Acta Pharmacologica Sinica (2009) 30: 78-89; doi:10.1038/aps.2008.4; published online 15th December 2008

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

Hirohiko SATO¹, Teruko TAKEO², Qiang LIU³, Kyoko NAKANO², Tomohiro OSANAI⁴, Sechiko SUGA¹, Makoto WAKUI^1,#, Jie WU^3,#,*

¹Department of Physiology, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan; ²Department of Medical Technology, Hirosaki University School of Health Sciences, Hirosaki 036-8564, Japan; ³Division of Neurology, Barrow Neurological Institute, Phoenix, AZ, 85013-4496, USA; ⁴Department of Internal Medicine, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan

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⁺]_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 Ca²⁺ concentrations ([Ca²⁺]_i) across two kinetic phases. A low concentration (400 µmol/L) of H₂O₂ induced a sustained elevation of [Ca²⁺]_i 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⁺]i elevation. A high concentration (1 mmol/L) of H₂O₂ induced an additional transient elevation of [Ca²⁺]_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 H₂O₂ but not by 400 µmol/L H₂O₂.

Conclusions: H₂O₂ mobilizes Ca²⁺ through two distinct mechanisms. In one, 400 µmol/L H₂O₂-induced sustained [Ca²⁺]i 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⁺]_i is mediated via activation of the PLC signaling pathway and subsequently, by mobilization of Ca²⁺ from intracellular Ca²⁺ stores.

Keywords:hydrogen peroxide; Ca²⁺ dynamics; non-selective cation channel; intracellular ATP; phospholipase C; hepatocyte; patch-clamp