Hydrogen peroxide mobilizes Ca^2＋ through two distinct mechanisms in rat hepatocytes

Aim： Hydrogen peroxide （H2O2） 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 PIP2. ATP concentrations were measured using the luciferin-luciferase method. Patch-clamp recordings were performed to evaluate membrane currents.
Results： H2O2 increased intracellular Ca^2＋ concentrations （Ca^2＋]i） across two kinetic phases. A low concentration （400 μmol/L） of H2O2 induced a sustained elevation of Ca^2＋]i that was reversed by removing extracellular Ca^2＋. H2O2 increased membrane currents consistent with intracellular ATP concentrations. The non-selective ATP-sensitive cation channel blocker amiloride inhibited HRO2-induced membrane current increases and Ca^2＋]i elevation. A high concentration （1 mmol/L） of H2O2 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 H2O2but not by 400 μmol/L H2O2.
Conclusions： H2O2 mobilizes Ca^2＋ through two distinct mechanisms. In one, 400 μmol/L H2O2-induced sustained Ca^2＋]i elevation is mediated via a Ca^2＋ influx mechanism, under which H2O2 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 H2O2-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.