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

AIM

Hydrogen peroxide (H2O2) 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+), 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 Ca2+ concentrations (Ca2+) across two kinetic phases. A low concentration (400 micromol/L) of H2O2 induced a sustained elevation of Ca2+ that was reversed by removing extracellular Ca2+. H2O2 increased membrane currents consistent with intracellular ATP concentrations. The non-selective ATP-sensitive cation channel blocker amiloride inhibited H2O2-induced membrane current increases and Ca2+ elevation. A high concentration (1 mmol/L)of H2O2 induced an additional transient elevation of Ca2+, 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 H2O2 but not by 400 micromol/L H2O2.

CONCLUSIONS

H2O2 mobilizes Ca2+ through two distinct mechanisms. In one, 400 micromol/L H2O2-induced sustained Ca2+ elevation is mediated via a Ca2+ 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 Ca2+ influx.In contrast, 1 mmol/L H2O2-induced transient elevation of Ca2+ is mediated via activation of the PLC signaling pathway and subsequently, by mobilization of Ca2+ from intracellular Ca2+ stores.