Differential NADPH- versus NADH-dependent superoxide production by phagocyte-type endothelial cell NADPH oxidase.
Cardiovasc Res
; 52(3): 477-86, 2001 Dec.
Article
em En
| MEDLINE
| ID: mdl-11738065
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Endotélio Vascular
/
Espécies Reativas de Oxigênio
/
Superóxidos
/
NADPH Oxidases
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Cardiovasc Res
Ano de publicação:
2001
Tipo de documento:
Article
País de afiliação:
Reino Unido