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Receptor-mediated endocytosis of ovalbumin by two carbohydrate-specific receptors in rat liver cells. The intracellular transport of ovalbumin to lysosomes is faster in liver endothelial cells than in parenchymal cells.

ABSTRACT: 1. The uptake of ovalbumin (OVA) in rat liver parenchymal cells (PC) and non-parenchymal cells was studied in vivo and in vitro in order to compare the cellular expression of glycoprotein receptors and the kinetics of intracellular transport of ligand endocytosed by these receptors. 2. Ovalbumin was labelled with 125I or with 125I-tyramine-cellobiose (125I-TC). By using 125I-TC-OVA the labelled degradation products were trapped in the cells. 3. 125I-TC-OVA was rapidly cleared from blood mainly by receptor-mediated uptake in the liver. At 30 min after injection, 50% of the ligand was recovered in the liver. The endothelial cells (EC) and the PC were the predominant cell types responsible for uptake. 4. The uptake in PC was strongly inhibited by asialo-orosomucoid (AOM), but not by mannan, indicating that the uptake in these cells was mediated by the galactose receptor and not by the mannose receptor. This finding is compatible with the observation that a proportion of the OVA contains terminal galactose residues in the carbohydrate moiety. 5. In vitro uptake of OVA in cultured EC was saturable and inhibited by mannan, mannose, fructose, N-acetylglucosamine, EDTA or monensin, but not by galactose or AOM. The uptake of OVA in these cells was therefore mediated by the mannose receptor. 6. To label the organelles involved in endocytosis in PC and EC, 125I-TC-OVA was injected intravenously together with an excess of either AOM or mannan. In this way the labelled ligand could be directed selectively to EC or PC respectively. Subcellular fractionation of total liver in sucrose and Nycodenz gradients revealed that in EC the intracellular transport of OVA is so fast that endocytosed ligand accumulates and thus increases the density of the lysosomes. Conversely, in PC transfer of ligand is slower, with the result that accumulation of undegraded ligand in the lysosomes does not occur. These findings are interpreted to mean that in EC the rate-limiting step of handling of endocytosed ligand is intralysosomal degradation, whereas in PC the rate-limiting step is transport of ligand to the lysosomes. 7. Altogether, these findings suggest that endocytosis of OVA by the liver EC and PC is mediated by mannose and galactose receptors respectively, and that the kinetics of intracellular transport of OVA differ in the two cell types.

SUBMITTER: Kindberg GM

PROVIDER: S-EPMC1131698 | biostudies-other | 1990 Aug

REPOSITORIES: biostudies-other

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Project description:Retinol-binding protein (RBP) was iodinated directly by radio-iodine substitution on the tyrosyl residues by the sodium hypochlorite (NaOCl) or the Enzymobead (EB) methods, or indirectly by linkage of 125I-tyramine-cellobiose (TC) or 125I-N-succinimidyl-3-(4- hydroxyphenyl)propionic acid ester (SHPP) adduct on to free amino residues of RBP. Binding, uptake and degradation of iodinated RBP were studied in isolated rat and rabbit liver parenchymal cells. The amount of ligand bound to cells at 4 degrees C was dependent on the type of labelling, in that the 125I-TC ligand was bound to a lesser extent than NaClO-labelled 125I-RBP, EB-labelled 125I-RBP and 125I-SHPP-RBP. At 37 degrees C, the 125I-SHPP-RBP and the EB-labelled 125I-RBP became cell-associated more rapidly than the other two ligands. The higher cell association at 37 degrees C than at 4 degrees C suggests that internalization of the ligand occurred at the higher temperature. The degradation of the ligands was also different. The EB-labelled 125I-RBP, the 125I-TC-RBP and the 125I-SHPP-RBP showed an apparent lag phase before a steady increase in acid-soluble radioactivity was observed. Much less of EB-labelled 125I-RBP and 125I-TC-RBP were degraded (about 6%) than of the other two ligands (about 16%) after 120 min. About 50% of the acid-soluble radioactivity in these experiments could be accounted for by degradation in the medium, suggesting that about half of the degradation observed was intracellular. The present study therefore shows that the different labelling techniques yield varying estimates of the cellular handling of RBP. In addition, a rapid release of RBP was observed in experiments where cells were pulsed with radioactive RBP at 4 degrees C, washed and incubated further at 37 degrees C. Between 50% and 70% was released after 5 min of incubation. By increasing the temperature during the pulse to 37 degrees C, or by lowering the temperature during the chase to 4 degrees C, much less RBP was released from the cells. These data suggest that the release process represents recycling of internalized ligand from an early endosome.

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Receptor-mediated endocytosis of ovalbumin by two carbohydrate-specific receptors in rat liver cells. The intracellular transport of ovalbumin to lysosomes is faster in liver endothelial cells than in parenchymal cells.

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