scholarly journals Purification and characterization of autophagosomes from rat hepatocytes

1998 ◽  
Vol 335 (2) ◽  
pp. 217-224 ◽  
Author(s):  
Per Eivind STRØMHAUG ◽  
Trond Olav BERG ◽  
Monica FENGSRUD ◽  
Per O. SEGLEN
Keyword(s):  
Endoplasmic Reticulum ◽  
Density Gradient ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Protein Markers ◽  
Intact Cells ◽  
Cytoskeletal Elements ◽  
Intermediate Density ◽  
Glucose Regulated Protein

To investigate the properties and intracellular origin of autophagosomes, a procedure for the purification and isolation of these organelles from rat liver has been developed. Isolated hepatocytes were incubated with vinblastine to induce autophagosome accumulation; the cells were then homogenized and treated with the cathepsin C substrate glycyl-l-phenylalanine 2-naphthylamide to cause osmotic disruption of the lysosomes. Nuclei were removed by differential centrifugation, and the postnuclear supernatant was fractionated on a discontinuous Nycodenz density gradient. The autophagosomes, recognized by their content of autophagocytosed lactate dehydrogenase (LDH), could be recovered in an intermediate-density fraction, free from cytosol and mitochondria. Finally, the autophagosomes were separated from the endoplasmic reticulum and other membranous elements by centrifugation in a Percoll colloidal density gradient, followed by flotation in iodixanol to remove the Percoll particles. The final autophagosome preparation represented a 24-fold purification of autophagocytosed LDH relative to intact cells, with a 12% recovery. The purified autophagosomes contained sequestered cytoplasm with a normal ultrastructure, including mitochondria, peroxisomes and endoplasmic reticulum in the same proportions as in intact cells. However, immunoblotting indicated a relative absence of cytoskeletal elements (tubulin, actin and cytokeratin), which may evade autophagic sequestration. The autophagosomes showed no enrichment in protein markers typical of lysosomes (acid phosphatase, cathepsin B, lysosomal glycoprotein of 120 kDa), endosomes (early-endosome-associated protein 1, cation-independent mannose 6-phosphate receptor, asialoglycoprotein receptor) or endoplasmic reticulum (esterase, glucose-regulated protein of 78 kDa, protein disulphide isomerase), suggesting that the sequestering membranes are not derived directly from any of these organelles, but rather represent unique organelles (phagophores).

A characterization of heam uptake and intracellular distribution by isolated hepatocytes

1985 ◽  
Vol 5 (7) ◽  
pp. 609-614 ◽  
Author(s):  
A. Lodola
Keyword(s):  
Endoplasmic Reticulum ◽  
Specific Binding ◽  
Isolated Hepatocytes ◽  
Intracellular Distribution ◽  
Distribution Data ◽  
Temperature Dependent ◽  
Rat Hepatocyte ◽  
Isolated Rat ◽  
Endoplasmic Reticulum Fraction

The uptake and intracellular distribution of haem by isolated rat hepatocyte suspensions was studied. An increase in cell haem content occurred after a challenge with 5, 10 or 20 μM haem, supplied as methaemalbumin. The rate of haem uptake was temperature dependent; no non-specific binding occurred. Intracellular haem distribution data are consistent with a rapid association of haem with the endoplasmic reticulum fraction prior to its accumulation in the cytosol and at the mitochondrion.

scholarly journals Characterization of the liver P2-purinoceptor involved in the activation of glycogen phosphorylase

1986 ◽  
Vol 240 (2) ◽  
pp. 367-371 ◽  
Author(s):  
S Keppens ◽  
H De Wulf
Keyword(s):  
Binding Sites ◽  
Glycogen Phosphorylase ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Rank Order ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
P2 Purinoceptors

Evidence has been presented for the existence in rat liver of P2-purinoceptors which are involved in the control of glycogenolysis. Isolated rat hepatocytes and purified liver plasma membranes have been used to study the binding of the ATP analogue adenosine 5′-[alpha- [35S]thio]triphosphate (ATP alpha [35S]) to these postulated P2-purinoceptors. The nucleotide analogue behaves as a full agonist for the activation of glycogen phosphorylase in isolated hepatocytes, 0.3 microM being required for half-maximal activation. Specific binding of ATP alpha [35S] to hepatocytes and plasma membranes occurs within 1 min and is essentially reversible. The analysis of the dose-dependency at equilibrium indicates the presence of binding sites with Kd of 0.23 microM with hepatocytes and Kd of 0.11 microM with plasma membranes. The relative affinities of 10 nucleotide analogues were deduced from competition experiments for ATP alpha [35S] binding to hepatocytes, and these correlated highly with their biological activity (activation of glycogen phosphorylase in hepatocytes). For all the agonists, binding occurs in the same concentration range as the biological effect. These data clearly suggest that the detected binding sites correspond to the physiological P2-purinoceptors involved in the regulation of liver glycogenolysis. The rank order of potency of some ATP analogues suggests that liver possesses the P2Y-subclass of P2-purinoceptors.

Characterization of glucagon and catecholamine effects on isolated sheep hepatocytes

1988 ◽  
Vol 255 (4) ◽  
pp. R539-R546
Author(s):  
C. Morand ◽  
C. Yacoub ◽  
C. Remesy ◽  
C. Demigne
Keyword(s):  
Rat Liver ◽  
Glycogen Phosphorylase ◽  
Plasma Membranes ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Cyclic Monophosphate ◽  
Sheep Liver ◽  
Beta Agonists ◽  
Rat Liver Cells

The purpose of this study was to characterize the glycogenolytic response to catecholamines and glucagon in isolated sheep hepatocytes. In this species, epinephrine appeared to exert its action on hepatic glycogenolysis by altering the cytosolic concentrations of both adenosine 3',5'-cyclic monophosphate (cAMP) and Ca2+. In contrast to results obtained in rat hepatocytes, glucagon failed to induce a rise in free cytosolic Ca2+ in sheep liver. Experiments on isolated hepatocytes or on liver plasma membranes showed that in sheep, glucagon was more efficient than epinephrine in promoting the production of cAMP. In the presence of glucagon or epinephrine, the activation of the glycogen phosphorylase a always appeared greater in sheep than in rat liver cells, whereas the variations in cellular cAMP were quite limited in sheep. The alpha 1- and beta-agonists (phenylephrine and isoproterenol) were alone as efficient as epinephrine in promoting phosphorylase a activation in sheep hepatocytes. All these results indicate the existence in sheep liver of a glycogen phosphorylase highly responsive to hormones.

scholarly journals Effects of analogues of ethanolamine and choline on phospholipid metabolism in rat hepatocytes

1977 ◽  
Vol 168 (3) ◽  
pp. 401-408 ◽  
Author(s):  
Björn Åkesson
Keyword(s):  
Molecular Species ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Phospholipid Metabolism ◽  
Control Points ◽  
Phospholipid Synthesis ◽  
Intact Cells

1. Analogues of ethanolamine and choline were incubated with different labelled precursors of phospholipids and isolated hepatocytes and the effects on phospholipid synthesis were studied. 2. 2-Aminopropan-1-ol and 2-aminobutan-1-ol were the most efficient inhibitors of [14C]ethanolamine incorporation into phospholipids, whereas the incorporation of [3H]choline was inhibited most extensively by NN-diethylethanolamine and NN-dimethylethanolamine. 3. When the analogues were incubated with [3H]glycerol and hepatocytes, the appearance of 3H in unnatural phospholipids indicated that they were incorporated, at least in part, via CDP-derivatives. The distribution of [3H]glycerol among molecular species of phospholipids containing 2-aminopropan-1-ol and 1-aminopropan-2-ol was the same as in phosphatidylethanolamine. In other phospholipid analogues the distribution of 3H was more similar to that in phosphatidylcholine. 4. NN-Diethylethanolamine stimulated both the conversion of phosphatidylethanolamine into phosphatidylcholine and the incorporation of [Me-14C]methionine into phospholipids. Other N-alkyl- or NN-dialkyl-ethanolamines also stimulated [14C]methionine incorporation, but inhibited the conversion of phosphatidylethanolamine into phosphatidylcholine. This indicates that phosphatidyl-NN-diethylethanolamine is a poor methyl acceptor, in contrast with other N-alkylated phosphatidylethanolamines. 5. These results on the regulation of phospholipid metabolism in intact cells are discussed with respect to the possible control points. They also provide guidelines for future experiments on the manipulation of phospholipid polar-headgroup composition in primary cultures of hepatocytes.

scholarly journals Characterization of molecules involved in protein translocation using a specific antibody.

1982 ◽  
Vol 92 (2) ◽  
pp. 579-583 ◽  
Author(s):  
D I Meyer ◽  
D Louvard ◽  
B Dobberstein
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Single Species ◽  
Ultrastructural Level ◽  
Subsequent Treatment ◽  
Intact Cells ◽  
Membrane Bound ◽  
Single Polypeptide

The vectorial translocation of nascent proteins through the membrane of the rough endoplasmic reticulum has been shown to require a specific membrane-bound protein whose cytoplasmic domain can be proteolytically cleaved and isolated as an active peptide of mol wt 60,000 (Meyer and Dobberstein, 1980, J. Cell Biol. 87:503-508). Rabbit antibodies raised against this peptide were used to further characterize the membrane-bound molecule. Immunoprecipitation of solubilized, radiolabeled rough microsomal proteins yielded a single polypeptide of mol wt 72,000, representing the membrane-bound protein from which the 60,000-mol wt peptide was proteolytically derived. The antibody could also be used to remove exclusively the 60,000-mol wt peptide, and thus the translocation activity, from elastase digests tested in a reconstituted system. Moreover, immunoprecipitation of elastase extracts alkylated with [14C] N-ethylmaleimide selected a single species of mol wt 60,000. Immunoprecipitation of in vivo radiolabeled proteins from the appropriate cell type yielded the 72,000-mol wt membrane protein irrespective of the duration of labeling, or if followed by a chase. Subsequent treatment with protease generated the 60,000-mol wt fragment. In addition, the antibody could be used to visualize reticular structures in intact cells which correspond to endoplasmic reticulum at the ultrastructural level. It is thus clear that one membrane component required in the vectorial translocation of nascent secretory (and membrane) proteins is a peptide of mol wt 72,000.

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.

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.

Sign in / Sign up

Export Citation Format

Share Document