Nitroarachidonic acid prevents NADPH oxidase assembly and superoxide radical production in activated macrophages

Abstract

Nitration of arachidonic acid (AA) to nitroarachidonic acid (AANO2) leads to anti-inflammatory intracellular activities during macrophage activation. However, less is known about the capacity of AANO2 to regulate the production of reactive oxygen species (ROS) under pro-inflammatory conditions. One of the immediate responses upon macrophage activation involves the production of superoxide radical (O2·−), due to the NADPH dependent univalent reduction of oxygen to O2·− by the phagocytic NADPH-oxidase isoform (NOX2), being the activity of NOX2 the main source of O2·− in monocytes/macrophages. Since NOX2 and AA pathways are connected, we propose that AANO2can modulate macrophage activation by inhibiting O2·− formation by NOX2. When macrophages were activated in the presence of AANO2, a significant inhibition of NOX2 activity was observed as evaluated by cytochrome c reduction, luminol chemiluminescence, Amplex Red fluorescence and flow cytometry; this process also occurs in physiological mimic conditions within the phagosomes. AANO2 decreased O2·− production in a dose-(IC50= 4.1 ± 1.8 μM AANO2) and time-dependent manner. The observed inhibition was not due to a decreased phosphorylation of the cytosolic subunits (e.g. p40phox and p47phox), as analyzed by immunoprecipitation and western blot. However, a reduction of the migration to the membrane of p47phox was obtained suggesting that the protective actions involve the prevention of the correct assembly of the active enzyme in the membrane. Finally, the observed in vitro effects were confirmed in an in vivo inflammatory model, where subcutaneous injection of AANO2 was able to decrease NOX2 activity in macrophages from thioglycolate treated mice.

Profiling and relative quantification of multiply nitrated and oxidized fatty acids

“…Although F 4 -neuroprostanes derived from Dha, like Aa derived F 2 -isoprostanes, have been related to various cardiovascular and neurodegenerative disorders [45,46], here we can only speculate about the beneficial effects of Dha-derived nitration products, as was shown for AaNO 2 [27,31,32] and all other singly nitrated fatty acids. Lipid peroxidation products usually posses proinflammatory properties [47]; however current data on biological activities of NO 2 -FA support their anti-inflammatory effects via both cGMPdependent and cGMP-independent mechanisms.…”

“…Lipid peroxidation products usually posses proinflammatory properties [47]; however current data on biological activities of NO 2 -FA support their anti-inflammatory effects via both cGMPdependent and cGMP-independent mechanisms. Although the overproduction of NO 2 -FA was observed during inflammation, a series of anti-inflammatory effects were reported for LaNO 2 [48][49][50] , OaNO 2 [15,24], and AaNO 2 [27,31,32]. The question about biological activities of multiply nitrated and nitro-oxidized PUFA remains open owing to the lack of the data on their biological availability.…”

“…Increased levels of nitrolinoleic acid (LaNO 2 ) were detected in mitochondria during ischemic preconditioning, whereas the level of nitrooleic acid (OaNO 2 ) remained unchanged, indicating possible selectivity of fatty acid nitration [18]. Under inflammatory conditions LaNO 2 , OaNO 2 , and nitroarachidonic acid (AaNO 2 ) posses anti-inflammatory activities [19][20][21], including activation of peroxisome-proliferator-activated receptors [22] and nuclear factor E2 related factor 2 [23,24], induction of heme oxygenase 1 [25], inhibition of nuclear factor κB, and inhibition of prostaglandin endoperoxide H synthase (PGHS) [26] and NADPH oxidase [27]. OaNO 2 decreased superoxide production in activated macrophages and pulmonary artery smooth muscle cells, demonstrating protective effects in a hypoxia-induced murine model of pulmonary hypertension [28].…”

“…Release of nitric oxide by AaNO 2 can induce cyclic GMP (cGMP)-dependent vasorelaxation and downregulate inducible nitric oxide synthase [31]. Additionally, AaNO 2 was shown to prevent NADPH oxidase assembly [27], regulate protein kinase C [32], and inhibit oxygenase activities of PGHS-1 and PGHS-2.…”

AKT/mTOR and c-Jun N-terminal kinase signaling pathways are required for chrysotile asbestos-induced autophagy

“…ROS production in A549 cells was separately measured by flow cytometry with the fluorescent dye dihydroethidium (DHE) and Amplex Red that have been used to quantify the intracellular oxidant production [23,24]. A549 cells were grown on 12-well plates.…”