Regulation of potassium channels in coronary smooth muscle by adenoviral expression of cytochrome P-450 epoxygenase

Epoxyeicosatrienoic acids (EETs) are endothelium-derived cytochrome P-450 (CYP) metabolites of arachidonic acid that relax vascular smooth muscle by large-conductance calcium-activated potassium ([BK.sub.Ca]) channel activation and membrane hyperpolarization. We hypothesized that if smooth muscle cells (SMCs) had the capacity to synthesize EETs, endogenous EET production would increase [BK.sub.Ca] channel activity. Bovine coronary SMCs were transduced with adenovirus coding the CYP Bacillus megaterium -3 (F87V) (CYP BM-3) epoxygenase that metabolizes arachidonic acid exclusively to 14(S),15(R)-EET. Adenovirus containing the cytomegalovirus promoter-Escherichia coli [beta]-galactosidase was used as a control. With the use of an anti-CYP BM-3 (F87V) antibody, a 124-kDa immunoreactive protein was detected only in CYP BM-3-transduced cells. Protein expression increased with increasing amounts of virus. When CYP BM-3-transduced cells were incubated with [[sup.14]C]arachidonic acid, HPLC analysis detected 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) and 14,15-EET. The identity of 14,15-EET and 14,15-DHET was confirmed by mass spectrometry. In CYP BM-3-transduced cells, methacholine ([10.sup.-5] M) increased 14,15-EET release twofold and [BK.sub.Ca] channel activity fourfold in cell-attached patches. Methacholine-induced increases in BKca channel activity were blocked by the CYP inhibitor 17-octadecynoic acid ([10.sup.-5] M). 14(S),15(R)-EET was more potent than 14(R), 15(S)-EET in relaxing bovine coronary arteries and activating [BK.sub.Ca] channels. Thus CYP BM-3 adenoviral transduction confers SMCs with epoxygenase activity. These cells acquire the capacity to respond to the vasodilator agonist by synthesizing 14(S), 15(R)-EET from endogenous arachidonic acid to activate [BK.sup.Ca] channels. These studies indicate that 14(S),15(R)-EET is a sufficient endogenous activator of [BK.sub.Ca], channels in coronary SMCs. endothelium-derived hyperpolarizing factor; arachidonic acid; vascular relaxation