Abstract
Previously we have demonstrated that 3′,4′-dihydroxyflavonol (DiOHF), a novel synthetic flavonol, protects against ischemia reperfusion injury in both heart and brain. In this study, we characterized the pharmacological effects of DiOHF on phagocytic and vascular NADPH oxidase. Superoxide release (lucigenin-enhanced chemiluminescence or cytochrome c reduction), NADPH oxidase activation (membrane translocation of p47phox), and subunit expression (real-time polymerase chain reaction and Western blot) were examined in differentiated HL-60 cells, human neutrophils, vascular endothelial and smooth muscle cells, and mouse aorta. DiOHF concentration dependently suppressed superoxide accumulation (EC50 = 8.4 ± 1.7 μM) in vascular smooth muscle cells, which appears to be attributable to its superoxide scavenging activity (EC50 = 6.1 ± 1.1 μM measured in a cell-free system). DiOHF had similar effects in HL-60 cells and isolated aortic rings. In HL-60 cells, but not endothelial or smooth muscle cells, DiOHF and quercetin (10 and 30 μM) significantly reduced the protein expression of p47phox, whereas p67phox was not altered. DiOHF did not affect phorbol ester-induced membrane translocation of either p47phox or protein kinase C in leukocytes. Our results suggest that suppression of NADPH oxidase-dependent superoxide accumulation may contribute to the cytoprotective actions of DiOHF during ischemia-reperfusion injury.
Footnotes
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This work was supported by Project Grants from the National Health and Medical Research Council of Australia and grants-in-aid from the National Heart Foundation of Australia. G.J.D. receives a National Health and Medical Research Council Principal Research Fellowship.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.131433.
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ABBREVIATIONS: ROS, reactive oxygen species; DiOHF, 3′,4′-dihydroxyflavonol; PKC, protein kinase C; DETCA, diethyldithiocarbamic acid; DPI, diphenyleneiodonium chloride; l-NAME, Nω-nitro-l-arginine methyl ester; NBT, nitroblue tetrazolium; 17-ODYA, 17-octadecynoic acid; PMA, phorbol 12-myristate 13-acetate; SOD, superoxide dismutase; DMSO, dimethylsulfoxide; RASMC, rat aortic smooth muscle cell; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; HMEC, human microvascular endothelial cell; PMN, polymorphonuclear cell(s); apoE0, apolipoprotein(E)-deficient mice; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ANOVA, analysis of variance; M40403, C21H35Cl2MnN5.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- The American Society for Pharmacology and Experimental Therapeutics
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