Department of Pharmacology, New York Medical College, Valhalla, New York 10595
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ABSTRACT |
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20-Hydroxyeicosatetraenoic acid
(20-HETE), a potent vasoconstrictor and mediator of the myogenic
response, is a major arachidonic acid metabolite in the
microvasculature of the rat kidney formed primarily by the cytochrome
P-450 (CYP) 4A isoforms, CYP4A1, CYP4A2, and CYP4A3. We
examined CYP4A isoform expression and 20-HETE synthesis in
microdissected interlobar, arcuate, and interlobular arteries; mRNA for
all CYP4A isoforms was identified by RT-PCR. Western blot analysis
indicated that the levels of CYP4A2/4A3-immunoreactive protein
increased with decreased arterial diameter, whereas those of
CYP4A1-immunoreactive protein remained unchanged. 20-HETE synthesis was
the highest in the interlobular arteries (17 ± 1.62 nmol · mg1 · h
1)
and, like CYP4A2/4A3-immunoreactive protein, decreased with increasing vessel diameter (4.5 ± 1.21, 2.65 ± 0.58, and
0.81 ± 0.14 nmol · mg
1 · h
1 in the
arcuate, interlobar, and segmental arteries, respectively). 20-HETE
synthesis in the renal artery and the abdominal aorta was undetectable.
The observed decreased immunoreactivity of NADPH-cytochrome P-450 (c) oxidoreductase with increased
arterial diameter provided a possible explanation for the decreased
capacity to generate 20-HETE in the large arteries. The increase in
CYP4A isoform expression and 20-HETE synthesis with decreasing diameter
along the preglomerular arteries and the potent biological activity of
20-HETE underscore the significance of 20-HETE as a modulator of renal hemodynamics.
rat kidney; arachidonic acid; NADPH-cytochrome P-450 (c) oxidoreductase; hydroxyeicosatetraenoic acid
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INTRODUCTION |
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THE
-HYDROXYLATION OF ARACHIDONIC acid to
20-hydroxyeicosatetraenoic acid (20-HETE) is catalyzed by enzymes of
the cytochrome P-450 4A isoform (CYP4A) gene family. This
subfamily encodes several CYP enzymes that are capable of hydroxylating
the terminal
-carbon and, to a lesser extent, the (
-1) position
of saturated and unsaturated fatty acids as well as various
prostaglandins (4). In the rat, four members of this
family have been identified, CYP4A1, CYP4A2, CYP4A3, and
CYP4A8, and messages for all four have been detected in the kidney
(17, 18, 29). These isoforms, although sharing 66-98% homology and a common catalytic activity, are localized to
different renal structures and are exposed to different regulatory mechanisms (4). Studies in our laboratory showed that
CYP4A expression and activity are segmented along the nephron
(20, 26). Ito et al. (14) examined the
expression of CYP4A isoforms in various nephron segments and
preglomerular arterioles microdissected from the kidneys of
Sprague-Dawley rats and demonstrated that CYP4A2, CYP4A3, and CYP4A8
mRNAs are constitutively expressed in all tissues examined, whereas
CYP4A1 mRNA is barely detectable. However, studies using CYP4A
isoform-specific antisense oligonucleotides suggested that CYP4A1
contributes significantly to renal 20-HETE synthesis in tubular and
vascular structures (33).
We found that despite their high homology, these isoforms display
different catalytic properties. In the presence of NADPH-cytochrome P-450 (c) oxidoreductase, the recombinant CYP4A1,
CYP4A2, and CYP4A3, but not CYP4idoA8, catalyzed arachidonic acid
-hydroxylation to 20-HETE with the highest catalytic efficiency
(Vmax/Km) for CYP4A1,
followed by CYP4A2 and CYP4A3. Moreover, CYP4A2 and CYP4A3 exhibited an
additional arachidonic acid-11,12-epoxidation activity [formation of
11,12-epoxyeicosatrienoic acid (EET)], whereas CYP4A1 operated solely
as an
-hydroxylase (24, 34). The ability of CYP4A2/4A3
to catalyze the formation of 20-HETE and 11,12-EET may be of great
significance to the regulation of vascular tone. 11,12-EET exhibits
biological activities opposite those of 20-HETE; it vasodilates blood
vessels and stimulates Ca2+-activated K+
channels in vascular smooth muscle cells (12). On the
other hand, 20-HETE has been shown to constrict renal blood vessels via
a mechanism that includes inhibition of the opening of
Ca2+-activated K+ channels in vascular smooth
muscle cells, cellular depolarization, and increased Ca2+
entry (8, 13, 15, 30, 36). Hence, the dual catalytic activity of these proteins may present a mechanism for balanced regulation of vascular tone. We herein describe CYP4A isoform-specific expression along the preglomerular arteries and evaluate the potential contribution of these CYP isoforms to 20-HETE synthesis.
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MATERIALS AND METHODS |
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Isolation and microdissection of renal arteries. Male Sprague-Dawley rats (7-9 wk old) were anesthetized with pentobarbital sodium (50 mg/kg body wt). For the isolation of a bulk preparation of renal microvessels, the aorta was cannulated below the renal arteries, and the kidneys were flushed with 10 ml of ice-cold Tyrode buffer, then with 5 ml of Tyrode solution containing 6% albumin and 1% Evan's blue solution. Kidneys were then excised, sliced into hemisections, and pushed through a 180-µm mesh screen. The vascular trees trapped on the screen were collected and digested using a solution of Tyrode containing 1 mg/ml collagenase, 1 mg/ml soybean trypsin inhibitor, 1 mg/ml dithiothreitol, and 1 mg/ml albumin, and the following nutrients: 10 µl/ml succinate, 10 µl/ml L-alanine, 1 µl/ml Na-lactate of 60% stock solution, and 10 µl/ml L-glutamate of 200 mM stock solution. Vessels were incubated for 40 min at 37°C with shaking while being superfused with O2. The enzyme solution containing the tissue was filtered through a 75-µm nylon filter. Digested tissue was resuspended in ice-cold Tyrode buffer, and vessels were collected using a microscope. The microvessels were homogenized in 100 µl of 10 mM KP buffer, pH 7.7, containing 240 mM sucrose, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 0.1% Nonidet P-40 and spun at 3,000 g for 5 min and then at 14,000 rpm in the microfuge for 15 min, and the supernatant was collected. Protein was measured using a Bio-Rad Protein Assay Kit.
To collect individual segments of the vascular tree, kidneys were excised, placed in ice-cold Tyrode buffer, and coronally sectioned. The renal papilla was removed to expose the microvessels. The segmental, interlobar, arcuate, and interlobular arteries were microdissected and freed from cortical and connective tissue. The purity of the microdissected microvessel preparation was determined by phase-contrast microscopy and by the absence of immunoreactive
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RT-PCR.
An RT reaction was performed using a first-strand cDNA synthesis kit
(Pharmacia Biotech, Milwaukee, WI). Briefly, RNA (10 ng) from the
microvessels was added to 15 µl of RT reaction mixture containing 45 mM Tris (pH 8.3), 68 mM KCl, 15 mM dithiothreitol, 9 mM
MgCl2, 0.08 mg/ml BSA, 1.8 mM 2-deoxynucleotide
5'-triphosphate, 40 pmol of either CYP4A1, 4A2, 4A3, 4A8, or -actin
backward primer, and Moloney murine leukemia virus RT. The reactions
were incubated for 1 h at 37°C. PCR was carried out in 25 µl
of PCR SuperMix (GIBCO BRL) containing 22 mM Tris · HCl (pH
8.4), 55 mM KCl, 1.65 mM MgCl2, 200 µM dGTP, 220 µM
dATP, 220 µM dTTP, 220 µM dCTP, 22 U of recombinant Taq
DNA polymerase/ml, 20 pmol of CYP4A1, 4A2, 4A3, 4A8, and
-actin
primer pairs, and 4 µl of first-strand synthesis reaction. PCR was
also performed using the CYP4A plasmids (100 ng) as a template for
positive controls. Reactions were cycled 30 times through a 5-min
denaturing step at 95°C, a 1-min annealing step at 60°C, and a
1-min extension step at 72°C. After the cycling procedure, a final
7-min elongation step at 72°C was performed. The CYP4A1, 4A2, 4A3,
4A8, and
-actin primers were designed to amplify 351-, 317-, 321-, 349-, and 764-bp fragments from each of the corresponding cDNAs. The
sequences of the primers were as follows: 5'-CTC TTA CTT CGG AGA ATG
GAG AA-3' (forward primer) and 5'-GAC TTG GAT ACC CTT GGG TAA AG-3'
(backward primer) for CYP4A1; 5'-AGA TCC AAA GCC TTA TCA ATC-3'
(forward primer) and 5'-CAG CCT TGG TGT AGG ACC-T-3' (backward primer)
for CYP4A2; 5'-CAA AGG CTT CTG GAA TTT ATC-3' (forward primer) and
5'-CAG CCT TGG TGT AGG ACC T-3' (backward primer) for CYP4A3; 5'-ATC CAG AGG TGT TTG ACC CTT AT-3' (forward primer) and 5'-AAT GAG ATG TGA
GCA GAT GGA GT-3' (backward primer) for CYP4A8; and 5'-TTG TAA CCA ACT
GGG ACG ATA TGG-3' (forward primer) and 5'-GAT CTT GAT CTT CAT GGT GCT
AGG-3' (backward primer) for
-actin. An aliquot (10 µl) of each
PCR reaction was separated on a 1.5% agarose gel, and PCR products
were stained with ethidium bromide.
Western blot analysis.
Homogenized microvessels were separated by electrophoresis on a large
(16 × 20-cm) 8% SDS-polyacrylamide gel at 25 mA, 4°C for
18-20 h. The protein samples were transferred electrophoretically to a nitrocellulose membrane in a transfer buffer consisting of 25 mM
Tris · HCl, 192 mM glycine, and 20% methanol (vol/vol). The
membranes were blocked with 10% nonfat dry milk in Tris-buffered solution (TBS) containing 10 mM Tris, 0.1% Tween 20, and 150 mM NaCl
for 2 h and then washed three times with TBS. The membranes were
then incubated overnight with goat anti-rat CYP4A1 polyclonal antibody (1:1,000; Gentest, Woburn, MA) at room temperature,
washed three times with TBS solution, and then incubated with 1:5,000 dilution of horseradish peroxidase-conjugated second antibody (Sigma)
for 1 h. After being washed three times with TBS, the membranes
were detected using the ECL Plus detection system (Amersham Life
Sciences). Membranes were stripped using a Re-Blot Western Recycling
Kit (Chemicon, Temacula, CA), and reprobed with either goat anti-rat
NADPH-P450 reductase polyclonal antibody (1:5,000; Gentest) for 2 h at room temperature, anti--GT polyclonal antibody (1:5,000; Sigma)
for 1 h at room temperature, or mouse anti-
-actin monoclonal
antibody (1:5,000; Sigma) for 2 h at room temperature.
Measurement of 20-HETE synthesis. Homogenates from bulk preparation or microdissected vessels (50-100 µg protein) were incubated with [1-14C]arachidonic acid (0.4 µCi, 7 nmol) and NADPH (1 mM) in 0.2 ml of potassium phosphate buffer (100 mM, pH 7.4) containing 10 mM MgCl2 for 30 min at 37°C. The reaction was terminated by acidification to pH 3.5-4.0 with 2 M formic acid, and metabolites were extracted with ethyl acetate. The final extract was evaporated under nitrogen, resuspended in 50 µl of methanol, and injected onto an 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, Tampa, FL). The identity of 20-HETE was confirmed by its comigration with an authentic standard. 20-HETE formation was estimated based on the specific activity of the added [1-14C]arachidonic acid and was expressed as nanomoles per milligram protein per hour.
20-HETE production in whole microvessels of preglomerular segments was determined by incubating 3-10 vessels with arachidonic acid (30 µM) in 1 ml of Tyrode buffer containing 1 mM NADPH, 10 µM indomethacin, and 10 µM NG-nitro-L-arginine methyl ester (L-NAME) for 1 h at 37°C. The reaction was terminated by acidification to pH 3.5-4.0 with 10 µl of 2 M formic acid. [20,20-2H2]20-HETE (3 ng) was added as an internal standard. The mixture was then extracted twice with 2 ml of ethyl acetate. The final extract was evaporated under nitrogen, resuspended in 30 µl of methanol, and subjected to reverse-phase HPLC as described above. Fractions coeluting with the 20-HETE standard were collected, evaporated to dryness, and derivitized to the pentafluorobenzyl bromide ester trimethylsilyl ether. Negative chemical ionization-gas chromatography/mass spectrometry (NCI-GC/MS) was performed on an HP5989A mass spectrometer (Hewlett-Packard) interfaced with a capillary gas chromatographic column (DB-1 fused silica, 10 m, 0.25-mm inner diameter, 0.25-µm film thickness, J&W Scientific, Rancho Cordova, CA) and programmed from 180-300°C at 25°C/min using helium as the carrier gas. Single ions were monitored with m/z 391, corresponding to the derivatized 20-HETE, and m/z 393 for the derivatized [20,20-2H2]20-HETE internal standard. Total 20-HETE in each sample was determined by comparison of the ratio of ion intensities (391:393) vs. a standard curve of derivatized 20-HETE/[20, 20-2H2]20-HETE molar ratio obtained from NCI-GC/MS analysis. After extraction, the vessels were collected and suspended in 100-300 µl 1 N NaOH, in which they were left overnight to dissolve. Any intact vessels left were manually ground using a glass rod. Protein concentration was determined using the Bio-Rad assay. Inhibitor studies were conducted using 6-(2-porpargyloxyphenyl)hexanoic acid (PPOH) and N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), which were diluted from ethanolic stock solutions with Tyrode buffer. The diluted solutions were added to the incubation mixture containing the vessels and preincubated for 10 min at 37°C. Arachidonic acid was then added, and the incubation was carried out at 37°C for 1 h. Control incubations included the vehicle of the inhibitor. Extraction and HPLC analysis were carried out as described above.Statistical analysis. Results were expressed as means ± SE, and significance was defined by P < 0.05 for all data. Data were analyzed by Student's t-test for paired observations as appropriate. The significance of the difference in mean values was evaluated by one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test).
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RESULTS |
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Bulk preparation of renal microvessels yields microvessel trees
that include all segments of the preglomerular arteries. Incubation of
homogenates from this preparation with 14C-labeled
arachidonic acid and NADPH resulted in the formation of 20-HETE (10.6 min) as the major metabolite. The rate of conversion of arachidonic
acid to 20-HETE amounted to 2.7 ± 0.3 nmol · mg1 · h
1
(n = 6). This same preparation readily converted
arachidonic acid to two other metabolites with the elution profile of
authentic 11,12-EET (15 min) and 11,12-dihydroxyeicosatrienoic acid
(9.2 min), the hydrolytic metabolite of 11,12-EET (Fig.
2).
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The bulk preparation of renal microvessels comprised a mixed population
of large and small vessels. In view of the differences in the
vasoconstrictor responses to 20-HETE between large and small vessels,
we thought differences might exist in the capacity of each to
make 20-HETE. We first examined CYP4A isoform expression in
microdissected arterial segments, namely, the large interlobar and small arcuate/interlobular vessels. Western blot analysis of these
segments revealed the presence of immunoreactive proteins with the
elution profile of the recombinant CYP4A1 and CYP4A2 (Fig.
3A). Importantly, there were
twofold greater levels of CYP4A-immunoreactive proteins in the small
arcuate and interlobular vessels compared with interlobar vessels (Fig.
3B). The bands produced by the interlobar artery coeluted
with the CYP4A1 standard, whereas the bands produced by the
arcuate/interlobular arteries coeluted with both the CYP4A1 and CYP4A2
standards. Correspondingly, measurement of 20-HETE synthesis,
determined as the rate of conversion of 14C-labeled
arachidonic acid to 20-HETE, showed a fivefold greater synthetic
capacity by the small-diameter arteries compared with the larger
diameter interlobar vessels (Fig. 3C).
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The above results suggest that the capacity to produce 20-HETE is
segmented along the preglomerular arterioles. We therefore proceeded to
evaluate relationships among vessel diameter, 20-HETE production, and
CYP4A expression. For this set of experiments, vessels were carefully
microdissected and the measurement of 20-HETE production was carried
out using the whole vessels so as to limit protein degradation and
inactivation as a result of homogenization. To increase sensitivity of
measurements, 20-HETE production was measured by GC/MS analysis.
As seen in Fig. 4, 20-HETE production in
the individual segments was indeed segmented. 20-HETE synthesis was the
highest in the interlobular arteries (17.3 ± 1.62 nmol · mg1 · h
1). Synthesis
greatly decreased with increasing diameter. The arcuate and interlobar
arteries showed 20-HETE production rates of 4.5 ± 1.21 and
2.65 ± 0.59 nmol · mg
1 · h
1,
respectively. Using this methodology, 20-HETE was detected in the
segmental artery, albeit at a synthesis level three times lower than
that in the interlobar artery. Both the renal artery and the abdominal
aorta did not exhibit detectable capacity to produce 20-HETE (Fig.
4).
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The segmentation detected with 20-HETE synthesis raised the question of
whether CYP4A expression is also segmented. RT-PCR analysis showed
expression of the CYP4A1 isoform in the aorta and renal artery, whereas
the CYP4A2, CYP4A3, and CYP4A8 isoforms were absent (Fig.
5A). The segmental artery
showed mRNA for the CYP4A1 and CYP4A2 isoforms only, whereas the
interlobar, arcuate, and interlobular arteries expressed mRNAs for the
CYP4A1, 4A2, 4A3, and 4A8 isoforms. Western blot analysis, using the
polyclonal CYP4A1 antibody, which cross-reacts with CYP4A2, CYP4A3, and
CYP4A8, revealed strong immunoreactive bands in the interlobar,
arcuate, and interlobular arteries as well as in the abdominal aorta.
Lower levels of CYP4A1-immunoreactive proteins were detected in the segmental artery, whereas in homogenates of the renal artery
CYP4A-immunoreactive protein was barely detectable (Fig.
6A). Densitometry analysis of
three separate blots indicated that CYP4A protein expression (normalized to -actin) is segmented and is the highest in the small-diameter interlobular arteries (Fig. 6D). We also
measured the expression of NADPH-cytochrome P-450
(c) oxidoreductase, an integral component of CYP
catalytic activity. As seen in Fig. 6B, NADPH-cytochrome
P-450 (c) oxidoreductase immunoreactive protein was detected in all segments; however, the levels were much higher in
the interlobular arteries, as indicated by densitometry analysis (Fig.
6D).
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Additional experiments were conducted to evaluate the contribution of
CYP4A isoforms to 20-HETE synthesis. The addition of 30 µM PPOH, the
CYP4A2/4A3-selective inhibitor, resulted in a 35% decrease of 20-HETE
synthesis in the interlobular arteries (Fig.
7). Similarly, PPOH inhibited 20-HETE
synthesis in the arcuate arteries by 62%. The interlobar arteries
remained insensitive to PPOH. Incubation of the vessels with 30 µM
DDMS, an inhibitor of arachidonate -hydroxylase activity,
significantly decreased 20-HETE production in the interlobular,
arcuate, and interlobar arteries by 48, 70, and 65%, respectively. On
the basis of previous studies showing that, although DDMS inhibits
20-HETE production by all recombinant CYP4A proteins, PPOH only
inhibits CYP4A2/4A3-catalyzed 20-HETE production
(24), it may be suggested that in the arcuate and
interlobular arteries, CYP4A1, CYP4A2, and CYP4A3 contribute to the
levels of 20-HETE.
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DISCUSSION |
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The present studies demonstrate that vascular 20-HETE synthesis in the kidney is segmented; the rate of 20-HETE production increases as arterial diameter decreases. It also reveals that whereas CYP4A1 expression is widely distributed, that of CYP4A2/4A3 is greatly increased with decreasing vessel diameter. Additional studies with the CYP4A isoform-specific inhibitor PPOH further suggested that 20-HETE synthesis in small-diameter arteries, including the arcuate and the interlobular segments of the preglomerular artery, is driven primarily by CYP4A1 and CYP4A2/4A3. The significance of these findings relates to the distinct catalytic properties of CYP isoforms as well as to the biological activity of 20-HETE.
The CYP4A enzymes are considered to be the major arachidonic acid
-hydroxylases in the rat kidney and thereby the primary contributors
of 20-HETE synthesis. These isoforms displayed distinct catalytic
properties that may impact on the extent to which 20-HETE is formed in
a given tissue as well as the extent to which its biological activity
is expressed. Several studies have shown that the CYP4A1 protein, in
its recombinant or purified form, is the most active arachidonic acid
-hydroxylase (2, 7, 32). Recombinant CYP4A1 displayed
the highest catalytic efficiency in catalyzing the oxidation of
arachidonic acid to 20-HETE, with a Km of 10 µM and Vmax of 9.47 min-1
(24). The purified protein showed similar
characteristics (32). Another feature that is typical of
CYP4A1 is its preference for catalyzing arachidonic acid at the
-carbon; the ratio of
to
-1 hydroxylation is ~6:1-10:1
(9, 24). On the other hand, recombinant CYP4A2 and CYP4A3
exhibit a catalytic efficiency toward
-hydroxylation of arachidonic
acid that is 10 and 40 times lower than that of CYP4A1, respectively
(24). More importantly, both CYP4A2 and CYP4A3, which
share a 96% amino acid sequence similarity (18),
catalyzed the formation of multiple arachidonic acid metabolites. These
proteins have the ability of catalyzing the epoxidation of arachidonic
acid at the 11,12 double bond (24). Moreover, the ratio of
to
-1 hydroxylation catalyzed by these proteins is
~3:1-2:1 (9). Thus a tissue endowed with CYP4A2
and/or CYP4A3 may produce significant amounts of 19-HETE and 11,12-EET
in addition to 20-HETE. This not only limits the amount of biologically
active 20-HETE but also impacts on the ability of 20-HETE to exert its full biological activity because other oxidation products may exhibit
biological activities that are either contrary or additive to that of
20-HETE.
To that end, 20-HETE has been extensively characterized as a potent vasoconstrictor of small arteries and arterioles in the rat kidney (1, 13). The mechanism underlying its vasoreactivity in small arteries and arterioles is believed to include the inhibition of large-conductance, calcium-activated potassium channels (36) and activation of L-type calcium channels (6) in vascular smooth muscle. In large arteries, including the aorta, 20-HETE has been shown to be metabolized by cyclooxygenase to 20-hydroxy-endoperoxides, which exhibit constrictor activity with a potency similar to that of arachidonic endoperoxides (5, 21). 19-HETE, which is produced by CYP4A1 and 4A2/4A3 at rates that are 10 and 25% that of 20-HETE, respectively, lacks significant vasoreactivity in rat renal arteries; however, it has been shown to be an antagonist of 20-HETE constrictor activity in rat interlobular arteries (1). On the other hand, 11,12-EET has been shown to dilate the rat renal arteries and arterioles and activate Ca2+-activated K+ channels in smooth muscle cells (12), effects opposite those of 20-HETE. 11,12-EET is considered as a major epoxide in the kidney and the primary epoxide in renal arteries (23, 34). Its synthesis is catalyzed by several CYP2C and 2J proteins (12, 28), but, more importantly, it also can be generated by CYP4A2 and CYP4A3 but not by CYP4A1 (12, 24). Thus the CYP4A isoform composition in a given vascular tissue could be an important determinant of the extent to which endogenous 20-HETE contributes to the regulation of vascular tone.
Our results also indicate that there is no apparent correlation between
CYP4A expression and 20-HETE synthesis. For example, the level of CYP4A
protein expression in the aorta was similar to that in the interlobular
or arcuate arteries. However, aortic tissue did not exhibit arachidonic
acid -hydroxylation; no 20-HETE could be detected. Such segmentation
may be accounted for by the presence of metabolizing enzymes such as
cyclooxygenase, which has been shown to metabolize 20-HETE in vascular
tissues (21). However, in this study measurements of
20-HETE production were carried out in the presence of indomethacin so
as to eliminate cyclooxygenase-dependent metabolism. One apparent
explanation is the lack of sufficient NADPH-cytochrome P-450
(c) oxidoreductase activity. The reductase is essential for
the catalytic activity of CYP proteins. More importantly, in vitro
studies have suggested that the amount of reductase necessary for
-hydroxylation of arachidonic acid by CYP4A1, CYP4A2, or CYP4A3 is
at least 4-10 times more than CYP protein on a molar basis
(27). In the present study, Western blot analyses clearly
demonstrated a gradual decrease in the protein levels of
NADPH-cytochrome P-450 (c) oxidoreductase as
blood vessel diameter increased, indicating a possible correlation between its level of expression and the ability of the tissue to
produce 20-HETE. A further possibility is the presence of other CYP4
proteins distinct from CYP4A isoforms with which our CYP4A1 polyclonal
antibodies cross-reacted. Recent reports indicate the presence of
CYP4F1, CYP4F4, and CYP4F5 in renal tissues (3, 16).
However, their ability to catalyze arachidonic acid
-hydroxylation has not been fully examined. These proteins, which share an ~40% amino acid sequence similarity to CYP4A proteins, by cross-reacting with the antibodies used in this study may increase the density of the
immunoreactive protein without contributing to 20-HETE synthesis.
Unfortunately, selective tools to distinguish between CYP4 proteins are
unavailable, and the high homology between them presents a significant
obstacle to achieve selectivity.
The age and the sex of the rat are also important parameters to
consider in an interpretation of the results of this study. Numerous
studies have demonstrated age-dependent differences in renal 20-HETE
synthesis and a distinct developmentally regulated CYP4A isoform
expression in the rat kidney (25, 10, 19, 20). The renal
CYP4A isoform expression is also sex dependent and androgen regulated
(11, 31, 35); in the male kidney, all four isoforms are
expressed with CYP4A2 as the major isoform, whereas in the female
CYP4A2 protein is undetectable. Our study was conducted in 7-wk-old
male Sprague-Dawley rats, the kidneys of which express all CYP4A
isoforms and demonstrate the highest rate of 20-HETE synthesis
(22). Preliminary results indicate that despite the
difference in CYP4A isoform expression between male and female rats,
renal microsomal 20-HETE synthesis was not different, i.e., 355 ± 27 and 330 ± 18 pmol · mg1 · min
1,
respectively. However, the sex-dependent CYP4A expression may functionally impact on 20-HETE synthesis in a specific site, i.e., microvessels. Further studies need to be done to elucidate
sex-dependent differences in synthesis and the function of 20-HETE in
the preglomerular arteries.
In summary, the results presented in this study indicate that CYP4A isoform expression and 20-HETE synthesis are segmented along the preglomerular arteries; the small-diameter interlobular arteries exhibited the highest capacity to generate 20-HETE, the highest levels of CYP4A-immunoreactive proteins including CYP4A1 and CYP4A2/4A3, as well as the highest levels of NADPH-cytochrome P-450 (c) oxidoreductase, a vital component of CYP-catalyzed oxidation reaction. Given the increase in both 20-HETE synthesis and CYP4A isoform expression along the preglomerular vessels and the potent biological activity of 20-HETE, the significance of 20-HETE as a modulator of renal hemodynamics is evident. Manipulation of its synthesis in an isoform-specific manner may be important for the regulation of vascular tone.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. Michal Balazy and Dr. Houli Jiang for technical support with the NCI-GC/MS.
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FOOTNOTES |
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This study was supported by National Heart, Lung, and Blood Institute Grant PO1-HL-34300 (to M. Laniado-Schwartzman) and by American Heart Association Grant 99-30277T (to M.-H. Wang).
Address for reprint requests and other correspondence: M. Laniado-Schwartzman, Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595 (E-mail: michal_schwartzman{at}nymc.edu).
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.
First published February 12, 2002;10.1152/ajprenal.00265.2001
Received 20 August 2001; accepted in final form 11 February 2002.
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