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1 Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; 2 Boehringer Ingelheim Pharmaceutical, Ridgefield, Connecticut 06877; and 3 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75482
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Epoxyeicosatrienoic acids (EETs) cause vascular relaxation by activating smooth muscle large conductance Ca2+-activated K+ (KCa) channels. EETs are metabolized to dihydroxyeicosatrienoic acids (DHETs) by epoxide hydrolase. We examined the contribution of 14,15-DHET to 14,15-EET-induced relaxations and characterized its mechanism of action. 14,15-DHET relaxed U-46619-precontracted bovine coronary artery rings but was approximately fivefold less potent than 14,15-EET. The relaxations were inhibited by charybdotoxin, iberiotoxin, and increasing extracellular K+ to 20 mM. In isolated smooth muscle cells, 14,15-DHET increased an iberiotoxin-sensitive, outward K+ current and increased KCa channel activity in cell-attached patches and inside-out patches only when GTP was present. 14,15-[14C]EET methyl ester (Me) was converted to 14,15-[14C]DHET-Me, 14,15-[14C]DHET, and 14,15-[14C]EET by coronary arterial rings and endothelial cells but not by smooth muscle cells. The metabolism to 14,15-DHET was inhibited by the epoxide hydrolase inhibitors 4-phenylchalcone oxide (4-PCO) and BIRD-0826. Neither inhibitor altered relaxations to acetylcholine, whereas relaxations to 14,15-EET-Me were increased slightly by BIRD-0826 but not by 4-PCO. 14,15-DHET relaxes coronary arteries through activation of KCa channels. Endothelial cells, but not smooth muscle cells, convert EETs to DHETs, and this conversion results in a loss of vasodilator activity.
endothelium-derived hyperpolarizing factor; epoxyeicosatrienoic acids; potassium channels; epoxide hydrolase
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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ENDOTHELIAL CELLS relax vascular smooth muscle through the release of a number of soluble mediators such as nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF) (4, 13, 22). Studies have implicated metabolites of arachidonic acid, K+, and hydrogen peroxide as mediators of the EDHF response (2, 7, 10, 16, 20, 26, 28). The mediator may vary with the vascular bed. In the coronary artery, evidence from a number of laboratories indicates that EDHF is an epoxyeicosatrienoic acid (EET), a cytochrome P-450 metabolite of arachidonic acid (2, 10, 12, 15, 16, 29). The coronary endothelium synthesizes four regioisomeric EETs, and agonists such as acetylcholine and bradykinin stimulate their release (2, 24, 29, 30). The endothelium-dependent relaxation and hyperpolarization of coronary smooth muscle to acetylcholine and bradykinin is blocked by inhibitors of cytochrome P-450 and by inhibitors of Ca2+-activated K+ (KCa) channels (2, 16). Like EDHF, EETs relax and hyperpolarize coronary smooth muscle. The relaxations to the EETs are independent of the endothelium (27, 29). The EETs open a large conductance KCa channel of the smooth muscle cell membrane, resulting in membrane hyperpolarization (2). The activation of the KCa channel by EETs involves a guanine nucleotide-binding protein, most likely Gs (11, 15, 18).
While most of these studies have been conducted in vitro in isolated coronary arteries, these conclusions have been confirmed in vivo in anesthetized dogs (21, 23, 35). In coronary microvessels, acetylcholine causes vasodilation that is not blocked by inhibitors of nitric oxide synthase or cyclooxygenase but eliminated by inhibitors of K+ channels. These findings implicated EDHF as a mediator of vasodilation in coronary microvessels. The nonnitric oxide, nonprostaglandin relaxation to acetylcholine was also blocked by inhibitors of cytochrome P-450. These experiments support the conclusion that a cytochrome P-450 metabolite mediates a portion of the coronary vasodilation in vivo.
EETs are hydrolyzed to dihydroxyeicosatrienoic acids (DHETs) by epoxide hydrolase (34, 36). The effects of DHETs on vascular tone vary. 5,6-EET relaxes the rat tail artery; however, 5,6-DHET is without effect (3). In contrast, 11,12-DHET relaxes porcine coronary arteries and is equipotent to 11,12-EET (33). Canine coronary microvessels are much more sensitive to the relaxing effects of 11,12-EET and 11,12-DHET, and 11,12-DHET is more potent than 11,12-EET (25). Inhibition of epoxide hydrolase to prevent DHET formation from EETs did not alter the EET-induced relaxation or the relaxations to bradykinin (34). Thus the conversion of EETs to DHETs is not required for relaxation. It is not known whether DHETs act by the same mechanism as EETs.
In the present study, we compared the vascular effects of 14,15-EET and 14,15-DHET and examined the role of 14,15-DHET in the action of 14,15-EET. 14,15-DHET relaxed the bovine coronary artery; however, 14,15-DHET was approximately fivefold less potent than 14,15-EET. Additional studies indicate that 14,15-DHET, like 14,15-EET, opens a large conductance KCa channel by a G protein-dependent mechanism (11, 15, 18). Endothelial cells, but not smooth muscle cells, converted 14,15-EET to 14,15-DHET. In bovine coronary arteries, the conversion of EET to DHET represents an inactivation pathway and causes a loss of activity.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Vascular reactivity of bovine coronary arteries. Bovine hearts were purchased from a local slaughterhouse, and the left anterior descending coronary artery was dissected and cleaned of connective tissue. Vessels were cut into 2-mm-diameter rings (3 mm width), and isometric tension measured as previously described (2, 18, 29). Vessels were stored in Krebs buffer consisting of (in mM) 119 NaCl, 4.8 KCl, 24 NaHCO3, 1.2 KH2PO4, 1.2 MgSO4, 11 glucose, 0.02 EDTA, and 3.2 CaCl2. In some studies, the K+ concentration was increased from 4.8 to 20 mM by substituting KCl for NaCl.
Basal tension was set at the length-tension maximum of 3.5 g and equilibrated for 1.5 h. KCl (40 mM) was added to the chamber until reproducible maximal contractions were maintained. U-46619 (10-20 nM), a thromboxane agonist, was used to precontract the vessels from basal tension to between 50% and 90% of the maximal KCl contraction. Cumulative additions of acetylcholine, 14,15-EET, 14,15-DHET, or 14,15-EET methyl ester (Me) were added to the chamber. Between concentration-response curves, the chambers were rinsed with fresh Krebs buffer, 40 mM KCl was administered to determine the maximum contraction, and the vessels were rinsed. Pretreatment with charybdotoxin (100 nM), iberiotoxin (50 nM), BIRD-0826 (10
5 M), 4-phenylchalcone oxide (4-PCO; 2.2 × 105 M), or vehicle occurred 10 min before precontraction
with U-46619. Tension is presented as a percentage of relaxation, where
100% relaxation is basal pre- U-46619 tension.
Isolation of coronary smooth muscle cells. Pieces of bovine heart (2 × 3 × 1 cm) were obtained from where the left anterior descending coronary artery branches. These pieces of heart were sliced into 300-µm sections, the microvessels were dissected, and smooth muscle cells were isolated as previously described (18, 19). The dissociated cells were stored at 4°C until they were placed in the recording chamber.
Patch-clamp methods.
Single channel K+ currents were recorded using the
patch-clamp technique (14) as previously described
(18, 19, 27). Whole cell, cell-attached, and inside-out
configurations were used to determine the effect of 14,15-DHET on
K+ currents in vascular smooth muscle cells. Patch pipettes
were made from borosilicate glass capillaries and had a tip resistance of 8-10 M
for single channel recording when filled with 145 mM KCl solution. Smooth muscle cells were placed in a 1-ml perfusion chamber that was mounted on the stage of a Nikon inverted microscope. After the tip of the pipette was positioned on a cell, a
high-resistance seal (5-15 G) was formed between the pipette
tip and the cell membrane by light suction. This configuration was used
for the cell-attached mode. For the whole cell mode, an additional
acute suction was applied to rupture the membrane under the pipette. Inside-out membrane patches were excised by lifting the pipette membrane complex to the air-solution interface.
60 mV. Cells were
dialyzed with a pipette solution that contained (in mM) 145 potassium
glutamate, 1 MgCl2, 10 HEPES, 1 EGTA, 1 Na2ATP,
and 0.05 Na2GTP and 100 nM ionized calcium (pH 7.2) and
perfused with a bath solution composed of (in mM) 145 NaCl, 4 KCl, 1 MgCl2, 10 glucose, 10 HEPES, and 2 CaCl2 (pH
7.4). Control trials were performed using a bath solution with 0.095% ethanol. Trials were performed in triplicate and averaged to estimate current amplitude. Subsequently, cells were perfused with a bath solution containing 14,15-DHET (10 µM) and/or iberiotoxin (100 nM).
The membrane capacitance of each cell was estimated by integrating the
capacitative current generated by a 20-mV hyperpolarizing pulse after
electronic cancellation of pipette-patch capacitance. Currents are
expressed in picoamperes per picofarad to normalize for differences in
cell membrane area between individual cells.
For single channel recordings in the cell-attached mode, the bath
solution contained (in mM) 145 KCl, 1.8 CaCl2, 1.1 MgCl2, 10 glucose, and 5 HEPES (pH 7.4), and the pipette
solution contained (in mM) 145 KCl, 1.8 CaCl2, 1.1 MgCl2, and 5 HEPES (pH 7.4). For single channel recordings
using the inside-out excised membrane patch, the bath solution
contained (in mM) 145 KCl, 1.1 MgCl2, 10 HEPES, and 2 EGTA
and 300 nM ionized calcium (pH 7.2). The pipette solution contained (in
mM) 145 KCl, 1.8 CaCl2, 1.1 MgCl2, and 10 HEPES
(pH 7.4). All patch-clamp experiments were performed at room
temperature, ~20°C. Channel open probability
(NPo) was used to represent the activity of the
KCa channels.
The current-voltage relationship was established by recording the
currents at patch membrane potentials of 60 to +60 mV. A
K+ channel with a conductance of 256 ± 5 pS was
identified (2, 18, 19, 27). This large conductance
KCa channel increases NPo with
depolarization and increases in calcium concentration. The
channel was blocked by tetraethylammonium chloride, iberiotoxin, and charybdotoxin.
To determine the effects of 14,15-DHET on the activity of
KCa channels, cell-attached patches were studied first. A
3-min control recording at a membrane potential of +40 mV was obtained after a tight seal was established. The bath solution was then rapidly
changed by flushing the perfusion chamber with 10 ml of the same
solution containing 14,15-DHET (0.01, 0.1, or 1 µM, n = 10), and a series of 3-min recordings was obtained. To determine whether a G protein was involved in the effect of 14,15-DHET on the
activity of the KCa channels, the inside-out patch modes
were used. After inside-out patches were established, a 3-min control recording was obtained at a membrane potential of +40 mV. The bath
solution was then rapidly changed by flushing the perfusion chamber
with 5-10 ml of the same solution containing 0.01, 0.1, or 1 µM
14,15-DHET (n = 4-5) with or without 0.5 mM GTP
and 1 mM ATP, and a second successive 3-min recording was obtained.
Metabolism of 14,15-[14C]EET-Me.
Bovine coronary endothelial cells and smooth muscle cells were cultured
in 75-cm2 flasks as previously described (30).
The culture medium was removed, and the cells washed twice with HEPES
buffer. 14,15-[14C]EET-Me (8,000 counts/min,
106 M) was incubated for 10 min at 37°C in HEPES buffer
(see above) in the presence and absence of rings of the coronary
artery, endothelial cells, or smooth muscle cells (29,
30). In some studies, the incubation time was varied, and in
others, the cells or tissue were pretreated with the epoxide hydrolase
inhibitors BIRD-0826 (105 M) or 4-PCO (2.2 × 105 M). After incubation, the media were removed, and
radioactive metabolites were extracted by solid phase extraction using
C-18 extraction columns (Varian) as previously described (2,
30). The extract was resolved into its components by
reverse-phase HPLC using a linear gradient from 50% acetonitrile in
water to 100% acetonitrile as previously described (2,
30). In parallel analyses, the migration times of known
standards (14,15-EET, 14,15-EET-Me, 14,15-DHET, and 14,15-DHET-Me) were
determined. Epoxide hydrolase activity was expressed as the conversion
of 14,15-EET-Me to 14,15-DHET-Me and 14,15-DHET.
Chemicals. Charybdotoxin and iberiotoxin were purchased from Research Biochemicals International (Natick, MA). U-46619 was purchased from Cayman Chemical (Ann Arbor, MI). BIRD-0826 was provided by Boehringer-Ingelheim Pharmaceutical. It inhibits soluble epoxide hydrolase (6) with an IC50 of 0.46 µM but did not affect microsomal epoxide hydrolase in concentrations up to 10 mM. Its solubility in 20 mM sodium phosphate buffer (pH 7.4) was 16.3 mg/ml. Collagenase and elastase were purchased from Worthington (Lakewood, NJ). 14,15-EET was prepared as previously described by the method of Corey et al. (2, 5). 14,15-EET-Me was prepared by treating 14,15-EET with ethereal diazomethane for 15 min at room temperature (30). The solvent was removed under a stream of nitrogen. The purity was determined by HPLC as described above. 14,15-DHET was prepared by acid hydrolysis of 14,15-EET. The 14,15-EET was treated with 0.1 N hydrochloric acid for 2 h at room temperature. The eicosanoids were extracted and purified by HPLC as described above. All other chemicals were purchased from Sigma (St. Louis, MO).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Vascular relaxations to 14,15-EET, 14,15-DHET, and 14,15-EET-Me.
14,15-EET relaxed the U-46619-precontracted bovine coronary artery in a
concentration-related manner as previously described (Fig.
1). Similar relaxation responses were
obtained with 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET (2,
29). 14,15-EET-Me also relaxed the coronary artery and was less
potent than 14,15-EET. The activity of the methyl ester may represent
conversion of 14,15-EET-Me to 14,15-EET by vascular cells
(31). 14,15-DHET also relaxed the bovine coronary artery,
as was previously indicated in the canine coronary artery (25,
33). However, in our studies, the concentration-response curve
for 14,15-DHET was shifted approximately fivefold to the right of the
curve for 14,15-EET with no change in maximal effect. Thus the
hydrolysis of the epoxide to a vicinal diol results in loss of potency
but retention of full agonist activity. The relaxations to 14,15-DHET
were inhibited by increasing the extracellular K+
concentration from 4.8 to 20 mM (Fig. 2).
Similarly, pretreatment with the KCa channel blocker
iberiotoxin (50 nM) blocked 14,15-DHET-induced relaxations. Another
KCa channel inhibitor, charybdotoxin (100 nM), shifted the
14,15-DHET concentration-response curve to the right by ~10-fold.
Similar effects of these inhibitors have been described for EETs
(2). These studies indicate a role for K+
channels in the relaxations to 14,15-DHET.
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Activation of KCa channels in coronary smooth muscle by
14,15-DHET.
Figure 3A shows tracings of
macroscopic, outward K+ currents that were generated by
10-mV depolarizing steps from 60 to +60 mV in an isolated smooth
muscle cell in the whole cell mode. The application of 14,15-DHET (10 µM) activated outward K+ currents by ~50%, and the
further application of iberiotoxin (100 nM) eliminated most of the
outward K+ current of this cell. The averaged data show
that 14,15-DHET significantly activated the current density (Fig.
3B). At +60 mV, 14,15-DHET increased current density by
43%. The subsequent addition of iberiotoxin blocked current density by
42% below control levels. Membrane capacitance, an indicator of cell
membrane area, averaged 20.5 ± 3.4 pF.
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8 to 105M
(NPo = 0.030 ± 0.011 for control and
0.026 ± 0.010 with 10 µM 14,15-DHET, n = 5). If
GTP (0.5 mM) and ATP (1 mM) were added to the bathing solution of
inside-out patches, 14,15-DHET increased KCa channel
opening (Fig. 5A). As with
cell-attached patches, 14,15-DHET caused a concentration-related
increase in NPo of inside-out patches incubated
with GTP without changing the current amplitude (Fig. 5, B
and C). The concentrations of 14,15-DHET that increased KCa channel activity were similar in cell-attached patches
and inside-out patches exposed to GTP. A similar dependence on GTP was
reported for 11,12-EET in inside-out patches (11, 15, 18).
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Metabolism of 14,15-[14C]EET-Me by coronary arteries
and vascular cells.
Incubation of coronary arterial rings or endothelial cells with
14,15-[14C]EET showed the same conversion of 14,15-DHET
as cell-free incubations (data not shown). Because fatty acid methyl
esters are taken up by cells and metabolized to free acids (17,
31), we incubated 14,15-[14C]EET-Me
(106 M) with coronary arteries and resolved the
radioactive metabolites by HPLC (Fig.
6B). Four radioactive peaks
were detected. The major product comigrated with 14,15-DHET. The minor
products comigrated with 14,15-EET and the methyl esters of 14,15-EET
and 14,15-DHET. When 14,15-[14C]EET-Me was incubated
under identical conditions in the absence of the coronary artery, the
major radioactive peak comigrated with 14,15-EET-Me and a minor peak
with 14,15-EET (Fig. 6A). Pretreatment with BIRD-0826
(105 M) or 4-PCO (2.2 × 105 M)
inhibited the conversion of 14,15-EET to 14,15-DHET by 86% and 93%,
respectively (Fig. 6C). These studies indicate that coronary arteries contain esterases that convert fatty acid methyl esters to
fatty acids and epoxide hydrolase to convert the epoxide to a vicinal
diol.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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EETs are endothelium-derived eicosanoids that mediate a portion of
the vasodilator effect of agonists such as bradykinin and acetylcholine
(2, 10, 12, 16, 29). They act on vascular smooth muscle to
open KCa channels, which results in membrane hyperpolarization and vasodilation. The activation of KCa
channels by EETs requires a G protein, Gs (11, 15,
18). These findings may indicate that EETs function as EDHFs.
EETs may be metabolized in the vasculature to DHETs by epoxide
hydrolase, to 16- or 18-carbon metabolites by 
-oxidation, or to
22-carbon metabolites by chain elongation (8, 9, 34). The
EETs may also be esterified into phospholipids and neutral lipids
(32, 34).
Interestingly, 14,15-DHET also relaxed the bovine coronary artery;
however, DHET was five times less potent than 14,15-EET. This finding
indicates that the vicinal diol can partially substitute for the
epoxide group. This finding confirms published studies that 14,15-DHET
is a potent vasorelaxant in porcine coronary arteries and canine
coronary microvessels (9, 25). In these studies, 11,12-EET
and 11,12-DHET were equipotent in relaxing porcine coronary arteries
(9). In contrast, 11,12-DHET relaxed coronary microvessels in concentrations as low as 1018 M and was 1,000 times
more potent than 11,12-EET (25). This study suggests that
the coronary microcirculation is more sensitive to the relaxant effect
of DHET. Like 14,15-EET, the relaxations to 14,15-DHET were blocked by
increasing the extracellular K+ concentration from 4.8 to
20 mM and by inhibitors of KCa channels (2, 16,
25). These findings implicate K+ channels in the
action of DHET. This conclusion was confirmed by directly measuring
whole cell K+ currents in isolated arteriolar smooth muscle
cells. 14,15-DHET increased outward K+ currents, which was
blocked by iberiotoxin. Similar results were reported for 14,15-EET
(1). Additionally, 14,15-DHET activated KCa
channels in a concentration-related manner in cell-attached patches.
When compared with our previous studies, 14,15-DHET was ~10-fold less
potent than 14,15-EET in opening KCa channels
(2). Also, like 14,15-EET, 14,15-DHET failed to open
KCa channels in inside-out patches unless GTP was added to
the bathing solution (11, 15, 18). Thus 14,15-DHET appears
to have the same mechanism of action as 14,15-EET, with both
eicosanoids requiring a G protein for KCa channel activation.
EETs are hydrolyzed to DHETs by epoxide hydrolase (8, 34, 36). When 14,15-EET was incubated with coronary arterial rings, there was little metabolism to 14,15-DHET (29). It was not clear whether this lack of metabolism was due to a failure of 14,15-EET uptake into the vascular cells or the absence of epoxide hydrolase. Because previous studies indicate that long-chain fatty acid methyl esters are taken up by cells and hydrolyzed to their fatty acids (17, 31), we examined the metabolism of 14,15-EET-Me by coronary arteries and coronary vascular cells. 14,15-[14C]EET-Me was taken up by vascular cells and metabolized mainly to 14,15-DHET and 14,15-DHET-Me and to a lesser extent to 14,15-EET. These biochemical studies indicate that the coronary artery contains an epoxide hydrolase to convert EET to DHET and esterases to convert methyl esters to free acids. The hydrolysis of EET-Me to EET occurred in both the endothelial cell and smooth muscle cell. However, only endothelial cells converted 14,15-EET to 14,15-DHET. This finding is consistent with the presence of epoxide hydrolase in coronary endothelial cells (8, 34). The hydrolysis of 14,15-EET to 14,15-DHET was inhibited by two epoxide hydrolase inhibitors, 4-PCO and BIRD-0826. Thus endothelial cells synthesize EETs and contain the epoxide hydrolase necessary for DHET formation (30).
14,15-EET-Me relaxed the coronary artery and was less active than 14,15-EET. The relaxations to 14,15-EET-Me were enhanced slightly by pretreatment with the epoxide hydrolase inhibitor BIRD-0826 but not by 4-PCO. The lack of effect of 4-PCO may be because it is a substrate for epoxide hydrolase and not active for the duration of the tension experiments. These data are consistent with 14,15-EET and to a lesser extent 14,15-DHET mediating a portion of the action of 14,15-EET-Me. Because 14,15-DHET was fivefold less potent than 14,15-EET in causing relaxation, it is not surprising that 14,15-EET-Me was less active than 14,15-EET if 14,15-DHET mediates a portion of its effect.
In bovine coronary arteries, the relaxations to acetylcholine are mediated in part by an EDHF (2). EETs appear to represent EDHF in these arteries. In the current study, the relaxations to acetylcholine were not affected by inhibition of epoxide hydrolase by BIRD-0826 or 4-PCO. Similarly, in porcine coronary arteries, 4-PCO failed to alter the relaxations to bradykinin (34). These studies would suggest that metabolism of endogenous EETs by epoxide hydrolase is not an important determinant of EET action.
In summary, 14,15-DHET, like 14,15-EET, relaxes the bovine coronary artery. However, 14,15-DHET was less active than 14,15-EET. DHET shares the same mechanism of action as EET. It acts by opening KCa channels through a G protein-dependent mechanism. This opening of K+ channels allows the efflux of K+ that hyperpolarizes the smooth muscle cell membrane and causes relaxation. The endothelial cell is the vascular source of EETs and the vascular source of epoxide hydrolase that converts EETs to DHETs (30). Because DHET is less active than EET, the metabolism of EETs to DHETs represents an inactivation pathway.
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ACKNOWLEDGEMENTS |
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The authors thank Gretchen Barg for secretarial assistance and Sarah Hittner and Michelle Warras for technical assistance.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants HL-51055 and GM-31278, American Heart Association-Northland Affiliate grant, and by the Robert A. Welch Foundation.
Address for reprint requests and other correspondence: W. B. Campbell, Dept. of Pharmacology and Toxicology, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: wbcamp{at}mcw.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpheart.00597.2001
Received 20 July 2001; accepted in final form 9 January 2002.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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