Srivastava I K, Rottenberg H, Vaidya A B
Department of Microbiology and Immunology, MCP Hahnemann School of Medicine, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19102-1192, USA.
J Biol Chem. 1997 Feb 14;272(7):3961-6. doi: 10.1074/jbc.272.7.3961.
At present, approaches to studying mitochondrial functions in malarial parasites are quite limited because of the technical difficulties in isolating functional mitochondria in sufficient quantity and purity. We have developed a flow cytometric assay as an alternate means to study mitochondrial functions in intact erythrocytes infected with Plasmodium yoelii, a rodent malaria parasite. By using a very low concentration (2 nM) of a lipophilic cationic fluorescent probe, 3,3'dihexyloxacarbocyanine iodide, we were able to measure mitochondrial membrane potential(DeltaPsim) in live intact parasitized erythrocytes through flow cytometry. The accumulation of the probe into parasite mitochondria was dependent on the presence of a membrane potential since inclusion of carbonyl cyanide m-chlorophenylhydrazone, a protonophore, dissipated the membrane potential and abolished the probe accumulation. We tested the effect of standard mitochondrial inhibitors such as myxothiazole, antimycin, cyanide and rotenone. All of them except rotenone collapsed the DeltaPsim and inhibited respiration. The assay was validated by comparing the EC50 of these compounds for inhibiting DeltaPsim and respiration. This assay was used to investigate the effect of various antimalarial drugs such as chloroquine, tetracycline and a broad spectrum antiparasitic drug atovaquone. We observed that only atovaquone collapsed DeltaPsim and inhibited parasite respiration within minutes after drug treatment. Furthermore, atovaquone had no effect on mammalian DeltaPsim. This suggests that atovaquone, shown to inhibit mitochondrial electron transport, also depolarizes malarial mitochondria with consequent cellular damage and death.
目前，由于在分离足够数量和纯度的功能性线粒体方面存在技术困难，研究疟原虫线粒体功能的方法相当有限。我们开发了一种流式细胞术检测方法，作为研究感染约氏疟原虫（一种啮齿动物疟原虫）的完整红细胞中线粒体功能的替代方法。通过使用极低浓度（2 nM）的亲脂性阳离子荧光探针3,3'-二己基氧杂羰花青碘化物，我们能够通过流式细胞术测量活的完整寄生红细胞中的线粒体膜电位（ΔΨm）。探针在寄生虫线粒体中的积累取决于膜电位的存在，因为加入质子载体羰基氰化物间氯苯腙会使膜电位消散并消除探针积累。我们测试了标准线粒体抑制剂如粘噻唑、抗霉素、氰化物和鱼藤酮的作用。除鱼藤酮外，所有这些抑制剂都会使ΔΨm崩溃并抑制呼吸作用。通过比较这些化合物抑制ΔΨm和呼吸作用的半数有效浓度（EC50）来验证该检测方法。该检测方法用于研究各种抗疟药物如氯喹、四环素和广谱抗寄生虫药物阿托伐醌的作用。我们观察到，只有阿托伐醌在药物处理后几分钟内就会使ΔΨm崩溃并抑制寄生虫呼吸作用。此外，阿托伐醌对哺乳动物的ΔΨm没有影响。这表明，已证明能抑制线粒体电子传递的阿托伐醌，也会使疟原虫线粒体去极化，从而导致细胞损伤和死亡。
