Evidence against the involvement of multiple radical generating sites in the expression of the vascular cell adhesion molecule-1.
The present study was undertaken to investigate the hypothesis that multiple oxygen radical generating systems contribute to the tumor necrosis factor (TNF) alpha-stimulated transcriptional activation of the vascular cell adhesion molecule (VCAM)-1 in endothelial cells. Experimental evidence has implicated the cytochrome P450 monooxygenase and a phagocyte type NADPH-oxidase as a source of oxygen radicals in these cells. We show here that endothelial cells exhibit cytochrome P450 activity by measuring the O-dealkylation of the exogenous substrate 7-ethoxyresorufin, but components of the phagocyte-type NADPH oxidase could not be demonstrated in endothelial cells. In that latter respect it was surprising that the NADPH oxidase inhibitor apocynin completely prevented the accumulation of VCAM-1 mRNA. However, we found that apocynin also acts as an inhibitor of cytochrome P450 activity in endothelial cells. Therefore the inhibitory effect of apocynin on the induction of VCAM-1 may no longer be used to demonstrate a role for the NADPH oxidase in this process. Furthermore, different cytochrome P450 inhibitors Co2+, metyrapone, SKF525a decreased the endothelial VCAM-1 expression stimulated by TNFalpha. Also under hypoxic conditions the expression of VCAM-1 was reduced. On this basis we assume that the oxygen dependent step in the intracellular signalling cascade underlying the TNFalpha stimulated transcriptional activation of VCAM-1 resides in the activity of a cytochrome P450 dependent monooxygenase. The finding that the phospholipase A2 inhibitor bromophenacylbromide inhibited the expression of VCAM-1 may indicate that arachidonic acid serves as a substrate for the cytochrome P450 monooxygenase reaction, but further research is needed to elucidate the particular cytochrome P450 family member mediating the expression of VCAM-1.
Pietersma
A
A
Department of Biochemistry, Cardiovascular Research Institute (COEUR), Erasmus University Rotterdam, The Netherlands.
de Jong
N
N
de Wit
L E
LE
Kraak-Slee
R G
RG
Koster
J F
JF
Sluiter
W
W
D000098:Acetophenones / Q000494:pharmacology*; D000478:Alkylation / Q000187:drug effects; D015152:Blotting, Northern; D015687:Cell Hypoxia; D002478:Cells, Cultured; D003035:Cobalt / Q000494:pharmacology; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004338:Drug Combinations; D004365:Drugs, Chinese Herbal / Q000494:pharmacology; D004579:Electron Transport / Q000187:drug effects; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005609:Free Radicals; D005786:Gene Expression Regulation / Q000187:drug effects*; D006035:Glycyrrhiza; D006801:Humans; D008797:Metyrapone / Q000494:pharmacology; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology; D009861:Onium Compounds / Q000494:pharmacology*; D010078:Oxazines / Q000378:metabolism; D010084:Oxidation-Reduction; D029596:Paeonia; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000494:pharmacology; D054467:Phospholipases A2; D016133:Polymerase Chain Reaction; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014471:Umbilical Veins; D019010:Vascular Cell Adhesion Molecule-1 / Q000096:biosynthesis* / Q000235:genetics
D000098:Acetophenones; D065607:Cytochrome P-450 Enzyme Inhibitors; D004338:Drug Combinations; D004365:Drugs, Chinese Herbal; D004791:Enzyme Inhibitors; D005609:Free Radicals; D009861:Onium Compounds; D010078:Oxazines; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; C038462:shakuyaku-kanzoh-toh; D003035:Cobalt; C007649:ethoxyresorufin; C007517:diphenyleneiodonium; C044943:pentoxyresorufin; D003577:Cytochrome P-450 Enzyme System; C056165:acetovanillone; D019255:NADPH Oxidases; D010741:Phospholipases A; D054467:Phospholipases A2; C006821:4-bromophenacyl bromide; D008797:Metyrapone