Sato Hirohiko, Takeo Teruko, Liu Qiang, Nakano Kyoko, Osanai Tomohiro, Suga Sechiko, Wakui Makoto, Wu Jie
Department of Physiology, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan.
Acta Pharmacol Sin. 2009 Jan;30(1):78-89. doi: 10.1038/aps.2008.4. Epub 2008 Dec 15.
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.
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.
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.
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.
肝移植过程中会产生过氧化氢（H₂O₂）。缺血/再灌注会引发氧化反应并导致细胞内Ca²⁺过载，从而损害肝细胞。我们的目标是确定这些过程的确切机制。
从大鼠中提取肝细胞。使用荧光成像测量细胞内Ca²⁺浓度（[Ca²⁺]i）、线粒体内膜电位和NAD(P)H水平。使用外源性磷脂酰肌醇-4,5-二磷酸（PIP₂）检测磷脂酶C（PLC）活性。使用荧光素-荧光素酶法测量ATP浓度。进行膜片钳记录以评估膜电流。
H₂O₂在两个动力学阶段增加细胞内Ca²⁺浓度（[Ca²⁺]i）。低浓度（400 μmol/L）的H₂O₂诱导[Ca²⁺]i持续升高，通过去除细胞外Ca²⁺可使其逆转。H₂O₂增加的膜电流与细胞内ATP浓度一致。非选择性ATP敏感性阳离子通道阻滞剂阿米洛利抑制H₂O₂诱导的膜电流增加和[Ca²⁺]i升高。高浓度（1 mmol/L）的H₂O₂诱导[Ca²⁺]i额外的瞬时升高，这被特异性PLC阻滞剂U73122消除，但通过去除细胞外Ca²⁺并未消除。1 mmol/L H₂O₂可增加PLC活性，但400 μmol/L H₂O₂则不会。
H₂O₂通过两种不同机制动员Ca²⁺。一种机制是，400 μmol/L H₂O₂诱导的[Ca²⁺]i持续升高是通过Ca²⁺内流机制介导的，在此机制下，H₂O₂通过氧化应激损害线粒体功能，减少细胞内ATP生成，进而打开ATP敏感性非特异性阳离子通道，导致Ca²⁺内流。相反，1 mmol/L H₂O₂诱导的[Ca²⁺]i瞬时升高是通过激活PLC信号通路介导的，随后通过从细胞内Ca²⁺储存中动员Ca²⁺来实现。
