pO2-dependent NO production determines OPPC activity in macrophages |
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| Authors: | Mary A. Robinson Stephen W. Tuttle Cynthia M. Otto Cameron J. Koch |
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| Affiliation: | 1. Department of Radiation Oncology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA;2. Department of Clinical Studies–Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA;3. Center for Sleep and Respiratory Neurobiology, University of Pennsylvania, Philadelphia, PA 19104, USA;1. Smooth Muscle Research Group, Department of Physiology and Pharmacology, Hotchkiss Brain Institute and Libin Cardiovascular Institute, University of Calgary, Alberta, Canada;2. Department of Physiology, Instituto de Biología y Genética Molecular, University of Valladolid, Valladolid, Spain;3. Smooth Muscle Research Group, Department of Biochemistry and Molecular Biology, Hotchkiss Brain Institute and Libin Cardiovascular Institute, University of Calgary, Alberta, Canada;3. Molecular Medicine Research Center and Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu 610041, China;4. Institute for Genomic Medicine and Shiley Eye Center, University of California at San Diego-La Jolla, California, 92093;5. Department of Bioengineering, University of California at San Diego, La Jolla, California 92093;6. Tong Ren Eye Hospital, Beijing, China;12. The Sichuan Provincial Key Laboratory for Human Disease Gene Study and The Institute of Laboratory Medicine, Sichuan Academy of Medical Sciences and Sichuan Provincial People''s Hospital, Chengdu, Sichuan 610072, China;1. Reproductive Medicine Center, The First Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu, China;2. Gynaecology and Obstetrics, Suzhou Ninth People''s Hospital, Suzhou 215000, Jiangsu, China;1. Beijing Institute of Respiratory Medicine, Capital Medical University, Beijing 100069, China;2. Department of Toxicology, Fourth Military Medical University, Xi’an 710032, China;3. Department of Toxicology, Bayer Pharma AG, 42096 Wuppertal, Germany;4. Department of Pharmacology Vascular Diseases, Cardiology & Hematology, Bayer Pharma AG, 42096 Wuppertal, Germany;5. Beijing Institute of Respiratory Medicine, Beijing Hospital, Ministry of Health, Beijing 100730, China |
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| Abstract: | Stimulated macrophages produce nitric oxide (NO) via inducible nitric oxide synthase (iNOS) using molecular O2, L-arginine, and NADPH. Exposure of macrophages to hypoxia decreases NO production within seconds, suggesting substrate limitation as the mechanism. Conflicting data exist regarding the effect of pO2 on NADPH production via the oxidative pentose phosphate cycle (OPPC). Therefore, the present studies were developed to determine whether NADPH could be limiting for NO production under hypoxia. Production of NO metabolites (NOx) and OPPC activity by RAW 264.7 cells was significantly increased by stimulation with lipopolysaccharide (LPS) and interferon γ (IFNγ) at pO2 ranging from 0.07 to 50%. OPPC activity correlated linearly with NOx production at pO2 > 0.13%. Increased OPPC activity by stimulated RAW 264.7 cells was significantly reduced by 1400 W, an iNOS inhibitor. OPPC activity was significantly increased by concomitant treatment of stimulated RAW 264.7 cells with chemical oxidants such as hydroxyethyldisulfide or pimonidazole, at 0.07 and 50% O2, without decreasing NOx production. These results are the first to investigate the effect of pO2 on the relationship between NO production and OPPC activity, and to rule out limitations in OPPC activity as a mechanism by which NO production is decreased under hypoxia. |
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