Characterization of endothelium‐derived hyperpolarizing factor as a cytochrome P450‐derived arachidonic acid metabolite in mammals.

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Abstract
1. In addition to nitric oxide (NO) and prostacyclin (PGI2) an as yet unidentified endothelium‐derived hyperpolarizing factor (EDHF) contributes to the dilator effect of bradykinin in different vascular beds. We have investigated the nature and mechanism of action of this factor in freshly isolated bovine and porcine coronary artery segments which were preconstricted with the thromboxane mimetic U46619 (9,11‐dideoxy‐11 alpha, 9 alpha‐epoxymethano‐prostaglandin F2 alpha, 10‐30 nM). 2. The concentration‐response curve of bradykinin was significantly shifted to the right after inhibition of NO synthesis with NG‐nitro‐L‐arginine (L‐NNA, 30 microM), whereas cyclo‐oxygenase blockade with diclofenac (1 microM) had no effect. Preconstriction of the segments with potassium chloride (40‐60 mM) completely abrogated the NO/PGI2‐independent dilator response to bradykinin. In sandwich bioassay experiments, both the luminal and abluminal release of NO, but not that of EDHF, was readily detectable. 3. Inhibitors of Ca(2+)‐activated K+ channels (K+Ca), such as apamin (1 microM) and tetrabutylammonium (TBA, 3 mM), strongly attenuated the EDHF‐mediated bradykinin‐induced relaxation, while glibenclamide (3 microM), an inhibitor of K+ATP channels, had no effect. 4. These relaxations were also significantly inhibited by the phospholipase A2 inhibitor, quinacrine (30 microM), and the cytochrome P450 inhibitors, SKF525a (30‐100 microM) and clotrimazole (100 microM). Moreover, incubation of endothelium‐denuded coronary artery rings with a cytochrome P450‐derived arachidonic acid metabolite, 11,12‐epoxyeicosatetraenoic acid, elicited a concentration‐dependent (1‐10 microM) dilatation which was abolished both in the presence of TBA (3 mM) and following preconstriction of the segments with potassium chloride instead of U46619.(ABSTRACT TRUNCATED AT 250 WORDS)

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