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 macrophage-like RAW 264.7 cells activated by bacterial lipopolysaccharide and interferon-gamma. Rates of NO(2)(-) and NO(3)(-) accumulation were determined 8-11 h after stimulation. The rate of NO synthesis was computed by using a reaction-diffusion model to correct NO(2)(-) and NO(3)(-) accumulation for physical loss of NO, whereas O(2)(-) synthesis was equated with NO(3)(-) formation. Rates of O(2)(-) synthesis determined by a spectrophotometric (cytochrome c) assay were in good agreement with those from NO(3)(-) accumulation and showed production of O(2)(-) to be detectable immediately, in contrast to the approximately 6 h time lag for NO. The assumption that NO(2)(-) and NO(3)(-) are stable end products of the extracellular oxidation of NO by O(2) and O(2)(-), respectively, was supported by the fact that NO(2)(-) and NO(3)(-) 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 NO(2)(-) and NO(3)(-) accumulation by an order of magnitude at the smallest depth. The principal finding was that the rates of NO(2)(-) and NO(3)(-) accumulation each remained nearly constant over the 4-fold range of liquid depths. Because greater depths should greatly facilitate the trapping of NO as NO(2)(-), 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 approximately 1 microM for all conditions examined, suggesting that regulation of NO synthase activity by NO might also have mediated the effect of liquid depth.