Expression of phagocyte NADPH oxidase components in human endothelial cells.
Low-level generation of reactive oxygen species (ROS) by endothelial cells in response to a variety of stimuli has been observed; however, the enzyme system responsible is unknown. Using a variety of techniques, we examined for components of the phagocyte superoxide-generating NADPH oxidase to elucidate whether this enzyme could be a source of endothelial-derived ROS. Superoxide generation on addition of 100 microM NAD(P)H to human umbilical vein endothelial cell (HUVEC) sonicates (using lucigenin-enhanced chemiluminescence) was partially inhibited on addition of the flavoenzyme inhibitor diphenyliodonium (IDP). Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated expression of gp91phox, p22phox, p67phox, and p47phox in four independent HUVEC isolates. Expression of p22phox was also confirmed by Northern blotting. RT-PCR for tumor necrosis factor-alpha was negative, indicating an absence of mononuclear cell contamination (a potential source of NADPH oxidase). Immunoperoxidase staining, using anti-p47phox (JW-1)- and anti-p67phox (JW-2)-specific antibodies, showed protein expression of these cytosolic components. However, heme spectroscopy failed to indicate the presence of the low-potential cytochrome b558. These data indicate that cultured human endothelial cells express both mRNA and protein for cytosolic components of the phagocyte superoxide-generating NADPH oxidase. However, because the cytochrome b558 heme could not be conclusively demonstrated, a contribution of the phagocyte NADPH oxidase to endothelial oxidant generation may be unlikely.
Department of Biochemistry, School of Medical Sciences, University of Bristol, United Kingdom.
S A
SA
Jones
V B
VB
O'Donnell
J D
JD
Wood
J P
JP
Broughton
E J
EJ
Hughes
O T
OT
Jones
D002478:Cells, Cultured; D003573:Cytochrome b Group / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010586:Phagocytes / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D003573:Cytochrome b Group; D010750:Phosphoproteins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1