Nitric oxide, oxygen, and superoxide formation and consumption in macrophages and colonic epithelial cells

Abstract

Knowledge of the rates at which macrophages and epithelial cells synthesize NO is critical for predicting the concentrations of NO and other reactive nitrogen species in colonic crypts during inflammation, and elucidating the linkage between inflammatory bowel disease, NO, and cancer. Macrophage-like RAW264.7 cells, primary bone marrow-derived macrophages (BMDM), and HCT116 colonic epithelial cells were subjected to simulated inflammatory conditions, and rates of formation and consumption were determined for NO, O(2), and O(2)(-). Production rates of NO were determined in either of two ways: continuous monitoring of NO concentrations in a closed chamber with corrections for autoxidation, or NO(2)(-) accumulation measurements in an open system with corrections for diffusional losses of NO. The results obtained using the two methods were in excellent agreement. Rates of NO synthesis (2.3 +/- 0.6 pmol s(-1) 10(6) cells(-1)), NO consumption (1.3 +/- 0.3 s(-1)), and O(2) consumption (59 +/- 17 pmol s(-1) 10(6) cells(-1) when NO is negligible) for activated BMDM were indistinguishable from those of activated RAW264.7 cells. NO production rates calculated from NO(2)(-) accumulation data for HCT116 cells infected with Helicobacter cinaedi (3.9 +/- 0.1 pmol s(-1) 10(6) cells(-1)) were somewhat greater than those of RAW264.7 macrophages infected under similar conditions (2.6 +/- 0.1 pmol s(-1) 10(6) cells(-1)). Thus, RAW264.7 cells have NO kinetics nearly identical to those of primary macrophages, and stimulated epithelial cells are capable of synthesizing NO at rates comparable to those of macrophages. Using these cellular kinetic parameters, simulations of NO diffusion and reaction in a colonic crypt during inflammation predict maximum NO concentrations of about 0.2 microM at the base of a crypt.

Figure 1

The four major fates of NO generated by cells in a typical plate culture experiment: oxidation to NO₃⁻ via intracellular pathways, oxidation to NO₂⁻ in the medium via Reaction 1, oxidation to NO₃⁻ in the medium via Reaction 2, or diffusional loss to the headspace. With macrophages (as shown), some of the O₂⁻ in the medium is produced by a membrane-bound NADPH oxidase; with colonic epithelial cells, that source of O₂⁻ is absent. The rate constants shown for each process are defined later, in connection with the kinetic models.

Figure 2

Measurement of NO synthesis, NO consumption, and O₂ consumption by bone marrow-derived macrophages (BMDM) and RAW264.7 cells. Shown are NO concentrations (black) and O₂ concentrations (gray) as a function of time for representative closed-chamber experiments. The linear decline in O₂ concentration in the absence of NO for unactivated macrophages (8 × 10⁶ cells) indicates that the rate of O₂ consumption is constant. Measurements with unactivated RAW264.7 and HCT116 cells resulted in similar plots (not shown). NO and O₂ concentrations were also measured for 8 × 10⁶ BMDM after activation by 12 h exposure to IFN-γ and LPS. The rate of O₂ consumption slowed as the NO concentration increased. The kinetics of NO synthesis and consumption are reflected in the plateau concentration for NO and the rate at which the plateau is reached. Plots of NO and O₂ concentrations measured for activated RAW264.7 cells were nearly identical (not shown). O₂ concentrations are also depicted for activated RAW264.7 cells after 1 h exposure to rotenone, a respiratory inhibitor; the reduced slope corresponds to about an 85% reduction in the overall rate of O₂ consumption.

Figure 3

Measurement of NO synthesis, NO consumption, and O₂ consumption by activated HCT116 cells stimulated with capsaicin and resveratrol. Results are shown for 7 × 10⁶ cells in the closed chamber. Induction of NO synthesis, as reflected by the increasing NO concentration, inhibited respiration in a manner similar to that seen with macrophages in Figure 2.

Figure 4

Nitrite and nitrate concentrations in the supernatant of HCT116 cells which were infected with H. cinaedi. Means ± SD are shown for n = 5. The initial cell numbers were 1 × 10⁶ for HCT116 and 1 × 10⁷ or 1 × 10⁸ for H. cinaedi, yielding multiplicity of infections (MOI) of 10 or 100. The times (24 or 48 h) correspond to the duration of exposure to the bacteria. The increased NO₂⁻ concentrations for MOI = 100 are evidence of NO synthesis by the HCT116 cells.

Figure 5

Comparison of NO₂⁻ concentrations for RAW264.7 cells and HCT116 cells infected with H. cinaedi. Means ± SD are shown for n = 5. See Figure 4 for additional explanation.

Figure 6

Rate of O₂⁻ production by primary macrophages (BMDM). Plotted are nmol of ferrocytochrome c detected as a function of time for representative experiments with 5 × 10⁵ cells and with culture medium only. Results are shown for cells activated by incubation with IFN-γ and LPS beginning at t= 0 in the absence or presence of SOD, unactivated cells, untreated medium, and medium with SOD. Experiments performed with RAW264.7 resulted in similar plots (not shown).

Figure 7

Predicted NO concentrations in an inflamed colonic crypt. Concentrations at the crypt center are plotted as a function of height above the base. The calculations assumed that k_c = 1.3 s⁻¹ for macrophages and 4.1 s⁻¹ for epithelial cells, based on the BMDM and HCT116 data in Table 2. The rate of NO synthesis used for macrophages was 2.3 pmol s⁻¹ (10⁶ cells)⁻¹ (Table 2); using the cell volume (v) in Table 1, this corresponds to a volumetric rate of 2.4 µM/s. Results are shown for three assumed rates of NO synthesis by epithelial cells: 0, 1.2, and 4.2 µM/s. The latter values correspond to the HCT116 data for capsaicin-resveratrol in Table 2 and H. cinaedi in Table 3, respectively, again using v from Table 1. All other parameter values were as given in Chin et al (2008).