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Vol. 284, Issue 3, 966-973, March 1998
Department of Pharmacology (M-H.W., E.B-S., B.A.Z., X.N., M.L.S.), New York Medical College, Valhalla, New York, and Departments of Biochemistry and Pharmacology (J.R.F., N.B.), University of Texas Southwestern Medical Center, Dallas, Texas
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Abstract |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We characterized the inhibitory activity of several acetylenic and
olefinic compounds on cytochrome P450 (CYP)-derived arachidonic acid
-hydroxylation and epoxidation using rat renal cortical microsomes
and recombinant CYP proteins. Among the acetylenic compounds,
6-(2-propargyloxyphenyl)hexanoic acid (PPOH) and
N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide were found to be
potent and selective inhibitors of microsomal epoxidation with
IC50 values of 9 and 13 µM, respectively. On the other
hand, 17-octadecynoic acid inhibited both -hydroxylation and
epoxidation of arachidonic acid with IC50 values of 7 and 5 µM, respectively. The olefinic compounds
N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) and
12,12-dibromododec-11-enoic acid (DBDD) exhibited a high degree of
selectivity inhibiting microsomal -hydroxylation with an
IC50 value of 2 µM, whereas the IC50 values
for epoxidation were 60 and 51 µM for DDMS and DBDD, respectively.
Studies using recombinant rat CYP4A isoforms showed that PPOH caused a
concentration-dependent inhibition of -hydroxylation and
11,12-epoxidation by CYP4A3 or CYP4A2 but had no effect on
CYP4A1-catalyzed -hydroxylase activity. On the other hand, DDMS
inhibited both CYP4A1- and CYP4A3- or CYP4A2-catalyzed arachidonic acid
oxidations. Inhibition of microsomal activity by PPOH, but not DDMS,
was time- and NADPH-dependent, a result characteristic of a
mechanism-based irreversible inhibitor. These studies provide
information useful for evaluating the role of the CYP-derived
arachidonic acid metabolites in the regulation of renal function and
blood pressure.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The
CYP monooxygenases constitute a major metabolic pathway for arachidonic
acid in the rat kidney. The CYP proteins are primarily localized to the
cortex with high concentrations in the proximal tubules, vasculature
and thick ascending limb of Henle's loop (TALH), where they metabolize
arachidonic acid mainly to 20-HETE as well as to 19-HETE and EETs
(Hardwick, 1991
; Omata et al., 1992; Dees et al.,
1982; Ma et al., 1993). Hydroxylation of arachidonic acid at
the -carbon to form 20-HETE is primarily catalyzed by isoforms of
the CYP4A family (Kimura et al., 1989a; Kimura et al., 1989b; Stromstedt et al., 1990; Laniado
Schwartzman et al., 1996). On the other hand, arachidonic
acid epoxidation is much less specific and can be carried out by
numerous CYP isoforms from different CYP gene families, including 1A1,
1A2, 2B1, 2B4, 2C2, 2C9, 2C11, 2C23, 2E1 and 2G1 (Laethem et
al., 1994; Laethem and Koop, 1992; Laethem et al.,
1993; Rifkind et al., 1995). In addition to having a broad
spectrum of substrate specificity, CYP isoforms also demonstrate a lack
of product specificity; i.e., one protein can catalyze the
oxidation of arachidonic acid at multiple sites. For example, we have
shown that CYP4A2 functions not only as an arachidonate -hydroxylase
but also as an arachidonate 11,12-epoxygenase (Wang et al.,
1996).
The potential role of the CYP-derived arachidonic acid metabolites in
the regulation of renal function and in hypertension has been
documented in numerous studies. 20-HETE, in vitro, is a
potent vasoconstrictor of renal arterioles (Ma et al., 1993; Imig et al., 1996; Zou et al., 1996c) and an
inhibitor of renal tubular transport and K+ channel
activity (Escalante et al., 1994; Wang and Lu, 1995). In vivo, 20-HETE affects renal vascular resistance,
autoregulation of renal blood flow and tubuloglomerular feedback
(Gebremedhin et al., 1993; Kauser et al., 1991;
Zou et al., 1994a; Zou et al., 1996a; Zou
et al., 1994b). In the kidney, EETs exhibit a broad and
contrasting spectrum of biological activities, including vasodilation and vasoconstriction (Carroll et al., 1992; Zou et
al., 1996b; Katoh et al., 1991) as well as inhibition
and stimulation of ion transport mechanisms (Harris et al.,
1990; Takahashi et al., 1990; Sakairi et al.,
1995; Hirt et al., 1989). Studies using different animal
models of hypertension and CYP inhibitors have implicated both
epoxidation and hydroxylation products of arachidonic acid in the renal
responses to changes in dietary salt levels and in hypertension
(Sacerdoti et al., 1989; Makita et al., 1994;
Imig et al., 1993; Zou et al., 1994c; Capdevila
et al., 1992; Brand-Schieber et al., 1996).
In order to elucidate the physiological and pathophysiological roles of
these arachidonic acid metabolites, it is important to inhibit one
CYP-catalyzed reaction selectively without affecting the others. The
fact that the CYP monooxygenases exist in many isoforms with relatively
high homology and broad substrate specificity complicates this task;
enzyme inhibitors are frequently nonspecific, and multiple isoforms
often demonstrate similar catalytic activities (Gonzalez, 1988). A
relatively new approach to inhibiting CYP activities selectively
involves the use of "suicide substrates" that were designed to
resemble the substrate and at the same time to inactivate the enzyme.
Inactivation is irreversible, and activity is restored on de
novo synthesis of the enzyme. Acetylenic derivatives, such as
17-ODYA, have been designed to be selective inhibitors of CYP-derived
fatty acid -hydroxylation (Muerhoff et al., 1989). The
effect of 17-ODYA on renal function has been studied extensively by Zou
et al. (1994c), who reported that infusion of 17-ODYA into either renal artery or cortical interstitium increased papillary blood
flow, urine flow and sodium excretion without affecting glomerular
filtration rate and cortical blood flow. However, because 17-ODYA
inhibited -hydroxylation and epoxidation of arachidonic acid with
similar potency (Zou et al., 1994c), it is difficult to sort
out the relative contribution of these metabolites to the overall
effect of the drug. Therefore, there is a need for selective inhibitors
that will expedite the determination of the role of each pathway in the
regulation of renal function and hypertension.
In the present study, we characterize the effect of several acetylenic
and olefinic fatty acid analogs on -hydroxylation and epoxidation of
arachidonic acid in rat renal cortical microsomes and on recombinant
rat CYP4A isoforms. This study provides information useful for
evaluating the role of the CYP-arachidonic acid metabolites in the
regulation of renal function and blood pressure.
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Materials. The following drugs and chemicals were used in this study: [1-14C]-arachidonic acid (56 mCi/mmol) (DuPont-New England Nuclear, Boston, MA), purified recombinant rat NADPH-CYP (c) oxidoreductase (OR) (specific activity, 58 µmol/min/mg) (Oxford Biomedical Research Inc., Oxford, MI), 17-ODYA (Cayman Chemical Co., Ann Arbor, MI), emulgen E911 (KAO Atlas, Tokyo, Japan), tissue culture and molecular biology reagents (Gibco-BRL, Gaithersburg, MD), Sf9 insect cells and the liposome DNA transfection kit (Invitrogen, San Diego, CA) and purified rat liver cytochrome b5 (specific activity, 40 nmol/mg) (Panvera Corp., Madison, WI). All solvents were HPLC grade.
Synthesis of CYP inhibitors. The structures of all compounds are shown in figure 1. The compounds PPOH, MS-PPOH, DDMS, DBDD and DPMS were synthesized and their structures confirmed by spectral analysis as described by Falck et al., in press.
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Tissue and microsome preparations.
Male Sprague-Dawley rats
(8 weeks old, Charles River, Wilmington, PA) were anesthetized with
pentobarbital sodium (50 mg/kg i.p.). Kidneys were removed, and the
cortex was dissected and homogenized in buffer containing 100 mM
Tris-HCl and 1.15% KCl, pH 7.4. Homogenates were centrifuged at
10,000 × g for 30 min. Microsomes were obtained by
centrifugation of the supernatant at 100,000 × g for
90 min and resuspended in 0.25 M sucrose buffer and stored at 
80°C.
Preparation of recombinant CYP4A cell membranes.
CYP4A
proteins were expressed in the baculovirus-Sf9 insect cell expression
system as described previously (Wang et al., 1996). Briefly,
CYP4A cDNAs were ligated into the Sma I site of the baculovirus expression vector, pVL1393. Transfection of the expression vector was
performed individually by an AcMNPV linear transfection kit (Invitrogen). AcMNPV DNA (1 µg) was mixed with 3 µg of
CYP4A-PVL1393 construct in a 5-ml polystyrene tube containing 1.5 ml of
serum-free Grace medium. A mixture of 30 µg of cationic liposomes and
1.5 ml of serum-free medium was added to the DNA, mixed and added into
cultured Sf9 cells dropwise. Transfected cells were incubated at 27°C
overnight, after which the medium was removed, fresh medium was added,
and the cells were incubated for 4 to 6 days. Identification of
recombinant viruses was done by visualization of Occ-negative plaques,
immunoblot and P450 spectral analysis. Recombinant viruses were
amplified in Sf9 cells. The titer for recombinant viruses was
determined by plaque assay. In order to prepare CYP4A recombinant cell
membranes, Sf9 cells were infected with CYP4A recombinant viruses and
cultured in the presence of hemin (4 µg/ml). After 72 h, the
cells were harvested, washed with 0.14 M NaCl in 50 mM potassium
phosphate (pH 7.2) and resuspended in sucrose buffer (50 mM potassium
phosphate, pH 7.4, and 0.5 M sucrose). Cell lysates were prepared by
brief sonication (4-5 bursts of 4-s duration) and subjected to
high-speed centrifugation (100,000 × g) for 60 min.
The membrane pellet was then resuspended in sucrose buffer and stored
at 80°C. Protein concentration was measured according to the method
of Bradford (BioRad; Melville, NY). CYP content was calculated from the
reduced CO-difference spectrum using an extinction coefficient of 91 mM
1 (Omura and Sato, 1964).
Incubation conditions, metabolite extraction and HPLC
separation.
Compounds (stock solution in ethanol) were diluted 10 to 20 fold with 100 mM potassium phosphate buffer, pH 7.5. The final concentration of ethanol in the incubations was 0.5% (v/v). At this
concentration of ethanol, there was no effect on the activities of
-hydroxylation and epoxidation of arachidonic acid. The diluted solution was then added into the incubation mixture consisting of
microsomes (150 µg of protein), 1 mM NADPH and buffer (100 mM
potassium phosphate and 10 mM MgCl2, pH 7.5). For the
recombinant CYP4A system, recombinant cell membranes containing the
expressed CYP4A isoform were mixed on ice with purified OR and
b5 at a molar ratio of 1:14:4, and the diluted inhibitor
solution at the indicated concentration, 1 mM NADPH and buffer (150 µl final volume) were added. Mixtures containing either microsomes or
recombinant proteins were preincubated at 37°C for 10 min.
[1-14C]-arachidonic acid (0.4 µCi, 7 nmol) was then
added and incubated at 37°C for 30 min. To determine whether
inhibition of arachidonic acid oxidation is reversible or irreversible,
a two-stage incubation protocol for time-dependent inhibition was
carried out. In the first-stage incubation, microsomes (1.5 mg) were
incubated at 37°C with 0.1 mM inhibitors either with or without 1 mM
NADPH (300 µl final volume). At given time intervals, an aliquot was diluted 10-fold by the addition of a second-stage assay mixture consisting of the same buffer, 0.9 mM NADPH and
[1-14C]-arachidonic acid (0.4 µCi, 7 nmol) in a total
volume of 0.4 ml. The reactions were continued for 20 min. The reaction
was terminated by acidification to pH 3.5 to 4.0 with 2 M formic acid, and the metabolites were extracted with ethyl acetate. The organic extract was evaporated under nitrogen, resuspended in methanol and
injected onto the HPLC column. Reverse-phase HPLC was performed on a
5-µm ODS-Hypersil column, 4.6 × 200 mm (Hewlett-Packard, Palo
Alto, CA) using a linear gradient ranging from
acetonitrile/water/acetic acid (50:50:0.1) to acetonitrile/acetic acid
(100:0.1) at a flow rate of 1 ml/min for 30 min. The elution profile of
the radioactive products was monitored by a flow detector (In/us System
Inc., Tampa, FL). The identity of each metabolite was confirmed by its comigration with an authentic standard. Specific activity in
nmol/mg/min was calculated from the added arachidonic acid, and results
were generally expressed as the mean ± S.E.M. of the percent of
the control activity. IC50 was determined by quantal
dose-response analysis.
COX assay. The effect of the various inhibitors on COX activity was measured using the purified ram seminal vesicles PGH1 synthase (Cayman Chemical Co., Ann Arbor, MI). The purified enzyme (10 units) was incubated with 1 nmol of arachidonic acid in 0.5 ml of incubation buffer (0.1 M Tris-HCl, pH 8, 1 mM EDTA, 2 mM phenol and 1 µM hematin) with or without PPOH (10 and 50 µM), MS-PPOH (15 and 50 µM), DBDD (2 and 20 µM), DDMS (2 and 20 µM), 17-ODYA (5 and 50 µM) and indomethacin (5 µM). Reaction mixtures were incubated in a 37°C shaking-water bath for 10 min before the addition of arachidonic acid and for 15 min thereafter. All samples were run in duplicate. Amounts of PGE2 were determined using a PGE2-EIA Kit (Cayman Chemical Co., Ann Arbor, MI) after 1000-fold dilution with Tris buffer.
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Because of the presence of epoxide hydrolase in microsomal
preparations (Oliw et al., 1982), EETs are readily converted
to their corresponding metabolites, dihydroxyeicosatrienoic acids (DHETs). We therefore considered epoxygenase activity as the sum of EET
and DHET formation. Both PPOH and MS-PPOH were designed to target
arachidonic acid epoxidation specifically. Addition of PPOH (1-50
µM) decreased microsomal arachidonate epoxygenase activity in a
concentration-dependent manner with an IC50 of 9 µM but
had almost no effect on -hydroxylation (fig.
2). Similar results were obtained with
MS-PPOH: no effect on microsomal arachidonic acid -hydroxylation
(20-HETE formation) and a concentration-dependent inhibition of EETs
and DHETs formation with an IC50 of 13 µM (data not
shown). These results indicate that PPOH and MS-PPOH specifically and
potently inhibit renal microsomal-derived epoxidation of arachidonic acid without affecting -hydroxylation.
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In order to examine the structure-function relationship of these
acetylenic compounds, we tested another aliphatic acetylenic fatty
acid, 17-ODYA. The effect of 17-ODYA on renal microsomal -hydroxylation and epoxidation of arachidonic acid is shown in figure 3. 17-ODYA was a very potent
inhibitor of both -hydroxylation and epoxidation of arachidonic acid
with similar IC50 values of 7 and 5 µM, respectively.
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In addition to these acetylenic compounds, we also examined some
dibromo-olefinic fatty acids (DBDD, DPMS and DDMS). Addition of DDMS to
the incubation mixture of renal microsomes caused a concentration-dependent inhibition of -hydroxylase activity with an
IC50 of 2 µM, but it caused only a weak inhibition of
epoxygenase activity with an IC50 of 60 µM (fig.
4). These results indicate that DDMS is a
specific and potent inhibitor of microsomal arachidonic acid
-hydroxylation. Both DDMS and DBDD demonstrated a pattern of
inhibition similar to that of DPMS with different potencies. The
IC50 values for all of inhibitors are summarized in
table 1.
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The specificity of these inhibitors was further examined by using
recombinant CYP4A isoforms. We have recently demonstrated that the
baculovirus-Sf9 cell-expressed CYP4A2 membranes possess dual catalytic
activity: -hydroxylation and 11,12-epoxidation (Wang et
al., 1996). Preliminary results indicated that CYP4A3 exhibits
similar catalytic activities (Nguyen et al., 1997). In contrast, the baculovirus-Sf9 cell-expressed CYP4A1 membranes (Nguyen
et al., 1997), as well as other forms of recombinant CYP4A1 (Alterman et al., 1995; Imaoka et al., 1989),
exhibit only arachidonic acid -hydroxylation activity. Given these
characteristics, we examined the effect of PPOH as an epoxygenase
inhibitor and that of DDMS as an -hydroxylase inhibitor on CYP4A3-
and CYP4A1-catalyzed arachidonic acid oxidations. The results are shown
in figures 5 and
6. PPOH at
30 µM inhibited both CYP4A3-mediated -hydroxylation and
11,12-epoxidation of arachidonic acid by 90%. In contrast, PPOH at the
same concentration had little effect on CYP4A1-catalyzed -hydroxylation of arachidonic acid (8% inhibition). On the other hand, DDMS inhibited both CYP4A1- and CYP4A3-catalyzed arachidonic acid
oxidations without differentiating between the two reactions (data not
shown).
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In order to characterize further the mechanisms of the inhibitory action of these inhibitors, we carried out a two-stage incubation. In the first stage, microsomes were preincubated with either acetylenic (PPOH) or olefinic (DDMS) compounds in the presence and absence of NADPH. Aliquots were then taken at different time-points and added to the incubation reaction, which contained 14C-arachidonic acid. Over a 15-min period in which PPOH was preincubated in the absence of NADPH, there was no time-dependent inhibition of DHET and EET formation (fig. 7A). However, when PPOH was preincubated in the presence of NADPH, there was a rapid, time-dependent decrease in the formation of DHETs and EETs. After a 15-min preincubation, the activity was significantly decreased by 60% (fig. 7A). The same approach was used to test the inhibitory mechanism of DDMS. As shown in figure 7B, neither preincubation time nor the presence of NADPH affected the degree of inhibition by DDMS.
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We further examined the effect of these compounds on COX activity by measuring PGH1 synthase-catalyzed conversion of arachidonic acid to PGE2. The results indicated that whereas this activity was readily inhibited by indomethacin (95% inhibition at 5 µM), it was unaffected by the various compounds tested in this study. DDMS, DBDD, DPMS and 17-ODYA at concentrations up to 20 µM had no effect on COX activity; however, PPOH and MS-PPOH at 50 µM inhibited COX activity by 20 and 40%, respectively. Nevertheless, these concentrations are much above the IC50 for epoxygenase activity, and thus they provide a window of selectivity in differentiating between CYP and COX activities.
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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It is well recognized that the CYP-derived eicosanoids constitute a new member of the arachidonic acid cascade with important implications in the regulation of physiological and pathophysiological processes. These metabolites are formed endogenously in various tissues and exert potent biological effects on cellular functions. Studies of their role in normal and diseased cells and tissues are impeded by the difficulty in selectively targeting their synthesis or their effects, because these metabolites are generated from multiple closely related proteins of the CYP superfamily. Consequently, the development of enzyme inhibitors that target specific isoforms/reactions should aid in the study of their pathophysiological roles.
We have synthesized a series of fatty acid/arachidonic acid analogs and
tested their potency and selectivity in inhibiting arachidonic acid
epoxidation and -hydroxylation reactions in rat renal microsomes.
Our study confirms that the widely used, terminal acetylenic 17-carbon
aliphatic compound 17-ODYA is a potent but nonselective inhibitor of
both arachidonic acid epoxidation and -hydroxylation in rat renal
microsomes. In contrast to 17-ODYA, the other terminal acetylenic
compounds, PPOH and MS-PPOH, selectively inhibited microsomal
arachidonic acid epoxidation with IC50 values of 9 and 13 µM, respectively, while having no effect on -hydroxylations at
concentrations up to 50 µM. The major structural difference among
these compounds is the presence of a benzene ring moiety in the PPOH
derivatives, which suggests that the benzene ring confers selectivity
toward the epoxidation reaction. To examine this possibility, Mancy
et al. (1996) have proposed a model for the active site of
CYP2C9, an arachidonate epoxygenase (Rifkind et al., 1995),
in which hydrophobic and/or 
- interactions may be important for
binding between the benzene ring of substrates and aromatic amino acid
residues of the protein. Alternatively, the -hydroxylases may have
sterically hindered binding sites that do not accommodate aryl
moieties. In a result similar to that observed with 17-ODYA, PPOH
inhibitory activity in renal microsomes increased with time and
required NADPH, which suggests the formation of an NADPH-dependent
intermediate that accounts for inactivation of the enzyme. Ortiz de
Montellano and Reich (1984) and Helvig et al. (1997) showed
that terminal acetylenes are CYP suicide-substrate inhibitors; they can
be further oxidized to a ketene moiety, which then results in
alkylation and inactivation of the CYP proteins. PPOH may fit in as a
suicide-substrate inhibitor. Furthermore, the time dependence and NADPH
dependence of PPOH inhibitory activity are consistent with a
mechanism-based irreversible inhibitor (Ortiz de Montellano and Reich,
1984; Muerhoff et al., 1989).
The acyclic compounds, i.e., DDMS, DBDD and DPMS, were
selective inhibitors of -hydroxylation. The rank order of inhibitory potency was DBDD = DDMS > DPMS, which suggests that a
carboxylic acid or methyl sulfimide with 12 carbons (DBDD and DDMS) may
be the best fit for the active site of the -hydroxylase CYP4A
isoforms. Indeed, we and others have demonstrated that recombinant
CYP4A proteins have a much higher lauric acid -hydroxylase activity than an arachidonic acid -hydroxylase activity (Wang et
al., 1996; Alterman et al., 1995; Imaoka et
al., 1989). Modification of the carboxyl group in DBDD to a methyl
sulfonate in DDMS did not change the potency or selectivity of the
inhibitory activity. This modification may be important for in
vivo studies, where blocking the carboxyl group renders the
compound resistant to 
-oxidation and makes the compound a more
effective inhibitor. Alonso-galicia et al. (1997)
administered DDMS locally into an isolated perfused renal arteriolar
preparation and systemically into anesthetized rats and demonstrated a
high selectivity for DDMS in inhibiting 20-HETE formation. However,
unlike 17-ODYA and PPOH, these acyclic dibromide derivatives did not
exhibit time- and NADPH-dependent inhibitory activity, a result that
emphasizes the importance of a terminal acetylenic bond in providing
the mechanisms for the enzyme's inactivation. These experiments also imply that inhibition by DDMS is reversible. Indeed, when microsomes were incubated with DDMS or DBDD, inhibition of 20-HETE formation could
be washed out (data not shown).
The dual catalytic activity of the recombinant CYP4A2/CYP4A3 proteins
as -hydroxylases and epoxygenases provides a means for examining
whether the inhibitors distinguish between these two reactions. The
results showed that PPOH caused a significant inhibition of
CYP4A3-catalyzed -hydroxylation and 11,12-epoxidation of arachidonic
acid but had no effect on CYP4A1-dependent -hydroxylase activity;
this suggests that PPOH inhibitory activity is isoform-specific. In
contrast to PPOH, DDMS potently inhibited both CYP4A1- and CYP4A3-catalyzed arachidonate -hydroxylation as well as
CYP4A3-catalyzed 11,12-epoxidation. Taken together, these results
suggest that CYP4A1 and CYP4A2/4A3 may have similar size or topology of
the active site, which allows the binding of common substrates such as
lauric and arachidonic acids. Conversely, there may be more space or
freedom for the orientation of substrates or inhibitors in the active
sites of CYP4A2/4A3 compared with those of CYP4A1. Furthermore, the
amino acid residues involved in the binding of substrates and
inhibitors may be quite different. In other words, it is possible that
the active site of CYP4A1 is more rigid than that of CYP4A2/4A3.
Indeed, Bambal et al. have proposed that the active site of
CYP4A1 is sterically hindered and rigid around the heme iron or ferryl
moiety, which may explain the prominent regioselectivity of
CYP4A1-catalyzed -hydroxylase activity (Bambal and Hanzlik, 1996)
and accounts for the differences between CYP4A1 and CYP4A2/4A3 that we
observed in catalytic activity and inhibitor sensitivity.
Previous studies by Capdevila et al. (1988) examined the
selectivity and potency of various arachidonic acid analogs in
inhibiting CYP-dependent arachidonic acid oxygenases. The results
demonstrated that these analogs were quite potent at low µM
concentrations in inhibiting rat liver microsomal CYP-dependent
oxygenase reactions without affecting ram seminal vesicle COX or
soybean lipoxygenase activities at concentrations of up to 100 µM.
However, these analogs did not significantly distinguish between the
different oxygenase reactions; i.e., they inhibited both
epoxidation and -hydroxylation of arachidonic acid to the same
extent. In the same study, the authors documented the potency and
selectivity of two imidazole derivatives, clotrimazole and
ketoconazole, in preferentially inhibiting arachidonate epoxygenases
(Capdevila et al., 1988). These imidazole derivatives are
being used extensively in studies to evaluate the physiological role of
arachidonate epoxides, especially as they relate to the control of
vascular tone and renal function (Makita et al., 1994;
Alkayed et al., 1996; Alonso-galicia et al.,
1997; Fulton et al., 1995). However, they do affect many CYP-dependent reactions as well as cellular processes unrelated to CYP
(Testa and Jenner, 1981; Alvarez et al., 1992).
Our study describes specific inhibitors that, at least in
vitro, can distinguish between CYP-catalyzed arachidonate
epoxidation and -hydroxylation reactions as well as between CYP4A
isoform-catalyzed reactions. Additional studies to examine their
potency and specificity in vivo, as well as thorough
examination of their selectivity with regard to other CYP-catalyzed
reactions, should accompany studies designed to evaluate the role of
the CYP-arachidonic acid metabolites in the regulation of renal
function and blood pressure.
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Acknowledgments |
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The authors thank Dr. Richard J. Roman (Department of Physiology, Medical College of Wisconsin, Milwaukee, WI) for providing the CYP4A1, 4A2, 4A3 and 4A8 cDNAs.
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Footnotes |
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Accepted for publication November 7, 1997.
Received for publication July 31, 1997.
1 Supported by NIH grants HLPO134300 and EY06513 (M.L.S.) and DK38226 (J.R.F.) and by a Grant-in-Aid (M.H.W.) from the American Heart Association (#970104).
2 Mong-Heng Wang and Elimor Brand-Schieber contributed equally to the development of this research paper.
Send reprint requests to: Dr. Michal Laniado Schwartzman, Department of Pharmacology, New York Medical College, Valhalla, NY 10595.
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Abbreviations |
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CYP, cytochrome P450; EET, epoxyeicosatrienoic acid; 20-HETE, 20-hydroxyeicosatrienoic acid; 17-ODYA, 17-octadecynoic acid; PPOH, 6-(2-propargyloxyphenyl)hexanoic acid; MS-PPOH, N-methanesulfonyl-6-(2-propargyloxyphenyl)hexanamide; DDMS, N-methylsulfonyl-12,12-dibromododec-11-enamide; DBDD, 12-12-dibromododec-11-enoic acid; DPMS, N-methylsulfonyl-15,15-dibromopentadec-14-enamide; SHR, spontaneously hypertensive rat; COX, cyclooxygenase.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics
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D. Li, Y. Wei, and W.-H. Wang Dietary K intake regulates the response of apical K channels to adenosine in the thick ascending limb Am J Physiol Renal Physiol, November 1, 2004; 287(5): F954 - F959. [Abstract] [Full Text] [PDF]
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K. Nithipatikom, E. R. Gross, M. P. Endsley, J. M. Moore, M. A. Isbell, J. R. Falck, W. B. Campbell, and G. J. Gross Inhibition of Cytochrome P450{omega}-Hydroxylase: A Novel Endogenous Cardioprotective Pathway Circ. Res., October 15, 2004; 95(8): e65 - e71. [Abstract] [Full Text] [PDF]
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S. J. Lee, C. S. Landon, S. J. Nazian, and J. R. Dietz Cytochrome P-450 metabolites in endothelin-stimulated cardiac hormone secretion Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2004; 286(5): R888 - R893. [Abstract] [Full Text] [PDF]
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F. Xu, J. R. Falck, P. R. Ortiz de Montellano, and D. L. Kroetz Catalytic Activity and Isoform-Specific Inhibition of Rat Cytochrome P450 4F Enzymes J. Pharmacol. Exp. Ther., March 1, 2004; 308(3): 887 - 895. [Abstract] [Full Text] [PDF]
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G. O. Ogungbade, L. A. Akinsanmi, H. Jiang, and A. O. Oyekan Role of epoxyeicosatrienoic acids in renal functional response to inhibition of NO production in the rat Am J Physiol Renal Physiol, November 1, 2003; 285(5): F955 - F964. [Abstract] [Full Text] [PDF]
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I. T. Udosen, H. Jiang, H. C. Hercule, and A. O. Oyekan Nitric oxide-epoxygenase interactions and arachidonate-induced dilation of rat renal microvessels Am J Physiol Heart Circ Physiol, November 1, 2003; 285(5): H2054 - H2063. [Abstract] [Full Text] [PDF]
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S. I. Pomposiello, J. Quilley, M. A. Carroll, J. R. Falck, and J. C. McGiff 5,6-Epoxyeicosatrienoic Acid Mediates the Enhanced Renal Vasodilation to Arachidonic Acid in the SHR Hypertension, October 1, 2003; 42(4): 548 - 554. [Abstract] [Full Text] [PDF]
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J.-I. Kaide, M.-H. Wang, J.-S. Wang, F. Zhang, V.R. Gopal, J. R. Falck, A. Nasjletti, and M. Laniado-Schwartzman Transfection of CYP4A1 cDNA increases vascular reactivity in renal interlobar arteries Am J Physiol Renal Physiol, January 1, 2003; 284(1): F51 - F56. [Abstract] [Full Text] [PDF]
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M. Medhora, J. Daniels, K. Mundey, B. Fisslthaler, R. Busse, E. R. Jacobs, and D. R. Harder Epoxygenase-driven angiogenesis in human lung microvascular endothelial cells Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H215 - H224. [Abstract] [Full Text] [PDF]
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J. C. Frisbee, K. G. Maier, and D. W. Stepp Oxidant stress-induced increase in myogenic activation of skeletal muscle resistance arteries in obese Zucker rats Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2160 - H2168. [Abstract] [Full Text] [PDF]
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R.-M. Gu and W.-H. Wang Arachidonic acid inhibits K channels in basolateral membrane of the thick ascending limb Am J Physiol Renal Physiol, September 1, 2002; 283(3): F407 - F414. [Abstract] [Full Text] [PDF]
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C. Sauvant, H. Holzinger, and M. Gekle Short-Term Regulation of Basolateral Organic Anion Uptake in Proximal Tubular OK cells: EGF Acts via MAPK, PLA2, and COX1 J. Am. Soc. Nephrol., August 1, 2002; 13(8): 1981 - 1991. [Abstract] [Full Text] [PDF]
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F. Zhang, M.-H. Wang, U.M. Krishna, J. R. Falck, M. Laniado-Schwartzman, and A. Nasjletti Modulation by 20-HETE of Phenylephrine-Induced Mesenteric Artery Contraction in Spontaneously Hypertensive and Wistar-Kyoto Rats Hypertension, December 1, 2001; 38(6): 1311 - 1315. [Abstract] [Full Text] [PDF]
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I. Fleming Cytochrome P450 and Vascular Homeostasis Circ. Res., October 26, 2001; 89(9): 753 - 762. [Abstract] [Full Text] [PDF]
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 R.-M. Gu, Y. Wei, J. R. Falck, U. M. Krishna, and W.-H. Wang Effects of protein tyrosine kinase and protein tyrosine phosphatase on apical K+ channels in the TAL Am J Physiol Cell Physiol, October 1, 2001; 281(4): C1188 - C1195. [Abstract] [Full Text] [PDF]
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Y. Wu, A. Huang, D. Sun, J. R. Falck, A. Koller, and G. Kaley Gender-specific compensation for the lack of NO in the mediation of flow-induced arteriolar dilation Am J Physiol Heart Circ Physiol, June 1, 2001; 280(6): H2456 - H2461. [Abstract] [Full Text] [PDF]
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A. Huang, D. Sun, M. A. Carroll, H. Jiang, C. J. Smith, J. A. Connetta, J. R. Falck, E. G. Shesely, A. Koller, and G. Kaley EDHF mediates flow-induced dilation in skeletal muscle arterioles of female eNOS-KO mice Am J Physiol Heart Circ Physiol, June 1, 2001; 280(6): H2462 - H2469. [Abstract] [Full Text] [PDF]
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M. P. Kunert, R. J. Roman, M. Alonso-Galicia, J. R. Falck, and J. H. Lombard Cytochrome P-450 {omega}-hydroxylase: a potential O2 sensor in rat arterioles and skeletal muscle cells Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1840 - H1845. [Abstract] [Full Text] [PDF]
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J. C. Frisbee, R. J. Roman, U. M. Krishna, J. R. Falck, and J. H. Lombard 20-HETE modulates myogenic response of skeletal muscle resistance arteries from hypertensive Dahl-SS rats Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1066 - H1074. [Abstract] [Full Text] [PDF]
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S. I. Pomposiello, M. A. Carroll, J. R. Falck, and J. C. McGiff Epoxyeicosatrienoic Acid-Mediated Renal Vasodilation to Arachidonic Acid Is Enhanced in SHR Hypertension, March 1, 2001; 37(3): 887 - 893. [Abstract] [Full Text] [PDF]
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R. Gu, Y. Wei, H. Jiang, M. Balazy, and W. Wang Role of 20-HETE in mediating the effect of dietary K intake on the apical K channels in the mTAL Am J Physiol Renal Physiol, February 1, 2001; 280(2): F223 - F230. [Abstract] [Full Text] [PDF]
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H. C. Hercule and A. O. Oyekan Role of NO and cytochrome P-450-derived eicosanoids in ET-1-induced changes in intrarenal hemodynamics in rats Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2000; 279(6): R2132 - R2141. [Abstract] [Full Text] [PDF]
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J. D. Imig Eicosanoid regulation of the renal vasculature Am J Physiol Renal Physiol, December 1, 2000; 279(6): F965 - F981. [Abstract] [Full Text] [PDF]
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D. Zhu, E. K. Birks, C. A. Dawson, M. Patel, J. R. Falck, K. Presberg, R. J. Roman, and E. R. Jacobs Hypoxic pulmonary vasoconstriction is modified by P-450 metabolites Am J Physiol Heart Circ Physiol, October 1, 2000; 279(4): H1526 - H1533. [Abstract] [Full Text] [PDF]
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A. Bhardwaj, F. J. Northington, J. R. Carhuapoma, J. R. Falck, D. R. Harder, R. J. Traystman, and R. C. Koehler P-450 epoxygenase and NO synthase inhibitors reduce cerebral blood flow response to N-methyl-D-aspartate Am J Physiol Heart Circ Physiol, October 1, 2000; 279(4): H1616 - H1624. [Abstract] [Full Text] [PDF]
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 M. A Carroll and J. C McGiff A new class of lipid mediators: cytochrome P450 arachidonate metabolites Thorax, October 1, 2000; 55(90002): 13S - 16. [Full Text]
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W. Kozak, M. J. Kluger, A. Kozak, M. Wachulec, and K. Dokladny Role of cytochrome P-450 in endogenous antipyresis Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2000; 279(2): R455 - R460. [Abstract] [Full Text] [PDF]
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D. Gebremedhin, A. R. Lange, T. F. Lowry, M. R. Taheri, E. K. Birks, A. G. Hudetz, J. Narayanan, J. R. Falck, H. Okamoto, R. J. Roman, et al. Production of 20-HETE and Its Role in Autoregulation of Cerebral Blood Flow Circ. Res., July 7, 2000; 87(1): 60 - 65. [Abstract] [Full Text] [PDF]
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J. C. Frisbee, J. R. Falck, and J. H. Lombard Contribution of cytochrome P-450 omega -hydroxylase to altered arteriolar reactivity with high-salt diet and hypertension Am J Physiol Heart Circ Physiol, May 1, 2000; 278(5): H1517 - H1526. [Abstract] [Full Text] [PDF]
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J. M. Lasker, W. B. Chen, I. Wolf, B. P. Bloswick, P. D. Wilson, and P. K. Powell Formation of 20-Hydroxyeicosatetraenoic Acid, a Vasoactive and Natriuretic Eicosanoid, in Human Kidney. ROLE OF CYP4F2 AND CYP4A11 J. Biol. Chem., February 11, 2000; 275(6): 4118 - 4126. [Abstract] [Full Text] [PDF]
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D. Zhu, M. Bousamra II, D. C. Zeldin, J. R. Falck, M. Townsley, D. R. Harder, R. J. Roman, and E. R. Jacobs Epoxyeicosatrienoic acids constrict isolated pressurized rabbit pulmonary arteries Am J Physiol Lung Cell Mol Physiol, February 1, 2000; 278(2): L335 - L343. [Abstract] [Full Text] [PDF]
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J. D. Imig, B. T. Pham, E. A. LeBlanc, K. M. Reddy, J. R. Falck, and E. W. Inscho Cytochrome P450 and Cyclooxygenase Metabolites Contribute to the Endothelin-1 Afferent Arteriolar Vasoconstrictor and Calcium Responses Hypertension, January 1, 2000; 35(1): 307 - 312. [Abstract] [Full Text] [PDF]
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J. C. McGiff and J. Quilley 20-HETE and the kidney: resolution of old problems and new beginnings Am J Physiol Regulatory Integrative Comp Physiol, September 1, 1999; 277(3): R607 - R623. [Abstract] [Full Text] [PDF]
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X. Nguyen, M.-H. Wang, K. M. Reddy, J. R. Falck, and M. L. Schwartzman Kinetic profile of the rat CYP4A isoforms: arachidonic acid metabolism and isoform-specific inhibitors Am J Physiol Regulatory Integrative Comp Physiol, June 1, 1999; 276(6): R1691 - R1700. [Abstract] [Full Text] [PDF]
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A. O. Oyekan, K. McAward, J. Conetta, L. Rosenfeld, and J. C. McGiff Endothelin-1 and CYP450 arachidonate metabolites interact to promote tissue injury in DOCA-salt hypertension Am J Physiol Regulatory Integrative Comp Physiol, March 1, 1999; 276(3): R766 - R775. [Abstract] [Full Text] [PDF]
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B. A. Rzigalinski, K. A. Willoughby, S. W. Hoffman, J. R. Falck, and E. F. Ellis Calcium Influx Factor, Further Evidence It Is 5,6-Epoxyeicosatrienoic Acid J. Biol. Chem., January 1, 1999; 274(1): 175 - 182. [Abstract] [Full Text] [PDF]
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J. D. Imig, E. W. Inscho, P. C. Deichmann, K. M. Reddy, and J. R. Falck Afferent Arteriolar Vasodilation to the Sulfonimide Analog of 11,12-Epoxyeicosatrienoic Acid Involves Protein Kinase A Hypertension, January 1, 1999; 33(1): 408 - 413. [Abstract] [Full Text] [PDF]
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J. T. Weber, B. A. Rzigalinski, and E. F. Ellis Traumatic Injury of Cortical Neurons Causes Changes in Intracellular Calcium Stores and Capacitative Calcium Influx J. Biol. Chem., January 12, 2001; 276(3): 1800 - 1807. [Abstract] [Full Text] [PDF]
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B. A. Escalante, J. C. McGiff, and A. O. Oyekan Role of cytochrome P-450 arachidonate metabolites in endothelin signaling in rat proximal tubule Am J Physiol Renal Physiol, January 1, 2002; 282(1): F144 - F150. [Abstract] [Full Text] [PDF]
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) and absence (
) of 1 mM NADPH were preincubated
for 1, 2, 5, and 15 min at 37°C. The respective controls were
microsomes and ethanol in the same preincubation condition. An aliquot
of reaction mixtures was then added to test tubes (10-fold dilution) containing [1-14C]-arachidonic acid (0.4 µCi), buffer
and 0.9 mM NADPH. The reactions were carried out for 30 min at 37°C.
Results are expressed as percent of control and are the mean ± S.E.M., n = 3.