J M Li1, A M Shah. 1. Department of Cardiology, Guy's, King's and St Thomas' School of Medicine, King's College London (Denmark Hill Campus), London, UK.
Abstract
OBJECTIVE: A poorly characterized phagocyte-type NADPH oxidase, which is reportedly NADH- rather than NADPH-dependent, is a major source of endothelial reactive oxygen species (ROS) production. We investigated the molecular nature of this oxidase and the characteristics of NADPH- versus NADH-dependent O(2)(-) production in endothelial cells of three different species. METHODS: NADPH oxidase expression in human, bovine and porcine endothelial cells was studied by RT-PCR and immunoblotting. O(2)(-) production was assessed by lucigenin chemiluminescence and cytochrome c reduction assay. RESULTS: The NADPH oxidase subunits p47-phox, p67-phox, p22-phox, gp91-phox, and rac1 were all expressed in endothelial cells. NADPH-dependent O(2)(-) production by endothelial cells was readily detectable using lucigenin 5 micromol/l, was minimally affected by increasing lucigenin dose up to 400 micromol/l, and was abolished by diphenyleneiodonium. In contrast, NADH-dependent O(2)(-) production was only detectable with lucigenin > or =50 micromol/l, increased substantially with higher lucigenin dose, and was unaffected by diphenyleneiodonium. Predominance of NADPH- over NADH-dependent O(2)(-) production was confirmed in cell homogenates and by cytochrome c reduction assay. CONCLUSION: Endothelial cells express all components of a phagocyte-type NADPH oxidase. Like the neutrophil enzyme, the endothelial oxidase is preferentially NADPH- rather than NADH-dependent. NADH-dependent O(2)(-) production appears to be an artefact related to the use of lucigenin doses > or =50 micromol/l.
OBJECTIVE: A poorly characterized phagocyte-type NADPH oxidase, which is reportedly NADH- rather than NADPH-dependent, is a major source of endothelial reactive oxygen species (ROS) production. We investigated the molecular nature of this oxidase and the characteristics of NADPH- versus NADH-dependent O(2)(-) production in endothelial cells of three different species. METHODS:NADPH oxidase expression in human, bovine and porcine endothelial cells was studied by RT-PCR and immunoblotting. O(2)(-) production was assessed by lucigenin chemiluminescence and cytochrome c reduction assay. RESULTS: The NADPH oxidase subunits p47-phox, p67-phox, p22-phox, gp91-phox, and rac1 were all expressed in endothelial cells. NADPH-dependent O(2)(-) production by endothelial cells was readily detectable using lucigenin 5 micromol/l, was minimally affected by increasing lucigenin dose up to 400 micromol/l, and was abolished by diphenyleneiodonium. In contrast, NADH-dependent O(2)(-) production was only detectable with lucigenin > or =50 micromol/l, increased substantially with higher lucigenin dose, and was unaffected by diphenyleneiodonium. Predominance of NADPH- over NADH-dependent O(2)(-) production was confirmed in cell homogenates and by cytochrome c reduction assay. CONCLUSION: Endothelial cells express all components of a phagocyte-type NADPH oxidase. Like the neutrophil enzyme, the endothelial oxidase is preferentially NADPH- rather than NADH-dependent. NADH-dependent O(2)(-) production appears to be an artefact related to the use of lucigenin doses > or =50 micromol/l.
Authors: S Wind; K Beuerlein; T Eucker; H Müller; P Scheurer; M E Armitage; H Ho; H H H W Schmidt; K Wingler Journal: Br J Pharmacol Date: 2010-10 Impact factor: 8.739
Authors: Yanfeng Ding; Yu-Long Li; Matthew C Zimmerman; Robin L Davisson; Harold D Schultz Journal: Cardiovasc Res Date: 2008-12-17 Impact factor: 10.787