CHARACTERIZATION OF ENDOTHELIUM-DERIVED HYPERPOLARIZING FACTOR AS A CYTOCHROME P450-DERIVED ARACHIDONIC-ACID METABOLITE IN MAMMALS

1. In addition to nitric oxide (NO) and prostacyclin (PGI(2)) an as yet unidentified endothelium-derive 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 F-2 alpha, 10-30 nM). 2. The concentration-response curve of bradykinin was significantly shifted to the right after inhibition of NO synthesis with N-G-nitro-L-arginine (L-NNA, 30 mu M), whereas cyclo-oxygenase blockade with diclofenac (1 mu M) had no effect. Preconstriction of the segments with potassium chloride (40-60 mM) completely abrogated the NO/PGI(2)-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 Ca2+-activated K+ channels (K-Ca(+)), such as apamin (1 mu M) and tetrabutylammonium (TBA, 3 mM), strongly attenuated the EDHF-mediated bradykinin-induced relaxation, while glibenclamide (3 mu M), an inhibitor of K-ATP(+) channels, had no effect. 4. These relaxations were also significantly inhibited by the phospholipase A(2) inhibitor, quinacrine (30 mu M), and the cytochrome P450 inhibitors, SKF525a (30-100 mu M) and clotrimazole (100 mu M). 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 mu M) dilatation which was abolished both in the presence of TBA (3 mM) and following preconstriction of the segments with potassium chloride instead of U46619. 5. These findings suggest that EDHF released by bradykinin is a cytochrome P450-derived arachidonic acid metabolite, presumably an epoxide. This factor seems to hyperpolarize the underlying smooth muscle cell layers by opening K-Ca(+) channels.