Expression of phagocyte nadph oxidase components in human
endothelial cells.
Jonesa, Simon A., Valerie B. O'donnell, Jonathan D. Wooda, Jonathan.
P. Broughton, Eleanor. J. Hughes & Owen. T. G. Jones.
Department of Biochemistry, School of Medical Sciences, University
of Bristol, Bristol BS8 1TD, U.K., Institute of Biochemistry and
Molecular Biology, University of Bern, CH-3012, Switzerland
APStracts 3:0117H, 1996.
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 in order to elucidate whether this enzyme
could be a source of endothelial-derived ROS. Superoxide generation
on addition of 100[mu]M NAD(P)H to 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 gp91-phox, p22-phox, p67-phox and p47-phox in four
independent human umbilical vein endothelial cell isolates.
Expression of p22-phox was also confirmed by northern blotting. RT
-PCR for TNF[alpha] was negative, indicating 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 expression of these
cytosolic components. However haem 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 since the cytochrome b558 haem
could not be conclusively demonstrated, a contribution of the
phagocyte NADPH oxidase to endothelial oxidant generation may be
unlikely.
Received 14 February 1995; accepted in final form 22 February
1996.
APS Manuscript Number H139-5.
Article publication pending Am. J. Physiol. (Heart Circ. Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 27 March 96