scholarly journals Isolation and subfractionation on ficoll gradients of adult rat hepatocytes. Size, morphology, and biochemical characteristics of cell fractions.

1975 ◽  
Vol 66 (1) ◽  
pp. 1-22 ◽  
Author(s):  
P Drochmans ◽  
J C Wanson ◽  
R Mosselmans
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Gradient Centrifugation ◽  
Parenchymal Cell ◽  
Liver Homogenate ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Isolated Cells ◽  
Adult Rat ◽  
Mean Diameter

The recirculating perfusion of adult rat liver with a Ca-++-free Hanks' solution produces a release of the adhesiveness of cells and a cleaving of the desmosomes. The addition of collagenase and hyaluronidase to the perfusion medium leads to complete dissociation of the liver tissue into a mixture of isolated cells and cell cords in which the hepatocytes remain connected with specific junctional differentiations, namely the gap and tight junctions. Individual cells are released by submitting the suspension of cell trabeculae to a gentle rolling. The gap junctions are ruptured at least in one of the two adjacent cells and remain generally attached to the other cell taking with them a small portion of cytoplasm. This technique of isolation of hepatocytes yields about 60-65% of the parenchymal cells contained in a liver; endothelial cells and other cells of the connective tissue are not recovered. The ultrastructural preservation of the isolated hepatocytes is excellent and the glucose-6-phosphatase activity, confined to the endoplasmic reticulum, appears unaltered in most cells. Protein, DNA and RNA recovery in the preparations of isolated hepatocytes is satisfactory, amounting to 70% of that found in liver homogenate; glycogen, the most labile component examined, is partly lost or degraded during the manipulations. Cell diameters measured by different methods confirm the preservation of the original volume of the in situ hepatocytes and the presence of more than one type of parenchymal cell. By submitting this heterogeneous cell population to an isopycnic density gradient centrifugation, two types of hepatocytes can be distinguished: the light hepatocytes, with a mean diameter of 20.5 mum and a mean density of 1.10, are characterized by an extended smooth-walled endoplasmic reticulum entrapping dispersed alpha-glycogen particles; the heavy hepatocytes, with a mean diameter of 19.0 mum and a mean density of 1.14, present a relatively reduced compartment of smooth endoplasmic reticulum, but large accumulations of glycogen. It is suggested that the cell fraction of low density is enriched in centrolobular cells and the high density fraction in perilobular hepatocytes.

scholarly journals Effect of insulin on ultrastructure and glycogenesis in primary cultures of adult rat hepatocytes

1977 ◽  
Vol 74 (3) ◽  
pp. 878-900 ◽  
Author(s):  
D Bernaert ◽  
JC Wanson ◽  
P Drochmans ◽  
A Popowski
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Primary Cultures ◽  
Active Form ◽  
Rat Hepatocytes ◽  
High Concentration ◽  
Trophic Effect ◽  
Adult Rat ◽  
Glycogenosis Type ◽  
High Concentrations

Insulin in the presence of high concentrations of glucose has a beneficial trophic effect on the development of primary cultures of hepatocytes. Compared to the situation observed in hormone-free control cultures, the flattening of the reaggregated hepatocytes is enhanced, and the reconstituted cell trabeculae are enlarged and tend to form a confluent monolayer after 3 days; the survival time is prolonged from 3 to 5 or 6 days. Ultrastructural modifications are also initiated by insulin; numerous glycogen particles appear after 24 h, in between the cisternae of the proliferated smooth endoplasmic reticulum. After 48 h, large amounts of glycogen are stored, and numerous polysomes are present. A small number of cells showed an increased synthesis of lipid droplets in the lumen of the smooth endoplasmic reticulum and form liposomes at the same time. After 72 h, cytolysomes filled with glycogen develop, simulating glycogenosis type II. Simultaneously, microtubules and microfilaments, closely related to numerous polysomes, appear in cytoplasmic extensions constituting undulating membranes. The biochemical data demonstrate that, in the absence of insulin, a high concentration of glucose stimulates glycogenesis and hinders glycogenolysis. This effect of glucose on polysaccharide synthesis is progressively lost. The addition of insulin to the culture induces after 48 and 72 h, a three- to fivefold increase of the glucose incorporation into glycogen, as compared to the controls. The presence of insulin is required to maintain the hepatocyte's capacity to store glycogen. Glycogen synthetase is converted into its active form under the influence of glucose. Insulin increases the rate of activation.

Morphological and biochemical analyses of changes in rat hepatocytes related to wine and ethanol consumption

1984 ◽  
Vol 42 ◽  
pp. 232-233
Author(s):  
S.M. Geyer ◽  
C.L. Mendenhall ◽  
J.T. Hung ◽  
E.L. Cardell ◽  
R.L. Drake ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Enzyme Activity ◽  
Malic Enzyme ◽  
Smooth Endoplasmic Reticulum ◽  
Rat Hepatocytes ◽  
Mature Male ◽  
Treatment Groups ◽  
Malic Enzyme Activity ◽  
Biochemical Analyses

Thirty-three mature male Holtzman rats were randomly placed in 3 treatment groups: Controls (C); Ethanolics (E); and Wine drinkers (W). The animals were fed synthetic diets (Lieber type) with ethanol or wine substituted isocalorically for carbohydrates in the diet of E and W groups, respectively. W received a volume of wine which provided the same gram quantity of alcohol consumed by E. The animals were sacrificed by decapitation after 6 weeks and the livers processed for quantitative triglycerides (T3), proteins, malic enzyme activity (MEA), light microscopy (LM) and electron microscopy (EM). Morphometric analysis of randomly selected LM and EM micrographs was performed to determine organellar changes in centrilobular (CV) and periportal (PV) regions of the liver. This analysis (Table 1) showed that hepatocytes from E were larger than those in C and W groups. Smooth endoplasmic reticulum decreased in E and increased in W compared to C values.

scholarly journals DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES

1971 ◽  
Vol 49 (2) ◽  
pp. 264-287 ◽  
Author(s):  
A. Leskes ◽  
P. Siekevitz ◽  
G. E. Palade
Keyword(s):  
Endoplasmic Reticulum ◽  
Phosphatase Activity ◽  
Rough Endoplasmic Reticulum ◽  
Specific Activity ◽  
Smooth Endoplasmic Reticulum ◽  
Rat Hepatocytes ◽  
Adult Level ◽  
Enzyme Specific Activity ◽  
Cytochemical Reaction ◽  
And Control

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.

Trypan blue dye uptake and lactate dehydrogenase in adult rat hepatocytes—Freshly isolated cells, cell suspensions, and primary monolayer cultures

In Vitro ◽  
1981 ◽  
Vol 17 (12) ◽  
pp. 1100-1110 ◽  
Author(s):  
Hugo O. Jauregui ◽  
Nancy T. Hayner ◽  
James L. Driscoll ◽  
Rhonda Williams-Holland ◽  
Milton H. Lipsky ◽  
...  
Keyword(s):  
Trypan Blue ◽  
Rat Hepatocytes ◽  
Blue Dye ◽  
Dye Uptake ◽  
Isolated Cells ◽  
Adult Rat ◽  
Monolayer Cultures ◽  
Adult Rat Hepatocytes ◽  
Primary Monolayer ◽  
Primary Monolayer Cultures

Biogenesis of Smooth Endoplasmic Reticulum in Hepatocytes of Phenobarbital Treated Rats

1974 ◽  
Vol 32 ◽  
pp. 308-309
Author(s):  
Joan A. Higgins
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Specific Activity ◽  
Smooth Endoplasmic Reticulum ◽  
Rat Hepatocytes ◽  
Membrane Formation ◽  
Drug Detoxification ◽  
Marked Proliferation

In response to intraperitoneal injections of phenobarbital there is a marked proliferation of smooth endoplasmic reticulum membranes (s.e.r.) of rat hepatocytes, with little change in other membranous organelles. This increased membrane formation is accompanied by a rise in the specific activity of the enzymes involved in drug detoxification initially in the rough endoplasmic reticulum (r.e.r.) followed by a rise in the s.e.r. There is also an increased accumulation of glycerophospholipid in the newly formed s.e.r.

Implication of mitochondrial dysfunction and cell death in cold preservation – warm reperfusion-induced hepatocyte injury

2006 ◽  
Vol 84 (5) ◽  
pp. 547-554 ◽  
Author(s):  
Marjolaine Duval ◽  
Catherine Plin ◽  
Aziz Elimadi ◽  
Diane Vallerand ◽  
Jean-Paul Tillement ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Cytochrome C ◽  
Mitochondrial Dysfunction ◽  
Isolated Hepatocytes ◽  
Mitochondrial Pathway ◽  
Liver Graft ◽  
Isolated Cells ◽  
Hepatocyte Injury ◽  
Effector Caspase

Cold ischemia – warm reperfusion (CI/WR) injury of liver transplantation involves hepatocyte cell death, the nature and underlying mechanisms of which remain unclear. Isolated hepatocytes and isolated perfused livers were used to determine the prevalence of necrosis and apoptosis as well as mitochondrial dysfunction. In isolated cells, propidium iodide and Hoechst 33342 staining showed a cold-storage, time-dependent increase in necrosis, whereas apoptosis was minimal even after 48 h of hypothermia. Nonetheless, a progressive loss of mitochondrial membrane potential was observed. Translocation of mitochondrial cytochrome c toward microsomes occurred within 24 h of CI/WR, with cytochrome c reaching the cytosol later. Mitochondria isolated from whole livers subjected to CI/WR also display reduced metabolic parameters and increased susceptibility to swelling. These events are associated with increased activity of major initiator (caspase 9) and effector (caspase 3) caspases. The results demonstrate that CI/WR induces mitochondrial dysfunction in isolated cells and in the whole organ; only in the latter is that sufficient to trigger the classical mitochondrial pathway of apoptosis. Our study also provides evidence for the involvement of endoplasmic reticulum stress in CI/WR hepatocyte injury. Combined protection of mitochondria and endoplasmic reticulum may thus represent an innovative therapeutic avenue to enhance liver graft viability and functional integrity.

Hormonal induction of malic enzyme in rat hepatocytes cultured on laminin-rich gels

1992 ◽  
Vol 8 (3) ◽  
pp. 235-242 ◽  
Author(s):  
D. J. Mann ◽  
A. J. Strain ◽  
E. Bailey
Keyword(s):  
Malic Enzyme ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Specific Expression ◽  
Adult Rat ◽  
Adult Rat Hepatocytes ◽  
Species Being ◽  
Rat Tail

ABSTRACT The levels of malic-enzyme mRNA and activity were determined in primary cultures of adult rat hepatocytes maintained on either rat-tail collagen or a laminin-rich substratum. Cells plated on laminin-rich gels exhibited substantially improved patterns of albumin and malic-enzyme expression when compared with cells maintained on rat-tail collagen. Moreover, hepatocytes plated on the laminin-rich matrix displayed marked malic-enzyme inducibility in response to tri-iodothyronine and dichloroacetate, especially in the presence of insulin. However, Northern blot analysis revealed that the ratio of the amounts of the two major malic-enzyme mRNA species (2.0 and 3.1 kb) was reversed when compared with that found in the liver in vivo, the altered levels of these two species being closer to those found in non-hepatic tissues. These findings indicate that, although the hormonal responsiveness of isolated hepatocytes maintained on laminin-rich gels is markedly improved, and approaches the degree of induction demonstrated in the liver in vivo, the mechanisms of control differ, indicating a loss of liver-specific expression.

scholarly journals Studies on the glycosylation of hydroxylysine residues during collagen biosynthesis and the subcellular localization of collagen galactosyltransferase and collagen glucosyltransferase in tendon and cartilage cells

1975 ◽  
Vol 152 (2) ◽  
pp. 291-302 ◽  
Author(s):  
Richard Harwood ◽  
Michael E. Grant ◽  
David S. Jackson
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Specific Activity ◽  
Smooth Endoplasmic Reticulum ◽  
Gradient Centrifugation ◽  
Subcellular Fractions ◽  
Anterior Lens Capsule ◽  
Cartilage Cells ◽  
Membrane Bound ◽  
And Cartilage

1. The glycosylation of hydroxylysine during the biosynthesis of procollagen by embryonic chick tendon and cartilage cells was examined. When free and membrane-bound ribosomes isolated from cells labelled for 4min with [14C]lysine were assayed for hydroxy[14C]lysine and hydroxy[14C]lysine glycosides, it was found that hydroxylation took place only on membrane-bound ribosomes and that some synthesis of galactosylhydroxy[14C]lysine and glucosylgalactosylhydroxy[14C]lysine had occurred on the nascent peptides. 2. Assays of subcellular fractions isolated from tendon and cartilage cells labelled for 2h with [14C]lysine demonstrated that the glycosylation of procollagen polypeptides began in the rough endoplasmic reticulum. 14C-labelled polypeptides present in the smooth endoplasmic reticulum and Golgi fractions were glycosylated to extents almost identical with the respective secreted procollagens. 3. Assays specific for collagen galactosyltransferase and collagen glucosyltransferase are described, using as substrate chemically treated bovine anterior-lens-capsule collagen. 4. When homogenates were assayed for the collagen glycosyltransferase activities, addition of Triton X-100 (0.01%, w/v) was found to stimulate enzyme activities by up to 45%, suggesting that the enzymes were probably membrane-bound. 5. Assays of subcellular fractions obtained by differential centrifugation for collagen galactosyltransferase activity indicated the specific activity to be highest in the microsomal fractions. Similar results were obtained for collagen glucosyltransferase activity. 6. When submicrosomal fractions obtained by discontinuous-sucrose-density-gradient-centrifugation procedures were assayed for these enzymic activities, the collagen galactosyltransferase was found to be distributed in the approximate ratio 7:3 between rough and smooth endoplasmic reticulum of both cell types. Similar determinations of collagen glucosyltransferase indicated a distribution in the approximate ratio 3:2 between rough and smooth microsomal fractions. 7. Assays of subcellular fractions for the plasma-membrane marker 5′-nucleotidase revealed a distribution markedly different from the distributions obtained for the collagen glycosyltransferase. 8. The studies described here demonstrate that glycosylation occurs early in the intracellular processing of procollagen polypeptides rather than at the plasma membrane, as was previously suggested.

Les systèmes membranaires de Scopulariopsis brevicaulis. II. Étude comparée de leur composition lipidique

1987 ◽  
Vol 33 (1) ◽  
pp. 78-81 ◽  
Author(s):  
Jean-Louis Fonvieille ◽  
Jane-Marie Touze-Soulet ◽  
Charles Montant
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Smooth Endoplasmic Reticulum ◽  
Gradient Centrifugation ◽  
Percoll Gradient ◽  
Mitochondrial Membranes ◽  
Isolation Method ◽  
Discontinuous Sucrose Gradient ◽  
Phospholipid Ratio ◽  
Scopulariopsis Brevicaulis

Biochemical studies of Scopulariopsis brevicaulis membranes (except mitochondrial membranes), in particular their lipid composition, were undertaken using plasma membranes and fractions containing smooth endoplasmic reticulum and Golgi apparatus membranes. The results obtained by two separation techniques, a continuous Percoll gradient and a discontinuous sucrose gradient centrifugation, were similar. Depending on the isolation method used, the plasma membrane contained 30 or 33% protein and 23 or 30% lipid of which 16.5 or 18% was phospholipid. The sterol/phospholipid ratio was 1.98 or 2.28. The endoplasmic reticulum + Golgi fraction contained 21 or 26.7% protein and 43 or 50% lipid of which 7 or 10% was phospholipid. The sterol/phospholipid ratio was 0.85 or 0.81.

scholarly journals Biogenesis of peroxisomes: immunocytochemical investigation of peroxisomal membrane proteins in proliferating rat liver peroxisomes and in catalase-negative membrane loops.

1989 ◽  
Vol 108 (6) ◽  
pp. 2221-2231 ◽  
Author(s):  
E Baumgart ◽  
A Völkl ◽  
T Hashimoto ◽  
H D Fahimi
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Smooth Endoplasmic Reticulum ◽  
Gradient Centrifugation ◽  
Normal Liver ◽  
Membrane System ◽  
Peroxisomal Membrane ◽  
Lowicryl K4m ◽  
Membrane Reservoir ◽  
Liver Peroxisomes

Treatment of rats with a new hypocholesterolemic drug BM 15766 induces proliferation of peroxisomes in pericentral regions of the liver lobule with distinct alterations of the peroxisomal membrane (Baumgart, E., K. Stegmeier, F. H. Schmidt, and H. D. Fahimi. 1987. Lab. Invest. 56:554-564). We have used ultrastructural cytochemistry in conjunction with immunoblotting and immunoelectron microscopy to investigate the effects of this drug on peroxisomal membranes. Highly purified peroxisomal fractions were obtained by Metrizamide gradient centrifugation from control and treated rats. Immunoblots prepared from such peroxisomal fractions incubated with antibodies to 22-, 26-, and 70-kD peroxisomal membrane proteins revealed that the treatment with BM 15766 induced only the 70-kD protein. In sections of normal liver embedded in Lowicryl K4M, all three membrane proteins of peroxisomes could be localized by the postembedding technique. The strongest labeling was obtained with the 22-kD antibody followed by the 70-kD and 26-kD antibodies. In treated animals, double-membraned loops with negative catalase reaction in their lumen, resembling smooth endoplasmic reticulum segments as well as myelin-like figures, were noted in the proximity of some peroxisomes. Serial sectioning revealed that the loops seen at some distance from peroxisomes in the cytoplasm were always continuous with the peroxisomal membranes. The double-membraned loops were consistently negative for glucose-6-phosphatase, a marker for endoplasmic reticulum, but were distinctly labeled with antibodies to peroxisomal membrane proteins. Our observations indicate that these membranous structures are part of the peroxisomal membrane system. They could provide a membrane reservoir for the proliferation of peroxisomes and the expansion of this intracellular compartment.

Sign in / Sign up

Export Citation Format

Share Document