Isolation and characterization of rough endoplasmic reticulum associated with mitochondria from normal rat liver

A subfraction of rough endoplasmic reticulum (RER) characterized by its close association with mitochondria (MITO) was isolated from low speed pellets of normal rat liver homogenate under defined ionic conditions. This fraction enriched in MITO-RER complexes contained 20% of cellular RNA, 20% of glucose-6-phosphatase and 47% of cytochrome c oxidase activities. Morphologically, the isolated MITO-RER complexes closely resembled physiological associations between the two organelles commonly seen in intact liver. Partial dissociation of RER from mitochondria of the MITO-RER fraction was achieved by either EDTA (0.5 mM) or by hypotonic/hypertonic treatment of MITO-RER complexes. With the latter procedure approx. 70% of RER (RERmito) with 50% of ribosomes still attached could be separated from the inner compartments of mitochondria. This RERmoto exhibited a higher glucose-6-phosphatase activity than RER isolated as rough microsomes from the postmitochondrial supernatant. Isopycnic centrifugation on linear metrizamide gradients revealed that the mitochondria-associated part of RER corresponds to the high density, ribosome-rich subfraction of rough microsomes isolated in cation-free sucrose solution. The combined data demonstrate that a morphologically and biochemically distinct portion of RER is associated with mitochondria and support the concept of considerable intracellular heterogeneities in distribution of enzymes and enzyme systems along the lateral plane of the endoplasmic reticulum membrane system.     

Phosphatase cytochemistry with cerium as trapping agent. Verification of acid phosphatase and glucose-6-phosphatase reactive sites

Lead is prevalently replaced by cerium as trapping agent in phosphatase cytochemistry to prevent non-specific precipitation. Recently, substrate specific but artefactual lead precipitates have been described in the nuclear envelope (NE) and rough endoplasmic reticulum (RER) due to a local matrix effect. In the present study a verification was carried out of the localization of acid phosphatase and glucose-6-phosphatase in the NE and RER of rat peritoneal macrophages and hepatocytes respectively with cerium. It appeared that precipitates of cerium phosphate in NE and RER of peritoneal macrophages do not represent sites of acid phosphatase activity but are due to the matrix effect. However, in rat hepatocytes these organelles demonstrate true reactive sites for glucose-6-phosphatase.     

Phosphatase cytochemistry with cerium as trapping agent

Summary Lead is prevalently replaced by cerium as trapping agent in phosphatase cytochemistry to prevent nonspecific precipitation. Recently, substrate specific but artefactual lcad precipitates have been described in the nuclear envelope (NE) and rough endoplasmic reticulum (RER) due to a local matrix effect. In the present study a verification was carried out of the localization of acid phosphatase and glucose-6-phosphatase in the NE and RER of rat peritoneal macrophages and hepatocytes respectively with cerium. It appeared that precipitates of cerium phosphate in NE and RER of peritoneal macrophages do not represent sites of acid phosphatase activity but are due to the matrix effect. However, in rat hepatocytes these organelles demonstrate true reactive sites for glucose-6-phosphatase.In honour of Professor van Duijn     

DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES : I. Glucose-6-Phosphatase Distribution In Situ

The distribution of glucose-6-phosphatase activity in rat hepatocytes during a period of rapid endoplasmic reticulum differentiation (4 days before birth-1 day after birth) was studied by electron microscope cytochemistry. Techniques were devised to insure adequate morphological preservation, retain glucose-6-phosphatase activity, and control some other possible artifacts. At all stages examined the lead phosphate deposited by the cytochemical reaction is localized to the endoplasmic reticulum and the nuclear envelope. At 4 days before birth, when the enzyme specific activity is only a few per cent of the adult level, the lead deposit is present in only a few hepatocytes. In these cells a light deposit is seen throughout the entire rough-surfaced endoplasmic reticulum. At birth, when the specific activity of glucose-6-phosphatase is approximately equal to that of the adult, nearly all cells show a positive reaction for the enzyme and, again, the deposit is evenly distributed throughout the entire endoplasmic reticulum. By 24 hr postparturition all of the rough endoplasmic reticulum, and in addition the newly formed smooth endoplasmic reticulum, contains heavy lead deposits; enzyme activity at this stage is 250% of the adult level. These findings indicate that glucose-6-phosphatase develops simultaneously within all of the rough endoplasmic reticulum membranes of a given cell, although asynchronously in the hepatocyte population as a whole. In addition, the enzyme appears throughout the entire smooth endoplasmic reticulum as the membranes form during the first 24 hr after birth. The results suggest a lack of differentiation within the endoplasmic reticulum with respect to the distribution of glucose-6-phosphatase at the present level of resolution.     

Genesis of unusual vesicles in rat periportal hepatocytes after administration of N-hydroxy-2-acetylaminofluorene

Administration of N-hydroxy-2-acetylaminofluorene (90 mumol/kg, iv) to rats results in damage to periportal hepatocytes. The most prominent ultrastructural changes are the appearance of numerous unusual vesicles of about 200 nm diameter and the proliferation of the endoplasmic reticulum to form fingerprint-like structures. In order to characterize these vesicles and to investigate their possible origin, their enzymatic activity was studied by ultrastructural enzyme histochemistry. The vesicles as well as the fingerprint-like structures exhibited glucose-6-phosphatase activity. Continuities between the vesicles and the membranes of both the RER and the SER were frequently seen. The vesicles lacked ATPase, 5'nucleotidase and catalase activity. From these results we conclude that the vesicles may be an unusual proliferation of the endoplasmic reticulum.     

Studies on vinblastine-induced autophagocytosis in mouse liver. IV. Origin of membranes

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 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.     

Chemical and Biochemical Changes in Subcellular Fractions of Guinea Pig Liver During Infection with Coxiella burneti

Livers of uninfected guinea pigs and of guinea pigs infected with Coxiella burneti were fractionated into smooth endoplasmic reticulum, rough endoplasmic reticulum (RER), pellet, and cell sap fractions. The ribonucleic acid (RNA) and protein of each fraction were determined, and the phosphorylase, glucose-6-phosphatase, and glucosyl transferase (glycogen synthetase) activities of each fraction were measured. Decreased RNA, protein, and enzyme activities were found in the RER and pellet fractions of infected livers, with the greatest differences in the RER. The evidence indicates a solubilization of the phosphorylase and synthetase, with the enzymes moving from the RER and glycogen-containing pellet fraction to the cell sap. The data suggest the RER as a target during Q fever.     

DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES : II. Glucose-6-Phosphatase in Rough Microsomes

Electron microscope cytochemical localization of glucose-6-phosphatase in the developing hepatocytes of fetal and newborn rats indicates that the enzyme appears simultaneously in all the rough endoplasmic reticulum of a cell, although asynchronously within the hepatocyte population as a whole. To confirm that the pattern of cytochemical deposits reflects the actual distribution of enzyme sites, a method to subfractionate rough endoplasmic reticulum was developed. The procedure is based on the retention of the cytochemical reaction product (precipitated lead phosphate) within freshly prepared rough microsomes reacted in vitro with glucose-6-phosphate and lead ions. Lead phosphate increases the density of the microsomes which have glucose-6-phosphatase activity and thereby makes possible their separation from microsomes lacking the enzyme; separation is obtained by isopycnic centrifugation on a two-step density gradient. The procedure was applied to rough microsomes isolated from rats at several stages during hepatocyte differentiation and the results obtained agree with those given by cytochemical studies in situ. Before birth, when only some of the cells react positively for glucose-6-phosphatase, only a commensurate proportion of the rough microsome fraction can be rendered dense by the enzyme reaction. At the time of birth and in the adult, when all cells react positively, practically all microsomes acquire deposit and become dense after reaction. Thus, the results of the microsome subfractionation confirm the cytochemical findings; the enzyme is evenly distributed throughout all the endoplasmic reticulum of a cell and there is no regional differentiation within the rough endoplasmic reticulum with respect to glucose-6-phosphatase. These findings suggest that new components are inserted molecule-by-molecule into a pre-existing structural framework. The membranes are thus mosaics of old and new molecules and do not contain large regions of entirely "new" membrane in which all of the components are newly synthesized or newly assembled.     

Developmental pattern of glucose-6-phosphatase activity in the small intestine of the mouse fetus.

The distribution of glucose-6-phosphatase (G6Pase) activity in the epithelium of the small intestine in mouse embryos (the last 4 days of gestation) was studied by electron microscope cytochemistry and by enzymatic assays. At 16 days, the lead phosphate deposited by the cytochemical reaction is localized on the rough endoplasmic reticulum (RER) and nuclear envelope of very few cells in the duodenum and jejunum. Positive cells are more frequently seen in the upper part of the developing villi. At 17 days of gestation, a tremendous burst in RER differentiation is noticed in all parts of the small intestine and concomitantly glycogen disappears. At 18 days of gestation all the principal cells of the intestinal mucosa show a well differentiated positive RER and the enzyme is also present in the smooth endoplasmic reticulum. Biochemically, G6Pase activity is detected in the proximal 2 thirds of the small intestine at 17 days of gestation and appears at 18 days in the last third. Afterwards the activity increases up until birth. These results suggest (1) that the endoplasmic reticulum differentiates very late in the intestinal mucosa of mouse embryos (2) that the differentiation with respect to G6Pase is asynchronous between the enterocytes, (3) that for a given cell all the cisternae of RER are involved in G6Pase synthesis at the same moment and (4) that the enterocytes of the duodenum differentiate sooner and faster that those of the jejunum and ileum.     

Effect of diabetes on the rat hepatic glucose-6-phosphatase system in endoplasmic reticulum subfractions

Diabetes-induced alterations in the activities of the components of the glucose-6-phosphatase system (i.e., the enzyme, the glucose-6-P translocase (T(1)), and the phosphate translocase (T(2)) were examined in smooth and rough subfractions of hepatic endoplasmic reticulum from streptozotocin-injected rats. A significant effect of diabetes on the maximal velocity of glucose-6-P hydrolysis by the enzyme was present in both endoplasmic reticulum subfractions (3.1-fold increase in rough endoplasmic reticulum; 3.8-fold increase in smooth endoplasmic reticulum). Based on latency values, diabetes did not result in a proportional increase in capacity of T(1) or T(2). In contrast to the control condition, the relationship between transport capacity and hydrolytic capacity was not significantly different in the two subfractions from diabetic animals. Elucidation of the effects of diabetes on the components of the glucose-6-phosphatase system associated with smooth and rough endoplasmic reticulum membranes enhances our understanding of the hepatic contribution to diabetic hyperglycemia.     

Phenobarbital-induced alterations in the activities of the transport and hydrolytic components of the glucose-6-phosphatase system in smooth and rough subfractions of the rat hepatic endoplasmic reticulum

We have examined the influence of the phenobarbital-induced proliferation of the hepatic endoplasmic reticulum (ER) on the activities of the components of the glucose-6-phosphatase system, i.e., the enzyme, the glucose-6-P translocase (T1), and the phosphate translocase (T2). Young male rats were injected ip twice daily for 4 days with 4 mg/100 g body wt of phenobarbital (PB) or an equivalent volume of saline solution. On the fifth day, the rats were killed and smooth (SER) and rough (RER) fractions of the ER were isolated from liver homogenates. Kinetic constants for glucose-6-P hydrolysis by the system and enzyme were determined and used to calculate the kinetic constants for glucose-6-P transport. T2 activity was approximated by assaying the pyrophosphatase activity at pH 6.0 in intact microsomes. Three times more SER protein was recovered from livers of PB-treated rats. PB-treatment did not alter total liver enzyme activity, but total liver T1 activity was decreased to 59% of the control value. Maximal specific activities of the system, enzyme and T1 were all reduced by PB treatment to 44% of control values in the RER and to 68% of control values in the SER. PB treatment reduced the apparent activity of T2 in RER and SER to 35 and 49% of the respective control values. In the SER from both groups of rats, T1 activity or apparent T2 activity divided by enzyme activity was about 55% of the corresponding ratio in the RER. Our analysis of these data suggests that the lower activities of T1 and T2 in the smooth ER are the results of suppression by some intrinsic component localized in the smooth membrane. Accordingly, the reduction in total liver T1 activity and, therefore, system activity in PB-treated rats reflects the redistribution of the glucose-6-P translocase from the RER to the more abundant SER membrane where it is less active. The possibility is discussed that a higher cholesterol content within the SER membrane is responsible for the lower transport activities.     

Consequences of recurrent phosphate trapping induced by repeated injections of 2-deoxy-D-galactose. Biochemical and morphological studies in rats.

2-Deoxy-D-galactose, in a dose of 3 mmol/kg, was administered intraperitoneally twice daily to young rats for periods up to 12 weeks. This dosage schedule resulted in recurrent phosphate trapping predominantly in liver. UTP deficiency was excluded by simultaneous uridine injections. Phosphate trapping was caused by the rapid accumulation of 2-deoxy-D-galactose 1-phosphate and was most pronounced in liver but also demonstrated in small intestine, brain, spleen, and thymus. The marked, although transient, drop in the hepatic content of inorganic phosphate triggered the catabolism of adenine nucleotides and a loss of ATP. Other metabolic pathways affected by phosphate deficiency include glycogenolysis and glycolysis. Increasing with time, repeated doses of the galactose analog led to retardation and arrest of growth, hepatomegaly, and splenomegaly. The average relative liver and spleen weights were elevated 2.5- and 4.5-fold, respectively, after 12 weeks of treatment. Liver damage was indicated by hyperbilirubinaemia and a progressive rise in the activity in plasma of sorbitol dehydrogenase, alkaline phosphatase, and gamma-glutamyltransferase. Examination by light and electron microscopy showed increasing numbers of vacuoles, surrounded by a single membrane, in hepatocytes, sinusoidal endothelial cells, and Kupffer cells. Focal cytoplasmic degeneration in hepatocytes was occasionally indicated by formation of autophagic vacuoles and finger print lysosomes. Hepatocytes of 2-deoxy-D-galactose-treated rats showed a dissociation and fragmentation of the rough endoplasmic reticulum. Sinusoidal endothelial cells and Kupffer cells were markedly enlarged, the latter contained a PAS-positive but amylase resistant substance. Extrahepatic changes included an increased occurrence of vacuolated cells in thymus. Phosphate trapping and its metabolic consequences are common phenomena in the experimental injury induced b 2-deoxy-D-galactose and in some hereditary diseases such as uridylyltransferase deficiency galactosaemia, fructose intolerance and glucose-6-phosphatase deficiency.     

Cytoplasmic changes in level and distribution of glucose-6-phosphatase activities from rat liver during diethylnitrosamine-induced carcinogenesis.

Glucose-6-phosphatase (EC 3.1.3.9) activities were determined in isolated microsomes, cytoplasmic smooth and rough membranes, ribosomes and free cytosol from rat liver undergoing carcinogenesis by diethylnitrosamine (DENA) and compared with cytoplasmic fractions isolated in parallel from healthy animals from the same age.With continuous administration of a low dose of DENA (2.6 mg/kg rat per day for 20 weeks in the drinking water) livers of carcinogen treated rats became heavier than the control livers but the body weight decreased. About 70% of total glucose-6-phosphatase activity could be detected in the microsomal fraction. While there was no significant difference in this activity in both animal groups up to the 4th week, glucose-6-phosphatase of cancerous liver showed a distinct decrease of activity compared with normal liver.During cancer induction this enzyme became more soluble, confirmed by the observation that it was detached from firmer structures of cytoplasm as rough membranes and polysomes and translocated to smooth membranes and the soluble cytoplasmic fraction successively. The corresponding increase in glucose-6-phosphatase activity in the 105 000 g supernatant appears to be due to the loss of enzyme activity in a distinct cytoplasmic membrane fraction. These data strongly suggest that in parallel with alteration of cytoplasmic membrane structures during carcinogen feeding glucose-6-phosphatase is detached from heavier components of the cytoplasm while total activity decreased. Possible mechanisms of these findings are discussed.     

Effect of praseodymium nitrate on hepatocytes and Kupffer cells in the rat.

Intravenous administration of the rare earth metal salt, praseodymium nitrate, induced hepatic damage in the rat, as assessed by morphologic examination (light and electron microscopy) and biochemical parameters (serum glutamic-pyruvic transaminase (EC 2.6.1.2) and glutamic-oxalacetic transaminase (EC 2.6.1.1) activity as well as hepatic triglyceride content). Praseodymium hepatotoxicity was only attained with lower doses (10, 20, or 40 mg/kg), whereas a larger dose (80 mg/kg) was inactive in this respect. As detected by electron microscopy, lower doses of the metal salt caused hepatocytic alterations consisting of degranulation and dilatation of rough endoplasmic reticulum, accumulation of smooth endoplasmic reticulum as well as numerous lipid droplets. No abnormalities were detected in the cell organelles following administration of a large dose of the metal salt; however, vacuoles containing markedly electron-dense material were seen in the cytoplasm of the hepatocytes and the sinusoidal Kupffer cells.     

The effect of hydrocortisone and cyclosporin A on bacillus Calmette-Guérin epitheloid cell granulomas

The injection of bacillus Calmette-Guérin (BCG) intradermally into the ear of guinea pigs leads to the formation in the draining lymph node of granulomas containing epithelioid cells with rough endoplasmic reticulum (RER) and an absence of phagocytosed material. BCG granulomas in hydrocortisone- or cyclosporin A (CsA)-treated animals contain mononuclear phagocytes with no RER. In CsA-treated animals, these cells contain fragments of phagocytosed organisms. CsA was given at two doses, 25 mg/kg orally and 50 mg/kg ip. The higher dose completely suppressed the delayed hypersensitivity (DH) response to purified protein derivative (PPD) but the lower dose did not affect the level of the DH response, but had a profound effect on epithelioid cell formation. The role of lymphokines in the maturation of the monocyte/macrophage to epithelioid cells is discussed.     

A rapid calcium precipitation method of recovering large amounts of highly pure hepatocyte rough endoplasmic reticulum.

We sought a rapid and non-ultracentrifugal method of recovering large amounts of highly pure rough endoplasmic reticulum (RER) membranes from livers. By substantially modifying a 20-year-old calcium precipitation technique, we obtained a RER fraction from rat liver and established its high degree of purity by quantitating classic membrane markers for different subcellular organelles. This RER fraction is highly enriched in four known proteins (or enzyme activities) required for lipoprotein assembly: apolipoprotein B, microsomal triglyceride transfer protein, acyl CoA:diacylglycerol acyltransferase, and acyl CoA:cholesterol acyltransferase, when compared to two classical RER markers, RNA and glucose-6-phosphatase. From one 10-12 g rat liver, we recover ten to twelve RER pellets of 1.5-1.6 cm in diameter containing approximately 110-125 mg of total protein, about half of which is sodium carbonate-releasable. By electron microscopy, these large RER pellets from rat livers are homogeneously comprised largely of non-vesiculated short strips of ribosome-rich membranes. This novel technique for isolating RER membranes from liver may provide a useful tool for future studies on the assembly of apolipoprotein B-containing lipoproteins as well as for research focused on mechanisms of secretory and membrane protein translation, translocation, and folding.     

Molecular pathology of glucose-6-phosphatase

It was known in the 1950s that hepatic microsomal glucose-6-phosphatase plays an important role in the regulation of blood glucose levels. All attempts since then to purify a single polypeptide with glucose-6-phosphatase activity have failed. Until recently, virtually nothing was known about the molecular basis of glucose-6-phosphatase or its regulation. Recent studies of the type 1 glycogen storage diseases, which are human genetic deficiencies that result in impaired glucose-6-phosphatase activity, have greatly increased our understanding of glucose-6-phosphatase. Glucose-6-phosphatase has been shown to comprise at least five different polypeptides, the catalytic subunit of glucose-6-phosphatase with its active site situated in the lumen of the endoplasmic reticulum; a regulatory Ca2+ binding protein; and three transport proteins, T1, T2, and T3, which respectively allow glucose-6-phosphate, phosphate, and glucose to cross the endoplasmic reticulum membrane. Purified glucose-6-phosphatase proteins, immunospecific antibodies, and improved assay techniques have led to the diagnosis of a variety of new type 1 glycogen storage diseases. Recent studies of the type 1 glycogen storage diseases have led to a much greater understanding of the role and regulation of each of the glucose-6-phosphatase proteins.     

ON THE RELATIONSHIP OF LIVER GLUCOSE-6-PHOSPHATASE TO THE PROLIFERATION OF ENDOPLASMIC RETICULUM IN PHENOBARBITAL INDUCTION

The differentiated effects of phenobarbital treatment on liver microsomal enzymes have been further studied. The relationship between the resulting decrease in the specific glucose-6-phosphatase activity and the enhancement of formation of endoplasmic reticulum membranes with high drug-hydroxylating activity has been investigated with biochemical and histochemical methods. Biochemically and histochemically demonstrable glucose-6-phosphatase activity was found to be present in all endoplasmic reticulum membranes, including the phenobarbital-induced smooth-surfaced proliferates, even though there was an over-all decrease in activity. Actinomycin D did not inhibit the decrease in glucose-6-phosphatase activity. The findings are discussed with reference to the enzyme-membrane relationship in phenobarbital induction.     

Effect of rehydration of rat liver tissue after water deprivation.

Male albino rats were deprived of water for 6 days, then they were allowed to drink tap water ad libitim. The structure of the liver was examined by light and electron microscopy, and the protein and dry matter contents, oxygen consumption and glucose-6-phosphatase activity of the liver were determined after rehydration. At 10 minutes, the mitochondria showed signs of division and a peculiar transformation of the cristae. At 60 minutes, the membranes of the rough endoplasmic reticulum were found to have proliferated. At 12 hours, the smooth-surfaced membranes showed hypertrophy and the bile canaliculi were distended. At 24 hours all rehydration induced organelle alterations were declining. The biochemical findings agreed well with the fine structural changes and both were indicative of an enchanced functional capacity of liver cells during rehydration.