EXPRESSION OF PHAGOCYTE NADPH OXIDASE COMPONENTS IN HUMAN ENDOTHELIAL-CELLS

Low-level generation of reactive oxygen species (ROS) by endothelial c ells in response to a variety of stimuli has been observed; however, t he enzyme system responsible is unknown. Using a variety of techniques , we examined for components of the phagocyte superoxide-generating NA DPH oxidase to elucidate whether this enzyme could be a source of endo thelial-derived ROS. Superoxide generation on addition of 100 mu M NAD (P)H to human umbilical vein endothelial cell (HUVEC) sonicates (using lucigenin-enhanced chemiluminescence) was partially inhibited on addi tion of the flavoenzyme inhibitor diphenyliodonium (IDP). Reverse tran scriptase-polymerase chain reaction (RT-PCR) demonstrated expression o f gp91(phox), p22(phox), p67(phox), and p47(phox) four independent HUV EC isolates. Expression of p22(phox) was also confirmed by Northern bl otting. RT-PCR for tumor necrosis factor-alpha was negative, indicatin g an absence of mononuclear cell contamination (a potential source of NADPH oxidase). Immunoperoxidase staining, using anti-p47(phox) (JW-1) - and anti-p67(phox) (JW-2)-specific antibodies, showed protein expres sion of these cytosolic components. However, heme spectroscopy failed to indicate the presence of the low-potential cytochrome b558. These d ata indicate that cultured human endothelial cells express both mRNA a nd protein for cytosolic components of the phagocyte superoxide-genera ting NADPH oxidase. However, because the cytochrome b558 heme could no t be conclusively demonstrated, a contribution of the phagocyte NADPH oxidase to endothelial oxidant generation may be unlikely.