From the Cardiovascular Division, Brigham and Women's Hospital and
Harvard Medical School, Boston, Massachusetts 02115, the
Department of Internal Medicine and Therapeutics, Osaka
University Graduate School of Medicine, Osaka 565-0871, Japan, and the
§ Laboratory of Pulmonary Pathobiology, NIEHS, National
Institutes of Health,
Research Triangle Park, North Carolina 27709
Received for publication, January 17, 2001, and in revised form, February 19, 2001
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ABSTRACT |
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The epoxyeicosatrienoic acids (EETs) are products
of cytochrome P450 (CYP) epoxygenases that have vasodilatory and
anti-inflammatory properties. Here we report that EETs have additional
fibrinolytic properties. In vascular endothelial cells, physiological
concentrations of EETs, particularly 11,12-EET, or overexpression of
the endothelial epoxygenase, CYP2J2, increased tissue plasminogen
activator (t-PA) expression by 2.5-fold without affecting plasminogen
activator inhibitor-1 expression. This increase in t-PA
expression correlated with a 4-fold induction in t-PA gene
transcription and a 3-fold increase in t-PA fibrinolytic activity and
was blocked by the CYP inhibitor, SKF525A, but not by the
calcium-activated potassium channel blocker, charybdotoxin,
indicating a mechanism that does not involve endothelial cell
hyperpolarization. The t-PA promoter is cAMP-responsive, and induction
of t-PA gene transcription by EETs correlated with increases in
intracellular cAMP levels and, functionally, with cAMP-driven promoter
activity. To determine whether increases in intracellular cAMP levels
were due to modulation of guanine nucleotide-binding proteins, we
assessed the effects of EETs on G The epoxyeicosatrienoic acids
(EETs)1 are important
vasoactive products of cytochrome P450 epoxygenases (1-3). The EETs
have properties similar to those of endothelium-derived
hyperpolarizing factor because they hyperpolarize and relax vascular
smooth muscle cells by activating calcium-sensitive potassium
(KCa2+) channels (4, 5). Recently, two CYP
epoxygenases, CYP2C8 and CYP2J2, have been identified in vascular
endothelial cells (1, 6). Although both enzymes produce EETs, the
regulation of CYP2C8 and CYP2J2 appears to be somewhat different, and
their products may exert additional nonvasodilatory effects. For
example, Fisslthaler et al. (1) have shown that treatment of
porcine coronary artery endothelial cells in vitro with
An interesting observation is that endogenous vasodilators such as
nitric oxide (NO) frequently possess anti-inflammatory (8),
antithrombotic (9), and antiproliferative properties (10). Indeed,
vasoconstriction, smooth muscle cell proliferation, inflammation, and
thrombosis are hallmarks of atherosclerotic vascular disease (11). In
particular, the rupture of atherosclerotic plaques with ensuing
occlusive thrombosis is the predominant feature underlying acute
coronary syndromes (12). Thus, it is possible that EETs may play an
additional role in the regulation of vascular hemostasis. Vascular
thrombosis is regulated, in part, by endogenous mediators such as the
proteolytic enzyme, tissue-type plasminogen activator (t-PA), and its
inhibitor, plasminogen activator inhibitor (PAI)-1 (13). The function
of t-PA is to convert plasminogen to a proteolytic enzyme, plasmin,
which digests fibrin-dependent blood clots. Thus,
thrombotic vascular complications may be attenuated by agents that
increase the secretion of t-PA and/or decrease the expression of PAI-1.
Indeed, the plasma level of t-PA has been shown to be inversely
correlated with the risk of myocardial infarction (14). The purpose of
this study, therefore, was to determine whether EETs could regulate
t-PA or PAI-1 expression and, if so, by what mechanism.
Reagents--
All standard culture reagents were obtained from
JRH Bioscience (Lenexa, KS). Ascorbic acid, creatinine phosphate,
phosphocreatine kinase, GTP Cell Culture--
Endothelial cells were harvested from human
saphenous veins and bovine aortas as described (8, 15). The cells were
cultured at 37 °C in a growth medium containing Dulbecco's modified
Eagle's medium) supplemented with 5 mM
L-glutamine, 10% fetal calf serum (Hyclone, Logan, UT),
and an antibiotic mixture of penicillin (100 units/ml), streptomycin
(100 mg/ml), and Fungizone (250 ng/ml). Relatively pure (>95%)
endothelial cell cultures were confirmed by Nomarski optical microscopy
(Olympus IX70, × 40 objective) and by immunofluorescence staining with
anti-factor VIII antibodies (Vector Laboratories, Inc., Burlingame,
CA). All passages were performed with a disposable cell scraper
(Costar), and only endothelial cells of less than six passages were
used. Confluent endothelial cells (~2 × 106 for
human and ~5 × 106 for bovine) were treated with
various concentrations of EETs for the indicated time intervals.
Measurements of EETs--
The method used to quantify endogenous
EETs present in bovine aortic endothelial cells was similar to
those used to quantify EETs in tissue preparations (7, 16, 17).
Briefly, 8 × 106 endothelial cells were frozen in
liquid nitrogen and homogenized in 15 ml of phosphate-buffered saline
containing triphenylphosphine (10 mg), and the homogenate was extracted
under acidic conditions with two volumes of chloroform/methanol (2:1)
and twice more with an equal volume of chloroform. The combined organic
phases were evaporated in tubes containing mixtures of
1-14C-labeled 8,9-, 11,12-, and 14,15-EET internal
standards (57 µCi/µmol; 30 ng each), which were synthesized from
[1-14C]arachidonic acid by nonselective epoxidation (18).
Saponification to recover phospholipid-bound EETs was followed by
silica column purification. The eluent, containing a mixture of
radiolabeled internal standards and total endogenous EETs, was resolved
into individual regioisomers by HPLC as described (7, 16, 17). For
analysis, aliquots of individual EET-PFB esters were dissolved in dodecane and analyzed by gas chromatography/mass spectrometry as
described (7, 16, 17). Quantifications were made by selected ion
monitoring at m/z 319 (loss of PFB from
endogenous EET-PFB) and m/z 321 (loss of PFB from
[1-14C]EET-PFB internal standard). The
EET-PFB/[1-14C]EET-PFB ratios were calculated from the
integrated values of the corresponding ion current intensities.
Measurement of t-PA Expression and Activity--
The amounts of
t-PA and PAI-I antigens expressed by endothelial cells were determined
using the ImulyseTM reagent kits from Biopool (Umea, Sweden) according
to the manufacturer's instructions. The t-PA activity in endothelial
cell supernatants was determined by a chromogenic assay
(Spectrolyse/Fibrin; Biopool). The t-PA in the supernatant from
conditioned cells was bound using a specific t-PA monoclonal antibody
(SP-322). Preliminary studies indicate that the SP-322 antibody
completely bound all of the t-PA without affecting subsequent t-PA
activity. The t-PA substrate consisting of plasminogen,
plasmin-sensitive chromogenic substrate, and co-factors for t-PA
activity (HEPES buffer, pH 8.5) were added to the bound t-PA, and
absorption was read at 405 nm. The standard curve was constructed using
human single-chain t-PA, which has been calibrated against an
international standard for t-PA (Lot 66570; NIBSC, Hartfordshire, UK).
Northern Blotting--
Equal amounts of total RNA (20 µg) were
separated by 1.2% formaldehyde-agarose gel electrophoresis,
transferred overnight onto Hybond nylon membranes (Amersham Pharmacia
Biotech) by capillary action, and baked for 2 h at 80 °C before
prehybridization. Radiolabeling of full-length human t-PA or PAI-1
cDNA was performed using random hexamer priming,
[ [35S]GTP
The following G protein antisera with their corresponding final
dilutions were added to the mixture: G Measurement of Intracellular cAMP Levels--
Confluent
endothelial cells were pretreated with 3-isobutyl-1-methyl-xanthine
(0.5 mM) for 30 min prior to stimulation with the indicated
concentrations of EETs for 5 min. Cells were scraped on ice, pelleted,
and resuspended in ice-cold 3-isobutyl-1-methyl-xanthine (0.5 mM), boiled for 3 min, and frozen at Transfection Studies--
The human t-PA promoter was obtained
by the standard PCR method using a human genomic library (Invitrogen)
as template. The following paired primers were used: sense,
5'-CGATCGGTACTTTCGGGATGATTCAAGAGGATTAC-3'; antisense,
5'-CGATCAGATCTGAAAGAAGAGGAGACAGACCCCAAG-3'. The PCR product (3.6 kilobase pairs) containing a 3.1-kilobase pair t-PA promoter was
obtained using the following PCR conditions (35 cycles): annealing at
48 °C for 2 min, elongation at 68 °C for 4 min, and denaturing at
94 °C for 30 s. The t-PA promoter was verified by sequence
analysis and corresponded exactly to the already published 5'-flanking
sequence of the t-PA gene (20). The t-PA promoter was then subcloned
into a luciferase reporter construct, pGL.2 (Promega, Madison, WI). For
cAMP-driven promoter activity, a heterologous promoter (pCRE-Luc,
Stratagene) containing four tandem cAMP-response element (4×-CRE)
linked to the luciferase reporter gene was used to assess
cAMP-dependent gene transcription. The pcDNA3.1/CYP2J2 expression plasmid was prepared as described (6).
Transient transfections in bovine aortic endothelial cells were
accomplished using the calcium phosphate precipitation method as
described (8). Preliminary studies with Data Analysis--
All values are expressed as mean ± S.E.
compared with controls and among separate experiments. Paired and
unpaired Student's t tests were employed to determine the
significance of changes in GTP binding and luciferase activities. A
significant difference was taken for p values of less than
0.05.
Cell Culture--
There were no observable effects of synthetic
EETs or 11,12-DHET, the hydration metabolite of 11,12-EET, on cell
number, morphology, or immunofluorescent staining for factor
VIII-related antigens. Cellular confluence and viability as determined
by trypan blue exclusion was maintained for all treatment conditions described.
Endothelial Arachidonic Acid Metabolism and Detection of Endogenous
EETs--
We have previously shown that bovine endothelial cells
metabolize radiolabeled arachidonic acid to epoxygenase products (EETs and DHETs) and that endothelial EET biosynthesis is augmented by
transfection with an expression vector containing the CYP2J2 cDNA
(6). Using a combination of HPLC and gas chromatography/mass spectrometry techniques, we detected EETs as endogenous constituents of
cultured bovine aortic endothelial cells (Table
I). Endothelial cells contained 7.98 ± 0.90 ng of EET per 107 cells. The 8,9-, 11,12-, and
14,15-EET regioisomers were each present in roughly equal amounts (40, 28, and 32% of total EETs, respectively). The labile 5,6-EET suffers
extensive decomposition during the extraction and purification process
used and therefore cannot be quantified. Transfection of endothelial
cells with the pcDNA3.1 vector containing the CYP2J2 cDNA
resulted in a significant 30% increase in total EETs that was due
largely due to increases in 11,12- and 8,9-EET (Table I). Treatment of
CYP2J2-transfected cells with SKF525A (100 µM)
significantly reduced total EETs to control levels. The documentation
of EETs in endothelial cells provides direct evidence to support the
epoxidation of endogenous arachidonic acid pools by endothelial
cytochrome P450s.
Effects of EETs on t-PA Expression and Activity--
In a
concentration-dependent manner, treatment of endothelial cells
with physiologically relevant concentrations of 5,6-, 8,9-, 11,12-, 14,15-EET, or 11,12-DHET (1-1000 nM, 24 h) increased t-PA protein levels (Fig. 1A).
Maximal increase was achieved with 11,12-EET at 100 nM.
Interestingly, 14,15- and 8,9-EET induced significantly less t-PA
expression than 11,12-EET, indicating differential bioactivity of
specific EET regioisomers. The increase in t-PA protein levels by
11,12-EET correlated with increases in t-PA steady-state mRNA
levels (Fig. 1B). Concentrations of 10 and 100 nM 11,12-EET increased t-PA mRNA levels by 40 ± 5 and 95 ± 6%, respectively (p < 0.05 for both,
n = 3). Consistent with effects on t-PA protein levels,
a higher concentration of 11,12-EET (i.e. 1000 nM) also did not produce any further increase in t-PA mRNA levels. Interestingly, the increase in t-PA mRNA
expression was not due to the membrane hyperpolarizing effects
of EETs, since the selective KCa2+ blocker,
charybdotoxin (100 nM), was unable to inhibit the increase
in t-PA expression by 11,12-EET.
The increase in t-PA expression by 11,12-EET correlated with a 2.8-fold
increase in t-PA fibrinolytic activity (Fig.
2A). Maximal t-PA fibrinolytic
activity was observed at 100 nM 11,12-EET. However, 10 nM 11,12-EET, which produced a smaller increase in t-PA
expression compared with that of 100 nM, did not produce significant increases in t-PA fibrinolytic activity. This may be due to
endogenous levels of PAI-1, which is known to bind and inhibit t-PA
(13). However, in contrast to t-PA, EETs have no effect on PAI-I
protein levels (Fig. 2B). These findings suggest that
treatment with EETs would favor fibrinolysis by increasing the
ratio of t-PA to PAI-1.
In a time-dependent manner, 11,12-EET (100 nM)
increased t-PA protein levels with maximal increase occurring after
24 h of exposure (Fig.
3A). This increase in t-PA
expression was attenuated by co-treatment with the translational
inhibitor, cycloheximide (CHX; 5 µmol/liter), indicating
that 11,12-EET does not promote the release of already synthesized t-PA
but that de novo protein synthesis was required. Since t-PA
expression is known to be induced by agents or conditions that increase
cAMP-dependent pathways (21-23), we investigated whether
the induction of t-PA expression by EETs was mediated by the
cAMP-dependent kinase such as PKA. Co-treatment with
the PKA inhibitor, H-89 (30 µM), almost completely inhibited EET-induced t-PA expression (Fig. 3B). Again, the
selective KCa2+ blocker, charybdotoxin (100 nM), was unable to inhibit the increase in t-PA expression
by 11,12-EET, suggesting a nonhyperpolarizing effect of EETs on t-PA
expression.
Effects of EETs and CYP2J2 on t-PA Gene Transcription--
Using a
3.1-kilobase pair human t-PA promoter linked to a luciferase reporter
(pt-PA.Luc), we found that 11,12-EET and, to a lesser extent,
11,12-DHET increased t-PA promoter activity in a
concentration-dependent manner (Fig.
4A). A maximal 4.3-fold increase in t-PA promoter activity by 11,12-EET occurred at a concentration of 100 nM with an EC50 value of
~10 nM. To determine whether overexpression of the
endothelial cytochrome P450 isoform, CYP2J2, which produces EETs, can
increase t-PA gene transcription, endothelial cells were transiently
co-transfected with pcDNA3.1/CYP2J2 cDNA. We have previously
shown that, compared with endothelial cells transfected with the empty
vector, CYP2J2-transfected endothelial cells produce epoxygenase
metabolites at a 2-3-fold higher rate and that this increase was
almost completely inhibited by SKF525A (6). Compared with transfection
with the empty vector (pcDNA3.1), transfection with CYP2J2 caused a
5.2-fold induction in t-PA promoter activity, which was almost
completely blocked by the nonspecific cytochrome P450 epoxygenase
inhibitor, SKF525A (Fig. 4B). The KCa2+ blocker charybdotoxin, however, had no
effect on EET-induced t-PA promoter activity. These findings suggest that CYP2J2-derived eicosanoids increase t-PA gene transcription.
Effects of EETs and CYP2J2 on cAMP-dependent Gene
Transcription--
The induction of t-PA gene transcription is
regulated, in part, by increases in intracellular cAMP (21-23). We
found that EETs, particularly 11,12-EET, increased intracellular cAMP
levels in a concentration-dependent manner (Fig.
5A). Treatment with 11,12-EET (100 nM) produced a 3-fold increase in intracellular cAMP
levels (3.1 ± 0.2 to 10.2 ± 0.7 pmol/500,000 cells;
p < 0.01, n = 3). To address the
question of whether the increase in intracellular cAMP by 11,12-EET can
lead to transactivation of cAMP-response element (CRE)-containing
promoters, we transfected endothelial cells with a heterologous
luciferase reporter construct containing four tandem CREs (pCRE-Luc).
Treatment of transfected endothelial cells with 11,12-EET (1-1000
nM, 12 h) increased pCRE-Luc activity by up to
6.8-fold (p < 0.01, n = 4), with
maximal effect occurring at a concentration of 100 nM (Fig.
5B). These findings suggest that increases in intracellular
cAMP levels and induction of cAMP-responsive trans-acting
factors mediate increases in t-PA expression by EETs.
In support of the role of cAMP in CRE-mediated gene transcription, we
found that the increase in pCRE-Luc activity by 11,12-EET (100 nM) was inhibited in a time-dependent manner by
inhibition of the cAMP-dependent kinase, PKA (Fig.
6A). Treatment with the PKA
inhibitor, H-89, decreased 11,12-EET-induced CRE promoter activity by
70% after 24 h. To determine whether endogenous EETs produced by
CYP2J2 can increase CRE promoter activity, we co-transfected endothelial cells with pcDNA3.1/CYP2J2. Compared with transfection with the empty pcDNA3.1 vector, overexpression of
pcDNA3.1/CYP2J2 increased CRE promoter activity by 7.3-fold after
12 h (Fig. 6B). This effect was almost completely
blocked in the presence of the cytochrome P450 inhibitor, SKF525A.
These findings suggest that CYP2J2-derived eicosanoids can functionally
increase CRE promoter activity.
Effects of EETs on G Proteins--
To determine the whether the
increase in cAMP is due to the effects of EETs on G proteins, we
measured G We have shown that EETs, particularly 11,12-EET, increase
endothelial t-PA expression and activity by a transcriptional mechanism involving activation of G The precise mechanism by which EETs alter membrane signal transduction
and increase t-PA expression is not known. Previous studies have shown
that 11,12-EET stimulates the ADP-ribosylation of a number of
intracellular proteins in vascular smooth muscle including
G The regulation of gene transcription by cAMP is mediated by
trans-acting factors, which bind to cAMP-response element
(CRE) (5'-ATGACGTCAT-3') of target genes (29). Although the consensus sequence for CRE is not present in the t-PA promoter, a functional CRE-like element has been identified, which acts synergistically with a
putative AP-2 binding site to induced t-PA gene transcription by
phorbol 12-myristate 13-acetate (22). Indeed, previous studies have
shown that cholera toxin and dibutyryl-cAMP can directly induce t-PA
gene transcription in rat hepatocytes (30, 31). Furthermore, we have
previously shown that low concentrations of alcohol enhance
isoproterenol-induced t-PA gene transcription via increase in cAMP
levels and induction of CRE promoter activity in vascular endothelial
cells (32). These studies, therefore, suggest that the activation of
CRE and CRE-binding protein(s) may play an important role in mediating
the induction of t-PA by EETs.
The vasodilatory effect of EETs, which resemble endothelium-derived
hyperpolarizing factor, is mediated by activation of
KCa2+ channels and subsequent hyperpolarization
of vascular smooth muscle membranes (4, 5). The anti-inflammatory
effect of EETs involves inhibition of cellular adhesion molecule
expression via inhibition of NF- The finding that EET induces t-PA may have important clinical
implications, since the etiology of acute coronary syndromes is thought
to be due to atherosclerotic plaque rupture with ensuing vascular
thrombosis and occlusion (11, 12). Our results, therefore, suggest that
EETs may lower the risks of cardiovascular disease by augmenting t-PA
levels and enhancing the overall fibrinolytic activity in the vascular
wall. However, in contrast to the fibrinolytic effect of EETs, other
cytochrome P450-derived eicosanoids have been shown to have opposing
effects. For example, 15-hydroxyeicosatrienoic acid, an arachidonic
acid metabolite that is produced via oxidation and ketoreduction, has
been shown to decrease t-PA expression (34). The concentrations of EETs
used in our study are within the range of physiological
concentrations of EETs measured in the bloodstream of healthy humans
and rats, which is ~30 nM (16). However, it should be
noted that EET levels in human and rat heart tissues are estimated to
be 6-7-fold higher (35) and may be even higher during vascular injury
and inflammation.
In summary, our findings suggest that EETs may play an important role
in regulating the fibrinolytic balance in the vessel wall. The potency
of EETs' anti-fibrinolytic effects suggests that the biological
actions of these cytochrome P450-derived eicosanoids, like those of the
prostaglandins and leukotrienes, may be mediated through specific cell
surface receptors. The identification and functional characterization
of putative EET receptor(s) would greatly aid future research in this field.
s and
G
i2. Treatment with EETs increased G
s,
but not G
i2, GTP-binding activity by 3.5-fold. These
findings indicate that EETs possess fibrinolytic properties through the
induction of t-PA and suggest that endothelial CYP2J2 may play an
important role in regulating vascular hemostasis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-naphthoflavone induces CYP2C8 expression, increases 11,12-EET
biosynthesis, and enhances endothelium-derived hyperpolarizing
factor-mediated coronary artery relaxation. However,
-naphthoflavone
does not induce the CYP2J subfamily enzymes (7). Furthermore, we have
recently reported that EETs possess anti-inflammatory properties
through the inhibition of the proinflammatory transcriptional factor, NF-
B (6). In contrast to their vasodilatory effects, the
anti-inflammatory effects of EETs do not involve endothelial cell
hyperpolarization. These studies, therefore, indicate that EETs have
multiple homeostatic effects on the vascular wall in addition to their
vasodilatory effects.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S, phenylmethylsulfonyl fluoride,
leupeptin, aprotinin, dithiothreitol, bovine serum albumin, ATP, GDP,
GTP, charybdotoxin, H-89, and SKF525A were purchased from Sigma.
Triphenylphosphine,
-bromo-2,3,4,5,6-pentafluorotoluene,
N,N-diisopropylethylamine, and
N,N-dimethylformamide were purchased from
Aldrich. [1-14C]arachidonic acid,
[
-32P]GTP (30 Ci/mmol), [35S]GTP
S
(1250 Ci/mmol), and [
-32P]CTP (3000 Ci/mmol), and the
polyclonal rabbit antiserum to G
s (RM/1) were
supplied by PerkinElmer Life Sciences. The polyclonal rabbit
antiserum P4 was raised against a decapeptide corresponding to the
COOH-terminal region of G
i2 and has been characterized previously (15). Protein molecular weight markers were purchased from Life Technologies, Inc. The chemiluminescence detection kit was
obtained from Amersham Pharmacia Biotech. The polyvinylidene difluoride
transfer membrane (pore size 0.2 µm) was obtained from Bio-Rad. The
heterologous 4×-CRE promoter luciferase reporter construct (pCRE-Luc)
was purchased from Stratagene (San Diego, CA).
-32P]CTP, and Klenow (Amersham Pharmacia Biotech).
The membranes were hybridized with the indicated probes overnight at
45 °C in a solution containing 50% formamide, 5× SSC (1× SSC:
0.15 M NaCl and 0.015 M sodium citrate), 2.5×
Denhardt's solution, 25 mM sodium phosphate buffer (pH
6.5), 0.1% SDS, and 250 µg/ml salmon sperm DNA. All Northern blots
were subjected to stringent washing conditions (0.2× SSC, 0.1% SDS at
65 °C) before autoradiography with intensifying screen at 80 °C
for 24-72 h. Equalization of RNA loading was assessed by ethidium
bromide staining of 18 S ribosomal RNA.
S Binding Assay--
Partially purified
membranes were prepared from endothelial cells as previously described
(19). Membrane proteins (30 µg) from control and EET-treated
endothelial cells were incubated for 30 min at 30 °C in a buffer
containing [35S]GTP
S (20 nM), GTP (2 µM), MgCl2 (5 mM), EGTA (0.1 mM), NaCl (50 mM), creatine phosphate (4 mM), phosphocreatine kinase (5 units), ATP (0.1 mM), dithiothreitol (1 mM), leupeptin (100 µg/ml), aprotinin (50 µg/ml), bovine serum albumin (0.2%), and
triethanolamine HCl (50 mM, pH 7.4). In some samples,
membrane was stimulated with the
2-adrenergic agonist,
UK14304 (10 nM), for 10 min. The assay was terminated after
30 min with excess unlabeled GTP
S (100 µM). Samples
were then resuspended in 100 µl of immunoprecipitation buffer
containing Triton X-100 (1%), SDS (0.1%), NaCl (150 mM), EDTA (5 mM), Tris-HCl (25 mM, pH 7.4),
leupeptin (10 µg), aprotinin (10 µg), and phenylmethylsulfonyl
fluoride (2 mM).
i2 (P4, 1:20) and
G
s (RM/1, 1:100). The samples were allowed to incubate
for 16 h at 4 °C with gentle mixing. The antibody-G protein
complexes were then incubated with 50 µl of protein A-Sepharose (1 mg/ml; Amersham Pharmacia Biotech) for 2 h at 4 °C, and the
precipitate was collected by centrifugation at 12,000 × g for 10 min. Preliminary studies indicated that all
G
i2 and G
s were completely precipitated
by this procedure because Western blot analysis of the supernatant with
the P4 and RM/1 antisera did not reveal their presence. The pellets were washed three times in a buffer containing HEPES (50 mM, pH 7.4), NaF (100 µM), sodium phosphate
(50 mM), NaCl (100 mM), Triton X-100 (1%), and
SDS (0.1%). The final pellet containing the immunoprecipitated
[35S]GTP
S-labeled G protein was counted in a liquid
scintillation counter (LS 1800; Beckman Instruments, Inc.). Nonspecific
activity was determined in the presence of excess unlabeled GTP
S (10 µM).
70 °C. The
intracellular cAMP level was determined using a radioimmunoassay kit
with radiolabeled cAMP (Amersham Pharmacia Biotech). A standard curve
was constructed using increasing amounts of unlabeled cAMP.
-galactosidase staining
indicate that the transfection efficiency was 12-15%. Bovine
endothelial cells (60-70% confluent) were transfected with 5 µg of
either pt-PA-Luc or pCRE-Luc and 1 µg of pCMV.
-galactosidase construct. Approximately 48 h after transfection, endothelial cells were treated with 11,12-EET and the protein kinase A (PKA) inhibitor, H-89, alone or in combination, for 12 h. The luciferase and
-galactosidase activities were determined by chemiluminescence (Dual-Light, Tropix, Bedford, MA) using a Berthold L9501 luminometer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Endogenous EETs in control and CYP2J2-transfected endothelial cells
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Fig. 1.
A, cell surface enzyme immunoassay
showing the effects of EET regioisomers and 11,12-DHET (1-1000
nM) on t-PA expression (percentage of basal level). The
differences between treatment with and without EETs or 11,12-DHET were
statistically significant compared with untreated controls (*,
p < 0.05; **, p < 0.01).
B, representative Northern analyses (20 µg of total
RNA/lane) showing the effects of 11,12-EET (10-100
nM) with and without charybdotoxin (100 nM) on
endothelial t-PA mRNA levels at 12 h. Equal loading conditions
were determined by the corresponding ethidium bromide-stained Northern
blot for 18 S ribosomal RNA. Each experiment was performed three times
with similar results.
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Fig. 2.
A, effect of 11,12-EET (10-1000
nM) on endothelial t-PA activity (IU/ml). The differences
between treatment with and without 11,12-EET were statistically
significant (*, p < 0.05). B, PAI-1
expression (percentage of basal level) in human endothelial cells
treated with EETs or DHET (1-1000 nM).
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Fig. 3.
A, time-dependent effect of
11,12-EET (100 nM) on t-PA expression (percentage of basal
level) with or without cycloheximide (CHX; 5 µg/ml). *,
p < 0.05 versus control (time 0); #,
p < 0.05 versus 11,12-EET. B,
effect of PKA inhibitor, H-89 (30 µM) and
KCa2+ channel blocker, charybdotoxin (100 nM), on increases in t-PA expression by 11,12-EET. *,
p < 0.05 versus control; #,
p < 0.05 versus 11,12-EET.
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[in a new window]
Fig. 4.
A,
concentration-dependent effect of 11,12-EET and 11,12-DHET
(1-1000 nM) on t-PA promoter activity at 12 h.
Transfection efficiency was standardized to -galactosidase activity,
and changes in the ratio of t-PA promoter activity to
-galactosidase
activity were calculated relative to basal activity (-fold induction).
(*, p < 0.05; **, p < 0.01 versus basal). B, effects of transfection with
empty vector (pcDNA3.1) or pcDNA3.1/CYP2J2 on t-PA promoter
activity in the presence or absence of cytochrome P450 epoxygenase
inhibitor, SKF-525A (100 µM) or charybdotoxin (100 nM). *, p < 0.05 versus
pcDNA3.1; #, p < 0.05 versus
CYP2J2-transfected cells.
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Fig. 5.
A, effects of EETs and DHET (1-1000
nM) on intracellular cAMP levels at 5 min. *,
p < 0.05; **, p < 0.01 versus control. B,
concentration-dependent effect of 11,12-EET on CRE promoter
activity. Transfection efficiency was standardized to -galactosidase
activity, and changes in the ratio of t-PA promoter activity to
-galactosidase activity were calculated relative to basal activity
(-fold induction). (*, p < 0.05; **, p < 0.01 versus no treatment).
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Fig. 6.
Time-dependent effect (0-24 h)
of 11,12-EET on CRE promoter activity (-fold induction) in the presence
or absence of the PKA inhibitor, H-89 (30 µM). *, p < 0.05;
**, p < 0.01 versus control (time 0). #,
p < 0.05 versus 11,12-EET. B,
the effect of CYP2J2 transfection with and without cytochrome P450
epoxygenase inhibitor, SKF525A, on CRE promoter activity (-fold
induction). *, p < 0.01 versus
pcDNA3.1; #, p < 0.05 versus
11,12-EET.
i2 and G
s GTP binding activities in membranes from endothelial cells treated with EETs. Treatment with 11,12-EET (10-1000 nM, 5 min) caused a
progressive increase in G
s GTP-binding activity with a
maximal 3-fold increase (6.1 ± 2.1 to 18.5 ± 1.2 fmol/min/mg, p < 0.01, n = 3)
occurring at a concentration of 100 nM (Fig.
7A). In contrast, the same membrane from 11,12-EET-treated cells did not demonstrate any change in
basal G
i2 GTP-binding activity. UK14304 stimulated G
i2 GTP-binding activity by ~7-fold (2.8 ± 0.1 to 19 ± 3 fmol/min/mg, p < 0.01, n = 3), which was unaffected by 11,12-EET treatment. Other EET regioisomers such as 5,6-EET, 14,15-EET, and 8,9-EET also
increased G
s GTP-binding activity, albeit to a lesser
extent compared with 11,12-EET, without affecting G
i2
activity (Fig. 7B). These findings suggest that the effects
of EETs on cAMP levels, CRE promoter activity, and t-PA
expression/activity are probably mediated by their stimulatory effects
on G
s.
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Fig. 7.
Concentration-dependent effect of
11,12-EET (A) or EET regioisomers (B)
(100 nM) on specific G protein activity as determined by
immunoprecipitation of [35S]GTP S
labeling of G
s and UK14304 (10 nM)-stimulated
[35S]GTP
S labeling of
G
i2 at 5 min. *,
p < 0.01 versus control (Cont;
untreated).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
s. The activation of
G
s leads to the stimulation of adenylyl cyclase,
increase in intracellular cAMP, and induction of
cAMP-dependent promoter activity. The effect of EETs on
G
s activity is rather specific, since EETs did not affect basal or UK14304-stimulated G
i2 activity. Because
G
i2 is specifically coupled to UK14304-stimulated
2-adrenergic receptor in vascular endothelial cells
(24), these results suggest that EETs selectively modulate
G
s activity. The net effect of EETs on G proteins,
therefore, is to favor receptor-mediated activation of adenylyl
cyclase. Indeed, inhibition of cAMP-dependent PKA inhibits
EET-induced t-PA expression and CRE promoter activity. These findings
are in agreement with previous studies that showed that arteriolar
vasodilation by sulfonimide analogs of 11,12-EET analogs is associated
with elevation in intracellular cAMP levels and increase in PKA
activity (25).
s (26, 27). Indeed, the binding of 14,15-EET to its
putative receptor in U937 cells leads to the increase in cAMP levels
and PKA activity and subsequently to a decrease in its receptor binding
(28). Thus, it is possible that EET-induced ADP-ribosylation of
G
s results in the activation of G
s
similar to that of cholera toxin. Although we cannot exclude the
additional possibility that EETs may induce similar changes in other G
proteins, these effects of EETs are unlikely given that
G
i2 activity was not affected. It is also possible that
EETs may induce changes in the level of G
s and
G
i2. However, under the conditions of our study, the
observed changes in G
s activity and cAMP levels occurred
within 5 min; which is somewhat rapid for a transcriptional effect on G
proteins. Nevertheless, the downstream effect of cAMP is dependent upon
de novo protein synthesis, since the protein synthesis
inhibitor, cycloheximide, was able to attenuate EET-induced t-PA expression.
B activation (6). The
antiproliferative effect of EETs has not been reported, and we do not
find that 11,12-EET inhibits platelet-derived growth factor-induced
smooth muscle cell or fibroblast proliferation.2 Our study
showed that EETs possess anti-fibrinolytic properties through the
induction of t-PA expression. By linking EETs to the fibrinolytic
pathway, another important nonvasodilatory action of EETs is suggested,
and the homeostatic role of EETs in the vascular wall is broadened
considerably because thrombosis is implicated in vascular occlusive
disease and atherosclerosis (11, 12). In this respect, EETs are similar
to endothelium-derived NO, which relaxes vascular smooth muscle (33),
inhibits NF-
B (8), and prevents platelet aggregation (9). The
mechanisms by which EETs exert these effects, however, are quite
distinct from that of NO, which suggests that cAMP- and
cGMP-dependent pathways may work in concert to regulate
vascular hemostasis.
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ACKNOWLEDGEMENTS |
---|
We thank A. Jetton and T. Murphy for critical reading of the manuscript, J. R. Falck for EET and DHET standards, and S. Degen and D. Luskatoff for human t-PA and PAI-1 cDNAs.
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FOOTNOTES |
---|
* Supported by National Institutes of Health (NIH) Grants HL-52233 and HL-48743 and the NIEHS (NIH) Division of Intramural Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ An Established Investigator of the American Heart Association. To whom correspondence should be addressed: Cardiovascular Division, Brigham & Women's Hospital, 221 Longwood Ave., LMRC-322, Boston, MA 02115. Tel.: 617-732-6538; Fax: 617-264-6336; E-mail: jliao@rics.bwh.harvard.edu.
Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M100439200
2 K. Node, X.-L. Ruan, J. Dai, S.-X. Yang, L. Graham, D. C. Zeldin, and J. K. Liao, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
EET, epoxyeicosatrienoic acid;
KCa2+, channel,
calcium-activated potassium channel;
CYP, cytochrome P450;
DHET, dihydroxyeicosatrienoic acid;
t-PA, tissue-type plasminogen activator;
PAI, plasminogen activator inhibitor;
CRE, cAMP-response element;
GTPS, guanosine 5'-
-thiotriphosphate;
PKA, protein kinase A;
PCR, polymerase chain reaction;
HPLC, high pressure liquid chromatography;
PFB, pentafluorobenzyl.
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