Nitrate reductase alters 3-nitrotyrosine accumulation and cell cycle progression in LPS + IFN-gamma-stimulated RAW 264.7 cells.

Nitrite (NO2-), an end product of nitrogen radical metabolism, has recently been shown to increase tyrosine nitration by activated leukocytes indicating that nitrite modulates the immune response. We investigated the hypothesis that nitrite may increase nitration of molecular targets within activated cells leading to altered cell cycle progression. Intracellular nitrite was increased by transfection of murine macrophage-like RAW 264.7 cells with the nitrate reductase gene obtained from barley. Nitrate reductase facilitates the conversion of nitrate to nitrite; thus when extracellular nitrate is present, intracellular nitrite will be increased. Results show that addition of KNO3 increases NO2- production and intracellular nitrotyrosine accumulation in the transfectant but not the parent. Inhibition of nitric oxide synthesis with L-NAME during activation with IFN-gamma + LPS reduced NO2- production to the same extent in both cell lines; however, cellular accumulation of nitrotyrosine was reduced by only 25% in the transfectant (P = 0.21) and 49% in the parent cell line (P = 0.007), suggesting that intracellular nitrite increased nitrotyrosine accumulation through a pathway not requiring NO synthesis, i.e., myeloperoxidase system. Approximately 15% of the transfected cells had 4n DNA content 24 h postactivation compared to < 1% of the parent cells. Increased DNA copy number was correlated to nitrotyrosine accumulation. These findings show that intracellular nitrite can increase accumulation of nitrotyrosine and that nitration is linked to cell cycle perturbation.