Hydrogen peroxide mobilizes Ca2+ through two distinct mechanisms in rat hepatocytes

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). CONCLUSION: 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