Nitric oxide diffusion to red blood cells limits extracellular, but not intraphagosomal, peroxynitrite formation by macrophages

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Macrophage-derived nitric oxide (•NO) participates in cytotoxic mechanisms against diverse microorganisms and tumor cells. These effects can be mediated by •NO itself or •NO-derived species such as peroxynitrite formed by its diffusion-controlled reaction with NADPH oxidase-derived superoxide radical anion (O2•−). In vivo, the facile extracellular diffusion of •NO as well as different competing consumption routes limit its bioavailability for the reaction with O2•− and, hence, peroxynitrite formation. In this work, we evaluated the extent by which •NO diffusion to red blood cells (RBC) can compete with activated macrophages-derived O2•− and affect peroxynitrite formation yields. Macrophage-dependent peroxynitrite production was determined by boron-based probes that react directly with peroxynitrite, namely, coumarin-7-boronic acid (CBA) and fluorescein-boronate (Fl-B). The influence of •NO diffusion to RBC on peroxynitrite formation was experimentally analyzed in co-incubations of •NO and O2•−-forming macrophages with erythrocytes. Additionally, we evaluated the permeation of •NO to RBC by measuring the intracellular oxidation of oxyhemoglobin to methemoglobin. Our results indicate that diluted RBC suspensions dose-dependently inhibit peroxynitrite formation, outcompeting the O2•− reaction. Computer-assisted kinetic studies evaluating peroxynitrite formation by its precursor radicals in the presence of RBC are in accordance with experimental results. Moreover, the presence of erythrocytes in the proximity of •NO and O2•--forming macrophages prevented intracellular Fl-B oxidation pre-loaded in L1210 cells co-cultured with activated macrophages. On the other hand, Fl-B-coated latex beads incorporated in the macrophage phagocytic vacuole indicated that intraphagosomal probe oxidation by peroxynitrite was not affected by nearby RBC. Our data support that in the proximity of a blood vessel, •NO consumption by RBC will limit the extracellular formation (and subsequent cytotoxic effects) of peroxynitrite by activated macrophages, while the intraphagosomal yield of peroxynitrite will remain unaffected.