© 1997 American Heart Association, Inc.
Articles |
Potentiation of Endothelium-Dependent Relaxation by Epoxyeicosatrienoic Acids
From the Departments of Internal Medicine (N.L.W., M.V., P.C., A.A.S.) and Biochemistry (X.F., T.L.K., A.A.S.), University of Iowa, Iowa City.
Correspondence to Dr Neal L. Weintraub, Department of Internal Medicine, Cardiovascular Division, E-329GH, University of Iowa College of Medicine, Iowa City, IA 52242.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract Epoxyeicosatrienoic acids (EETs) are potent endothelium-derived vasodilators formed from cytochrome P-450 metabolism of arachidonic acid. EETs and their diol products (DHETs) are also avidly taken up by endothelial cells and incorporated into phospholipids that participate in signal transduction. To investigate the possible functional significance of EET and DHET incorporation into cell lipids, we examined the capacity of EETs and DHETs to relax porcine coronary arterial rings and determined responses to bradykinin (which potently activates endothelial phospholipases) before and after incubating the rings with these eicosanoids. 14,15-EET and 11,12-EET (5 µmol/L) produced 75±9% and 52±4% relaxation, respectively, of U46619-contracted rings, whereas 8,9-EET and 5,6-EET did not produce significant relaxation. The corresponding DHET regioisomers produced comparable relaxation responses. Preincubation with 14,15-EET, 11,12-EET, 14,15-DHET, and 11,12-DHET augmented the magnitude and duration of bradykinin-induced relaxation, whereas endothelium-independent relaxations to aprikalim and sodium nitroprusside were not potentiated. Pretreatment with 2 µmol/L triacsin C (an inhibitor of acyl coenzyme A synthases) inhibited [3H]14,15-EET incorporation into endothelial phospholipids and blocked 11,12-EET– and 14,15-DHET–induced potentiation of relaxation to bradykinin. Exposure of [3H]14,15-EET–labeled endothelial cells to the Ca2+ ionophore A23187 (2 µmol/L) resulted in a 4-fold increased release of EET and DHET into the medium. We conclude that incorporation of EETs and DHETs into cell lipids results in potentiation of bradykinin-induced relaxation in porcine coronary arteries, providing the first evidence that incorporated EETs and DHETs are capable of modulating vascular function.
Key Words: epoxyeicosatrienoic acid • porcine coronary artery • dihydroxyeicosatrienoic acid • arachidonic acid • acyl coenzyme A synthase
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Vascular endothelial cells produce several EDRFs that are capable of regulating vascular tone. These include NO, derived from the action of NO synthase on L-arginine; PGI2, a product of cyclooxygenase-mediated arachidonic acid metabolism; and EDHF, an unidentified factor or factors that produce vasorelaxation by activating K+ channels.1 2 3 Recently, EDHF in some blood vessels was proposed to be an EET metabolite of arachidonic acid.4 5 6 7 8 The four EET regioisomers, 5,6-, 8,9-, 11,12-, and 14,15-EET, are formed from arachidonic acid by the action of cytochrome P-450 epoxygenase enzymes.9 10 11 The production of EETs by blood vessels and vascular endothelial cells has recently been demonstrated.7 12 13 14 In several studies, EETs were observed to relax blood vessels by activating KCa channels.4 7 15 16 Moreover, inhibitors of cytochrome P-450 enzyme activity were reported to block EDHF-mediated relaxations in isolated blood vessels from several species and in the perfused rat heart.4 5 6 7 8 17
One property that distinguishes EETs from vasoactive prostaglandins and leukotrienes is their close structural similarity to the precursor arachidonic acid. Like arachidonic acid, EETs occur predominantly esterified to phospholipids in plasma,18 heart,19 liver,20 21 and kidney.22 Moreover, porcine aortic endothelial cells avidly take up EETs; saturation does not occur even at EET concentrations of up to 5 µmol/L.23 The uptake of EET is very rapid, reaching a maximum in just 15 to 30 minutes. Furthermore, a high percentage of the EET is incorporated into PC and PI, phospholipids that participate in endothelial signal transduction. These observations suggest that preformed EETs may be stored in endothelial cells and influence vascular function in several ways. For example, EETs present in cell membrane phospholipids may alter membrane function, ion transport, or lipid-dependent cell signaling pathways.11 5,6-EET was reported to enhance NO production by stimulating Ca2+ influx into vascular endothelial cells24 ; thus, the release of incorporated EETs during endothelial cell activation may also amplify EDRF production. Finally, preformed EETs released from endothelial cells after stimulation of phospholipid hydrolysis by agonists such as bradykinin, and perhaps by other stimuli such as shear stress, might be transferred to underlying smooth muscle cells, thereby promoting vasorelaxation through the activation of KCa channels.4 7 15 16
Besides becoming rapidly incorporated into cell lipids, EETs are rapidly converted by cellular epoxide hydrolases to their respective DHET regioisomers,23 25 which can themselves be taken up by vascular endothelial cells and incorporated into cell lipids.26 In several previous studies, DHETs were found to lack vasoactive properties.27 28 29 However, we recently observed that 11,12-DHET is vasoactive in the porcine coronary artery, producing a similar amount of relaxation as its parent EET.30 Thus, it is possible that the incorporation of DHETs into endothelial cell lipids may also influence vascular function.
The biological relevance of EET incorporation into cell lipids is suggested by the recent identification of EETs in lipid extracts from human platelets31 and cardiac tissue.19 However, whether such incorporated EETs or DHETs affect cellular function has not been determined. In the present study, we investigated the capacity of EETs and their diol products to relax porcine coronary arteries, and we examined responses to bradykinin (a receptor agonist that potently activates endothelial phospholipases) before and after incubating coronary arterial rings with EETs and DHETs.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
EETs and DHETs were purchased from Cayman Chemical Co. U46619, PGF2
, bradykinin, acetylcholine, sodium
nitroprusside, PDBu, L-NAME, indomethacin, and
L-glutamine were purchased from Sigma Chemical Co. ODYA and
triacsin C were purchased from Biomol, Inc. MEM nonessential amino
acids, MEM vitamin solution, HEPES, and trypsin were obtained from
GIBCO; FBS from HyClone Laboratories; fatty acid–free bovine serum
albumin from Miles Laboratories, Inc; and gentamicin from
Schering Corp. Radiolabeled arachidonic acid was
obtained from American Radiolabeled Chemicals Inc and Amersham Corp,
and phospholipid standards were obtained from Avanti Polar Lipids.
Radiolabeled 14,15-EET was obtained from Dupont–New England Nuclear
Corp, and Whatman LK5D silica gel TLC plates and silica gel G TLC
plates were purchased from Alltech Associates, Inc.
Measurement of Porcine Coronary Arterial
Ring Vasoactivity
Coronary arteries were dissected from pig hearts
immediately after removal at a local slaughterhouse, placed into
ice-cold KRB solution (composed of [mmol/L] NaCl 118.3, KCl
4.7, CaCl2 2.5, MgSO4 1.2,
KH2PO4 1.2, NaHCO3 26, and glucose
11.1, aerated with 95% O2/5% CO2), and
prepared for experimentation as described
previously.30 32 33 34 The arterial rings were
contracted several times with KCl (60 mmol/L) until a
maximum level of tension was achieved. Each ring was then contracted
with a thromboxane mimetic, U46619, to 40% to 80% of the
maximal tension obtained with KCl (60 mmol/L). When tension
stabilized, bradykinin (0.3 to 100 nmol/L) was administered in a
cumulative fashion. The baths were rinsed, and after tension returned
to baseline, the rings were contracted again with U46619 to a level of
tension similar to that of the previous contraction. When a stable
level of tension was achieved, one of the EET or DHET regioisomers
(0.05 to 5 µmol/L) was administered in a cumulative
fashion. Thirty minutes after introduction of the last EET or DHET dose
(final concentration, 5 µmol/L), the organ chambers were
rinsed with six exchanges of fresh KRB solution to remove EET or DHET
that had not been taken up by the tissue. After an additional 30
minutes, the rings were recontracted with U46619 to a level of tension
similar to that of the previous contractions, and when a stable level
of tension was achieved, bradykinin (0.3 to 100 nmol/L) was
administered in a cumulative fashion. When used, L-NAME (100
µmol/L) and indomethacin (10
µmol/L) were introduced into the organ chambers 45 minutes
before the initial contractions and were thereafter included for the
duration of the experiments. Triacsin C was added to the organ chambers
20 minutes before contracting the rings with U46619; the compound was
not added again after the administration of 11,12-EET or 14,15-DHET and
rinsing of the organ chambers. The final concentrations of all vehicles
were <0.1%.
In some experiments, protocols were performed exactly as described
above, except that rings were contracted with acetylcholine (0.03 to
1.0 µmol/L), PGF2 (0.02 to
2.0 µmol/L), or KCl (22 to 28 mmol/L) instead
of U46619. To contract the rings with KCl, a KRB solution was prepared
in which 20 mmol/L KCl was isotonically substituted for
NaCl. Additional KCl from a 3 mol/L stock solution was added to
the organ chambers to achieve 40% to 80% of the tension obtained with
KCl (60 mmol/L). When the rings were contracted with PDBu
(9 to 100 nmol/L) to test responses to bradykinin, 11,12-EET, or
14,15-EET, repetitive responses could not be obtained because of the
failure of rings to return to basal tension after washout of PDBu.
Mechanical disruption of the endothelium was achieved immediately after rings were cut from arteries by inserting the rings onto the tips of watchmaker's forceps and rolling them back and forth over a gauze wetted with KRB solution. The endothelium was considered to be denuded when maximal relaxation to bradykinin was <10%, as described previously.32 33 Responses to bradykinin and sodium nitroprusside (30 to 300 nmol/L) in endothelium-denuded U46619-contracted rings were determined before and after treatment with 11,12-EET or 14,15-EET, as described above. Relaxation responses were expressed as the percent decrease from the agonist-induced tension, as described previously.30 32 33 34
Cell Culture and Incubations
Porcine coronary artery endothelial
cells were isolated as reported previously34 and grown in
medium 199 supplemented with MEM nonessential amino acids, MEM vitamin
solution, 15 mmol/L HEPES, 2 mmol/L
L-glutamate, 50 µmol/L gentamicin, and 10%
FBS. The cultures were maintained until confluent at 37°C in a
humidified atmosphere containing 5% CO2. Stocks were
subcultured weekly by trypsinization, and before experimentation the
cells between passages 3 and 7 were grown to confluence on six-well
plates. All experiments were conducted with medium 199 supplemented as
described above but containing 0.1 µmol/L bovine serum
albumin instead of 10% FBS.
Cells were incubated in 1 mL of fresh medium containing 14,15-EET (0.5 µmol/L) and [3H]14,15-EET1 (0.3 µCi) for 1 hour. To investigate EET incorporation into cell lipids, the medium was removed, and after they were washed twice with fresh medium containing no EET, the cells were harvested by scraping into methanol, a process that does not cause appreciable hydrolysis of tissue lipids,35 and transferred to test tubes. In some experiments, cells were pretreated with triacsin C (2 µmol/L) or its vehicle, DMSO, for 30 minutes before incubation with [3H]14,15-EET. To investigate the release of incorporated EET, cells were incubated with EET for 1 hour as described above, after which the cells were washed with fresh medium containing no EET. The medium was then replaced with fresh medium containing either A23187 (2 µmol/L) or vehicle (DMSO). Thirty minutes after the application of A23187 or vehicle, the incubation was terminated by removing the medium and washing the cells twice with 2 mL ice-cold PBS, and the cells were harvested and transferred to test tubes. The amount of radioactivity in the medium was determined by scintillation counting. The final concentrations of DMSO and ethanol in the medium (in all experiments) were <0.1%.
Analyses of Cell Lipids
Cell lipids were extracted and analyzed by TLC as
described previously,30 36 37 38 with the following
modifications: 6 mL of chloroform/methanol (2:1) followed by 2 mL of
acidified saline (4 mmol/L HCl in 0.09% NaCl) was used to
extract cell lipids, and neutral lipids were separated on silica gel G
plates with hexane/ethyl ether/acetic acid/methanol
(85:20:2:2).37 Some of the cell lipid extracts were
saponified as described previously30 and assayed by
reverse-phase HPLC, as described below.
Assay of Incubation Medium
After incubation of porcine coronary artery
endothelial cells with [3H]14,15-EET, the
incubation medium was extracted and separated by reverse-phase HPLC as
described previously,30 except that a Gilson 302 pump plus
2050 UV detector and a Gilson 715 gradient controller were used, and
the acetonitrile gradient was increased from 25% to 100% over 60
minutes at a flow rate of 0.7 mL/min.
Incubation of Porcine Coronary Arterial Rings
With [1-14C]Arachidonic Acid
Porcine coronary arterial rings were
incubated in 1 mL KRB solution at 37°C in 5% CO2. After
2 hours, the KRB solution was exchanged with fresh solution containing
triacsin C (2 µmol/L) or vehicle. Twenty minutes later,
the KRB solution was removed, and fresh solution containing triacsin C
or its vehicle plus U46619 (10 nmol/L) was applied in order to
duplicate, as much as possible, the conditions under which ring
vasoactivity was examined. After 20 minutes, the KRB solution was
exchanged for fresh solution containing U46619, triacsin C or its
vehicle, and 2.6 µmol/L
[1-14C]arachidonic acid. After 30
minutes, the KRB solution was removed, and the rings were washed with
PBS (4°C) and transferred to test tubes. Six milliliters of
chloroform/methanol (2:1) was added, and the tubes were mixed and
stored overnight at -20°C. The following day, 2 mL acidified saline
was added, and the rings were vortexed and maintained at 4°C for 2
hours. Cell lipids were extracted and separated by TLC as described
above, except that phospholipids were separated using
chloroform/methanol/glacial acetic acid/Millipore–filtered
water (50:50:2:1.5), and neutral lipids were separated using
heptane/ethyl ether/glacial acetic acid (75:60:1.5). The values were
converted to picomoles, calculated from the specific activity of the
[1-14C]arachidonic acid with which the
rings were incubated, and normalized to ring wet weight.
Statistical Analysis
All data are expressed as mean±SEM. The effects of treatment
with EETs and DHETs on bradykinin-induced relaxation were determined by
repeated-measures ANOVA with a Greenhouse-Geisser 
correction
(SAS-STAT [SAS Instititutes], procedure GLM). When the interaction
term (ie, concentration of bradykininxtreatment with EETs or DHETs)
was not significant, analysis of the main effect of treatment
(ie, overall relaxation to bradykinin) was performed. The half-maximal
effective concentration (EC50) of bradykinin was calculated
for each concentration-response curve before and after treatment with
the individual EET and DHET regioisomers. The EC50 values
(expressed as -log[M]) before and after treatment were
analyzed by paired Student's t tests. All other
data were also analyzed by paired Student's t
tests. Values of P
.05 were considered to be statistically
significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Vasoactivity of EETs in Porcine Coronary Arteries
The effects of the EET regioisomers (0.05 to 5.0 µmol/L) on endothelium-intact porcine coronary arterial rings submaximally contracted with a thromboxane mimetic, U46619 (0.5 to 30 nmol/L), were examined. 14,15-EET and 11,12-EET produced concentration-dependent relaxations exceeding that produced by the vehicle (ethanol), whereas 8,9- and 5,6-EET produced minimal relaxations (Fig 1
).
|
In some experiments, 11,12-EET or 14,15-EET was administered to endothelium-denuded porcine coronary arterial rings contracted with U46619. Both 11,12-EET and 14,15-EET produced relaxations of denuded rings that were similar in magnitude and duration to those resulting from administration to endothelium-intact rings (71±6% and 65±5%, respectively, at 5 µmol/L; n=4 for each group), as was previously reported for bovine and canine coronary arteries.12 13
In separate experiments, the effects of 14,15-EET and 11,12-EET
on endothelium-intact porcine coronary artery
rings contracted with agonists other than U46619 were examined. Both
compounds relaxed rings contracted with
PGF2, but neither produced significant
relaxations of rings contracted with acetylcholine, KCl, or PDBu (Table 1). In fact, small contractions were
frequently observed when either compound was administered to rings
contracted with KCl or when 14,15-EET was administered to rings
contracted with acetylcholine. In contrast to these findings,
bradykinin, in the presence of indomethacin (10
µmol/L) and L-NAME (100 µmol/L) to inhibit
prostaglandin and NO production,
respectively,32 relaxed porcine coronary rings
contracted with either acetylcholine or PDBu.
|
The effects of 17-ODYA, a cytochrome P-450 enzyme inhibitor,17 on bradykinin-induced relaxation of endothelium-intact U46619-contracted rings were examined. These experiments were conducted in the presence of indomethacin (10 µmol/L) and L-NAME (100 µmol/L) to inhibit prostaglandin and NO production, respectively.32 After the initial determination of bradykinin-induced relaxation, rings were treated with 50 µmol/L 17-ODYA for 30 minutes and then recontracted with U46619. When tension stabilized, bradykinin was applied in a concentration-dependent fashion. Under these conditions, bradykinin-induced relaxation was unaltered by treatment with 17-ODYA (73±4% [before 17-ODYA] versus 76±7% [after 17-ODYA] at 100 nmol/L bradykinin, n=4).
Effects of EETs on Endothelium-Dependent Relaxation
to Bradykinin
Relaxation responses produced by the EETs were more sustained (Fig 2, top) than the brief relaxations
produced by bradykinin before exposure to EETs (Fig 2, middle). After
the administration of EETs to endothelium-intact
U46619-contracted rings, the organ chambers were rinsed thoroughly to
remove any EET that had not been taken up by the tissues. After 30
minutes, the rings were contracted with U46619 to a level of tension
similar to that of the preceding contractions, and bradykinin-induced
relaxation was determined and compared with the initial (pre-EET)
bradykinin-induced response. Contractions elicited by U46619 after
exposure to the EETs did not differ from the pre-EET contractions (n=4
to 8 for each regioisomer, data not shown). However, after treatment
with 14,15-EET or 11,12-EET, bradykinin-induced relaxation was enhanced
in magnitude and duration (EC50 values were 8.4±0.1
[before 14,15-EET] versus 8.8±0.0 [after 14,15-EET] and 8.4±0.2
[before 11,12-EET] versus 8.9±0.0 [after 11,12-EET],
P<.03, respectively) (Fig 2, bottom; Fig 3, top and middle). In contrast to these
findings, bradykinin-induced relaxation was not potentiated by 8,9-EET
(EC50 values were 8.7±0.0 [before 8,9-EET] versus
8.6±0.0 [after 8,9-EET], P=.8) (Fig 3, bottom). Likewise,
bradykinin-induced relaxation was not potentiated by 5,6-EET (n=4, not
shown) or the EET vehicle (n=4, not shown).
|
|
In some experiments, responses to bradykinin and sodium nitroprusside were determined in endothelium-denuded U46619-contracted rings before and after administering 11,12-EET or 14,15-EET. Bradykinin failed to relax endothelium-denuded rings before or after treatment with either 11,12-EET or 14,15-EET (n=6, not shown), and relaxation to sodium nitroprusside was not potentiated after treatment with 14,15-EET (52±10% [before 14,15-EET] versus 46±5% [after 14,15-EET] at 30 nmol/L sodium nitroprusside, n=4).
The effects of treatment with 11,12-EET and 14,15-EET on relaxation to aprikalim (0.03 to 3 µmol/L) in endothelium-intact rings were also determined. These experiments were performed exactly as described previously, except that the rings were relaxed with aprikalim rather than bradykinin before and after treatment with 14,15-EET or 11,12-EET. Unlike relaxation to bradykinin, relaxation to aprikalim (which hyperpolarizes vascular smooth muscle cells by activating ATP-sensitive K+ channels39 ) was not potentiated after treatment with 14,15-EET or 11,12-EET (n=4 for each group, not shown).
Role of NO in EET-Induced Potentiation of Relaxation to
Bradykinin
We investigated whether 14,15-EET or 11,12-EET could have
potentiated bradykinin-induced relaxation by enhancing NO
production. To selectively examine bradykinin-induced
NO-mediated relaxation, endothelium-intact rings were
preincubated with indomethacin (10
µmol/L) to inhibit prostaglandin
production and contracted with KCl (22 to 28
mmol/L) to inhibit EDHF-mediated relaxation, as described
previously.32 33 34 Under these conditions,
bradykinin-induced relaxation of the porcine coronary artery is
mediated solely by NO.32 33 34 40 41 Responses to bradykinin
were determined before and after the administration of 14,15-EET or
11,12-EET to indomethacin-pretreated KCl-contracted
rings. Under these conditions, treatment with 14,15-EET (n=4, not
shown) or 11,12-EET (Fig 4, top) did not
potentiate bradykinin-induced relaxation (EC50 values were
8.5±0.1 [before 11,12-EET] versus 8.6±0.1 [after 11,12-EET],
P=.4).
|
To confirm that NO production is not essential for
EET-induced potentiation of relaxation to bradykinin,
endothelium-intact rings were preincubated with L-NAME
(100 µmol/L) to inhibit NO synthase
activity32 33 34 and with indomethacin
(10 µmol/L). Bradykinin-induced relaxation of
U46619-contracted rings was then determined before and after the
administration of 14,15-EET or 11,12-EET, as described above. Despite
inhibition of NO synthase, 14,15-EET (n=4, not shown) and 11,12-EET
(Fig 4
, bottom) still potentiated bradykinin-induced relaxation
(EC50 values were 8.0±0.1 [before 11,12-EET] versus
8.5±0.2 [after 11,12-EET], P<.01).
Vascular Effects of DHETs in Porcine Coronary Arteries
We examined the effects of DHETs on
endothelium-intact U46619-contracted porcine
coronary arterial rings and investigated whether
exposure to DHETs also results in potentiation of
endothelium-dependent relaxation to bradykinin. Similar
to the results obtained with the EETs, 14,15-DHET and 11,12-DHET
produced relaxations of U46619-contracted porcine coronary
arterial rings which exceeded that produced by the vehicle,
whereas 8,9-DHET produced minimal relaxation (Fig 5, top). DHET-induced relaxations were
sustained in duration, like those produced by EETs. Responses to
5,6-DHET were not examined because of the instability of this compound
in aqueous solutions.
|
After exposure to 14,15-DHET (Fig 5, bottom) or 11,12-DHET (n=6, not
shown), bradykinin-induced relaxation was potentiated (EC50
values were 8.4±0.2 [before 14,15-DHET] versus 9.2±0.2 [after
14,15-DHET], P<.05). In contrast, as was observed with
8,9-EET (see Fig 2, bottom), exposure to 8,9-DHET did not potentiate
bradykinin-induced relaxation (n=4, not shown).
Importance of EET and DHET Incorporation Into Cell Lipids to
the Potentiation of Bradykinin-Induced Relaxation
To investigate whether EETs and DHETs must be incorporated into
cell lipids to potentiate bradykinin-induced relaxation, we pretreated
endothelium-intact rings with an inhibitor
of acyl-CoA synthases, triacsin C,42 before exposure to
11,12-EET and 14,15-DHET. Acyl-CoA synthases catalyze the conversion of
free fatty acids to acyl-CoA thioesters, the metabolically
active forms of fatty acids that participate in numerous cellular
processes, including phospholipid synthesis and
remodeling.43 After the initial determination of
bradykinin-induced relaxation, triacsin C (2 µmol/L) was
introduced into the organ chambers, the rings were contracted with
U46619, and 11,12-EET or 14,15-DHET (0.05 to 5.0 µmol/L)
was administered as described previously. The organ chambers were then
rinsed with six exchanges of KRB solution, and bradykinin-induced
relaxation was determined and compared with the initial (pre-EET or
-DHET) bradykinin-induced relaxation. Pretreatment with triacsin C
prevented the 11,12-EET–induced potentiation (Fig 6, top) and attenuated the
14,15-DHET–induced potentiation (Fig 6, bottom) of relaxation to
bradykinin (EC50 values were 8.0±0.2 [before 11,12-EET]
versus 8.0±0.1 [after 11,12-EET] and 8.4±0.1 [before 14,15-DHET]
versus 8.7±0.1 [after 14,15-DHET]; P=.5 and
P=.1, respectively). In separate control experiments,
triacsin C was administered as described above to
endothelium-intact rings that were contracted with
U46619 but not subsequently exposed to 11,12-EET or 14,15-DHET. Under
these conditions, triacsin C did not alter bradykinin-induced
relaxation (n=5, not shown).
|
Next, we investigated the efficacy of triacsin C to block
[3H]14,15-EET incorporation into porcine coronary
artery endothelial cell lipids. Cultured porcine
coronary artery endothelial cells were
pretreated for 30 minutes with triacsin C (2 µmol/L) or
its vehicle and then incubated with [3H]14,15-EET for 1
hour. The medium was removed, and the cell lipids were extracted and
analyzed by TLC. Triacsin C inhibited both the total uptake and
the amount of [3H]14,15-EET incorporated into the PI, PC,
and PE fractions (Fig 7).
|
To confirm that triacsin C also blocks fatty acid incorporation into
freshly harvested arterial tissue, we determined the
effects of triacsin C on
[1-14C]arachidonic acid incorporation
into porcine coronary arterial rings. Similar to
the results observed with [3H]14,15-EET in cultured
endothelial cells, triacsin C (2 µmol/L)
inhibited total [1-14C]arachidonic acid
uptake into porcine coronary arterial rings and the
amount of [1-14C]arachidonic acid
incorporated into phospholipids (Table 2). Triacsin C also inhibited
[1-14C]arachidonic acid incorporation
into triglycerides. In contrast, the amount of
[1-14C]arachidonic acid present as
free fatty acids was unaffected by triacsin C, suggesting that the
compound did not nonspecifically inhibit fatty acid transport into the
tissue.
|
Release of Incorporated EETs From Vascular Endothelial
Cells
To investigate whether incorporated EETs are released from
endothelial cells by agonists that stimulate
endothelial phospholipase activity, porcine
coronary artery endothelial cells were
incubated with [3H]14,15-EET for 1 hour and then washed
to remove EET that had not been taken up by the cells. Cell lipids were
then extracted in some experiments and analyzed by TLC or
saponified and analyzed by HPLC. In other experiments, the
cells were treated with A23187 (2 µmol/L) or its vehicle
for 30 minutes, after which the medium was removed, assayed for
radioactivity, and analyzed by HPLC. After treatment with
A23187 or vehicle, the cell lipids were also extracted and
analyzed by TLC. After a 1-hour incubation with
[3H]14,15-EET, over half of the cell-associated
radioactivity was contained in the PC fraction (Fig 8, top). The remainder of the
incorporated radioactivity was contained in PI and PE. HPLC
analysis of the saponified cell lipids indicated that 
90%
of the incorporated radioactivity consisted of
[3H]14,15-EET (Fig 8, bottom), whereas <5% consisted of
[3H]14,15-DHET.
|
Treatment of [3H]14,15-EET–labeled cells with A23187 for
30 minutes resulted in a 4-fold increase in the release of radioactive
products into the media (Fig 9, top).
Calculations indicate that >95% of the released radioactivity was
derived from PC and PI (n=3, not shown). HPLC analysis of the
media demonstrated that 66% of the released radioactivity coeluted
with a peak previously identified by gas chromatography
coupled with mass spectrometry as
[3H]14,15-DHET23 (Fig 9, bottom), whereas
the remainder of the released radioactivity consisted of
[3H]14,15-EET.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, we found that 14,15-EET, 11,12-EET, and their diol products relaxed porcine coronary arterial rings precontracted with U46619 and that relaxation to bradykinin, an endothelium-dependent vasodilator, was potentiated after the rings were exposed to 14,15-EET, 11,12-EET, 14,15-DHET, and 11,12-DHET. Both the magnitude and duration of bradykinin-induced relaxation were enhanced and matched the values observed when EETs or DHETs were directly applied. Moreover, the same order of regiospecificity was observed for potentiation and for the EET- or DHET-induced relaxations. We recently showed that these same doses of bradykinin potently stimulate phospholipase activity in porcine coronary arteries.34 Together, these findings suggest that the potentiation of bradykinin-induced relaxation resulted from the release of EETs and/or DHETs, perhaps after their incorporation into endothelial phospholipids.
Alternatively, the potentiation of bradykinin-induced relaxation may have resulted from an effect of EETs or DHETs on the vascular smooth muscle rather than endothelial cells. Fang et al44 observed that EETs can be incorporated into porcine aortic smooth muscle cell lipids. However, treatment with EETs did not alter U46619-induced contractions, and bradykinin failed to relax endothelium-denuded rings after exposure to EETs. Furthermore, relaxations produced by two endothelium-independent relaxing agonists, aprikalim and sodium nitroprusside, were not potentiated by EETs. We did not investigate whether incorporation of EETs into cell lipids might potentiate endothelium-independent relaxation mediated by activation of KCa channels. Also, our experimental preparation did not enable us to selectively examine the effects of smooth muscle cell EET incorporation on bradykinin-induced relaxation. Thus, further studies are required to definitively address the importance of vascular smooth muscle cell EET and DHET incorporation to the potentiation of bradykinin-induced relaxation.
EETs are rapidly converted by vascular endothelial and smooth muscle cells to DHETs,23 44 which can also become incorporated into endothelial cell lipids.26 In noncoronary blood vessels, DHETs were reported to be devoid of vasoactive properties.27 28 29 However, we recently observed that 11,12-DHET is vasoactive in porcine coronary arteries, producing an amount of relaxation similar to that with 11,12-EET.30 In the present study, we found that 14,15-DHET also produced substantial relaxation of U46619-contracted porcine coronary arterial rings, whereas 8,9-DHET did not; 5,6-DHET was not tested because of the chemical lability of this diol. Moreover, exposure to 14,15-DHET or 11,12-DHET potentiated the endothelium-dependent relaxation to bradykinin. Pretreatment with triacsin C attenuated the 14,15-DHET–induced potentiation of relaxation to bradykinin, suggesting that the potentiation was dependent on incorporation of 14,15-DHET into cell lipids through an acyl-CoA synthase–dependent pathway. These observations extend our previous findings and confirm that DHETs are capable of regulating vascular tone in coronary arteries. Interestingly, both EET- and DHET-induced vasorelaxations were regioisomeric specific and reflected structural polarities; ie, the eicosanoids with the epoxide and hydroxyl moieties farthest removed from the carboxyl group eluted first on a reverse-phase HPLC column45 and possessed the greatest amount of vasoactivity. In contrast, all four EET regioisomers produce equivalent amounts of relaxation of U46619-contracted canine and bovine coronary arteries.12 13 However, 5,6-EET potently dilates rat intestinal microvessels, whereas 14,15-EET produces little effect.27 Likewise, in cat cerebral microvessels, the only EET demonstrated to produce potent dilation was 5,6-EET, an effect that was attributed to cyclooxygenase-dependent free radical production.46 Recently, nanomolar concentrations of 11(R),12(S)-EET were observed to hyperpolarize and relax renal preglomerular arterioles, whereas 11(S),12(R)-EET and 14,15-EET were far less potent.16 Thus, in blood vessels and in platelets,47 48 the effects of EETs may be both regioisomeric and stereoisomeric specific.
The similarities between the vascular effects of the EET and DHET regioisomers observed in the present study suggest that conversion to DHETs could contribute to EET-induced relaxations in porcine coronary arteries. Campbell et al7 recently reported that the conversion of EETs to DHETs in bovine coronary arteries occurs slowly in relation to the rapid activation of KCa channels by EETs, implying that conversion to DHETs is not responsible for the EET-induced KCa activation. However, in the present study, twice as much 14,15-DHET as 14,15-EET accumulated in the medium after stimulation of [3H]14,15-EET–labeled cells with A23187 for 30 minutes. This observation suggests that after the exogenous administration or endogenous release of EET, the intracellular DHET concentration may rise rapidly enough to contribute to the EET-induced response. Further studies are required to determine the role of conversion to DHET in EET-induced relaxation of porcine coronary arteries.
It is unlikely that the EET-induced potentiation of relaxation to bradykinin was due to enhanced production or activity of NO. This possibility was suggested by a recent report that 5,6-EET can activate Ca2+ influx into vascular endothelial cells, leading to stimulation of NO synthase activity and hence NO production.24 However, 14,15-EET and 11,12-EET failed to potentiate bradykinin-induced NO-mediated relaxation of KCl-contracted rings. Moreover, the EET-induced potentiation of U46619-contracted rings was not prevented by treatment with L-NAME (100 µmol/L), an inhibitor of NO synthase. Since the latter experiments were performed in the presence of indomethacin, these results also suggest that the EET-induced potentiation was not due to enhanced production of vasodilator prostaglandins.
In the presence of NO synthase and cyclooxygenase inhibition, bradykinin relaxes porcine coronary arteries by stimulating the release of EDHF.3 40 41 In a recent study, bradykinin-induced EDHF-mediated relaxation of porcine coronary arteries was reported to be blocked by SKF525a (proadifen) and clotrimazole, inhibitors of cytochrome P-450 enzymes, leading the authors to postulate that relaxation attributed to EDHF in porcine coronary artery is mediated by EETs.4 However, in two other studies, bradykinin-induced EDHF-mediated relaxation of porcine coronary arteries was not blocked by proadifen, clotrimazole, or 7-ethoxyresorufin.32 33 Recently, relaxation induced by A23187 in porcine coronary arteries treated with NO synthase and cyclooxygenase inhibitors was not blocked by the cytochrome P-450 inhibitors econazole or protoporphyrin IX.49 In the present study, 17-ODYA (50 µmol/L), a selective inhibitor of cytochrome P-450 enzymes,17 also did not block bradykinin-induced EDHF-mediated relaxation. These results suggest that unlike EDHF in bovine coronary artery7 49 and in perfused rat heart,5 17 EDHF in porcine coronary artery is resistant to inhibitors of cytochrome P-450 enzymes. Furthermore, in the presence of NO synthase and cyclooxygenase inhibition, bradykinin relaxed porcine coronary arterial rings contracted with acetylcholine or PDBu, whereas the EETs failed to produce significant relaxations of rings contracted with either of these agonists. Although the reasons for these differences in relaxant properties are unclear, these observations suggest that the mechanisms of EET- and EDHF-induced vasorelaxations differ in the porcine coronary artery. Taken together, these results suggest that EDHF in the epicardial porcine coronary artery is most likely not an EET.
In summary, incubation of porcine coronary arterial rings with EETs and DHETs resulted in regioisomeric-specific potentiation of relaxation to bradykinin. The capacity of EETs and DHETs to potentiate bradykinin-induced relaxation paralleled the vasorelaxing potency of the individual regioisomers. Inhibition of acyl-CoA synthase activity blocked the incorporation of EET into endothelial cell lipids and attenuated the EET- and DHET-induced potentiation of relaxation to bradykinin, suggesting that the potentiation is dependent on the incorporation of EETs and DHETs into cell lipids. Thus, unlike vasoactive prostaglandins and leukotrienes, EETs and DHETs may be stored in cell lipids to subsequently modulate vascular tone.
|
Selected Abbreviations and Acronyms |
|---|
|
-nitro-L-arginine methyl ester
|
Acknowledgments |
|---|
This study was supported by grant HL-49264 from the National Heart, Lung, and Blood Institute, National Institutes of Health, and by an American Heart Association Clinician Scientist Award (No. 96004540) to Dr Weintraub. The authors gratefully acknowledge Ruzicka's Meat Processing in Solon, Iowa, for supplying the porcine coronary arteries.
|
Footnotes |
|---|
1 [3H]14,15-EET: the 3H is present at carbons 5, 6, 8, 9, 11, 12, 14, and 15. The 14,15- refers to the location of the epoxide group.
Received November 25, 1996; accepted May 30, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980;288:373-376.[Medline] [Order article via Infotrieve]
2. Moncada S, Gryglewski R, Bunting S, Vane JR. An enzyme isolated from arteries transforms prostaglandin endoperoxides into an unstable substance that inhibits platelet aggregation. Nature. 1976;263:663-665.[Medline] [Order article via Infotrieve]
3. Beny J-L, Brunet PC. Neither nitric oxide nor nitroglycerin accounts for all the characteristics of endothelially mediated vasodilatation of pig coronary arteries. Blood Vessels. 1988;25:308-311.[Medline] [Order article via Infotrieve]
4.
Hecker M, Bara AT, Bauersachs J, Busse R.
Characterization of endothelium-derived hyperpolarizing
factor as a cytochrome P450-derived arachidonic acid
metabolite in mammals. J Physiol (Lond). 1994;481:407-414.
5. Bauersachs J, Hecker M, Busse R. Display of the characteristics of endothelium-derived hyperpolarizing factor by a cytochrome P450-derived arachidonic acid metabolite in the coronary microcirculation. Br J Pharmacol. 1994;113:1548-1553.[Medline] [Order article via Infotrieve]
6. Lischke V, Busse R, Hecker M. Selective inhibition by barbiturates of the synthesis of endothelium-derived hyperpolarizing factor in the rabbit carotid artery. Br J Pharmacol. 1995;115:969-974.[Medline] [Order article via Infotrieve]
7.
Campbell WB, Gebremedhin D, Pratt PF, Harder
DR. Identification of epoxyeicosatrienoic acids as
endothelium-derived hyperpolarizing factors.
Circ Res. 1996;78:415-423.
8.
Chen G, Cheung DW. Modulation of
endothelium-dependent
hyperpolarization and relaxation to acetylcholine
in rat mesenteric artery by cytochrome P450 enzyme activity.
Circ Res. 1996;79:827-833.
9. Fitzpatrick FA, Murphy RC. Cytochrome P-450 metabolism of arachidonic acid: formation and biological actions of `epoxygenase'-derived eicosanoids. Pharmacol Rev. 1988;40:229-241.[Medline] [Order article via Infotrieve]
10. Capdevila JH, Falck JR, Estabrook RW. Cytochrome P450 and the arachidonate cascade. FASEB J. 1992;6:731-736.[Abstract]
11. Oliw EH, Bylund J, Herman C. Bisallylic hydroxylation and epoxidation of polyunsaturated fatty acids by cytochrome P450. Lipids. 1996;31:1003-1021.[Medline] [Order article via Infotrieve]
12.
Rosolowsky M, Falck JR, Willerson JT, Campbell
WB. Synthesis of lipoxygenase and
epoxygenase products of arachidonic
acid by normal and stenosed canine coronary arteries.
Circ Res. 1990;66:608-621.
13.
Rosolowsky M, Campbell WB. Role of
PGI2 and epoxyeicosatrienoic acids in relaxation of bovine
coronary arteries to arachidonic acid.
Am J Physiol. 1993;264:H327-H335.
14. Rosolowsky M, Campbell WB. Synthesis of hydroxyeicosatetraenoic (HETEs) and epoxyeicosatrienoic acids (EETs) by cultured bovine artery endothelial cells. Biochim Biophys Acta. 1996;1299:267-277.[Medline] [Order article via Infotrieve]
15.
Gebremedhin D, Ma Y-H, Falck JR, Roman RJ, VanRollins
M, Harder DR. Mechanism of action of cerebral
epoxyeicosatrienoic acids on cerebral arterial smooth
muscle. Am J Physiol. 1992;263:H519-H525.
16.
Zou A-P, Fleming JT, Falck JR, Jacobs ER, Gebremedhin
D, Harder DR, Roman RJ. Stereospecific effects of
epoxyeicosatrienoic acids on renal vascular tone and
K+-channel activity. Am J Physiol. 1996;270:F822-F832.
17. Fulton D, Mahboubi K, McGiff JC, Quilley J. Cytochrome P450-dependent effects of bradykinin in the rat heart. Br J Pharmacol. 1995;114:99-102.[Medline] [Order article via Infotrieve]
18. Karara A, Wei S, Spady D, Swift L, Capdevila JH, Falck JR. Arachidonic acid epoxygenase: structural characteristics and quantification of epoxyeicosatrienoates in plasma. Biochem Biophys Res Commun. 1992;182:1320-1325.[Medline] [Order article via Infotrieve]
19.
Wu S, Moomaw CR, Tomer KB, Falck JR, Zeldin DC.
Molecular cloning and expression of CYP2J2, a human cytochrome P450
arachidonic acid epoxygenase highly
expressed in human heart. J Biol Chem. 1996;271:3460-3468.
20. Capdevila J, Pramanik B, Napoli JL, Manna S, Falck JR. Arachidonic acid epoxidation: epoxyeicosatrienoic acids are endogenous constituents of rat liver. Arch Biochem Biophys. 1984;231:511-517.[Medline] [Order article via Infotrieve]
21.
Karara A, Dishman E, Falck JR, Capdevila JH.
Endogenous epoxyeicosatrienoyl-phospholipids.
J Biol Chem. 1991;266:7561-7569.
22. Karara A, Dishman E, Jacobson H, Falck JR, Capdevila JH. Stereochemical analysis of the endogenous epoxyeicosatrienoic acids of human kidney cortex. FEBS Lett. 1990;268:227-230.[Medline] [Order article via Infotrieve]
23. VanRollins M, Kaduce TL, Knapp HR, Spector AA. 14,15-Epoxyeicosatrienoic acid metabolism in endothelial cells. J Lipid Res. 1993;34:1931-1942.[Abstract]
24.
Graier WF, Simecek S, Sturek M. Cytochrome P450
mono-oxygenase-regulated signalling of Ca2+
entry in human and bovine endothelial cells.
J Physiol (Lond). 1995;482:259-274.
25.
Catella F, Lawson JA, Fitzgerald DJ, Fitzgerald
GA. Endogenous biosynthesis of
arachidonic acid epoxides in humans: increased
formation in pregnancy-induced hypertension. Proc Natl
Acad Sci U S A. 1990;87:5893-5897.
26.
VanRollins M, Kaduce TL, Fang X, Knapp HR, Spector
AA. Arachidonic acid diols produced by
cytochrome P-450 monooxygenases are incorporated into
phospholipids of vascular endothelial cells.
J Biol Chem. 1996;271:14001-14009.
27.
Proctor KG, Falck JR, Capdevila J. Intestinal
vasodilation by epoxyeicosatrienoic acids: arachidonic
acid metabolites produced by a cytochrome P450
monooxygenase. Circ Res. 1987;60:50-59.
28. Carroll MA, Schwartzman M, Capdevila J, Falck JR, McGiff JC. Vasoactivity of arachidonic acid epoxides. Eur J Pharmacol. 1987;138:281-283.[Medline] [Order article via Infotrieve]
29.
Pfister SL, Falck JR, Campbell WB. Enhanced
synthesis of epoxyeicosatrienoic acids by cholesterol-fed
rabbit aorta. Am J Physiol. 1991;261:H843-H852.
30.
Fang X, Kaduce TL, Weintraub NL, VanRollins M, Spector
AA. Functional implications of a newly characterized pathway of
11,12-epoxyeicosatrienoic acid metabolism in
arterial smooth muscle. Circ Res. 1996;79:784-793.
31.
Zhu Y, Brand Schieber E, McGiff JC, Balazy M.
Identification of arachidonate P-450 metabolites in human
platelet phospholipids. Hypertension. 1995;25:854-859.
32.
Weintraub NL, Joshi SN, Branch CA, Stephenson AH,
Sprague RS, Lonigro AJ. Relaxation of porcine coronary
artery to bradykinin: role of arachidonic acid.
Hypertension. 1994;23:976-981.
33. Lonigro AJ, Weintraub NL, Branch CA, Stephenson AH, McMurdo L, Sprague RS. Endothelium-dependent relaxation to arachidonic acid in porcine coronary artery: is there a fourth pathway? Pol J Pharmacol. 1994;46:567-577.[Medline] [Order article via Infotrieve]
34.
Weintraub NL, Stephenson AH, Sprague RS, McMurdo L,
Lonigro AJ. Relationship of arachidonic acid
release to porcine coronary artery relaxation.
Hypertension. 1995;26:684-690.
35. Spector AA, Kaduce TL, Hoak JC, Fry GL. Utilization of arachidonic and linoleic acids by cultured human endothelial cells. J Clin Invest. 1981;68:1003-1011.
36.
Folch JM, Lees M, Sloane Stanley GH. A simple
method for the isolation and purification of total lipids from animal
tissues. J Biol Chem. 1957;226:497-509.
37.
Spector AA, Kaduce TL, Hoak JC, Czervionke RL.
Arachidonic acid availability and prostacyclin
production by cultured human endothelial
cells. Arteriosclerosis. 1983;3:323-331.
38. Bell ME, Peterson RG, Eichberg J. Metabolism of phospholipids in peripheral nerve from rats with chronic streptozotocin-induced diabetes: increased turnover of phosphatidylinositol 4,5-bis phosphate. J Neurochem. 1982;39:192-200.[Medline] [Order article via Infotrieve]
39.
Nelson MT, Quayle JM.
Physiological roles and properties of potassium
channels in arterial smooth muscle. Am J
Physiol. 1995;268:C799-C822.
40.
Cowan CL, Cohen RA. Two mechanisms mediate
relaxation by bradykinin of pig coronary artery: NO-dependent
and NO-independent responses. Am J Physiol. 1991;261:H830-H835.
41.
Nagao T, Vanhoutte PM.
Hyperpolarization as a mechanism for
endothelium-dependent relaxations of the porcine
coronary artery. J Physiol (Lond). 1992;445:355-367.
42. Tomoda H, Igarashi K, Omura S. Inhibition of acyl Co-A synthetase by triacsins. Biochim Biophys Acta. 1987;921:595-598.[Medline] [Order article via Infotrieve]
43.
Korchak HM, Kane LH, Rossi MW, Corkey BE. Long
chain acyl coenzyme A and signaling in neutrophils.
J Biol Chem. 1994;269:30281-30287.
44. Fang X, VanRollins M, Kaduce TL, Spector AA. Epoxyeicosatrienoic acid metabolism in arterial smooth muscle cells. J Lipid Res. 1995;36:1236-1246.[Abstract]
45. VanRollins M, Kochanek PK, Evans RW, Schiding JK, Nemoto EM. Optimization of epoxyeicosatrienoic acid syntheses to test their effects on cerebral blood flow in vivo. Biochim Biophys Acta. 1995;1256:263-274.[Medline] [Order article via Infotrieve]
46.
Ellis EF, Police RJ, Yancey L, McKinney JS, Amruthesh
SC. Dilation of cerebral arterioles by cytochrome
P-450 metabolites of arachidonic acid.
Am J Physiol. 1990;259:H1171-H1177.
47.
Fitzpatrick FA, Ennis MD, Baze ME, Wynalda MA, McGee
JE, Liggett WF. Inhibition of cyclooxygenase
activity and platelet aggregation by epoxyeicosatrienoic acids:
influence of stereochemistry. J Biol Chem. 1986;261:15334-15338.
48.
VanRollins M. Epoxygenase
metabolites of docosahexaenoic and eicosapentaenoic acids inhibit
platelet aggregation at concentrations below those affecting
thromboxane synthesis. J Pharmacol Exp
Ther. 1995;274:798-804.
49. Graier WF, Holzmann S, Hoebel BG, Kukovetz WR, Kostner GM. Mechanisms of L-NG nitroarginine/indomethacin-resistant relaxation in bovine and porcine coronary arteries. Br J Pharmacol. 1996;119:1177-1186.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  D. J. Behm, A. Ogbonna, C. Wu, C. L. Burns-Kurtis, and S. A. Douglas Epoxyeicosatrienoic Acids Function as Selective, Endogenous Antagonists of Native Thromboxane Receptors: Identification of a Novel Mechanism of Vasodilation J. Pharmacol. Exp. Ther., January 1, 2009; 328(1): 231 - 239. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. C. Hercule, W.-H. Schunck, V. Gross, J. Seringer, F. P. Leung, S. M. Weldon, A. Ch. da Costa Goncalves, Y. Huang, F. C. Luft, and M. Gollasch Interaction Between P450 Eicosanoids and Nitric Oxide in the Control of Arterial Tone in Mice Arterioscler Thromb Vasc Biol, January 1, 2009; 29(1): 54 - 60. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Nayeem, S. M. Poloyac, J. R. Falck, D. C. Zeldin, C. Ledent, D. S. Ponnoth, H. R. Ansari, and S. J. Mustafa Role of CYP epoxygenases in A2AAR-mediated relaxation using A2AAR-null and wild-type mice Am J Physiol Heart Circ Physiol, November 1, 2008; 295(5): H2068 - H2078. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Minuz, H. Jiang, C. Fava, L. Turolo, S. Tacconelli, M. Ricci, P. Patrignani, A. Morganti, A. Lechi, and J. C. McGiff Altered Release of Cytochrome P450 Metabolites of Arachidonic Acid in Renovascular Disease Hypertension, May 1, 2008; 51(5): 1379 - 1385. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Spector and A. W. Norris Action of epoxyeicosatrienoic acids on cellular function Am J Physiol Cell Physiol, March 1, 2007; 292(3): C996 - C1012. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Bellien, R. Joannides, M. Iacob, P. Arnaud, and C. Thuillez Evidence for a basal release of a cytochrome-related endothelium-derived hyperpolarizing factor in the radial artery in humans Am J Physiol Heart Circ Physiol, April 1, 2006; 290(4): H1347 - H1352. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Fang, S. Hu, B. Xu, G. D. Snyder, S. Harmon, J. Yao, Y. Liu, B. Sangras, J. R. Falck, N. L. Weintraub, et al. 14,15-Dihydroxyeicosatrienoic acid activates peroxisome proliferator-activated receptor-{alpha} Am J Physiol Heart Circ Physiol, January 1, 2006; 290(1): H55 - H63. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. You, E. M. Golding, and R. M. Bryan Jr. Arachidonic acid metabolites, hydrogen peroxide, and EDHF in cerebral arteries Am J Physiol Heart Circ Physiol, September 1, 2005; 289(3): H1077 - H1083. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Pozzi, I. Macias-Perez, T. Abair, S. Wei, Y. Su, R. Zent, J. R. Falck, and J. H. Capdevila Characterization of 5,6- and 8,9-Epoxyeicosatrienoic Acids (5,6- and 8,9-EET) as Potent in Vivo Angiogenic Lipids J. Biol. Chem., July 22, 2005; 280(29): 27138 - 27146. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Gauthier, E. M. Edwards, J. R. Falck, D. S. Reddy, and W. B. Campbell 14,15-Epoxyeicosatrienoic Acid Represents a Transferable Endothelium-Dependent Relaxing Factor in Bovine Coronary Arteries Hypertension, April 1, 2005; 45(4): 666 - 671. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Zhao, A. Dey, O. P. Romanko, D. W. Stepp, M.-H. Wang, Y. Zhou, L. Jin, J. S. Pollock, R. C. Webb, and J. D. Imig Decreased epoxygenase and increased epoxide hydrolase expression in the mesenteric artery of obese Zucker rats Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2005; 288(1): R188 - R196. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Fang, N. L. Weintraub, R. B. McCaw, S. Hu, S. D. Harmon, J. B. Rice, B. D. Hammock, and A. A. Spector Effect of soluble epoxide hydrolase inhibition on epoxyeicosatrienoic acid metabolism in human blood vessels Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2412 - H2420. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Nithipatikom, E. R. Gross, M. P. Endsley, J. M. Moore, M. A. Isbell, J. R. Falck, W. B. Campbell, and G. J. Gross Inhibition of Cytochrome P450{omega}-Hydroxylase: A Novel Endogenous Cardioprotective Pathway Circ. Res., October 15, 2004; 95(8): e65 - e71. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. L. Nerheim, J. L. Meier, M. A. Vasef, W.-G. Li, L. Hu, J. B. Rice, D. Gavrila, W. E. Richenbacher, and N. L. Weintraub Enhanced Cytomegalovirus Infection in Atherosclerotic Human Blood Vessels Am. J. Pathol., February 1, 2004; 164(2): 589 - 600. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Fornage, E. Boerwinkle, P. A. Doris, D. Jacobs, K. Liu, and N. D. Wong Polymorphism of the Soluble Epoxide Hydrolase Is Associated With Coronary Artery Calcification in African-American Subjects: The Coronary Artery Risk Development In Young Adults (CARDIA) Study Circulation, January 27, 2004; 109(3): 335 - 339. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. L. Kaduce, X. Fang, S. D. Harmon, C. L. Oltman, K. C. Dellsperger, L. M. Teesch, V. R. Gopal, J. R. Falck, W. B. Campbell, N. L. Weintraub, et al. 20-Hydroxyeicosatetraenoic Acid (20-HETE) Metabolism in Coronary Endothelial Cells J. Biol. Chem., January 23, 2004; 279(4): 2648 - 2656. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Oltman, N. L. Kane, F. J. Miller Jr., A. A. Spector, N. L. Weintraub, and K. C. Dellsperger Reactive oxygen species mediate arachidonic acid-induced dilation in porcine coronary microvessels Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2309 - H2315. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zhao, D. M. Pollock, D. C. Zeldin, and J. D. Imig Salt-Sensitive Hypertension After Exposure to Angiotensin Is Associated With Inability to Upregulate Renal Epoxygenases Hypertension, October 1, 2003; 42(4): 775 - 780. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. I. Pomposiello, J. Quilley, M. A. Carroll, J. R. Falck, and J. C. McGiff 5,6-Epoxyeicosatrienoic Acid Mediates the Enhanced Renal Vasodilation to Arachidonic Acid in the SHR Hypertension, October 1, 2003; 42(4): 548 - 554. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Gauthier, S. G. Jagadeesh, J. R. Falck, and W. B. Campbell 14,15-Epoxyeicosa-5(Z)-Enoic-mSI: A 14,15- and 5,6-EET Antagonist in Bovine Coronary Arteries Hypertension, October 1, 2003; 42(4): 555 - 561. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zhao, D. M. Pollock, E. W. Inscho, D. C. Zeldin, and J. D. Imig Decreased Renal Cytochrome P450 2C Enzymes and Impaired Vasodilation Are Associated With Angiotensin Salt-Sensitive Hypertension Hypertension, March 1, 2003; 41(3): 709 - 714. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. W. Miller, P. V. G. Katakam, H.-C. Lee, C. D. Tulbert, D. W. Busija, and N. L. Weintraub Arachidonic Acid-Induced Vasodilation of Rat Small Mesenteric Arteries Is Lipoxygenase-Dependent J. Pharmacol. Exp. Ther., January 1, 2003; 304(1): 139 - 144. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Medhora, J. Daniels, K. Mundey, B. Fisslthaler, R. Busse, E. R. Jacobs, and D. R. Harder Epoxygenase-driven angiogenesis in human lung microvascular endothelial cells Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H215 - H224. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Falck, U. M. Krishna, Y. K. Reddy, P. S. Kumar, K. M. Reddy, S. B. Hittner, C. Deeter, K. K. Sharma, K. M. Gauthier, and W. B. Campbell Comparison of vasodilatory properties of 14,15-EET analogs: structural requirements for dilation Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H337 - H349. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Fang, N. L. Weintraub, C. L. Oltman, L. L. Stoll, T. L. Kaduce, S. Harmon, K. C. Dellsperger, C. Morisseau, B. D. Hammock, and A. A. Spector Human coronary endothelial cells convert 14,15-EET to a biologically active chain-shortened epoxide Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2306 - H2314. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Bauersachs, M. Christ, G. Ertl, U.R. Michaelis, B. Fisslthaler, R. Busse, and I. Fleming Cytochrome P450 2C expression and EDHF-mediated relaxation in porcine coronary arteries is increased by cortisol Cardiovasc Res, June 1, 2002; 54(3): 669 - 675. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-K. Chen, J. Capdevila, and R. C. Harris Heparin-binding EGF-like growth factor mediates the biological effects of P450 arachidonate epoxygenase metabolites in epithelial cells PNAS, April 30, 2002; 99(9): 6029 - 6034. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 T. Lu, T. Hoshi, N. L Weintraub, A. A Spector, and H.-C. Lee Activation of ATP-sensitive K+ channels by epoxyeicosatrienoic acids in rat cardiac ventricular myocytes J. Physiol., December 15, 2001; 537(3): 811 - 827. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Fleming Cytochrome P450 and Vascular Homeostasis Circ. Res., October 26, 2001; 89(9): 753 - 762. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Lu, P. V G Katakam, M. VanRollins, N. L Weintraub, A. A Spector, and H.-C. Lee Dihydroxyeicosatrienoic acids are potent activators of Ca2+-activated K+ channels in isolated rat coronary arterial myocytes J. Physiol., August 1, 2001; 534(3): 651 - 667. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. M. Faraci, C. G. Sobey, S. Chrissobolis, D. D. Lund, D. D. Heistad, and N. L. Weintraub Arachidonate dilates basilar artery by lipoxygenase-dependent mechanism and activation of K+ channels Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2001; 281(1): R246 - R253. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Zhang, C. L. Oltman, T. Lu, H.-C. Lee, K. C. Dellsperger, and M. VanRollins EET homologs potently dilate coronary microvessels and activate BKCa channels Am J Physiol Heart Circ Physiol, June 1, 2001; 280(6): H2430 - H2440. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Rastaldo, N. Paolocci, A. Chiribiri, C. Penna, D. Gattullo, and P. Pagliaro Cytochrome P-450 metabolite of arachidonic acid mediates bradykinin-induced negative inotropic effect Am J Physiol Heart Circ Physiol, June 1, 2001; 280(6): H2823 - H2832. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Benoit, B. Renaudon, D. Salvail, and E. Rousseau EETs relax airway smooth muscle via an EpDHF effect: BKCa channel activation and hyperpolarization Am J Physiol Lung Cell Mol Physiol, May 1, 2001; 280(5): L965 - L973. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. H. Zink, C. L. Oltman, T. Lu, P. V. G. Katakam, T. L. Kaduce, H.-C. Lee, K. C. Dellsperger, A. A. Spector, P. R. Myers, and N. L. Weintraub 12-Lipoxygenase in porcine coronary microcirculation: implications for coronary vasoregulation Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H693 - H704. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Yu, F. Xu, L. M. Huse, C. Morisseau, A. J. Draper, J. W. Newman, C. Parker, L. Graham, M. M. Engler, B. D. Hammock, et al. Soluble Epoxide Hydrolase Regulates Hydrolysis of Vasoactive Epoxyeicosatrienoic Acids Circ. Res., November 24, 2000; 87(11): 992 - 998. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-K. Chen, J. Capdevila, and R. C. Harris Overexpression of C-terminal Src Kinase Blocks 14,15-Epoxyeicosatrienoic Acid-induced Tyrosine Phosphorylation and Mitogenesis J. Biol. Chem., April 28, 2000; 275(18): 13789 - 13792. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. Capdevila, J. R. Falck, and R. C. Harris Cytochrome P450 and arachidonic acid bioactivation: molecular and functional properties of the arachidonate monooxygenase J. Lipid Res., February 1, 2000; 41(2): 163 - 181. [Abstract] [Full Text]
|
|
|
|
|
|
X. Fang, T. L. Kaduce, M. VanRollins, N. L. Weintraub, and A. A. Spector Conversion of epoxyeicosatrienoic acids (EETs) to chain-shortened epoxy fatty acids by human skin fibroblasts J. Lipid Res., January 1, 2000; 41(1): 66 - 74. [Abstract] [Full Text]
|
|
|
|
|
|
X. Fang, N. L. Weintraub, L. L. Stoll, and A. A. Spector Epoxyeicosatrienoic Acids Increase Intracellular Calcium Concentration in Vascular Smooth Muscle Cells Hypertension, December 1, 1999; 34(6): 1242 - 1246. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. L. Weintraub, X. Fang, T. L. Kaduce, M. VanRollins, P. Chatterjee, and A. A. Spector Epoxide hydrolases regulate epoxyeicosatrienoic acid incorporation into coronary endothelial phospholipids Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H2098 - H2108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-C. Lee, T. Lu, N. L Weintraub, M. VanRollins, A. A Spector, and E. F Shibata Effects of epoxyeicosatrienoic acids on the cardiac sodium channels in isolated rat ventricular myocytes J. Physiol., August 15, 1999; 519(1): 153 - 168. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. R. Hutcheson, A. T. Chaytor, W. H. Evans, and T. M. Griffith Nitric Oxide–Independent Relaxations to Acetylcholine and A23187 Involve Different Routes of Heterocellular Communication : Role of Gap Junctions and Phospholipase A2 Circ. Res., January 22, 1999; 84(1): 53 - 63. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Oltman, N. L. Weintraub, M. VanRollins, and K. C. Dellsperger Epoxyeicosatrienoic Acids and Dihydroxyeicosatrienoic Acids Are Potent Vasodilators in the Canine Coronary Microcirculation Circ. Res., November 2, 1998; 83(9): 932 - 939. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Kaley Novel Vasodilator Released by Retinal Tissue Circ. Res., October 5, 1998; 83(7): 772 - 773. [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
W. B. Campbell, C. Deeter, K. M. Gauthier, R. H. Ingraham, J. R. Falck, and P.-L. Li 14,15-Dihydroxyeicosatrienoic acid relaxes bovine coronary arteries by activation of KCa channels Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1656 - H1664. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||