Figure 6
From: Somatic cell hemoglobin modulates nitrogen oxide metabolism in the human airway epithelium
Carbon monoxide (CO) prevents NO oxidation in human primary airway epithelial cell culture medium but does not affect cell viability or NOS activity. (A) Nitrate accumulation in the extracellular medium did not differ between subjects (PCD vs wt, p = NS for each pair-wise comparison). Surprisingly, cytomix (10 ng/uL each of TNFα, IFNγ and LPS for 4 h) did not increase NO3− in these primary pseudostratified ALI cultures, in contrast to previous reports from our group and others20,21 regarding submerged cell lines. This argues against iNOS being the principal source of nitrogen oxides in this system. However, CO (1 ppm; 10 min) completely inhibited NO3− production overall (n = 21 positive vs n = 30 negative, p < 0.001). For the pair-wise comparisons, the inhibition effect of CO was significant in PCD without cytomix (p = 0.001, Tukey p = 0.002) and WT with cytomix (p < 0.001, Tukey p < 0.001) after Tukey’s multiple correction; was significant in PCD with cytomix before Tukey’s correction (p = 0.017, Tukey p = 0.067), and insignificant in WT without cytomix (p = 0.355, Tukey p = 0.827). (B) Carbon monoxide (as above) also inhibited NO2− accumulation in the medium (4 h, as above; p < 0.001). For the pair-wise comparison, CO significantly inhibited NO2− production in PCD without cytomix (p = 0.009, Tukey p = 0.035) and WT without cytomix (p < 0.001, Tukey p < 0.001) after Tukey’s correction; and significantly inhibited NO2− in WT with cytomix before Tukey’s correction (p = 0.035, Tukey p = 0.131), while no significant difference was observed in PCD with cytomix (p = 0.071, Tukey p = 0.255).” (C, D) Though CO can also bind heme in NOS22 the effect of NOS substrate, l-arginine (1 mM), to increase intracellular nitrogen oxides NO3− (C) and NO2− (D) was completely ablated by excess NOS inhibitor nitro-L-arginine methyl ester (L-NAME; 10 mM), but not by CO (NO3−: L-NAME vs CO; n = 5; p = 0.019); (NO2–2; L-NAME vs CO; n = 5; p = 0.0015). (E, F) Note that 10 min of CO pretreatment had no effect on the viability of these cells in culture, either by phase contrast imaging (E) or by Triton blue exclusion (F).
