Studies on vinblastine-induced autophagocytosis in mouse liver

Summary The origin of the limiting membranes of autophagic vacuoles (AV) in mouse hepatocytes was studied by cytochemical techniques. Autophagocytosis was induced by an intraperitoneal injection of vinblastine (50 mg/kg). The marker enzymes used were adenosine triphosphatase for the plasma membrane, glucose-6-phosphatase for the endoplasmic reticulum and thiamine pyrophosphatase for the Golgi apparatus and the endoplasmic reticulum. All the three enzymes showed a characteristic localization in both control and vinblastine-treated hepatocytes. The space between the limiting membranes of a few apparently newly formed AV's showed weak glucose-6-phosphatase activity. Neither adenosine triphosphatase nor thiamine pyrophosphatase activities were observed on or between the AV membranes. It was suggested that endoplasmic reticulum membranes may be used as a source of AV membranes in hepatocytes. The lack of glucose-6-phosphatase activity in the limiting membranes even of most of the newly formed AV's suggests a transformation process of the membranes destined to form AV, during which the enzyme activity characteristic for endoplasmic reticulum may disappear from them.     

Studies on vinblastine-induced autophagocytosis in mouse liver

Summary The origin of the membranes of autophagic vacuoles (AV) and acquisition of acid phosphatase into AV's were studied in vinblastine-induced autophagocytosis (VBL, 50 mg/kg, i.p.) in mouse hepatocytes. Using unbuffered OsO₄, very intense staining was observed in the outer cisternae of the Golgi apparatus and also frequently in the cavity between the double membranes obviously destined to form AV's as well as in the cavity between the double membranes of newly formed AV's. There may occur a transformation process in the membranes limiting an AV analogous to that observed at the Golgi cisternae. The transformation of the outer AV membrane occurs independently of fusion with lysosomes. Inosine diphosphatase activity was localized within the cisternae and on the membranes of the endoplasmic reticulum and occasionally within the innermost cisterna of the Golgi apparatus. The results together with the unbuffered OsO₄-staining pattern suggest that the membranes of most AV's are derived from the transformed smooth surfaced cisternae of the endoplasmic reticulum which do not have inosine diphosphatase activity. Acid phosphatase activity was localized in lysosomes, occasionally within the innermost cisternae of the Golgi apparatus, between the double membranes of a few newly formed AV's and within most older single membrane-limited AV's. VBL did not prevent the fusion of lysosomes with AV's.This research was supported by grants from the Ellen and Artturi Nyyssönen Foundation and the Heikki and Hilma Honkanen Foundation     

Studies on vinblastine-induced autophagocytosis in mouse liver

Summary The origin and the structure of the limiting membranes of autophagic vacuoles (AV) in mouse hepatocytes was studied using cytochemical techniques. Autophagocytosis was induced by an intraperitoneal injection of viblastine (50 mg/kg). Imidazole-buffered osmium tetroxide impregnation was used as a marker for unsaturated fatty acids, and uranyl-lead-copper impregnation for the, determination of possible connections of AV membranes with the other cellular membranes.AV membranes stained strongly with both techniques. The staining pattern of AV membranes differed from that of the other cellular membranes. AV's were frequently seen to fuse with vesicles containing very low density lipoprotein particles. No other connections of AV membranes with other cellular membranes were observed. The results suggest that if pre-existing cellular membranes are used in AV formation some kind of transformation must occur in these membranes during AV formation. The content of unsaturated fatty acids appears to be high in AV membranes.     

Studies on vinblastine-induced autophagocytosis in mouse liver. II. Origin of membranes and acquisition of acid phosphatase

The origin of the membranes of autophagic vacuoles (AV) and acquisition of acid phosphatase into AV's were studied in vinblastine-induced autophagocytosis (VBL, 50 mg/kg, i.p.) in mouse hepatocytes. Using unbuffered OsO4, very intense staining was observed in the outer cisternae of the Golgi apparatus and also frequently in the cavity between the double membranes obviously destined to form AV's as well as in the cavity between the double membranes of newly formed AV's. There may occur a transformation process in the membranes limiting an AV analogous to that observed at the Golgi cisternae. The transformation of the outer AV membrane occurs independently of fusion with lysosomes. Inosine diphosphatase activity was localized within the cisternae and on the membranes of the endoplasmic recticulum and occasionally within the innermost cisterna of the Golgi apparatus. The results together with the unbuffered OsO4-staining pattern suggest that the membranes of most AV's are derived from the transformed smooth surfaced cisternae of the endoplasmic reticulum which do not have inosine diphosphatase activity. Acid phosphatase activity was localized in lysosomes, occasionally within the innermost cisternae of the Golgi apparatus, between the double membranes of a few newly formed AV's and within most older single membranes of a few newly formed AV's and within most older single membrane-limited AV's. VBL did not prevent the fusion of lysosomes with AV's.     

Studies on vinblastine-induced autophagocytosis in mouse liver. V. A cytochemical study on the origin of membranes

The origin and the structure of the limiting membranes of autophagic vacuoles (AV) in mouse hepatocytes was studied using cytochemical techniques. Autophagocytosis was induced by an intraperitoneal injection of vinblastine (50 mg/kg). Imidazole-buffered osmium tetroxide impregnation was used as a marker for unsaturated fatty acids, and uranyl-lead-copper impregnation for the determination of possible connections of AV membranes with the other cellular membranes. AV membranes stained strongly with both techniques. The staining pattern of AV membranes differed from that of the other cellular membranes. AV's were frequently seen to fuse with vesicles containing very low density lipoprotein particles. No other connections of AV membranes with other cellular membranes were observed. The results suggest that if pre-existing cellular membranes are used in AV formation some kind of transformation must occur in these membranes during AV formation. The content of unsaturated fatty acids appears to be high in AV membranes.     

Studies on vinblastine-induced autophagocytosis in mouse liver. III. A quantitative study

The microtubule inhibitor vinblastine (25 mg/kg, i.p.) induces autophagocytosis in mouse hepatocytes. The formation of autophagic vacuoles, their contents, and other cellular changes after vinblastine injection in hepatocytes, were studied by light and electron microscopic morphometric analysis. The volume density of autophagic vacuoles increased significantly during the experimental period (24 h). This increase was due to the significant increase in their number, which was approximately 5-fold 4 h, 12 h and 24 h after vinblastine injection. The mean volume of the autophagic vacuoles increased significantly 1 h after vinblastine injection, at which time the formation of new autophagic vacuoles was at its greatest. There was an accumulation of single membrane-limited, obviously older autophagic vacuoles in the cytoplasm. Their volume density was at its maximum 12 h after injection, suggesting a retarded turnover of autophagic vacuoles. The segregation of cytoplasmic components into autophagic vacuoles may not be selective after vinblastine injection. The injurious effects of vinblastine were evident both in light and electron microscopic studies. In the parenchymal cells the Golgi cisternae were dilated and disorganized and the volume density of the Golgi apparatus was significantly decreased 12 h after vinblastine injection. The volume density of lysosomes was increased during the 12 h after vinblastine injection. Vesicles containing very low density lipoprotein particles accumulated in the cytoplasm so that their volume density was significantly increased during the entire experimental period. Vinblastine apparently interfered with the transport and secretion of the very low density lipoproteins from the parenchymal cells.     

Studies on cellular autophagocytosis : Vinblastine-induced autophagy in the rat liver

Administration of vinblastine (5 mg/kg) in two doses intraperitoneally to rats induced a prominent formation of autophagic vacuoles in rat liver parenchymal cells. Four hours after the first injection of vinblastine, clearly recognizable organelles were seen inside these vacuoles, and by 12 h the cells were filled with residual body type of lysosomes. As compared with glucagon administration, vinblastine was followed by a far greater degree of autophagy, and therefore, offers an excellent model for future studies on autophagy.     

Catechol-O-methyl transferase in mouse liver plasma membranes.

Catechol-o-methyl transferase is usually localized predominantly in the cytosol fraction of cells, but fractionation of mouse liver showed plasma membranes contain ～ 70% of the total enzyme activity and have a specific activity ～ 10x greater than the cytosol fraction. Treatment of the membrane fraction with Lubrol-PX solubilized 47% of the membrane protein and 95% of the enzyme activity. A comparison of Lubrol-solubilized enzyme and [³H]norepinephrine binding activities in a variety of experimental conditions suggest binding is not related to interaction with the active site of catechol-o-methyl transferase. Isoelectric focusing of solubilized membrane proteins showed the enzyme has an isoelectric pH of 4.5-4.8.     

Vinblastine-induced autophagocytosis: effects on liver glycogen

The possible similarities of the mechanism by which vinblastine induces autophagocytosis in liver were compared with the known effects of glucagon in glucagon-induced autophagocytosis. A single intraperitoneal injection of vinblastine produced a wave of autophagocytosis in less than 0.5 h in mouse hepatocytes. Liver glycogen content decreases simultaneously and blood glucose first increased and then decreased below control values. Both liver cAMP concentration and the activity of glycogen phosphorylase remained unchanged. These findings provide evidence that the induction of autophagocytosis after vinblastine injection is not mediated by cAMP. The increased degradation of glycogen may occur in the lysosomal system by means of increased autophagocytosis.     

Studies on plasma membranes XVII. On the chemical composition of plasma membranes prepared from rat and mouse liver and hepatomas

Summary Plasma membranes were isolated under hypotonic conditions from rat and mouse livers and five hepatomas, i.e. one rather anaplastic rat hepatoma (and its subline) and three well-differentiated mouse hepatomas. All these membranes contained some 25% protein soluble in 0.15m NaCl. Evidence is presented that this protein is mainly, if not exclusively of nonmembranous origin. Protein/phospholipid P (P=phosphorus) ratios did not differ significantly for the various plasma membrane species except the rat-hepatoma subline, which showed a markedly lower ratio and was thus identified. Hepatoma membranes contained more P of a nonphospholipid nature than did liver membranes and to this increase contributed in all instances an increased RNA content and in some cases also an increased DNA content. The presence of DNA in these plasma membranes is artefactual, but that of RNA is more complicated. Artefactually, Ca²⁺-associated RNA of low mol wt and soluble in 0.15m NaCl, and residual RNA (genuine?, in liver membranes less than 1% in respect of protein) have been demonstrated. The increase in hepatoma-membrane RNA is attributed to the ribosomal RNA of the few microsomal vesicles which are structurally connected with these plasma membranes. The sialic acid content and the percentage of neuraminidase-resistant sialic acid of hepatoma as compared with liver membranes was either similar or changed, depending on the hepatoma strain. Gelfiltration of trypsin-released peptides of liver plasma membranes showed hexosamine and hexose to be confined to the sialic acidcontaining fractions. In spite of quantitative differences among fractions, the relative contents of the three carbohydrates in the combined fractions were (about) similar to those in intact liver membranes. Similar experiments with the rat-hepatoma membranes showed a changed carbohydrate expression.     

Cytochemical studies on induced autophagocytosis in mouse exocrine pancreas

1. The origin of the limiting membranes of autophagic vacuoles (AVs) in mouse pancreatic acinar cells was studied in vinblastine-induced autophagocytosis. 2. The marker enzymes used were adenosine triphosphatase, lipase, inosine diphosphatase and thiamine pyrophosphatase. The following impregnation techniques were used: unbuffered osmium tetroxide impregnation, imidazole-buffered osmium tetroxide impregnation and uranyl-lead-copper impregnation. 3. Only a weak lipase activity was observed between the limiting membranes of a few AVs. The AV membranes were stained heavily with all impregnation techniques used. 4. The origin of AV membranes seems to be same in mouse liver and exocrine pancreas in vinblastine-induced autophagocytosis.     

Studies on mouse liver urate oxidase

Summary The fine localization of urate oxidase was investigated with immunoferritin technique directly applied to ultrathin sections of fixed and frozen mouse liver tissue. The ferritin particles indicating the urate oxidase antigen were localized in microbodies, cisternae of rough- and smooth-surfaced endoplasmic reticulum (ER), and Golgi vacuoles and vesicles. In ER the particles were abundantly observed in dilated terminal portions. In addition, Golgi lamellae were slightly stained comparing with the vacuoles and vesicles. The staining with ferritin particles was inhibited by the treatment of unconjugated anti-urate oxidase before ferritin conjugate staining. From these results, the formation of microbody was discussed.     

Studies on mouse liver urate oxidase

Summary The localization of urate oxidase in mouse liver was investigated by fluorescent antibody technique. The fluorescence was observed in the cytoplasm and fine granules scattering throughout the cytoplasm of liver cells. The diffuse cytoplasmic fluorescence around the central vein was somewhat stronger than that of the medial and outer zone of hepatic lobule. Nuclei of the liver cell and stellate cell of Kuppfer were not stained.     

Studies on mouse liver urate oxidase

Summary Distribution of urate oxidase in subcellular components such as nuclei, mitochondria, lysosomes, microsomes, and cell sap, was investigated by both enzymatic and immunochemical methods. The subcellular components were prepared from mouse liver homogenate by differential centrifugation and the resulting microbody-rich mitochondrial fraction was fractionated by sucrose density gradient centrifugation. The enzymatically determined urate oxidase was distributed mainly in mitochondrial and lysosome fractions. The immunochemically assayed urate oxidase antigen was localized in mitochondrial, lysosome, and microsome fractions. The antigen to enzyme ratio was 1.0 in the mitochondrial and lysosome fractions, and about 2.0 in the microsome fraction.Sucrose density gradient centrifugation of the mitochondrial fraction indicated that the urate oxidase antigen was distributed around three density bands of 1.07, 1.15, and 1.24. The main band (1.24) was consistent with the microbody fraction. From these results, it was suggested that a precursor protein (proenzyme) might be located in the microsome fraction.This work was supported in part by a grant 777007 from the Ministry of Education, Japan, in 1972.     

Cellular autophagocytosis in mouse seminal vesicle cells in vitro

Cellular autophagocytosis was observed in mouse seminal vesicle cells incubated in vitro up to 8 h in medium 199 or Krebs-Ringer bicarbonate buffer. During the first 2 h of incubation, early forms of autophagic vacuoles were seen in the cells, advanced forms containing degraded material began to cumulate later. After 6--8 h, early vacuoles occurred sparsely, while advanced forms were detected in a great number. During the first 2 h of incubation, we often observed smooth surfaced membrane pairs between the cisternae of rough surfaced endoplasmic reticulum resembling isolating membranes of autophagic vacuoles. They varied in size and shape from short, straight cisternae to long, curved ones, almost completely encircling areas of the cytoplasm. Based on these observations, we propose a tentative scheme of the formation of autophagic vacuoles, viz., the short, straight cisternae would represent the first stage in the development of an autophagic vacuole, while the curved sack-like forms are interpreted as successive steps leading to the complete sequestration of an area of the cytoplasm.