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 (EC(50) = 8.4 +/- 1.7 microM) in vascular smooth muscle cells, which appears to be attributable to its
superoxide scavenging activity (EC(50) = 6.1 +/- 1.1 microM 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 microM) 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.