Hydrogen peroxide mobilizes Ca2+ through two distinct mechanisms in rat hepatocytes
2009
Aim: Hydrogen peroxide (H 2 O 2 ) is produced during liver transplantation. Ischemia/reperfusion induces oxidation and causes intracellular Ca 2+ overload, which harms liver cells. Our goal was to determine the precise mechanisms of these processes.
Methods: Hepatocytes were extracted from rats. Intracellular Ca 2+ concentrations ([Ca 2+ ] i ), inner mitochondrial membrane potentials and NAD(P)H levels were measured using fluorescence imaging. Phospholipase C (PLC) activity was detected using exogenous PIP 2 . ATP concentrations were measured using the luciferin-luciferase method. Patch-clamp recordings were performed to evaluate membrane currents.
Results: H 2 O 2 increased intracellular Ca 2+ concentrations ([Ca 2+ ] i ) across two kinetic phases. A low concentration (400 μmol/L) of H 2 O 2 induced a sustained elevation of [Ca 2+ ] i that was reversed by removing extracellular Ca 2+ . H 2 O 2 increased membrane currents consistent with intracellular ATP concentrations. The non-selective ATP-sensitive cation channel blocker amiloride inhibited H 2 O 2 -induced membrane current increases and [Ca 2+ ] i elevation. A high concentration (1 mmol/L) of H 2 O 2 induced an additional transient elevation of [Ca 2+ ] i , which was abolished by the specific PLC blocker U73122 but was not eliminated by removal of extracellular Ca 2+ . PLC activity was increased by 1 mmol/L H 2 O 2 but not by 400 μmol/L H 2 O 2 .
Conclusions: H 2 O 2 mobilizes Ca 2+ through two distinct mechanisms. In one, 400 μmol/L H 2 O 2 -induced sustained [Ca 2+ ] I elevation is mediated via a Ca 2+ influx mechanism, under which H 2 O 2 impairs mitochondrial function via oxidative stress, reduces intracellular ATP production, and in turn opens ATP-sensitive, non-specific cation channels, leading to Ca 2+ influx. In contrast, 1 mmol/L H 2 O 2 -induced transient elevation of [Ca 2+ ] i is mediated via activation of the PLC signaling pathway and subsequently, by mobilization of Ca 2+ from intracellular Ca 2+ stores.
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