Effect of liquid depth on the synthesis and oxidation of nitric oxide in macrophage cultures

The effect of liquid depth on the synthesis of NO and O-2(-) was studied in murine macrophagelike RAW 264.7 cells activated by bacterial lipopolysaccharide and interferon-gamma. Rates of NO2- and NO3- accumulation were determined 8-11 h after stimulation. The rate of NO synthesis was computed by using a reaction-diffusion model to correct NO2- and NO 3 accumulation for physical loss of NO, whereas O-2(-) synthesis was equated with NO formation. Rates of O-2(-) synthesis determined by a spectrophotometric (cytochrome c) assay were in good agreement with those from NO3- accumulation and showed production of O-2(-) to be detectable immediately, in contrast to the similar to6 h time lag for NO. The assumption that NO2- and NO3- are stable end products of the extracellular oxidation of NO by O-2 and O-2, respectively, was supported by the fact that NO2- and NO3- concentrations remained constant in the presence of unstimulated cells or stimulated cells where NO synthesis was inhibited. Data were obtained for media depths ranging from 1 to 4 mm. The physical loss of NO was found to be quite significant, exceeding NO2- and NO3- accumulation by an order of magnitude at the smallest depth. The principal finding was that the rates of NO2- and NO3- accumulation each remained nearly constant over the 4-fold range of liquid depths. Because greater depths should greatly facilitate the trapping of NO as NO2-, this implies that NO synthesis decreased markedly with increasing depth. In contrast, O-2(-) synthesis remained approximately constant. Oxygen availability is likely to have affected NO synthesis, in that diffusional limitations will yield the lowest cellular O-2 concentrations when the liquid depth is greatest and NO synthesis is known to decrease when O-2 levels are reduced. Concentrations of NO near the cells were calculated to remain at similar to1 muM for all conditions examined, suggesting that regulation of NO synthase activity by NO might also have mediated the effect of liquid depth.