© 2000 American Heart Association, Inc.
Molecular Medicine |
Soluble Epoxide Hydrolase Regulates Hydrolysis of Vasoactive Epoxyeicosatrienoic Acids
From the Department of Biopharmaceutical Sciences (Z.Y., F.X., L.M.H., D.L.K.), School of Pharmacy, and Department of Physiological Nursing (M.M.E.), School of Nursing, University of California, San Francisco, Calif; Department of Entomology (C.M., A.J.D., J.W.N., B.D.H.), University of California, Davis, Calif; and the Division of Intramural Research (C.P., L.G., D.C.Z.), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC.
Correspondence to Deanna L. Kroetz, PhD, Department of Biopharmaceutical Sciences, 513 Parnassus Box 0446, San Francisco, CA 94143-0446. E-mail deanna{at}itsa.ucsf.edu
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Abstract |
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Abstract—The cytochrome P450-derived epoxyeicosatrienoic acids (EETs) have potent effects on renal vascular reactivity and tubular sodium and water transport; however, the role of these eicosanoids in the pathogenesis of hypertension is controversial. The current study examined the hydrolysis of the EETs to the corresponding dihydroxyeicosatrienoic acids (DHETs) as a mechanism for regulation of EET activity and blood pressure. EET hydrolysis was increased 5- to 54-fold in renal cortical S9 fractions from the spontaneously hypertensive rat (SHR) relative to the normotensive Wistar-Kyoto (WKY) rat. This increase was most significant for the 14,15-EET regioisomer, and there was a clear preference for hydrolysis of 14,15-EET over the 8,9- and 11,12-EETs. Increased EET hydrolysis was consistent with increased expression of soluble epoxide hydrolase (sEH) in the SHR renal microsomes and cytosol relative to the WKY samples. The urinary excretion of 14,15-DHET was 2.6-fold higher in the SHR than in the WKY rat, confirming increased EET hydrolysis in the SHR in vivo. Blood pressure was decreased 22±4 mm Hg (P<0.01) 6 hours after treatment of SHRs with the selective sEH inhibitor N,N'-dicyclohexylurea; this treatment had no effect on blood pressure in the WKY rat. These studies identify sEH as a novel therapeutic target for control of blood pressure. The identification of a potent and selective inhibitor of EET hydrolysis will be invaluable in separating the vascular effects of the EET and DHET eicosanoids.
Key Words: epoxyeicosatrienoic acids • dihydroxyeicosatrienoic acids • cytochrome P450 • soluble epoxide hydrolase • hypertension
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Introduction |
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Eicosanoids produced by renal cytochromes P450 (CYPs) have potent effects on vascular tone and tubular ion and water transport and have been implicated in the control of blood pressure.1 The major products of CYP-catalyzed arachidonic acid metabolism are regio- and stereoisomeric epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE). The EETs are further metabolized by soluble (sEH) and microsomal (mEH) epoxide hydrolases to form the corresponding dihydroxyeicosatrienoic acids (DHETs). CYP enzymes can also catalyze the formation of 19-HETE and other subterminal HETEs in smaller quantities.1
In the renal microcirculation 20-HETE produces potent vasoconstriction by inhibiting large-conductance, calcium-activated potassium channels leading to vascular smooth muscle depolarization.2 In contrast, both vasodilator and vasoconstrictor properties have been attributed to the EETs, depending on the vascular bed and EET regioisomer.3 4 5 Vasodilatory properties of the EETs have been associated with increased open-state probability of calcium-activated potassium channels and hyperpolarization of the vascular smooth muscle.6 Indeed, a CYP2C epoxygenase catalyzes the formation of 11,12-EET that acts as an endothelial-derived hyperpolarizing factor in coronary arteries.7 Recently, the EETs were shown to have anti-inflammatory effects in endothelial cells, suggesting that these eicosanoids also play an important role in vascular inflammation.8 Hydrolysis of the EETs to the corresponding DHETs generally is regarded as one mechanism whereby the biological effects of the EETs are attenuated or eliminated.9 However, the DHETs themselves have inherent effects on vascular tone,5 10 and their role in the regulation of blood pressure cannot be dismissed.
Alterations in renal vascular resistance are evident in the spontaneously hypertensive rat (SHR) and are proposed to play an important role in resetting the pressure-natriuresis relationship and the development of hypertension.11 The CYP eicosanoids are recognized as potent mediators of vascular tone and have been implicated in these vascular changes. Recent studies indicate that renal 20-HETE and EET formation and urinary excretion are increased in the SHR relative to the normotensive Wistar-Kyoto (WKY) rat.12 13 14 Whether the increase is causative or compensatory is unknown. The opposing effects of the EET and HETE eicosanoids on vascular tone make it difficult to predict their effect in vivo and emphasize the importance of understanding the factors that regulate their formation and degradation. Modulation of CYP enzyme activities in these rats is associated with changes in blood pressure15 16 ; however, the utility of this approach has been limited by the ability of multiple CYP isoforms to catalyze these reactions and by the general lack of selectivity of available CYP inhibitors and inducers. A novel alternative for modulating the levels of the vasoactive EET and DHET eicosanoids is regulation of EET hydrolysis to the corresponding vicinal diols. The regulation of EET hydrolysis in the SHR kidney and the effect of modulation of EH activity on regulation of blood pressure are explored in the present study. Both in vitro and in vivo evidence demonstrate that increased blood pressure is associated with increased sEH expression and EET hydrolysis in the SHR. Furthermore, inhibition of EET hydrolysis with a tight binding sEH inhibitor can reverse the hypertensive phenotype in the SHR. These data also describe novel inhibitors for characterizing the distinct biological properties of the EET and DHET eicosanoids.
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Materials and Methods |
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Animals
Male SHR and WKY rats 3 to 13 weeks of age were purchased from Charles River Laboratories (Wilmington, Mass). The University of California San Francisco Committee on Animal Research approved all animal use. Rats were anesthetized with methoxyflurane, and kidneys were dissected and removed as described previously.13 14 For the sEH inhibition studies, groups of 8-week-old male SHRs and WKY rats were treated daily for 1 to 4 days with a 3 mg/kg IP dose of N,N'-dicyclohexylurea (DCU) in a 1.5:1 mixture of corn oil and DMSO. Systolic blood pressure was measured at room temperature by a photoelectric tail-cuff system (model 179, IITC, Inc) for up to 4 days after the dose of inhibitor. Blood pressures are reported as the average of 3 separate readings during a 30-minute period. Urine was collected for 24 hours immediately after a dose of DCU or vehicle for quantification of DHET and EET excretion. Similar inhibition studies were performed with equimolar doses of N-cyclohexyl-N'-dodecylurea, N-cyclohexyl-N'-ethylurea, and dodecylamine.
Renal Microsomal Arachidonic Acid Metabolism
and EET Hydrolysis
In vitro determination of arachidonic acid
epoxygenase activity was performed as described
previously.13 16
Hydrolysis of [1-14C]EETs was measured in
WKY and SHR renal fractions as
described.9 Detailed
descriptions of these procedures are contained in the online data
supplement.
Western Immunoblotting
Renal and hepatic microsomes and cytosol (4 to 10
µg) were separated on an 8% SDS-polyacrylamide gel and transferred
to nitrocellulose in Towbin’s buffer. Primary antibodies used in these
studies were a rabbit anti-mouse sEH
antiserum17 and a rabbit
anti-rat mEH antiserum kindly provided by Drs Franz Oesch and Michael
Arand (University of Mainz, Mainz, Germany). A second rabbit anti-mouse
sEH antiserum was recently prepared in our laboratory against
recombinant mouse sEH expressed in insect cell culture. Western blots
were incubated with a 1:1000- (mEH) or 1:2000-fold (sEH) dilution of
the primary antibody followed by a 1:2000-fold dilution of horseradish
peroxidase-conjugated goat anti-rabbit IgG. Immunoreactive proteins
were visualized using an ECL detection kit (Amersham Life
Science).
RNase Protection Assays
RNase protection assays for quantification of sEH
mRNA levels were performed as described previously using a riboprobe
corresponding to 383 to 775 bp of the
cDNA.14
DHET Urinary Excretion
Methods used to quantify endogenous EETs and DHETs
present in rat urine were described
previously18 and in detail
in the online data supplement.
Other Enzyme Assays
Activities of mEH and sEH were determined using
cis-stilbene oxide (cSO) and
trans-1,3-diphenylpropene oxide
(tDPPO), respectively, exactly as
described.19 20
Inhibition of recombinant sEH by DCU was measured using
tDPPO.21
Statistics
Statistical significance of differences between mean
values was evaluated by a one-way ANOVA or a Student’s
t test. Significance was set at
a P value of
<0.05.
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
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Results |
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Renal EET Hydrolysis
A dramatic increase in DHET formation in incubations of arachidonic acid with SHR renal cortical microsomes relative to the WKY samples (data not shown) led us to hypothesize that EET hydrolysis may be altered in this experimental model of hypertension and that EH activity may be an important determinant of blood pressure regulation. Both mEH and sEH are expressed in most tissues and species, although constitutive levels of sEH in both hepatic and extrahepatic tissues are low in the rat.22 Higher rates of EET hydrolysis in cytosol versus microsomes and preference of sEH for endogenous arachidonate epoxide pools are consistent with the majority of EET hydrolysis being catalyzed by sEH.9 Direct hydrolysis of the regioisomeric EETs was measured in S9 fractions (containing both the soluble and microsomal forms of EH) from WKY and SHR renal cortex. The hydrolysis of 8,9-, 11,12-, and 14,15-EET in S9 fractions from both WKY and SHR kidneys was measurable (Figure 1
), with a significant increase in hydrolysis in the
SHR relative to the WKY. For example, 8,9- and 11,12-EET hydrolysis
rates were 5- to 15-fold higher in the SHR than the WKY, and 14,15-EET
hydrolysis was as much as 54-fold higher in the SHR. These data also
showed a distinct preference of sEH for the 14,15-EET regioisomer. In
the SHR kidney hydrolysis of 14,15-EET was 10-fold higher than that of
8,9- and 11,12-EET. Similar studies were also performed using a mixture
of unlabeled EETs and detection by gas chromatography–mass
spectrometry (GC-MS). The hydrolysis ratio from these studies was
20:7:1 for 14,15-EET:11,12-EET:8,9-EET (data not shown). This is
consistent with the preference observed with the individual
regioisomers
(Figure 1). One explanation for an intermediate level of
hydrolysis of the 11,12-EET when measured in the mixture and similar
hydrolysis of the 11,12- and 8,9-EETs when measured separately is that
the relative concentrations of the isomers change during the course of
the incubation with the EET mixture, thereby affecting the competition
of these 3 substrates for the enzyme.
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) and SHR (•) renal S9 fractions
with [1-14C]EETs (50 µmol/L).
Metabolites were extracted from the incubation mixture and quantified
by HPLC with radiometric detection. Hydrolysis rates for each age and
strain from 2 separate animals are shown, and the line is drawn through
the average
value.
Epoxide Hydrolase Expression
To investigate the possibility that altered EH
expression is responsible for the differences in EET hydrolysis in the
SHR and WKY rat renal microsomes and S9 fractions, we measured EH
protein levels in these samples. Control studies demonstrated that the
rabbit anti-mouse sEH antisera cross-reacted with rat sEH but not mEH,
and that the rabbit anti-rat mEH antisera did not cross-react with rat
sEH (data not shown). mEH was abundantly expressed in the renal
microsomes at relatively constant levels throughout development, and
there was no evidence of altered expression of mEH in the SHR kidney
(Figure 2A). sEH was detected easily in SHR cortical
microsomes but not in the corresponding WKY samples
(Figure 2A). Quantitation of the immunoreactive protein bands
indicated that levels of sEH protein in the SHR microsomes were 6- to
90-fold higher than the corresponding levels in the WKY microsomes.
This differential pattern of sEH expression in the WKY and SHR
microsomes was confirmed using a second antibody against the enzyme
(online Figure 1
; available in the online data supplement at
http://www.circresaha.org).
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The high levels of expression of sEH in the SHR microsomes
were limited to the renal cortex
(Figure 2B). sEH was hardly detectable in SHR outer medulla
and liver microsomes by Western blot. In contrast, relatively high
levels of sEH were detected in SHR cortex, outer medulla, and liver
cytosol
(Figure 2B). In the WKY rats, the level of sEH protein was
uniformly low in both microsomes and cytosol from the kidney and liver.
sEH protein in the normotensive Sprague-Dawley rat kidney was also
hardly detectable. Increased sEH expression in SHR versus WKY rats
provides an explanation for the increased EET hydrolysis in the SHR
kidney and the absence or very low levels of 14,15-EET hydrolysis, the
preferred sEH substrate,9 in
the WKY kidney.
The mechanistic basis for the dramatic difference in sEH
protein levels between WKY and SHR kidneys was investigated with the
use of RNase protection assays. sEH RNA was detected in both WKY and
SHR kidneys with a 5- to 12-fold increase in expression in the SHR
samples
(Figure 3). This is consistent with the corresponding
increase in sEH protein levels in the SHR kidney
(Figure 2). In addition to the major protected fragment of
predicted size, a slightly smaller fragment was detected in all the SHR
samples. Additional smaller protected fragments were also found in the
SHR but not in the WKY kidneys. This raises the possibility that a
second mRNA with a structure very similar to the cloned sEH is also
expressed in the SHR kidney at much higher levels than in the WKY
kidney.
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Epoxide Hydrolase Activity
Increased sEH activity in the SHR kidney was confirmed
independently using the sEH substrate tDPPO. Consistent with the
Western blots, there was a 26-fold increase in tDPPO hydrolysis in the
SHR cortical cytosol relative to that in the WKY rat cortical cytosol
(online Table 1; available in the online data supplement at
http://www.circresaha.org). The corresponding difference in the
microsomal fraction was 32-fold. Hydrolysis of tDPPO was also
significantly higher in SHR versus WKY rat liver microsomes and
cytosol. In contrast, mEH activity, as measured by cSO hydrolysis, was
increased only slightly in SHR kidney cortex and liver microsomes and
liver cytosol relative to the WKY rat (online Table 1). The sEH
activity in microsomes as a fraction of the total cellular sEH activity
was 2% to 9%, consistent with a low level of cytosolic contamination
of microsomes.
DHET Urinary Excretion
Urinary excretion of DHETs was measured to evaluate
whether increased sEH expression and EET hydrolysis in the SHR was also
apparent in vivo. Urine was collected in untreated 4- and 8-week-old
SHR and WKY rats, and their DHET excretion rates are shown in
Figure 4. The excretion rates were similar for the 4- and
8-week-old animals, and the reported numbers are averages from all
samples of a given strain. The excretion of 14,15-DHET was 2.6-fold
higher in the SHR relative to the WKY rat, consistent with the
increased EET hydrolysis and sEH expression in SHR kidney. In contrast,
the 8,9- and 11,12-DHET urinary excretion in the SHR and WKY rats were
similar.
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Inhibition of sEH and Effect on Blood
Pressure
A tight binding sEH specific inhibitor,
DCU,21 was used to reduce
sEH activity in vivo and to determine the effect of decreased EET
hydrolysis on blood pressure. DCU was administered to 8-week-old SHRs
daily for 4 days, and urinary DHET excretion was measured during the 24
hours immediately after the third dose. The dose of DCU was based on in
vitro estimates of inhibitory potency and previous studies in the
mouse.21 A significant 65%
decrease in 14,15-DHET urinary excretion occurred in the DCU-treated
rats, and a corresponding 30% increase in 14,15-EET urinary excretion
relative to vehicle-treated controls
(Figure 5), consistent with DCU-mediated inhibition of sEH in
vivo. The excretion of total epoxygenase-derived products (EETs and
DHETs) was decreased from 2020 pg/mg creatinine in the vehicle-treated
animals to 1237 pg/mg creatinine in the DCU-treated rats
(P<0.05). This inhibition of
14,15-DHET excretion was accompanied by a significant decrease in blood
pressure measured in conscious animals 3 to 5 hours after the fourth
dose (data not shown). Systolic blood pressure decreased from 128±5
mm Hg in the vehicle-treated rats to 102±5 mm Hg
(P<0.01) in the DCU-treated
animals.
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A study of the time course of the effect of a single dose of
DCU (3 mg/kg) demonstrated that the antihypertensive effect in the SHR
was acute
(Figure 6A). Blood pressure was decreased 22±4 mm Hg 6
hours after DCU treatment
(P<0.01) and returned to
baseline levels by 24 hours after the dose. DCU had no effect on blood
pressure in the WKY rats
(Figure 6B). This is consistent with the very low levels of
sEH protein in the WKY kidney. Several additional structurally related
inhibitors were also studied in the SHR.
N-Cyclohexyl-N'-dodecylurea
is a sEH inhibitor with similar potency to DCU
(IC50 with mouse sEH=0.05±0.01 compared with
0.09±0.01 µmol/L for DCU; C. Morisseau and B. Hammock, unpublished
data, 2000). A single dose of
N-cyclohexyl-N'-dodecylurea
significantly decreased systolic blood pressure 12±2 mm Hg 6 hours
after the dose, and similar to DCU, blood pressure returned to normal
by 24 hours after the dose (online Figure 2A). The
N-cyclohexyl-N'-ethylurea
analog is a weak sEH inhibitor (IC50 with mouse
sEH=51.7±0.7 µmol/L; C. Morisseau and B. Hammock, unpublished data,
2000) and had no effect on blood pressure in the SHR (online Figure 2B). Likewise, the selective mEH inhibitor dodecylamine also had no
effect on blood pressure (online Figure 2C). Collectively, these data
suggest that the effect of DCU and
N-cyclohexyl-N'-dodecylurea
on blood pressure is related to their ability to inhibit sEH and EET
hydrolysis in vivo.
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The decreased 14,15-DHET excretion after treatment with DCU
was consistent with a decrease in renal sEH activity. Inhibition of EET
hydrolysis by DCU was confirmed in incubations of renal S9 fractions
with the regioisomeric EETs
(Figure 7). A dose-dependent inhibition of EET hydrolysis by
DCU was apparent for all 3 regioisomers. DCU had the most significant
effect on the hydrolysis of 8,9-EET, inhibiting this reaction with an
IC50 of 0.086±0.014 µmol/L. The corresponding
IC50 values for inhibition of 11,12- and
14,15-EET hydrolysis were 0.54±0.08 and 0.45±0.16 µmol/L,
respectively. Because of the large difference in hydrolysis of the
regioisomeric EETs by sEH, it was not possible to use the same amount
of enzyme to measure an IC50 for DCU inhibition
for these substrates. At a concentration of enzyme half of that used
for 8,9- and 11,12-EET, the IC50 for 14,15-EET
is similar to that for 11,12-EET and 5-fold higher than that for
8,9-EET. Thus, at a given concentration of DCU, sEH hydrolysis of
14,15-EET will be inhibited to a lesser degree than that of the other
two regioisomers, consistent with the higher preference of the enzyme
for this isomer. At concentrations up to 25 µmol/L, DCU had no effect
on CYP epoxygenase or 
-hydroxylase activity (data not shown), and
previous studies from our laboratory have shown that DCU does not
inhibit mEH.21 The potent
inhibition of sEH by DCU was confirmed with purified recombinant
rat sEH. DCU inhibited sEH-catalyzed tDPPO hydrolysis with a
Ki of 34
nmol/L (online Figure 3). This is similar to the
Ki
values for DCU with human (30 nmol/L) and murine (26 nmol/L)
sEH.21
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Discussion |
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EET and DHET eicosanoids are recognized as potent mediators of vascular tone and have been implicated in the pathogenesis of hypertension.1 5 11 23 The data presented herein provide substantial evidence of a primary defect in sEH expression in the SHR kidney, and of an antihypertensive effect of sEH inhibition. Accordingly, inhibition of EET hydrolysis increased EET levels and decreased DHET levels in vivo. Whether the antihypertensive effect is caused by a protective effect of EETs and/or a deleterious effect of the DHETs cannot be discerned. Indeed, the ratio of these eicosanoids may be the major determinant of their effect on vascular tone and blood pressure. The fact that DHETs are increased in SHR and that blood pressure is reduced coincident with decreased DHET formation suggests that the primary defect in renal sEH expression identified in the SHR kidney is causal and that high levels of DHETs are detrimental. These data provide evidence that the DHETs may be important regulators of blood pressure and identify sEH as a novel therapeutic target for the treatment of hypertension. Whether increased levels of DHETs are sufficient to increase blood pressure or whether they modulate the response to other factors involved in vascular reactivity and blood pressure regulation remains to be determined.
Progress in understanding the role of the EET and DHET eicosanoids in the regulation of vascular tone is limited by the lack of selective and stable inhibitors and mimics. In many cases, in vitro and in situ studies do not separate the effects of the EETs from those of the corresponding DHETs, making it difficult to assign specific properties to these eicosanoids. EETs are generally considered antihypertensive because of their vasodilatory properties; however, vasoconstrictor effects have also been attributed to the EETs, depending on the vascular bed and species that are studied. After systemic administration to rats, both 5,6- and 8,9-EET caused a dose-dependent vasoconstriction and decrease in glomerular filtration rate which was cyclooxygenase-dependent.3 10 In contrast, studies using a rat juxtamedullary nephron preparation characterized 11(R),12(S)-EET as a potent vasodilator.5 Similar to the EETs, the effect of the DHETs on vascular tone is highly dependent on the vascular bed, species, and experimental system that is studied.5 24 25 26 For example, 11,12-DHET produces a vasodilatory effect on porcine coronary artery rings and a vasoconstrictive effect on rat interlobular and afferent arterioles, whereas 5,6-DHET attenuates the vasoconstrictive effect of 5,6-EET.5 26 The novel sEH inhibitors described herein will be valuable reagents for further dissecting the vascular effects of the EETs and DHETs both in vivo and in vitro.
The hydrolysis of each of the major regioisomeric EETs was increased dramatically in the SHR relative to the WKY rat because of increased sEH expression in the SHR kidney. This increased hydrolysis results in altered levels of the vasoactive EET and DHET eicosanoids that are implicated in the control of blood pressure. Although these data suggest that increased EET hydrolysis is an important regulator of blood pressure, it is clearly not the sole mechanism for the SHR phenotype. In the SHR, blood pressure rises steadily between 4 and 13 weeks of age, in contrast to the relatively constant expression of sEH during this period. EET hydrolysis is also highest in prehypertensive SHR, suggesting that these changes in sEH expression might be most important in the early development of hypertension. Several lines of evidence support a major role for sEH in the hydrolysis of EETs in the SHR model of hypertension. First, studies with human and rabbit liver and rabbit lung indicate a 10-fold higher EET hydration rate in cytosol than in microsomes, and EET hydrolysis is catalyzed by purified sEH.9 Second, increased hydrolysis of EETs in SHR renal subcellular fractions compared with the WKY samples was consistent with the dramatic increase in sEH expression in the SHR kidney. Finally, the hydrolysis of 14,15-EET was absent or very low in the WKY microsomes and S9 fractions that have only minimal expression of sEH protein. This is consistent with the strong preference of purified sEH for the 14,15-EET regioisomer9 and the preference of the EH in renal S9 fractions for 14,15-EET. Although the hydrolysis of all the regioisomeric EETs was increased in SHR renal fractions, only 14,15-DHET urinary excretion was increased in vivo. This in vivo-in vitro discrepancy most likely reflects sequential metabolism of the DHETs or limiting efflux mechanisms that may operate only in the intact cell or organ.
These data may provide a link between the pathophysiological regulation of blood pressure in rats and humans.27 During pregnancy 8,9- and 11,12-DHET excretion is increased significantly, and excretion of 11,12- and 14,15-DHET is increased even further in patients with pregnancy-induced hypertension. This increase is most dramatic for 14,15-DHET, the major product of sEH-catalyzed EET hydrolysis.9 The present results in the SHR support the hypothesis that altered sEH expression may be responsible for the hypertension observed in pregnant women. In this regard, the substantial interindividual variation in sEH expression in humans is well established.28 29 Furthermore, we have recently discovered several single nucleotide polymorphisms within the human sEH gene that result in amino acid substitutions (D.C. Zeldin, unpublished data, 2000). The functional characteristics of these polymorphisms, and their frequency distribution in normal and hypertensive populations are currently being investigated.
The in vitro and in vivo evidence for increased EET
formation and hydrolysis in the SHR kidney suggested that accelerated
elimination of EETs may contribute to the hypertensive phenotype in the
SHR, and it raised the question of whether sEH may be a novel
therapeutic target for the treatment of hypertension. Initial studies
with the selective tight binding sEH inhibitor
DCU21 and several structural
analogs support this approach. Inhibition of sEH by DCU in vivo was
evident from urinary EET and DHET excretion measurements and was
associated with a 15% reduction in systolic blood pressure. This
antihypertensive effect was acute, with a maximal decline in blood
pressure 6 hours after a dose and complete recovery to baseline levels
by 24 hours after the dose. The antihypertensive effects of DCU are
attributed to its inhibition of EET hydrolysis in vivo, because it has
no effect in WKY rats with very low expression of sEH; another sEH
inhibitor with similar potency also decreases blood pressure.
Similarly, a weak sEH inhibitor and a selective mEH inhibitor have no
effect on blood pressure. Recently, inhibition of arachidonic acid
-hydroxylase (CYP4A) activity with a mechanism-based CYP inhibitor
has effectively lowered blood pressure in the
SHR.16 Combined inhibition
of CYP4A, resulting in decreased levels of the vasoconstrictive 20-HETE
eicosanoid, and sEH, leading to increased levels of the vasodilatory
EETs, would be predicted to cause either an additive or synergistic
reduction in blood pressure. Inhibition of related CYP isoforms is a
potential limitation of -hydroxylase inhibition, but is of little
concern with sEH inhibition in light of the recent development of
potent and specific urea, carbamate, and amide inhibitors of
sEH.21 The possibility of
similar pharmacological changes in sEH activity in human hypertensive
populations is compelling. Identification of individuals with elevated
sEH activity and lower EET levels may prove useful in designing the
most effective antihypertensive therapy.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In summary, we have described an increased expression of sEH in the SHR kidney that leads to increased EET hydrolysis. Increased hydrolysis of the antihypertensive EETs is proposed as an important determinant of the hypertensive phenotype in this model. Consistent with this hypothesis is the reduction of blood pressure associated with significant inhibition of EET hydrolysis. These data suggest that the modulation of sEH may have therapeutic uses for the treatment of hypertension. Selective sEH inhibitors also may have more general clinical applications in altering processes where in vivo levels of EETs and DHETs are involved, and will be invaluable in characterizing the vasoactive effects of the EETs and DHETs.
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Acknowledgments |
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This work was supported in part by National Institutes of Health grants R29 HL53994 (D.L.K.), R01 ES02710 (B.D.H.), and F32 ES05808 (A.J.D.), the National Institute of Environmental Health Sciences (NIEHS) Division of Intramural Research (D.C.Z.), the NIEHS Superfund Basic Research Program at the University of California Davis (P42 ES04699), and an Amgen-American Liver Foundation Postdoctoral Fellowship to A.J.D. We acknowledge Elizabeth Murphy, Tom Eling, and M. Almira Correia for their careful review of this manuscript.
Received April 18, 2000; revision received September 26, 2000; accepted September 27, 2000.
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References |
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L. A. Cowart, S. Wei, M.-H. Hsu, E. F. Johnson, M. U. Krishna, J. R. Falck, and J. H. Capdevila The CYP4A Isoforms Hydroxylate Epoxyeicosatrienoic Acids to Form High Affinity Peroxisome Proliferator-activated Receptor Ligands J. Biol. Chem., September 13, 2002; 277(38): 35105 - 35112. [Abstract] [Full Text] [PDF]
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 J. W. Newman, T. Watanabe, and B. D. Hammock The simultaneous quantification of cytochrome P450 dependent linoleate and arachidonate metabolites in urine by HPLC-MS/MS J. Lipid Res., September 1, 2002; 43(9): 1563 - 1578. [Abstract] [Full Text] [PDF]
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F. Xu, W. O. Straub, W. Pak, P. Su, K. G. Maier, M. Yu, R. J. Roman, P. R. Ortiz De Montellano, and D. L. Kroetz Antihypertensive effect of mechanism-based inhibition of renal arachidonic acid omega -hydroxylase activity Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2002; 283(3): R710 - R720. [Abstract] [Full Text] [PDF]
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I. Fleming To Move or Not To Move?: Cytochrome P450 Products and Cell Migration Circ. Res., May 17, 2002; 90(9): 936 - 938. [Full Text] [PDF]
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B. B. Davis, D. A. Thompson, L. L. Howard, C. Morisseau, B. D. Hammock, and R. H. Weiss Inhibitors of soluble epoxide hydrolase attenuate vascular smooth muscle cell proliferation PNAS, February 6, 2002; (2002) 261710799. [Abstract] [Full Text] [PDF]
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J. D. Imig, X. Zhao, J. H. Capdevila, C. Morisseau, and B. D. Hammock Soluble Epoxide Hydrolase Inhibition Lowers Arterial Blood Pressure in Angiotensin II Hypertension Hypertension, February 1, 2002; 39(2): 690 - 694. [Abstract] [Full Text] [PDF]
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 R. J. Roman P-450 Metabolites of Arachidonic Acid in the Control of Cardiovascular Function Physiol Rev, January 1, 2002; 82(1): 131 - 185. [Abstract] [Full Text] [PDF]
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I. Fleming Cytochrome P450 and Vascular Homeostasis Circ. Res., October 26, 2001; 89(9): 753 - 762. [Abstract] [Full Text] [PDF]
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B. Yang, L. Graham, S. Dikalov, R. P. Mason, J. R. Falck, J. K. Liao, and D. C. Zeldin Overexpression of Cytochrome P450 CYP2J2 Protects against Hypoxia-Reoxygenation Injury in Cultured Bovine Aortic Endothelial Cells Mol. Pharmacol., August 1, 2001; 60(2): 310 - 320. [Abstract] [Full Text] [PDF]
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 I. Fleming and R. Busse Vascular cytochrome P450 in the regulation of renal function and vascular tone: EDHF, superoxide anions and blood pressure Nephrol. Dial. Transplant., July 1, 2001; 16(7): 1309 - 1311. [Full Text] [PDF]
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X. Fang, T. L. Kaduce, N. L. Weintraub, S. Harmon, L. M. Teesch, C. Morisseau, D. A. Thompson, B. D. Hammock, and A. A. Spector Pathways of Epoxyeicosatrienoic Acid Metabolism in Endothelial Cells. IMPLICATIONS FOR THE VASCULAR EFFECTS OF SOLUBLE EPOXIDE HYDROLASE INHIBITION J. Biol. Chem., April 27, 2001; 276(18): 14867 - 14874. [Abstract] [Full Text] [PDF]
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D. C. Zeldin Epoxygenase Pathways of Arachidonic Acid Metabolism J. Biol. Chem., September 21, 2001; 276(39): 36059 - 36062. [Full Text] [PDF]
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B. B. Davis, D. A. Thompson, L. L. Howard, C. Morisseau, B. D. Hammock, and R. H. Weiss Inhibitors of soluble epoxide hydrolase attenuate vascular smooth muscle cell proliferation PNAS, February 19, 2002; 99(4): 2222 - 2227. [Abstract] [Full Text] [PDF]
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