© 2001 by European Society of Cardiology
Copyright © 2001, European Society of Cardiology
Differential NADPH- versus NADH-dependent superoxide production by phagocyte-type endothelial cell NADPH oxidase
Department of Cardiology, Guy’s, King’s and St Thomas’ School of Medicine, King’s College London (Denmark Hill Campus), Bessemer Road, London SE5 9PJ, UK
* Tel.: +44-20-7346-3865; fax: +44-20-7346-4771 ajay.shah{at}kcl.ac.uk
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 O2– 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. O2– 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 O2– production by endothelial cells was readily detectable using lucigenin 5 µmol/l, was minimally affected by increasing lucigenin dose up to 400 µmol/l, and was abolished by diphenyleneiodonium. In contrast, NADH-dependent O2– production was only detectable with lucigenin 
50 µmol/l, increased substantially with higher lucigenin dose, and was unaffected by diphenyleneiodonium. Predominance of NADPH- over NADH-dependent O2– 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 O2– production appears to be an artefact related to the use of lucigenin doses 50 µmol/l.
KEYWORDS Cell culture/isolation; Endothelial function; Free radicals; Gene expression
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