Differential NADPH- versus NADH-dependent superoxide production by phagocyte-type endothelial cell NADPH oxidase.
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.


CONCLUSIONS
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.
Li
J M
JM
Department of Cardiology, Guy's, King's and St Thomas' School of Medicine, King's College London (Denmark Hill Campus), London, UK.
Shah
A M
AM
D000166:Acridines; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000201:enzymology*; D006801:Humans; D015151:Immunoblotting; D008163:Luminescent Measurements; D009245:NADH Dehydrogenase / Q000378:metabolism; D019255:NADPH Oxidases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D009861:Onium Compounds; D010586:Phagocytes / Q000201:enzymology; D012333:RNA, Messenger / Q000032:analysis; D017382:Reactive Oxygen Species / Q000032:analysis / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013045:Species Specificity; D013481:Superoxides / Q000032:analysis / Q000378:metabolism*; D013552:Swine
D000166:Acridines; D009861:Onium Compounds; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013481:Superoxides; C033472:10,10'-dimethyl-9,9'-biacridinium; C007517:diphenyleneiodonium; D019255:NADPH Oxidases; D009245:NADH Dehydrogenase