From the Departments of Biochemistry,
§ Internal Medicine, and ¶ Molecular Analysis Facility,
College of Medicine, University of Iowa, Iowa City, Iowa 52242 and
the
Department of Entomology and Cancer Research Center,
University of California, Davis, California 95616
Received for publication, December 28, 2000, and in revised form, January 31, 2001
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ABSTRACT |
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Epoxyeicosatrienoic acids (EETs) are
products of cytochrome P-450 epoxygenase that possess important
vasodilating and anti-inflammatory properties. EETs are converted to
the corresponding dihydroxyeicosatrienoic acid (DHET) by soluble
epoxide hydrolase (sEH) in mammalian tissues, and inhibition of sEH has
been proposed as a novel approach for the treatment of hypertension. We
observed that sEH is present in porcine coronary endothelial cells
(PCEC), and we found that low concentrations of
N,N'-dicyclohexylurea (DCU), a selective sEH inhibitor,
have profound effects on EET metabolism in PCEC cultures. Treatment
with 3 µM DCU reduced cellular conversion of 14,15-EET to
14,15-DHET by 3-fold after 4 h of incubation, with a concomitant
increase in the formation of the novel Epoxyeicosatrienoic acids
(EETs)1 are arachidonic acid
metabolites produced by cytochrome P-450 epoxygenases (1, 2). EETs are
formed by endothelial cells and produce vasodilation in a number of
vascular beds, including the coronary circulation (2-4). This occurs
through hyperpolarization of the vascular smooth muscle cells by
activation of Ca2+-activated K+ channels,
suggesting that EETs function as endothelium-derived hyperpolarizing
factors (4-7). In addition, EETs are taken up and incorporated into
phospholipids by endothelial cells (8), a process that may contribute
to the endothelium-dependent vascular relaxation response
(9). Incorporation of EETs into phospholipids also may play a role in
their effects on cytokine-induced expression of adhesion molecules
(10), Ca2+ signaling (11), and tyrosine kinase activity in
the endothelium (12). Although a great deal is known about the
biological actions of EETs in the vasculature, relatively little is
known about the biochemical pathways that mediate EET uptake and
metabolism in endothelial cells.
A major pathway for EET metabolism in many tissues is hydration of the
epoxide group to a diol, forming the corresponding dihydroxyeicosatetraenoic acid (DHET), a reaction mediated by an
epoxide hydrolase (13, 14). The two most studied forms of epoxide
hydrolase and the two that have been shown to metabolize arachidonic
acid epoxides in mammals are a soluble epoxide hydrolase (sEH) and a
microsomal epoxide hydrolase (14-16). Considerable interest has
recently centered on sEH because of results suggesting that it is a
potential target for the treatment of hypertension. Disruption of the
sEH gene was found to lower systolic blood pressure in male mice (17).
Likewise, N,N'-dicyclohexylurea (DCU), a recently discovered
selective sEH inhibitor that has a Ki = 30 nM for recombinant human sEH (18), reduced blood pressure in the spontaneously hypertensive rat (19). Both of these studies demonstrated that the anti-hypertensive effects were associated with reduced conversion of EETs to DHETs in the kidney (17, 19).
One potential mechanism whereby inhibition of sEH could reduce blood
pressure is by enhancing the amount of EET available for incorporation
into endothelial phospholipids. Thus, whereas EETs were shown to be
avidly taken up and incorporated into endothelial cell phospholipids,
DHETs appeared to be released preferentially from the cells (8). In
keeping with this notion, we have previously observed that
4-phenylchalcone oxide (4-PCO), a nonspecific inhibitor of epoxide
hydrolases (20), blocked conversion of 14,15-EET to 14,15-DHET and
increased 14,15-EET incorporation in porcine coronary artery
endothelial cell (PCEC) phospholipids (21). Consequently,
endothelium-dependent relaxation responses were augmented
in coronary arteries. Taken together, these findings suggest that
epoxide hydrolase inhibition might beneficially affect vascular
function by enhancing the amount of EET incorporated into endothelial
cells. Moreover, these reports suggest that sEH functions to
enzymatically degrade EETs and thereby terminate their biological activity.
On the other hand, several recent studies have challenged the view that
sEH is simply a degradation pathway for EETs. For example, whereas
DHETs were originally believed to be devoid of vasoactive properties
(22-24), more recent reports indicate that they are capable of
dilating conduit coronary arteries and coronary microvessels (9, 25,
26). Also, DHETs are incorporated into endothelial cell phospholipids,
albeit less avidly than the precursor EETs (27). Finally, porcine
coronary artery smooth muscle cells were found to metabolize 11,12-DHET
through a The purpose of the present study was to delineate the pathways of EET
uptake and metabolism in endothelial cells during sEH inhibition. We
have identified two novel pathways of EET metabolism in endothelial
cells that could have important implications regarding the mechanisms
by which sEH inhibition lowers blood pressure. Moreover, elucidation of
these pathways provides important new insight into the mechanisms by
which cytochrome P-450 epoxygenase products may regulate vascular function.
Synthesis of
[3H]EET--
[5,6,8,9,11,12,14,15-3H]Arachidonic
acid ([3H]AA, 55 Ci/mmol, American Radiolabeled
Chemicals, St. Louis, MO) was mixed with AA (Cayman Chemical, Ann
Arbor, MI) at a final concentration of 0.2 µCi/nmol. After removing
the solvent, the fatty acid was methylated with 12% BF3 in
methanol for 45 min at 50 °C. Following extraction, the AA methyl
ester was incubated with 3-chloroperoxybenzoic acid in methylene
chloride for 1.5 h at 0 °C (28). The reaction was stopped by
addition of 50 µl of methyl sulfide, and the mixture was allowed to
stand at room temperature for 20 min. The products were resuspended in
methylene chloride and washed first with NaCl-saturated water and then
water. The [3H]EET methyl esters were isolated and
purified by reverse-phase high performance liquid chromatography (HPLC)
using a gradient that started at 65% acetonitrile, 35% water, was
ramped to 85% acetonitrile, 15% water over 55 min, and then was taken
to 100% acetonitrile at 60 min and held constant for 5 min. Fractions containing the purified [3H]11,12- and
14,15-EET2 methyl esters were
hydrolyzed with 50 µl of 2 N NaOH in 500 µl of methanol
at 50 °C for 1 h, and the [3H]EETs were extracted
from the mixture with water-saturated ethyl acetate (28). After drying
under N2, the [3H]EETs were suspended in 95%
ethanol and stored at Cell Culture and Incubation--
PCEC were isolated and grown in
modified Medium 199 supplemented with 10% fetal bovine serum as
described previously (9, 21). The cultures were maintained until
confluent at 37 °C in a humidified atmosphere containing 5%
CO2. Stocks were subcultured weekly by trypsinization, and
cultures were used for experiments between passages 3 and 7. Experiments were carried out with confluent monolayers using modified
Medium 199 containing 0.1 µM bovine serum albumin.
Prior to incubation, the radiolabeled EET or AA was mixed with the
corresponding non-radiolabeled compound obtained from Cayman Chemical
to obtain the substrate concentration necessary for each experiment and
a specific activity of 0.1 µCi/nmol. The PCEC were incubated
initially in 1 ml of medium containing either DCU dissolved in dimethyl
sulfoxide or the same amount of dimethyl sulfoxide alone as a control.
The DCU was purchased from Aldrich, and a liquid chromatography
combined with mass spectrometry (LC/MS) analysis and melting point
measurement verified the purity. After 45 min, the radiolabeled
substrate was added and the incubation continued. The medium was
removed at the end of the incubation period, and the cells were washed
twice and harvested by scraping into methanol. In some studies, the
initial incubation medium was removed, and after washing, the cells
were incubated for an additional 20 min in 1 ml of fresh medium
containing either 3 µM DCU and 2 µM calcium
ionophore A23187 or the ionophore alone.
Assay of Incubation Medium--
The radioactivity remaining in
the medium after the incubation was measured by liquid scintillation
counting. Lipids contained in the medium were extracted twice with 4 ml
of ice-cold ethyl acetate saturated with water; the extracts were
combined; the solvent was evaporated under N2; and the
residue was dissolved in acetonitrile. The lipids were separated by
reverse-phase HPLC using a Gilson dual pump gradient system equipped
with model 306 pumps, a model 117 Dual wavelength UV detector, a model
231 XL automatic sample injector (Gilson Medical Electronics, Inc.,
Middleton, WI), and a 3 µm 4.6 × 150-mm Spherisorb
C18 column obtained from Alltech (Deerfield, IL). The
elution profile consisted of water adjusted to pH 3.4 with phosphoric
acid and an acetonitrile gradient that increased from 27.5 to 100%
over 60 min at a flow rate of 0.7 ml/min (29). The distribution of
radioactivity was measured by combining the column with scintillator
solution and passing the mixture through an on-line flow detector
(IN/US Systems, Inc., Tampa, FL).
Analyses of Cell Lipids--
The lipids were extracted from the
PCEC with a 2:1 mixture of chloroform/methanol. After the phases were
separated and the solvent removed under N2, the lipids were
dissolved in 200 µl of chloroform/methanol, and an aliquot of this
mixture was dried under N2 and assayed for radioactivity in
a liquid scintillation spectrometer (25). To determine the distribution
of the radioactivity in the cell lipids, aliquots of the extract were
separated by TLC on Whatman LK5D silica gel plates obtained from
Alltech Associates (Deerfield, IL) with a solvent system of chloroform,
methanol, 40% methylamine (65:35:5, v/v/v). The radioactivity
contained in the separated lipids was assayed using a Radiomatic gas
flow proportional scanner with automatic peak search and integration (25, 29). Authentic phospholipid standards purchased from Avanti Polar
Lipids (Naperville, IL) were added to each chromatogram and visualized
by staining (30).
To separate and identify the radiolabeled fatty acid derivatives
incorporated in the PCEC lipids, additional aliquots of the extract
were hydrolyzed for 1 h at 50 °C in 0.5 ml of methanol containing 50 µl of 0.2 N NaOH and 10% H2O.
After the pH was adjusted to 7.2 with ice-cold
H3PO4/H2O (1:50, v/v), the free
fatty acids were extracted twice with 5 ml of ice-cold ethyl acetate
saturated with H2O. The solvent was removed under
N2, and the lipids were dissolved in acetonitrile and
separated by reverse-phase HPLC (29).
Identification of EET Metabolites--
A Hewlett-Packard 1100 MSD LC/MS system was used to separate and identify the metabolites
(31). HPLC was carried out on a Supelco C18 5 µm 4.6 × 150 mm DiscoveryTM column with mobile phase solvents
consisting of water/formic acid (100:0.03 v/v) (solvent A) and
acetonitrile (solvent B) at a flow rate of 0.7 ml/min. The gradient was
maintained at 30% solvent B for the first 2 min and then linearly
ramped to 57% solvent B at 20 min, 65% solvent B at 40 min, 70%
solvent B at 45 min, and 95% solvent B at 50 min. Negative ion
electrospray was used with the fragmentor voltage set at 140 V in order
to produce in-source collision-induced decompositions (CID).
N2 nebulizing gas was maintained at 60 bar, whereas the
N2 drying gas was set to a flow rate of 10 liters/min at
350 °C. Data were processed with the Hewlett-Packard Chemstation
software program.
To confirm the identification of the metabolites, additional studies
were done with
[5,6,8,9,11,12,14,15-2H8]14,15-EET
([2H8]14,15-EET, Biomol Research
Laboratories, Inc., Plymouth Meeting, PA). The PCEC cultures were
incubated initially with 10 µM DCU for 45 min, and then 5 µM [2H8]14,15-EET was added and
the incubation continued for 5 h. After the media and cells were
separated, the lipids were extracted from each and were analyzed by
LC/MS as indicated above.
Epoxide Hydrolase Activity--
Harvested PCEC were suspended in
1 ml of chilled 0.1 M sodium phosphate buffer, pH 7.4, containing 1 mM EDTA, phenylmethylsulfonyl fluoride, and
dithiothreitol. The cells were disrupted using a Polytron homogenizer
at 9,000 rpm for 30 s; the homogenate was centrifuged at
9,000 × g for 10 min at 4 °C; and the supernatant solution was used as the enzyme extract. Protein concentration was
measured using the Pierce BCA assay. Epoxide hydrolase activity was
measured using racemic
[3H]trans-1,3-diphenylpropene oxide (tDPPO), a
selective substrate for the enzyme (32). The tDPPO was synthesized by
reducing the chemical precursor with [3H]sodium
borohydride (PerkinElmer Life Sciences) to give a final specific
activity of 50 mCi/mmol (32). After the tDPPO was diluted with
unlabeled material, 1 µl of a 5 mM solution of
[3H]tDPPO in dimethylformamide was added to 100 µl of
enzyme preparation in 0.1 M sodium phosphate buffer, pH
7.4, containing 0.1 mg/ml albumin. The final tDPPO concentration was 50 µM. After incubating at 30 °C for 15 min, the reaction
was quenched by addition of 60 µl of methanol and 200 µl of
isooctane. These solvents extracted the remaining epoxide from the
aqueous phase, and the quantity of radioactive diol present in the
aqueous phase was measured by liquid scintillation counting. Each assay
was performed in triplicate.
Statistical Analysis--
The data are expressed as means ± S.E. Values were analyzed by Student's t test for
unpaired data. Probability values of 0.05 or less were considered to be
statistically significant.
Determination of Epoxide Hydrolase Activity--
The 9000 × g supernatant solution from the PCEC homogenate was
incubated with [3H]tDPPO, a substrate that is specific
for sEH (32). The preparation produced 2.1 nmol
min Effect of sEH Inhibition on 14,15-EET Metabolism--
To
investigate the role of sEH in endothelial EET metabolism, we
determined whether DCU, a potent and selective inhibitor of recombinant
sEH (18), would inhibit the conversion of EET to DHET in intact PCEC.
The cultures were incubated for either 75 min or 4 h with 2 µM [3H]14,15-EET, with or without 3 µM DCU, and the distribution of radiolabeled products in
the culture medium was determined by reverse-phase HPLC. Representative
chromatograms are shown in Fig. 1.
14,15-DHET accounted for 85% of the radioactivity contained in the
medium of control cultures after a 75-min incubation, and less than
10% remained as EET (Fig. 1A). By contrast, when DCU was
added to the medium, most of the radioactivity remained as 14,15-EET
and only 13% was converted to 14,15-DHET after 75 min (Fig.
1B). Small amounts of two additional radiolabeled products, designated X and Y with retention times of 25.6 and 31 min,
respectively, were detected in the medium of the cultures treated with
DCU. When the incubation was extended to 4 h, 14,15-DHET accounted for >95% of the radioactivity in the medium (Fig. 1C).
However, when DCU was present during the 4-h incubation, EET remained
the most abundant radiolabeled compound in the medium, X and Y
increased, and DHET accounted for only 35% of the radioactivity (Fig.
1D).
Additional experiments further investigated the
time-dependent effect of DCU and the effect of increasing
DCU concentrations on [3H]14,15-EET metabolism. After
incubation, the medium obtained from each culture was assayed by HPLC,
and the differences in radiolabeled metabolite production are
illustrated in Fig. 2. The inhibition of
14,15-DHET formation approached a maximum at 3 µM DCU
when the PCEC were incubated with 2 µM
[3H]14,15-EET for 75 min (Fig. 2A), and the
calculated IC50 of DCU was 0.23 µM.
Radiolabeled DHET accumulation reached a maximum in 1-2 h when no
inhibitor was present. However, it increased much more slowly when 3 µM DCU was present, and 50% less 14,15-DHET was formed
at the end of the 8-h incubation (Fig. 2B). The amounts of
radiolabeled metabolites X and Y in the medium during the 8-h incubation with DCU are shown in Fig. 2C. Product Y formed
more rapidly, reached a maximum at 4 h, and then declined, whereas product X increased progressively and was more abundant at the end of
the 8-h incubation.
Identification of Metabolites--
The three main radiolabeled
compounds that accumulated in media containing DCU were identified by
LC/MS (Fig. 3). In-source CID was
utilized to generate additional structural information. The mass
spectra of deuterium-labeled metabolites isolated from the medium
following incubation of PCEC cultures with
[2H8]14,15-EET also were obtained to confirm
the assignments to the fragment ions in the spectra of the
metabolites.
The mass spectrum of 14,15-EET contains a (M
The (M
Both the in-source CID mass spectra of metabolite X (Fig.
3E) and its deuterated analog (Fig. 3F) show
abundant (M
These findings indicate that in addition to being converted to diols by
sEH, 14,15-EET can undergo Effect of sEH Inhibition on 14,15-EET Incorporation into
Cells--
A series of experiments was done to determine whether
inhibition of sEH would affect the amount of 14,15-EET taken up and retained by the PCEC. The effects of DCU concentration and time of
incubation on the total amount of radiolabeled 14,15-EET present in the
cell lipids are shown in Fig. 4. DCU
produced a dose-dependent increase in uptake of 2 µM [3H]14,15-EET, with the maximum
accumulation occurring at 1 µM DCU in a 75-min incubation
(Fig. 4A). During the course of an 8-h incubation, the
amount incorporated by the control cells reached a maximum at 1 h
and then declined (Fig. 4B). The amount contained in the
cells was greater when 3 µM DCU was present throughout the 8-h incubation; the maximum accumulation did not occur until 4 h, and the incorporation at 8 h was 5 times greater than in the
control cultures.
Analysis by TLC indicated that the endothelial phospholipids contained
more than 90% of the radioactivity incorporated by the PCEC (Fig.
5). The phospholipid fractions containing
the radioactivity were phosphatidylcholine (PC),
phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Similar
distributions, PC > PE > PI, occurred in the control and
DCU-treated cultures. When the DCU concentration was raised, however,
the amounts of radioactivity in PC and PE increased, whereas the amount
in PI did not change appreciably (Fig. 5A). The time
dependence of the distribution was examined when the PCEC cultures were
incubated with 3 µM DCU. The incorporation of 14,15-EET
into PE and PI reached a steady state level within 1-2 h, whereas the
amount in PC continued to increase during the first 4 h and then
declined (Fig. 5B).
Radiolabeled Compounds in Cell Lipids--
The extent to which the
14,15-EET incorporated by the PCEC was chemically modified was
determined by HPLC analysis of the hydrolyzed cell lipid extract. The
results of an experiment in which the PCEC were incubated with 2 µM [3H]14,15-EET for 4 h are shown in
Fig. 6. In the control cultures, only
32% of the radioactivity contained in the PCEC lipids remained as
14,15-EET, whereas 30% was converted to 14,15-DHET (Fig.
6A). A compound containing 24% of radioactivity, designated
T, eluted with a retention time of 53 min, and two other compounds with retention times of 33 and 35.4 min, designated Q and R, respectively, contained smaller amounts of radioactivity.
The distribution when 3 µM DCU was present during the
incubation is shown in Fig. 6B. As indicated by the
difference in the scale of the y axis, much more
radiolabeled material was contained in the hydrolyzed cell lipid
extract when DCU was present. Metabolite T was the most abundant
product and accounted for 40% of the incorporated radioactivity, and
36% was present as 14,15-EET, 7% as metabolite R, and less than 2%
as 14,15-DHET. A metabolite with a retention time of 48 min, designated
S, accounted for 14% of the radioactivity. This product was not
detected in the control cultures. In addition, 10,11-epoxy-16:2, a
metabolite that accumulated in the medium (designated as product Y in
Figs. 1D and 2C), accounted for 2% of the
radioactivity in the cells. Product Q, which was present in the control
cells, was not detected in the cells incubated with DCU. Thus, taken
together, these results indicate that after a 4-h incubation of
endothelial cells with 14,15-EET, most of the incorporated
radioactivity is in the form of metabolites of 14,15-EET, and the
profile of incorporated metabolites is altered by inhibition of
sEH.
Identification of Metabolites in Cells--
The in-source CID mass
spectra of metabolites T, S, and Q and their deuterated analogs
obtained from the PCEC lipids in a corresponding incubation with
[2H8]14,15-EET are shown in Fig.
7. Although an in-source CID mass spectrum of metabolite R also was obtained, we have not been able to
assign a structure for this compound.
The fragmentations of the 2H8-labeled products
confirm the assignments to the fragment ions in the spectra of the
14,15-EET metabolites. Specifically, a comparison of the in-source CID
mass spectrum of metabolite T (Fig. 7A) and its deuterated
counterpart (Fig. 7B) indicates that the masses of the
(M
The fragmentation pattern of metabolite Q differs from that of the
other products. The (M
These findings show that 14,15-EET can be elongated by endothelial
cells and that the process is augmented in the presence of sEH
inhibition. Moreover, the results indicate that elongation products of
14,15-EET and its metabolites can be incorporated into endothelial phospholipids.
Effect of sEH Inhibition on EET Release from Cells--
Previous
studies indicated that PCEC rapidly release some of the EET
incorporated in cell phospholipids if the cultures are incubated with a
calcium ionophore (9). The material is released into the medium as the
free acid (9, 21). Since sEH inhibition increased the incorporation of
14,15-EET and altered the distribution of 14,15-EET metabolites
contained in the PCEC lipids, we investigated whether the amount or
type of products released would be affected by the presence of DCU.
PCEC cultures were incubated for 4.5 h with 2 µM
[3H]14,15-EET, with or without 3 µM DCU.
After the medium was removed, the cultures were washed and incubated
for 20 min in medium containing 2 µM ionophore A23187.
The PCEC treated with DCU released 4.1 times more radiolabeled products
(128 ± 5 pmol as compared with 31 ± 2 pmol,
n = 3, p < 0.05; the pmol values are
calculated from the specific radioactivity of the
[3H]14,15-EET used to load the cells). The radiolabeled
material was present in the medium as the free acid. The greater
release is consistent with the fact that the PCEC treated with DCU
contained 4.8 times more radiolabeled material in phospholipids at the
start of the incubation with the calcium ionophore.
HPLC analysis of the medium was done to determine whether DCU also
affected the composition of the radiolabeled material released from the
cells (Fig. 8). Only 5% of the
radioactivity released by the control cultures remained as 14,15-EET
during the 20 min of incubation with the ionophore, and 90% was
converted to DHET (Fig. 8A). By contrast, 95% of the
radioactivity released by the PCEC treated with 3 µM DCU
remained as EET (Fig. 8B).
Effect of DCU on 11,12-EET Metabolism--
Additional studies were
done with 11,12-EET to determine whether DCU would produce similar
effects with another EET regioisomer. HPLC data showing the effects of
3 µM DCU on the radiolabeled compounds present in the
medium during incubation of PCEC with 2 µM
[3H]11,12-EET are shown in Fig.
9. 11,12-DHET accounted for 21% of the
radioactivity contained in the medium of control cultures after 75 min
of incubation, and 65% remained as EET (Fig. 9A). DCU
reduced the conversion of 11,12-EET to 11,12-DHET. After 75 min, DHET
accounted for only 5% of the radiolabeled material in the medium when
DCU was present (Fig. 9B). 11,12-DHET accounted for 60% of
the radioactivity in the medium when the incubation was extended to
4.5 h, and only 3% remained as 11,12-EET (Fig. 9C).
When DCU was present throughout the 4.5-h incubation, 30% of the
medium radioactivity remained as 11,12-EET, and only 20% was converted
to 11,12-DHET (Fig. 9D). In addition, a metabolite designated J with a retention time of 32 min accounted for 5% of the
radioactivity in the 75 min of incubation with DCU. The amount of
metabolite J increased to 20% after 4.5 h. LC/MS analysis of
compound J with in-source CID indicated a structure of
7,8-epoxyhexadecadienoic acid (7,8-epoxy-16:2) (data not shown).
HPLC analysis of the hydrolyzed cell lipids at the end of the 4-h
incubation indicated that the control cultures contained 440 ± 8 pmol of [3H]11,12-EET (n = 3), whereas the cultures incubated with 3 µM DCU
contained 640 ± 13 pmol, an increase of 45% (p < 0.05).
Effect of DCU on AA Metabolism--
We also examined the effect of
DCU on AA uptake to determine whether the increased incorporation of
EETs into PCEC lipids might be due to an effect of DCU unrelated to sEH
inhibition. PCEC cultures were incubated for 4 h with 2 µM [3H]AA and 3 µM DCU, and
control cultures were similarly incubated without DCU. Treatment with
DCU did not appreciably alter the amount of [3H]AA
incorporated into the cells (1090 ± 54 pmol in the control cultures as compared with 1260 ± 51 pmol in those treated with DCU, n = 3, p > 0.05). Furthermore,
about 45% of radioactivity remaining in the medium of both cultures
was converted to prostaglandin E2 and 6-keto-prostaglandin
F1 In the present study, we identified three distinct but interactive
pathways of EET metabolism in coronary endothelial cells: (a) conversion of 14,15-EET to 14,15-DHET by sEH is the
prevailing pathway; (b) both 14,15-EET and 14,15-DHET can be
incorporated into membrane phospholipids and converted to elongation
products; (c) when the sEH is inhibited, 14,15-EET is
converted to chain-shortened epoxide metabolites through a
In a previous study, we showed that treatment of endothelial
cells with 4-PCO blocked conversion of 14,15-EET to 14,15-DHET but did
not stimulate the formation of When the endothelial sEH was inhibited by DCU, 14,15- and
11,12-EET were converted to chain-shortened epoxy-fatty acids. These products accumulated primarily in the extracellular fluid. No such
products have been identified in any previous studies in which
endothelial cells were incubated with EETs. Similar EET metabolites
were, however, observed in a previous study with human skin
fibroblasts, and data with mutant fibroblasts indicated that they are
formed by peroxisomal Cultured human skin fibroblasts contain very little epoxide hydrolase
activity and convert only trace amounts of EETs to DHETs (29). Based on
this, we conclude that The present findings also demonstrate for the first time that 14,15-EET
and its derivatives can undergo chain elongation by endothelial cells.
Three of the products formed by chain elongation were identified by
LC/MS. 16,17-Epoxy-22:3, the elongation product of 14,15-EET, was
formed by the control cultures but accumulated in increased amounts in
PCEC treated with DCU. Thus, as observed regarding In addition to blocking conversion to 14,15-DHET and enhancing the
formation of The novel metabolites that were incorporated into the cells treated
with DCU, including 16,17-epoxy-22:3 which was the most abundant
product, were not released from the cells in response to stimulation
with a calcium ionophore. Thus, 95% of the radioactivity released when
the DCU-treated cells were incubated with the calcium ionophore was in
the form of 14,15-EET. The reason that the EET metabolites were not
released from the cells is unclear. However, given their apparent
propensity to be retained in endothelial phospholipids, accumulation of
these compounds could have an important modulating effect on cell
signaling pathways.
As noted with 14,15-EET, the conversion of 11,12-EET to 11,12-DHET was
reduced when DCU was present. More 11,12-EET accumulated in the PCEC,
but the magnitude of the increase in intracellular 11,12-EET content
was considerably smaller than that with 14,15-EET under these
conditions. This probably is due to the fact that the endothelial sEH
was less active against 11,12-EET than with 14,15-EET, a finding
consistent with enzymatic results and data from the spontaneously
hypertensive rat indicating that 14,15-EET is a better substrate than
11,12-EET for sEH (13, 19). The reduced metabolism of 11,12-EET by
endothelial sEH may account for the fact that it has more potent
anti-inflammatory properties in the vascular wall than 14,15-EET
(10).
In summary, these findings suggest that in addition to being
metabolized by sEH, EETs are substrates for -oxidation products
10,11-epoxy-16:2 and 8,9-epoxy-14:1. DCU also markedly enhanced the
incorporation of 14,15-EET and its metabolites into PCEC lipids. The
most abundant product in DCU-treated cells was 16,17-epoxy-22:3, the
elongation product of 14,15-EET. Another novel metabolite,
14,15-epoxy-20:2, was present in DCU-treated cells. DCU also caused a
4-fold increase in release of 14,15-EET when the cells were stimulated
with a calcium ionophore. Furthermore, DCU decreased the conversion of
[3H]11,12-EET to 11,12-DHET, increased 11,12-EET
retention in PCEC lipids, and produced an accumulation of the partial
-oxidation product 7,8-epoxy-16:2 in the medium. These findings
suggest that in addition to being metabolized by sEH, EETs are
substrates for
-oxidation and chain elongation in endothelial cells
and that there is considerable interaction among the three pathways.
The modulation of EET metabolism by DCU provides novel insight into the
mechanisms by which pharmacological or molecular inhibition of sEH
effectively treats hypertension.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-oxidation pathway, thereby generating a 16-carbon diol
that also possessed vasodilating activity (25). These reports indicate
that epoxide hydrolase can, in some instances, generate biologically
active EET metabolites. Moreover, the latter study raised the
possibility that enzymes other than epoxide hydrolases might be capable
of metabolizing EETs in vascular cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C. Purity was checked by isocratic
normal phase HPLC with a solvent consisting of 90%
n-heptane and 10% of a mixture of n-heptane,
isopropyl alcohol, and acetic acid (100:5:0.2, v/v/v). Only
samples of >98% purity were used for the cell studies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 mg protein
1 of
radiolabeled diol metabolite, demonstrating that the coronary artery
endothelial cells contain sEH activity. This finding is consistent with previous studies indicating that PCEC cultures convert 14,15-EET and 8,9-EET to the corresponding DHETs (21).
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Fig. 1.
Effect of DCU on [3H]14,15-EET
products present in the culture medium. PCEC were incubated with
either 3 µM DCU dissolved in dimethyl sulfoxide or
a corresponding amount of dimethyl sulfoxide for 45 min. This medium
was removed and replaced with fresh medium containing 3 µM DCU in dimethyl sulfoxide and 2 µM
[3H]14,15-EET or dimethyl sulfoxide and 2 µM [3H]14,15-EET (control), and the
incubation was continued for either 75 min (left panel) or
4 h (right panels). After incubation, the medium was
removed and lipids extracted and analyzed for radioactivity by HPLC
with an on-line flow scintillation counter. Radiochromatograms from a
single culture are shown, but similar results were obtained from two
additional cultures in each case: A, 75 min
control; B, 75 min with 3 µM DCU;
C, 4 h control; D,
4 h with 3 µM DCU. The identifications of compounds
X and Y are described in the text, and mass spectra of the compounds
are shown in Fig. 3.
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Fig. 2.
Time- and concentration-dependent
formation of radiolabeled 14,15-EET products in the culture
medium. PCEC were incubated with 2 µM
[3H]14,15-EET and DCU where indicated, and the medium was
analyzed for radioactivity as described in Fig. 1. The time of
incubation in A was 75 min. B and C,
the DCU concentration was 3 µM. The picomole values are
calculated from the specific activity of [3H]14,15-EET
added to the cultures. Each point is the average of results obtained
from two separate cultures, and both values were within 10%
agreement.
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Fig. 3.
Mass spectra of the main products and their
deuterated analogs contained in the incubation medium. The spectra
were obtained by LC/MS with in-source CID. In order to obtain
sufficient quantities of products for structural identification, it was
necessary to utilize 75-cm2 PCEC cultures. The time of
incubation was 4 h, and DCU was present in the culture medium. A
similar incubation was done with
[2H8]14,15-EET to obtain the corresponding
deuterated compounds. In both cases, HPLC analysis indicated a pattern
of metabolites similar to that shown in Fig. 1D. The mass
spectra correspond to the following: A, the 14,15-EET
present in the culture medium at the end of the incubation;
B, [2H8]14,15-EET present in the
culture medium at the end of the incubation; C, compound Y;
D, the deuterated analog of compound Y; E,
compound X; F, the deuterated analog of compound X. The * in
the structural diagrams of the deuterated compounds indicate the
carbons that contain 2H.
H)
ion of m/z 319, an ion of m/z 301 due to loss of
one water molecule, and an ion of m/z 219 from fragmentation
of the carbon chain through the oxirane ring (31, 33). The fragment ion
of m/z 257, which is of lesser importance, could be the
result of loss of water and either loss of CO2 or loss of a
neutral alkene and H2 via charge remote fragmentation (31).
The identity of the compound, which appeared to be 14,15-EET based on
HPLC retention time (Fig. 1), was confirmed by the mass spectrum (Fig.
3A) and that of the corresponding deuterated analog (Fig.
3B).
H)
and fragment ions that are analogous to
those of 14,15-EET are labeled in the mass spectra of metabolites Y and X. These ions were utilized to determine the molecular weight, if an
epoxide is present, and the location of the epoxide ring in the carbon
chain. Comparison of the in-source CID mass spectra of metabolite Y
(Fig. 3C) and its deuterated analog (Fig. 3D) indicates that
the masses of the (M
H)
ions
(m/z 265 and 272) differ by 7 daltons. The fragment ions corresponding to the loss of water (m/z 247 and 254) also
differ by 7 Da, but the fragment ions that are the result of carbon
chain cleavage at the epoxide ring (m/z 165 and 171) differ
by only 6 Da. This confirms that due to the in-source CID the (M
H)
ions are fragmenting via cleavage of the carbon
chain at the epoxide ring, and one deuterium is retained by the neutral
loss. Thus, the structure of metabolite Y is consistent with a
structure of 10,11-epoxyhexadecadienoic acid (10,11-epoxy-16:2).
H)
ions of m/z 239 and
245, ions due to loss of water (m/z 221 and 227), and ions
due to carbon chain cleavage at the oxirane ring (m/z 139 and 144). The data suggest a structure of 8,9-epoxytetradecaneoic acid
(8,9-epoxy-14:1).
-oxidation by endothelial cells, with
each cycle removing two carbons from the carboxyl end of the molecule.
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Fig. 4.
Time- and concentration-dependent
effects of DCU on 14,15-EET uptake by PCEC. The cultures were
incubated with 2 µM [3H]14,15-EET for
various times as described in Fig. 1. The time of incubation in the DCU
concentration study was 75 min (A), and the DCU
concentration in the time-dependent study was 3 µM (B). After removal of medium and washing
the cells with buffer, the cell lipids were extracted, and an aliquot
was assayed for radioactivity. The picomole values are calculated from
specific activity of [3H]14,15-EET added to the cultures.
Each point is the average of results obtained from two separate
cultures, and both values were within 10% agreement.
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Fig. 5.
Time- and concentration-dependent
effects of DCU on 14,15-EET incorporation into PCEC phospholipids.
The incubation and analysis was done as described in Fig. 4, except
that the cell lipid extracts were separated by TLC, and the
radioactivity contained in each of the phospholipid fractions was
determined. The picomole values are calculated from specific activity
of [3H]14,15-EET added to the cultures. Each point is the
average of results obtained from two separate cultures, and both values
were within 10% agreement.
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Fig. 6.
Radiolabeled 14,15-EET products present in
the cell lipids. The PCEC were incubated with 2 µM
[3H]-14,15-EET for 4 h in the absence (A)
or presence (B) of 3 µM DCU. After incubation,
the cell lipids were extracted, hydrolyzed by saponification, and
analyzed for radioactivity by reverse-phase HPLC as described in Fig.
1. Radiochromatograms from a single culture are shown, but similar
results were obtained from two additional cultures in both cases. The
identifications of compounds Q, S, and T are described in the text, and
mass spectra of these compounds are shown in Fig. 7. Compound R was not
been identified.
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Fig. 7.
Mass spectra of 14,15-EET metabolites and
their deuterated analogs contained in the cell lipids. The
incubation conditions and lipid extraction from the PCEC were the same
as described in Fig. 6, and the mass spectra were obtained by LC/MS
with in-source CID. Deuterated compounds were obtained from the
hydrolyzed cell lipid extract following incubation of PCEC cultures
with [2H8]14,15-EET. The mass spectra
correspond to the following: A, compound T; B,
the deuterated analog of compound T; C, compound S;
D, the deuterated analog of compound S; E,
compound Q; and F, the deuterated analog of compound Q (the
compounds are designated as indicated in Fig. 6). The * in the
structural diagrams of the deuterated compounds indicates the carbons
that contain 2H.
H)
ions (m/z 347 and 355) differ by
8 Da. The fragment ions corresponding to the loss of water
(m/z 329 and 337) also differ by 8 Da, but the fragment ions
that are the result of carbon chain cleavage at the epoxide ring
(m/z 247 and 254) differ by only 7 Da. Thus, the structure
of metabolite T is consistent with 16,17-epoxydocosatrienoic acid
(16,17-epoxy-22:3), the chain elongation product of 14,15-EET. Both the
in-source CID mass spectra of metabolite S (Fig. 7C) and its
deuterated analog (Fig. 7D) show abundant (M
H)
ions of m/z 321 and 329, ions due to loss
of water (m/z 303 and 311), and ions due to carbon chain
cleavage at the oxirane ring (m/z 221 and 228). These data
suggest a structure of 14,15-epoxyeicosadienoic acid
(14,15-epoxy-20:2).
H)
ions (m/z
365 and 373) fragment to lose first one molecule of water and then
another (m/z 347 and 329 in Fig. 7E;
m/z 355 and 337 in Fig. 7F, the corresponding
deuterated derivative). The mass difference between fragment ions,
which in the previous spectra was thought to indicate epoxide groups,
is only 6 Da (m/z 235 and 241 in Fig. 7, E and
F, respectively), indicating that the metabolite is not an
epoxy fatty acid. However, it is consistent with a structure of
16,17-dihydroxydocosatrienoic acid (16,17-diOH-22:3) (34), the chain
elongation product of 14,15-DHET.
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Fig. 8.
Effect of DCU on radiolabeled products
released into the medium in response to calcium ionophore A23187.
The PCEC were pretreated with vehicle or 3 µM DCU for 45 min and then incubated with 2 µM
[3H]14,15-EET for 4.5 h as described in Fig. 1.
After washing, the radiolabeled cells were incubated for an additional
20 min with 2 µM A23187, without (A) or with
(B) 3 µM DCU. The medium was collected and
assayed for radiolabeled products as described in Fig. 1.
Radiochromatograms from a single culture are shown, but similar results
were obtained from two additional cultures in each case.
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Fig. 9.
Effect of DCU on [3H]11,12-EET
products present in the culture medium. PCEC were incubated as
described in Fig. 1, except that the radiolabeled substrate was 2 µM [3H]11,12-EET. The incubations were for
either 75 min (left panels) or 4.5 h (right
panels), and the medium was analyzed for radioactivity by HPLC
with an on-line flow scintillation counter. Radiochromatograms from a
single culture are shown, but similar results were obtained from two
additional cultures in each case: A, 75 min
control; B, 75 min with 3 µM DCU;
C, 4.5 h control; D,
4.5 h with 3 µM DCU.
, indicating that DCU did not affect the production or
release of AA metabolites from the cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-oxidation process, and the
-oxidation products can be further
metabolized to elongation products.
-oxidation products in the medium or
elongation products in the cells (21). The most likely reason that
these novel pathways of 14,15-EET metabolism were detected in the
present study is that DCU is a more effective and longer lasting
inhibitor of sEH than 4-PCO. This permitted the incubations to be
carried out for up to 8 h in the present study, as compared with
1 h in the study with 4-PCO. Because 4-PCO produces transient
inhibition of epoxide hydrolase, and as a benzylic epoxide 4-PCO is
somewhat unstable, especially in the presence of glutathione (18, 35),
the compound proved to be of limited utility in elucidating the effects
of epoxide hydrolase inhibition on EET metabolism and incorporation. In
contrast, DCU, a substituted urea derivative, is a potent, selective
inhibitor of recombinant human and murine sEH (18). Also, DCU is a
slowly reversible sEH inhibitor that has a duration of action long
enough to be useful in vivo. Therefore, DCU was suitable to
perform a detailed analysis of the effects of sEH inhibition on
endothelial EET metabolism.
-oxidation (29). The initial metabolites that
accumulated in the medium, 10,11-epoxy-16:2 from 14,15-EET and
7,8-epoxy-16:2 from 11,12-EET, are formed after two
-oxidation
cycles (36, 37). These compounds contain a
4,5 double
bond that must be removed through the action of 2,4-dienoyl CoA
reductase before
-oxidation can continue (37). This is presumed to
be a slow step that allows some of the 16-carbon CoA intermediates to
accumulate, be hydrolyzed by a thiolase, and be released from the cells
as the fatty acid (29, 38, 39).
-Oxidation eventually continued as
indicated by the subsequent decline of 10,11-epoxy-16:2 and
accumulation of 8,9-epoxy-14:1, a process that also occurs during the
-oxidation of arachidonic acid (38, 40). Although the
-oxidation
should proceed further (37), we could not detect any additional
chain-shortened metabolites. This may be because the incubations were
not carried out for a long enough period or these products are degraded
too rapidly to accumulate.
-oxidation is an alternative pathway for EET
metabolism that occurs when conversion to DHET is limited. Peroxisomal
-oxidation is the main metabolic pathway in endothelial cells for
other types of fatty acid biomediators, including 15- and
12-hydroxyeicosatetraenoic acid (HETE) and 13-hydroxyoctadecadienoic acid (41-43). However, the present results indicate that this is not
the primary pathway for EET metabolism in the coronary endothelium, since appreciable amounts of EET are channeled into the
-oxidation pathway only when DHET formation is inhibited. Channeling of 14,15-EET into the
-oxidation pathway also occurred with
N-cyclohexyl-N'-dodecylurea, a urea derivative
with different substituents than DCU that is approximately ten times
more potent against recombinant human and mouse sEH (data not shown).
Although EET
-oxidation might function as a degradative pathway, it
is distinctly possible that one or more of the epoxy-fatty acid
products could have metabolic or functional effects. In support of this
possibility, the
-oxidation product of 11,12-DHET was found to
produce vasodilation of porcine coronary arteries (25).
-oxidation, more
14,15-EET is channeled into the elongation pathway when sEH is
inhibited, and conversion to 14,15-DHET is decreased. A small amount of
14,15-epoxy-20:2 also accumulated when the cultures were treated with
DCU. This product probably was formed by elongation of
10,11-epoxy-18:2, the 18-carbon intermediate produced by
-oxidation.
Although not observed by HPLC, we detected the formation of very small
amounts of 12,13-epoxy-18:2 and deuterated 12,13-epoxy-18:2 by
LC/MS.3 Like the mass
spectrum of deuterated 14,15-epoxy-20:2 (Fig. 7D), the mass
spectrum of the deuterated 10,11-epoxy-18:2 contained a (M
H)
ion with 8 deuterium atoms, whereas the (M
H)
ion of deuterated 8,9-epoxy-16:2 contains only 7 deuterium atoms (Fig. 3D). Therefore, analogous to the
formation of eicosatrienoic acid from arachidonic acid (44),
14,15-epoxy-20:2 appears to be formed primarily by conversion of
14,15-EET to an 18-carbon intermediate that is subsequently elongated.
The 14,15-DHET elongation product, 16,17-dihydroxy-22:3, also was
detected in small amounts in the control cells, demonstrating that the
coronary endothelium is capable of elongating DHETs when they
accumulate. No information is presently available as to whether any of
these products have biological activity.
-oxidation and elongation products of 14,15-EET, treatment with DCU resulted in a marked and sustained accumulation of
14,15-EET and its metabolites in endothelial phospholipids. Such
findings likely help to explain recent reports that targeted disruption
of the sEH gene lowers systolic blood pressure in male mice (17), and
that a substantial decrease in blood pressure occurs when spontaneously
hypertensive rats are treated with DCU (19). For example, incorporation
of EETs may alter the membrane properties of the endothelium or,
because the increase occurs in PC and PI, affect signaling pathways
within the endothelium. Furthermore, the augmented release of EETs
following stimulation with calcium ionophore in DCU-treated cells may
prolong smooth muscle hyperpolarization and thereby enhance
endothelium-dependent vasorelaxation (9). These findings may
provide novel insight into the cellular mechanisms by which
pharmacological or molecular inhibition of sEH can effectively treat
hypertension (17, 19).
-oxidation and chain elongation pathways present in endothelial cells. The observation that
diol metabolites and
-oxidation products of the epoxides also can
undergo chain elongation suggests that there is considerable interaction among the three metabolic pathways. The emergence of
-oxidation and chain elongation when sEH is inhibited suggests that
these processes function primarily as alternative pathways of EET
metabolism. Whereas the
-oxidation products are largely excreted
from the cells, the elongation products are predominantly incorporated
into endothelial phospholipids. The modulation of EET metabolism,
incorporation, and release by sEH inhibition in the endothelium could
help to explain the reductions in blood pressure produced by DCU in
spontaneously hypertensive rats and by sEH-knockout in male mice (17,
19).
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FOOTNOTES |
---|
* This study was supported by National Institutes of Health Program Project Grants HL49264 and HL62984 (to A. A. S. and N. L. W.), by an American Heart Association Heartland Affiliate Beginning Grant-in-aid 0060413Z (to X. F.), by an American Heart Association Clinician-Scientist Award 96004540 (to N. L. W.), by NIEHS Grant R01 ES02710, the NIEHS Superfund Basic Research Program P42 ES04699, and NIEHS Center P30 ES05707 (to B. D. H.) from the National Institutes of Health, and by National Institutes of Health Training Grant HL07013 (to D. A. T.).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.
To whom correspondence should be addressed: Dept. of
Biochemistry, 4-403 BSB, University of Iowa, Iowa City, IA 52242. Tel.: 319-335-7913; Fax: 319-335-9570; E-mail:
arthur-spector@uiowa.edu.
Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc. M011761200
2 The 3H is present at carbon 5,6,8,9,11,12,14, and 15. The 11,12- and 14,15- refer to the location of the epoxide group.
3 T. L. Kaduce, L. M. Teesch, X. Fang, and A. A. Spector, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: EETs, epoxyeicosatrienoic acid(s); DHETs, dihydroxyeicosatrienoic acid(s); sEH, soluble epoxide hydrolase; 4-PCO, 4-phenylchalcone oxide; PCEC, porcine coronary artery endothelial cells; DCU, N,N'-dicyclohexylurea; AA, arachidonic acid; HPLC, high performance liquid chromatography; LC/MS, liquid chromatography combined with mass spectrometry; TLC, thin layer chromatography; CID, collision-induced decompositions; [2H8]14, 15-EET, [5,6,8,9,11,12,14,15-2H8]14,15-EET; tDPPO, trans-1,3-diphenylpropene oxide; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol.
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