Jordani M C, Santos A C, Prado I M, Uyemura S A, Curti C
Department of Physics and Chemistry, School of Pharmaceutical Sciences, University of São Paulo, Ribeirão Preto, Brazil.
Mol Cell Biochem. 2000 Jul;210(1-2):153-8. doi: 10.1023/a:1007185825101.
To assess the mechanism by which mitochondrial permeability transition (MPT) is induced by the nonpolar carboxylic acids, we investigated the effects of flufenamic acid (3'-trifluoromethyl diphenylamine-2-carboxylic acid, FA) on mitochondrial respiration, electrical transmembrane potential difference (delta psi), osmotic swelling, Ca2+ efflux, NAD(P)H oxidation and reactive oxygen species (ROS) generation. Succinate-energized isolated rat liver mitochondria incubated in the absence or presence of 10 microM Ca2+, 5 microM ruthenium red (RR) or 1 microM cyclosporin A (CsA) were used. The dose response-curves for both respiration release and delta psi dissipation were nearly linear, presenting an IC50 of approximately 10 microM and reaching saturation within 25-50 microM, indicating that FA causes mitochondrial uncoupling by a protonophoric mechanism. Within this same concentration range FA showed the ability to induce MPT in energized mitochondria incubated with 10 microM Ca2+, followed by delta psi dissipation and Ca2+ efflux, and even in deenergized mitochondria incubated with 0.5 mM Ca2+. ADP, Mg2+, trifluoperazine (TFP) and N-ethylmaleimide (NEM) reduced the extent of FA-promoted swelling in energized mitochondria by approximately one half, whereas dithiothreitol (DTT) slightly enhanced it. NAD(P)H oxidation and ROS generation (H2O2 production) by mitochondria were markedly stimulated by FA; these responses were partly prevented by CsA, suggesting that they may be implicated as both a cause and effect of FA-induced MPT. FA incubated with mitochondria under swelling assay conditions caused a decrease of approximately 40% in the content of protein thiol groups reacting with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The present results are consistent with a ROS-intermediated sensitization of MPT by a direct or indirect FA interaction with inner mitochondrial membrane at a site which is in equilibrium with the NAD(P)H pool, namely thiol groups of integral membrane proteins.
为了评估非极性羧酸诱导线粒体通透性转换（MPT）的机制，我们研究了氟芬那酸（3'-三氟甲基二苯胺-2-羧酸，FA）对线粒体呼吸、跨膜电位差（δψ）、渗透性肿胀、Ca2+外流、NAD(P)H氧化和活性氧（ROS）生成的影响。使用在不存在或存在10μM Ca2+、5μM钌红（RR）或1μM环孢素A（CsA）的情况下孵育的琥珀酸供能的分离大鼠肝线粒体。呼吸释放和δψ耗散的剂量反应曲线几乎呈线性，IC50约为10μM，在25-50μM范围内达到饱和，表明FA通过质子载体机制导致线粒体解偶联。在相同浓度范围内，FA显示出能够在与10μM Ca2+孵育的供能线粒体中诱导MPT，随后δψ耗散和Ca2+外流，甚至在与0.5 mM Ca2+孵育的去能线粒体中也能诱导MPT。ADP、Mg2+、三氟拉嗪（TFP）和N-乙基马来酰亚胺（NEM）使供能线粒体中FA促进的肿胀程度降低了约一半，而二硫苏糖醇（DTT）则略有增强。FA显著刺激线粒体的NAD(P)H氧化和ROS生成（H2O2产生）；这些反应部分被CsA阻止，表明它们可能既是FA诱导MPT的原因也是结果。在肿胀测定条件下与线粒体孵育的FA导致与5,5'-二硫代双（2-硝基苯甲酸）（DTNB）反应的蛋白质巯基含量降低约40%。目前的结果与通过FA与线粒体内膜在与NAD(P)H池平衡的位点（即整合膜蛋白的巯基）直接或间接相互作用而导致的ROS介导的MPT致敏作用一致。
