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

¹Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin; ²Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas; and ³Department of Medicine and Biochemistry, Vanderbilt University, Nashville, Tennessee

Submitted 17 May 2005 ; accepted in final form 1 September 2005

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_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_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 -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 [¹⁴C]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^–5 M) increased 14,15-EET release twofold and BK_Ca channel activity fourfold in cell-attached patches. Methacholine-induced increases in BK_Ca channel activity were blocked by the CYP inhibitor 17-octadecynoic acid (10^–5 M). 14(S),15(R)-EET was more potent than 14(R),15(S)-EET in relaxing bovine coronary arteries and activating BK_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_Ca channels. These studies indicate that 14(S),15(R)-EET is a sufficient endogenous activator of BK_Ca channels in coronary SMCs.

endothelium-derived hyperpolarizing factor; arachidonic acid; vascular relaxation

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