Wortmannin-sensitive trafficking steps in the endocytic pathway in rat liver endothelial cells.

Liver endothelial cells (LECs) play an important homoeostatic role by removing potentially harmful macromolecules from blood. The extremely efficient endocytosis in LECs makes these cells an interesting model for the study of the involvement of phosphoinositides in the different steps of the endocytic process. In the present investigation we have studied the effect of wortmannin, an inhibitor of phosphatidylinositol kinases, on uptake, recycling and intracellular transport of (125)I-labelled ovalbumin, which is taken up in LECs via mannose-receptor-mediated endocytosis. Wortmannin was found to inhibit both uptake and degradation of ovalbumin. Further studies indicated that the reduced uptake via the mannose receptor was due both to a reduction of the number of surface receptors and a reduction in the rate of receptor-ligand internalization. Transport of ligand from endosomes to lysosomes was prevented, leading to increased recycling of internalized ligand. Wortmannin treatment released the Rab5 effector EEA1 from the endosomes and caused reduced size of early endosomes.

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 inhibitors of the vacuolar proton pump on hepatic heterophagy and autophagy.

Bafilomycin A(1) (BAF) and concanamycin A (ConcA) are selective inhibitors of the H(+)-ATPases of the vacuolar system. We have examined the effects of these inhibitors on different steps in endocytic pathways in rat hepatocytes, using [(125)I]tyramine-cellobiose-labeled asialoorosomucoid ([(125)I]TC-AOM) and [(125)I]tyramine-cellobiose-labeled bovine serum albumin ([(125)I]TC-BSA) as probes for respectively receptor-mediated endocytosis and pinocytosis (here defined as fluid phase endocytosis). The effects of BAF and ConcA were in principle identical, although ConcA was more effective than BAF. The main findings were as follows. (1) BAF/ConcA reduced the rate of uptake of both [(125)I]TC-AOM and [(125)I]TC-BSA. The reduced uptake of [(125)I]TC-AOM was partly due to a redistribution of the asialoglycoprotein receptors (ASGPR) such that the number of surface receptors was reduced approximately 40% without a change in the total number of receptors. (2) BAF/ConcA at the same time increased retroendocytosis (recycling) of both probes. The increased recycling of the ligand ([(125)I]TC-AOM) is partly a consequence of the enhanced pH in endosomes, which prevents dissociation of ligand. (3) It was furthermore found that the ligand remained bound to the receptor in presence of BAF/ConcA and that the total amount of ligand molecules internalized in BAF/ConcA-treated cells was only slightly in excess of the total number of receptors. These data indicate that reduced pH in endosomes is the prime cause of receptor inactivation and release of ligand in early endosomes. (4) Subcellular fractionation experiments showed that [(125)I]TC-AOM remained in early endosomes, well separated from lysosomes in sucrose gradients. The fluid phase marker, [(125)I]TC-BSA, on the other hand, seemed to reach a later endosome in the BAF/ConcA-treated cells. This organelle coincided with lysosomes in the gradient, but hypotonic medium was found to selectively release a lysosomal enzyme (beta-acetylglucosaminidase), indicating that even [(125)I]TC-BSA remained in a prelysosomal compartment in the BAF/ConcA-treated cells. (5) Electron microscopy using horseradish peroxidase (HRP) as a fluid phase marker verified that BAF/ConcA inhibited transfer of material from late endosomes ('multivesicular bodies'). (6) BAF/ConcA led to accumulation of lactate dehydrogenase (LDH) in autophagic vacuoles, but although the drugs partly inhibited fusion between autophagosomes and lysosomes a number of autolysosomes was formed in the presence of BAF/ConcA. This observation explains the reduced buoyant density of lysosomes (revealed in sucrose density gradients). In conclusion, BAF/ConcA inhibit transfer of endocytosed material from late endosomes to lysosomes, but do not at the same time prevent fusion between autophagosomes and lysosomes.

Internalization and stepwise degradation of heparan sulfate proteoglycans in rat hepatocytes.

Intracellular transport and degradation of membrane anchored heparan sulfate proteoglycans (HSPGs) were studied in cultured rat hepatocytes labeled with [35S]sulfate and [3H]glucosamine. Pulse chase experiments showed that membrane anchored HSPGs were constitutively transported to the cell surface after completion of polymerization and modification of the glycosaminoglycan chains in the Golgi apparatus. The intact HSPGs had a relatively short residence time at the cell surface and in non-degrading compartments (T(1/2) approximately 2-3 h), while [35S]sulfate labeled degradation products were found in lysosomes, and to a lesser extent in late endosomes. These degradation products which were free heparan sulfate chains with little or no protein covalently attached, were approximately half the size of the original glycosaminoglycan chains and were the only degradation intermediate found in the course of HSPG catabolism in these cells. In cells incubated in the presence of the microtubule perturbant vinblastine, or in the presence of the vacuolar ATPase inhibitor bafilomycin A1, and in cells incubated at 19 degrees C, the endocytosed HSPGs were retained in endosomes and no degradation products were detected. Disruption of lysosomes with glycyl-phenylalanine 2-naphthylamide (GPN) revealed a GPN resistant degradative compartment with both intact and partially degraded HSPGs. This compartment probably corresponds to late endosomes. Treatment of hepatocytes with the thiol protease inhibitor leupeptin inhibited the final degradation of the protein moiety of the HSPGs. The protein portion seems to be degraded completely before the glycosaminoglycan chains are cleaved. The degradation of the glycosaminoglycan chains is rapid and complete with one observable intermediate.

Distribution of liposome-encapsulated iodixanol in rat liver cells.

Distribution of liposome-encapsulated [(125)I]iodixanol in different types of liver cells following intravenous injection was studied in rats. The data showed that liposome-encapsulated [(125)I]iodixanol was rapidly taken up by the liver; after 15 min, radioactivity corresponding to nearly 25% of the injected radioactivity could be recovered therein. After 4 hr, approximately 60% of the injected radioactivity was in the liver. One week after injection, nearly 30% of the encapsulated radioactivity could still be recovered in the liver. Liposome-encapsulated [(125)I]iodixanol was taken up both by hepatocytes and the Kupffer cells. On a per cell basis, the uptake of liposome-encapsulated [(125)I]iodixanol in Kupffer cells was more than 10-fold greater than that in hepatocytes, while the contribution of liver endothelial cells to uptake was negligible. Osmotic protection studies showed that iodixanol does not readily diffuse across lysosomal membranes, indicating that loss of iodixanol from the liver probably occurred by recycling rather than by diffusion across phagolysosomal and plasma membranes.

Endocytosed ricin and asialoorosomucoid follow different intracellular pathways in hepatocytes.

Earlier studies have suggested that fluid phase endocytosis in rat hepatocytes takes place via a clathrin-independent mechanism [1,2]. This observation suggests that a relatively large amount of plasma membrane outside coated pits may be involved in hepatic endocytosis. Ricin, which binds to galactose residues on glycoproteins and glycolipids, has, in this report, been used as a general marker for the plasma membrane of hepatocytes. The endocytosis of ricin was compared with that of asialoorosomucoid (AOM) which is taken up exclusively via clathrin-coated pits. Hypertonic medium has been shown to inhibit uptake via coated pits more effectively than clathrin-independent uptake [3-5]. It was found, in this study, that the addition of 100 mM sucrose to the incubation medium inhibited the uptake of 125I-tyramine-cellobiose-asialoorosomucoid (125I-TC-AOM) more extensively than that of 125I-tyramine-cellobiose-ricin (125I-TC-ricin), compatible with the notion that the two probes are internalised via different mechanisms. Subcellular fractionation experiments indicated that 125I-TC-ricin entered a denser endocytic organelle than that receiving 125I-TC-AOM. To determine whether the separation of the two probes was due to a different transport kinetics (i.e. that 125I-TC-ricin is transported more rapidly to a later, denser compartment than 125I-TC-AOM) the cells were incubated at 18 degreesC to allow a slower internalisation/transport of the labelled probes. The results obtained showed, again, that the early endosomes containing 125I-TC-ricin were significantly denser than those containing 125I-TC-AOM. We also employed the horseradish peroxidase (HRP)-diaminobenzidine (DAB) density shift technique of Courtoy et al. [6] to determine whether 125I-TC-ricin and 125I-TC-AOM were in separate endosomes early after their uptake. The results showed that early endosomes containing 125I-TC-AOM were density shifted whereas those containing 125I-TC-ricin were unaffected by the density shift procedure. The use of probes labelled with 125I-TC allowed us to identify compartments involved in the degradation of 125I-TC-AOM and 125I-TC-ricin, by measuring acid soluble radioactivities in the gradient fractions. It was found that 125I-TC-ricin was degraded mainly in endosomes, whereas 125I-TC-AOM, as expected, was degraded mainly in lysosomes.

Density shift of endocytic vesicles induced by uptake of colloidal gold particles in rat hepatocytes.

The aim of this study was to demonstrate the influence of endocytosed asialoorosomucoid gold complexes (ASOR-Au10, 10 = diameter of gold particles) on the density of endocytic compartments in rat hepatocytes when fractionated on linear sucrose gradients. We show that by loading hepatocytes with ASOR-Au10 for different time intervals, organelles containing gold particles are denser than organelles containing unconjugated asialoorosomucoid (ASOR). This density shift was observed in endosomes, which were loaded with a short pulse (15 minutes) of ASOR- Au10. Also lysosomes, loaded with ASOR-Au10 for 120 minutes at 37 degrees C, display a similar density shift.

Involvement of early and late lysosomes in the degradation of mannosylated ligands by rat liver endothelial cells.

The intracellular transport and degradation of endocytosed mannosylated albumin (Man-BSA) was studied in cell cultures of rat liver endothelial cells by subcellular fractionation, fluorescence microscopy, and electron microscopy. The ligand used for subcellular fractionation experiments was labeled with 125I-labeled tyramine cellobiose or 131I-labeled tyramine cellobiose. The labeled degradation products are trapped in the degradative compartments and may therefore serve as markers for these compartments. Cell fractionation was performed using Nycodenz gradients. The cell fractionation experiments demonstrated that the ligand sequentially occupied three compartments of increasing density. After 15 min it was mainly found in large cisternal organelles that banded in the gradient at about 1.09 g/ml. These organelles were rab5 positive and showed a peripher distribution in the fluorescence microscope. Degradation of ligand started after 30-60 min and this coincided with its transfer to a electron lucent vesicle with a density of 1.12 g/ml. After > 1 h, degradation products started to accumulate in perinuclear, electron-dense lysosomes that banded in the gradient at 1.15 g/ml. The density distribution of lysosomal beta-acetylglucosaminidase coincided with the densest organelle. The results obtained show that the degradation of ligand takes place sequentially in two types of lysosomes. The early lysosome is an electronlucent vesicle of low density, whereas the terminal lysosome is an electron-dense organelle with higher density and a more perinuclear distribution. The main degradation of the ligand takes place in the early lysosome. The transfer of ligand and degradation products from the early to the late lysosome is slow. Texas red-labeled ovalbumin (OVA) coincided with lysosomes labeled with OVA-Bodipy 24 h in advance only after 4-6 h.

Characterization of two distinct pathways of endocytosis of ricin by rat liver endothelial cells.

We have studied the characteristics of internalization and intracellular transport of ricin via two distinct pathways in rat liver endothelial cells (EC), i.e., via binding to mannose receptors and surface galactosyl-residues, respectively. Treatments that inhibit endocytosis from coated pits, i.e., hyperosmolarity and acidification of the cytoplasm, decreased uptake via mannose receptors much more than uptake via galactosyl-residues, indicating that mannose receptors are largely internalized from coated pits, whereas internalization via galactosyl-residues is to a significant extent independent of coated pits. Uptake of ricin via mannose receptors was strongly inhibited by NH4Cl and monensin, and accordingly, NH4Cl protected the cells against ricin intoxication via mannose receptors. On the other hand, uptake via galactosyl-residues was not significantly inhibited by NH4Cl or monensin, and NH4Cl even sensitized the cells to intoxication via this pathway. Brefeldin A, which did not affect ricin uptake, protected the cells against ricin intoxication via either pathway. Protein synthesis in the EC was efficiently inhibited by ricin, even after very short periods of uptake at low ricin concentrations. The onset of protein synthesis inhibition was more rapid upon internalization of ricin via mannose receptors than via galactosyl-residues. Also, ricin internalized via mannose receptors was more efficiently transported from endosomes to lysosomes than ricin internalized via galactosyl-residues. Partial blocking of the galactosyl-binding sites of ricin caused a reduction in the extent of recycling of ricin from endosomes to the cell surface (retroendocytosis), indicating that binding of ricin to membrane galactosyl-residues, which is relatively stable at the slightly acidic pH of endosomes, is an important determinant of the intracellular handling of ricin. We suggest that the observed difference in the transport from endosomes to lysosomes between the two internalization pathways is related to the different stability of the two binding mechanisms at endosomal pH.