Inhibition of protein phosphatase activity and changes in protein phosphorylation following acetaminophen exposure in cultured mouse hepatocytes

Protein phosphorylation was determined in cultured mouse hepatocytes expose d to an hepatotoxic concentration of acetaminophen (APAP) for selected time s up to 12 h. Cultures were radiolabled with P-32-orthophosphoric acid and the cell extracts were analyzed by 2D gel electrophoresis and autoradiograp hy. APAP exposure selectively increased the phosphorylation state of protei ns of molecular weight 22, 25, 28, and 59 kDa and decreased the phosphoryla tion of a 26-kDa protein. Evidence is presented that these changes (1) are dependent on cytochrome P-450 activation of APAP; (2) occur well before enz yme leakage in this in vitro model; (3) are not likely attributed to GSH de pletion alone; (4) are in part mimicked by okadaic acid, calyculin A, and c antharidic acid, three structurally distinct inhibitors of protein phosphat ases 1 and 2A; and (5) are paralleled by a decline in protein phosphatase a ctivity. The physiological consequences of protein phosphatase inactivation could be significant in APAP overdose since these enzymes are involved in the dephosphorylation of regulatory proteins that control many cell functio ns. This study also provides the first evidence for disruption in signal tr ansduction pathways as a response to or component of APAP-induced hepatic i njury. (C) 1998 Academic Press.