20-125Iodo-14,15-Epoxyeicosa-5(Z)-enoic Acid: a High-Affinity
Radioligand Used to Characterize the Epoxyeicosatrienoic Acid
Antagonist Binding Site□
Yuenmu Chen, John R. Falck, Venugopal R. Tuniki, and William B. Campbell
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin (Y.C., W.B.C.); and
Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas (J.R.F.,
Received June 22, 2009; accepted September 16, 2009
Epoxyeicosatrienoic acids (EETs) are endothelium-derived me-
tabolites of arachidonic acid. They relax vascular smooth mus-
cle by membrane hyperpolarization. These actions are inhibited
by the EET antagonist, 14,15-epoxyeicosa-5(Z)-enoic acid
(14,15-EE5ZE). We synthesized 20-125iodo-14,15-EE5ZE (20-
125I-14,15-EE5ZE), a radiolabeled EET antagonist, and charac-
terized its binding to cell membranes. 14,15-EET (10?9-10?5M)
caused a concentration-related relaxation of the preconstricted
bovine coronary artery and phosphorylation of p38 in U937
cells that were inhibited by 20-125I-14,15-EE5ZE. Specific 20-
125I-14,15-EE5ZE binding to U937 cell membranes reached
equilibrium within 5 min and remained unchanged for 30 min.
The binding was saturable and reversible, and it exhibited KD
and Bmaxvalues of 1.11 ? 0.13 nM and 1.13 ? 0.04 pmol/mg
protein, respectively. Guanosine 5?-O-(3-thio)triphosphate (10
?M) did not change the binding, indicating antagonist binding
of the ligand. Various EETs and EET analogs (10?10-10?5M)
competed for 20-125I-14,15-EE5ZE binding with an order of
potency of 11,12-EET ? 14,15-EET ? 8,9-EET ? 14,15-EE5ZE ?
15-hydroxyeicosatetraenoic acid ? 14,15-dihydroxyeicosatrie-
noic acid. 8,9-Dihydroxyeicosatrienoic acid and 11-hydroxyei-
cosatetraenoic acid did not compete for binding. The soluble
and microsomal epoxide hydrolase inhibitors (1-cyclohexyl-3-
dodecyl-urea, elaidamide, and 12-hydroxyl-elaidamide) and
cytochrome P450 inhibitors (sulfaphenazole and proadifen) did
not compete for the binding. However, two cytochrome P450
mide (MS-PPOH) and miconazole competed for binding with Ki
of 1558 and 315 nM, respectively. Miconazole and MS-PPOH,
but not proadifen, inhibited 14,15-EET-induced relaxations.
These findings define an EET antagonist’s binding site and
support the presence of an EET receptor. The inhibition of
binding by some cytochrome P450 inhibitors suggests an al-
ternative mechanism of action for these drugs and could lead to
new drug candidates that target the EET binding sites.
Epoxyeicosatrienoic acids (EETs) are cytochrome P450 ep-
oxygenase metabolites of arachidonic acid (Capdevila et al.,
1981; Spector and Norris, 2007). Four EET regioisomers
(14,15-, 11,12-, 8,9-, and 5,6-EET) are synthesized. They are
actively metabolized by ?-oxidation and epoxide hydration in
mammalian cells and tissues (Spector et al., 2004). EETs
function as endothelium-derived hyperpolarization factor in
the cardiovascular system (Campbell et al., 1996; Fisslthaler
et al., 1999; Campbell and Falck, 2007), but also have effects
on the immune (Node et al., 1999; Liu et al., 2005) and
neuronal systems (Inceoglu et al., 2007, 2008; Terashvili
et al., 2008). They cause vasodilation, mitogenesis, angiogen-
esis, inhibition of inflammation, fibrinolysis, and antinoci-
ception (Spector and Norris, 2007). These functions are at-
This work was supported by the National Institutes of Health National
Heart, Lung and Blood Institute [Grant HL-51055]; the National Institutes of
Health National Institute of General Medical Sciences [Grant GM-31278]; and
the Robert A. Welch Foundation.
Article, publication date, and citation information can be found at
S The online version of this article (available at http://jpet.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: EET, epoxyeicosatrienoic acid; 14,15-EE8ZE, 14(S),15(R)-cis-epoxy-eicosa-8(Z)-enoic acid; 14,15-EE5ZE, 14(S),15(R)-cis-
epoxyeicosa-5(Z)-enoic acid; EH, epoxide hydrolase; 20-I-14,15-EE8ZE, 20-iodo-14,15-epoxyeicosa-8(Z)-enoic acid; OTs, 20-tosyl; 14,15-EET-
mSA, 14,15-Epoxyeicosatrienoyl-methylsulfonamide; 15-HETE, 15-hydroxyeicosatetraenoic acid; 14,15-DHET, 14,15-dihydroxyeicosatrienoic
acid; 14,15-DHE5ZE, 14,15-dihydroxy-eicosa-5(Z)-enoic acid; 8,9-DHET, 8,9-dihydroxyeicosatrienoic acid; 11-HETE, 11-hydroxyeicosatetraenoic
acid; MS-PPOH, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide; GTP?S, guanosine 5?-O-(3-thio)triphosphate; MAP, mitogen-activated
protein; PPAR, peroxisomal proliferator-activated receptor; U46619, 9–11-dideoxy-11?,9a-epoxymethano-prostaglandin F2?; NS1619, 1,3-
dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one; BKCa, large-conductance Ca2?-activated K?chan-
nel; KATP, ATP-sensitive K?channel.
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics
JPET 331:1137–1145, 2009
Vol. 331, No. 3
Printed in U.S.A.
tributed, but not limited, to several signal transduction
pathways including G protein coupling to large-conductance,
calcium-activated potassium (BKCa) channels (Li and Camp-
bell, 1997), nuclear factor ?B (Node et al., 1999), epidermal
growth factor receptor-Src-kinase (Chen et al., 1999), mito-
gen-activated protein (MAP) kinases (Fleming et al., 2001),
and phosphatidylinositol 3-kinase (Chen et al., 2001).
Although many downstream molecules and pathways have
been identified, the initiation step in EET signaling path-
ways is still not clear. Low-affinity EET-binding proteins
have been proposed to mediate EET action. These binding
proteins include fatty acid-binding protein (Widstrom et al.,
2001), peroxisomal proliferator-activated receptor (PPAR)-?
(Cowart et al., 2002), PPAR-? (Liu et al., 2005), and ATP-
sensitive K channels (Lu et al., 2006). Although EETs may
exert some actions through some of these proteins, the mi-
cromolar affinity of EETs for these proteins cannot explain
physiological responses that occur with nanomolar concen-
trations of EETs. High-affinity EET binding proteins or re-
ceptors still require identification.
Several lines of evidence suggest that EETs act through a
specific binding site. Falck et al. (2003a) tested a series of
14,15-EET analogs for their ability to relax the bovine coro-
nary artery. 14(S),15(R)-cis-Epoxyeicosa-8Z-enoic acid was
the simplest structure with full agonist activity. The require-
ment for a specific stereoisomer of the epoxide suggested a
specific binding site for the EET. On vascular smooth muscle,
14,15-EET that was tethered to silica beads could not enter
the cell but inhibited aromatase activity to a similar extent
as 14,15-EET (Snyder et al., 2002). Thus, 14,15-EET acted on
the cell surface and not intracellularly. A high-affinity EET
binding site was described in intact cells and membrane
preparations from guinea pig mononuclear cells and human
U937 cells. By use of [3H]14,15-EET as a radioligand, specific
and saturable binding with a KDof 5.7 nM was determined in
guinea pig monocytes and a KDof 13.84 nM in U937 cells
(Wong et al., 1993, 1997, 2000). This binding site was further
defined in the cell membranes by Yang et al. (2008) by use of
EE8ZE). 20-125I-14,15-EE8ZE bound U937 membranes in a
specific, saturable, and reversible manner with a KDof 11.8
nM. EET analogs, but not prostaglandins or lipoxygenase
metabolites, displaced the 14,15-EET radioligands from their
binding site. This binding site was down-regulated by cAMP-
protein kinase A pathway activation and GTP?S suggesting
a possible G protein-coupled receptor (GPCR) (Wong et al.,
1997; Yang et al., 2008). These studies suggested that EETs
act via a cell surface receptor. U937 cells are good
model systems for studying a high-affinity EET binding
Radiolabeled ligands have been key tools for receptor iden-
tification, signal transduction pathway investigation, drug
discovery, and mapping amino acid residues in ligand bind-
ing sites.3H-Labeled ligands, in general, have low specific
activity and are expensive to synthesize (Wong et al., 1993,
synthesized (Yang et al., 2007, 2008), but antagonist radio-
ligands are traditionally favored in drug screening. Further-
more, antagonists are proposed to occupy a different, but
overlapping, binding pocket than agonists. An antagonist
EET radioligand may be used to map an antagonist’s binding
pocket of the EET-binding protein(s). Here, we have modified
125I-Labeled EET agonist ligands have been
the structure of the first EET antagonist, 14,15-epoxyeicosa-
5Z-enoic acid (14,15-EE5ZE), synthesized, and characterized
the first EET antagonist radioligand, 20-125I-14,15-EE5ZE
(Gauthier et al., 2002).
Materials and Methods
Synthesis of 20-125I-14,15-EE5ZE. 20-125I-14,15-EE5ZE is syn-
thesized from the corresponding 20-tosyl (OTs)-14,15-EE5ZE as re-
ported previously (Prestwich et al., 1988; Yang et al., 2008). The
syntheses of nonradiolabeled (cold) 20-I-14,15-EE5ZE and 20-OTs-
14,15-EE5ZE are described in the Supplemental Data (Mosset et al.,
1989; Cai et al., 2006; Yang et al., 2008). Here, the synthesis of
20-125I-14,15-EE5ZE is described. To 2 mCi in 20 ?l of carrier-free
Na125I (0.8 nmol; 17.4 Ci/mg) was added 20 ?l of NaI in acetone (6.4
?g) and 40 ?l of 20-OTs-14,15-EE5ZE in acetone (640 nmol). The
reaction was carried out at 37°C for 4 days, with shaking 2 to 3 times
daily, and stopped by 10 ?l of a saturated Na2S2O3solution. The
reaction mixture was added to a Bio-Sil A (Bio-Rad Laboratories,
Hercules, CA) silicic acid column. The column was then eluted by 2
volumes of hexane/ethyl acetate (90%:10%) and 2 volumes of hexane/
ethyl acetate (80%:20%). The eluent was dried under N2and purified
by high-performance liquid chromatography with use of a C18 re-
verse-phase column (Nucleosil; 5 ?M; 4.6 ? 250 mm; Phenomenex,
Torrance, CA). A linear gradient of 50 to 100% solvent B in solvent A
(solvent B: acetonitrile/glacial acetic acid ? 999:1; solvent A: water)
over 40 min was used to elute 20-125I-14,15-EE5ZE. 20-I-14,15-
EE5ZE was used as a chromatographic standard and detected in the
column effluent by UV absorbance at 205 nm. The specific activity of
20-125I-14,15-EE5ZE was 47.69 Ci/mmol.
Culture of U937 Cells. U937 cells were cultured in suspension in
RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 10% fetal
bovine serum (HyClone Laboratories, Logan, UT), 25 mM HEPES, 2
mM L-glutamine, and 1 mM sodium pyruvate, 100 U/ml penicillin,
100 ?g/ml streptomycin, and 0.25 ?g/ml amphotericin B (Yang et al.,
2007, 2008). Culture medium was changed every 2 to 3 days. Cells
were cultured at 37°C in a 5% CO2in air-humidified atmosphere and
harvested after reaching a density of 5 to 10 ? 105cells/ml.
Measurement of Phospho-p38 and p38 in U937 Cells. U937
cells (106cells/ml) were suspended in phosphate-buffered saline con-
taining SKF525a (10 ?M), triascin C (20 ?M), and 12-(3-adamantan-
1-yl-ureido)-dodecanoic acid (1 ?M) to inhibit cytochrome P450, es-
terification, and epoxide hydrolase (EH), respectively (Yang et al.,
2008). Cells were incubated for 10 min at 37°C with vehicle, 14,15-
EET (100 nM), 20-I-14,15-EE5ZE (10–1000 nM), or 14,15-EET and
20-I-14,15-EE5ZE (10–1000 nM). Subsequently, the cell suspension
was centrifuged for 5 min at 4°C. The cell pellet was resuspended in
lysis buffer (150 mM NaCl, 10 mM HEPES, 1 mM EDTA, 1 mM
EGTA, 1 mM Na2S2O5, pH 7.5, containing 1% Triton X-100 and
Roche protease inhibitor mix) and incubated for 10 min on ice.
Proteins were separated by electrophoresis, and phospho-p38 and
p38 were detected by Western immunoblotting.
Western Blotting. U937 cellular lysates were mixed with reduc-
ing buffer and heated at 95°C for 10 min to denature proteins (Yang
et al., 2008). The above samples were separated by electrophoresis on
a 12% polyacrylamide Redi-Gel (Bio-Rad Laboratories) and trans-
ferred to a nitrocellulose membrane (Bio-Rad Laboratories) for im-
munoblotting with anti-phospho-p38 antibody (Cell Signaling Tech-
nology, Danvers, MA). The nitrocellulose membrane was reprobed
with anti-p38 antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
Vascular Reactivity of Bovine Coronary Arteries. Fresh bo-
vine hearts were obtained from a local slaughterhouse. The left
anterior descending branch of coronary artery was dissected, cleaned
of connective tissue, and cut into 3-mm-long rings of 1.5- to 3.0-mm
diameter (Campbell et al., 1996; Falck et al., 2003a). The arterial
rings were suspended in a water-jacketed tissue chamber containing
Krebs’ buffer (119 mM NaCl, 4.8 mM KCl, 24 mM NaHCO3, 0.2 mM
Chen et al.
KH2PO4, 0.2 mM MgSO4, 11 mM glucose, 0.02 mM EDTA, and 3.2
mM CaCl2) in 5% CO2and 95% O2environment at 37°C. Ring
tension was recorded with a model FT-03C force transducer (Grass
Instruments, Milford, MA), ETH-400 bridge amplifier, and MacLab
8e A/D converter controlled by a Macintosh computer. The arterial
rings were stretched gradually to a tension of 3.5g and equilibrated
for 1.5 h. KCl (40–60 mM) was repeatedly added and washed away
until reproducible stable contractions were reached. The thrombox-
ane mimetic U46619 (20 nM) was added to increase basal contraction
to 50 to 75% KCl. Increasing concentrations of 14,15-EET or the
BKCachannel activator, NS1619, were added and relaxations re-
corded. To block the 14,15-EET effects, rings were preincubated with
vehicle, 20-I-14,15-EE5ZE (10 ?M), proadifen (20 ?M), miconazole
(20 ?M), or MS-PPOH (20 ?M) for 10 min, and the 14,15-EET
relaxation was recorded. Similar experiments using miconazole (20
?M) and MS-PPOH (20 ?M) were repeated with the BKCachannel
opener NS1619 as the agonist (Gauthier et al., 2002). Results are
expressed as the percentage of relaxation of the U46619-treated
rings, with 100% relaxation representing basal tension.
U937 Membrane Preparation. Cell and membrane prepara-
tions were kept in ice or in the cold room. Cells were pooled and
centrifuged at 1000 rpm for 5 min (Yang et al., 2007, 2008). Cell
pellets were combined, washed with 10 ml of phosphate-buffered
saline, pH 7.4, twice, and resuspended with Hanks’ balanced salt
solution containing protease inhibitor cocktail (Roche Diagnostics,
Indianapolis, IN). After sonicating for 20 s, the lysate was centri-
fuged at 1000g for 10 min. The supernatants were centrifuged at
110,000g for 45 min, and the pellet was resuspended in binding
buffer consisting of 10 mM HEPES, 5 mM CaCl2. 5 mM MgCl2, and
5 mM EGTA, pH 7.4. Protein concentration was determined by the
Bradford method (Bio-Rad Laboratories).
binding assays were performed with a Brandel 48-well harvester
system (Brandel Inc., Gaithersburg, MD) at 4°C (Yang et al., 2007,
2008). Binding was determined in triplicate and repeated on three to
four membrane preparations. Fifty micrograms of protein was incu-
bated in binding buffer (see U937 Membrane Preparation for compo-
sition) with various concentrations of 20-125I-14,15-EE5ZE for vari-
ous times. The binding was stopped by filtration through GF/A glass
filter paper. After washing five times with 3 ml of binding buffer
each, the radioactivity on the filter paper was counted by a ?-scin-
tillation counter. Nonspecific binding was measured in the presence
of 20 ?M 14,15-EE5ZE. Specific binding was calculated from total
binding minus nonspecific binding. The data were analyzed using
Prism software as reported previously (Yang et al., 2007, 2008).
Time course of binding was determined by incubating 2.9 nM
radioligand with the membranes for various times (0–30 min) (Yang
et al., 2008). Saturation of binding was carried out by use of a 15-min
incubation time with different concentrations of the radioligand. To
determine the reversibility of ligand binding, 1 or 20 ?M 11,12-EET
was incubated with membranes for various times (0–60 min) after
10 min of preincubation with radioligand (2.9 nM). For ligand com-
petition, 20-125I-14,15-EE5ZE (1–2 nM) was incubated in presence of
different concentrations of competing ligands for 15 min. Binding
obtained in the presence of vehicle was defined as 100%. To deter-
mine the effect of GTP?S on ligand binding, the membranes were
preincubated with 10 ?M GTP?S or vehicle for 15 min before incu-
bation with various concentrations of the radioligand for 15 min.
Statistical Analysis. The data are expressed as means ? S.E.M.
Statistical evaluation of the data were performed by a one-way
analysis of variance followed by the Student-Newman-Keuls multi-
ple comparison test when significant differences were present. P ?
0.05 was considered statistically significant.
Chemical Structures of EETs, EET Analogs, Cyto-
chrome P450 Inhibitors, and Epoxide Hydrolase Inhib-
itors. Figure 1A shows the structures of EET regioisomers,
EET analogs, cytochrome P450 inhibitors, and epoxide hy-
drolase inhibitors that were studied.
Synthesis of 20-125I-14,15-EE5ZE. Cumulative synthesis
and structure-activity relationships have revealed the basic
Fig. 1. Chemical structures of EETs, EET analogs, cytochrome P450 inhibitors, and EH inhibitors. CDU, 1-cyclohexyl-3-dodecyl-urea.
20-125I-14,15-EE5ZE: EET Antagonist Radioligand
structural requirements for EET agonist and antagonist ac-
tivity (Gauthier et al., 2002, 2003; Falck et al., 2003a, 2003b).
14,15-EE8ZE has all of the structural features of a full ago-
nist whereas 14,15-EE5ZE is the first EET receptor antago-
nist. We have previously synthesized a125I-labeled EET ag-
onist, 20-125I-14,15-EE8ZE (Yang et al., 2008). In a similar
manner, we synthesized 20-125I-14,15-EE5ZE as a radiola-
Antagonist Activity of 20-I-14,15-EE5ZE. We tested
whether 20-I-14,15-EE5ZE is an antagonist similar to 14,15-
EE5ZE in rings of bovine coronary arteries. 14,15-EET re-
laxed U46619 preconstricted bovine coronary artery rings
with EC50value of approximately 2 ?M (Fig. 2A). Pretreat-
ment with 10 ?M 20-I-14,15-EE5ZE reduced 14,15-EET-in-
duced relaxations. These results indicate that 20-I-14,15-
EE5ZE inhibits the action of 14,15-EET.
To further confirm the antagonist activity of 20-I-14,15-
EE5ZE, 14,15-EET-induced p38 MAP kinase activity was
monitored in U937 cells with immunoblotting. The phosphor-
ylation of p38 in U937 cells was stimulated in concentration-
dependent manner by 14,15-EET (0.1–100 nM) (Fig. 3A). In
contrast, the inactive EET thiirane analog (0.1–100 nM) did
not alter p38 phosphorylation, indicating specific activation
of p38 by 14,15-EET (Falck et al., 2003a). The effect of 20-I-
14,15-EE5ZE was tested on 14,15-EET-induced p38 phos-
phorylation (Fig. 3, B and C). 20-I-14,15-EE5ZE decreased
14,15-EET-stimulated p38 phosphorylation at concentra-
tions from 1 to 1000 nM. This result indicates that 20-I-
14,15-EE5ZE is an antagonist of 14,15-EET in U937 cells.
Characterization of 20-125I-14,15-EE5ZE Binding on
U937 Membranes. Figure 4, A and C, shows the time- and
concentration-dependent binding of 20-125I-14,15-EE5ZE to
U937 cell membranes. The half-time of association was 0.9
min at 2.9 nM 20-125I-14,15-EE5ZE (Fig. 4A). The specific
binding reached equilibrium within 5 min and remained
unchanged up to 30 min. Equilibrium binding was performed
at an incubation time of 15 min with increasing concentra-
tion of radioligand. Specific binding increased with radioli-
gand and was saturable (Fig. 4, B and C). Nonspecific binding
increased linearly with increasing concentrations of the ra-
dioligand. Scatchard analysis of the saturable binding sug-
gested a single-site binding model (Fig. 4D) (r2? 0.95).
Binding affinity KDwas 1.11 ? 0.13 nM, and Bmaxwas 1.13 ?
0.04 pmol/mg (n ? 4). If we assume association rate constant
kon? 6.4 ? 106M?1s?1for 20-125I-14,15-EE5ZE, the disso-
ciation rate constant koffwill be 0.007 s?1calculated from koff
? KD? konand the t1/2is 99 s from t1/2? ln 2/koff. This
antagonist radioligand has 10 times higher affinity than the
agonist radioligand, 20-125I-14,15-EE8ZE (KD? 11.8 nM and
Bmax? 5.8 pmol/mg, n ? 5) (Yang et al., 2008). This differ-
ence in KDvalues between the agonist and antagonist li-
gands was statistically significant (p ? 0.0057). The Bmax
Fig. 2. Effect of 20-I-14,15-EE5ZE and cytochrome
P450 inhibitors on 14,15-EET- and NS1619-induced
relaxation of bovine coronary arteries. Bovine coro-
nary artery rings were preconstricted with U46619
and treated with increasing concentrations of 14,15-
EET (A, B, C, E) or NS-1619 (D, F) in the presence
of vehicle or 20-I-14,15-EE5ZE (1 ? 10?5M) (A),
proadifen (2 ? 10?5M) (B), MS-PPOH (2 ? 10?5M)
(C, D) or miconazole (2 ? 10?5M) (E, F). Each value
represents the mean ? S.E.M. ?, p ? 0.01.
Chen et al.
values also differ with the two ligands. The antagonist radio-
ligand may bind different populations of receptors than the
To test whether the binding is reversible, 20-125I-14,15-
EE5ZE (2.9 nM) was incubated with U937 membranes for 10
min to establish equilibrium. 11,12-EET (1 or 20 ?M) was
then added to compete for binding. The incubations were
stopped at different times from 20 s to 1 h. Figure 5A shows
that 11,12-EET replaced the radioligand completely within
0.5 h and with 50% displacement of the ligand in less than 10
min. The rate of displacement of 20-125I-14,15-EE5ZE was
slower than with 20-125I-14,15-EE8ZE, which was less than 1
min (Yang et al., 2008). The slower dissociation time contrib-
utes to the higher affinity for the antagonist radioligand.
These data also indicate that 20-125I-14,15-EE5ZE binding to
U937 membranes is reversible and the same binding site is
occupied by 11,12-EET.
Previous experiments suggested that the EET receptor in
U937 membranes might be a GPCR because GTP?S blocked
the binding of the agonist radioligand 20-125I-14,15-EE8ZE
(Yang et al., 2008). 20-125I-14,15-EE5ZE binding to U937
membranes was determined in the present or absence of 10
?M GTP?S. The specific binding did not differ in the presence
or absence of GTP?S (Fig. 5B). This experiment further in-
dicates that 20-I-14,15-EE5ZE is a antagonist.
Competition for 20-125I-14,15-EE5ZE binding to U937 cell
membranes was performed using three EETs, several EET
structural analogs, cytochrome P450 inhibitors, and EH in-
hibitors (Zou et al., 1994; Harder et al., 1995; Wang et al.,
1998; Morisseau et al., 1999; Falck et al., 2003a). Figure 6, A
and B, and Table 1 show the rank order and Kivalues of
(11,12-EET ? 14,15-EET ? 8,9-EET ? 14,15-EET-mSA ?
14,15-EE5ZE ? 15-HETE ? 14,15-DHET ? thiirane ?
14,15-DHE5ZE). 8,9-DHET and 11-HETE did not displace
20-125I-14,15-EE5ZE from its binding site (Table 1). These
results suggest that the binding site is specific for three EETs
(8,9-EET, 11,12-EET, and 14,15-EET) but not for 15-HETE,
14,15-DHET, thiirane of 14,15-EET, or 14,15-DHE5ZE. This
competition rank order of EETs and EET analogs is similar
to the rank order previously report for 20-125I-14,15-EE8ZE
suggesting the same binding site (Yang et al., 2008).
Cytochrome P450s metabolize arachidonic acid to EETs
and EH metabolizes EETs to DHETs (Morisseau et al., 1999;
Spector et al., 2004). Several of the inhibitors of these en-
zymes have structures similar to fatty acids and the EETs.
For this reason, cytochrome P450 and EH inhibitors were
also tested. The cytochrome P450 inhibitors sulfaphenazole
and proadifen and the soluble and microsomal EH inhibitors
1-cyclohexyl-3-dodecyl-urea, elaidamide, and 12-hydroxyl-
elaidamide did not compete with 20-125I-14,15-EET, indicat-
ing that this binding site is not a cytochrome P450 or EH
(Table 1). Three cytochrome P450 inhibitors inhibited 20-
125I-14,15-EE5ZE binding to U937 membranes. Miconazole
and MS-PPOH inhibit with Kiof 315 and 1558 nM, respec-
tively (Fig. 6B). In contrast, ketoconazole is less effective in
inhibiting binding with approximately 50% inhibition at the
highest concentration tested, 50 ?M. These findings suggest
that the three compounds are cytochrome P450 and EET
receptor dual inhibitors with principle structures unrelated
to EETs or EET analogs. To test this possibility, we examined
their effects of 14,15-EET-induced relaxation of coronary ar-
teries. 14,15-EET caused a concentration-related relaxation
of preconstricted arterial rings (Fig. 2). Both miconazole (20
?M) and MS-PPOH (20 ?M) inhibited the EET-induced re-
laxations (Fig. 2, E and C). In contrast, proadifen (20 ?M)
was without effect (Fig. 2B). NS1619, a BKCachannel opener,
relaxed coronary arteries in a concentration-related manner
(Gauthier et al., 2002) (Fig. 2, D and F). The relaxations to
NS1619 were not altered by MS-PPOH but were reduced
slightly, and significantly, by miconazole at the highest con-
centrations of NS1619. Thus, the blockade of 14,15-EET-
induced relaxations by MS-PPOH is due to inhibition of EET
binding to its receptor and not inhibition of the BKCachan-
nel. The blockade of the EET relaxations by miconazole is
predominantly due to inhibition of EET binding; however, a
component is due to a reduction in BKCachannel activation.
Phospho p38 >
14,15-EET [100 nM]-+++++
20-I [nM]--1 10 100 1000
14,15-EET [100 nM]-
10 100 1000
Phospho p38/Total p38
Phospho p38/Total p38
0 0.11 10100
Fig. 3. Effect of 20-I-14,15-EE5ZE on 14,15-EET-stimulated p38 phos-
phorylation in U937 cells. Western immunoblotting of phosphorylated
p38 and total p38. A, U937 cells were treated with various concentrations
of 14,15-EET or 14,15-thiirane. B, U937 cells were treated without 100
nM 14,15-EET (lanes 1 and 2) or with 100 nM 14,15-EET combined with
different concentrations of 20-I-14,15-EE5ZE (lanes 3–6). The U937 cell
proteins were separated through SDS-polyacrylamide gel electrophore-
sis, transferred onto nitrocellulose membrane, and immunoblotted with
anti-phospho-p38 antibody (top row) or anti-p38 antibody (bottom row).
C, summary of four independent experiments with the same experimen-
tal protocol as B. The results were expressed as ratio of phospho-p38 over
total p38 (mean ? S.E.M., n ? 4). ?, p ? 0.05 compared with no treat-
ment; ??, p ? 0.05 compared with 14,15-EET.
20-125I-14,15-EE5ZE: EET Antagonist Radioligand
EETs are synthesized by the vascular endothelium and
have a number of cardiovascular actions (Rosolowsky and
Fig. 4. Time- and concentration-dependent bind-
ing of 20-125I-14,15-EE5ZE to U937 membranes.
A, time-dependent binding of 20-125I-14,15-
EE5ZEto U937 membranes.
EE5ZE (2.9 nM) was incubated with 50 ?g of
total U937 membrane for indicated times at 4°C
(n ? 4). Specific binding was determined in the
presence of 20 ?M 14,15-EE5ZE. B, effect of
nonspecific, and specific binding (n ? 4). C, spe-
cific binding of 20-125I-14,15-EE5ZE expanded
from B. D, Scatchard analysis of data from B.
Each value represents the mean ? S.E.M.
(n ? 4).
Fig. 5. 20-125I-14,15-EE5ZE binding to U937 membranes. A, reversibil-
ity: U937 membranes were incubated with 2.9 nM 20-125I-14,15-EE5ZE
for 10 min to reach binding equilibrium. 11,12-EET (1 or 20 ?M) was
added, and binding was terminated at the indicated times. Specific bind-
ing was determined. B, effect of GTP?S. Membranes were preincubated
with or without 10 ?M GTP?S for 15 min. Indicated concentrations of
20-125I-14,15-EE5ZE were added, and the incubation was continued for
15 min. The specific binding was determined. Each value represents the
mean ? S.E.M. (n ? 4).
Fig. 6. Inhibition of 20-125I-14,15-EE5ZE binding to U937 membranes by
EETs (A), EET analogs (B), and cytochrome P450 inhibitors (C). 20-125I-
14,15-EE5ZE (2.9 nM) was incubated with increasing concentrations of
EETs, EET analogs or inhibitors, and U937 membrane for 15 min. Spe-
cific binding was determined in the presence or absence of 20 ?M 14,15-
EE5ZE. Specific binding obtained in the presence of vehicle represents
100% binding. Each value represents the mean ? S.E.M. (n ? 4).
Chen et al.
Campbell, 1996; Campbell and Falck, 2007; Spector and Nor-
ris, 2007). They have been implicated as endogenous media-
tors of vasodilation, cardioprotection, and angiogenesis and
inhibitors of inflammation, thrombosis, and platelet aggre-
gation (Node et al., 1999; Krotz et al., 2003; Gauthier et al.,
2007; Gross et al., 2008). Considering these diverse actions, it
is important to understand the mechanism of action of the
In screening a series of 14,15-EET analogs for relaxation of
bovine coronary artery rings, we discovered that 14,15-
EE5ZE blocked the relaxations by all four regioisomeric
EETs (Gauthier et al., 2002). However, this analog did not
block the relaxations to iloprost, sodium nitroprusside, or the
potassium channel openers, NS1619 or bimikalim, or the
contractions to potassium chloride, the thromboxane mimetic
U46619 or 20-HETE. Furthermore, 14,15-EE5ZE displaced
20-125I-14,15-EE8ZE from its binding site on membranes of
U937 cells with a Kisimilar to 14,15-EET (Ki? 37 nM for
14,15-EE5ZE and 40 nM for 14,15-EET) (Yang et al., 2008).
These studies suggested that 14,15-EE5ZE was a selective
EET antagonist. Because the iodo group approximates the
size of a methyl group and previous studies permitted addi-
tion of a 20-iodo group to 14,15-EE8ZE without changing
biological activity (Prestwich et al., 1988; Yang et al., 2008),
we synthesized 20-I-14,15-EE5ZE as a possible EET antag-
onist and tested its activity. For this purpose, 14,15-EET
relaxed the preconstricted bovine coronary artery. 20-I-
14,15-EE5ZE, like 14,15-EE5ZE, inhibited 14,15-EET-in-
duced relaxations. Activation of p38 MAP kinase by EETs
was reported in endothelial and smooth muscle cells (Flem-
ing et al., 2001). Likewise, we showed that 14,15-EET in-
duces p38 MAP kinase phosphorylation in a concentration-
related manner in U937 cells. The inactive thiirane analog of
14,15-EET did not alter the formation of phospho-p38 (Falck
et al., 2003a). The 14,15-EET-induced increase in phospho-
p38 was blocked in a concentration-related manner by 20-I-
14,15-EE5ZE. The consequence of EET activating p38 MAP
kinase in U937 cell was not studied further. These studies
confirmed that 20-I-14,15-EE5ZE, like 14,15-EE5ZE, is an
We synthesized and characterized 20-125I-14,15-EE5ZE as
an antagonist radioligand. It showed specific, saturable bind-
ing to U937 membranes, and the specific binding was re-
versed by the addition of an excess of 11,12-EET. The antag-
onist radioligand bound with higher affinity than did the
agonist radioligand. 20-125I-14,15-EE5ZE had a KDof 1.11
nM, whereas 20-125I-14,15-EE8ZE had a KDof 11.8 nM
(Yang et al., 2008). The reason for the lower KDfor the
antagonist radioligand than the agonist radioligand is the
faster kon(0.5 versus 6.4 ? 106M?1s?1for agonist versus
antagonist) and slower koff(0.06 versus0.007 s?1for agonist
versus antagonist) (Yang et al., 2008). The higher affinity of
20-125I-14,15-EE5ZE is an advantage over previously studied
radioligands providing a higher sensitivity for ligand binding
The binding of 20-125I-14,15-EE5ZE was displaced by
EETs and some EET analogs. The active EET agonists and
EET antagonist displaced the radioligand with a lower Ki
than the inactive analogs or EET metabolites (Falck et al.,
2003a, 2003b). Likewise, Wong et al. (1993) demonstrated
that 14,15-EET displaced [3H]14,15-EET binding to mono-
cytes, but the ligand was not displaced by thromboxane,
platelet-activating factor, leukotriene B4, or leukotriene D4.
Inceoglu et al. (2007) showed that the EETs did not alter
binding to neurokinin, cannabinoid, benzodiazepine, or dopa-
mine receptors. It is interesting that the ranking order of the
Kivalues of EETs and EET analogs was similar with 20-125I-
14,15-EE8ZE and 20-125I-14,15-EE5ZE, suggesting that the
agonist and antagonist radioligands label the same binding
protein on U937 membranes.
Several lines of evidence suggest that the EET binding site
is a GPCR. 11,12-EET increased KCachannel activity in
cell-attached patches of coronary artery smooth muscle cells
(Li and Campbell, 1997). However, in inside-out patches of
the same cells, the EET was without effect unless GTP was
added to the bath. The ability of 11,12-EET to increase KCa
channel activity in inside-out patches with GTP was inhib-
ited by the G protein antagonist GDP?S or an anti-Gs?
antibody (Li and Campbell, 1997). These studies indicate
that 11,12-EET activates KCachannels via a membrane-
delimited mechanism involving activation of a G protein,
possibly Gs?. Likewise, EETs increase GTP?S binding to
Gs, but not Gi, to endothelial cell membranes (Node et al.,
2001). Radioligand-binding studies confirmed these findings.
The binding of 20-125I-14,15-EE8ZE to membranes of U937
was decreased in a concentration-related manner by the GTP
analog, GTP?S (Yang et al., 2008). Because this ligand is an
EET agonist, the decreased binding indicates that the EET
binding site is coupled to a G protein. In contrast, 20-125I-
14,15-EE5ZE is an EET antagonist, and, as would be pre-
dicted with an antagonist, GTP?S did not affect binding of
the radioligand. These results further supported the notion
that the EET receptor is coupled to a G protein.
Cytochrome P450 inhibitors and EH inhibitors are com-
monly used to estimate the contribution of endogenous EETs
to physiological or pathological processes (Harder et al.,
1995; Fisslthaler et al., 1999; Campbell and Falck, 2007;
Gauthier et al., 2007; Spector and Norris, 2007). As a result,
we wondered if these drugs altered binding of 20-125I-14,15-
EE5ZE to its binding site. None of the EH inhibitors com-
peted with 20-125I-14,15-EE5ZE for binding to U937 mem-
branes. Thus, despite their lipid character and the ability of
Comparison of Kiof different EETs, EET analogs, cytochrome P450
inhibitors, and EH inhibitors.
Ki(95% Confidence Intervals)
?5 ? 104
?5 ? 104
?5 ? 104
?2 ? 104
?2 ? 104
?2 ? 104
?2 ? 104
?2 ? 104
?5 ? 104
20-125I-14,15-EE5ZE: EET Antagonist Radioligand
some EH inhibitors to activate PPARs (Liu et al., 2005), they
are without effect on EET binding, which supports their
activity as specific EH inhibitors. The cytochrome P50 inhib-
itors proadifen and sulfaphenazole also failed to alter bind-
ing. Thus, this high-affinity binding site for 20-125I-14,15-
EE5ZE is unique from other previous known lipid receptors
and EET-related enzymes.
Three cytochrome P450 inhibitors, miconazole, MS-PPOH,
and ketoconazole, displace 20-125I-14,15-EE5ZE from the
EET receptor. They represent the first group of ligands struc-
turally unrelated to the EETs. Miconazole inhibits cyto-
chrome P450 and EET binding with similar Kivalues (300
and 315 nM, respectively) (Zou et al., 1994; Harder et al.,
1995). MS-PPOH has a lower Kivalue for inhibition of EET
binding than for inhibition of cytochrome P450 (1.6 and 13
?M, respectively) (Wang et al., 1998). Ketoconazole was the
least effective inhibitor of EET binding and inhibited binding
by approximately 50% at 50 ?M. The Kifor ketoconazole
inhibition of cytochrome P450 epoxygenase is approximately
10 ?M (Zou et al., 1994; Harder et al., 1995). Thus, these
structures differ widely in their specificity for cytochrome
P450 and EET binding. To determine whether binding pre-
dicts EET antagonist activity, we tested the effects of these
inhibitors on 14,15-EET-induced relaxation of the bovine cor-
onary artery. Both MS-PPOH and miconazole inhibited the
relaxations to 14,15-EET, whereas proadifen, which did not
affect binding, was without effect on EET relaxations. MS-
PPOH did not alter the relaxations to the BKCachannel
opener NS1619, indicating that it does not block BKCachan-
nel activation. Although miconazole (10 ?M) failed to alter
BKCachannel activity (Campbell et al., 1996), miconazole (20
?M) partially inhibited the relaxations to NS1619. However,
this degree of BKCachannel inhibition could not account for
the blockade of EET-induced relaxation by miconazole. Thus,
MS-PPOH acts as an EET antagonist, and miconazole acts
predominantly as an EET antagonist. The structural differ-
ences in these nonlipid inhibitors may lead to the design of
EET receptor ligands with improved water solubility and
kinetic properties that are useful for future animal and hu-
man applications. These dual cytochrome P450 inhibitors
and EET receptor ligands, and possibly other cytochrome
P450 inhibitors, inhibit EET signaling pathways at two dif-
ferent sites of action. Careful interpretation of previous pub-
lications using these dual inhibitor/ligands may be needed.
Variations on the structures of miconazole and/or MS-PPOH
may lead to the identification of specific, noneicosanoid EET
antagonists devoid of cytochrome P450 inhibition. Such an-
tagonists may be useful for studies in vivo and provide new
insights into the endogenous roles of the EETs.
We thank Daniel Goldman and Sarah Christian for technical
assistance, Dr. Kathryn Gauthier for review of the manuscript and
suggestions, Gretchen Barg for secretarial assistance, and Drs.
Bruce Hammock and Christophe Morisseau of the University of
California at Davis for the EH inhibitors.
Cai G, Zhu W, and Ma D (2006) A sequential reaction process to assemble polysub-
stituted indolizidines, quinolzidines and quinolzidine analogues. Tetrahedron 62:
Campbell WB and Falck JR (2007) Arachidonic acid metabolites as endothelium-
derived hyperpolarizing factors. Hypertension 49:590–596.
Campbell WB, Gebremedhin D, Pratt PF, and Harder DR (1996) Identification of
epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circ Res
Capdevila J, Chacos N, Werringloer J, Prough RA, and Estabrook RW (1981) Liver
microsomal cytochrome P-450 and the oxidative metabolism of arachidonic acid.
Proc Natl Acad Sci U S A 78:5362–5366.
Chen JK, Capdevila J, and Harris RC (2001) Cytochrome P450 epoxygenase metab-
olism of arachidonic acid inhibits apoptosis. Mol Cell Biol 21:6322–6331.
Chen JK, Wang DW, Falck JR, Capdevila J, and Harris RC (1999) Transfection of an
active cytochrome P450 arachidonic acid epoxygenase indicates that 14,15-
epoxyeicosatrienoic acid functions as an intracellular second messenger in re-
sponse to epidermal growth factor. J Biol Chem 274:4764–4769.
Cowart LA, Wei S, Hsu MH, Johnson EF, Krishna MU, Falck JR, and Capdevila JH
(2002) The CYP4A isoforms hydroxylate epoxyeicosatrienoic acids to form high
affinity peroxisome proliferator-activated receptor ligands. J Biol Chem 20:35105–
Falck JR, Krishna UM, Reddy YK, Kumar PS, Reddy KM, Hittner SB, Deeter C,
Sharma KK, Gauthier KM, and Campbell WB (2003a) Comparison of the vasodi-
latory properties of 14,15-EET analogs: Structural requirements for dilation. Am J
Physiol Heart Circ Physiol 284:H337–H349.
Falck JR, Reddy LM, Reddy YK, Bondlela M, Krishna UM, Ji Y, Sun J, and Liao JK
(2003b) 11,12-Epoxyeicosatrienoic acid (11,12-EET): Structural determinants for
inhibition of TNF-alpha-induced VCAM-1 expression. Bioorg Med Chem Lett 13:
Fisslthaler B, Popp R, Kiss L, Potente M, Harder DR, Fleming I, and Busse R. (1999)
Cytochrome P450 2C is an EDHF synthase in coronary arteries. Nature 401:493–
Fleming I, Fisslthaler B, Michaelis UR, Kiss L, Popp R, and Busse R (2001) The
coronary endothelium-derived hyperpolarizing factor (EDHF) stimulates multiple
signalling pathways and proliferation of vascular cells. Pflugers Arch 442:511–
Gauthier KM, Deeter C, Krishna UM, Reddy YK, Bondlela M, Falck JR, and Camp-
bell WB (2002) 14,15-Epoxyeicosa-5(Z)-enoic acid: a selective epoxyeicosatrienoic
acid antagonist that inhibits endothelium-dependent hyperpolarization and relax-
ation in coronary arteries. Circ Res 90:1028–1036.
Gauthier KM, Jagadeesh SG, Falck JR, and Campbell WB (2003) 14,15-Epoxyeicosa-
5(Z)-enoic-mSI: a 14,15- and 5,6-EET antagonist in bovine coronary arteries.
Gauthier KM, Yang W, Gross GJ, and Campbell WB (2007) Roles of epoxyeicosa-
trienoic acids in vascular regulation and cardiac preconditioning. J Cardiovasc
Gross GJ, Gauthier KM, Moore J, Falck JR, Hammock BD, Campbell WB, and
Nithipatikom K (2008) Effects of the selective EET antagonist, 14,15-EEZE, on
cardioprotection produced by exogenous and endogenous EETs in the canine heart.
Am J Physiol Heart Circ Physiol 294:H2838–H2844.
Harder DR, Campbell WB, and Roman RJ (1995) Role of cytochrome P-450 enzymes
and metabolites of arachidonic acid in the control of vascular tone. J Vasc Res
Inceoglu B, Jinks SL, Ulu A, Hegedus CM, Georgi K, Schmelzer KR, Wagner K,
Jones PD, Morisseau C, and Hammock BD (2008) Soluble epoxide hydrolase and
epoxyeicosatrienoic acids modulate two distinct analgesic pathways. Proc Natl
Acad Sci U S A 105:18901–18906.
Inceoglu B, Schmelzer KR, Morisseau C, Jinks SL, and Hammock BD (2007) Soluble
epoxide hydrolase inhibition reveals novel biological functions of epoxyeicosatrie-
noic acids (EETs). Prostaglandins Other Lipid Mediat 82:42–49.
Krotz F, Riexinger T, Buerkle MA, Nithipatikom K, Gloe T, Sohn HY, Campbell WB,
and Pohl U (2003) Membrane potential-dependent inhibition of platelet adhesion
to endothelial cells by epoxyeicosatrienoic acids. Arterioscler Thromb Vasc Biol
Li PL and Campbell WB (1997) Epoxyeicosatrienoic acids activate K? channels in
coronary smooth muscle through guanine nucleotide binding protein. Circ Res
Liu Y, Zhang Y, Schmelzer K, Lee TS, Fang X, Zhu Y, Spector AA, Gill S, Morisseau
C, Hammock BD, et al. (2005) The antiinflammatory effect of laminar flow: the role
of PPAR? epoxyeicosatrienoic acids and soluble epoxide hydrolase. Proc Natl Acad
Sci U S A 102:16747–16752.
Lu T, Ye D, Wang X, Seubert JM, Graves JP, Bradbury JA, Zeldin DC, and Lee HC
(2006) Cardiac and vascular KAPTchannels in rats are activated by endogenous
epoxyeicosatrienoic acids through different mechanisms. J Physiol 575:627–644.
Morisseau C, Goodrow MH, Dowdy D, Zheng J, Greene JF, Sanborn JR, and Ham-
mock BD (1999) Potent urea and carbamate inhibitors of soluble epoxide hydro-
lases. Proc Natl Acad Sci U S A 96:8849–8854.
Mosset P, Gree R, and Falck JR (1989) Synthesis of two intermediate phosphonium
salts for 5,20- and 15,20-diHETEs. Synth Commun 19:645–658.
Node K, Huo Y, Ruan X, Yang B, Spiecker M, Ley K, Zeldin DC, and Liao JK (1999)
Anti-inflammatory properties of cytochrome P450 epoxygenase-derived eico-
sanoids. Science 285:1276–1279.
Node K, Ruan XL, Dai J, Yang SX, Graham L, Zeldin DC, and Liao JK (2001)
Activation of G?s mediates induction of tissue-type plasminogen activator gene
transcription by epoxyeicosatrienoic acids. J Biol Chem 276:15983–15989.
Prestwich GD, Eng WS, Robles S, Vogt RG, Wis ´niewski JR, and Wawrzen ´czyk C
(1988) Synthesis and binding affinity of an iodinated juvenile hormone. J Biol
Rosolowsky M and Campbell WB (1996) Synthesis of hydroxyeicosatetraenoic acids
(HETEs) and epoxyeicosatrienoic acids (EETs) by cultured bovine coronary artery
endothelial cells. Biochim Biophys Acta 1299:267–277.
Snyder GD, Krishna UM, Falck JR, and Spector AA (2002) Evidence for a membrane
site of action for 14,15-EET on expression of aromatase in vascular smooth muscle.
Am J Physiol Heart Circ Physiol 283:H1936–H1942.
Chen et al.
Spector AA, Fang X, Snyder GD, and Weintraub NL (2004) Epoxyeicosatrienoic acids
(EETs): metabolism and biochemical function. Prog Lipid Res 43:55–90.
Spector AA and Norris AW (2007) Action of epoxyeicosatrienoic acids on cellular
function. Am J Physiol Cell Physiol 292:C996–C1012.
Terashvili M, Tseng LF, Wu HE, Narayanan J, Hart LM, Falck JR, Pratt PF, and
Harder DR (2008) Antinociception produced by 14,15-epoxyeicosatrienoic acid is
mediated by the activation of beta-endorphin and met-enkephalin in the rat
ventrolateral periaqueductal gray. J Pharmacol Exp Ther 326:614–622.
Wang MH, Brand-Schieber E, Zand BA, Nguyen X, Falck JR, Balu N, and Schwartz-
man ML (1998) Cytochrome P450-derived arachidonic acid metabolism in the rat
kidney: characterization of selective inhibitors. J Pharmacol Exp Ther 284:966–
Widstrom RL, Norris AW, and Spector AA (2001) Binding of cytochrome P450
monooxygenase and lipoxygenase pathway products by heart fatty acid-binding
protein. Biochemistry 40:1070–1076.
Wong PY, Lai PS, and Falck JR (2000) Mechanism and signal transduction of
14(R),15(S)-epoxyeicosatrienoic acid (14,15-EET binding in guinea pig monocytes.
Prostaglandins Other Lipid Med. 62:321–333.
Wong PY, Lai PS, Shen SY, Belosludtsev YY, and Falck JR (1997) Post-receptor
signal transduction and regulation of 14(R), 15(S)-epoxyeicosatrienoic acid (14,15-
EET) binding in U-937 cells. J Lipid Med Cell Signal 16:155–169.
Wong PY, Lin KT, Yan YT, Ahern D, Iles J, Shen YS, Bhatt RK, and Falck JR (1993)
14(R), 15(S)-Epoxyeicosatrienoic acid receptor in guinea pig mononuclear cell
membranes. J Lipid Mediat 6:199–208.
Yang W, Holmes BB, Gopal VR, Kishore RV, Sangras B, Yi XY, Falck JR, and
Campbell WB (2007) Characterization of 14,15-epoxyeicosatrienoyl-sulfonamides
as 14,15-epoxyeicosatrienoic acid agonists: Use for studies of metabolism and
ligand binding. J Pharmacol Exp Ther 321:1023–1031.
Yang W, Tuniki VR, Anjaiah S, Falck JR, Hillard CJ, and Campbell WB (2008)
Characterization of epoxyeicosatrienoic acid binding site in U937 membranes
using a novel radiolabeled agonist, 20–125I-14,15-epoxyeicosa-8(Z)-enoic acid.
J Pharmacol Exp Ther 324:1019–1027.
Zou AP, Ma YH, Sui ZH, Ortiz de Montellano PR, Clark JE, Masters BS, and Roman
RJ (1994) Effects of 17-octadecynoic acid, a suicide-substrate inhibitor of cyto-
chrome P450 fatty acid omega-hydroxylase, on renal function in rats. J Pharmacol
Exp Ther 268:474–481.
Address correspondence to: Dr. William B. Campbell, Medical College of
Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. E-mail:
20-125I-14,15-EE5ZE: EET Antagonist Radioligand