Nitric oxide protects rat hepatocytes against reperfusion injury mediated by the mitochondrial permeability transition
Nitric oxide protects rat hepatocytes against reperfusion injury mediated by the mitochondrial permeability transition
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DOI：
10.1002/hep.20197
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发表时间：
2004-06-01
期刊：
影响因子：
13.5
通讯作者：
Lemasters, JJ
中科院分区：
文献类型：
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作者：
Kim, JS;Ohshima, S;Lemasters, JJ
我们研究了一氧化氮(NO)在模拟缺血/再灌流后对肝细胞杀伤的影响，并研究了NO触发细胞保护的信号因子。培养的大鼠肝细胞在pH 6.2的缺氧Krebs-Ringer-HEPES缓冲液中孵育4小时，在pH 7.4的条件下复氧2小时。在复氧过程中，一些肝细胞暴露于NO供体(S-亚硝基-N-乙酰青霉胺等)、cGMP类似物(8-溴-鸟苷-3，5-cGMP[8-溴-cGMP])和cGMP依赖的蛋白激酶抑制物(KT5823)的组合中。用碘化丙啶荧光法测定细胞存活率。共聚焦显微镜观察内膜通透性和线粒体去极化。Snap，而不是氧化SNAP，在再灌流过程中增加cGMP，减少细胞杀伤。其他NO供体和8-BR-cGMP也阻止了细胞杀伤。鸟苷酸环化酶和cGMP依赖的激酶抑制均可阻断NO的细胞保护作用。然而，5-羟基癸酸和二氮嗪-线粒体K-ATP通道调节剂-不影响NO依赖的细胞保护或再灌注损伤。在复氧过程中，共聚焦显微镜显示线粒体复极化，随后是去极化、内膜通透性和细胞死亡。在SNAP或8-Br-cGMP存在的情况下，再灌注后线粒体的复极得以维持，阻止了内膜通透性和细胞死亡。在分离的大鼠肝线粒体中，cGMP类似物在胞浆提取物和三磷酸腺苷的存在下阻断了钙离子诱导的线粒体通透性转变(MPT)，这一作用被KT5823逆转。综上所述，NO通过鸟苷酸环化酶和cGMP依赖的激酶信号通路阻止MPT依赖的缺血肝细胞在再灌注后的坏死性杀伤，这些事件可能代表了NO在预适应中的细胞保护作用的靶点。
We investigated the effects of nitric oxide (NO) on hepatocellular killing after simulated ischemia/reperfusion and characterized signaling factors triggering cytoprotection by NO. Cultured rat hepatocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 4 hours and reoxygenated at pH 7.4 for 2 hours. During reoxygenation, some hepatocytes were exposed to combinations of NO donors (S-nitroso-N-acetylpenicillamine [SNAP] and others), a cGMP analogue (8-bromoguanosine-3,5-cGMP [8-Br-cGMP]), and a cGMP-dependent protein kinase, inhibitor (KT5823). Cell viability was determined by way of propidium iodide fluorometry. Inner membrane permeabilization and mitochondrial depolarization were monitored by confocal microscopy. SNAP, but not oxidized SNAP, increased cGMP during reperfusion and decreased cell killing. Other NO donors and 8-Br-cGMP also prevented cell killing. Both guanylyl cyclase and cGMP-dependent kinase inhibition blocked the cytoprotection of NO. However, 5-hydroxydecanoate and diazoxide-mitochondrial K-ATP channel modulators-did not affect NO-dependent cytoprotection or reperfusion injury. During reoxygenation, confocal microscopy showed mitochondrial repolarization, followed by depolarization, inner membrane permeabilization, and cell death. In the presence of either SNAP or 8-Br-cGMP, mitochondrial repolarization was sustained after reperfusion preventing inner membrane permeabilization and cell death. In isolated rat liver mitochondria, a cGMP analogue in the presence of a cytosolic extract and adenosine triphosphate blocked the Ca2+-induced mitochondrial permeability transition (MPT), an effect that was reversed by KT5823. In conclusion, NO prevents MPT-dependent necrotic killing of ischemic hepatocytes after reperfusion through a guanylyl cyclase and cGMP-dependent kinase signaling pathway, events that may represent the target of NO cytoprotection in preconditioning.