Hydrogen peroxide mobilizes Ca2+ through two distinct mechanisms in rat hepatocytes.
OBJECTIVE
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 micromol/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 micromol/L H2O2.


CONCLUSIONS
H2O2 mobilizes Ca2+ through two distinct mechanisms. In one, 400 micromol/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.
Sato
Hirohiko
H
Department of Physiology, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan.
Takeo
Teruko
T
Liu
Qiang
Q
Nakano
Kyoko
K
Osanai
Tomohiro
T
Suga
Sechiko
S
Wakui
Makoto
M
Wu
Jie
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology*; D010084:Oxidation-Reduction; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D026941:Sodium Channel Blockers / Q000494:pharmacology; D012966:Sodium Cyanide / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism
D004791:Enzyme Inhibitors; D016877:Oxidants; D026941:Sodium Channel Blockers; D000584:Amiloride; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D010738:Type C Phospholipases; D012966:Sodium Cyanide; D002118:Calcium