G M Kindberg

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.

Intracellular transport of asialoglycoproteins in rat hepatocytes. Evidence for two subpopulations of lysosomes.

The intracellular transport and degradation of asialoorosomucoid (AOM) in isolated rat hepatocytes was studied by means of subcellular fractionation in Nycodenz gradients. The asialoglycoprotein was labelled by covalent attachment of a radioiodinated tyramine-cellobiose adduct ( [125I]TC) which leads to labelled degradation products being trapped intracellularly and thus serving as markers for the degradative organelles. The ligand was initially (1 min) in a slowly sedimenting (small) vesicle and subsequently in larger endosomes. Acid-soluble, radioactive degradation products were first found in a relatively light lysosome whose distribution coincided in the gradient with that of the larger endosome. Later (30 min) degradation products were found in denser lysosomes which banded in the same region of the gradient as the lysosomal enzyme, beta-acetylglucosaminidase. Colchicine, monensin and leupeptin all inhibited degradation of [125I]tyramine-cellobiose asialoorosomucoid ( [125I]TC-AOM) and reduced the formation of degradation products in both the light and the dense lysosomes. In presence of monensin and colchicine no undegraded ligand was seen in the dense lysosome, suggesting that uptake in these vesicles was inhibited. Leupeptin allowed accumulation of undegraded ligand in the dense lysosome. Therefore, transfer from light to dense lysosomes is not dependent on degradation as such. In the presence of monensin two peaks of undegraded ligand were found in the gradients. It seems possible that in the monensin-sensitive endosomes, dissociation of the ligand-receptor complex is inhibited, allowing ligand to recycle with the receptors in small vesicles.

Separation of endocytic vesicles in Nycodenz gradients

The endocytosis of 125I-labeled asialofetuin by rat hepatocytes was studied using Nycodenz/sucrose gradients. It was shown in pulse chase experiments that the ligand endocytosed initially (after 1/2 to 1 min) was in small, slow-sedimenting vesicles of similar sizes. The vesicles containing the ligand increased in size, and after about 2.5 min 20-30% of the ligand was recovered in larger, faster-sedimenting vesicles. After 15 min almost all internalized ligand was recovered in the fast-sedimenting vesicles. The initial, small endocytic vesicles and the later, larger endocytic vesicles have similar buoyant densities; the maturation of the endosomes can only be revealed by rate sedimentation, not by isopycnic centrifugation. Dissociation of ligand from receptor was found to occur in the larger, faster-sedimenting vesicles. The presence of ammonia inhibited the increase in size of the ligand-containing endosomes. The methods employed here offer the possibility of obtaining endocytic vesicles at various stage of their development for further studies.

Renal Uptake and Degradation of Trapped-Label Insulin

In order to study the kinetics of insulin degradation in the kidneys and liver, insulin was labelled by a trapped-label procedure and injected into rats. In contrast to conventional 125I-insulin, the trapped-label preparation allows quantitative measurements of the extent of degradation in vivo because the final degradation products do not leave the cells. One hour after injection, the amount of radioactivity in the kidneys from a trace dose of trapped-label insulin was 10 times higher that from conventionally labelled insulin; over 80% of the increase was due to low molecular weight degradation products which were retained in the kidneys. The amount of acid-precipitable radioactivity in the blood was the same for both labelled preparations, indicating that their rates of clearance were similar. In the kidney, we detected no degradation products of molecular weight intermediate between intact insulin and the end products of proteolysis. After 2 h, 33% of the injected dose remained in the kidneys and only 13% in the liver. Over 80% of the renal radioactivity was sedimentable in an isotonic density gradient, indicating that intact insulin, as well as degradation products in the cells, were enclosed within membrane-bound vesicles.

Clearance of purified human liver γ-glutamyltransferase after intravenous injection in the rat

Abstract The clearance of γ-glutamyltransferase was studied by injecting the purified human liver enzyme intravenously in the rat. The results show a biphasic clearance, with a rapid initial rate of removal. The initial uptake is more rapid for neuraminidase-treated GT. Liver accounts for the bulk organ uptake and the enzyme is almost exclusively taken up into the parenchymal cells. We suggest that the uptake of circulating GT is receptor mediated, most likely by the galactose receptor of the parenchymal cells.

Intracellular transport of ovalbumin afterin vivo endocytosis in rainbow trout liver

AbstractThe intracellular handing of a mannose-terminated glycoprotein taken up in rainbow trout liver cells by receptor-mediated endocytosis has been studied. The intracellular transport and degradation of ovalbumin (OA) were studied by means of subcellular fractionation in Nycodenz gradients and by differential centrifugation following intravenous injection of the ligand. By using OA labelled with125I-tyramine cellobiose (125I-TC), the subcellular distribution of labelled degradation products could be studied, since they are trapped intracellularly in the organelle where the degradation takes place. 125I-TC-OA was shortly after injection (45 min) localized in a homogenous population of endosomes. Labelled degradation products firs appeared in an organelle with the same density distribution as the endosomes. In livers homogenized 2h after injection the degradation products appeared in organelles with increasing size and density. After 24h, the degradation products were recovered in at least two populations of lysosomes with a distribution profile which coincided with that of the lysosomal enzyme β-acetylglucosaminidase.The heterogeneous distribution of the late degradation products seemed not to be due to uptake of ligand in different liver cell types as only the parenchymal liver cells took up labelled OA after intravenous injection of the ligand.

Intracellular transport and degradation of chylomicron remnants in rat liver cells after in vivo endocytosis

The intracellular transport and degradation of in vivo endocytosed chylomicron remnants labelled with 125I in the protein moiety was studied in rat liver cells by means of subcellular fractionation in Nycodenz and sucrose density gradients. Initially, the radioactivity was located in low-density endosomes and was sequentially transferred to light and dense lysosomes. Data from gel filtration of the light and dense lysosomal fractions showed radioactive material with a molecular weight of about 1000-2000, representing short peptide fragments or amino acids which remain attached to iodinated tyramine cellobiose. In addition, undegraded apoproteins accumulated in both types of lysosome. Our data suggest that endocytosed chylomicron remnant apoproteins are first located in low-density endosomes and are sequentially transferred to light and dense lysosomes. Furthermore, the degradation process starts in the light lysosomes.

Enhanced activity of lysosomal proteases in activated rat hepatic stellate cells is associated with a concomitant increase in the number of the mannose-6-phosphate/insulin-like growth factor II receptor

Activated hepatic stellate cells (HSCs) play a central role during hepatic tissue repair through their influence on extracellular matrix remodelling. We have determined whether the activity levels of cathepsin B and D are affected by in vitro activation of rat HSCs, and whether the enzymes were released from the cells. Furthermore, given the important role of the mannose‐6‐phosphate/insulin‐like growth factor II receptor (M6P/IGF‐IIR) in the intracellular transport of lysosomal enzymes, we have examined whether changes in the activity of these proteases were associated with parallel changes in the level of the M6P/IGF‐IIR. The activity of cathepsin B and D increased ∼4 times between 2 and 8 days of HSC culture. This result was supported by analysing mRNA expression by RT‐PCR. The cells released the enzymes into the culture medium, amounting to ∼10% of the cell‐associated activity over 24 h. The release of enzymes was not affected by reducing medium pH from 7.4 to 6.2, indicating that the enzymes were transported to the medium independently of the M6P/IGF‐II‐R. The released cathepsin B was mostly in the inactive proenzyme form. HSC activation led to a particularly large increase in M6P/IGF‐IIR expression. A large proportion of the receptors was located on the cell surface and was found to be very suitable for measuring endocytosis of 125I‐IGF‐II. The results show that the endocytic activity increased in parallel with the increase in surface receptors and activity of lysosomal enzymes. Degradation of the ligand was reduced by inhibitors of lysosomal proteases and therefore took place in lysosomes.

Autophagy and Protein Degradation in Rat Hepatocytes, Studied by Means of Subcellular Fractionation in Nycodenz Gradients

Cellular autophagy is inducible by reduction in nutrient supply or by the accumulation of macromolecules which are rendered ineffective or potentially toxic [1]. The process may be a response to cell derangements and may serve to degrade cytoplasmic constituents during remodelling of tissues.

Intracellular Degradation of Asialoglycoproteins in Rat Hepatocytes Studied by Fractionation in Nycodenz Gradients

Information about the intracellular transport of endocytosed ligands may be obtained by subcellular fractionation studies. We have used this approach to follow the intracellular path of asialoglycoproteins in isolated rat liver parenchymal cells (PC). In these studies asialoorosomucoid labeled with125 I-tyramine cellobiose (125I-TC-AOM) was used. When ligands labeled in this way are degraded, the labeled degradation products are trapped in the lysosomes as they do not penetrate the lysosomal membrane. Therefore, in subcellular fractionation studies the labeled degradation products may serve as markers for the organeltes in which they are formed.