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

Aim:Hydrogen peroxide (H"2O"2) is produced during liver transplantation. Ischemia/reperfusion induces oxidation and causes intracellular Ca²⁺ overload, which harms liver cells. Our goal was to determine the precise mechanisms of these processes.Methods:Hepatocytes were extracted from rats. Intracellular Ca²⁺ concentrations ([Ca²⁺](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"2O"2 increased intracellular Ca ²⁺ concentrations ([Ca²⁺](i)) across two kinetic phases. A low concentration (400 μmol/L) of H"2O"2 induced a sustained elevation of [Ca²⁺](i) that was reversed by removing extracellular Ca²⁺. H"2O"2 increased membrane currents consistent with intracellular ATP concentrations. The non-selective ATP-sensitive cation channel blocker amiloride inhibited H"2O"2-induced membrane current increases and [Ca²⁺](i) elevation. A high concentration (1 mmol/L) of H"2O"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 Ca²⁺. PLC activity was increased by 1 mmol/L H"2O"2 but not by 400 μmol/L H"2O"2.Conclusion:H"2O"2 mobilizes Ca²⁺ through two distinct mechanisms. In one, 400 μmol/L H"2O"2-induced sustained [Ca²⁺](i) elevation is mediated via a Ca²⁺ influx mechanism, under which H"2O"2 impairs mitochondrial function via oxidative stress, reduces intracellular ATP production, and in turn opens ATP-sensitive, non-specific cation channels, leading to Ca²⁺ influx. In contrast, 1 mmol/L H"2O"2-induced transient elevation of [Ca ²⁺](i) is mediated via activation of the PLC signaling pathway and subsequently, by mobilization of Ca²⁺ from intracellular Ca ²⁺ stores.