Nitroarachidonic acid prevents NADPH oxidase assembly and superoxide radical production in activated macrophages |
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| Institution: | 1. Division of Endocrinology, Diabetes & Metabolism, Leonard M. Miller School of Medicine, University of Miami, Miami, FL, USA;2. Department of Neurology, Leonard M. Miller School of Medicine, University of Miami, Miami, FL, USA;3. Department of Epidemiology and Public Health, Leonard M. Miller School of Medicine, University of Miami, Miami, FL, USA;4. Evelyn F. McKnight Brain Institute, Leonard M. Miller School of Medicine, University of Miami, Miami, FL, USA;5. Stroke Division, Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, NY, USA;6. Department of Epidemiology, Mailman Public School of Health, Columbia University, New York, NY, USA;7. Neuroscience Program, Leonard M. Miller School of Medicine, University of Miami, Miami, FL, USA;1. Department of Medicine, Harborview Medical Center, University of Washington, Seattle, Washington;2. Department of Critical Care Medicine, Safar Center for Resuscitation Research and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania;3. Department of Pharmacy and Therapeutics, Center for Clinical Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania;4. Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania;5. Department of Biostatistics, University of Washington, Seattle, Washington;6. Department of Health Services, University of Washington, Seattle, Washington;7. Department of Emergency Medicine, Harborview Medical Center, University of Washington, Seattle, Washington;1. Lipid Clinic, Endocrinology and Nutrition Service, Hospital Clínic, Institut d''Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain;2. CIBER Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III (ISCIII), Spain;3. Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain;4. Cardiovascular Institute, Hospital Clínico San Carlos, Madrid, Spain;5. Hospital Universitario Quirón, Universidad Europea de Madrid, Spain;6. Hospital Universitario Montepríncipe, Madrid, Spain;7. The Zena and Michael A. Wiener Cardiovascular Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States;8. IIS, Fundación Jiménez Díaz, Madrid, Spain;9. Hospital Universitario Central de la Defensa Gómez Ulla, Madrid, Spain |
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| 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 under proinflammatory 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), the activity of NOX2 being the main source of O2?? in monocytes/macrophages. Because the NOX2 and AA pathways are connected, we propose that AANO2 can 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 under 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 in 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, in which subcutaneous injection of AANO2 was able to decrease NOX2 activity in macrophages from thioglycolate-treated mice. |
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