Nitric oxide, oxygen, and superoxide formation and consumption in macrophage cultures

To examine the potential for generating toxic nitrogen oxides during the immune response, rates of formation and consumption of NO, O-2, and O-2(-) were measured in murine macrophage-like RAW264.7 cells. Cellular kinetic parameters for NO and O-2 were obtained by monitoring their time-dependent concentrations in a closed chamber, and net cellular synthesis of O-2(-) was quantified from ferricytochrome c reduction in cultures where NO synthesis was inhibited. Also measured was the photosensitive generation of O-2(-) in the culture media. Unactivated cells (without NO synthesis) had an O-2 consumption rate of 32 +/- 3 pmol s(-1) (10(6) cells)(-1), typical of mammalian cells. Also typical was that adding NO rapidly and reversibly inhibited respiration. Activated cells synthesized NO at a rate of 4.9 +/- 0.6 pmol s(-1) (10(6) cells)(-1). When NO synthesis was inhibited, they consumed three times as much O-2 as unactivated cells [108 +/- 17 pmol s(-1) (10(6) cells)(-1)]; however, O-2 consumption of activated cells exposed to 1 mu M NO was calculated to be comparable to that of NO-free unactivated cells. Rates, of intracellular NO consumption were small, implying that enzymatic consumption does little to limit net NO synthesis by macrophages. Accounting for O-2(-) generation in the culture media resulted in net rates of cellular O-2(-) synthesis smaller than previously reported; the rate was 6% of NO synthesis in activated cells and was undetectable in unactivated cells.