From the Departments of Medicine and
¶ Biochemistry, Vanderbilt University, Nashville, Tennessee 37232 and the § Department of Biochemistry, University of
Texas Southwestern, Dallas, Texas 75235
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ABSTRACT |
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A common feature of most isolated cell systems is low or undetectable levels of bioactive cytochrome P450. We therefore developed stable transfectants of the renal epithelial cell line, LLCPKcl4, that expressed an active regio- and enantioselective arachidonic acid (AA) epoxygenase. Site-specific mutagenesis was used to convert bacterial P450 BM-3 into an active regio- and stereoselective 14S,15R-epoxygenase (F87V BM-3). In clones expressing F87V BM-3 (F87V BM-3 cells), exogenous AA induced significant 14S,15R-epoxyeicosatrienoic acid (EET) production (241.82 ng/108 cells, >97% of total EETs), whereas no detectable EETs were seen in cells transfected with vector alone. In F87V BM-3 cells, AA stimulated [3H]thymidine incorporation and increased cell proliferation, which was blocked by the tyrosine kinase inhibitor, genistein, by the phosphatidylinositol 3 (PI-3) kinase inhibitors, wortmannin and LY294002, and by the mitogen-activated protein kinase kinase inhibitor, PD98059. AA also induced tyrosine phosphorylation of extracellular signal-regulated kinase (ERK) and PI-3 kinase that was inhibited by the cytochrome P450 BM-3 inhibitor, 17-ODYA. Epidermal growth factor (EGF) increased EET production in F87V BM-3 cells, which was completely abolished by pretreatment with either 17-ODYA or the phospholipase A2 (PLA2) inhibitor, quinacrine. Compared with vector-transfected cells, F87 BM-3 transfected cells demonstrated marked increases in both the extent and sensitivity of DNA synthesis in response to EGF. These changes occurred in the absence of significant differences in EGF receptor expression. As seen with exogenous AA, EGF increased ERK tyrosine phosphorylation to a significantly greater extent in F87V BM-3 cells than in vector-transfected cells. Furthermore, in these control cells, neither 17-ODYA nor quinacrine inhibited EGF-induced ERK tyrosine phosphorylation. On the other hand, in F87V BM-3 cells, both inhibitors reduced ERK tyrosine phosphorylation to levels indistinguishable from that seen in cells transfected with vector alone.
These studies provide the first unequivocal evidence for a role for the
AA epoxygenase pathway and endogenous EET synthesis in EGF-mediated
signaling and mitogenesis and provide compelling evidence for the
PLA2-AA-EET pathway as an important
intracellular-signaling pathway in cells expressing high levels of
cytochrome P450 epoxygenase.
Release of arachidonic acid secondary to phospholipase
A2 activation is a well recognized cellular response to a
variety of growth factors, hormones, and cytokines (1, 2). Metabolites of arachidonic acid play important roles as intracellular second messengers, with well documented autocrine effects of cyclooxygenase and lipoxygenase metabolites (1, 3-5). There is now abundant suggestive evidence that arachidonate metabolites generated by cytochrome P450, the third pathway of arachidonic acid metabolism, may
also serve as second messengers (6). These compounds have been
implicated in the regulation of peptide secretion, distal nephron
Na+ fluxes, cell Ca2+ influx, and activation of
Ca2+-dependent K+ channels (6).
However, previous studies have relied upon the use of inhibitors of
limited selectivity for cP450 or upon the exogenous administration of
relatively high concentrations of cP450 arachidonate metabolites; in
addition, a significant limitation to studies of
EET1 mechanisms and sites of
actions is the well documented loss and redistribution of cP450
isoforms that occur soon after the initiation of cell culture. To
overcome these limitations, we have used stable transfection of a
mammalian proximal tubule-like cell line, LLCPKcl4, with F87V BM-3, an
active and regio- and stereoselective 14S,15R arachidonic acid expoxygenase of bacterial origin containing a cP450
domain fused to a cP450 reductase domain (7). We report studies of the
mechanism of EET-dependent EGF-mediated mitogenesis and
provide unequivocal evidence that in these cells, cP450 arachidonic acid metabolite production increases in response to agonist activation, and cP450 arachidonic acid metabolites can serve as intracellular second messengers to mediate signal transduction and functional responses.
Antibodies and Chemicals--
Polyclonal rabbit anti-BM-3
antibodies were purified using protein G chromatography (7). Polyclonal
and monoclonal anti-phosphotyrosine antibodies were purchased from
(San Francisco, CA).
Monoclonal anti-PI-3 kinase antibody and anti-EGF receptor antibody
were from Transduction Laboratories (Lexington, KY). Polyclonal
anti-ERK antibodies and protein A-agarose beads were from Santa Cruz
Biotechnology (Santa Cruz, CA). Arachidonic acid was obtained from
NuCheck-Prep, Inc. (Elysian, MN). The sulfonimide analog of 14,15-EET
was synthesized as described previously (8). EGF (receptor grade) was
purchased from Collaborative Research (Bedford, MA). Genistein,
wortmannin, PD98059, and quinacrine were from Calbiochem. 17-ODYA was
from Biomol (Plymouth Meeting, PA). All other chemicals were from Sigma.
Cell Culture--
LLCPKcl4, an established proximal tubule
epithelial cell line derived from pig kidney (9), was routinely
cultured in Dulbecco's modified Eagle's medium/F-12 medium
supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin,
and 10% fetal calf serum (Hyclone Laboratories, Logan, UT) at 37 °C
in a 5% CO2 incubator. The medium was changed every 2-3 days.
Stable Transfection of Mutant BM-3--
The entire coding
region of mutant BM-3 (F87V BM-3) cDNA was cloned into the
HindIII and XbaI sites in the mammalian
expression vector pCB6+. 1 µg/ml F87V BM-3 cDNA in
pCB6+ or empty pCB6+ vector alone was used for
stable transfection. Cultured LLCPKcl4 cells were grown to 80%
confluence and were transfected using LipofectAMINE (Life Technologies,
Inc.). After 6 h, the DNA-LipofectAMINE mixture was removed and
the cells incubated in medium containing 10% fetal calf serum for
24 h, then cultured for 7 passages in medium containing 600 µg/ml of G418. G418-resistant clones were then isolated and screened
by immunoblotting with the affinity purified polyclonal antibody
against BM-3. Two of the clones that expressed detectable levels of
immunoreactive F87V BM-3, C9, and C23 (Fig. 1A), along with
LLCPKcl4 cells transfected with vector alone, were used for subsequent
functional studies.
Immunoprecipitation and Western Blot Analysis--
Quiescent
LLCPKcl4 cells were treated with arachidonic acid and/or other drugs as
indicated, then washed twice with ice-cold Ca2+/Mg2+-free phosphate-buffered saline and
lysed on ice for 30 min with radioimmune precipitation buffer (8). Cell
lysates were clarified at 10,000 × g for 15 min at
4 °C, and protein concentrations were determined by the
bicinchoninic acid assay (Pierce). Tyrosine-phosphorylated proteins
were immunoprecipitated with anti-phosphotyrosine antibodies, immune
complexes captured with protein A-agarose beads, washed four times with
wash buffer (20 mM HEPES, pH 7.2, 100 mM NaCl, 0.1% Triton X-100, 10% glycerol, and 100 µM
Na3VO4), and eluted by boiling in sample buffer.
Total cell lysates or anti-phosphotyrosine immunoprecipitates, as
indicated in the corresponding data, were subjected to 7.5-15% SDS-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes, probed with the indicated primary antibody and
the appropriate secondary antibody conjugated with biotin, incubated
with preformed avidin-biotin-horseradish peroxidase complex using a
commercially available kit (ABC kit; Pierce), and the immune complexes
detected by a peroxidase-catalyzed enhanced chemiluminescence detection
system (ECL; Amersham, United Kingdom).
Endogenous EET Production Measurement and Stereochemical
Analysis--
Quiescent C9, C23, and empty vector-transfected cells
were treated with or without 30 µM arachidonic acid, 10 nM EGF, and/or indicated inhibitors for the indicated
times, scraped with a 1:1 mixture of media and CH3OH, and
then mixed with two volumes of CHCl3 containing 1 mM triphenylphosphine, and an equimolar mixture of
synthetic 14C-labeled 8,9-, 11,12-, and 14,15-EET (55-56
mCi/mmol, 30 ng each). After acidification, the samples were extracted
twice and the organic phases evaporated under argon. To the resulting
residue, 0.5 ml of 0.4 N KOH in 80% CH3OH was
added and the mixture incubated at 50 °C for 60 min. Acidification
was followed by extraction into ethyl ether and chromatography in
SiO2 as described (10). The EETs were resolved into
14,15-EET, and a mixture of 8,9- and 11,12-EET by reversed-phase
high-pressure liquid chromatography, and then derivatized to the
corresponding pentafluorobenzyl (PFB) esters by reaction with
pentafluorobenzyl bromine. Aliquots of the purified EET-PFB
regioisomers were individually dissolved in dodecane and analyzed and
quantified by NICI/gas chromatography/mass spectrometry, utilizing
CH4 as reagent gas, as described (10).
For stereochemical analysis, samples of enzymatically derived
[14C]14,15-epoxyeicosatrienoic acid (25 µg, 1 µCi/µmol) and of synthetic 14R,15S-EET and
14S,15R-EET were catalytically hydrogenated over PtO2 and esterified using excess pentafluorobenzyl bromine,
as described (10). The resulting PFB esters were purified by
reversed-phase high-pressure liquid chromatography and, after solvent
evaporation, the optical antipodes of the purified
PFB-14,15-epoxyeicosatrienoic acid were resolved by high-pressure
liquid chromatography on a Chiralcel OD column (4.6 × 250 mm)
(J.T. Baker Inc.) with an isocratic mixture of 0.11% isopropanol,
99.89% n-hexane at 1 ml/min with UV monitoring at 210 nm.
The retention times for the PFB esters of synthetic
14R,15S- and 14S,15R-EET
were 70.6 and 78.9 min, respectively (7).
[3H]Thymidine Incorporation
Assay--
Subconfluent cells in 24-well plates were made quiescent
with serum-free medium. Agonists and antagonists were routinely added to the quiescent cells in triplicate and incubated for 19 h,
followed by addition of 2 µCi/ml of [3H]thymidine to
pulse the cells for an additional 2 h. Cells were then washed four
times with ice-cold phosphate-buffered saline, precipitated twice (30 min each time on ice) with ice-cold 10% trichloroacetic acid, briefly
washed once with ice-cold ethanol, then lysed with 0.2 N
NaOH, 0.5% SDS lysis buffer, incubated at 37 °C for at least 30 min, and radioactivity of incorporated [3H]thymidine was
determined by liquid scintillation spectrometry (Beckman). Results were
plotted as the number of counts/min/well. Each experimental data point
represents triplicate or duplicate wells from at least four different
experiments. For determination of cell proliferation, cells were plated
in replicate dishes at a density of 105 cells in 60-mm
dishes in Dulbecco's modified Eagle's/F12 + 10% fetal calf serum.
24 h later, the medium was changed to Dulbecco's modified
Eagle's/F12 + 0.2% fetal calf serum and 5 µM
arachidonic acid.
Statistics--
Data are presented as means ± S.E. for at
least four separate experiments (each in triplicate or duplicate).
Unpaired Student's t test was used for statistical analysis
and for multiple group comparisons, analysis of variance and Bonferroni
t tests were used. A value of p < 0.05 compared with control was considered statistically significant.
Previous studies indicated that synthetic 14,15-EET was a potent
mitogen for renal epithelial cells and that the mitogenic effects of
this EET were mediated, at least in part, by Src kinase and the
initiation of a tyrosine kinase phosphorylation cascade (8). To
reproduce more physiologic conditions in which the putative lipid
mediator is synthesized endogenously from endogenous AA pools, we have
used a novel strategy to investigate the role of the 14,15-AA
epoxygenase in endogenous EET production and cell signaling. LLCPKcl4
cells, a well characterized proximal tubule cell line with undetectable
endogenous cP450 expression levels, were stably transfected with a
mutant form of bacterial cP450 BM-3. cP450 BM-3, isolated from
Bacillus megaterium, is a single polypeptide containing
fused cP450 and cP450 reductase domains that catalyzes
NADPH-dependent AA oxidation as a self-contained catalytic
unit (7). Replacement of phenylalanine 87 for valine converts this
protein into a regio- and stereoselective
14S,15R-epoxygenase (14S,15R-EET, 99% of total products, 98%
optical purity) (7). We identified two clones, C9 and C23, that
expressed immunoreactive F87V BM-3 proteins (Fig.
1A).
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
RESULTS
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Fig. 1.
A, expression of F87V BM-3 protein in
the transfected LLCPKcl4 cells. LLCPKcl4 cells
were transfected with the entire coding region of F87V BM-3 cDNA
using LipofectAMINE (Life Technologies, Inc.). Sixty-three
G418-resistant clones were isolated and screened by Western
immunoblotting with the affinity purified polyclonal antibody against BM-3. C9, C21, C22, C23, C24, and C25 were representative
G418-resistant clones of these isolated 63 clones. M was the pooled
G418-resistant multiple clone cells used for selecting single-cell
clones. B, EET production following administration of
exogenous arachidonic acid. C9, C23, and empty vector-transfected cells
(empty vector) were rendered quiescent with serum-free
medium for 24 h and treated with or without 30 µM
arachidonic acid for 30 min, then scraped into the culture medium, and
EET production was measured as described under "Experimental
Procedures." C, DNA synthesis. Arachidonic acid
induced [3H]thymidine incorporation in the F87V
BM-3-transfected clones, C9 and C23, but not in cells transfected with
empty vector alone (n = 3-7). D, cell
counts. In cells grown in the absence of other exogenous growth
factors, the addition of arachidonic acid (5 µM)
increased cell proliferation in C23 but not in cells transfected with
the empty vector.
As shown in Fig. 1B, in the absence of exogenous arachidonic acid, EET levels were low or undetectable in quiescent C9 or C23 or in control vector-transfected cells. Following incubation with exogenous arachidonic acid (30 µM), EET production increased significantly in both C9 (67.28 ng/108 cells) and C23 (241.82 ng/108 cells), whereas there was no increase in the cells transfected with empty vector alone. The greater increase in EET production in C23 was consistent with the higher levels of immunoreactive protein expression in this clone (Fig. 1A). The predominant EET present in arachidonic acid-treated F87V BM-3-transfected cells was 14S,15R-EET (>97% optical purity), indicating enzymatic production by the expressed F87V BM-3 (Table I).
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Together with increased EET production, the AA addition stimulated [3H]thymidine incorporation in a concentration-dependent manner in the F87V BM-3-transfected cells but not in cells transfected with the empty vector (Fig. 1C). In the absence of added growth factors, 5 µM arachidonic acid also stimulated cell proliferation in F87V BM-3-transfected cells but not in vector-transfected cells (Fig. 1D). There were no differences in growth rate in the absence of exogenous arachidonic acid administration (not shown). Similar to what was previously observed in LLCPKcl4 cells in response to exogenous administration of 14,15-EET (8), arachidonic acid stimulation of [3H]thymidine incorporation in F87V BM-3-transfected cells was blocked by the tyrosine kinase inhibitor, genistein (11) (Fig. 2A), by the PI-3 kinase inhibitors wortmannin (12) and LY294002 (13) (not shown), and by the MEK inhibitor, PD 98059 (14) (Fig. 2B), suggesting an important role for PI-3 kinase and mitogen-activated protein kinases (ERKs) in the EET-mediated mitogenic response.
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Arachidonic acid produced significant increases in tyrosine phosphorylation of ERK in F87V BM-3 cells (Fig. 2C) and also increased tyrosine phosphorylation of the p85 subunit of PI-3 kinase (not shown). On the other hand, no such effects were observed in vector-transfected cells. Pretreatment of the cells with 17-ODYA (15), which is a potent inhibitor of BM-3 P450 enzyme activity2 did not alter the tyrosine phosphorylation of ERKs by exogenously administered 14,15-EET in either empty vector-transfected cells or F87V BM-3-transfected LLCPKcl4 cells. However, 17-ODYA inhibited completely ERK tyrosine phosphorylation induced by arachidonic acid in F87V BM-3 cells (Fig. 2D).
To study hormonal regulation of EET production in these transfected cells and to investigate the role of this pathway in EGF signaling, LLCPKcl4 cells were exposed to EGF, a documented mitogen for these cells (8). EGF/EGF receptor interactions markedly increased EET production to 387.59 ng/108 cells in C23, whereas minimal increases were seen in empty vector-transfected LLCPKcl4 cells (Fig. 3A). The EGF-induced EET production in C23 was completely inhibited by 17-ODYA, and the PLA2 inhibitor, quinacrine, completely abolished the EGF-stimulated EET production in these cells (Fig. 3A).
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EGF-stimulated [3H]thymidine incorporation was significantly greater in the F87V BM-3-transfected LLCPKcl4 cells than in cells transfected with empty vector alone (Fig. 3B). F87V BM-3 transfection resulted in (a) increased sensitivity to EGF, i.e. stimulation of [3H]thymidine incorporation could be observed at lower concentrations of EGF in the transfected cells; and (b) augmentation of the mitogenic potency, i.e. maximal stimulation seen with 1 µM EGF was greater in the F87V BM-3-transfected LLCPKcl4 cells. These effects appeared to be associated with the changes in EGF-signaling efficiency, because no changes in the levels of immunoreactive EGF receptor were observed after cell transfection (Fig. 3C), whereas administration of EGF to F87V BM-3 cells increased ERK tyrosine phosphorylation to a significantly greater extent than in cells transfected with the vector alone (Fig. 3D). In cells transfected with the vector alone, as well as in wild type LLCPKcl4 cells (not shown), 17-ODYA had no effect on EGF-induced ERK tyrosine phosphorylation. Similarly, quinacrine did not affect EGF-induced ERK tyrosine phosphorylation in the vector-transfected cells (Fig. 3D). In contrast, either 17-ODYA or quinacrine reduced EGF-stimulated ERK tyrosine phosphorylation in the F87V BM-3-transfected LLCPKcl4 cells to levels not different from that observed in the empty vector-transfected cells (Fig. 3D).
A further indication of intracellular responses induced by endogenously produced EETs in the F87V BM-3-transfected cells was the finding that administration of arachidonic acid to these cells induced tyrosine phosphorylation of the EGF receptor (Fig. 4). These results are consistent with our previous findings of increased EGF receptor tyrosine phosphorylation following administration of exogenous 14,15-EET to wild type LLCPKcl4 cells (8).
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DISCUSSION |
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Cytochrome P450 epoxygenase catalyzes the NADPH-dependent epoxidation of arachidonic acid to 5,6-, 8,9-, 11,12-, and 14,15-EET, in a regio- and stereoselective manner (16). The early demonstration of endogenous EET biosynthesis in several rat and human organs (6, 16) and of the presence of EETs in human plasma and urine (17, 18) established this reaction as a formal metabolic pathway and provided further credence to its postulated functional roles. Previous studies have indicated that EETs, or their dihydroxy derivatives, have potent biological activities, including modulation of vascular tone (19), glomerular hemodynamics (20, 21), and regulation of mitogenesis (22). EETs have been suggested to be an endothelial-derived hyperpolarizing factor (23). In addition, recent studies have also suggested that EETs may serve as intracellular second messengers in vasculature (24) and in epithelia (25).
Most studies implicating a role for EETs as intracellular second messengers have been based on the effects of cP450 inhibitors and/or the use of synthetic EETs. The cP450 inhibitors currently available do not selectively inhibit epoxygenase activity, so these studies cannot definitively rule out the possibility that other cP450 arachidonate metabolites, other cellular activities of cP450, or even non-cP450-derived arachidonate metabolites, mediate the observed effects. Although reproduction of the effect with the addition of exogenous EETs is suggestive evidence, the high concentrations of exogenously administered EETs required to elicit biological responses has also raised the possibility of nonspecific effects. In addition, it is well documented that most lipid mediators are only one component of what are usually complex signaling processes that involve, in addition to the temporally and spatially controlled biosynthesis of the putative lipid mediator, the coordinated participation of additional signaling pathways to elicit the indicated cellular response. Therefore, addition of synthetic EETs or other lipid moieties bypasses the integrated cellular response seen in response to EGF or similar agonists. To overcome these limitations of previous studies, the present studies used stable transfection of a cP450 that has been genetically engineered to be a regio- and stereoselective epoxygenase to investigate the potential role of EETs as intracellular second messengers.
The findings in the present study demonstrate that 14,15-EET can serve as an intracellular second messenger for EGF in cells expressing epoxygenase activity and that these signaling mechanisms involve the activation of kinase-associated mitogenic pathways. Endogenous production of EETs significantly augmented the mitogenic effects of EGF, indicated both by augmented stimulation of early signaling responses such as mitogen-activated protein kinase and PI-3 kinase and by increases in DNA synthesis. In F87V BM-3-transfected cells, administration of exogenous arachidonic acid activated a tyrosine kinase cascade and increased [3H]thymidine incorporation. This activation required conversion of the arachidonic acid to 14S,15R-EET, because addition of 17-ODYA blocked 14,15-EET production and subsequent cellular responses. The lack of significant EET formation in the absence of either exogenous arachidonic acid or agonist stimulation, and the inhibition of EGF-mediated EET production and functional activity by PLA2 inhibition, indicate that, as with most enzymes of the AA cascade, the rate-limiting step for the cP450 pathway is substrate availability.
In addition to autophosphorylation following ligand binding, there has been increasing evidence for tyrosine phosphorylation of intrinsic tyrosine kinase receptors by activation of seven-transmembrane receptors (26-28). These G-protein receptor-mediated tyrosine-phosphorylated proteins use growth factor receptors such as EGF receptor and platelet-derived growth factor receptor as "scaffolds" (29, 30); unlike the sequence of events occurring when these receptors bind their ligands, they are not activated by autophosphorylation in this process but are tyrosine phosphorylated by Src-like kinases. Src, GRB2, and SOS may be associated via interactions with Shc, which interacts with SH2 domains of the receptor. It is therefore of interest that, together with our previous findings (8), our results indicate that the endogenous AA epoxygenase metabolite, 14,15-EET, also appears to activate Src kinase and initiate a tyrosine phosphorylation cascade that uses the EGF receptor as a scaffold and results in mitogen-activated protein kinase activation. cP450 arachidonic acid metabolites have been implicated as second messengers for a number of agonists that activate seven-transmembrane receptors, such as angiotensin II, bradykinin, and parathyroid hormone (31-33); whether EETs are also involved in activation of tyrosine kinase-mediated pathways by these agonists has not yet been determined.
The biological effects of prostaglandins, leukotrienes, and other polar arachidonic acid metabolites are largely mediated through specific cell surface G-protein-coupled receptors; in contrast, the mechanisms of activation of the less polar cP450 arachidonate metabolites is unclear. There have been reports of a specific binding site for 14,15-EET in monocytes (34, 35). In addition, recent studies have indicated that EETs activated calcium-activated K+ channels in vascular smooth muscle cells. EETs did not activate channels when added directly to cytoplasmic surface of excised inside-out patches but required intermediate signaling steps involving G-proteins (36, 37). It is also possible that these compounds may directly activate or modulate enzymatic activity or could become incorporated into phospholipid and thereby modulate function. The availability of cultured cells with robust functional epoxygenase activity should provide a valuable tool for elucidation of the mechanisms of action of EETs.
In summary, the lack of significant functional cP450 activity in
cultured cell systems has contributed to the failure heretofore to
appreciate fully the signaling capabilities of these compounds. The
present studies provide direct and definitive evidence that cP450
arachidonate metabolites can serve as intracellular second messengers
in cells expressing functional cP450 activity and should stimulate
further research to explore the range of functions and molecular
mechanisms of cP450 arachidonate metabolites.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK38226 (to R. C. H., J. C., and J. R. F.) and funds from the Department of Veterans Affairs (to R. C. H.).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.
To whom correspondence should be addressed: Div. of Nephrology,
S 3322, MCN, Vanderbilt University School of Medicine, Nashville, TN
37232. Tel.: 615-343-0030; Fax: 615-343-7156; E-mail: Ray.Harris{at}mcmail.vanderbilt.edu.
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ABBREVIATIONS |
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The abbreviations used are: EET, epoxyeicosatrienoic acid; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; PFB, pentafluorobenzyl; PLA2, phospholipase A2.
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REFERENCES |
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