Endothelium-independent, ouabain-sensitive relaxation of bovine coronary arteries by EETs

Endothelium-derived hyperpolarizing factor (EDHF) is released in response t o agonists such as ACh and bradykinin and regulates vascular smooth muscle tone. Several studies have indicated that ouabain blocks agonist-induced, e ndothelium-dependent hyperpolarization of smooth muscle. We have demonstrat ed that epoxyeicosatrienoic acids (EETs), cytochrome P-450 metabolites of a rachidonic acid, function as EDHFs. To further test the hypothesis that EET s represent EDHFs, we have examined the effects of ouabain on the electrica l and mechanical effects of 14,15- and 11,12-EET in bovine coronary arterie s. These arteries are relaxed in a concentration-dependent manner to 14,15- and 11,12-EET (EC50 = 6 X 10(-7) M), bradykinin (EC50 = 1 X 10(-9) M), sod ium nitroprusside (SNP; EC50 = 2 X 10(-7) M), and bimakalim (BMK; EC50 = 1 X 10(-7) M). 11,12-EET-induced relaxations were identical in vessels with a nd without an endothelium. Potassium chloride (1-15 X 10(-3) M) inhibited [ H-3] ouabain binding to smooth muscle cells but failed to relax the arterie s. Ouabain (10(-5) to 10(-4) M) increased basal tone and inhibited the rela xations to bradykinin, 11,12-EET, and 14,15- EET, but not to SNP or BMK. Ba rium (3 X 10(-5) M) did not alter EET-induced relaxations and ouabain plus barium was similar to ouabain alone. Resting membrane potential (E-m) of is olated smooth muscle cells was -50.2 +/- 0.5 mV. Ouabain (3 X 10(-5) and 1 X 10(-4) M) decreased E-m (-48.4 +/- 0.2 mV), whereas 11,12-EET (10(-7) M) increased E-m (-59.2 +/- 2.2 mV). Ouabain inhibited the 11,12-EET-induced i ncrease in E-m. In cell-attached patch clamp studies, 11,12-EET significant ly increased the open-state probability (NPo) of a calcium-activated potass ium channel compared with control cells (0.26 +/- 0.06 vs. 0.02 +/- 0.01). Ouabain did not change NPo but blocked the 14,15-EET-induced increase in NP o. These results indicate that: 1) EETs relax coronary arteries in an endot helium- independent manner, 2) unlike EETs, potassium chloride does not rel ax the coronary artery, and 3) ouabain inhibits bradykinin- and EET-induced relaxations as has been reported for EDHF. These findings provide further evidence that EETs are EDHFs.