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

Macrophage-derived nitric oxide ((NO)-N-center dot) participates in cytotoxic mechanisms against diverse microorganisms and tumor cells. These effects can be mediated by (NO)-N-center dot itself or (NO)-N-center dot-derived species such as peroxynitrite formed by its diffusion-controlled reaction with NADPH oxidase-derived superoxide radical anion (O-2(center dot-)). In vivo, the facile extracellular diffusion of (NO)-N-center dot as well as different competing consumption routes limit its bioavailability for the reaction with O-2(center dot-) and, hence, peroxynitrite formation. In this work, we evaluated the extent by which (NO)-N-center dot diffusion to red blood cells (RBC) can compete with activated macrophages derived O-2(center dot-) 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)-N-center dot diffusion to RBC on peroxynitrite formation was experimentally analyzed in co incubations of (NO)-N-center dot and O-2(center dot-) -forming macrophages with erythrocytes. Additionally, we evaluated the permeation of (NO)-N-center dot to RBC by measuring the intracellular oxidation of oxyhernoglobin to methemoglobin. Our results indicate that diluted RBC suspensions close dependently inhibit peroxynitrite formation, outcompeting the O-2(center dot-) 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)-N-center dot and O-2(center dot-) 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)-N-center dot 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. (C) 2015 Published by Elsevier Inc.