scholarly journals Glucagon stimulation of ruthenium red-insensitive calcium ion transport in developing rat liver

1981 ◽  
Vol 194 (2) ◽  
pp. 541-549 ◽  
Author(s):  
P H Reinhart ◽  
F L Bygrave
Keyword(s):  
Endoplasmic Reticulum ◽  
Cyclic Amp ◽  
Rat Liver ◽  
Adult Life ◽  
Ruthenium Red ◽  
Enzyme Analysis ◽  
Marker Enzyme ◽  
Transport Activity ◽  
Electron Microscopic ◽  
Metabolic Role

The maturation of glucagon-stimulated Ruthenium Red-insensitive Ca2+-transport activity was determined in livers of rats ranging in age from 5 days preterm to 10 weeks of adult life. Previous indications are that this activity is confined to vesicles derived mainly from the endoplasmic reticulum. Perinatal-rat liver contains near-adult values of Ruthenium Red-insensitive Ca2+-transport activity, and exhibits large transient increases in the rate of this activity at two stages of development, immediately after birth, and at 2-5 days after birth. The administration of glucagon to foetal rats, at developmental stages after 19.5 days of gestation (2.5 days before birth), results in a large stable increase (greater than 100%) of Ca2+-transport activity in a subsequently isolated ‘heavy’ microsomal fraction. That this fraction was enriched in vesicles derived from the rough endoplasmic reticulum was indicated by both an electron-microscopic examination and a marker-enzyme analysis of the subcellular fractions. The administration of glucagon into newborn animals only hours old does not enhance further the initial rate of Ca2+-transport activity, and from day 1 to 10 weeks after birth the administration of the hormone results in the moderate enhancement of Ca2+ transport. Experiments with cyclic AMP and inhibitors of phosphodiesterase activity suggest that cyclic AMP plays a key role in the enhancement by glucagon of Ruthenium Red-insensitive Ca2+ transport, and arguments are presented that this transport system has an important metabolic role in the redistribution of intracellular Ca2+ in liver tissue.

scholarly journals Two fractions of rough endoplasmic reticulum from rat liver. I. Recovery of rapidly sedimenting endoplasmic reticulum in association with mitochondria.

1977 ◽  
Vol 72 (3) ◽  
pp. 714-725 ◽  
Author(s):  
G C Shore ◽  
J R Tata
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Structural Damage ◽  
Gradient Centrifugation ◽  
Mitochondrial Protein ◽  
Enzyme Analysis ◽  
Marker Enzyme

Low-speed centrifugation (640 g) of rat liver homogenates, prepared with a standard ionic medium, yielded a pellet from which a rapidly sedimenting fraction of rough endoplasmic reticulum (RSER) was recovered free of nuclei. This fraction contained 20-25% of cellular RNA and approximately 30% of total glucose-6-phosphatase (ER marker) activity. A major portion of total cytochrome c oxidase (mitochondrial marker) activity was also recovered in this fraction, with the remainder sedimenting between 640 and 6,000 g. Evidence is provided which indicates that RSER may be intimately associated with mitochondria. Complete dissociation of ER from mitochondria in the RSER fraction required very harsh conditions. Sucrose density gradient centrifugation analysis revealed that 95% dissociation could be achieved when the RSER fraction was first resuspended in buffer containing 500 mM KCl and 20 mM EDTA, and subjected to shearing. Excluding KCl, EDTA, or shearing from the procedure resulted in incomplete separation. Both electron microscopy and marker enzyme analysis of mitochondria purified by this procedure indicated that some structural damage and leakage of proteins from matrix and intermembrane compartments had occurred. Nevertheless, when mitochondria from RSER and postnuclear 6,000-g pellet fractions were purified in this way fromanimals injected with [35S]methionine +/- cycloheximide, mitochondria from the postnuclear 6,000-g pellet were found to incorporate approximately two times more cytoplasmically synthesized radioactive protein per milligram mitochondrial protein (or per unit cytochrome c oxidase activity) than did mitochondria from the RSER fraction. Mitochondria-RSER associations, therefore, do not appear to facilitate enhanced incorporation of mitochondrial proteins which are newly synthesized in the cytoplasm.

scholarly journals The subcellular location, maturation and response to increased plasma glucagon of Ruthenium Red-insensitive calcium-ion transport in rat liver

1978 ◽  
Vol 174 (3) ◽  
pp. 1021-1030 ◽  
Author(s):  
Fyfe L. Bygrave ◽  
Charmaine J. Tranter
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Adult Life ◽  
Subcellular Location ◽  
Maximal Activity ◽  
Mitochondrial Fraction ◽  
Ruthenium Red ◽  
Maximal Response ◽  
Transport Activity ◽  
Adult Rats

1. The subcellular distribution and maturation of Ruthenium Red-insensitive Ca2+ transport activity were determined in livers of rats ranging in age from 3 days pre-term to 10 weeks of adult life and compared with those of glucose 6-phosphatase, 5′-nucleotidase and Ruthenium Red-sensitive Ca2+ transport. Initial rates of Ruthenium Red-insensitive Ca2+ transport were highest in those fractions enriched in glucose 6-phosphatase, i.e. the microsomal fraction; this fraction was devoid of Ruthenium Red-sensitive Ca2+ transport activity. Although the heaviest fraction (nuclear) contained significant amounts of 5′-nucleotidase activity it was devoid of Ruthenium Red-insensitive Ca2+ transport activity. 2. Foetal rat liver contain minimal amounts of Ruthenium Red-insensitive Ca2+ transport activity, glucose 6-phosphatase and 5′-nucleotidase activities. These begin to be expressed concomitantly soon after birth; Ruthenium Red-insensitive Ca2+ transport is maximal by 3 to 4 days and remains so for up to at least 10 weeks of adult life. Glucose 6-phosphatase also reaches a peak at 3–4 days, but then rapidly decreases to approach adult values. Maximal activity of 5′-nucleotidase in the microsomal and nuclear fractions is seen about 4–6 days after birth; this enzyme activity remains increased for up to about 10 days and then falls, but not as rapidly as glucose 6-phosphatase. It is tentatively suggested that the bulk of the Ruthenium Red-insensitive Ca2+ transport is attributable to the system derived from the endoplasmic reticulum. 3. Administration of glucagon to adult rats enhances by 2–3-fold the initial rate of Ruthenium Red-insensitive Ca2+ transport in the intermediate but not the microsomal fraction. The hormone-induced effect is fully suppressed by co-administration of puromycin, is dose-dependent with half-maximal response at approx. 1μg of glucagon/100g body wt. and time-dependent exhibiting a half-maximal response about 1h after administration of the hormone. 4. Ruthenium Red-insensitive Ca2+ transport in the post-mitochondrial fraction of foetal liver also responds to the administration in situ of glucagon. The response, which also is prevented by co-administration of puromycin, is maximal in those foetuses nearing term. The suggestion is made that these effects of the hormone on Ruthenium Red-insensitive Ca2+ transport are an integral part of the physiological network in the liver cell.

scholarly journals Re-examination of the subcellular localization of thyroxine 5′-deiodination in rat liver

1979 ◽  
Vol 180 (2) ◽  
pp. 273-279 ◽  
Author(s):  
D Auf Dem Brinke ◽  
R D Hesch ◽  
J Köhrle
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Subcellular Localization ◽  
Rat Liver ◽  
Marker Enzyme ◽  
Cytochrome C Reductase ◽  
Substrate Saturation ◽  
Single Enzyme ◽  
Optimal Ph ◽  
Nadph Cytochrome C Reductase

We describe the existence of at least two thyroxine 5′-deiodinases in rat liver. They co-fractionate with NADPH-cytochrome c reductase, the marker enzyme for membranes of the endoplasmic reticulum. Subcellular-localization studies of the most active microsomal thyroxine 5′-deiodinase were performed under substrate saturation and at optimal pH 6.8. This enzyme was a Km(app.) of about 3 microM-thyroxine and a Vmax. of about 8 ng of tri-iodothyronine/min per mg of protein. Our study confirms in part the earlier reports of microsomal localization of thyroxine 5′-deiodination. However, this process is not mediated by only a single enzyme.

scholarly journals A procedure for light and electron microscopic intracellular immunolocalization of collagen and fibronectin in rat liver.

1985 ◽  
Vol 33 (5) ◽  
pp. 407-414 ◽  
Author(s):  
B Clement ◽  
M Rissel ◽  
S Peyrol ◽  
Y Mazurier ◽  
J A Grimaud ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Type Iii Collagen ◽  
Collagen Types ◽  
Electron Microscopic ◽  
Experimental Conditions ◽  
The Matrix ◽  
Matrix Components ◽  
Indirect Immunoperoxidase

Experimental conditions have been designed that permit both extracellular and intracellular immunolocalization of various collagen types and fibronectin in rat liver. The procedure involves paraformaldehyde fixation by perfusion of the organ, use of saponin as a membrane permeabilizing agent, and visualization of the matrix components by indirect immunoperoxidase. Intracellular demonstration of collagens was particularly sensitive to the composition of the fixative and the duration of fixation. Hepatocytes contained fibronectin and types I and IV collagen, whereas fat-storing and endothelial cells evidenced type III collagen in addition. All the components were specifically located in the endoplasmic reticulum and/or the Golgi apparatus.

Incorporation in vivo of [32P]orthophosphate and [Me-3H]choline into rough microsomal, Golgi, and plasma membranes of rat liver

1977 ◽  
Vol 55 (8) ◽  
pp. 876-885 ◽  
Author(s):  
Patricia L. Chang ◽  
John R. Riordan ◽  
Mario A. Moscarello ◽  
Jennifer M. Sturgess
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Marker Enzyme ◽  
Golgi Membranes ◽  
Endomembrane Flow

To study membrane biogenesis and to test the validity of the endomembrane flow hypothesis, incorporation of 32P and [Me-3H]choline in vivo into membranes of the rat liver was followed. Rough microsomal, Golgi-rich, and plasma membrane fractions were monitored with marker enzyme assays and shown with morphometric analysis to contain 82% rough microsomes, at least 70% Golgi complexes, and 88% plasma membranes, respectively. Membrane subfractions from the rough microsomal and Golgi-rich fractions were prepared by sonic disruption.At 5 to 30 min after 32P injection, the specific radioactivity of phosphatidylcholine was higher in the rough microsomal membranes than in the Golgi membranes. From 1 to 3 h, the specific activity of phosphatidylcholine in Golgi membranes became higher and reached the maximum at about 3 h. Although the plasma membrane had the lowest specific radioactivity throughout 0.25–3 h, it increased rapidly thereafter to attain the highest specific activity at 5 h. Both rough microsomal and plasma membranes reached their maxima at 5 h.The specific radioactivity of [32P]phosphatidylethanolamine in the three membrane fractions was similar to that of [32P]phosphatidylcholine except from 5 to 30 min, when the specific radioactivity of phosphatidylethanolamine in the Golgi membranes was similar to the rough microsomal membranes.At 15 min to 5 h after [Me-3H]choline injection, more than 90% of the radioactivity in all the membranes was acid-precipitable. The specific radioactivities of the acid-precipitated membranes, expressed as dpm per milligram protein, reached the maximum at 3 h. After [Me-3H]choline injection, the specific radioactivity of phosphatidylcholine separated from the lipid extract of the acid-precipitated membranes (dpm per micromole phosphorus) did not differ significantly in the three membrane fractions. The results indicated rapid incorporation of choline into membrane phosphatidylcholine by the rough endoplasmic reticulum, Golgi, and plasma membranes simultaneously.The data with both 32P and [Me-3H]choline precursors did not support the endomembrane flow hypothesis. The Golgi complexes apparently synthesized phosphatidylethanolamine and incorporated choline into phosphatidylcholine as well as the endoplasmic reticulum. The results are discussed with relevance to current hypotheses on the biogenesis and transfer of membrane phospholipids.

Multivalent interaction between asialofetuin and plasma membrane preparations from the rat liver

1980 ◽  
Vol 58 (12) ◽  
pp. 1414-1420 ◽  
Author(s):  
Maria T. Debanne ◽  
Erwin Regoeczi ◽  
Mark W. C. Hatton
Keyword(s):  
Rat Liver ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Theoretical Models ◽  
Underlying Mechanism ◽  
Marker Enzyme ◽  
Electron Microscopic ◽  
Microscopic Appearance ◽  
Wide Range ◽  
Electron Microscopic Appearance

Binding of bovine asialofetuin by rat liver plasma membranes was studied using different techniques for the separation of the free and bound forms of the glycoprotein and also different approaches to measure nonspecific binding. The membrane preparations had the electron microscopic appearance of a mixture of lamellae and vesicles and their lipid:protein ratios and marker enzyme profiles fell within the range of values available from the literature. The binding capacity was approximately 15 pmol of asialofetuin per milligram of membrane protein.Scatchard plots of the values obtained over a wide range of concentrations (4.8–12.6 μg asialofetuin per 30 μg membrane protein) after incubation at 22 °C showed pronounced non-linearity which, in combination with evaluations according to other theoretical models, was referable to heterogeneity of binding. In sharp contrast, after incubation at 4 °C the Scatchard plot was linear. This difference is interpreted as the expression of a functional, rather than a chemical, heterogeneity in asialofetuin binding. The underlying mechanism is thought to be competition of galactose groups for binding sites with the result that the number of bonds varies between the galactose groups of a bound asialofetuin molecule and the hepatic lectin, depending on the concentration of the glycoprotein in the incubation mixture.

scholarly journals Relationship between peroxisomes and endoplasmic reticulum investigated by combined catalase and glucose-6-phosphatase cytochemistry.

1981 ◽  
Vol 29 (11) ◽  
pp. 1263-1272 ◽  
Author(s):  
H Shio ◽  
P B Lazarow
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Rat Liver ◽  
Reaction Products ◽  
Lead Phosphate ◽  
Electron Microscopic ◽  
Phosphatase Cytochemistry ◽  
Electron Microscopic Cytochemistry ◽  
Cellular Compartments

The theoretical advantages of electron microscopic cytochemistry were utilized to look for evidence of possible connections between peroxisomes and the endoplasmic reticulum in rat liver. Established cytochemical procedures for catalase (peroxisomes) and glucose-6-phosphatase (endoplasmic reticulum) were carried out, and evidence was sought of diffusion of reaction products between the organelles. No such diffusion was observed: lead phosphate was found in the endoplasmic reticulum and in the nuclear envelope but not in peroxisomes; oxidized diaminobenzidine (DAB) was seen only in peroxisomes. In addition, both types of cytochemistry were carried out on the same tissue. The two kinds of reaction product could be distinguished by virtue of their different electron opacities. No mixing of the two reaction products was observed. These results do not support the hypothesis that peroxisomes and endoplasmic reticulum may be connected; rather, they support the idea that the two organelles exist as separate cellular compartments.

Electron microscopic tomography of rat-liver mitochondria and their interactions with the endoplasmic reticulum

BioFactors ◽  
1998 ◽  
Vol 8 (3-4) ◽  
pp. 225-228 ◽  
Author(s):  
Carmen A. Mannella ◽  
Karolyn Buttle ◽  
Bimal K. Rath ◽  
M. Marko
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Electron Microscopic

scholarly journals 3,4,3,4'-Tetrachlorobiphenyl-Induced Effects in the Rat Liver. II. Electron Microscopic Autoradiographic Localization of H-TCB

1989 ◽  
Vol 17 (4_part_2) ◽  
pp. 782-788 ◽  
Author(s):  
Stephen K. Durham ◽  
Abraham Brouwer
Keyword(s):  
Endoplasmic Reticulum ◽  
Endothelial Cell ◽  
Rat Liver ◽  
Kupffer Cell ◽  
Liver Cell ◽  
Morphologic Change ◽  
Cell Type ◽  
Sequential Order ◽  
Electron Microscopic ◽  
Morphological Alterations

Recent results (3) indicate that 200 mg 3,4,3′,4′-tetrachlorobiphenyl induces hepatomegaly accompanied by significant decreases in serum and hepatic retinoid content and hepatocyte morphologic alterations of proliferated and vesiculated endoplasmic reticulum and megamitochondria with paracrystalline inclusions. There was also an associated change in the number, size, and distribution of lipid droplets in hepatocytes and fat-storing cells. Electron microscopic autoradiographic techniques were utilized to determine the cellular and subcellular distribution of 3H-3,4,3′,4′-tetrachlorobiphenyl (3H-TCB) in the adult rat liver and determine if there is any relationship between subcellular morphologic change and radiolabel localization. Adult female WAG/Rij rats received a single intraperitoneal injection of 200 mg TCB/kg containing 1.85 mCi of 3H-TCB and were sacrificed at 1, 3, 7, and 14 days following exposure. The vast majority of 3H-TCB-derived radioactivity was located in the hepatocyte at all time points examined, ranging from 79–86% of the total number of autoradiographic grains counted over the liver cells. Sequential order of radiolabel localization per liver cell type at 1, 3, and 7 days was hepatocyte > > > Kupffer cell > fat-storing cell > endothelial cell. At day 14, the sequential order of radiolabel localization per liver cell type was hepatocyte > > > fat-storing cell > Kupffer cell > endothelial cell, which indicates that there was some shift movement of label over time. The lipid droplet, mitochondria, and endoplasmic reticulum were the subcellular structures or organelles of hepatocytes having the highest number of 3H-TCB-derived grains at all time periods examined. The predominant morphological alterations induced following TCB intoxication were observed in these organelles. The results of this study suggests that there is an association between TCB localization and morphologic change induced in mitochondria and endoplasmic reticulum of hepatocytes following TCB exposure.

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