title: Nitric oxide, oxygen, and superoxide formation and consumption in macrophage cultures.
abstract: To examine the potential for generating toxic nitrogen oxides during the immune response, rates of formation and consumption of NO, O2, and O2- were measured in murine macrophage-like RAW264.7 cells. Cellular kinetic parameters for NO and O2 were obtained by monitoring their time-dependent concentrations in a closed chamber, and net cellular synthesis of O2- was quantified from ferricytochrome c reduction in cultures where NO synthesis was inhibited. Also measured was the photosensitive generation of O2- in the culture media. Unactivated cells (without NO synthesis) had an O2 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 O2 as unactivated cells [108+/-17 pmol s-1 (10(6) cells)-1]; however, O2 consumption of activated cells exposed to 1 microM 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 O2- generation in the culture media resulted in net rates of cellular O2- synthesis smaller than previously reported; the rate was 6% of NO synthesis in activated cells and was undetectable in unactivated cells.
authors.affiliation: Department of Chemical Engineering and Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Nitesh
authors.initials: N
authors.lastname: Nalwaya
authors.forename: William M
authors.initials: WM
authors.lastname: Deen
mesh_terms: D000818:Animals; D002460:Cell Line; D045304:Cytochromes c / Q000378:metabolism; D008262:Macrophage Activation / Q000502:physiology; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis*; D010084:Oxidation-Reduction; D010101:Oxygen Consumption / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism*
chemical_list: D013481:Superoxides; D009569:Nitric Oxide; D045304:Cytochromes c