Endothelium-derived hyperpolarizing factor in human internal mammary artery is 11,12-epoxyeicosatrienoic acid and causes relaxation by activating smooth muscle BK_Ca channels

Background - Left internal mammary arteries (LIMAs) synthesize endothelium-derived hyperpolarizing factor (EDHF), a short-lived K⁺ channel activator that persists after inhibition of nitric oxide (NO) and prostaglandin synthesis. EDHF hyperpolarizes and relaxes smooth muscle cells (SMCs). The identity of EDHF in humans is unknown. We hypothesized that EDHF (1) is 11,12-epoxyeicosatrienoic acid (11,12-EET); (2) is generated by cytochrome P450-2C, CYP450-2C; and (3) causes relaxation by opening SMC large-conductance Ca²⁺-activated K⁺ channels (BK_Ca). Methods and Results - The identity of EDHF and its mechanism of action were assessed in 120 distal human LIMAs and 20 saphenous veins (SVs) obtained during CABG. The predominant EET synthesized by LIMAs is 11,12-EET. Relaxations to exogenous 11,12-EET and endogenous EDHF are of similar magnitudes. Inhibition of EET synthesis by chemically distinct CYP450 inhibitors (17-octadecynoic acid, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide), or a selective EET antagonist (4,15-epoxyeicosa-5(Z)-enoic acid) impairs EDHF relaxation. 11,12-EET activates a BK_Ca current and hyperpolarizes LIMA SMCs. Inhibitors of BK_Ca but not inward-rectifier or small-conductance K_Ca channels abolish relaxation to endogenous EDHF and exogenous 11,12-EET. BK_Ca and CYP450-2C mRNA and proteins are more abundant in LIMAs than in SVs, perhaps explaining the lack of EDHF activity of the SV. Laser capture microdissection and quantitative RT-PCR demonstrate that BK_Ca channels are primarily in vascular SMCs, whereas the CYP450-2C enzyme is present in both the endothelium and SMCs. Conclusions - In human LIMAs, EDHF is 11,12-EET produced by an EDHF synthase CYP450-2C and accounting for ≈40% of net endothelial relaxation. 11,12-EET causes relaxation by activating SMC BK_Ca channels.