Characterization of 14,15-epoxyeicosatrienoyl-sulfonamides as 14,15-epoxyeicosatrienoic acid agonists: Use for studies of metabolism and ligand binding

Article abstract of DOI:10.1124/jpet.107.119651

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid. EETs mediate numerous biological functions. In coronary arteries, they regulate vascular tone by the activation of smooth muscle large-conductance, calcium-a

Full text of DOI:10.1124/jpet.107.119651

0022-3565/07/3213-1023–1031$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics
JPET 321:1023–1031, 2007
Vol. 321, No. 3
119651/3204397
Printed in U.S.A.
Characterization of 14,15-Epoxyeicosatrienoyl-Sulfonamides as
14,15-Epoxyeicosatrienoic Acid Agonists: Use for Studies of
Metabolism and Ligand Binding
Wenqi Yang, Blythe B. Holmes, V. Raj Gopal, R. V. Krishna Kishore, Bhavani Sangras,
Xiu-Yu Yi, J. R. Falck, and William B. Campbell
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin (W.Y., B.B.H., X.-Y.Y.,
W.B.C.); and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas (V.R.G., R.V.K.K.,
B.S., J.R.F.)
Received January 10, 2007; accepted February 26, 2007
ABSTRACT
Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxy- methylsulfonamide (10 ␮M), and abolished by 20 mM extracel-
genase metabolites of arachidonic acid. EETs mediate numer- lular Kϩ. 14,15-EET-PISA is metabolized to 14,15-dihydroxyei-
ous biological functions. In coronary arteries, they regulate cosatrienoyl-PISA by soluble epoxide hydrolase in bovine
vascular tone by the activation of smooth muscle large-con- coronary arteries and U937 cells but not U937 cell membrane
ductance, calcium-activated potassium (BKCa) channels to fractions. 14,15-EET-P125ISA binding to human U937 cell
cause hyperpolarization and relaxation. We developed a series membranes was time-dependent, concentration-dependent,
of 14,15-EET agonists, 14,15-EET-phenyliodosulfonamide and saturable. The specific binding reached equilibrium by 15
(14,15-EET-PISA), 14,15-EET-biotinsulfonamide (14,15-EET- min at 4°C and remained unchanged up to 30 min. The esti-
BSA), and 14,15-EET-benzoyldihydrocinnamide-sulfonamide mated Kd and Bmax were 148.3 Ϯ 36.4 nM and 3.3 Ϯ 0.5
(14,15-EET-BZDC-SA) as tools to characterize 14,15-EET me- pmol/mg protein, respectively. These data suggest that 14,15-
tabolism and binding. Agonist activities of these analogs were EET-PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA are full
characterized in precontraced bovine coronary arterial rings. All 14,15-EET agonists. 14,15-EET-P125ISA is a new radiolabeled
three analogs induced concentration-dependent relaxation and tool to study EET metabolism and binding. Our results also
were equipotent with 14,15-EET. Relaxations to these analogs provide preliminary evidence that EETs exert their biological
were inhibited by the BKCa channel blocker iberiotoxin (100 effect through a membrane binding site/receptor.
nM), the 14,15-EET antagonist 14,15-epoxyeicosa-5(Z)-enoyl-
Epoxyeicosatrienoic acids (EETs) are cytochrome P450 ep- rived hyperpolarizing factors in the coronary circulation
oxygenase metabolites of arachidonic acid. There are four
regioisomers of EET: 5,6-, 8,9-, 11,12-, and 14,15-EET (Cap-
devila et al., 1981; Rosolowsky and Campbell, 1996; Nithipa-
tikom et al., 2001). They play key roles in regulating vascular
tone and homeostasis. Previous studies from our lab and
other groups have characterized EETs as endothelium-de-
(Rosolowsky and Campbell, 1993; Fulton et al., 1995; Camp-
bell et al., 1996; Fisslthaler et al., 1999). Vascular endothe-
lium synthesizes and releases EETs in response to bradyki-
nin, acetylcholine, arachidonic acid, flow, or cyclic stretch
(Pinto et al., 1987; Campbell et al., 1996; Rosolowsky and
Campbell, 1996; Fisslthaler et al., 1999; Gauthier et al.,
2005; Huang et al., 2005). EETs then activate smooth muscle
membrane large-conductance, calcium-activated potassium
(BKCa) channels to cause hyperpolarization and vascular
relaxation (Campbell et al., 1996; Gauthier et al., 2005). In
addition, EETs are involved in other biological functions
This work was supported by the National Institutes of Health (Grants
HL-51055 and GM-31278) and by the Robert A. Welch Foundation.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.107.119651.
ABBREVIATIONS: EET, epoxyeicosatrienoic acid; BKCa, large-conductance, calcium-activated potassium channel; sEH, soluble epoxide hydro-
lase; DHET, dihydroxyeicosatrienoic acid; 14,15-EET-PISA, 14,15-EET-phenyliodosulfonamide; 14,15-EET-BSA, 14,15-EET-biotinsulfonamide;
14,15-EET-BZDC-SA, 14,15-EET-benzoyldihydrocinnamide-sulfonamide; THF, tetrahydrofuran; NHS, N-hydroxysuccinimide; TLC, thin layer
chromatography; HPLC, high-performance liquid chromatography; IBTX, iberiotoxin; 14,15-EEZE-mSA, 14,15-epoxyeicosa-5(Z)-enoyl-methyl-
sulfonamide; HBSS, Hanks’ balanced salt solution; BCA, bovine coronary artery; CDU, 1-cyclohexyl-3-dodecylurea; MS, mass spectrometry;
14,15-EET-mSA, 14,15-EET-methylsulfonamide; U46619, 9,11-dideoxy-9␣,11␣-methanoepoxy-prostaglandin F2␣; HMPA, hexamethyl phosphor-
amide; DMF, N,N-dimethylformamide.
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Yang et al.
including anti-inflammation (Node et al., 1999), antipyresis der et al., 2002). For 14,15-EET-PISA, a benzyl ring that may
(Kozak et al., 2000), angiogenesis (Michaelis and Fleming, be iodinated with [125I] or [127I] is attached to the sulfon-
2006), and fibrinolysis (Node et al., 2001). Mechanisms in- amide group. For 14,15-EET-BSA and 14,15-EET-BZDC-SA,
volved in EET bioactivities include activation of Kϩ channels a biotin or BZDC moiety is covalently attached to the sulfon-
(Campbell et al., 1996; Yang et al., 2005), G-protein ADP- amide group through a benzyl linker. We characterized
ribosylation (Li et al., 1999), protein kinase A, and tyrosine 14,15-EET-P125ISA as a radioactive tracer to study EET
kinase pathways (Node et al., 2001; Michaelis and Fleming, metabolism and as a radioligand for studying the binding
2006). To maintain full agonist vascular activity of 14,15- kinetics of the putative EET receptor/binding site. It was
EET in bovine coronary arteries, key structural components used as a model for studying the metabolism of other EET-
including the carbon-1 (C-1) carboxyl group, the 8,9-double sulfonamides.
bond, the 14(S),15(R)-cis epoxide group, and a 14-carbon
distance between the carboxyl and the epoxide group are
Materials and Methods
required (Falck et al., 2003a; Gauthier et al., 2004). Likewise,
specific structural components are required for 11,12-EET-
mediated inhibition of vascular cell adhesion molecule-1 ex-
pression (Falck et al., 2003b). Such structure-activity rela-
tionships indicate a possible lipid-protein binding event that
requires precise conformation of the EET molecules. Numer-
ous studies have indicated that the EET action site may be
the plasma membrane. 11,12-EET activates coronary smooth
muscle BKCa channels in inside-out patches where a small
portion of plasma membrane was excised (Li and Campbell,
1997). In aortic smooth muscle cells, a membrane-imperme-
able 14,15-EET derivative was equipotent as 14,15-EET in
inhibiting aromatase activity, indicating that the EET acts
on the cell surface (Snyder et al., 2002). Wong et al. (1993,
2000) reported protein binding sites in both intact guinea pig
monocytes and mononuclear cell membranes by radioligand
binding studies using [3H]14,15-EET. [3H]14,15-EET bind-
ing to guinea pig monocytes was sensitive to mild protease
treatment, which suggests a protein identity of the 14,15-
Preparation of 14,15-EET-PISA, 14,15-EET-BZDC-SA, and
14,15-EET-BSA
Preparation of 14,15-EET-PISA (5) (Fig. 1, Scheme 1). Syn-
thesis of N-hydroxysuccinimide-ester 2. N-Hydroxysuccinimide (115
mg, 1 mmol), dried azeotropically using anhydrous toluene and then
under high vacuum for 2 h, was added to a solution of 14,15-EET (1)
(132 mg, 0.412 mmol) in dry THF (8 ml) at room temperature under
an argon atmosphere followed by N,NЈ-dicyclohexylcarbodiimide (94
mg, 0.453 mmol). After stirring overnight, the solvent was removed
in vacuo, and the residue was purified by SiO2 column chromatog-
raphy eluting with hexanes/EtOAc (70:30) to give N-hydroxysuccin-
imide (NHS)-ester 2 (151 mg, 88%). TLC: Rf ϳ 0.30 (50% EtOAc/
hexanes); 1H NMR (CDCl3, 300 MHz) ␦ 0.92 (t, J ϭ 7.0 Hz, 3H),
1.32–1.60 (m, 8H), 1.82 (quintet, J ϭ 7.3 Hz, 2H), 2.17–2.22 (m, 3H),
2.37–2.45 (m, 1H), 2.62 (t, J ϭ 7.3 Hz, 2H), 2.80–2.88 (m, 8H),
2.92–2.99 (m, 2H), 5.34–5.54 (m, 6H).
Synthesis of stannane 3. A mixture of 3-bromobenzenesulfonamide
(Sigma-Aldrich Co., St. Louis, MO; 1 g, 4.23 mmol), tetrakis(triph-
enylphosphine)palladium(0) (0.24 g, 0.209 mmol), and bis(tributy-
EET binding site. However, a membrane receptor/binding ditin) (4.3 g, 7.42 mmol) was stirred at room temperature in a sealed
pressure tube under an argon atmosphere in anhydrous CH3CN (25
ml) and toluene (5 ml) for 10 min, then warmed to 95°C. After 48 h,
the reaction mixture was cooled to room temperature, concentrated
in vacuo, and the residue was purified by silica gel column chroma-
tography to yield 3 (870 mg, 51%). TLC: Rf ϳ 0.32 (30% EtOAc/
hexanes); 1H NMR (CDCl3, 400 MHz) ␦ 0.88 (t, J ϭ 7.4 Hz, 9H), 1.11
(app t, J ϭ 7.4 Hz, 6H), 1.25–1.36 (m, 6H), 1.42–1.58 (m, 6H), 5.08 (s,
2H), 7.42–7.47 (m, 1H), 7.66 (dd, JH,H ϭ 8.3 Hz, JSn,H ϭ 34.5 Hz, 1H),
7.85 (dd, J ϭ 1.8, 8.4 Hz, 1H), 8.02 (dd, JH,H ϭ 1.8 Hz, JSn,H ϭ 34.5
Hz, 1H).
protein for EETs has not been purified or cloned. Tools to
identify the putative EET binding protein/receptor are
needed.
EETs are rapidly taken up into cells when applied exog-
enously (Snyder et al., 2002). Whether this uptake is through
simple diffusion or a protein transporter is unclear. EETs are
actively metabolized by multiple enzyme pathways. The ma-
jor pathways are hydrolysis of the epoxide groups by soluble
epoxide hydrolase (sEH) to the corresponding vicinal-dihy-
droxyeicosatrienoic acids (DHETs), ␤-oxidation that results
in chain-shortened products, and esterification into mem-
brane phosphoglycerolipids (Spector et al., 2004). These stud-
ies were done with 14C- or 3H-labeled EETs synthesized from
radiolabeled arachidonic acid. At present, no commercial
company markets radiolabeled EETs. The synthesis of radio-
labeled EETs from radiolabeled arachidonic acid is inefficient
and costly because: 1) yields cannot exceed 50%, or diep-
oxides are formed; and 2) chemical methods produce all four
EET regioisomers in varying amounts (Corey et al., 1979;
Synthesis of sulfonamide 4. A solution of 3-(tri-n-butylstannanyl)-
benzenesulfonamide (3) (80 mg, 0.179 mmol) and 4-(dimethylami-
no)pyridine (20 mg, 0.157 mmol) in dry HMPA (1.5 ml) was cannu-
lated into a solution of NHS-ester 2 (60 mg, 0.143 mmol) in HMPA
(2.5 ml) under an argon atmosphere. After stirring for 10 min at
room temperature, the reaction mixture was warmed to 65°C and
allowed to stir for 16 h, then quenched by the addition of water (10
ml). The mixture was extracted with EtOAc (3 ϫ 15 ml), and the
combined extracts were washed with water, brine, dried over
Na2SO4, and concentrated in vacuo. The residue was purified by
SiO2 column chromatography to give stannane 4 (38 mg, 36%). TLC:
Rosolowsky and Campbell, 1993). Thus, there is a need to Rf ϳ 0.52 (30% EtOAc/hexanes); 1H NMR (CDCl3, 300 MHz) ␦ 0.86–
0.93 (m, 12H), 1.11 (app t, J ϭ 7.4 Hz, 6H), 1.26–1.39 (m, 10H),
1.47–1.68 (m, 12H), 2.04 (dd, J ϭ 7.0, 7.0 Hz, 2H), 2.17–2.26 (m, 3H),
2.43–2.51 (m, 1H), 2.69–2.91 (m, 4H), 3.02–3.12 (m, 2H), 5.20–5.61
(m, 6H), 7.40–7.48 (m, 1H), 7.70 (dd, JH,H ϭ 8.4, JSn,H ϭ 34.5 Hz,1H),
7.96 (dd, J ϭ 1.8, 8.4 Hz,1H), 8.08 (dd, JH,H ϭ 1.8 Hz, JSn,H ϭ 34.5
Hz, 1H), 8.99 (br s,1H).
Synthesis of iodide 5. A solution of sodium iodide (40 mg, 0.027
mmol) in water (1.0 ml) was added to a 0°C solution of stannane 4 (20
mg, 0.027 mmol) in 95% EtOH (4 ml) under an argon atmosphere
followed by H2O2 (30% aq. soln, 24 ␮l, 0.213 mmol) and peracetic acid
produce radiolabeled EETs efficiently, cheaply and with high
specific activity for studies of EET transport, metabolism,
and binding.
To serve these purposes, we developed and characterized a
series of stable 14,15-EET agonists, 14,15-EET-PISA, 14,15-
EET-BSA, and 14,15-EET-BZDC-SA (see Fig. 2). These ana-
logs contain an N-acylsulfonamide group instead of a termi-
nal carboxylic acid. The N-acylsulfonamide group has a
similar pKa as a carboxyl group and obviates ␤-oxidation and
phospholipid esterification (Backes and Ellman, 1994; Sny- (32% in AcOH, 26 ␮l, 0.213 mmol). The reaction mixture was

14,15-EET-Sulfonamides: Stable 14,15-EET Agonists
1025
Fig. 1. Synthesis of 14,15-EET-sulfonamides in Schemes 1,
2, and 3.
warmed to room temperature. After 2 h, the reaction mixture was water (3 ml), extracted with EtOAc (3 ϫ 15 ml), and the combined
quenched with saturated aqueous sodium thiosulfate (1 ml) and organic extracts were washed with water, brine, dried over Na2SO4,
extracted with EtOAc (3 ϫ 15 ml). The combined organic extracts and concentrated in vacuo. The residue was purified by silica gel
were washed with water, brine, dried over Na2SO4, and concentrated column chromatography to yield 8 (13 mg, 30%). TLC: Rf ϳ 0.42 (20%
in vacuo. The residue was purified by silica gel column chromatog- EtOAc/CH2Cl2); 1H NMR (CDCl3, 400 MHz) ␦ 0.90 (t, J ϭ 7.0 Hz,
raphy to give 5 (9 mg, 58%). TLC: Rf ϳ 0.32 (30% EtOH/hexanes); 1H 3H), 1.31–1.62 (m, 8H), 1.63 (quintet, J ϭ 7.3 Hz, 2H), 2.41–2.53 (m,
NMR (CDCl3, 400 MHz) ␦ 0.92 (t, J ϭ 7 Hz, 3H), 1.32–1.69 (m, 1H), 1H), 2.71–2.92 (m, 6H), 3.02–3.20 (m, 4H), 5.20–5.60 (m, 6H), 7.34 (d,
2.04 (dd, J ϭ 7.0, 7.0 Hz, 2H), 2.18–2.26 (m, 3H), 2.46–2.54 (m, 1H),
J ϭ 8.2 Hz, 2H), 7.48 (t, J ϭ 7.6 Hz, 2H), 7.56–7.68 (m, 3H), 7.73 (d,
2.70–2.94 (m, 4H), 3.06–3.17 (m, 2H), 5.20–5.60 (m, 6H), 7.27 (dd, J ϭ 7.9 Hz, 2H), 7.78 (d, J ϭ 7.9 Hz, 2H), 7.88 (d, J ϭ 8.8 Hz, 2H),
J ϭ 8.4, 8.4 Hz, 1H), 7.95 (dd, J ϭ 1.8, 8.4 Hz, 1H), 7.96 (dd, J ϭ 1.8, 8.21 (br s, 1H), 9.35 (br s, 1H).
8.4 Hz, 1H), 8.05 (dd, J ϭ 1.8, 8.4 Hz, 1H), 8.33 (dd, J ϭ 1.8, 1.8 Hz,
1H), 9.37 (br s, 1H).
Preparation of 14,15-EET-BSA (10) (Fig. 1, Scheme 3). Syn-
thesis of acid 9. 4-Carboxybenzenesulfonamide (Aldrich Chemical
Preparation of 14,15-EET-BZDC-SA (8) (Fig. 1, Scheme 2). Co., Milwaukee, WI; 130 mg, 0.358 mmol), dried azeotropically with
Synthesis of aniline 6. n-Butyl lithium (0.21 ml of a 2.5 M solution in anhydrous benzene and then dried under high vacuum for 2 h, was
hexanes, 0.539 mmol) was added slowly to a Ϫ78°C solution of dissolved in anhydrous THF (6 ml) and HMPA (1.5 ml) and cooled to
4-aminobenzenesulfonamide (93 mg, 0.539 mmol) in anhydrous THF Ϫ78°C under an argon atmosphere. To this, n-butyl lithium (0.57 ml
(8 ml) and HMPA (2 ml) under an argon atmosphere. After 5 min, a
of a 2.5 M solution in hexanes, 0.862 mmol) was added, and after 10
solution of NHS-ester 2 (150 mg, 0.359 mmol) in THF (4 ml) was min, a solution of 2 (180 mg, 0.431 mmol) in THF (4 ml) was slowly
added at Ϫ78°C, and then the mixture was slowly warmed to room added. The reaction mixture was then slowly warmed to room tem-
temperature. After stirring overnight, the reaction mixture was perature. After stirring overnight, the reaction mixture was
quenched with saturated aq. NH4Cl (5 ml) and extracted with EtOAc quenched with saturated aqueous NH4Cl solution (15 ml), extracted
(3 ϫ 20 ml). The combined organic extracts were washed with water, with EtOAc (3 ϫ 30 ml), and the combined organic extracts were
brine, dried over Na2SO4, and concentrated in vacuo. The residue washed with water, brine, dried over Na2SO4, and concentrated in
was purified by SiO2 column chromatography to give 6 (120 mg, vacuo. The residue was purified by silica gel column chromatography
71%). TLC: Rf ϳ 0.34 (50% EtOAc/hexanes); 1H NMR (CDCl3, 400 to give acid 9 (85 mg, 71%). TLC: Rf ϳ 0.23 (50% EtOAc/hexanes); 1H
MHz) ␦ 0.92 (t, J ϭ 7.0 Hz, 3H), 1.32–1.60 (m, 8H), 1.63 (quintet, J ϭ NMR (CDCl3, 400 MHz) ␦ 0.91 (t, J ϭ 7.0 Hz, 3H), 1.34–1.60 (m, 8H),
7.3 Hz, 2H), 2.03 (dd, J ϭ 7.3, 7.3 Hz, 2H), 2.18–2.26 (m, 2H), 2.02–2.12 (m, 2H), 2.26 (t, J ϭ 8.5 Hz, 2H), 2.42–2.49 (m, 1H),
2.42–2.49 (m, 1H), 2.71–2.89 (m, 4H), 3.01–3.07 (m, 2H), 4.31 (br s, 2.71–2.89 (m, 4H), 3.07–3.16 (m, 2H), 5.23–5.57 (m, 6H), 8.14 (d, J ϭ
2H), 5.25–5.54 (m, 6H), 6.65 (d, J ϭ 8.6 Hz, 2H), 7.80 (d, J ϭ 8.6 Hz, 8.4 Hz, 2H), 8.23 (d, J ϭ 8.6 Hz, 2H).
Synthesis of biotin 10. To a Ϫ78°C solution of 9 (50 mg, 0.099
2H), 9.01 (br s, 1H).
Synthesis of BZDC 8. A solution of 4-(benzoyl)benzenepropanoic mmol) in dry CH2Cl2 (3 ml) under an argon atmosphere were added
acid NHS ester (Kort et al., 2000) (7) (22.3 mg, 0.063 mmol) and Et3N 1-methylpiperidine (Aldrich; 12 ␮l, 0.099 mmol) followed by isobutyl
(13 ␮l, 0.126 mmol) in DMF (2 ml) was added dropwise to a solution chloroformate (Aldrich; 13 ␮l, 0.099 mmol). After stirring for 45 min
of 6 (20 mg, 0.042 mmol) in anhydrous DMF (2 ml) followed by
at the same temperature, the reaction mixture was warmed to
4-(dimethylamino)pyridine (7.7 mg, 0.063 mmol) under an argon Ϫ25°C, stirred for another 30 min, and then a solution of biotin
atmosphere. After 48 h, the reaction mixture was quenched with hydrazide (Sigma-Aldrich Co.; 100 mg, 0.39 mmol) in DMF (1 ml,

1026
Yang et al.
prewarmed to ϳ80°C to dissolve) was added. The reaction mixture
was slowly warmed to room temperature over 1 h. After stirring at
room temperature overnight, the reaction mixture was diluted with
CH2Cl2 (10 ml), filtered, washed with water, and concentrated in
vacuo. The residue was purified by SiO2 PTLC to give 10 (15 mg,
38%) and recovered starting material (30 mg). TLC: Rf ϳ 0.45 (10%
MeOH/CH2Cl2); 1H NMR (CDCl3, 300 MHz) ␦ 0.90 (t, J ϭ 6.7 Hz,
3H), 1.28–1.42 (m, 6H), 1.43–1.84 (m, 10H), 1.99–2.10 (m, 2H), 2.25
(apparent t, J ϭ 7.0 Hz, 2H), 2.28–2.33 (m, 2H), 2.36 (apparent t, J ϭ
6.8 Hz, 2H), 2.68–2.78 (m, 4H), 2.84 (apparent t, J ϭ 5.8 Hz, 2H),
2.91–3.01 (m, 2H), 3.18–3.28 (m, 1H), 3.32 (br s, 1H, D2O exchange-
able), 3.90 (d, J ϭ 6.4 Hz, 1H, D2O exchangeable), 4.30–4.65 (m, 1H),
4.48–4.54 (m, 1H), 5.26–5.40 (m, 4H), 5.42–5.60 (m, 2H), 8.04 (d, J ϭ
8.5 Hz, 2H), 8.10 (d, J ϭ 8.5 Hz, 2H).
Culture of U937 Cells and Membrane Preparation
U937 cells were cultured in suspension in RPMI 1640 medium
(Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine se-
rum (Hyclone, South Logan, UT), 25 mM HEPES, 2 mM L-glutamine,
1 mM sodium pyruvate, 100 U/ml penicillin, 100 ␮g/ml streptomycin,
and 0.25 ␮g/ml amphotericin B. The cultures were maintained at a
density of 5 to 10 ϫ 105 cells/ml at 37°C in a humidified atmosphere
of 5% CO2 in air. Membrane extraction was modified from previously
reported protocol (Wong et al., 1993). Cells were collected by centrif-
ugation at 1000g for 2 min and washed twice with Hanks’ balanced
salt solution (HBSS) containing 5 mM CaCl2, 5 mM MgCl2, and
protease inhibitor cocktail (Roche Diagnostics, Mannheim, Ger-
many). The pellet was resuspended with HBSS (without CaCl2 or
MgCl2) containing protease inhibitors and sonicated for 20-s bursts
on ice five times at setting 4. The homogenate was centrifuged at
1000g for 10 min at 4°C to remove unbroken cells. The supernatant
was then centrifuged at 150,000g for 45 min at 4°C. The pellet
represents the membrane fraction and was resuspended in binding
buffer consisting of 10 mM HEPES, 5 mM CaCl2, 5 mM MgCl2, and
5 mM EDTA, pH 7.4.
Synthesis of 14,15-EET-P125ISA and 14,15-DHET-P125ISA
To synthesize 14,15-EET-P125ISA, carrier-free Na125I (2 ␮l, pH
9–10, 200 ␮Ci, 17.4 Ci/mg) (GE Healthcare, Arlington Heights, IL)
was mixed with NaI (1 ␮l, 1.08 mg in 10 ml H2O). 14,15-EET-
stannane (4) (10 ␮g in 43 ␮l of ethanol) was added to the NaI solution
followed by H2O2 (10.67 nmol in 1.25 ␮l of H2O) and peracetic acid
(2.69 nmol in 1.68 ␮l of acetic acid) at 4°C (Fig. 1, scheme 1). The
reaction mixture was warmed to room temperature and allowed to
react for 45 min. On quenching with saturated aqueous Na2S2O3
(250 ␮l), the aqueous layer was extracted with ethyl acetate (2 ϫ 300
␮l). The combined organic extracts were washed with H2O (300 ␮l)
and 0.9% NaCl (300 ␮l) and dried under N2. The residues were
purified by reverse-phase high-performance liquid chromatography
(HPLC) as described previously (Campbell et al., 1996). The extracts
were redissolved in 100 ␮l of acetonitrile/glacial acetic acid (999:1)
and 100 ␮l of distilled H2O and resolved on a Nucleosil C18 reverse-
phase column (5 ␮m, 4.6 ϫ 250 mm; Phenomenex, Torrance, CA).
Solvent A was distilled H2O, and solvent B was acetonitrile/glacial
acetic acid (999:1). A linear gradient from 50% solvent B in solvent A
to 94% solvent B within 40 min was used at a flow rate of 1 ml/min.
Column effluent corresponding to synthetic 14,15-EET-PISA was
collected, extracted, and dried under N2.
Bovine Coronary Artery Homogenate Preparation
Bovine coronary arteries (BCAs) were dissected as described
above, cut into small pieces, and homogenized with a glass homog-
enizer in binding buffer containing miconazole (10 ␮M; Sigma-Al-
drich Co.) and protease inhibitor cocktail (Roche) at 4°C. Unbroken
tissue and cell debris were removed by centrifugation at 6000g for 5
min at 4°C. The supernatant represents the BCA homogenate that
contains membrane and cytosolic fractions.
Tissue Incubation of 14,15-EET-P125ISA
14,15-EET-P125ISA was incubated and extracted using the follow-
ing protocols: 1) 14,15-EET-P125ISA (30–33 nM) was incubated with
500 ␮g of BCA homogenate in binding buffer for 1 h at 30°C. The
metabolites were extracted by ethyl acetate. In some incubations, 1
␮M 1-cyclohexyl-3-dodecylurea (CDU), an sEH inhibitor (Morisseau
et al., 2002), was included. 2) 14,15-EET-PISA (10 ␮M), with tracer
amounts of 14,15-EET-P125ISA, was incubated with BCA rings (1 g)
in Krebs’ buffer bubbled with 95% O2-5% CO2 for 15 min at 37°C. The
metabolites were harvested by solid-phase extraction using the
BondElut C18 octadecylsilica column (Varian Inc., Harbor City, CA)
as described previously (Nithipatikom et al., 2001). 3) 14,15-EET-
P125ISA (35 nM) was incubated with U937 cells (800,000 cells) in 200
␮l of HBSS for 15 min at 37°C. The metabolites were extracted by
liquid-liquid extraction using 500 ␮l of chloroform/methanol (1:2)
followed by the addition of 167 ␮l of chloroform and 150 ␮l of H2O.
The organic phase was collected and dried under N2. 4) 14,15-EET-
P125ISA (1 nM) was incubated with 50 ␮g of U937 cell membrane
proteins in 200 ␮l of binding buffer for 15 min at 4°C. In some
studies, incubations were performed with buffer alone or boiled
membranes. The metabolites were extracted by the same liquid-
liquid extraction protocol as for intact U937 cell incubations. The
extracts were then analyzed by reverse-phase HPLC as described
above. The column effluent was collected in 0.5-ml fractions, and
radioactivity was measured by a Packard Cobra-II Auto-Gamma
counter (PerkinElmer Life and Analytical Sciences, Boston, MA).
To synthesize 14,15-DHET-P125ISA, 14,15-EET-P125ISA was dis-
solved in HEPES buffer containing 10 mM HEPES, 150 mM NaCl, 5
mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5.5 mM glucose, pH 7.4.
Recombinant human sEH (35 ␮g) was added and incubated for 30
min at 37°C. The residues were extracted with ethyl acetate and
purified by HPLC as described above. Specific activities of 14,15-
EET-P125ISA and 14,15-DHET-P125ISA were 257.5 Ci/mmol.
Vascular Reactivity of Bovine Coronary Arteries
Bovine hearts were purchased from a local slaughterhouse. The
left anterior descending coronary artery was dissected and cleaned of
connective tissue. Arteries of 2-mm diameter were cut into rings
(3-mm width) and suspended on a pair of stainless hooks in a 6-ml
water-jacketed organ chamber in Krebs’ buffer consisting of 119 mM
NaCl, 4.8 mM KCl, 24 mM NaHCO3, 0.2 mM KH2PO4, 0.2 mM
MgSO4, 11 mM glucose, 0.02 mM EDTA, and 3.2 mM CaCl2. The
buffer was equilibrated with 95% O2-5% CO2 and maintained at
37°C. Tension was continuously recorded as described previously
(Campbell et al., 1996). In brief, submaximal concentrations of the
thromboxane-mimetic U46619 (10–20 nM; Cayman Chemical Co.,
Ann Arbor, MI) were administered to contract the vessels to 50 to
75% of KCl-induced contraction. Cumulative concentrations of 14,15-
Mass Spectrometry Analysis
To identify the 14,15-EET-PISA metabolite, 14,15-EET-PISA was
EET, 14,15-EET-PISA, 14,15-EET-BSA, or 14,15-EET-BZDC-SA incubated with BCA homogenate and extracted as described above.
were added to the chamber. In some studies, the arteries were The HPLC effluent (21 min) containing the 14,15-EET-PISA metab-
incubated with iberiotoxin (IBTX; 100 nM; Sigma-Aldrich Co.), 14,15- olite was collected, extracted with cyclohexane/ethyl acetate (50:50),
epoxyeicosa-5(Z)-enoyl-methylsulfonamide (14,15-EEZE-mSA; 10 ␮M), and further analyzed by mass spectrometry (MS). The MS analysis
or vehicle for 10 min before U46619 contraction (Gauthier et al., 2003). used an Agilent 1100 MSD mass spectrometer (Agilent Technologies,
In the high extracellular potassium ([Kϩ]o) studies, Kϩ was increased to Palo Alto, CA) with electrospray ionization. The residues were intro-
20 mM by substitution of Naϩ. Results are expressed as percent relax- duced into the electrospray chamber by flow injection. Solvent A was
ation with 100% relaxation representing basal tension.
water, and solvent B was acetonitrile with both solvents containing

14,15-EET-Sulfonamides: Stable 14,15-EET Agonists
1027
0.005% glacial acetic acid. Fifty percent of each solvent was used at and 14,15-EET-BZDC-SA relaxed these arteries with an
a flow rate of 0.2 ml/min, and data were collected for 3 min. Detection
was in the negative ion mode. Results are expressed as relative
abundance, with the most abundant ion set as 100%.
EC50 of 1 ␮M and maximal relaxation of 84.5 Ϯ 7.5, 89.6 Ϯ
3.9, and 92.9 Ϯ 5.0% at 10 ␮M, respectively (Fig. 3A). We
tested the effect of Kϩ channel inhibition on these analog-
mediated relaxations. Increasing extracellular Kϩ from 4 to
20 mM eliminated relaxations to 14,15-EET-PISA, 14,15-
EET-BSA, and 14,15-EET-BZDC-SA (maximum relaxation ϭ
7.8 Ϯ 12.8, 8.6 Ϯ 6.3, and 4.5 Ϯ 3.2%, respectively, Fig. 3,
B–D). The BKCa channel blocker, IBTX (100 nM), markedly
attenuated the 14,15-EET-PISA, 14,15-EET-BSA, and 14,15-
EET-BZDC-SA-induced relaxations (maximum relaxation ϭ
Radioligand Binding
U937 membranes (50 ␮g) were incubated with 14,15-EET-P125ISA
in the presence of 20 ␮M 14,15-EEZE-mSA (nonspecific binding) or
an equivalent amount of ethanol (total binding) at 4°C with shaking.
For the time course study, 1 nM 14,15-EET-P125ISA was incubated
at various times (1–30 min). For the concentration-dependent study,
increasing concentrations of 14,15-EET-P125ISA (0.1–150 nM) were
incubated for 15 min. Incubations (total volume, 200 ␮l) were per-
formed in binding buffer and were terminated by filtration through
GF/A glass filter paper on a Brandel harvester (Brandel Inc., Gaith-
ersburg, MD). Filters were washed four times with 5 ml of ice-cold
binding buffer and counted by a Packard Cobra-II Auto-Gamma
counter. Specific binding was defined as total binding minus nonspe-
cific binding. The Kd (equilibrium dissociation constant) and Bmax
(maximal binding site density) values are determined from satura-
tion studies using nonlinear regression to fit the data to the single-
site binding equation using Prism software (GraphPad Inc., San
Diego, LA).
Statistical Analysis
Data are expressed as means Ϯ S.E.M. Statistical evaluation was
performed by a one-way analysis of variance followed by the Student-
Newman-Keuls multiple comparison test when significant differ-
ences were present. P Ͻ 0.05 was considered statistically significant.
Results
Previously, we characterized 14,15-EET-methylsulfon-
amide (14,15-EET-mSA; structure shown in Fig. 2) as a full
agonist of 14,15-EET (Falck et al., 2003a). The N-acylmeth-
ylsulfonamide group has a similar pKa as the carboxyl group
(Backes and Ellman, 1994) and is a commonly used substi-
tute for a carboxyl group (Imig et al., 2001; Snyder et al.,
2002). Based on this knowledge, we developed 14,15-EET-
PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA, which con-
tain an iodine, biotin, and BZDC group, respectively, attach-
ing to the sulfonamide group through a benzyl linker (Fig. 2).
In bovine coronary arteries precontracted with U46619,
14,15-EET causes concentration-dependent relaxation with
an EC50 of 1 ␮M and a maximum relaxation averaging 80 to
94% at 10 ␮M. Likewise, 14,15-EET-PISA, 14,15-EET-BSA,
Fig. 3. Effects of 14,15-EET and 14,15-EET-sulfonamides on vascular
tone of U46619 preconstricted bovine coronary arteries. A, relaxations to
14,15-EET, 14,15-EET-PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA.
B to D, effects of the BKCa channel inhibitor, IBTX (100 nM); the EET
antagonist, 14,15-EEZE-mSA (10 ␮M); or increased extracellular Kϩ (20
mM) on 14,15-EET-PISA (B), 14,15-EET-BSA (C), and 14,15-EET-
BZDC-SA (D) relaxations. ء, significantly different from control, P Ͻ 0.05.
Fig. 2. Structures of 14,15-EET and 14,15-EET-sulfonamides.

1028
Yang et al.
39.3 Ϯ 12.8, 20.8 Ϯ 2.6, and 17.3 Ϯ 6.1%, respectively, Fig. 3, presence of 1 ␮M CDU, an sEH inhibitor, the production of
B–D). We next examined the effect of 14,15-EEZE-mSA, a peak 21 was inhibited. The peak 21/peak 29 ratio was re-
14,15-EET antagonist (Gauthier et al., 2003), on analog- duced to 0.22 (Fig. 4C). Similar metabolism was observed
mediated relaxation. Pretreatment with 14,15-EEZE-mSA with 90 min incubation and similar inhibition was obtained
(10 ␮M) inhibited 14,15-EET-PISA, 14,15-EET-BSA, and using another sEH inhibitor, adamantyl dodecanoic acid
14,15-EET-BZDC-SA relaxations (maximal relaxation ϭ urea (1 ␮M) (data not shown). This suggests that peak 21 is
23.5 Ϯ 9.1, 21.4 Ϯ 13.1, and 34.7 Ϯ 8.6%, Fig. 3, B–D). These likely the sEH metabolite of 14,15-EET-PISA, 14,15-DHET-
results demonstrate that 14,15-EET-PISA, 14,15-EET-BSA, PISA. To identify this metabolite, peak 21 was collected,
and 14,15-EET-BZDC-SA caused vascular relaxation by re-extracted, and analyzed by negative electrospray ioniza-
mechanisms similar to 14,15-EET, i.e., they cause relaxation tion MS. The most abundant ion in the mass spectrum of this
by activating BKCa channels, and the relaxations are inhib- peak was at m/z 602.1 (M-H) (Fig. 4E). This indicated a
ited by the 14,15-EET antagonist.
molecular weight of 603, which is consistent with the molec-
14,15-EET is metabolized through three major pathways ular weight of 14,15-DHET-PISA. Thus, peak 21 was identi-
including ␤-oxidation, epoxide hydration, and phospholipid fied as 14,15-DHET-PISA. We then tested 14,15-EET-
esterification. 14,15-EET-mSA, the parent compound of P125ISA metabolism by bovine coronary arterial rings using
14,15-EET-PISA, does not undergo ␤-oxidation or incorpora- conditions under which the vascular activities were tested.
tion into membrane lipids (Snyder et al., 2002). It is reason- After a 15-min incubation at 37°C, the majority of radioac-
able to predict that 14,15-EET-mSA derived analogs are like- tivity eluted at 29 min, and a small portion of radioactivity
wise not metabolized by these pathways. However, the 14,15- was converted into peak 21. The peak 21/peak 29 ratio was
epoxide group is susceptible to hydration. We used 14,15- 0.18 (Fig. 4D). These data indicate that 14,15-EET-PISA is
EET-P125ISA as a radioactive tracer to study its metabolism hydrolyzed to 14,15-DHET-PISA by sEH in both BCA homog-
in BCA homogenates. 14,15-EET-P125ISA was incubated enate and arterial rings. The arterial rings showed less con-
with buffer or BCA homogenate in the presence and absence version. There was no evidence of the metabolism of 14,15-
of 1 ␮M CDU for 1 h at 30°C. The metabolites were extracted EET-PISA by ␤-oxidation.
and resolved on reverse-phase HPLC. With buffer, 94.8% of
Similar results were obtained when 14,15-EET-P125ISA
radioactivity was extracted into the organic phase. The was incubated with U937 cells for 15 min at 37°C (Fig. 5A).
HPLC chromatogram showed one single peak at 29 min The major metabolite eluted at 29 min, and a small radioac-
(peak 29), which comigrates with the synthetic 14,15-EET- tive product eluted at 21 min. Only the 29-min radioactive
PISA standard (Fig. 4A). After 30-min incubation with BCA metabolite was observed when the cells were incubated at
homogenate at 30°C, an additional more polar metabolite 4°C (data not shown). Thus, U937 cells metabolized 14,15-
migrating at 21 min (peak 21) was produced. The radioactiv- EET-PISA to 14,15-DHET-PISA, as did coronary arteries.
ity ratio of peak 21 to peak 29 was 1.27 (Fig. 4B). In the Next, we investigated the stability of 14,15-EET-P125ISA
Fig. 4. Metabolism of 14,15-EET-P125ISA by BCA
homogenate and arterial rings. A to C, HPLC chro-
matographs of extracted 14,15-EET-P125ISA metab-
olites after incubation with buffer, BCA homogenate
(500 ␮g) in the presence or absence of CDU (1 ␮M) for
30 min at 30°C, respectively. D, HPLC chromato-
graph of extracted 14,15-EET-P125ISA metabolites
after incubation with arterial rings for 15 min at
37°C. E. Negative ion electrospray ionization MS
spectrum of 14,15-EET-PISA metabolite (peak 21).

14,15-EET-Sulfonamides: Stable 14,15-EET Agonists
1029
Fig. 6. 14,15-EET-P125ISA binding to U937 membrane protein. A, time
course of 14,15-EET-P125ISA binding. 14,15-EET-P125ISA (1 nM) was
incubated with 50 ␮g of protein at 4°C for various time (1–30 min). n ϭ
2. B, concentration-dependent 14,15-EET-P125ISA binding. Increasing
concentrations of 14,15-EET-P125ISA (0.1–150 nM) were incubated with
50 ␮g of protein at 4°C for 15 min. n ϭ 2–7. C, 14,15-DHET-P125ISA
binding to U937 membranes. Increasing concentrations of 14,15-DHET-
P125ISA (2.5–100 nM) were incubated with 50 ␮g of protein at 4°C for 15
min. n ϭ 2–3.
Fig. 5. Metabolism of 14,15-EET-P125ISA by U937 cells and U937 mem-
brane protein. A, 14,15-EET-P125ISA (35 nM) was incubated with intact
U937 cells (800,000 cells) for 15 min at 37°C. B to D, 14,15-EET-P125ISA
(1 nM) was incubated with buffer (B), boiled U937 membrane protein (C),
or U937 membrane protein (50 ␮g, D) for 15 min at 4°C. HPLC chromato-
graphs of extracted 14,15-EET-P125ISA metabolites from the incubations
were shown.
remained stable until 30 min (Fig. 6A). Then, we tested the
saturability of the specific 14,15-EET-P125ISA binding. Various
concentrations of 14,15-EET-P125ISA (0.1–150 nM) were incu-
bated with U937 membrane (50 ␮g of protein) at 4°C for 15 min.
14,15-EET-P125ISA-specific binding was concentration-depen-
dent and reached an apparent plateau at 100 nM (Fig. 6B). The
data were fit to a one-site saturation model, and the predicted
Kd and Bmax were 148.3 Ϯ 36.4 nM and 3.3 Ϯ 0.5 pmol/mg
protein, respectively. To confirm the specificity of this binding
site to 14,15-EET-PISA, the binding of 14,15-DHET-P125ISA to
U937 membranes was determined. Specific binding of 14,15-
DHET-P125ISA to U937 membranes was very low with a max-
imum binding of 0.3 pmol/mg protein (Fig. 6C). The data poorly
fit with saturation models. This suggests that 14,15-DHET-
PISA does not specifically bind to U937 membranes.
Therefore, the 14,15-epoxide group is required for 14,15-
EET-P125ISA binding to U937 membranes. Taken to-
gether, binding of 14,15-EET-P125ISA to U937 membranes
is specific, time-dependent, concentration-dependent, and
saturable.
under the experimental conditions of radioligand binding.
After incubation with U937 membranes at 4°C for 15 min,
the majority of the radioactivity was extracted with an or-
ganic solvent, and the extraction efficiency was 93.6 Ϯ 1.0%
(n ϭ 3). When 14,15-EET-P125ISA was incubated with boiled
and control U937 membranes, the percentage of radioactivity
in the organic phase was 95.8 Ϯ 0.1 and 95.6 Ϯ 0.2%, respec-
tively (n ϭ 3). The HPLC chromatograms showed a single
peak eluting at 29 min (Fig. 5, B–D). These results indicate
that 14,15-EET-P125ISA is metabolically stable under the
conditions for radioligand binding.
To determine whether 14,15-EET-P125ISA is a radioligand
useful for the characterization of 14,15-EET binding, we used
membrane fractions from U937 cells, a cell line that was re-
ported to contain an EET binding site (Wong et al., 1997). To
test whether 14,15-EET-P125ISA binds to U937 membrane in a
time-dependent manner, 1 nM 14,15-EET-P125ISA was incu-
bated with U937 membranes (50 ␮g of protein) at 4°C for 1 to 30
min. The binding of 14,15-EET-P125ISA to U937 membranes
Discussion
Our present study developed and characterized a series of
was rapid and specific. Equilibrium was reached in 15 min and stable 14,15-EET-mSA derivatives, 14,15-EET-PISA, 14,15-

1030
Yang et al.
EET-BSA, and 14,15-EET-BZDC-SA, as tools to study EET agonist with high specific activity, low production cost, and
efficient yields.
transport, metabolism, and binding. The analogs caused con-
centration-dependent relaxation of bovine coronary arteries
and retained full agonist potency and activity compared with
14,15-EET. Attachment of a bulky group at the C1 of 14,15-
EET did not alter its vascular activity. The relaxations to the
analogs were blocked by inhibition of Kϩ efflux by 20 mM
extracellular Kϩ and BKCa channel blockade by iberiotoxin.
These data suggest that these agonists, like 14,15-EET, me-
diate vascular relaxation by activation of BKCa channels and
membrane hyperpolarization. 14,15-EEZE-mSA, a known
14,15-EET antagonist, inhibited the relaxations (Gauthier et
al., 2003). It is likely that these agonists bind to the same
binding site or receptor as 14,15-EET and act through the
similar cellular mechanisms as 14,15-EET.
EET-mediated activation of vascular smooth muscle BKCa
channels requires active Gs␣ protein and may involve ADP-
ribosylation of Gs␣ (Li and Campbell, 1997; Li et al., 1999).
Likewise, Gs␣ is required for 11,12-EET-mediated activation
of BKCa channels expressed in human embryonic kidney 293
cells (Fukao et al., 2001). Because G proteins characteristi-
cally couple to membrane receptors, it is likely that EETs
initiate their signaling events by binding to a G protein-
coupled receptor. Node et al. (2001) showed that 11,12-EET
stimulated GTP-binding activity of Gs␣ in human saphenous
vein endothelial cells and activated endothelial t-PA gene
expression through a cAMP-dependent mechanism. Taken
together, this suggests that EET may mediate their cellular
events through a G protein-coupled receptor signaling path-
way. However, characterization and purification of such a
membrane receptor/binding site have been limited due to
lack of proper tools. In addition, the mechanisms of EETs
uptake and secretion by cells are unclear. Many eicosanoids
such as prostaglandins (Kanai et al., 1995), leukotrienes
(Leier et al., 1994), and arachidonic acid (Krischer et al.,
1998) have been described to enter and leave cells through
transporter-facilitated diffusion. It is unknown whether a
similar transporter is present for EETs.
␤-Oxidation and phospholipid esterification of 14,15-EET
require the C-1 carboxyl group. Other routes of 14,15-EET
metabolism include sEH hydration, cytochrome P450 ␻-hy-
droxylation (Cowart et al., 2002), and glutathione conjuga-
tion (Spearman et al., 1985) and are independent of the C-1
carboxyl group. Therefore, 14,15-EET-P125ISA is a useful
tool to study C1-carboxyl-independent metabolism of 14,15-
EET. Like 14,15-EET, 14,15-EET-PISA is hydrolyzed into its
corresponding 14,15-DHET-PISA. This metabolism is effec-
tively inhibited by sEH inhibitors. Among the tested tissues,
the hydration was most active in BCA homogenate where
sEH is exposed, markedly less active in BCA rings and U937
cells where sEH is intracellular and less accessible, and
absent in U937 membrane fractions where sEH is not local-
ized. These data indicate that 14,15-EET-PISA is actively
metabolized into 14,15-DHET-PISA by sEH.
Previously in the human leukemic monocyte lymphoma
cell line U937 cells, a 14,15-EET binding site was character-
ized by whole-cell radioligand binding using [3H]14,15-EET.
The Kd and Bmax were 13.84 Ϯ 2.58 nM and 3.54 Ϯ 0.28
pmol/106 cells, respectively (Wong et al., 1997). We used
U937 cell membrane fraction to test 14,15-EET-P125ISA
binding. 14,15-EET-P125ISA remained stable and unchanged
when it was incubated with U937 membrane proteins at 4°C
for 15 min. In agreement with the [3H]14,15-EET whole-cell
binding data, 14,15-EET-P125ISA bound to U937 membranes
in a time-dependent, concentration-dependent, and saturable
manner. Interestingly, the binding of 14,15-EET-P125ISA to
U937 membranes was with a lower affinity and lower recep-
tor density. 14,15-EET-P125ISA binding to U937 membranes
reached equilibrium by 15 min and remained unchanged to
30 min at 4°C. The Kd was 148.3 Ϯ 36.4 nM, which was
10-fold higher than that observed with [3H]14,15-EET in
whole cells. The Bmax of 14,15-EET-P125ISA binding to U937
membranes was 3.3 Ϯ 0.5 pmol/mg protein, which was lower
than that observed in whole cells. The reason for these dis-
crepant data could be due to esterification of [3H]14,15-EET
into phospholipids or conjugation to cellular components.
This could contribute to the observed binding in whole cells,
whereas these processes are absent or less active in mem-
brane fractions. It is also possible that intracellular compo-
nents maintain the binding sites in a conformation that fa-
vors binding to the ligand. This intracellular factor is missing
in extracted membranes, which may change the receptor
conformation to a ligand-unfavorable form. In other words, a
smaller portion of the receptors in the membrane fractions
than whole cell is available for the ligand, which results in a
decreased receptor density. In addition, ligands differ in af-
finity. The addition of the sulfonamide group to C-1 may
reduce the affinity for the binding site. The variability be-
tween U937 cell lines could also account for the discrepan-
cies. The 14,15-EET-P125ISA binding requires the 14,15-ep-
oxide because its hydrolyzed product, 14,15-DHET-P125ISA,
did not bind to U937 membranes. Taken together, our bind-
ing data validate the use of 14,15-EET-P125ISA as a radiola-
beled tool to study 14,15-EET binding.
We designed 14,15-EET-mSA derivatives as tools to study
EET transport, metabolism, and binding. Each agonist con-
tains a functional group, i.e., a biotin, a photolinking reagent,
or a radioiodinated group. With aid of convenient avidin
conjugates such as agarose beads and fluorophore conju-
gates, 14,15-EET-BSA can be used to pull down an EET
binding protein or image 14,15-EET binding sites. Upon ul-
traviolet radiation, 14,15-EET-BZDC-SA cross-links with
molecules in close proximity. This reaction may add a tag to
the target molecule, which may be detected by mass spectro-
metric analysis. 14,15-EET-PISA can be radiolabeled with
[
125I] to provide a new radioactive 14,15-EET agonist. Radio-
active EET analogs are valuable tools to study their metab-
olism and binding.
Currently, radiolabeled 14,15-EET is made by epoxidation
of 14C- or 3H-labeled arachidonic acid, which results in all
four EET regioisomers and less than 25% production of
14,15-EET (Corey et al., 1979; Rosolowsky and Campbell,
1993). In contrast, radioiodination of 14,15-EET-P125ISA
from 14,15-EET-stannane is specific and efficient. In addi-
tion, [125I] has higher specific activity than [3H], which re-
sults in increased sensitivity for radioligand binding assays.
EETs are key regulators of vascular tone and active medi-
ators in pathophysiologic conditions such as anti-inflamma-
tion, fibrinolysis, angiogenesis, and ischemic reperfusion pro-
Therefore, 14,15-EET-P125ISA is a radioactive 14,15-EET tection (Seubert et al., 2004; Nithipatikom et al., 2006).

14,15-EET-Sulfonamides: Stable 14,15-EET Agonists
1031
Kozak W, Kluger MJ, Kozak A, Wachulec M, and Dokladny K (2000) Role of
cytochrome P-450 in endogenous antipyresis. Am J Physiol 279:R455–R460.
Krischer SM, Eisenmann M, and Mueller MJ (1998) Transport of arachidonic acid
across the neutrophil plasma membrane via a protein-facilitated mechanism.
Biochemistry 37:12884–12891.
However, whether they initiate their signaling through bind-
ing to specific protein(s)/receptor(s) is unclear. 14,15-EET-
PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA are 14,15-
EET agonists that serve as tools to identify and purify EET
binding protein(s). 14,15-EET-P125ISA, in particular, is a
radioactive tracer to study EET metabolism and a radioli-
gand to characterize EET binding kinetics.
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Acknowledgments
We thank Dr. Bruce Hammock for the kind gift of recombinant
sEH and sEH inhibitors, Dr. Kathy Gauthier for review of the manu-
script, and Gretchen Barg for secretarial assistance.
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Address correspondence to: Dr. William B. Campbell, Department of Phar-
macology and Toxicology, Medical College of Wisconsin, 8701 Watertown
Plank Road, Milwaukee, WI 53226. E-mail: wbcamp@mcw.edu