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 ye t unidentified endothelium-derive hyperpolarizing factor (EDHF) contri butes 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 shif ted to the right after inhibition of NO synthesis with N-G-nitro-L-arg inine (L-NNA, 30 mu M), whereas cyclo-oxygenase blockade with diclofen ac (1 mu M) had no effect. Preconstriction of the segments with potass ium chloride (40-60 mM) completely abrogated the NO/PGI(2)-independent dilator response to bradykinin. In sandwich bioassay experiments, bot h the luminal and abluminal release of NO, but not that of EDHF, was r eadily detectable. 3. Inhibitors of Ca2+-activated K+ channels (K-Ca()), such as apamin (1 mu M) and tetrabutylammonium (TBA, 3 mM), strong ly attenuated the EDHF-mediated bradykinin-induced relaxation, while g libenclamide (3 mu M), an inhibitor of K-ATP(+) channels, had no effec t. 4. These relaxations were also significantly inhibited by the phosp holipase A(2) inhibitor, quinacrine (30 mu M), and the cytochrome P450 inhibitors, SKF525a (30-100 mu M) and clotrimazole (100 mu M). Moreov er, incubation of endothelium-denuded coronary artery rings with a cyt ochrome P450-derived arachidonic acid metabolite, 11,12-epoxyeicosatet raenoic acid, elicited a concentration-dependent (1-10 mu M) dilatatio n which was abolished both in the presence of TBA (3 mM) and following preconstriction of the segments with potassium chloride instead of U4 6619. 5. These findings suggest that EDHF released by bradykinin is a cytochrome P450-derived arachidonic acid metabolite, presumably an epo xide. This factor seems to hyperpolarize the underlying smooth muscle cell layers by opening K-Ca(+) channels.