Konno N, Kako K J
Department of Physiology, Faculty of Medicine, University of Ottawa, Ont., Canada.
Can J Physiol Pharmacol. 1991 Nov;69(11):1705-12. doi: 10.1139/y91-253.
Hydrogen peroxide (H2O2) and hypochlorite (HOCl) cause a variety of cellular dysfunctions. In this study we examined the effects of these agents on the electrical potential gradient across the inner membrane of mitochondria in situ in isolated rat heart myocytes. Myocytes were prepared by collagenase digestion and incubated in the presence of H2O2 or HOCl. Transmembrane electrical gradients were measured by distribution of [3H]triphenylmethylphosphonium+, a lipophilic cation. The particulate fraction was separated from the cytosolic compartment first by permeabilization using digitonin, followed by rapid centrifugal sedimentation through a bromododecane layer. We found that the mitochondrial membrane potential (161 +/- 7 mV, negative inside) was relatively well maintained under oxidant stress, i.e., the potential was decreased only at high concentrations of HOCl and H2O2 and gradually with time. The membrane potential of isolated rat heart mitochondria was affected similarly by H2O2 and HOCl in a concentration- and time-dependent manner. High concentrations of oxidants also reduced the cellular ATP level but did not significantly change the matrix volume. When the extra-mitochondrial free calcium concentration was increased in permeabilized myocytes, the transmembrane potential was decreased proportionally, and this decrease was potentiated further by H2O2. These results support the view that heart mitochondria are equipped with well-developed defense mechanisms against oxidants, but the action of H2O2 on the transmembrane electrical gradient is exacerbated by an increase in cytosolic calcium.
过氧化氢（H2O2）和次氯酸盐（HOCl）会引发多种细胞功能障碍。在本研究中，我们检测了这些试剂对分离的大鼠心脏心肌细胞中线粒体内膜跨膜电位梯度的影响。通过胶原酶消化制备心肌细胞，并在H2O2或HOCl存在的情况下进行孵育。使用亲脂性阳离子[3H]三苯基甲基鏻离子的分布来测量跨膜电位梯度。首先通过使用洋地黄皂苷进行透化处理，将颗粒部分与胞质部分分离，然后通过溴十二烷层进行快速离心沉降。我们发现，在氧化应激下，线粒体膜电位（161±7 mV，内膜为负）相对保持良好，即仅在高浓度的HOCl和H2O2下以及随时间逐渐降低。分离的大鼠心脏线粒体的膜电位受到H2O2和HOCl的类似影响，呈浓度和时间依赖性。高浓度的氧化剂也会降低细胞内ATP水平，但不会显著改变线粒体基质体积。当透化处理的心肌细胞中外线粒体游离钙浓度增加时，跨膜电位会成比例降低，并且H2O2会进一步增强这种降低。这些结果支持这样的观点，即心脏线粒体具备完善的抗氧化防御机制，但胞质钙的增加会加剧H2O2对跨膜电位梯度的作用。
