okadaic-acid and naringin

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.

Topics: Amino Acids; Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Autophagy; Electrophoretic Mobility Shift Assay; Enzyme Activation; Enzyme Inhibitors; Flavanones; Male; Microcystins; Multienzyme Complexes; Okadaic Acid; Peptides, Cyclic; Phosphorylation; Protein Serine-Threonine Kinases; Protein Subunits; Rats; Rats, Wistar; Ribonucleotides; Toxins, Biological

Glycine N-methyltransferase (GNMT) is an abundant cytosolic enzyme that catalyses the methylation of glycine into sarcosine, coupled with conversion of the methyl donor, S -adenosylmethionine (AdoMet), into S -adenosylhomocysteine (AdoHcy). GNMT is believed to play a role in monitoring the AdoMet/AdoHcy ratio, and hence the cellular methylation capacity, but regulation of the enzyme itself is not well understood. In the present study, treatment of isolated rat hepatocytes with the protein phosphatase inhibitor okadaic acid, was found to induce an overphosphorylation of GNMT, as shown by proteomic analysis. The analysis comprised two-dimensional gel electrophoretic separation of (32)P-labelled phosphoproteins and identification of individual protein spots by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. The identity of GNMT was verified by N-terminal Edman sequencing of tryptic peptides. Chromatographic separation of proteolytic peptides and (32)P-labelled amino acids suggested that GNMT was phosphorylated within a limited region, and only at serine residues. GNMT phosphorylation could be suppressed by naringin, an okadaic acid-antagonistic flavonoid. To assess the possible functional role of GNMT phosphorylation, the effect of okadaic acid on hepatocytic AdoMet and AdoHcy levels was examined, using HPLC separation for metabolite analysis. Surprisingly, okadaic acid was found to have no effect on the basal levels of AdoMet or AdoHcy. An accelerated AdoMet-AdoHcy flux, induced by the addition of methionine (1 mM), was likewise unaffected by okadaic acid. 5-Aminoimidazole-4-carboxamide riboside, an activator of the hepatocytic AMP-activated protein kinase, similarly induced GNMT phosphorylation without affecting AdoMet and AdoHcy levels. Activation of cAMP-dependent protein kinase by dibutyryl-cAMP, reported to cause GNMT phosphorylation under cell-free conditions, also had little effect on hepatocytic AdoMet and AdoHcy levels. Phosphorylation of GNMT would thus seem to play no role in regulation of the intracellular AdoMet/AdoHcy ratio, but could be involved in other GNMT functions, such as the binding of folates or aromatic hydrocarbons.

Topics: Aminoimidazole Carboxamide; Animals; Bucladesine; Cell-Free System; Enzyme Inhibitors; Flavanones; Flavonoids; Glycine; Glycine N-Methyltransferase; Hepatocytes; Methyltransferases; Okadaic Acid; Peptide Fragments; Phosphorylation; Rats; Ribonucleosides; S-Adenosylhomocysteine; S-Adenosylmethionine

To identify phosphoproteins that might play a role in naringin-sensitive hepatocellular cytoskeletal disruption and apoptosis induced by algal toxins, hepatocyte extracts were separated by gel electrophoresis and immunostained with a phosphothreonine-directed antibody. Use of dilute (5%) polyacrylamide gels containing 6 m urea allowed the resolution of one very large (approximately 500-kDa) okadaic acid- and naringin-sensitive phosphoprotein, identified by tryptic fingerprinting, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and immunostaining as the cytolinker protein, plectin. The naringin-sensitive phosphorylation induced by okadaic acid and microcystin-LR probably reflected inhibition of a type 2A protein phosphatase, whereas the naringin-resistant phosphorylation induced by calyculin A, tautomycin, and cantharidin probably involved a type 1 phosphatase. Okadaic acid caused a collapse of the plectin-immunostaining bile canalicular sheaths and the general cytoskeletal plectin network into numerous medium-sized plectin aggregates. Inhibitors of protein kinase C, cAMP-dependent protein kinase, or Ca(2+)/calmodulin-dependent kinase II had moderate or no protective effects on plectin network disruption, whereas naringin offered 86% protection. Okadaic acid induced a naringin-sensitive phosphorylation of AMP-activated protein kinase (AMPK), the stress-activated protein kinases SEK1 and JNK, and S6 kinase. The AMPK-activating kinase (AMPKK) is likely to be the target of inhibition by naringin, the other kinases serving as downstream components of an AMPKK-initiated signaling pathway.

Topics: Animals; Enzyme Inhibitors; Flavanones; Flavonoids; Hepatocytes; Intermediate Filament Proteins; Okadaic Acid; Peptide Mapping; Phosphorylation; Plectin; Protein Kinase Inhibitors; Rats; Rats, Wistar; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

The protein phosphatase-inhibitory algal toxins, okadaic acid and microcystin-LR, induced overphosphorylation of keratin and disruption of the keratin cytoskeleton in freshly isolated rat hepatocytes. In hepatocyte cultures, the toxins elicited DNA fragmentation and apoptotic cell death within 24 h. All these toxin effects could be prevented by the grapefruit flavonoid, naringin. The cytoprotective effect of naringin was apparently limited to normal hepatocytes, since the toxin-induced apoptosis of hepatoma cells, rat or human, was not prevented by the flavonoid.

Topics: Animals; Antioxidants; Apoptosis; Bacterial Toxins; Cell Death; Cells, Cultured; Cytoskeleton; Flavanones; Flavonoids; Fruit; Humans; Liver; Marine Toxins; Microcystins; Okadaic Acid; Peptides, Cyclic; Phosphoprotein Phosphatases; Rats; Rats, Wistar; Tumor Cells, Cultured

The regional selectivity and mechanisms underlying the toxicity of the serine/threonine protein phosphatase inhibitor okadaic acid (OA) were investigated in hippocampal slice cultures. Image analysis of propidium iodide-labeled cultures revealed that okadaic acid caused a dose- and time-dependent injury to hippocampal neurons. Pyramidal cells in the CA3 region and granule cells in the dentate gyrus were much more sensitive to okadaic acid than the pyramidal cells in the CA1 region. Electron microscopy revealed ultrastructural changes in the pyramidal cells that were not consistent with an apoptotic process. Treatment with okadaic acid led to a rapid and sustained tyrosine phosphorylation of the mitogen-activated protein kinases ERK1 and ERK2 (p44/42(mapk)). The phosphorylation was markedly reduced after treatment of the cultures with the microbial alkaloid K-252a (a nonselective protein kinase inhibitor) or the MAP kinase kinase (MEK1/2) inhibitor PD98059. K-252a and PD98059 also ameliorated the okadaic acid-induced cell death. Inhibitors of protein kinase C, Ca2+/calmodulin-dependent protein kinase II, or tyrosine kinase were ineffective. These results indicate that sustained activation of the MAP kinase pathway, as seen after e.g., ischemia, may selectively harm specific subsets of neurons. The susceptibility to MAP kinase activation of the CA3 pyramidal cells and dentate granule cells may provide insight into the observed relationship between cerebral ischemia and dementia in Alzheimer's disease.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Antioxidants; Apoptosis; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Enzyme Inhibitors; Flavanones; Flavonoids; Genistein; Hippocampus; Indole Alkaloids; Male; Microscopy, Electron; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nerve Degeneration; Neurons; Okadaic Acid; Organ Culture Techniques; Phosphoric Monoester Hydrolases; Propidium; Protein Kinase Inhibitors; Protein Kinases; Rats; Rats, Wistar; Staurosporine; Sulfonamides

In isolated rat hepatocytes, the protein phosphatase inhibitor okadaic acid exerts a strong inhibitory effect on autophagy, which can be partially overcome by certain protein kinase inhibitors like the isoflavone genistein. To see if other, more specific okadaic acid antagonists could be found among the flavonoids, 55 different flavonoids were tested for their effect on okadaic acid-inhibited autophagy, measured as the sequestration of electroinjected [3H]raffinose. Naringin (naringenin 7-hesperidoside) and several other flavanone and flavone glycosides (prunin, neoeriocitrin, neohesperidin, apiin, rhoifolin, kaempferol 3-rutinoside) offered virtually complete protection against the autophagy-inhibitory effect of okadaic acid. Unlike genistein, these compounds had little or no autophagy-inhibitory effect of their own. Their innocuousness appeared to be related to glycosylation, because the corresponding aglycones (naringenin, eriodictyol, hesperetin, apigenin, kaempferol) were all inhibitory, in particular apigenin (80% inhibition at 100 microM). Naringin, the most potent okadaic acid-antagonistic flavonoid, gave half-maximal protection at 5 microM and maximal effect at 100 microM. Naringin also prevented the okadaic acid-induced inhibition of endogenous, autophagic lysosomal protein degradation and of receptor-mediated asialoglycoprotein uptake and degradation. Naringin and other okadaic acid-antagonistic flavonoids may be useful tools in the study of intracellular protein phosphorylation and could have potential therapeutic value as protectants against pathological hyperphosphorylations, environmental toxins, or side effects of chemotherapeutic drugs.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Antioxidants; Autophagy; Cells, Cultured; Dose-Response Relationship, Drug; Endocytosis; Ethers, Cyclic; Flavanones; Flavonoids; Genistein; Isoflavones; Isoquinolines; Kinetics; Liver; Male; Okadaic Acid; Phosphoprotein Phosphatases; Piperazines; Protein Kinase Inhibitors; Rats; Rats, Wistar