Helge Tolleshaug

Uptake and degradation of 125I-labelled asialo-fetuin by isolated rat hepatocytes.

125I-Labelled asialo-fetuin was taken up by isolated rat hepatocytes by a saturable process. Half maximum uptake was seen at about 3 - 10(-8) M asialo-fetuin. Non-parenchymal liver cells did not take up asialo-fetuin in vitro. Rate of uptake of asialo-fetuin exceeded rate of degradation at all concentrations of asialo-fetuin tested. Asialo-fetuin consequently accumulated in the cells until the extracellular supply was exhausted. Asialo-fetuin degradation could be studied without concurrent uptake by incubating cells, previously exposed to asialo-fetuin, in asialo-fetuin-free medium. Degradation, as evidenced by increase in acid-soluble radioactivity, was inhibited by NH4Cl and chloroquine. The change with time in the intracellular distribution pattern of radioactivity in cells that had been exposed to 125I-labelled asialo-fetuin for 10 min was examined by means of differential centrifugation. Initially, the radioactivity was found mostly in the microsomal fraction. 60 min after the exposure to labelled protein, the distribution pattern of radioactivity resembled that of the lysosomal enzyme beta-acetylglucosaminidase. The possibility that asialo-fetuin digestion takes place in lysosomes is discussed.

Chloroquine reduces the number of asialo-glycoprotein receptors in the hepatocyte plasma membrane

Abstract Rat hepatocytes which had been incubated with chloroquine in concentrations ranging from 0.2 to 1.0 mM showed markedly reduced ability to take up asialo-glycoproteins. Chloroquine had no effect on the actual binding of asialo-orosomucoid to the receptor on hepatocytes. Chloroquine caused a progressive reduction in the binding capacity of the plasma membrane for asialo-glycoproteins; in hepatocytes that had been exposed to 1.0 mM chloroquine for 30 min at 37°C, the binding capacity of the plasma membrane was reduced to 15 per cent of controls. The results support the idea that the asialo-glycoprotein receptors of hepatocytes are internalized regardless of binding of ligand.

The effects of colchicine and cytochalasin B on uptake and degradation of asialo-glycoproteins in isolated rat hepatocytes.

Abstract We have studied the effects of colchicine, an inhibitor of microtubular function, and cytochalasin B (CB), an inhibitor of microfilaments, on the uptake and degradation of asialo-glycoproteins in isolated rat hepatocytes. CB inhibited degradation only, while colchicine inhibited uptake as well as degradation. When the two were combined, no additive effect on degradation was found. The inhibition of uptake by colchicine could be accounted for by a reduction in the binding capacity of the plasma membrane for asialo-glycoprotein. The intracellular distribution of endocytosed asialo-glycoprotein was examined by isopycnic centrifugation in sucrose gradients. The results suggest that in cells treated with colchicine or CB, access of the endocytosed protein to the lysosomes is impeded.

Hepatic clearance of Sonazoid perfluorobutane microbubbles by Kupffer cells does not reduce the ability of liver to phagocytose or degrade albumin microspheres

This study has been performed to examine which cells are responsible for the hepatic clearance of the new ultrasound contrast agent Sonazoid and to study whether uptake of these gas microbubbles disturbs the function of the cells involved. Sonazoid was injected into rats and perfused fixed livers were studied by electron microscopy, which revealed that the Sonazoid microbubbles were exclusively internalised in Kupffer cells, i.e. by the macrophages located in the liver sinusoids, and not by parenchymal, stellate or endothelial cells. This is the first demonstration of intact phagocytosed gas microbubbles within Kupffer cells. Uptake of the Sonazoid perfluorobutane microbubbles by the Kupffer cells following injection of a dose corresponding to 20× the anticipated clinical dose for liver imaging did not result in measurable changes in the uptake and degradation of radioactively labelled albumin microspheres previously shown to be a useful indicator marker for Kupffer cell phagocytosis.

The effects of ammonium ions and chloroquine on uptake and degradation of 125I-labeled asialo-fetuin in isolated rat hepatocytes

Abstract The effects of NH 4 + and chloroquine on the uptake, the degradation and the intracellular distribution of 125 I-labeled asialo-fetuin in isolated rat hepatocytes were investigated. Both ammonium ions and chloroquine inhibited asialo-fetuin degradation; chloroquine in high concentrations reduced uptake as well. Both compounds reduced the equilibrium density of the lysosomes in sucrose gradients. Labeled asialo-fetuin did not seem to accumulate in the lysosomes of cells exposed to NH 4 + or chloroquine. The possibility that both compounds retard the fusion between lysosomes and phagosomes is discussed.

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.

Intracellular localization and degradation of asialofetuin in isolated rat hepatocytes

Analysis by isopycnic and differential centrifuging of the intracellular distribution of radioactivity following uptake of 125I-labelled asialofetuin by isolated rat hepatocytes showed that during incubations up to 1 h, most of the radioactivity was associated with structures which had a subcellular distribution pattern different from both the lysosomes and the plasma membrane. The latter two organelles were followed by means of enzyme markers. Ca2+ is necessary for the binding of asialofetuin to the plasma membrane, and it was also possible to differentiate between asialofetuin bound to the plasma membrane and that contained in intracellular structures by removing Ca2+ from the medium (by EGTA). Such experiments showed that asialofetuin became rapidly internalized. Practically all the labelled protein was located intracellularly in cells that had been incubated with asialofetuin for more than 30 min. When incubations were carried out for more than 1 h a peak appeared in the radioactivity distribution in the same place as the peak of activity of lysosomal marker enzymes. However, degradation of asialofetuin takes place in the lysosomes and this starts before the labelled protein can be found in the lysosomal fractions. Our data suggest that the rate-determining step in the cellular handling of asialofetuin is the transport of endocytized protein from the endocytic vesicles to the lysosomes.

Binding and internalization of asialo-glycoproteins by isolated rat hepatocytes.

Abstract 1. 1. Binding of asialo-fetuin and asialo-orosomucoid to (and their dissociation from) the receptor on isolated hepatocytes has been studied at 10°C. The reaction between the asialo-glycoproteins and the receptor was found to be reversible, even though the approach to equilibrium was very slow. The rate constant (k1) for the association reaction was 7.4·104M−1sec−1 for asialo-orosomucoid and 2.9 · 104 M−1 sec−1 for asialo-fetuin. The dissociation rate constant (k−1) was the same for both proteins, 6·10−6sec−1. At 10°C the rate constant for internalization was 3.1 · 10−5sec−1. 2. 2. The rate constant for internalization was measured at 37°C, using initial concentrations of 1 nM and 23 nM asialo-orosomucoid, and 0.03–0.5 nM asialo-fetuin. The results of both sets of measurements agreed very well, giving a rate constant of 3.7·10−3 sec−1, corresponding to a half-life of 3 min for the occupied receptor on the cell surface. This result indicates that cooperative effects between occupied receptors do not affect the internalization of either asialo-orosomucoid or asialo-fetuin. 3. 3. The uptake of asialo-orosomucoid was studied in cells in which protein synthesis had been blocked with cycloheximide. Even though the amount of asialo-orosomucoid which was taken up into each cell corresponded to at least twice the number of receptor molecules, no effect of cycloheximide on the uptake reaction was observed. This proved that the receptors are not degraded in the lysosomes along with the asialo-glycoprotein.

The effect of leupeptin on intracellular digestion of asialofetuin in rat hepatocytes

Abstract The effect of leupeptin on the intracellular distribution of asialofetuin, endocytosed by isolated rat hepatocytes, was studied. By means of sucrose gradient centrifugation it was found that leupeptin led to accumulation of undegraded 125I-labeled asialofetuin both in lysosomes and in an organelle of lower density (probably an endocytic vesicle). To decide whether the protease inhibitor interfered with the uptake of asialofetuin into lysosomes we studied its effect on the intracellular distribution of [14C]sucrose-asialofetuin. Acid-soluble radioactivity formed from [14C]sucrose-asialofetuin is trapped within the lysosomes and the rate of uptake of this ligand in the lysosomes can therefore be studied. Using [14C]sucrose-asialofetuin it was found that leupeptin, in addition to inhibiting proteolysis inside the lysosomes, retards the transport of asialofetuin into these organelles. Reduced uptake of asialofetuin into lysosomes was seen only after incubating the cells with leupeptin for more than about 30 min. The leupeptin effect on the transport of asialofetuin may therefore be secondary to accumulation of undegraded substrates inside the lysosomes.

Uptake and degradation of bovine testes β-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.