Trond Berg

Transport and storage of vitamin A

The requirement of vitamin A (retinoids) for vision has been recognized for decades. In addition, vitamin A is involved in fetal development and in the regulation of proliferation and differentiation of cells throughout life. This fat-soluble organic compound cannot be synthesized endogenously by humans and thus is an essential nutrient; a well-regulated transport and storage system provides tissues with the correct amounts of retinoids in spite of normal fluctuations in daily vitamin A intake. An overview is presented here of current knowledge and hypotheses about the absorption, transport, storage, and metabolism of vitamin A. Some information is also presented about a group of ligand-dependent transcription factors, the retinoic acid receptors, that apparently mediate many of the extravisual effects of retinoids.

Clathrin-dependent endocytosis.

The process by which clathrin-coated vesicles are produced involves interactions of multifunctional adaptor proteins with the plasma membrane, as well as with clathrin and several accessory proteins and phosphoinositides. Here we review recent findings highlighting new insights into mechanisms underlying clathrin-dependent endocytosis.

Isolation and Characterization of Rat Liver Amphisomes: EVIDENCE FOR FUSION OF AUTOPHAGOSOMES WITH BOTH EARLY AND LATE ENDOSOMES

Amphisomes, the autophagic vacuoles (AVs) formed upon fusion between autophagosomes and endosomes, have so far only been characterized in indirect, functional terms. To enable a physical distinction between autophagosomes and amphisomes, the latter were selectively density-shifted in sucrose gradients following fusion with AOM-gold-loaded endosomes (endosomes made dense by asialoorosomucoid-conjugated gold particles, endocytosed by isolated rat hepatocytes prior to subcellular fractionation). Whereas amphisomes, by this criterion, accounted for only a minor fraction of the AVs in control hepatocytes, treatment of the cells with leupeptin (an inhibitor of lysosomal protein degradation) caused an accumulation of amphisomes to about one-half of the AV population. A quantitative electron microscopic study confirmed that leupeptin induced a severalfold increase in the number of hepatocytic amphisomes (recognized by their gold particle contents; otherwise, their ultrastructure was quite similar to autophagosomes). Leupeptin caused, furthermore, a selective retention of endocytosed AOM-gold in the amphisomes at the expense of the lysosomes, consistent with an inhibition of amphisome-lysosome fusion. The electron micrographs suggested that autophagosomes could undergo multiple independent fusions, with multivesicular (late) endosomes to form amphisomes and with small lysosomes to form large autolysosomes. A biochemical comparison between autophagosomes and amphisomes, purified by a novel procedure, showed that the amphisomes were enriched in early endosome markers (the asialoglycoprotein receptor and the early endosome-associated protein 1) as well as in a late endosome marker (the cation-independent mannose 6-phosphate receptor). Amphisomes would thus seem to be capable of receiving inputs both from early and late endosomes.

Phagosome dynamics and function

Phagocytosis of microorganisms and other particles is mediated most efficiently by receptors such as Fc‐receptors (FcR) and complement‐receptors (C3R). Interaction between these receptors and ligands on the particle results in signal transduction events that lead to actin polymerisation and phagosome formation. The phagosome then undergoes a maturation process whereby it transforms into a phagolysosome. Phagosome maturation depends on interactions (fusion events) with early and late endosomes as well as with lysosomes. The fusion processes are regulated by small GTP‐binding proteins and other proteins that are also involved in fusion processes in the endocytic pathway. Although most phagocytosed microorganisms are killed in the lysosome, some pathogens have developed survival strategies and are able to live in the harsh conditions in the phagolysosome or interfere with the maturation process and thereby evade destruction by acid hydrolases. BioEssays 22:255–263, 2000.

In Vitro Fusion of Phagosomes with Different Endocytic Organelles from J774 Macrophages

We describe novel biochemical and electron microscopy assays to investigate in vitro fusion of latex bead phagosomes with three different endocytic organelle fractions from J774 macrophages. After formation, early phagosomes fuse avidly with early and late endosomes and for a longer period of time with lysosomes, but they subsequently become fusion-incompetent. The fusion of early, but not late, phagosomes with all three endocytic fractions could be significantly stimulated by Rab5. In contrast to other cell types investigated, this Rab is uniquely enriched on both early and late endosomes in J774 macrophages. Moreover, exogenous Rab5 stimulates homotypic fusion between both sets of organelles. This was shown by a quantitative electron microscopy fusion assay that can directly assay fusion between any combination of morphologically defined organelles. By the same approach, we discovered an unexpected Rab5-stimulatable fusion between early and late endosomes in J774, but not in BHK cells. Thus, in J774 cells both Rab5 and the endocytic pathway seem to have evolved additional functions not yet seen in nonphagocytic cells.

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.

Uptake and degdradation of formaldehyde-treated 125I-labeled human serum albumin in rat liver cells in vivo and in vitro

Abstract The uptake of formaldehyde-treated 125 I-labelled human serum albumin in rat hepatocytes and nonparenchymal liver cells was measured in vivo and in vitro. Isolated liver cells were prepared by treating the perfused liver with collagenase. Purified hepatocytes and nonparenchymal cells were obtained by differential centrifugation. Human serum albumin was found to be taken up exclusively or almost exclusively by nonparenchymal cells in vitro and in vivo (after intravenous injection). The maximal rate of human serum albumin-uptake in vitro was comparable to that in vivo. Nonparenchymal cells degraded human serum albumin in vitro as indicated by release of trichloroacetic acid-soluble radioactivity. Degradation started about 20–30 min after addition of human serum albumin to cells and rate of degradation was proportional to rate of uptake. Human serum albumin-degradation could be studied without interference of concurrent uptake by separating cells that had been preincubated with human serum albumin from the medium and then reincubating them with human serum albumin-free medium. The lag phase before human serum albumin-degradation starts and the inhibitory effect of chloroquine on degradation indicate that human serum albumin is degraded in lysosomes. The data obtained show that enzymatically prepared nonparenchymal liver cells retain their endocytic activity in vitro. Denatured human serum albumin should be useful both as a marker for rat liver macrophages and for the study of intracellular proteolysis in these cells.

Distribution of lysosomal enzymes between parenchymal and Kupffer cells of rat liver.

1. 1. Suspensions of rat liver cells were prepared by perfusing the liver in vitro with solutions containing collagenase and hyaluronidase. About 90% of the liver mass was recovered in the cell suspension. 2. 2. Parenchymal cells were prepared by differential centrifugation or by magnetic removal of iron-loaded Kupffer cells. About 60% of the liver mass was regained in the parenchymal fractions. Specific activity of β glucoronidase (EC 3.2.1.31) was about the same in the parenchymal cells and in liver homogenates. Specific activity of acid deoxyribonuclease (EC 3.1.4.6) in parenchymal cells was about 65% of that in liver homogenates. 3. 3. Kupffer cells were separated from parenchymal cells either by incubating the original cell suspension with pronase, which destroys the parenchymal cells, or by magnetic means. 70 and 40% of the Kupffer cells in the original cell suspension could be recovered by the pronase method and the magnet method, respectively. Iron-loaded cells were contaminated with parenchymal cells. Acid deoxyribonuclease and β-glucuronidase were found to be selectively concentrated in Kupffer cells prepared with pronase. Specific activities of β-glucuronidase and acid deoxyribonuclease were, respectively, 3.6 and 13.6 times higher in pronase-prepared Kupffer cells than in parenchymal cells. Only acid deoxyribonuclease was found to be increased in Kupffer cells isolated by means of a magnet (about twice as high as in parenchymal cells).

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.

Induction of tryptophan oxygenase in primary rat liver cell suspensions by glucocorticoid hormone

Abstract The effect of dexamethasone phosphate on tryptophan oxygenase activity has been studied in suspensions of enzymatically prepared rat liver cells. The hormone was found to induce a 2–3 fold increase in enzyme activity after incubating the cells for 7 h in Krebs-Ringer phosphate solution at 37 °C. Purified parenchymal cells and a mixture of parenchymal cells and reticuloendothelial cells gave comparable results, indicating that the hormonal effect was on the parenchymal cells.