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

Abstract

Endothelium-derived hyperpolarizing factor (EDHF) is released in response to agonists such as ACh and bradykinin and regulates vascular smooth muscle tone. Several studies have indicated that ouabain blocks agonist-induced, endothelium-dependent hyperpolarization of smooth muscle. We have demonstrated that epoxyeicosatrienoic acids (EETs), cytochrome P-450 metabolites of arachidonic acid, function as EDHFs. To further test the hypothesis that EETs represent EDHFs, we have examined the effects of ouabain on the electrical and mechanical effects of 14,15- and 11,12-EET in bovine coronary arteries. These arteries are relaxed in a concentration-dependent manner to 14,15- and 11,12-EET (EC50 = 6 × 10−7 M), bradykinin (EC50 = 1 × 10−9 M), sodium nitroprusside (SNP; EC50 = 2 × 10−7 M), and bimakalim (BMK; EC50 = 1 × 10−7 M). 11,12-EET-induced relaxations were identical in vessels with and without an endothelium. Potassium chloride (1–15 × 10−3 M) inhibited [3H]ouabain binding to smooth muscle cells but failed to relax the arteries. Ouabain (10−5 to 10−4 M) increased basal tone and inhibited the relaxations to bradykinin, 11,12-EET, and 14,15-EET, but not to SNP or BMK. Barium (3 × 10−5 M) did not alter EET-induced relaxations and ouabain plus barium was similar to ouabain alone. Resting membrane potential ( E m) of isolated smooth muscle cells was −50.2 ± 0.5 mV. Ouabain (3 × 10−5 and 1 × 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 increase in E m. In cell-attached patch clamp studies, 11,12-EET significantly increased the open-state probability ( NP o) of a calcium-activated potassium channel compared with control cells (0.26 ± 0.06 vs. 0.02 ± 0.01). Ouabain did not change NP o but blocked the 14,15-EET-induced increase in NP o. These results indicate that: 1) EETs relax coronary arteries in an endothelium-independent manner, 2) unlike EETs, potassium chloride does not relax 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.