Hepatocytic autophagy, measured as the sequestration of electroinjected, cytosolic [3H]raffinose into sedimentable autophagic vacuoles, is markedly inhibited by epinephrine through an α1-adrenergic, receptor-mediated mechanism involving release of Ca2+ from the endoplasmic reticulum. More effective Ca2+-releasing agents like thapsigargin and tBHQ suppress autophagy completely. Autophagy is also inhibited by protein phosphatase inhibitors like okadaic acid, the potency of which suggests the involvement of a type 2A protein phosphatase (PP2A) in autophagy control. The effect of okadaic acid can be reversed by a calmodulin antagonist (W-7) and by inhibitors (KN-62, K-252a, KT-5926) of Ca2+/calmodulin-dependent protein kinase II (CaMK-II), indicating that this protein kinase is responsible for autophagy-inhibitory protein phosphorylations. Okadaic acid, furthermore, causes an extensive disruption of the hepatocytic cytoskeleton which becomes evident as structural disassembly of the cell corpses following electrodisruption of the plasma membrane. The cytoskeletal disruption can be specifically prevented by the autophagy-protective protein kinase inhibitors. One explanation of these observations would be that autophagic activity is dependent upon the integrity or formation of cytoskeletal elements that assemble or disassemble according to a dynamic phosphorylation balance governed by CaMK-II and PP2A.
