© 1998 by European Society of Cardiology
Copyright © 1998, European Society of Cardiology
Expression of a functional neutrophil-type NADPH oxidase in cultured rat coronary microvascular endothelial cells
aDepartment of Cardiology, University of Wales College of Medicine, Cardiff CF4 4XN, UK
bDepartment of Pharmacology and Therapeutics, University of Wales College of Medicine, Cardiff CF4 4XN, UK
* Corresponding author. Tel.: +44 (1222) 74 23 38; Fax: +44 (1222) 74 35 00; E-mail: shaham2@cf.ac.uk
Objectives: The production of reactive oxygen species (e.g., superoxide) by endothelial cells is relevant to tissue injury during ischemia-reperfusion, and may also play a role in intracellular signaling pathways. However, the molecular identities of the enzymes responsible for endothelial superoxide production are poorly defined, although xanthine oxidase, NADH/NADPH oxidoreductases and nitric oxide synthase are among proteins suggested to contribute. Recent studies suggest that an NADH/NADPH oxidase similar to that found in neutrophils is an important source of superoxide in vascular smooth muscle. Methods: We investigated whether a phagocyte-type NADH/NADPH oxidase complex is present in rat cultured coronary microvascular endothelial cells. The expression of NADPH oxidase components was studied by RT-PCR and Western blot analyses, while functional activity was assessed by measurement of superoxide production by lucigenin-enhanced chemiluminescence. Results: The major component of the phagocyte-type NADH/NADPH oxidase complex, a cytochrome b558 heterodimer, was shown to be present both at mRNA and protein levels, using oligonucleotide primers designed from published neutrophil and vascular smooth muscle sequences and anti-neutrophil antibodies respectively. Functional activity of the enzyme was also confirmed by NADPH-evoked superoxide production in cell homogenates, which was inhibited either by the superoxide chelator Tiron or by diphenyleneiodonium, an inhibitor of the oxidase. Conclusions: A functional phagocyte-type NADPH oxidase is expressed in coronary microvascular endothelial cells, where it may contribute to the physiological and/or pathophysiological effects of reactive oxygen species. These data, together with reports of the presence of a similar oxidase in other non-phagocytic cell types, suggest that this enzyme complex is widely expressed in many tissues where it may subserve signaling and other functions.
KEYWORDS Experimental; Vasculature; Molecular biology/biochemistry; Endothelial function; Endothelial factors; Free radicals; Gene expression; Signal transduction
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