ANALYSIS OF THE MEMBRANE-POTENTIAL OF RAT-LIVER AND MOUSE-LIVER MITOCHONDRIA BY FLOW-CYTOMETRY AND POSSIBLE APPLICATIONS
ANALYSIS OF THE MEMBRANE-POTENTIAL OF RAT-LIVER AND MOUSE-LIVER MITOCHONDRIA BY FLOW-CYTOMETRY AND POSSIBLE APPLICATIONS
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DOI：
10.1111/j.1432-1033.1990.tb15632.x
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发表时间：
1990-12-12
期刊：
影响因子：
--
通讯作者：
BROWN, SC
中科院分区：
文献类型：
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作者：
PETIT, PX;OCONNOR, JE;BROWN, SC
洗涤和纯化的大鼠或小鼠肝线粒体表现出高膜完整性和代谢活性的流式细胞术进行了研究。 阳离子亲脂性探针，罗丹明123，番红O和花青衍生物，3，3 '-dihexyloxadibocyanine碘化物，在电泳过程中的积累/再分配进行了研究，是一致的负的内膜电位的产生。 一个例外是壬基吖啶橙子，它通过其烃链通过疏水相互作用自发地结合到线粒体膜上。 通电纯化的线粒体与电位染料染色表现出更高的荧光和人口的同质性比非通电或断电（尼日利亚加缬氨霉素）线粒体。 相反，在非通电或断电条件下，线粒体群体表现出与残余膜电位相关的荧光强度异质性;两个亚群是明显的，一个是低荧光的，这可能与线粒体的自发荧光有关（加上非特异性染料结合）和显示高荧光的第二群体。用罗丹明123（一种经典使用的染料）染色的大鼠肝线粒体提供了它们在光散射性质和膜电位相关荧光方面的异质性的证据。 纯化的大鼠肝线粒体由于内源性底物建立的膜电位而产生的荧光表明线粒体群体相对于内源性底物水平的异质性。角光散射增加，并提供了一些原始信息的形状和修改的内部线粒体构象大鼠肝脏线粒体群体的异质性，从结构，代谢，（内源性底物的存在）和功能因此，可以通过流式细胞术分析来证明活性和非活性线粒体群体分散的观点。（大鼠肝线粒体），和铵毒性的影响（小鼠肝线粒体）。 这些结果是有希望的，并在神经病变的研究开辟了新的视角。
Washed and purified rat- or mouse-liver mitochondria exhibiting high membrane integrity and metabolic activity were studied by flow cytometry. The electrophoretic accumulation/redistribution of cationic lipophilic probes, rhodamine 123, safranine O and a cyandine derivative, 3,3'-dihexyloxadicarbocyanine iodide, during the energization process was studied and was consistent with the generation of a negative internal membrane potential. An exception to this was nonylacridine orange which spontaneously bound to the mitochondrial membrane by hydrophobic interactions via its hydrocarbon chain. Energized purified mitochondria stained with potentiometric dyes exhibited both higher fluorescence and population homogeneity than the non-energized or deenergized (nigericin plus valinomycin) mitochondria. By contrast, under non-energized or deenergized conditions, the mitochondrial population exhibited fluorescence intensity heterogeneity related to the residual membrane potential; two subpopulations were evident, one of low fluorescence which may be related to the autofluorescence of the mitochondria (plus non-specific dye binding) and a second population which exhibited high fluorescence.Flow cytometry of the unpurified, simply washed, rat-liver mitochondria stained with rhodamine 123, a classically used dye, provided evidence of their heterogeneity in terms of light-scattering properties and membrane-potential-related fluorescence. One third of the washed mitochondria were found to be non-functional by such assays.The fluorescence of purified rat-liver mitochondria due to the membrane potential built up by endogenous substrates indicates heterogeneity of the mitochondrial population with respect to levels of endogeneous substrates.The low-angle light scattering increases upon energization and provides some original information about the shape and modification of the inner mitochondrial conformation accompanying the energization.The heterogeneity of the rat liver mitochondrial population, from a structural, metabolic (existence of endogenous substrates) and functional (active and non-active mitochondrial population dispersion) point of view could thus be demonstrated by flow-cytometry analysis.Two animal models were examined with regard to the alteration of the mitochondrial membrane potential under the effects of drugs (rat-liver mitochondria), and the effects of ammonium toxicity (mouse-liver mitochondria). These results are promising and open new perspectives in the study of mitochondriopathies.