Cytochrome P-450 4A isoform expression and 20-HETE synthesis in renal preglomerular arteries

Jackleen S. Marji, Mong-Heng Wang, and Michal Laniado-Schwartzman

Department of Pharmacology, New York Medical College, Valhalla, New York 10595


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 · mg-1 · 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE omega -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 omega -carbon and, to a lesser extent, the (omega -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 omega -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 omega -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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 gamma -glutamyl transpeptidase (gamma -GT), a specific marker for the proximal tubule brush border (Fig. 1), as previously described (33).


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Fig. 1.   Purity of the renal microvessels. A: microdissected renal vessels seen under a phase-contrast microscope. Magnification: ×4. B: Western blot analysis of gamma -glutamyl transpeptidase (gamma -GT). Cortex (COX; 20 µg), proximal tubules (PT; 20 µg), and microvessels (MV; 25 and 10 µg) were subjected to SDS-PAGE followed by immunoblot analysis with anti-gamma -GT IgG.

For metabolic studies, the vessels were collected in a glass tube containing Tyrode buffer as described below. For RT-PCR, the microvessels were collected and homogenized in TRIzol reagent (Life Technologies) for RNA extraction. For immunoblot assays, homogenate was prepared by rapid freezing in liquid nitrogen and manual homogenization, using a ground-glass homogenizer. The renal arteries and abdominal aortas were also dissected for experimentation.

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 beta -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 beta -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 beta -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 beta -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-gamma -GT polyclonal antibody (1:5,000; Sigma) for 1 h at room temperature, or mouse anti-beta -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).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 · mg-1 · 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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Fig. 2.   Representative reverse-phase HPLC profile of metabolites formed by renal microvessels. Homogenates of renal microvessel (100 µg protein) were incubated with [1-14C]arachidonic acid (AA; 0.4 µCi, 7 nmol) and NADPH (1 mM) for 30 min at 37°C. Radiolabeled metabolites were extracted and separated by HPLC as described in MATERIALS AND METHODS. DHET, dihydroxyeicosatrienoic acid; EET, epoxyeicosatrienoic acid.

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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Fig. 3.   Cytochrome P-450 4A isoform (CYP4A) protein expression and 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis in microdissected preglomerular arteries. A: representative Western immunoblot of renal preglomerular arteries. Homogenate (20 µg of protein) of interlobular/arcuate (SV) and interlobar (LV), and the membrane fraction from Sf9 cells expressing CYP4A1 (4A1; 0.05 µg) and CYP4A2 (4A2; 3 µg) proteins were subjected to SDS-PAGE, followed by immunoblot analysis using goat anti-rat CYP4A1 antibody (1:5,000). B: densitometry analysis of 3 separate blots. Results are the means ± SE. *P < 0.05. C: 20-HETE synthesis in renal preglomerular vessels. Homogenates of interlobular/arcuate (SV; 50 µg protein) and interlobar (LV; 100 µg protein) were incubated with [1-14C]arachidonic acid (0.4 µCi, 7 nmol) and NADPH (1 mM) for 30 min at 37°C as described in MATERIALS AND METHODS. 20-HETE production was determined based on amount of radioactivity recovered and the specific activity of the added arachidonic acid. Values are means ± SE expressed as nmol 20- HETE · mg protein-1 · h-1; n = 4. *P < 0.05.

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 · mg-1 · 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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Fig. 4.   20-HETE production in microdissected renal preglomerular arteries, the renal artery, and the abdominal aorta. Whole vessels were incubated with 30 µM arachidonic acid in the presence of 1 mM NADPH, 10 µM indomethacin, and 10 µM NG-nitro-L-arginine methyl ester (L-NAME) in a final volume of 1 ml for 1 h at 37°C. 20-HETE was separated by reverse-phase HPLC, and the amount was determined by gas chromatography/mass spectrometry analysis as described in MATERIALS AND METHODS. Values are means ± SE expressed as nmol 20-HETE · mg protein-1 · h-1; n = 7 for the interlobular, arcuate, and interlobar arteries and n = 4 for the segmental and renal arteries and for the aorta. ND, not detectable.

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 beta -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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Fig. 5.   A: RT/PCR analysis of total RNA in the abdominal aorta, renal arteries, segmental arteries, interlobar arteries, arcuate arteries, and interlobular arteries using the CYP450 4A isoform-specific primers and beta -actin primers. Total RNA was extracted, and 10 ng were amplified with the CYP4A isoform-specific primers or beta -actin primers. RT-PCR products were separated on an agarose gel and stained with ethidium bromide. For each CYP4A isoform, 100 ng of the CYP4A plasmid were amplified with its respective primer pair to serve as an internal control. B: specificity of the CYP4A isoform-specific primers. CYP4A plasmids (10 ng) were amplified using 20 pmol of the CYP4A primer pairs; lane 1, amplification with CYP4A1-specific primers (351 bp); lane 2, amplification with CYP4A2-specific primers (317 bp); lane 3, amplification with CYP4A3-specific primers (321 bp). PCR products were separated on an agarose gel and stained with ethidium bromide. C: ethidium bromide staining of total RNA. Total RNA was extracted and heated to 65°C for 10 min, and 500 ng of RNA were run on an agarose gel and stained with ethidium bromide.



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Fig. 6.   Representative immunoblots of CYP4A1 (A), NADPH-cytochrome P-450 (c) oxidoreductase (B), and beta -actin (C) expression in microdissected preglomerular arteries, renal artery, and abdominal aorta. Microsomes from clofibrate-induced liver (CF; 5 µg, lane 1), CYP4A1 recombinant protein (4A1; 0.1 µg), CYP4A2 recombinant protein (4A2; 5 µg), purified reductase (CPR; 1 µg), and 20 µg of homogenate from interlobular, arcuate, interlobar, segmental artery, renal artery, and abdominal aorta were separated on an 8% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and blotted with goat anti-rat CYP4A1 (1:5,000), goat anti-rat reductase (1:5,000), or rabbit anti-rat beta -actin antibody (1:5,000). Immunoreactive proteins were detected using the ECL Plus detection system. D: densitometric analysis of CYP4A and NADPH-cytochrome P-450 (c) oxidoreductase normalized to beta -actin. Values are means ± SE of 3 separate blots and expressed as arbitrary units.

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 omega -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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Fig. 7.   Effect of 6-(2-porpargyloxyphenyl)hexanoic acid (PPOH) and N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) on 20-HETE production in renal preglomerular microvessels. Whole vessels were preincubated with 30 µM PPOH or DDMS for 10 min at 37°C. Arachidonic acid (30 µM), NADPH (1 mM), indomethacin (10 µM), and L-NAME (10 µM) were subsequently added to the mixture, and the reaction was carried out for 1 h at 37°C, as described in MATERIALS AND METHODS. Values are means ± SE; n = 4. *P < 0.05 compared with vehicle control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 omega -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 omega -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 omega -carbon; the ratio of omega  to omega -1 hydroxylation is ~6:1-10:1 (9, 24). On the other hand, recombinant CYP4A2 and CYP4A3 exhibit a catalytic efficiency toward omega -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 omega  to omega -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 omega -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 omega -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 omega -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 · mg-1 · 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.


    ACKNOWLEDGEMENTS

The authors thank Dr. Michal Balazy and Dr. Houli Jiang for technical support with the NCI-GC/MS.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alonso-Galicia, M, Falck JR, Reddy KM, and Roman RJ. 20-HETE agonists and antagonists in the renal circulation. Am J Physiol Renal Physiol 277: F790-F796, 1999[Abstract/Free Full Text].

2.   Alterman, MA, Chaurasia CS, Lu P, Hardwick JP, and Hanzlik RP. Fatty acid discrimination and omega -hydroxylation by cytochrome P450 4A1 and cytochrome P4504A1/NADPH-P450 reductase fusion protein. Arch Biochem Biophys 320: 289-296, 1995[ISI][Medline].

3.   Chen, L, and Hardwick JP. Identification of a new P450 subfamily, CYP4F1, expressed in rat hepatic tumors. Arch Biochem Biophys 300: 18-23, 1993[ISI][Medline].

4.   Claire, AE, and Simpson M. The cytochrome P450 4 (CYP4) family. Gen Pharmac 28: 351-359, 1997[Medline].

5.   Escalante, B, Sessa WC, Falck JR, Yadagiri P, and Schwartzman ML. Vasoactivity of 20-hydroxyeicosatetraenoic acid is dependent on metabolism by cyclooxygenase. J Pharmacol Exp Ther 248: 229-232, 1988[Abstract].

6.   Gebremedhin, D, Lange AR, Narayanan J, Aebly MR, Jacobs ER, and Harder DR. Cat cerebral arterial smooth muscle cells express cytochrome P450 4A2 enzyme and produce the vasoconstrictor 20-HETE which enhances L-type Ca2+ current. J Physiol 507: 771-781, 1998[Abstract/Free Full Text].

7.   Gibson, GG. Comparative aspects of the mammalian cytochrome P450 IV gene family. Xenobiotica 19: 1123-1148, 1989[ISI][Medline].

8.   Harder, DR, Gebremedhin D, Narayanan J, Jefcote C, Falck JR, Campbell WB, and Roman R. Formation and action of a P-450 4A metabolite of arachidonic acid in cat cerebral microvessels. Am J Physiol Heart Circ Physiol 266: H2098-H2107, 1994[Abstract/Free Full Text].

9.   Hoch, U, Zhang Z, Kroetz DL, and Ortiz de Montellano PR. Structural determination of the substrate specificities and regioselectivities of the rat and human fatty acid omega-hydroxylases. Arch Biochem Biophys 373: 63-71, 2000[ISI][Medline].

10.   Imaoka, S, and Funae Y. Hepatic and renal cytochrome P450s in spontaneously hypertensive rats. Biochim Biophys Acta 1074: 209-213, 1991[ISI][Medline].

11.   Imaoka, S, Yamazoe Y, Kato R, and Funae Y. Hormonal regulation of rat cytochrome P450s by androgen and pituitary. Arch Biochem Biophys 299: 179-184, 1992[ISI][Medline].

12.   Imig, JD Epoxyeicosatrienoic acids: biosynthesis, regulation, and actions. Methods Mol Biol 120: 173-192, 1999[Medline].

13.   Imig, JD, Zou AP, Stec DE, Harder DR, Falck JR, and Roman RJ. Formation and actions of 20-hydroxyeicosatetraenoic acid in rat renal arterioles. Am J Physiol Regulatory Integrative Comp Physiol 270: R217-R227, 1996[Abstract/Free Full Text].

14.   Ito, O, Alonso-Galicia M, Hopp KA, and Roman RJ. Localization of cytochrome P-450 4A isoforms along the rat nephron. Am J Physiol Renal Physiol 274: F395-F404, 1998[Abstract/Free Full Text].

15.   Kauser, K, Clark JE, Masters BS, Ortiz de Montellano PR, Ma YH, Harder DR, and Roman RJ. Inhibitors of cytochrome P450 attenuate the myogenic response of dog renal arcuate arteries. Circ Res 68: 1154-1163, 1991[Abstract].

16.   Kawashima, H, Kusunose E, Thompson CM, and Strobel HW. Protein expression, characterization, and regulation of CYP4F4 and CYP4F5 cloned from rat brain. Arch Biochem Biophys 347: 148-154, 1997[ISI][Medline].

17.   Kimura, S, Hanioka N, Matsunaga E, and Gonzalez FJ. The rat clofibrate-inducible CYP4A gene subfamily. I. Complete intron and exon sequence of the CYP4A1 and CYP4A2 genes, unique exon organization, and identification of a conserved 19-bp upstream element. DNA 8: 503-516, 1989[ISI][Medline].

18.   Kimura, S, Hardwick JP, Kozak CA, and Gonzalez FJ. The rat clofibrate-inducible CYP4A gene subfamily. II. cDNA sequence of IVA3, mapping of the Cyp4a locus to mouse chromosome 4, and coordinate and tissue-specific regulation of the CYP4A genes. DNA 8: 517-525, 1989[ISI][Medline].

19.   Kroetz, DL, Huse LM, Thuresson A, and Grillo MP. Developmentally regulated expression of the CYP4A genes in the spontaneously hypertensive rat kidney. Mol Pharmacol 52: 362-372, 1997[Abstract/Free Full Text].

20.   Laniado Schwartzman, M, da-Silva JL, Lin F, Nishimura M, and Abraham NG. Cytochrome P450 4A expression and arachidonic acid omega -hydroxylation in the kidney of the spontaneously hypertensive rat. Nephron 73: 652-663, 1996[ISI][Medline].

21.   Laniado-Schwartzman, M, Falck JR, Yadagiri P, and Escalante B. Metabolism of 20-hydroxyeicosatetraenoic acid by cyclooxygenase: formation and identification of novel endothelium-dependent vasoconstrictor metabolites. J Biol Chem 264: 1162-1165, 1989.

22.   Lin, F, Abraham NG, and Laniado-Schwartzman M. Arachidonic acid omega -hydroxylation and cytochrome P450 4A expression in the rat kidney. In: Advances in Prostaglandin, Thromboxane and Leukotriene Research, edited by Samuelsson B, and Paoletti R.. New York: Raven, 1995.

23.   Ma, YH, Schwartzman ML, and Roman RJ. Altered renal P-450 metabolism of arachidonic acid in Dahl salt-sensitive rats. Am J Physiol Regulatory Integrative Comp Physiol 267: R579-R589, 1994[Abstract/Free Full Text].

24.   Nguyen, X, Wang MH, Reddy KM, Falck JR, and Schwartzman ML. Kinetic profile of the rat CYP4A isoforms: arachidonic acid metabolism and isoform-specific inhibitors. Am J Physiol Regulatory Integrative Comp Physiol 276: R1691-R1700, 1999[Abstract/Free Full Text].

25.   Omata, K, Abraham NG, Escalante B, and Laniado-Schwartzman M. Age-related changes in renal cytochrome P-450 arachidonic acid metabolism in spontaneously hypertensive rats. Am J Physiol Renal Fluid Electrolyte Physiol 262: F8-F16, 1992[Abstract/Free Full Text].

26.   Omata, K, Abraham NG, and Laniado-Schwartzman M. Renal cytochrome P-450 arachidonic acid metabolism: localization and hormonal regulation in SHR. Am J Physiol Renal Fluid Electrolyte Physiol 262: F591-F599, 1992[Abstract/Free Full Text].

27.   Reed, CJ, Lock EA, and De Matteis F. NADPH: cytochrome P450 reductase in olfatory epithelium. Relevance to cytochrome P450-dependent reactions. Biochem J 240: 585-592, 1986[ISI][Medline].

28.   Scarborough, PE, Ma J, Qu W, and Zeldin DC. P450 subfamily CYP2J and their role in the bioactivation of arachidonic acid in extrahepatic tissues. Drug Metab Rev 31: 205-234, 1999[ISI][Medline].

29.   Stromstedt, M, Hayashi SI, Zaphiropoulos PG, and Gustafsson JA. Cloning and characterization of a novel member of the cytochrome P450 subfamily IVA in rat prostate. DNA 9: 567-577, 1990.

30.   Sun, CW, Alonso-Galicia M, Taheri MR, Falck JR, Harder DR, and Roman RJ. Nitric oxide-20-hydroxyeicosatetraenoic acid interaction in the regulation of K+ channel activity and vascular tone in renal arterioles. Circ Res 83: 1069-1079, 1998[Abstract/Free Full Text].

31.   Sundseth, SS, and Waxman DJ. Sex-dependent expression and clofibrate inducibility of cytochrome P450 4A fatty acid omega -hydroxylases. J Biol Chem 267: 3915-3921, 1993[Abstract/Free Full Text].

32.   Tanaka, S, Imaoka S, Kusunose E, Kusunose M, Maekawa M, and Funae Y. omega - and (omega -1) Hydroxylation of arachidonic acid, lauric acid and prostaglandin A1 by multiple forms of cytochrome P450 purified from rat hepatic microsomes. Biochim Biophys Acta 1043: 177-181, 1990[ISI][Medline].

33.   Wang, MH, Guan H, Nguyen X, Zand B, Nasjletti A, and Laniado-Schwartzman M. Contribution of cytochrome P-450 4A1 and 4A2 to vascular 20-hydroxyeicosatetraenoic acid synthesis in the rat kidney. Am J Physiol Renal Physiol 276: F246-F253, 1999[Abstract/Free Full Text].

34.   Wang, MH, Stec DE, Balazy M, Mastyugin V, Yang CS, Roman RJ, and Laniado Schwartzman M. Cloning, sequencing and cDNA-directed expression of the rat renal CYP4A2: arachidonic acid omega -hydroxylation and 11,12-epoxidation by CYP4A2 protein. Arch Biochem Biophys 336: 240-250, 1996[ISI][Medline].

35.   Webb, SJ, Xiao GH, Geoghegan TE, and Prough RA. Regulation of CYP4A expression in rat by dehydroepiandrosterone and thyroid hormone. Mol Pharmaco 49: 276-287, 1996[ISI].

36.   Zou, AP, Fleming JT, Falck JR, Jacobs ER, Gebremedhin D, Harder DR, and Roman RJ. 20-HETE is an endogenous inhibitor of the large-conductance Ca2+-activated K+ channel in renal arterioles. Am J Physiol Regulatory Integrative Comp Physiol 270: R228-R237, 1996[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 283(1):F60-F67
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