Receptor-mediated endocytosis of insulin by cultured endothelial cells.

Label-fracture immunochemistry and pre-embedding indirect immunocytochemistry were applied to investigate insulin uptake by endothelial cells. Freeze fracture replicas showed that a small percentage of native insulin receptors are associated with non-coated pits (4%) and coated pits (2%). After warming, receptor bound insulin became increasingly associated with such endocytotic vesicles. After 2 min the percentage of detectable insulin associated with non-coated and coated pits increased to 16% and 8%, respectively. Pre-embedding immunocytochemical localization of insulin gave results consistent with those obtained from the label-fracture studies. Both non-coated and coated vesicles appeared labelled after 5 min of warming. Non-coated vesicles contained 25% of the cell associated insulin while 9% was associated with coated pits and vesicles. After 10 min of warming, 9% of label was located in non-coated vesicles and 7% in coated vesicles. A large proportion (29%) of the label was found in tubular-vesicular endosomes at this time. After 15 min of warming, 30% of the remaining cell-associated gold label was found in multivesicular bodies. These experiments demonstrate that insulin uptake by endothelium is mediated by both coated and non-coated vesicles and that, once internalized, insulin is routed through endosomal pathways that primarily result in transcytosis.     

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.     

Fluid endocytosis in isolated rat parenchymal and non-parenchymal liver cells

Fluid endocytosis in rat liver parenchymal (hepatocytes) and non-parenchymal cells was studied by measuring uptake of [¹²⁵I]polyvinylpyrrolidone (PVP). Radioactive sucrose preparations were also tested but turned out to be unsuitable because of impurities of radioactive glucose and fructose. Fluid endocytosis was temperature dependent without any transition temperature. The rate of endocytosis was inhibited by inhibitors of the glycolytic and the respiratory pathway. Colchicine, but not cytochalasin B, inhibited the uptake of [¹²⁵I]PVP in hepatocytes. Therefore, intact microtubuli, but not microfilaments may be required for normal rate of fluid endocytosis in hepatocytes. Colchicine reduced the rate of fluid endocytosis in the non-parenchymal liver cells. Subcellular fractionation by isopycnic centrifugation in sucrose gradients indicated that [¹²⁵I]PVP taken up by the hepatocytes accumulated in the lysosomes. The rate of uptake expressed as volume of fluid internalized per unit time (endocytic index) was calculated to 0.08 μl/h/10⁶ cells for hepatocytes and 0.07 μl/h/10⁶ cells for non-parenchymal liver cells.     

Receptor-mediated endocytosis of aldehyde-modified proteins by sinusoidal liver cells

Formaldehyde-treated serum albumin (f-Alb) is known to be endocytosed by sinusoidal lever cells via a receptor-mediated mechanism. The receptor purified from rat livers exhibited a molecular weight of 125,000, consisting of two glycoprotein components with molecular weights of 53,000 and 30,000, respectively. Experiments using antireceptor antibody demonstrated that the f-Alb receptor is distinct from the receptor that mediates endocytotic uptake of acetylated low-density lipoprotein, but they share a common property of being inhibited by several polyanions, suggesting that polyanion-sensitivity might play an important role in the scavenger function of simusoidal liver cells. Studies on the ligand specificity of this receptor revealed that a covalent modification by formaldehyde of a limited number of lysine residues in albumin has led to the formation of a receptor-recognition domain(s). Furthermore, in addition to formaldehyde, the ligand activity was also generated with albumin modified by other aliphatic aldehydes, such as glycoaldehyde and glyceraldehyde. This phenomenon was extended to several proteins other than albumin. These data suggest therefore that the f-Alb receptor originally described as being specific for albumin modified by formaldehyde may play a general role as a scavenger receptor for aldehyde-modified proteins.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

The mechanism of pantothenate transport by rat liver parenchymal cells in primary culture

The mechanism of pantothenate transport across the plasma membrane was investigated with initial velocity studies of [14C]pantothenate uptake and efflux in rat liver parenchymal cells maintained in primary culture. At 116 mM sodium, double-reciprocal plots of the initial velocity of uptake versus [pantothenate] were linear from 0.3 to 36.5 microM pantothenate and gave an apparent Km,pant of 11 +/- 2 microM. The rate of pantothenate uptake at 0 [sodium] was about 14% of the rate at 116 mM sodium, and the reciprocal of the apparent Km,pant was a linear function of [sodium]. Vmax obtained by extrapolation to infinite [pantothenate] was independent of [sodium]. Ouabain, gramicidin D, cyanide, azide, and 2,4-dinitrophenol inhibited uptake, but preloading cells with pantothenate did not. Pantothenate derivatives or carboxylic acids were only weak inhibitors of uptake. Efflux was measured in cells preloaded with [14C]pantothenate. The apparent Km for efflux was 85 +/- 29 microM, and the rate of efflux was unaffected by addition of pantothenate, sodium, ouabain, gramicidin D, or 2,4-dinitrophenol to the external medium. These features are consistent with a mechanism for pantothenate transport in which sodium and pantothenate are cotransported in a 1:1 ratio on a carrier highly specific for pantothenate; sodium decreases the apparent Km for pantothenate, and a sodium-carrier complex forms only on the intracellular side of the membrane.     

Receptor-mediated endocytosis of enterokinase by rat liver. Preliminary characterisation of low-density endosomes

The endocytosis of enterokinase by rat hepatocytes has been studied both in a perfused liver system and in the intact, anaesthetised animal. 10 min after administration of the enzyme, only 70% of the activity was cleared by the perfused liver, whereas clearance was total in the intact animal. In both cases, about 85% of the internalised enzyme co-purified with the smooth microsomes and virtually all (more than 90%) of the catalytic activity was latent and could only be detected in the presence of detergent. After 10 min, 22.5% of the activity remained with the sinusoidal plasma membrane in the case of the perfused liver, while for the intact animal this figure was only 10%, confirming the more efficient clearance of enterokinase in the intact animal. Further subcellular fractionation showed that in the anaesthetised animal 8% of the internalised enzyme was associated with a low-density Golgi-like endosomal compartment (prepared from the mitochondrial pellet), whereas the corresponding value for the perfused liver was only 2.5%. Enterokinase specific activity was also up to 50-times greater in the low-density endosomes prepared from the intact animal. A second low-density Golgi-like compartment (purified from the smooth microsomes) also contained latent enterokinase, which together with the endosomes derived from the mitochondria accounted for 20% of the total enterokinase internalised by the liver 10 min after its administration to the intact animal. The passage of enterokinase through these two low-density compartments was shown not to be synchronous with its passage through the peripheral (sinusoidal membrane) and internal endosomes (smooth microsomes). There were qualitative differences in marker enzymes and polypeptide composition between the mitochondria and microsome-derived low-density endosomes. The sub-fractionation of low-density fractions on shallow sucrose gradients revealed a complex enzyme and polypeptide heterogeneity both between and within fractions. There was an apparent density-dependent separation of enterokinase from galactosyltransferase and the asialoglycoprotein receptor which was coincident with marked changes in the polypeptide composition of the endosomal membranes, particularly in the 30-45 kDa range.     

Receptor-mediated endocytosis of transferrin by Sertoli cells of the rat

Binding of 125I-transferrin (125I-Tf) to the plasma membrane of Sertoli cells and its endocytosis were analyzed by means of light- and electron-microscope quantitative radioautography. Five minutes after 125I-Tf was injected into the interstitial space of the testis, a strong labeling of the basal aspect of the seminiferous epithelium was observed in light-microscope radioautographs. Injection of the same dose of 125I-Tf plus a 200-fold excess of cold transferrin resulted in a marked diminution of the radioautographic reaction, indicating that the initial strong labeling with radiolabeled transferrin was specific. These results were consistent with the localization of immunoreactive fluorescence of transferrin receptor at the base of the seminiferous epithelium. In electron-microscope radioautographs of tubules collected at 5 min after injection, the membrane of Sertoli cells facing the basement membrane was well labeled with 125I-Tf. At 15 and 30 min, the plasma membrane was less intensely labeled, but the silver grains were then seen overlying multivesicular bodies with an electron-lucent matrix, identified as endosomes. This population of endosomes was always seen at a short distance from the basal membrane of Sertoli cells. At 90 min, no more labeling of the plasma membrane, endosomes, or any other cytoplasmic component was observed. Isolated seminiferous tubules and Sertoli cells labeled with 125I-Tf at 4 degrees C were rinsed and reincubated in a label-free medium at 37 degrees C for various periods of time from 5 to 90 min. A radioactive protein precipitated by trichloroacetic acid, presumably intact transferrin, was released from the tubules into the incubating medium; when measured, it was found to increase rapidly from 5 to 45 min and stabilize thereafter. These results suggest that transferrin was internalized by receptor-mediated endocytosis, reached endosomes, and then was released to the extratubular space. When native ferritin (NF), a tracer for fluid-phase endocytosis, was infused within the lumen of seminiferous tubules and 125I-Tf was simultaneously injected into the interstitial space, both markers rapidly reached different populations of endosomes. Endosomes labeled with NF, scattered throughout the cytoplasm, evolved with time into dense multivesicular bodies and secondary lysosomes, whereas radiolabeled transferrin reached only the endosomes located in the basal cytoplasm of Sertoli cells. The latter thus appeared to be principally involved in the uptake and recycling of transferrin.     

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.     

Effect of colchicine on the intracellular transport of secretory proteins in rat liver parenchymal cells. Immunocytochemical observations

We studied the effects of colchicine on the intracellular transport of secretory proteins in rat liver parenchymal cells using the direct immunoenzyme technique. Livers were perfusion-fixed 0.5, 1, and 2 h after injection of colchicine. Vibratome sections of the fixed liver were stained using peroxidase-conjugated Fab' of anti-albumin or anti-fibrinogen. By light microscopy, reaction deposits showing albumin and fibrinogen were observed in the cytoplasmic granules of hepatocytes. Such stained granules decreased 30 min after injection, but later increased gradually and crowded in the cytoplasm. The Golgi complex stained for the proteins decreased after 30 min but increased in the juxtanuclear region after 60 min. The analysis of serial sections showed that colchicine severely disturbed the spatial relationship between the Golgi apparatus and the bile canaliculus. We obtained similar results by electron microscopy; a positive reaction for albumin and fibrinogen was observed in a small number of the cytoplasmic granules after 30 min. After 1 h of treatment, most of the Golgi complexes were fragmented and lost their stacked cisternae. However, they reappeared accompanied with vacuolated cisternae and secretory granules, which were partially stained for albumin and fibrinogen. After 2 h, the secretory granules positive for both proteins accumulated further. Some of them lined a long the plasma membrane, and others made a cluster in the cytoplasm. The profiles showing exocytosis were very rarely seen. These results showed that in the first 30 min, colchicine primarily disturbs partially the Golgi assembly but does not affect the post Golgi secretory pathway much. Later, the drug affects both the post Golgi pathway and the Golgi assembly, and it causes a marked accumulation of secretory granules.     

Fluid phase endocytosis of [125I]iodixanol in rat liver parenchymal, endothelial and Kupffer cells

Endocytosis of [125I]iodixanol was studied in vivo and in vitro in rat liver cells to determine fluid phase endocytic activity in different liver cells (hepatocytes, Kupffer cells and endothelial cells). The Kupffer cells were more active in the uptake of [l25I]iodixanol than parenchymal cells or endothelial cells. Inhibition of endocytic uptake via clathrin-coated pits (by potassium depletion and hypertonic medium) reduced uptake of [125I]iodixanol much more in Kupffer cells and endothelial cells than in hepatocytes. To gain further information about the importance of clathrin-mediated fluid phase endocytosis, the expression of proteins known to be components of the endocytic machinery was investigated. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, endothelial cells and Kupffer cells were found to express approximately fourfold more rab4, rab5 and rab7 than parenchymal cells, while clathrin was expressed at a higher level in endothelial cells than in Kupffer cells and hepatocytes. Using electron microscopy it was shown that liver endothelial cells contained approximately twice as many coated pits per membrane unit than the parenchymal and Kupffer cells, thus confirming the immunoblotting results concerning clathrin expression. Electron microscopy on isolated liver cells following fluid phase uptake of horseradish peroxidase (HRP) showed that HRP-containing organelles had a different morphology in the different cell types: In the liver endothelial cells HRP was in small, tubular endosomes, while in Kupffer cells HRP was mainly found in larger structures, reminiscent of macropinosomes. Parenchymal cells contained HRP in small vacuolar endosomes with a punctuated distribution. In conclusion, we find that the Kupffer cells and the endothelial cells have a higher pinocytic activity than the hepatocytes. The hepatocytes do, however, account for most of the total hepatic uptake. The fluid phase endocytosis in liver endothelial cells depends mainly on clathrin-mediated endocytosis, while the parenchymal cells have additional clathrin-independent mechanisms that may play an important role in the uptake of plasma membrane components. In the Kupffer cells the major uptake of fluid phase markers seems to take place via a macropinocytic mechanism.     

Effects of chloroquine on lysosomes and endocytosis by liver cells in vivo.

1. Chloroquine accumulation in rat liver after a single and repeated drug administration and lysosomal changes resembling some symptoms of lysosomal storage diseases were observed. 2. Repeated chloroquine treatment of rats resulted in increased activity of liver lysosomal enzymes acid phosphatase and beta-galactosidase and a significant enhancement of the activities of cathepsin D and cysteine proteinases were found. 3. No changes in the activity of liver macrophages (as assessed by the colloidal carbon clearance test) or in fluid-phase endocytosis of the marker 125I-polyvinyl-pyrrolidone by hepatocytes in vivo were found.     

Zinc uptake by isolated rat liver parenchymal cells.

Primary cultures of rat liver parenchymal cells maintained as a monolayer in serum-free culture medium were used to investigate the characteristics of zinc accumulation in vitro. Liver parenchymal cells accumulated zinc by a temperature-dependent, saturable process that was inhibited by cyanide, azide, oligomycin, N-ethylmaleimide and iodoacetamide. Cadmium reversibly inhibited zinc accumulation in both serum-free and serum-containing media. Gel filtration chromatographic studies showed that recently accumulated intracellular zinc was present as a low molecular weight complex smaller than metallothionein, the zinc storage protein, but larger than individual amino acids. The quantity of zinc accumulated was affected by preincubation of the cells with various hor?ONES. Dexamethasone, prednisone and prednisolone each increased zinc uptake by 40--50% when either insulin or glucagon was also present. Hydrocortisone, cortisone and sex steroids did not influence zinc accumulation. Removal of the polypeptide hormones from the medium abolished the stimulatory effect of the synthetic glucocorticoid steroid hormones on zinc accumulation.     

Receptor-mediated endocytosis of testicular transferrin by germinal cells of the rat testis

Summary The present study examines events of the Sertoli cell iron delivery pathway following the secretion of diferric testicular transferrin (tTf) into the adluminal compartment of the rat seminiferous epithelium. The unidirectional secretion of tTf by Sertoli cells was verified, in vivo, and it was shown that this protein is internalized by adluminal germ cells. It was further determined by Scatchard analysis that this internalization was mediated by high affinity transferrin binding sites on the surface of round spermatids, numbering 1453/cell and displaying a K_d=0.6×10^-9 M. Northern blot analysis of RNA isolated from adluminal germ cells, namely spermatocytes, round spermatids and elongating spermatids, indicated that these cells expressed Tf receptor mRNA and ferritin mRNA in levels inversely related to their stage of maturation. Finally it was determined that following binding and internalization in round spermatids, Tf became associated with the endosomal compartment and was recycled back to the cell surface. This study illustrates the immediate fate of tTf once it is secreted by the Sertoli cell. Thus, diferric tTf binds of Tf receptor on the surface of adluminal germ cells, is internalized by receptor-mediated endocytosis and the apo Tf-Tf receptor complex is recycled back to the cell surface where apotTf is released into the adluminal fluid.     

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.     

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.     

Rapid transport of fatty acids from rat liver endothelial to parenchymal cells after uptake of cholesteryl ester-labeled acetylated LDL

Acetylated low-density lipoprotein (acetyl-LDL) radiolabeled in the oleate moiety of cholesteryloleate was injected into rats. Isolation of the various liver cell types at different times after acetyl-LDL injection by a low-temperature procedure allowed the intrahepatic metabolism of the oleate moiety to be followed in vivo. The cholesteryloleate radioactivity is rapidly cleared from the circulation and at 5 min after injection recovered into parenchymal and endothelial liver cells, mainly as cholesteryloleate ester. At longer time intervals after injection, the amount of cholesteryl esters associated with the endothelial cells was sharply decreased and the [14C]oleate was redistributed within the liver and mainly recovered in the parenchymal cells. The cholesteryl ester initially directly taken up by the parenchymal cells was also rapidly hydrolysed but, in contrast to the endothelial cells, the [14C]oleate remained inside the cells and was incorporated into triacylglycerols and phospholipids. The 14C radioactivity in parenchymal cells taken up between 5 and 30 min after injection of the cholesteryl [14C]oleate-labeled acetyl-LDL (transported as oleate from endothelial cells), followed a similar metabolic route as the amount which was directly associated to parenchymal cells. The data indicate that the liver and, in particular, the liver endothelial cell has the full capacity to rapidly catabolize modified lipoproteins. In this catabolism, the liver functions as an integrated organ in which fatty acids, formed from cholesteryl esters in endothelial cells, are rapidly transported to parenchymal cells, indicating the concept of metabolic cooperation between the various liver cell types.     

Morphological studies on endocytosis of chondroitin sulphate proteoglycan by rat liver endothelial cells

Summary Endocytosis via the hyaluronic acid/chondroitin sulphate receptor of rat liver endothelial cells was studied ultrastructurally, by use of a probe consisting of chondroitin sulphate proteoglycan attached to 15-nm gold particles. The probe bound to the surface of the cells exclusively in coated regions of the plasma membrane. Internalization at 37° C took place in less than one minute during which time interval the bound probe was transferred to coated vesicles. Further transfer to lysosomes was delayed in association with an accumulation of probes in a prelysosomal compartment consisting of large vacuoles in which probes lined the inner aspect of the membrane. Transport to lysosomes occurred only after a lag phase of at least 40–60 min at 37° C.Abbreviations CS chondroitin sulphate - CSPG chondroitin sulphate proteoglycan - CSPG-Au CSPG-gold complex - EM electronmicroscopical or electron microscopy - HA hyaluronic acid - KC Kuppfer cells - LEC liver endothelial cells - PC parenchymal cells - RES reticuloendothelial system     

Intracellular transport of endocytosed proteins in rat liver endothelial cells.

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.     

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.