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

William B. Campbell, Blythe B. Holmes, John R. Falck, Jorge H. Capdevila, Kathryn M. Gauthier

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

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 (BKCa) channel activation and membrane hyperpolarization. We hypothesized that if smooth muscle cells (SMCs) had the capacity to synthesize EETs, endogenous EET production would increase BKCa 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 β-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 BKCa channel activity fourfold in cell-attached patches. Methacholine-induced increases in BKCa 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 BKCa channels. These studies indicate that 14(S),15(R)-EET is a sufficient endogenous activator of BKCa channels in coronary SMCs.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume290
Issue number1
DOIs
StatePublished - Jan 2006

Fingerprint

Potassium Channels
Cytochrome P-450 Enzyme System
Smooth Muscle
Smooth Muscle Myocytes
Arachidonic Acid
Large-Conductance Calcium-Activated Potassium Channels
Methacholine Chloride
Adenoviridae
Galactosidases
Bacillus megaterium
14,15-epoxy-5,8,11-eicosatrienoic acid
Cytomegalovirus
Vasodilator Agents
Vascular Smooth Muscle
Endothelium
Mass Spectrometry
Coronary Vessels
Proteins
High Pressure Liquid Chromatography
Escherichia coli

Keywords

  • Arachidonic acid
  • Endothelium-derived hyperpolarizing factor
  • Vascular relaxation

ASJC Scopus subject areas

  • Physiology

Cite this

Regulation of potassium channels in coronary smooth muscle by adenoviral expression of cytochrome P-450 epoxygenase. / Campbell, William B.; Holmes, Blythe B.; Falck, John R.; Capdevila, Jorge H.; Gauthier, Kathryn M.

In: American Journal of Physiology - Heart and Circulatory Physiology, Vol. 290, No. 1, 01.2006.

Research output: Contribution to journalArticle

@article{a86a250cab624bb6bd15b15ea44b6b6c,
title = "Regulation of potassium channels in coronary smooth muscle by adenoviral expression of cytochrome P-450 epoxygenase",
abstract = "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 (BKCa) channel activation and membrane hyperpolarization. We hypothesized that if smooth muscle cells (SMCs) had the capacity to synthesize EETs, endogenous EET production would increase BKCa 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 β-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 BKCa channel activity fourfold in cell-attached patches. Methacholine-induced increases in BKCa 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 BKCa channels. These studies indicate that 14(S),15(R)-EET is a sufficient endogenous activator of BKCa channels in coronary SMCs.",
keywords = "Arachidonic acid, Endothelium-derived hyperpolarizing factor, Vascular relaxation",
author = "Campbell, {William B.} and Holmes, {Blythe B.} and Falck, {John R.} and Capdevila, {Jorge H.} and Gauthier, {Kathryn M.}",
year = "2006",
month = "1",
doi = "10.1152/ajpheart.00516.2005",
language = "English (US)",
volume = "290",
journal = "American Journal of Physiology - Heart and Circulatory Physiology",
issn = "0363-6135",
publisher = "American Physiological Society",
number = "1",

}

TY - JOUR

T1 - Regulation of potassium channels in coronary smooth muscle by adenoviral expression of cytochrome P-450 epoxygenase

AU - Campbell, William B.

AU - Holmes, Blythe B.

AU - Falck, John R.

AU - Capdevila, Jorge H.

AU - Gauthier, Kathryn M.

PY - 2006/1

Y1 - 2006/1

N2 - 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 (BKCa) channel activation and membrane hyperpolarization. We hypothesized that if smooth muscle cells (SMCs) had the capacity to synthesize EETs, endogenous EET production would increase BKCa 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 β-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 BKCa channel activity fourfold in cell-attached patches. Methacholine-induced increases in BKCa 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 BKCa channels. These studies indicate that 14(S),15(R)-EET is a sufficient endogenous activator of BKCa channels in coronary SMCs.

AB - 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 (BKCa) channel activation and membrane hyperpolarization. We hypothesized that if smooth muscle cells (SMCs) had the capacity to synthesize EETs, endogenous EET production would increase BKCa 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 β-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 BKCa channel activity fourfold in cell-attached patches. Methacholine-induced increases in BKCa 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 BKCa channels. These studies indicate that 14(S),15(R)-EET is a sufficient endogenous activator of BKCa channels in coronary SMCs.

KW - Arachidonic acid

KW - Endothelium-derived hyperpolarizing factor

KW - Vascular relaxation

UR - http://www.scopus.com/inward/record.url?scp=33644815636&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33644815636&partnerID=8YFLogxK

U2 - 10.1152/ajpheart.00516.2005

DO - 10.1152/ajpheart.00516.2005

M3 - Article

VL - 290

JO - American Journal of Physiology - Heart and Circulatory Physiology

JF - American Journal of Physiology - Heart and Circulatory Physiology

SN - 0363-6135

IS - 1

ER -