New insight into the NO-mediated signaling to modulate neutrophil free radical generation

Abstract

Neutrophils (PMNs) and nitric oxide (NO) play an important role in pathogen killing as well as in various pathological conditions associated with inflammation, hypoxia/ischemia and reoxygenation injury by generating reactive oxygen (ROS) and reactive nitrogen (RNS) species. Previous work from this lab has shown that NO modulates neutrophil NADPH-oxidase (NOX) activity and thus modulates ROS production. The present study has been undertaken to assess signalling mechanisms involved in NO mediated effect on PMNs by flowcytometry (DCF-DA, DHE, propidium iodide, CBA Flex array), biochemical (H₂O₂, hypochlorous acid [HOCl], nitryl chloride [NO₂Cl], dityrosine) and molecular (Western blotting) measurements. NO donors, SNP and SNAP (10 µM to 1 mM) inhibited the NADPH-oxidase activity but enhanced DCF-DA fluorescence in a concentration and time dependent manner, with no adverse effect on the cell viability. The effect was significantly blocked by NOS inhibitors and was partially blocked by ERK and MPO inhibitors. NO at the concentrations used seems to activate futile cycle generating peroxynitrite (ONOO)/NO₃⁻, which subsequently uncouples NOS and generate superoxide radicals and H₂O₂. H₂O₂ thus formed leads to the formation of HOCl by MPO catalysis, which further reacts with nitrite (NO₂⁻) to generate NO₂Cl, a more potent oxidant and nitrating agent. The results obtained thus suggest that NO mediated augmentation in neutrophil free radical production involves ERK signalling.