimental and control group. The transport of retinoid from the parenchymal to the nonparenchymal cells was not found to be significantly retarded in the ethanol-fed rats. Despite the reduction in total retinoid level in liver, the concentrations of unesterified retinol and retinyl oleate were increased in the ethanol fed rats. Hepatic retinol esterification was not significantly affected in the ethanol-fed rats. Since our study has demonstrated that liver uptake of chylomicron retinyl ester is not impaired in the ethanol-fed rat, we suggest that liver retinoid metabolism may be increased.

Acyltransferases↗

PubMed1984

Separation of endocytic vesicles in Nycodenz gradients.

The endocytosis of 125I-labeled asialofetuin by rat hepatocytes was studied using Nycodenz/sucrose gradients. It was shown in pulse chase experiments that the ligand endocytosed initially (after 1/2 to 1 min) was in small, slow-sedimenting vesicles of similar sizes. The vesicles containing the ligand increased in size, and after about 2.5 min 20-30% of the ligand was recovered in larger, faster-sedimenting vesicles. After 15 min almost all internalized ligand was recovered in the fast-sedimenting vesicles. The initial, small endocytic vesicles and the later, larger endocytic vesicles have similar buoyant densities; the maturation of the endosomes can only be revealed by rate sedimentation, not by isopycnic centrifugation. Dissociation of ligand from receptor was found to occur in the larger, faster-sedimenting vesicles. The presence of ammonia inhibited the increase in size of the ligand-containing endosomes. The methods employed here offer the possibility of obtaining endocytic vesicles at various stage of their development for further studies.

Ammonium Chloride↗

PubMed1984

Uptake of mannose-terminated glycoproteins in isolated rat liver cells. Evidence for receptor-mediated endocytosis in hepatocytes.

Even though most of the hepatic binding capacity for mannose-terminated glycoproteins has previously been shown to reside in the hepatocytes (not in the non-parenchymal cells), detailed evidence for the specific uptake of mannose-terminated ligands has been lacking. In the present studies, yeast invertase, a large glycoprotein (Mr 270 000) containing about 50% mannose, was shown to be taken up into hepatocytes by receptor-mediated endocytosis. The uptake was saturable and could be specifically inhibited by mannosides or by a Ca2+ chelator. The asialo-glycoprotein receptor was not involved. The low-Mr (13 000) ligand ribonuclease B, which contains a single high-mannose glycan, was not taken up by hepatocytes; however, it was taken up as fast as invertase by non-parenchymal liver cells. After injection of 131I-invertase into a rat in vivo, about one-half of the labelled protein was recovered in the hepatocytes. On a per-cell basis, each endothelial cell contained 3-4 times as much radioactivity as did the hepatocytes. On fractionation of hepatocytes in sucrose gradients, invertase showed a different intracellular distribution from that of asialo-fetuin, in that invertase moved much faster into that region of the gradient where the lysosomes were recovered. This indicates that invertase and asialo-fetuin are not transported intracellularly by identical mechanisms.

Animals↗

PubMed1984

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.

Animals↗

PubMed1984

Lymphatic absorption and transport of retinol and vitamin D-3 from rat intestine. Evidence for different pathways.

The lymphatic absorption and transport of retinol and vitamin D-3 from rat intestine has been studied. When rats were cannulated in the intestinal lymph duct and given an intraduodenal bolus of [3H]retinol and 14C-labelled vitamin D-3, 14C-labeled vitamin D-3 appeared later in the intestinal lymph than [3H]retinol and the rate of absorption of vitamin D-3 was still maximal at a time when that of retinol had declined. Both vitamins were absorbed via the lymphatic route in association with chylomicrons. Almost all the retinol was esterified, while vitamin D-3 appeared in the chylomicrons as free vitamin D-3. In vitro incubations and in vivo studies using hepatectomized and normal rats showed that the retinyl ester was a relatively nonexchangeable component of the chylomicrons and their remnants. Hence, all the vitamin A followed the remnants in their clearance from plasma. In contrast, significant amounts of vitamin D-3 were transferred from the chylomicrons to other plasma fractions. Therefore, only a fraction of this vitamin may be removed in association with the chylomicron remnants.

Animals↗

PubMed1984

Endocytosis of formaldehyde-treated serum albumin via scavenger pathway in liver endothelial cells.

Denatured or modified proteins (including albumin and low-density lipoprotein) are catabolized in vitro via scavenger receptors. We have studied the distribution of formaldehyde-denatured albumin in rat liver cells after intravenous injection of tracer doses of the protein. At 12 min after injection, most of the formaldehyde-denatured albumin (about 70% of the injected dose) was recovered in liver endothelial cells. Furthermore, isolated liver endothelial cells in suspension and in surface culture took up formaldehyde-denatured albumin by receptor-mediated endocytosis. Our data indicate that the scavenger receptor in liver is mainly located on the endothelial cells. Implications for the catabolism of low-density lipoproteins are discussed.

Animals↗

PubMed1984

Newly administered [3H]retinol is transferred from hepatocytes to stellate cells in liver for storage.

We have recently shown that newly administered vitamin A (retinol) is initially taken up by the parenchymal cells of the liver, and subsequently (within 1-2 h) transferred to non-parenchymal liver cells (NPC) (Blomhoff et al., ref. [10]). In the present study we have separated the NPC by different methods to determine the cell type responsible for this uptake of [3H]retinol. When liver cells were prepared between 5 and 18 h after intraduodenal administration of [3H]retinol, the radioactive retinol was recovered mainly in the stellate cells. Other liver cells (i.e., hepatocytes, endothelial cells and Kupffer cells) contained only small amounts of [3H]retinol. Further, fluorescence microscopy studies indicated that stellate cells contain large quantities of retinol. Our results show that newly administered [3H]retinol, which is initially located in the hepatocytes, is transferred to the stellate cells and stored there.

Animals↗

PubMed1984

Preparation of isolated liver endothelial cells and Kupffer cells in high yield by means of an enterotoxin.

A new method for preparing non-parenchymal rat liver cells (NPC) is described. The liver cell suspension, prepared by perfusing the liver with collagenase, was treated with enterotoxin from Clostridium perfringens for 15 min. The enterotoxin made the parenchymal cells leaky, and these cells could be separated from the NPC by centrifugation in a solution containing Nycodenz (20%, w/v). During the centrifugation, the NPC floated, while the parenchymal cells sedimented. The yield of NPC per liver (200 g rat) was about 250 X 10(6) cells. The NPC were further separated into endothelial cells, Kupffer cells and stellate cells by centrifugal elutriation. This method was particularly useful for preparing endothelial cells in high yield (100 X 10(6) cells per liver). Intravenously injected formaldehyde-treated albumin was selectively taken up by the endothelial cells. Isolated endothelial cells in suspension as well as in surface culture maintained their ability to endocytose this ligand.

Animals↗

PubMed1984

Effect of dextran and modified dextrans on the uptake and degradation of beta-galactosidase in isolated liver cells.

Conjugation of beta-galactosidase with either dextran, methylated dextran or acetylated dextran had only a small effect on uptake of the enzyme in isolated rat parenchymal and nonparenchymal liver cells. Conjugation of beta-galactosidase with dextran or the modified dextrans, reduced the intracellular degradation of the enzyme by up to about 45%. Methylated dextran had less effect than unmodified dextran or acetylated dextran on reducing the intracellular degradation of beta-galactosidase.

Animals↗

PubMed1983

Monensin inhibits receptor-mediated endocytosis of asialoglycoproteins in rat hepatocytes.

Isolated rat liver parenchymal cells incubated in the presence of monensin exhibited a reduced uptake of 125I-asialofetuin (125I-AF). Binding studies indicated that the effect was due to a rapid reduction in the number of active surface receptors for the asialoglycoprotein. Monensin had no effect on receptor internalization, but apparently interrupted the recycling of receptors back to the cell surface. Monensin also inhibited the degradation of 125I-AF previously bound to the cells; this inhibition was probably not due to a direct effect on intralysosomal proteolysis, as no lysosomal accumulation of undegraded ligand could be demonstrated in subcellular fractionation studies by means of sucrose gradients. It is more likely that monensin inhibits transfer of the labelled ligand from endocytic vesicles to lysosomes, as indicated by the accumulation of radioactivity in the former and by the ability of monensin to prevent the normally observed time-dependent increase in the buoyant density of endocytic vesicles. Whereas the effect of monensin on binding and uptake of asialofetuin was reversible, the effect on asialofetuin degradation could not be reversed.

Animals↗