Chandel N S, Budinger G R, Choe S H, Schumacker P T
Department of Medicine, Section of Pulmonary and Critical Care, the University of Chicago, Chicago, Illinois 60637, USA.
J Biol Chem. 1997 Jul 25;272(30):18808-16. doi: 10.1074/jbc.272.30.18808.
We previously reported that hepatocytes exhibit a reversible suppression of respiration during prolonged hypoxia (PO2 = 20 torr for 3-5 h). Also, isolated bovine heart cytochrome c oxidase undergoes a reversible decrease in apparent Vmax when incubated under similar conditions. This study sought to link the hypoxia-induced changes in cytochrome oxidase to the inhibition of respiration seen in intact cells. Hepatocytes incubated at PO2 = 20 torr exhibited decreases in respiration and increases in [NAD(P)H] after 2-3 h that were reversed upon reoxygenation (PO2 = 100 torr). Respiration during hypoxia was also inhibited when N,N,N',N'-tetramethyl-p-phenylenediamine (0.5 mM) and ascorbate (5 mM) were used to reduce cytochrome c, suggesting that cytochrome oxidase was partially inhibited. Similarly, liver submitochondrial particles revealed a 44% decrease in the apparent Vmax of cytochrome oxidase after hypoxic incubation. In hepatocytes loaded with tetramethylrhodamine ethyl ester (10 nM) to quantify mitochondrial membrane potential, acute hypoxia (<30 min) produced no change in fluorescence, consistent with the absence of an acute change in respiration. However, fluorescence increased during acute reoxygenation after prolonged hypoxia, suggesting an increase in potential. The control exhibited by NADH over mitochondrial respiration was not altered during hypoxia. Thus, changes in the Vmax of cytochrome oxidase during prolonged hypoxia correlate with the changes in respiration and mitochondrial potential. This suggests that the oxidase functions as an oxygen sensor in the intact hepatocyte.
我们之前报道过，在长时间缺氧（PO2 = 20托，持续3 - 5小时）期间，肝细胞的呼吸作用会出现可逆性抑制。此外，分离的牛心脏细胞色素c氧化酶在类似条件下孵育时，其表观Vmax会出现可逆性降低。本研究旨在将缺氧诱导的细胞色素氧化酶变化与完整细胞中观察到的呼吸抑制联系起来。在PO2 = 20托下孵育的肝细胞，2 - 3小时后呼吸作用降低，[NAD(P)H]增加，复氧（PO2 = 100托）后这些变化会逆转。当使用N，N，N'，N'-四甲基对苯二胺（0.5 mM）和抗坏血酸（5 mM）还原细胞色素c时，缺氧期间的呼吸作用也受到抑制，这表明细胞色素氧化酶被部分抑制。同样，肝脏亚线粒体颗粒在缺氧孵育后，细胞色素氧化酶的表观Vmax降低了44%。在用四甲基罗丹明乙酯（10 nM）加载以量化线粒体膜电位的肝细胞中，急性缺氧（<30分钟）未导致荧光变化，这与呼吸作用无急性变化一致。然而，长时间缺氧后的急性复氧过程中荧光增加，表明电位升高。在缺氧期间，NADH对线粒体呼吸的控制作用未改变。因此，长时间缺氧期间细胞色素氧化酶Vmax的变化与呼吸作用和线粒体电位的变化相关。这表明该氧化酶在完整肝细胞中充当氧传感器。
