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.     

Uptake of thymidine into isolated rat hepatocytes. Evidence for two transport systems

Thymidine transport was studied in isolated rat hepatocytes. In these cells no phosphorylation of the substrate by thymidine kinase occurred subsequent to transport. Results from studies of the concentration-dependent uptake of thymidine indicated two transport systems with about 80-fold differences in their kinetic constants. These systems were denoted as high affinity [Km = 5.3 micron, V = 0.47 pmol/(10(6) cells X s)] and low affinity systems [Km = 480 micron, V = 37.6 pmol/(10(6) cells X s)]. From intracellular to extracellular distribution ratios of [3H]thymidine it could be concluded that the uptake by the high affinity system was a concentrative process while the transport by the low affinity system was non-concentrative. The uptake of [3H]-thymidine by the high affinity system could only be inhibited by unlabeled thymidine. In contrast, all other nucleosides tested (uridine, 2'-deoxycytidine, and 2'-deoxyguanosine) were equally effective in inhibiting the low affinity system competitively. The results would suggest that in hepatocytes lacking phosphorylation by thymidine kinase, thymidine is taken up by a high and a low affinity system working in tandem. The high affinity system seems to be an active transport process with narrow substrate specificity. Thymidine uptake by the low affinity system is a facilitated diffusion process. This system is considered to be a common transport route for nucleosides of different structures.     

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.     

Degradation of asialoglycoproteins mediated by the galactosyl receptor system in isolated hepatocytes. Evidence for two parallel pathways

The ability of rat hepatocytes to degrade internalized surface-bound 125I-asialoorosomucoid (ASOR) was determined by measuring the appearance of acid-soluble radioactivity at 37 degrees C. The degradation kinetics were biphasic in cells previously equilibrated at 37 degrees C for 1 h or cultured for 24 h. Degradation began immediately and was linear for at least 20 min after which the rate increased to a steady state value 3-4 times greater than the initial rate. We previously showed that hepatocytes have two functionally distinct populations of galactosyl receptors that mediate ligand dissociation by two kinetically different pathways (Weigel, P. H., Clarke, B. L., and Oka, J. A. (1986) Biochem. Biophys. Res. Commun. 140, 43-50). The activity of one receptor population, designated State 2 galactosyl receptors, can be reversibly modulated by incubating cells between 22 and 37 degrees C and is not expressed on the surface of freshly isolated cells. When 125I-ASOR was prebound to freshly isolated cells at 4 degrees C and degradation was assessed subsequently at 37 degrees C, the kinetics were monophasic, not biphasic. Degradation of the surface-bound 125I-ASOR began immediately and was greater than 90% complete by 6 h. Freshly isolated cells were incubated at temperatures between 22 and 37 degrees C, chilled to 4 degrees C, allowed to pre-bind 125I-ASOR, and then incubated at 37 degrees C. As the State 2 galactosyl receptor population increased, the kinetics of degradation became progressively more biphasic and the rate of the delayed degradation process increased. This effect could be reversed in cells in culture or in suspension by down-modulating surface receptor activity at temperatures below 37 degrees C; only the degradation process appearing after a 20-min lag was affected. Degradation in both pathways is an apparent first order process with identical rate constants (kappa = 0.006 min-1, t1/2 = 116 min). We conclude that there are two separate pathways by which asialoglycoproteins are degraded. The major "classic" pathway mediated by State 2 galactosyl receptors occurs after a 20-min lag and the minor pathway mediated by State 1 galactosyl receptors begins immediately with no detectable lag.     

Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes: receptor-positive and receptor-negative endosomes

We have used combinations of subcellular fractionation, specific cytochemical tracers, and quantitative immunoadsorption to determine when, where, and in which intracellular structure internalized asialoglycoproteins (ASGPs) are segregated from their receptor. All membrane vesicles containing the receptor (R+ vesicles) were quantitatively immunoadsorbed from crude microsomes with Staphylococcus aureus cells and affinity-purified anti-ASGP receptor. Using this assay, we varied the time and temperature of exposure of perfused livers to 125I-asialoorosomucoid (125I-ASOR) and followed the movement of ligand from R+ to R- vesicles. After 2.5 min at 37 degrees C, 98% of the internalized ligand could be immunoadsorbed and thus was in R+ vesicles. Over the next 12 min of continuous 37 degrees C perfusion with 125I-ASOR, an increasing fraction of the ligand was not immunoadsorbed and therefore was present in R- vesicles. A maximum of 30% of the ligand could be found in R- vesicles (14-44 min). When livers were maintained at 16 degrees C, ligand was internalized but remained in R+ vesicles. Furthermore, ligand accumulating in R- vesicles at 37 degrees C remained there when livers were cooled to 16 degrees C. R- endosomes could be separated from R+ endosomes by flotation on sucrose density gradients and visualized by the presence of sequestered ASOR-horseradish peroxidase (ASOR-HRP). These structures resembled those labeled by ASOR-HRP in situ: R+ vesicles were relatively dense (1.12 g/cc), frequently tubular or spherical and small (100-nm diam), corresponding to the peripheral and internal tubular endosomes; R- structures were of lower density (1.09 g/cc), large (400-nm diam), and resembled internal multivesicular endosomes (MVEs). Endocytosed ASOR-HRP was found in both the peripheral and internal tubular endosomes in situ under conditions where 95% of the ligand was present in R+ vesicles by immunoadsorption, whereas MVEs containing ASOR-HRP were predominant in situ when ligand was found in R- vesicles and were often in continuity with the tubular internal endosomes. All of these results suggest that complete segregation of ligand and receptor occurs after arrival in the Golgi-lysosome region of the hepatocyte and that MVEs are R- and represent the final prelysosomal compartment.     

Intracellular and transcellular transport of secretory component and albumin in rat hepatocytes

The intra- and transcellular transports of hepatic secretory and membrane proteins were studied in rats in vivo using [3H]fucose and [35S]cysteine as metabolic precursors. Incorporated radioactivity in plasma, bile, and liver subcellular fractions was measured and the labeled proteins of the Golgi complex, bile, and plasma were separated by SDS PAGE and identified by fluorography. 3H-radioactivity in Golgi fractions peaked at 10 min postinjection (p.i.) and then declined concomitantly with the appearance of labeled glycoproteins in plasma. Maximal secretion of secretory fucoproteins from Golgi occurred between 10 and 20 min p.i. In contrast, the clearance of labeled proteins from Golgi membrane subfractions occurred past 30 min p.i., indicating that membrane proteins leave the Golgi complex at least 30 min later than the bulk of content proteins. A major 80,000-dalton form of secretory component (SC) was identified in the bile by co-precipitation with (IgA)2 by an anti-IgA antibody. An antibody (raised in rabbit) against the biliary 80,000-dalton peptide recognized two larger forms (116,000 and 94,000 dalton), presumably precursors, in Golgi membranes. A comparative study of kinetics of transport of 35S-SC and 35S-albumin showed that albumin peaked in bile at approximately 45 min p.i., whereas the SC peak occurred at 80 min p.i., suggesting that the transit time differs for plasma and membrane proteins that are delivered to the bile canaliculus.     

Intracellular distribution of tetracyclines in rat hepatocytes

Distribution of tetracyclines, such as oxytetracycline, morphocycline, tetracycline, doxicycline and methacycline in the liver cells of rats was studied. The ratio of the subcellular structures, i. e. nuclei, mitochondria and microsomes and the liquid phase containing the drugs in the dissolved state in the system studied was close to the natural ratio of the hepatocyte organoids and cytoplasm. Distribution of tetracyclines in the subcellular fractions was not uniform. The nuclei did not absorb the drugs. The role of microsomes in drug absorption was insignificant. The mitochondria bound the highest amounts of the drugs and defined the characteristics of their intracellular distribution. The amounts of the drugs in the active form remaining in the cytoplasm after their contact with organoids were low. At the same time there was observed a a definite activating effect of the cytoplasm components on the antibiotics contained in it.     

Intracellular transport of cholesteryl esters from lysosomes to cytoplasm in macrophages

The mechanism through which nonmembranous lipid inclusion bodies consisting of cholesteryl esters accumulate in the cytoplasm was studied. Most lipid inclusion bodies in macrophages after 24 h incubation with anisotropic cholesteryl oleate liquid crystals were surrounded by a limiting membrane. The limiting membrane, however, could not be observed after further incubation for 48 h in the presence of esterastin, which is known to be an inhibitor of lipase and esterase. Under these conditions, the levels of hydrolysis and re-esterification of cholesteryl esters were less than 15% and 5% of the control ones, respectively. These results suggest that the inclusion bodies were transferred from lysosomes to the cytoplasm, with partial hydrolysis of cholesteryl esters, in addition to through the pathway via microsomes.     

Receptor-mediated endocytosis of asialoglycoproteins by rat liver hepatocytes: biochemical characterization of the endosomal compartments

The endocytic compartments of the asialoglycoprotein (ASGP) pathway in rat hepatocytes were studied using a combined morphological and biochemical approach in the isolated perfused liver. Use of electron microscopic tracers and a temperature-shift protocol to synchronize ligand entry confirmed the route of ASGP internalization observed in our previous in vivo studies (1) and established conditions under which we could label the contents of successive compartments in the pathway for subcellular fractionation studies. Three endosomal compartments were demonstrated in which ASGPs appear after they enter the cell via coated pits and vesicles but before they reach their site of degradation in lysosomes. These three compartments could be distinguished by their location within the hepatocyte, by their morphological appearance in situ, and by their density in sucrose gradients. The distributions of ASGP receptors, both accessible and latent (revealed by detergent permeabilization), were also examined and compared with that of ligand during subcellular fractionation. Most accessible ASGP receptors co-distributed with conventional plasma membrane markers. However, hepatocytes contain a substantial intracellular pool of latent ASGP binding sites that exceeds the number of cell surface receptors and whose presence is not dependent on ASGP exposure. The distribution of these latent ASGP receptors on sucrose gradients (detected either immunologically or by binding assays) was coincident with that of ligand sequestered within the early endosome compartments. In addition, both early endosomes and the membrane vesicles containing latent ASGP receptors had high cholesterol content, because both shifted markedly in density upon exposure to digitonin.     

Sulfate transport by rat liver lysosomes

Sulfate transport was examined using membrane vesicles (pH 7.0 inside) prepared from rat liver lysosomes. Sulfate uptake was dependent upon external pH with increased uptake at lower buffer pH. The Km for uptake was 160 microM at pH 5.0 while at pH 7.0, a lower affinity system with a Km of 1.4 mM was present. The protonophore carbonyl cyanide m-chlorophenylhydrazone increased uptake at pH 5.0 while valinomycin/KCl had no effect. In contrast, at pH 7.0, valinomycin-induced changes in membrane potential stimulated uptake. Countertransport of sulfate at pH 7.0 was inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonic acid, and a variety of anions: SO4(2-) greater than MoO4(2-) greater than Cl- greater than HPO4- greater than HCO3-. Trans-stimulation of sulfate uptake at pH 7.0 was observed with MoO4(2-) and, to a lesser extent, with S2O3(2-) while Cl-, HPO4-, and HCO3- had little effect. However, chloride loading of vesicles resulted in marked stimulation of sulfate uptake at pH 5.0. It appears that sulfate and protons exit lysosomes in exchange for chloride by a specific, pH-regulated anion transport system.     

Evidence for two Ca2(+)-mobilizing purinoceptors on rat hepatocytes.

Aequorin measurements of cytosolic free Ca2+ in single rat hepatocytes show that ADP and ATP, thought to act through the same P2Y purinoceptor, elicited very different responses in the majority of cells tested. ADP invariably induced transients of short duration (approx. 9 s), whereas ATP induced either similar transients or transients with a much longer duration (approx. 49 s). We explain this variability in terms of two separate purinoceptors on rat hepatocytes, one of which responds to either ATP or ADP to generate free-Ca2+ transients of short duration, and the other responds to ATP only, with transients of longer duration.     

Characteristics of taurine transport in rat liver lysosomes

Taurine (2-aminoethanesulfonic acid) is a unique sulfur amino acid derivative that has putative nutritional, osmoregulatory, and neuroregulatory roles and is highly concentrated within a variety of cells. The permeability of Percoll density gradient purified rat liver lysosomes to taurine was examined. Intralysosomal amino acid analysis showed trace levels of taurine compared to most other amino acids. Taurine uptake was Na(+)-independent, with an overshoot between 5-10 minutes. Trichloroacetic acid extraction studies and detergent lysis confirmed that free taurine accumulated in the lysosomal space. Kinetic studies revealed heterogeneous uptake with values for Km1 = 31 +/- 1.82 and Km2 greater than 198 +/- 10.2 mM. The uptake had a pH optimal of 6.5 and was stimulated by the potassium specific ionophore valinomycin. The exodus rate was fairly rapid, with a t1/2 of 5 minutes at 37 degrees C. Analog inhibition studies indicated substrate specificity similar to the plasma membrane beta-alanine carrier system, with inhibition by beta-alanine, hypotaurine, and taurine. alpha-Alanine, 2-methylaminoisobutyric acid (MeAIB), and threonine were poor inhibitors. No effects were observed with sucrose and the photoaffinity derivative of taurine NAP-taurine [N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonate]. In summary, rat liver lysosomes possess a high Km system for taurine transport that is sensitive to changes in K+ gradient and perhaps valinomycin induced diffusional membrane potential. These features may enable lysosomes to adapt to changing intracellular concentrations of this osmotic regulatory substance.     

The relationship between autophagy and the intracellular degradation of asialoglycoproteins in cultured rat hepatocytes

The relationship between autophagy and the intracellular distribution of endocytosed asialoorosomucoid was studied in cultured rat hepatocytes. Overt autophagy was induced by shifting the cells to a minimal salt medium. Incubation in minimal salt medium led to the formation of buoyant lysosomes at the expense of denser lysosomes manifested as a dual distribution of these organelles in Nycodenz gradients. Asialoorosomucoid was labeled with 125I-tyramine cellobiose. The labeled degradation products formed from this ligand are trapped at the site of degradation and may therefore serve as markers for the subgroup of lysosomes involved in the degradation. In control cells the degradation of the ligand was initiated in a light prelysosomal compartment and continued in denser lysosomes. In cells with high autophagic activity, the degradation of labeled asialoorosomucoid took place exclusively in a buoyant group of lysosomes. These results suggest that degradation of endocytosed ligand takes place in the same secondary lysosomes as substrate sequestered by autophagic mechanisms. These light lysosomes represent a subgroup of active lysosomes which are gradually recruited from dense bodies. Data are also presented that indicate that insulin may prevent the change in buoyant density brought about by incubation in deficient medium.     

Turnover of the surface proteins and the receptor for serum asialoglycoproteins in primary cultures of rat hepatocytes

The turnover of plasma membrane proteins in primary rat hepatocyte cultures was examined by following the loss of polypeptides labeled in situ by lactoperoxidase-catalyzed iodination using 125I and 131I. Most plasma membrane proteins had similar rates of degradation, having a half-life of approximately 85 h. By in situ labeling via lactoperoxidase-catalyzed iodination, as well as metabolically labeling cells with L-[35S]methionine, the asialoglycoprotein receptor, a plasma membrane constituent, was identified and shown to exist in three forms which were structurally related. The turnover of receptor on the cell surface was examined by following the loss of iodinated cell surface receptor, while the turnover of total cellular receptor, including both surface and internally localized receptor was assayed by following the loss of receptor labeled metabolically with [35S]methionine. The turnover rate in both cases was approximately 20 h. Receptor-mediated endocytosis of asialoglycoproteins had no effect on the turnover of the plasma membrane proteins or receptor. Based on estimates of the rate of metabolism of the asialoglycoprotein ligand relative to the turnover rate of the receptor, we conclude each molecule of receptor can deliver about 1,000 molecules of ligand to the lysosome to be degraded.     

Metabolism of asialoglycoproteins in cultured rat hepatocytes: evidence for receptor mediated uptake and degradation which is not feed-back regulated

1. Cultured rat hepatocytes took up and degraded asialofetuin by a saturable mechanism. 2. The uptake was specific for desialylated fetuin; native fetuin and was taken up to a much lesser extent. 3. Degradation was inhibited by lysosomotropic agents; both uptake and degradation were, however, unaffected by cycloheximide. 4. The uptake was also unaffected by incubating the cells for 24 hr in the presence of large extracellular concentrations of unlabelled asialofetuin. 5. The data suggest that the asialoglycoprotein receptor is recycled and not subject to down-regulation.     

Neutral-sugar transport by rat liver lysosomes.

Transport of D-glucose was studied in Percoll-gradient-purified rat liver lysosomes. D-Glucose uptake had a Km of 22 mM and a t1/2 of approx. 30 s. D-Fucose, 2-deoxyglucose and methyl alpha-glucoside were the most effective competitors for uptake of D-glucose, although D-galactose, D-mannose, D-xylose and L-fucose also appeared to compete for uptake. L-Glucose was a poor competitor for uptake. No competition was observed with N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-glucuronic acid, N-acetylneuraminic acid, D-glucosamine or the amino acids L-glycine, L-lysine and L-proline. Uptake was unaffected by N-ethylmaleimide, dithiothreitol, KCl, NaCl, ATP/Mg or alteration of buffer pH. D-Glucose efflux from lysosomes was temperature-dependent, with a Q10 of 2.3, and was inhibited by cytochalasin B. Counter-transport could not be demonstrated. In contrast, L-fucose uptake had a Km of 65 mM and was largely unaffected by 5 M excess of neutral D-sugars. Both uptake and efflux of L-fucose were inhibited by cytochalasin B. It appears that lysosomes possess a facilitated transport system for D-glucose and perhaps other neutral D-sugars that is discrete from transport systems for acetylated and acidic sugars.     

Receptor-mediated introduction of pepstatin-asialofetuin conjugate into lysosomes of rat hepatocytes

Pepstatin was linked through a carboxyl group to asialofetuin (PS-ASF). An analysis by separation of hepatocytes from nonparenchymal cells showed that PS-ASF was taken up by hepatocytes, following intravenous injection into rats. After the injection of PS-ASF, pepstatin concentration in the liver reached a maximum at 2 h and then decreased. In an analysis by differential centrifugation of the liver homogenate from rats injected with PS-ASF, pepstatin showed a lysosomal type subcellular distribution pattern. Isolation studies of tritosomes clearly demonstrated the exclusive accumulation of pepstatin within the lysosomes of livers from rats given PS-ASF (at 2 h after administration). Pepstatin contained in tritosomes was in a free form, as determined by column chromatography of Sephadex G-15. The activity of cathepsin D in the livers was markedly inhibited in rats given PS-ASF. However, the treatment of rats with PS-ASF had no effect on the hepatic lysosomal degradation of endocytosed FITC-labeled asialofetuin (FITC-ASF). Introduction of PS-ASF into the hepatocytes was followed by the immediate and time-dependent excretion of free pepstatin into the bile. Quantification of pepstatin excreted into the bile revealed that the biliary excretion route can account for the disappearance of pepstatin from the liver.     

Identification of two subpopulations of thyroid lysosomes: relation to the thyroglobulin proteolytic pathway.

Using a combination of differential centrifugation and isopycnic centrifugation in Percoll gradients, we obtained a highly purified preparation of thyroid lysosomes [Alquier, Guenin, Munari-Silem, Audebet & Rousset (1985) Biochem. J. 232, 529-537] in which we identified thyroglobulin. From this observation, we postulated that the isolated lysosome population could be composed of primary lysosomes and of secondary lysosomes resulting from the fusion of lysosomes with thyroglobulin-containing vesicles. In the present study, we have tried to characterize these lysosome populations by (a) subfractionation of purified lysosomes using iterative centrifugation on Percoll gradients and (b) by functional studies on cultured thyroid cells. Thyroglobulin analysed by soluble phase radioimmunoassay, Western blotting or immunoprecipitation was used as a marker of secondary lysosomes. The total lysosome population separated from other cell organelles on a first gradient was centrifuged on a second Percoll gradient. Resedimented lysosomes were recovered as a slightly asymmetrical peak under which the distribution patterns of acid hydrolase activities and immunoreactive thyroglobulin did not superimpose. This lysosomal material (L) was separated into two fractions: a light (thyroglobulin-enriched) fraction (L2) and a dense fraction (L1). L1 and L2 subfractions centrifuged on a third series of Percoll gradients were recovered as symmetrical peaks at buoyant densities of 1.12-1.13 and 1.08 g/ml, respectively. In each case, protein and acid hydrolase activities were superimposable. The specific activity of acid phosphatase was slightly lower in L2 than in L1. In contrast, the immunoassayable thyroglobulin content of L2 was about 4-fold higher than that of L1. The overall polypeptide composition of L, L1 and L2 analysed by polyacrylamide-gel electrophoresis was very similar, except for thyroglobulin which was more abundant in L2 than in either L or L1. The functional relationship between L1 and L2 lysosome subpopulations has been studied in cultured thyroid cells reassociated into follicles. Thyroid cells, prelabelled with 125I-iodide to generate 125I-thyroglobulin, were incubated in the absence of in the presence of inhibitors of intralysosomal proteolysis. The fate of 125I-thyroglobulin, and especially its appearance in the lysosomal compartment, was studied by Percoll gradient fractionation and immunoprecipitation. Treatment of prelabelled thyroid cells with chloroquine and leupeptin induced the accumulation of immunoprecipitable 125I-thyroglobulin into a lysosome fraction corresponding to the L2 subpopulation. In control cells, in which intralysosomal proteolysis was n