Inhibition of hepatocytic autophagy by okadaic acid and other protein phosphatase inhibitors

Autophagy, measured as the sequestration of electroinjected [ 3 H]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 Ca 2+ /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.