EVIDENCE AGAINST THE INVOLVEMENT OF MULTIPLE RADICAL GENERATING SITESIN THE EXPRESSION OF THE VASCULAR CELL-ADHESION MOLECULE-1

The present study was undertaken to investigate the hypothesis that mu ltiple oxygen radical generating systems contribute to the tumor necro sis factor (TNF) a-stimulated transcriptional activation of the vascul ar cell adhesion molecule (VCAM)-1 in endothelial cells. Experimental evidence has implicated the cytochrome P450 monooxygenase and a phagoc yte type NADPH-oxidase as a source of oxygen radicals in these cells. We show here that endothelial cells exhibit cytochrome P450 activity b y measuring the O-dealkylation of the exogenous substrate 7-ethoxyreso rufin, but components of the phagocyte-type NADPH oxidase could not be demonstrated in endothelial cells. In that latter respect it was surp rising 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 stimu lated by TNF alpha. Also under hypoxic conditions the expression of VC AM-1 was reduced. On this basis we assume that the oxygen dependent st ep in the intracellular signalling cascade underlying the TNF alpha st imulated transcriptional activation of VCAM-1 resides in the activity of a cytochrome P450 dependent monooxygenase. The finding that the pho spholipase A(2) inhibitor bromophenacylbromide inhibited the expressio n of VCAM-1 may indicate that arachidonic acid serves as a substrate f or the cytochrome P450 monooxygenase reaction, but further research is needed to elucidate the particular cytochrome P450 family member medi ating the expression of VCAM-1.