β-lapachone-mediated WST1 Reduction as Indicator for the Cytosolic Redox Metabolism of Cultured Primary Astrocytes

Abstract

Electron cycler-mediated extracellular reduction of the water-soluble tetrazolium salt 1 (WST1) is frequently used as tool for the determination of cell viability. We have adapted this method to monitor by determining the extracellular WST1 formazan accumulation the cellular redox metabolism of cultured primary astrocytes via the NAD(P)H-dependent reduction of the electron cycler β-lapachone by cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1). Cultured astrocytes that had been exposed to β-lapachone in concentrations of up to 3 µM remained viable and showed an almost linear extracellular accumulation of WST1 formazan for the first 60 min, while higher concentrations of β-lapachone caused oxidative stress and impaired cell metabolism. β-lapachone-mediated WST1 reduction was inhibited by the NQO1 inhibitors ES936 and dicoumarol in a concentration-dependent manner, with half-maximal inhibition observed at inhibitor concentrations of about 0.3 µM. β-lapachone-mediated WST1 reduction depended strongly on glucose availability, while mitochondrial substrates such as lactate, pyruvate or ketone bodies allowed only residual β-lapachone-mediated WST1 reduction. Accordingly, the mitochondrial respiratory chain inhibitors antimycin A and rotenone hardly affected astrocytic WST1 reduction. Both NADH and NADPH are known to supply electrons for reactions catalysed by cytosolic NQO1. Around 60% of the glucose-dependent β-lapachone-mediated WST1 reduction was prevented by the presence of the glucose-6-phosphate dehydrogenase inhibitor G6PDi-1, while the glyceraldehyde-3-phosphate dehydrogenase inhibitor iodoacetate had only little inhibitory potential. These data suggest that pentose phosphate pathway-generated NADPH, and not glycolysis-derived NADH, is the preferred electron source for cytosolic NQO1-catalysed reductions in cultured astrocytes.

Modulation of Pyruvate Export and Extracellular Pyruvate Concentration in Primary Astrocyte Cultures

“…1 – 3 ). To test for a potential release of pyruvate from astrocytes that had been exposed to other metabolic substrates than glucose, the cells were incubated for 5 h in 250 µL glucose-free buffer that had been supplemented with 5 mM of other hexoses or known mitochondrial substrates [ 18 , 33 , 60 ]. Compared to glucose-fed astrocytes, almost identical extracellular pyruvate concentrations were found for cells that had been exposed to mannose or lactate, but the cell established also in the presence of fructose, sorbitol or alanine extracellular pyruvate concentrations that were significantly higher than those determined for the substrate-free incubation (None) (Fig.…”

Consequences of a 2-Deoxyglucose Exposure on the ATP Content and the Cytosolic Glucose Metabolism of Cultured Primary Rat Astrocytes

“…An additional pathway that may contribute to the disappearance of 2DG6P from cultured astrocytes is the oxidation In panel (c), the significance of differences (t-test) between the data obtained for incubations in the absence and the presence of 2DG is indicated by # p < 0.05 and ## p < 0.01 of 2DG6P by G6PDH, as 6-phospho-2-deoxygluconate has been found in the brain of 2DG-exposed rats [4]. We have previously shown that cellular NADPH regeneration via the PPP can be studied by monitoring the NQO1-dependent reduction of extracellular WST1 to its formazan in the presence of the redox cycler β-lapachone [26,42]. In the presence of 2DG the glucose-independent WST1 reduction was almost doubled in a concentration-dependent manner by 2DG, suggesting that 2DG6P acts as substrate of G6PDH in living astrocytes, thereby providing electrons via NADPH for the WST1 reduction.…”

“…To test for the consequences of a high cellular concentration of 2DG6P on the NADPH regeneration by the PPP of astrocytes, the PPP-and NADPH-dependent WST1 reduction was investigated in the absence and the presence of glucose in the experimental paradigm previously described In panel (c), the significance of differences (t-test) between data from incubations in the absence or the presence of 2DG for the given incubation periods (black vs. green and red vs. yellow) is indicated by # p < 0.05, ## p < 0.01 and ### p < 0.001. Significant differences between data for the two different incubations periods of each condition (with or without 2DG preincubation, black vs. red and green vs. yellow) are indicated by + p < 0.05 and ++ p < 0.01 [26,42]. After the 30 min preincubation of astrocytes in the presence of 2DG, the cells contained a specific 2DG6P content of 130.6 ± 9.1 nmol/mg.…”

“…The WST1 formazan content in the incubation media was determined as previously described in detail [26]. The 50 µL samples collected for the given time periods were diluted with water to a total volume of 200 µL in wells of a microtiter plate and the WST1 formazan absorbance was measured at 450 nm in a microtiter plate reader (Multiscan Sky, Thermo Fisher, Darmstadt, Germany) as previously reported [26,27]. The extracellular WST1 formazan concentration was normalized to the initial protein content of the respective cultures to obtain the specific WST1 formazan content.…”

Analysis of the Effects of Pentose Phosphate Pathway Inhibition on the Generation of Reactive Oxygen Species and Epileptiform Activity in Hippocampal Slices

“…In contrast, the specific inhibitor G6PDi−1 prevented neuronal death caused by the expression of mutated SOD1 or TDP43 proteins in a model of amyotrophic lateral sclerosis, indicating a beneficial effect of G6PDH activity on cells in pathological conditions [42]. Results from a study on cultured astrocytes demonstrated that G6PDi−1 (10 µM) inhibited 60% of the supply of electrons for a NQO1-catalysed β-lapachone-mediated reduction in the water-soluble tetrazolium salt 1, suggesting that NADPH, derived from PPP, is the main source of electrons for cytosolic NAD(P)H quinone oxidoreductase 1 (NQO1) [43]. Importantly, another study from this group provided compelling evidence that G6PDi−1 efficiently and specifically inhibits astrocytic G6PDH without affecting other oxidoreductases, indicating that G6PDi−1 is a suitable and specific tool for inhibiting G6PDH and PPP-dependent processes [44].…”