Oxidative Stress Induces the Phosphorylation of NAD+ to NADP+ by NAD Kinase in Cultured Primary Rat Astrocytes

Astrocytes have important functions in the metabolism and antioxidative defence of the brain. Three redox pairs and the ratio of the reduced and oxidized partners in each pair are essential for astrocytic redox metabolism, GSx (glutathione (GSH) plus glutathione disulfide (GSSG)), NADx (NADH plus NAD⁺) and NADPx (NADPH plus NADP⁺). In order to elucidate the interactions between the three redox pairs in astrocytes, we first analysed the basal levels of the six redox co-substrates for cultured primary rat astrocytes by using sensitive and specific enzymatic cycling assays. In untreated cultures, the basal specific contents of GSx, NADPx and NADx were 44.7 ± 8.2 nmol/mg protein, 0.64 ± 0.09 nmol/mg protein and 2.91 ± 0.40 nmol/mg protein, with the reduced co-substrates accounting for 97 ± 3%, 37 ± 14% and 28 ± 10% of the total amounts, respectively. Exposure of cultured astrocytes to oxidative stress (100 µM H₂O₂ in the presence of the pentose-phosphate pathway inhibitor G6PDi-1) caused a rapid and severe but transient oxidation of GSH to GSSG. This increase was accompanied by a doubling of the total pool of NADPx on the expense of the cellular NADx pool, suggesting that NAD⁺ was phosphorylated to NADP⁺ under these conditions. Testing for NAD kinase (NADK) activity in lysates of cultured astrocytes revealed that the enzyme is present with a specific v_max activity of around 1 nmol/(min x mg protein) and has K_M-values of 1.30 ± 0.19 mM for NAD⁺ and 2.71 ± 0.18 mM for ATP. Preincubation of astrocytes with thionicotinamide, the precursor for the cellular synthesis of the NADK inhibitor thio-NADP, prevented the transient oxidative stress-induced phosphorylation of NAD⁺ to NADP⁺. These data demonstrate that the NADPx pool can be increased in cultured astrocytes during oxidative stress by NADK-mediated phosphorylation of NAD⁺, providing experimental evidence for an additional interaction of the main astrocytic redox pairs during oxidative stress.