The Menadione-Mediated WST1 Reduction by Cultured Astrocytes Depends on NQO1 Activity and Cytosolic Glucose Metabolism
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ORIGINAL PAPER
The Menadione-Mediated WST1 Reduction by Cultured Astrocytes Depends on NQO1 Activity and Cytosolic Glucose Metabolism Eric Ehrke1,2 · Johann Steinmeier1,2 · Karsten Stapelfeldt1,3 · Ralf Dringen1,2 Received: 15 April 2019 / Revised: 3 December 2019 / Accepted: 5 December 2019 © Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract 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 K ic 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. Keywords Astrocytes · Glucose · Metabolism · NAD(P)H · WST1
Introduction Tetrazolium salts are widely used for biochemical and cell biological test systems. These compounds are weaklycolored but are reduced by cellular electron donors to their Special Issue: In Honor of Prof. Juan Bolanos. * Ralf Dringen dringen@uni‑bremen.de http://www.uni-bremen.de/dringen 1
Center for Biomolecular Interactions Bremen (CBIB), Faculty 2 (Biology/Chemistry), University of Bremen, P.O. Box 330440, 28334 Bremen, Germany
2
Center for Environmental Research and Sustainable Technology (UFT), University of Bremen, Bremen, Germany
3
Institute for Biophysics, University of Bremen, Bremen, Germany
brightly colored formazan products which can be easily detected by their strong absorbance [1]. One of the bestknown tetrazolium salts is 3-(4,5-dimethylthiazol-2-yl)2,5
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