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首页> 外文期刊>Protoplasma: An International Journal of Cell Biology >Trans-plasma membrane electron transport: a cellular assay for NADH- and NADPH-oxidase based on extracellular, superoxide-mediated reduction of the sulfonated tetrazolium salt WST-1
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Trans-plasma membrane electron transport: a cellular assay for NADH- and NADPH-oxidase based on extracellular, superoxide-mediated reduction of the sulfonated tetrazolium salt WST-1

机译：跨质膜电子传输：基于细胞外超氧化物介导的磺化四唑盐WST-1还原的NADH和NADPH氧化酶的细胞分析

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Plasma membrane NADH-oxidase of mammalian cells is usually assayed biochemically in isolated plasma membranes by measuring its ability to oxidise NADH or to reduce oxygen to water. Lack of a convenient cellular assay has greatly limited the study of NADH-oxidase. the physiological significance of which remains uncertain. Recently, we demonstrated that the novel cell-impermeative sulfonated tetrazolium salt WST-1 (2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-terrazo monosodium salt), used in conjunction with an intermediate electron acceptor, was reduced extracellularly suggesting involvement of a component of the trans-plasma membrane electron transport system in WST-1 reduction. In this study we provide evidence that WST-1 is reduced at the external surface of the plasma membrane by an NADH-oxidase, and that reduction is primarily mediated by superoxide. Thus, WST-1 reduction was extensively inhibited by superoxide dismutase and by the potent NADH-oxidase inhibitor resiniferatoxin. Dihydrocapsaicin and capsaicin which are less potent inhibitors of NADI-oxidase also inhibited WST-1 reduction, bur the impermeative SH-blocking reagent para-chloromercuriphenylsulfonic acid and trypsin, both of which are known to inhibit NADH-ferricyanide reductase but not NADH oxidase, had little effect on WST-1 reduction. Human peripheral blood neutrophils activated by phorbol myristate acetate efficiently reduced WST-1. This reduction was inhibited by 95% by superoxide dismutase but was unaffected by resiniferatoxin indicating a distinct mechanism of reduction by neutrophil NADPH-oxidase. Metabolic inhibitors were used to investigate putative involvement of cytosolic NADH in WST-1 reduction. Mitochondrial inhibitors such as cyanide and thenoyltrifluoroacetone, and to a lesser extent azide and rotenone, stimulated WST-1 reduction by Jurkat cells whereas inhibitors of glucose uptake and glycolysis were inhibitory. These results are explained by respiratory inhibitors having a sparing effect on cytosolic NADH levels and by glycolytic inhibitors lowering NADH. We conclude that WST-1 is reduced extracellularly by plasma membrane NADH-oxidase by a mechanism involving superoxide production. WST-1 is also efficiently reduced by the plasma membrane NADPH-oxidase of activated neutrophils. [References: 23]
机译：哺乳动物细胞的质膜NADH氧化酶通常在离体的质膜中进行生化分析，方法是测量其氧化NADH或将氧气还原为水的能力。缺乏便利的细胞测定法极大地限制了NADH-氧化酶的研究。其生理意义仍然不确定。最近，我们证明了新型的不渗透细胞的磺化四唑盐WST-1（2- [4-碘苯基] -3- [4-硝基苯基] -5- [2,4-二磺苯基] -2H-terrazo一钠盐），与中间电子受体结合使用的WST-1还原剂在细胞外被还原，表明跨质膜电子传输系统的成分参与WST-1还原。在这项研究中，我们提供证据表明WST-1在膜的外表面被NADH-氧化酶还原，并且还原主要是由超氧化物介导的。因此，超氧化物歧化酶和有效的NADH-氧化酶抑制剂resiniferatoxin广泛抑制了WST-1的减少。较弱的NADI氧化酶抑制剂二氢辣椒素和辣椒素也抑制WST-1的降低，渗透性的SH封闭剂对氯汞铜苯磺酸和胰蛋白酶都已知抑制NADH-铁氰化物还原酶但不抑制NADH氧化酶。对减少WST-1的影响很小。佛波醇肉豆蔻酸酯乙酸盐激活的人外周血中性粒细胞有效降低WST-1。该减少被超氧化物歧化酶抑制了95％，但不受树脂毒素的影响，表明中性粒细胞NADPH氧化酶具有明显的减少机制。代谢抑制剂被用于研究细胞质NADH参与WST-1减少的推测。线粒体抑制剂如氰化物和壬基三氟丙酮，以及较小程度的叠氮化物和鱼藤酮，可刺激Jurkat细胞降低WST-1，而葡萄糖摄取和糖酵解抑制剂则具有抑制作用。这些结果可以通过对细胞溶质NADH水平有少量影响的呼吸抑制剂和降低NADH的糖酵解抑制剂来解释。我们得出的结论是，WST-1在细胞外被质膜NADH-氧化酶通过一种涉及超氧化物生成的机制还原。活化的中性粒细胞的质膜NADPH-氧化酶还可以有效降低WST-1。 [参考：23]

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