Expression of functional neutrophil-type NADPH oxidase in cultured rat coronary microvascular endothelial cells.
OBJECTIVE
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.
Bayraktutan
U
U
Department of Cardiology, University of Wales College of Medicine, Cardiff, UK.
Draper
N
N
Lang
D
D
Shah
A M
AM
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000201:enzymology*; D003573:Cytochrome b Group / Q000235:genetics / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology*; D008833:Microcirculation; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D012689:Sequence Homology, Nucleic Acid; D013481:Superoxides / Q000037:antagonists & inhibitors; D013552:Swine; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors
D003573:Cytochrome b Group; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D013481:Superoxides; C036888:cytochrome b558; D014969:Xanthine Oxidase; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases