36 - Quantitative Relationship between NADPH Depletion and Inhibition of NOX2-Induced Superoxide Radical Anion

Abstract

Nicotinamide adenine dinucleotide (NAD + ) is an essential cofactor controlling cell metabolism and survival. NAD + is recycled in cells at rates controlled by nicotinamide phosphoribosyl transferase (NAMPRT). The pharmacological inhibition of NAMPRT by FK866 diminishes intracellular concentration of redox couples NAD + /NADPH and NADP + /NADPH. Here we examined the quantitative relationship between inhibition of NAMPRT and superoxide-producing activity of NOX2 from PMA activated macrophage RAW 264.7 cells. Cells were treated with 10nM FK866 for 24 h showed PI staining negative and survival >80%. Superoxide (O 2 ●– ) in PMA activated cells was maximally stimulated with 100-400 ng/ml. 2-Hydroxyethidium, 2-OHE + in both supernatant and cytosolic fractions was decreased in cells pre-treated with 10nM FK866 for 24 h. This inhibition was rescued with10µM NMN co-treatment. The NOX inhibitor, VAS2870 (10-100µM) decreased O 2 ●– release although toxicity was evident. FK866 did not alter the protein expression of NOX subunits, p91phox and p47phox. HPLC analysis of FK866 treated cells showed diminished intracellular levels of NAD + and NADPH and to a lesser extent NADH, NADP + . A dose-dependent effects of FK866 (1-20nM) indicated that after 24h incubation FK866 at concentrations of 10nM led to a decrease of ~50% of NADPH (from 2.2±0.2 to 1.2±0.2 nmol/mg protein) eliciting a reduction of 2-OHE + >70%. Increasing FK to 20nM showed small effects compared to 10nM treatment. An inhibition of 50% of 2-OHE+ formation was attained with 5nM FK866 treatments indicating that NADPH EC50 for NOX2 ~1.6nmol/mg protein. These results indicate that NOX2 activity is inhibited by depletion of physiological levels of NADPH which could be highly significant in the regulation of peroxynitrite and other oxidant production.