Nitric Oxide Inactivates NADPH Oxidase in Pig Neutrophils by Inhibiting Its Assembling Process

Abstract

Nitric oxide (NO) is now recognized as one of the key mediators in many physiological and pathological processes (see reviews in Refs. 1 and 2). NO is also known to be a multifunctional molecule, one function of which is to inactivate biologically important enzymes such as mitochondrial respiratory enzymes and GAPDH, which play important roles in energy production (3, 4), ribonucleotide reductase, which is the key enzyme for protein synthesis (5), and the superoxide-generating enzymes, NADPH oxidase (6) and xanthine oxidase (7). Particularly important is the effect of NO on NADPH oxidase, because under conditions such as inflammation, the accumulation of phagocytes is a common feature and the induction of NO synthase has been shown. It is plausible that increased formation of NO interferes with the activity of NADPH oxidase and reduces superoxide (O 2 . ) production (8, 9). Despite the importance of the NO effect on NADPH oxidase, no detailed study has been carried out since the initial report by Clancy et al. (6) in which inhibition of O 2 . generation by NO was demonstrated. The underlying mechanism was suggested to be the direct interaction of NO on the membrane components of NADPH oxidase (6). The NADPH oxidase of phagocytes is a multi-component electron transport system, in which activation requires the assembly of three cytosolic regulatory proteins (p47 phox , p67 phox , and Rac1/Rac2) to membrane-bound cytochrome b 558 (10, 11). Cytochrome b 558 is postulated to be a membranebound flavocytochrome with six-coordinated low spin heme and FAD as redox centers. The electrons provided by NADPH are thought to be transferred in a linear sequence, NADPH 3 FAD 3 heme (Fe 3ϩ ) 3 O 2 . The heme in cytochrome b 558 is assumed to be the terminal electron donor in the production of O 2. from molecular oxygen due to its unusually low redox potential, Ϫ245 mV (12). Although the most plausible site of NADPH oxidase attacked by NO was suggested to be in membrane protein(s) (6), a detailed analysis of these effects has not been performed. Considering that nitrosyl-iron complex easily forms in heme-containing enzymes (3), the heme structure of cytochrome b 558 and the electron flux from substrate (NADPH) to redox centers, FAD and low spin heme, in NADPH oxidase should be examined to clarify the effects of NO on its O 2 . -generating activity.In the present study, we examined the effects of NO on electron fluxes in neutrophil NADPH oxidase. Under aerobic conditions the effects of NO on O 2 . -generating activity of NADPH oxidase (reaction 1) was examined by the cytochrome c reduction method. In this study, we also employed the solubilized NADPH oxidase obtained from stimulated cells and measured its O 2 . -generating activity in the presence of NO.Under anaerobic conditions the effects of NO on the electron transfer reaction in each redox center was examined: NADPH 3 FAD 3 exogenous electron acceptor, cytochrome c (reaction 2) and NADPH 3 FAD 3 cytochrome b 558 (reaction 3). Under both aerobic and anaerobic conditions, the...

Tissue kallikrein infusion prevents cardiomyocyte apoptosis, inflammation and ventricular remodeling after myocardial infarction

“…Nitric oxide is capable of inhibiting neutrophil superoxide anion production via a direct action on the membrane components of NADPH oxidase and the assembly of NADH/NADPH oxidase subunits [25]. However, NO has been considered to be a double-edged sword [26,27].…”

Nitric oxide‐mediated augmentation of neutrophil reactive oxygen and nitrogen species formation: Critical use of probes

“…Neutrophils have also been coupled commonly with inflammatory conditions, which are mostly associated with the accumulation of NO and its metabolites. Various reports demonstrated NOmediated biphasic regulation (14,16,17), stimulation (15,22), and even inhibition (18,20) of PMNs reactive species formation. These outcomes could be due to the differences in probes used to detect ROS/RNS, which exhibit different reactivity, specificity, and selectivity, and some probes even interact with NO (25,26).…”

“…Studies from this laboratory as well as from others have demonstrated a biphasic role of NO on the reactive species formation in PMNs in response to arachidonic acid, fMLP, and PMA stimulation (14)(15)(16)(17). Low concentrations (10-100 nM) of NO potentiated reactive species generation by augmented ADP-ribosylation, intracellular calcium, and activation of ERK MAPK pathway, but higher concentrations (1 lM-1 mM) attenuated ROS generation through feed back inhibition of NADPH-oxidase and superoxide scavenging effect and activation of the guanylate cyclase-cyclic GMP pathway from the rat and human PMNs (15,16,(18)(19)(20). Indeed, NO suppressed NADPH oxidase-dependent superoxide production by S-nitrosylation of p47 subunit in human endothelial cells (21).…”

“…), absorbance (NBT, cytochrome c reduction), or electron paramagnetic resonance (EPR; by utilizing spin traps, DMPO/ DEPMPO) (15,16,(18)(19)(20)23,24). The ROS/RNS species have different sensitivity/specificity for these probes/methods like O 2 2 (DHE, lucigenin, NBT, cytochrome c reduction, and DEPMPO), H 2 O 2 (DCF, DHR, luminol, and Scopoletin), ONOO 2 (DCF, DHR, and luminol), OH (DCF, luminol, and DMPO), HOCl (DCF, DHR, and luminol), etc.…”

S-nitrosylation: Physiological regulation of NF-κB

“…Other proven or likely targets for nitrosylation in the NF-B pathway include the epidermal growth factor and src tyrosine kinases (24,25), which activate NF-B through p21 ras ; tyrosine phosphatases (26); and NADPH oxidase (27), known to activate NF-B through IKK (presumably by means of the oxidative inactivation of phosphatase). Given the multiple loci of S-nitrosylation in NF-Brelated pathways, the effects of S-nitrosylation may be considered analogous to those of phosphorylation, with a large number of regulatory permutations that combine ultimately to optimize cellular responses.…”