The reduction of water-soluble tetrazolium salts (WSTs) is frequently used to determine the metabolic integrity and the viability of cultured cells. Recently, we have reported that the electron cycler menadione can efficiently connect intracellular oxidation reactions in cultured astrocytes with the extracellular reduction of WST1 and that this menadione cycling reaction involves an enzyme. The enzymatic reaction involved in the menadione-dependent WST1 reduction was found strongly enriched in the cytosolic fraction of cultured astrocytes and is able to efficiently use both NADH and NADPH as electron donors. In addition, the reaction was highly sensitive towards dicoumarol with Kic values in the low nanomolar range, suggesting that the NAD(P)H:quinone oxidoreductase 1 (NQO1) catalyzes the menadione-dependent WST1 reduction in astrocytes. Also, in intact astrocytes, dicoumarol inhibited the menadione-dependent WST1 reduction in a concentration-dependent manner with half-maximal inhibition observed at around 50 nM. Moreover, the menadione-dependent WST1 reduction by viable astrocytes was strongly affected by the availability of glucose. In the absence of glucose only residual WST1 reduction was observed, while a concentration-dependent increase in WST1 reduction was found during a 30 min incubation with maximal WST1 reduction already determined in the presence of 0.5 mM glucose. Mannose could fully replace glucose as substrate for astrocytic WST1 reduction, while other hexoses, lactate and the mitochondrial substrate β-hydroxybutyrate failed to provide electrons for the cell-dependent WST1 reduction. These results demonstrate that the menadione-mediated WST1 reduction involves cytosolic NQO1 activity and that this process is strongly affected by the availability of glucose as metabolic substrate.

中文翻译：

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培养的星形胶质细胞甲萘醌介导的 WST1 减少取决于 NQO1 活性和胞质葡萄糖代谢。


水溶性四唑盐 (WST) 的还原经常用于确定培养细胞的代谢完整性和活力。最近，我们报道了电子循环仪甲萘醌可以有效地将培养的星形胶质细胞中的细胞内氧化反应与WST1的细胞外还原反应联系起来，并且这种甲萘醌循环反应涉及一种酶。研究发现，参与甲萘醌依赖性 WST1 还原的酶促反应在培养的星形胶质细胞的胞质部分中高度富集，并且能够有效地利用 NADH 和 NADPH 作为电子供体。此外，该反应对 Kic 值在低纳摩尔范围内的双香豆素高度敏感，表明 NAD(P)H:醌氧化还原酶 1 (NQO1) 催化星形胶质细胞中甲萘醌依赖性 WST1 还原。此外，在完整的星形胶质细胞中，双香豆素以浓度依赖性方式抑制甲萘醌依赖性 WST1 减少，在 50 nM 左右观察到半最大抑制。此外，活星形胶质细胞的甲萘醌依赖性 WST1 减少受到葡萄糖可用性的强烈影响。在不存在葡萄糖的情况下，仅观察到残留的 WST1 减少，而在 30 分钟孵育期间发现 WST1 减少呈浓度依赖性增加，并且在存在 0.5 mM 葡萄糖的情况下已确定最大 WST1 减少。甘露糖可以完全替代葡萄糖作为星形胶质细胞WST1还原的底物，而其他己糖、乳酸和线粒体底物β-羟基丁酸未能为细胞依赖性WST1还原提供电子。 这些结果表明，甲萘醌介导的 WST1 还原涉及胞质 NQO1 活性，并且该过程受到葡萄糖作为代谢底物的可用性的强烈影响。