المؤلفون: Hamid R. Samari, Lise Holden, Michael T. N. Møller, Tonje Asmyhr, Per Ottar Seglen

المصدر: The Biochemical journal. 386(Pt 2)

مصطلحات موضوعية: Male, Microcystins, Phosphatase, Electrophoretic Mobility Shift Assay, AMP-Activated Protein Kinases, Protein Serine-Threonine Kinases, Biochemistry, Peptides, Cyclic, chemistry.chemical_compound, AMP-activated protein kinase, Multienzyme Complexes, Okadaic Acid, Autophagy, Animals, Amino Acids, Enzyme Inhibitors, Phosphorylation, Rats, Wistar, Protein kinase A, Molecular Biology, Toxins, Biological, biology, Chemistry, AMPK, Cell Biology, Okadaic acid, Ribonucleotides, Aminoimidazole Carboxamide, Cell biology, Rats, Enzyme Activation, Protein Subunits, Flavanones, biology.protein, Tautomycin, Research Article

الوصف: Autophagic activity in isolated rat hepatocytes is strongly suppressed by OA (okadaic acid) and other PP (protein phosphatase)-inhibitory toxins as well as by AICAR (5-aminoimidazole-4-carboxamide riboside), a direct activator of AMPK (AMP-activated protein kinase). To investigate whether AMPK is a mediator of the effects of the toxin, a phosphospecific antibody directed against the activation of phosphorylation of the AMPK alpha (catalytic)-subunit at Thr172 was used to assess the activation status of this enzyme. AICAR as well as all the toxins tested (OA, microcystin-LR, calyculin A, cantharidin and tautomycin) induced strong, dose-dependent AMPKalpha phosphorylation, correlating with AMPK activity in situ (in intact hepatocytes) as measured by the AMPK-dependent phosphorylation of acetyl-CoA carboxylase at Ser79. All treatments induced the appearance of multiple, phosphatase-sensitive, low-mobility forms of the AMPK alpha-subunit, consistent with phosphorylation at several sites other than Thr172. The flavonoid naringin, an effective antagonist of OA-induced autophagy suppression, inhibited the AMPK phosphorylation and mobility shifting induced by AICAR, OA or microcystin, but not the changes induced by calyculin A or cantharidin. AMPK may thus be activated both by a naringin-sensitive and a naringin-resistant mechanism, probably involving the PPs PP2A and PP1 respectively. Neither the Thr172-phosphorylating protein kinase LKB1 nor the Thr172-dephosphorylating PP, PP2C, were mobility-shifted after treatment with toxins or AICAR, whereas a slight mobility shifting of the regulatory AMPK beta-subunit was indicated. Immunoblotting with a phosphospecific antibody against pSer108 at the beta-subunit revealed a naringin-sensitive phosphorylation induced by OA, microcystin and AICAR and a naringin-resistant phosphorylation induced by calyculin A and cantharidin, suggesting that beta-subunit phosphorylation could play a role in AMPK activation. Naringin antagonized the autophagy-suppressive effects of AICAR and OA, but not the autophagy suppression caused by cantharidin, consistent with AMPK-mediated inhibition of autophagy by toxins as well as by AICAR.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::61da67fee638da6847323507e6b4c034Test
https://pubmed.ncbi.nlm.nih.gov/15461583Test

المؤلفون: Per E. Stromhaug, Trond Berg, Kristian Berg, Per Ottar Seglen

المصدر: The Biochemical journal. 321

مصطلحات موضوعية: Male, Radiation-Sensitizing Agents, Porphyrins, Endosome, Biochemistry, chemistry.chemical_compound, Cytosol, In vivo, Lactate dehydrogenase, Phagosomes, Animals, Rats, Wistar, Molecular Biology, chemistry.chemical_classification, biology, Leupeptin, Autophagy, Acid phosphatase, Cell Biology, Rats, Enzyme, chemistry, Liver, biology.protein, Lysosomes, Subcellular Fractions, Research Article

الوصف: A photoactivatable porphyrin, tetra(4-sulphonatophenyl)porphine (TPPS4), was shown to accumulate in rat hepatocytes as a linear function of dose after intravenous injection, and to localize predominantly in hepatocytic lysosomes. A major fraction of the lysosomal enzymes acid phosphatase and N-acetyl-β-d-glucosaminidase was inactivated by TPPS4 after 20 h of contact with the drug in vivo in the absence of photoactivation. On exposure of isolated hepatocytes to light, photoactivated TPPS4 caused additional inactivation of the lysosomal enzymes as well as inactivation of intralysosomal lactate dehydrogenase (LDH), a cytosolic enzyme that accumulated in lysosomes as a result of autophagy during a 2 h incubation of hepatocytes at 37 °C in the dark (in the presence of the proteinase inhibitor leupeptin to prevent degradation of intralysosomal LDH). Photoactivation of TPPS4 also induced lysosomal rupture, with a loss of lysosomal enzymes, autophagocytosed LDH, endocytosed 125I-tyramine-cellobiose-asialo-orosomucoid and TPPS4 from the lysosomes. However, LDH-containing autophagosomes, accumulated in the presence of vinblastine (a microtubule inhibitor used to prevent the fusion of lysosomes with autophagosomes or endosomes), were not affected by TPPS4. TPPS4 may thus be useful as a selective lysosomal (or endosomal) perturbant in the study of autophagic–endocytic–lysosomal interactions.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::1cdec3921b82f8902d0f2be55b1d2fd7Test
https://pubmed.ncbi.nlm.nih.gov/9003422Test

المؤلفون: Per Ottar Seglen, P. B. Gordon, H. Hoyvik

المصدر: The Biochemical journal. 283

مصطلحات موضوعية: Male, Sucrose, Glycoside Hydrolases, Endocytic cycle, Vacuole, Biology, Endocytosis, Tritium, Vinblastine, Biochemistry, chemistry.chemical_compound, Raffinose, Lysosome, medicine, Autophagy, Animals, Asparagine, Carbon Radioisotopes, Molecular Biology, Cells, Cultured, beta-Fructofuranosidase, Rats, Inbred Strains, Cell Biology, Cell biology, Rats, Kinetics, Invertase, medicine.anatomical_structure, chemistry, Liver, Lysosomes, Research Article

الوصف: In isolated rat hepatocytes electroloaded with [14C]sucrose, autophaged sugar accumulated in lysosomes under control conditions, and in prelysosomal autophagic vacuoles (amphisomes) in the presence of asparagine, an inhibitor of autophagic-lysosomal fusion. Endocytic uptake of the sucrose-cleaving enzyme invertase resulted in rapid and complete degradation of autophaged sucrose in both amphisomes and lysosomes. Pre-accumulated sucrose was degraded equally well in both compartments, regardless of amphisomal-lysosomal flux inhibition by asparagine, suggesting that endocytic entry into the autophagic pathway can take place both at the lysosomal and at the amphisomal level. The completeness of sucrose degradation by endocytosed invertase furthermore indicates that all lysosomes involved in autophagy can also engage in endocytosis. Endocytosed invertase reached the amphisomes even when autophagy was blocked by 3-methyladenine, and autophaged sucrose reached this compartment even when endocytic influx was blocked by vinblastine, suggesting that amphisomes may exhibit some degree of permanence independently of either pathway.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::593cdfa4a315c24d76d168ff441cd148Test
https://pubmed.ncbi.nlm.nih.gov/1575680Test

المؤلفون: Helge Tolleshaug, P. B. Gordon, Per Ottar Seglen

المصدر: The Biochemical journal. 232(3)

مصطلحات موضوعية: Male, Sucrose, Liver cytology, Digitonin, Mitochondria, Liver, Mitochondrion, Biology, In Vitro Techniques, Biochemistry, chemistry.chemical_compound, Phagocytosis, Lysosome, medicine, Autophagy, Animals, Amino Acids, Molecular Biology, Phagosome, Adenine, Rats, Inbred Strains, Cell Biology, Rats, medicine.anatomical_structure, chemistry, Liver, Hepatocyte, Lysosomes, Research Article

الوصف: In isolated rat hepatocytes, electroinjected [14C]sucrose is sequestered both by mitochondria and by autophagosomes/lysosomes. Radioactivity can be selectively extracted from the latter organelles by low concentrations of digitonin, thereby providing a specific bioassay for autophagic sequestration. By including a digitonin extraction step in the assay procedure, autophagic [14C]sucrose sequestration could be shown to be virtually completely (greater than 90%) suppressed by the autophagy inhibitor 3-methyladenine (10 mM), whereas mitochondrial sugar uptake was unaffected. An amino acid mixture likewise suppressed autophagic sequestration very strongly, while having no detectable effect on the mitochondria.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::a2900ed63a7bd6c0117e4728b666452fTest
https://pubmed.ncbi.nlm.nih.gov/4091820Test