Inhibition of hepatocytic autophagy by okadaic acid and other protein phosphatase inhibitors.
Autophagy, measured as the sequestration of electroinjected [3H]raffinose or endogenous lactate dehydrogenase, was inhibited in isolated rat hepatocytes by the protein phosphatase inhibitors okadaic acid, calyculin A and microcystin-LR. Okadaic acid, the most potent inhibitor, suppressed autophagy almost completely at 15 nM, suggesting inhibition of a protein phosphatase of type 2A. Okadaic acid had no effect on ATP levels, protein synthesis or cellular viability at this concentration, but caused a disruption of the hepatocytic cytoskeleton and a consequent reduction in organelle sedimentability, potentially interfering with the autophagy assay unless the necessary precautions are taken. Lysosomal (propylamine-sensitive) degradation of endogenous protein was inhibited by okadaic acid, whereas non-lysosomal (propylamine-resistant) degradation was unaffected. The autophagy-inhibitory effect of okadaic acid was not affected by inhibitors of cAMP-dependent protein kinase or protein kinase C (H-7, H-89, calphostin C) but eliminated by the non-specific inhibitor K-252a and its analogues (KT-5720, KT-5823, KT-5926) and by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II. Protein phosphorylation by this kinase would thus seem to play a role in regulation of the autophagic-lysosomal degradation pathway.
Department of Tissue Culture, Norwegian Radium Hospital, Oslo.
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Holen
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Gordon
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Seglen
D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D003599:Cytoskeleton / Q000187:drug effects; D004734:Energy Metabolism / Q000187:drug effects; D004988:Ethers, Cyclic / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D008387:Marine Toxins; D019319:Okadaic Acid; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D011494:Protein Kinases / Q000502:physiology; D011506:Proteins / Q000378:metabolism; D011887:Raffinose / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D014617:Vacuoles / Q000187:drug effects
D004988:Ethers, Cyclic; D008387:Marine Toxins; D010080:Oxazoles; D011506:Proteins; D019319:Okadaic Acid; C059041:calyculin A; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D011887:Raffinose