Contribution of cytochrome P-450 4A1 and 4A2 to vascular 20-hydroxyeicosatetraenoic acid synthesis in rat kidneys

Mong-Heng Wang, Hui Guan, Xuandai Nguyen, Barbara A. Zand, Alberto Nasjletti, and Michal Laniado-Schwartzman

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


    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

20-Hydroxyeicosatetraenoic acids (20-HETE), a biologically active cytochrome P-450 (CYP) metabolite of arachidonic acid in the rat kidney, can be catalyzed by CYP4A isoforms including CYP4A1, CYP4A2, and CYP4A3. To determine the contribution of CYP4A isoforms to renal 20-HETE synthesis, specific antisense oligonucleotides (ODNs) were developed, and their specificity was examined in vitro in Sf9 cells expressing CYP4A isoforms and in vivo in Sprague-Dawley rats. Administration of CYP4A2 antisense ODNs (167 nmol · kg body wt-1 · day-1 iv for 5 days) decreased vascular 20-HETE synthesis by 48% with no effect on tubular synthesis, whereas administration of CYP4A1 antisense ODNs inhibited vascular and tubular 20-HETE synthesis by 52 and 40%, respectively. RT-PCR of microdissected renal microvessel RNA indicated the presence of CYP4A1, CYP4A2, and CYP4A3 mRNAs, and a CYP4A1-immunoreactive protein was detected by Western analysis of microvessel homogenates. Blood pressure measurements revealed a reduction of 17 ± 6 and 16 ± 4 mmHg in groups receiving CYP4A1 and CYP4A2 antisense ODNs, respectively. These studies implicate CYP4A1 as a major 20-HETE synthesizing activity in the rat kidney and further document the feasibility of using antisense ODNs to specifically inhibit 20-HETE synthesis and thereby investigate its role in the regulation of renal function and blood pressure.

arachidonic acid; antisense oligonucleotides; preglomerular microvessels; proximal tubules; blood pressure


    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

PREVIOUS STUDIES HAVE established that 20-hydroxyeicosatetraenoic acid (20-HETE), the omega -hydroxylation product of arachidonic acid, is the principal arachidonic acid metabolite formed in tubular and vascular structures of the rat renal cortex and outer medulla (11, 22). The synthesis of 20-HETE is age dependent and is higher in kidneys obtained from spontaneously hypertensive rats (21). Numerous studies have demonstrated that 20-HETE is endowed with potent biological activities and have provided evidence that it contributes to the regulation of renal vascular and tubular functions and to the control of arterial pressure (18, 20, 35, 36).

The omega -hydroxylation of fatty acids, including arachidonic acid, is catalyzed by enzymes of the CYP4A family. In the rat, four isoforms have been identified: CYP4A1, CYP4A2, CYP4A3, and CYP4A8 (14, 15). Messages for all four of these isoforms have been identified in the kidney (28). These isoforms, although sharing 66-98% homology and a common catalytic activity, i.e., hydroxylation of fatty acids at the omega -carbon, are localized to different renal structures and are exposed to different regulatory mechanisms. For example, whereas CYP4A2 is preferentially expressed in the outer-medullary/thick ascending limb of Henle's loop region, CYP4A1 and CYP4A3 are highly expressed in the proximal tubules (9, 13). In addition, CYP4A2 is believed to be the major CYP4A isoform expressed in the renal microvasculature, a major site of 20-HETE synthesis and action (11). Renal CYP4A1 and CYP4A3 can be induced by hypolipidimic drugs such as clofibrate, whereas CYP4A2 is thought to be constitutively expressed, especially in male rats (10, 14, 15, 29). The renal expression of these isoforms is age dependent; CYP4A1 and CYP4A3 proteins are detectable in the fetus, and their levels gradually increase from birth until ~9 wk of age, followed by a progressive decline to very low levels in adults. CYP4A2 protein is undetectable until 5 wk, but then the levels increase so that in adult male rats it is the major isoform detected in the kidney (16, 17).

The role of each of the CYP4A proteins in the generation of endogenous 20-HETE in the rat kidney is yet to be determined. Studies with purified and recombinant proteins characterize some of the catalytic activities and substrate specificities of the rat CYP4A isoforms (1, 3, 7, 27, 30). Recent studies in our laboratory, using baculovirus-expressed recombinant CYP4A proteins, demonstrated that CYP4A1, CYP4A2, and CYP4A3, but not CYP4A8, exhibit significant arachidonic acid omega -hydroxylating activity. Moreover, CYP4A2 and CYP4A3, which share 97% homology, have similar catalytic properties, both catalyzing arachidonic acid omega -hydroxylation and 11,12-epoxidation. In contrast, CYP4A1 functions solely as an arachidonate omega -hydroxylase with a turnover rate 20 times higher than that of CYP4A2 or CYP4A3 (unpublished data). Because of the high sequence homology among CYP4A isoforms, overlapping catalytic activities, and the similar sensitivity to metabolic inhibitors, it is difficult to evaluate the contribution of each of these isoforms to the synthesis of 20-HETE in the kidney.

Antisense oligonucleotides (ODNs) have been widely used as specific inhibitors of gene expression. They offer the possibility of blocking the expression of a particular gene without any changes in the function of other genes. Several investigators used antisense ODNs to inhibit specific CYP activities in vitro and in vivo with significant success (5, 32). In the present study, we designed antisense ODNs against CYP4A1 and CYP4A2/4A3 expression and evaluated their effectiveness for achieving isoform-specific inhibition of 20-HETE synthesis in vitro and in vivo.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Materials. [1-14C]arachidonic acid (56 mCi/mmol) was obtained from DuPont-New England Nuclear (Boston, MA). Purified recombinant rat NADPH-cytochrome P-450 (c) oxidoreductase (OR; EC 1.6.2.4) (specific activity, 58 µmol · min-1 · mg-1) was purchased from Oxford Biomedical Research (Oxford, MI). Emulgen E911 was obtained from KAO Atlas (Tokyo, Japan). Tissue culture and molecular biology reagents were purchased from Life Technologies (Gaithersburg, MD). Sf9 insect cells and the liposome DNA transfection kit were from Invitrogen (San Diego, CA). Purified recombinant rat liver cytochrome b5 (specific activity, 40 nmol/mg) was purchased from Panvera (Madison, WI). All solvents were HPLC grade.

Preparation of recombinant CYP4A cell membranes. CYP4A proteins were expressed in the baculovirus-Sf9 insect cell expression system as described previously (33). CYP4A recombinant Sf9 cell membranes were prepared after infection with the recombinant virus and incubation in the presence of hemin (4 µg/ml) for 72 h followed by centrifugation at 100,000 g for 60 min of cell lysates as described (33). The membrane pellets were resuspended in sucrose buffer (50 mM potassium phosphate, pH 7.4, and 0.5 M sucrose) and stored at -80°C. Protein concentration was determined according to the method of Bradford (Bio-Rad, Melville, NY). Cytochrome P-450 content was calculated from the reduced CO-difference spectrum using an extinction coefficient of 91 mM-1 (23).

Preparation and selection of ODNs. Antisense and scrambled ODNs were targeted to bases -3 to +21 of the CYP4A1 and CYP4A2 cDNAs (15) encompassing the ATG translation site codon. The scrambled ODNs were designed to have the same base composition as the antisense ODNs. All ODN sequences were aligned to the DNA database (GenBank) using the MacVector Sequence Analysis Software. The phosphorothioate derivatives of the selected ODNs were synthesized and purified by Gene Link (Thornwood, NY). The following ODNs were used: antisense for CYP4A2 (4A2-AS), 5'-GCT-AAA-TAC-AGA-GAA-ACC-CAT-GGT-3'; scrambled ODN for CYP4A2 (4A2-S), 5'-CAG-ACC-GCA-GGA-CTA-AAT-AGA-TAT-3'; antisense for CYP4A1 (4A1-AS), 5'-CAG-TGC-AGA-GAC-GCT-CAT-GGT-3' (21 bases) and 5'-CAG-TGC-AGA-GAC-GCT-CAT-3' (18 bases); scrambled ODN for CYP4A1 (4A1-S), 5'-CTG-ACC-GCA-GCA-GGA-CTT-AGA-TGG-3'. The potency and specificity of antisense ODNs were tested in the baculovirus/Sf9 cell system. Briefly, Sf9 cells (1 × 105 cells/dish) were infected with CYP4A recombinant viruses in the presence of hemin (4 µg/ml). Various concentrations of liposome-encapsulated antisense ODNs, scrambled ODNs, or liposome alone were added into 3-cm culture dishes at 3, 24, 48, and 72 h after infection. The amount of liposomes was fixed at 1 µg per each 0.06 nmol of ODNs. Twenty-four hours after the last treatment, cells were harvested, and membranes were prepared for analyses.

Preparation of liposomes. Cationic liposomes composed of dimethyldioctadecylammonium bromide and L-alpha -dioleylphosphatidylethanolamine (2:5, wt/wt; Avanti Polar Lipid, Alabaster, AL) were dissolved in 1 ml chloroform, and the solvent was evaporated under N2 gas at room temperature. Liposomes were prepared by resuspending the lipids in 9 ml of sterile deionized water and sonication on ice until the solution was almost clear (19).

Animal treatment. Male Sprague-Dawley rats (Harlan, 7 wk old; n = 4 per group) anesthetized with pentobarbital sodium (60 mg/kg ip) were instrumented with two polyethylene (PE-50) catheters. One catheter was placed into the femoral artery for blood pressure measurement, and the other one was placed into the femoral vein for administration of ODNs. The rats were allowed 24 h to recover before commencing treatment. Liposome-encapsulated antisense ODNs or scrambled ODNs (1 µg/0.06 nmol ODNs) were injected daily as a bolus at doses of 167 nmol · kg body wt-1 · day-1 for 5 days. Additional rats were treated with liposomes mixed with the ODN vehicle (water). In some experiments, rats were injected with two antisense preparations, in combination, each at a dose of 100 nmol · kg body wt-1 · day-1. On day 6, after the onset of treatment, the rats were killed, and tissues (liver and kidney) were removed for preparation of microsomes and isolation of preglomerular vessels and proximal tubules as previously described (33, 34). The purity of each preparation was ascertained by phase-contrast microscopy and by the presence or absence of immunoreactive gamma -glutamyl transpeptidase (gamma -GT), a specific marker for the proximal tubule brush border (2). The microvessel preparation was estimated to be 98% pure by phase-contrast microscopy (Fig. 1A). Furthermore, Western analysis of microvessel homogenates with antibody against gamma -GT revealed no immunoreactive protein in the microvessel preparation. In contrast, proximal tubular homogenates showed a strong immunoreactive band (Fig. 1B). To examine the specificity of these ODNs, CYP2E1-derived p-nitrophenol hydroxylation and cytochrome P-450 (c) reductase activity were measured as previously described (33).


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Fig. 1.   Purity of the renal microvessel preparation. A: phase-contrast microscopy of microdissected renal microvessel. Renal microvessels were prepared as described under METHODS. Renal microvessels appear as blue-filled branching structures. Magnification, ×20. B: Western blot analysis of gamma -glutamyl transpeptidase (gamma -GT). Twenty micrograms of microsomal proteins from proximal tubules (lane 5) and varying concentration of renal microvessel homogenate proteins (2.5, 5, 10, 20 µg, lanes 1-4) were subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblot analysis with anti-gamma -GT IgG as described under METHODS.

Arachidonic acid metabolism. Microsomes prepared from different tissues (150 µg) were preincubated with [1-14C]arachidonic acid (0.4 µCi, 7 nmol) in 100 mM potassium phosphate buffer, pH 7.4, containing 10 mM MgCl2 for 3 min at 37°C. NADPH (1 mM) and the reaction mixture with a final volume of 0.4 ml was incubated for 30 min at 37°C. For recombinant CYP4A proteins, CYP4A-expressed Sf9 cell membranes (10 pmol P-450) were mixed with purified OR and cytochrome b5 at a molar ratio of 1:14:4 in a final volume of 150 µl buffer and incubated for 3 min on ice followed by incubation with [1-14C]arachidonic acid (0.4 µCi, 7 nmol) for an additional 3 min before adding NADPH (1 mM). Incubations were carried out 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 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, Tampa, FL). The identity of each metabolite was confirmed by its comigration with an authentic standard.

Western blot analysis. Goat anti-rat CYP4A1 antibody was purchased from Gentest (Woburn, MA). Although this polyclonal antibody preparation cross-reacts with all CYP4A proteins, its sensitivity for detecting CYP4A1 protein is ~30 times higher than that of CYP4A2 protein and 2 and 9 times greater than CYP4A3 and CYP4A8 proteins, respectively. CYP4A-expressed Sf9 cell membranes were separated by electrophoresis on an 8% SDS-polyacrylamide gel at 25 mA/gel at 4°C for 18-20 h, and immunoblotting using either phosphoimaging or alkaline phosphatase detection systems was performed. Microsomes prepared from microvessels and proximal tubules were treated with SDS reducing buffer containing 62.5 mM Tris · HCl, pH 6.8, 10% glycerol, 2% SDS, 5% beta -mercaptoethanol, and 0.125% (wt/vol) bromophenol blue, then further incubated in boiling water for 4 min. SDS gel electrophoresis was performed, and immunoreactive CYP4A proteins were detected as described above. Antibody against gamma -GT was kindly provided by Dr. R. P. Hughey, University of Pittsburgh Medical School. Western analysis for gamma -GT was performed as described previously (2).

RT-PCR. Renal microvessels (3 mm in length) were microdissected and connective tissue was removed under microscope. The microvessels were transferred by microbeads (0.5-1 mm in diameter) into 10 µl of a buffer containing 2% (vol/vol) Triton X-100, 5 mM DTT, and 1,600 U/ml of RNasin (Promega, Madison, WI). The microvessels were immediately frozen on dry ice and stored in -80°C until RT-PCR reaction was performed. The total RNA of the whole kidney was extracted using TRIzol reagent (Life Technologies). The RNA pellet was resuspended in nuclease-free water. A reverse transcription reaction was performed using a First-strand cDNA synthesis kit (Pharmacia Biotech, Milwaukee, WI). Briefly, RNA (3 µg) from whole kidney or 10 µl of microvessel permeability solution was added to 15 µl reverse transcription reaction mixture containing 45 mM Tris (pH 8.3), 68 mM KCl, 15 mM DTT, 9 mM MgCl2, 0.08 mg/ml BSA, 1.8 mM dNTP, 40 pmol of either CYP4A1, 4A2, or 4A3 backward primer, and Moloney murine leukemia virus reverse transcriptase. The reactions were incubated for 1 h at 37°C and terminated by heating to 95°C for 5 min. PCR was carried out in a 100-µl reaction mixture containing 50 mM Tris · HCl (pH 9.0), 20 mM NH4SO4, 3 mM MgCl2, 200 µM dNTPs, 20 pmol of specific CYP4A1, 4A2, and 4A3 primer pairs, and 10 µl of first strand synthesis reaction from whole kidney or microvessels. PCR was also performed using the CYP4A plasmids (100 ng) as a template for positive controls. Samples containing Ampliwax (Perkin-Elmer, Branchburg, NJ) were denatured by heating to 80°C for 8 min, cooled to 2°C for 2 min, and heated to 22°C for 2 min to solidify the wax. Tf1 DNA polymerase (1 U; Epicentre Technologies, Madison, WI) was added to initiate a "hot-start" reaction. Reactions were cycled 30 times through a 1-min denaturing step at 95°C, a 1-min annealing step at 50°C, and a 1-min extension step at 70°C. After the cycling procedure, a final 10-min elongation step at 70°C was performed. The specific CYP4A1, 4A2, and 4A3 primers were designed to amplify 827-, 317-, and 321-bp fragments from each of the corresponding cDNAs. Additional nested PCR amplification was performed for CYP4A1 (300-bp expected fragment size). The sequences of the primers used were as follows: CYP4A1, 5'-GTA TCC AAG TCA CAC TCT CCA-3' (forward primer), 5'-CAG GAC ACT GGA CAC TTT ATT G-3' (backward primer for 827 bp), 5'-CAG AGG TGT TTG ACC CTT CCA G-3' (forward primer, nested PCR), 5'-CCA TCA AGG TTG TGA CCG TTG-3' (backward primer, nested PCR for 300 bp); CYP4A2, 5'-AGA TCC AAA GCC TTA TCA ATC-3' (forward primer), 5'-CAG CCT TGG TGT AGG ACC T-3' (backward primer); CYP4A3, 5'-CAA AGG CTT CTG GAA TTT ATC-3' (forward primer), 5'-CAG CCT TGG TGT AGG ACC T-3' (backward primer). An aliquot (12 µl) of each PCR reaction was separated by 1.5% agarose gel, and PCR products were stained with ethidium bromide. The PCR products were transferred from the agarose gel to nylon membranes (Hybond-N, Amersham) by the capillary method for 18 h, ultraviolet cross-linked to the membrane, and prehybridized for 30 min at 65°C in a rapid-hybridization buffer (Amersham). Southern hybridization was performed using 32P-labeled CYP4A cDNA probes (Rediprimer Kit, Amersham).


    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Antisense ODNs were designed to specifically recognize the ATG region of the CYP4A isoforms. The 4A2-antisense ODN contained 24 nucleotides and by computer analysis showed no homology to CYP4A1 or CYP4A8 mRNA sequences. However, it did recognize CYP4A3 mRNA; the homology between these two isoforms in their coding regions is 97%. The 4A2-scrambled ODN contained the same base composition and showed by computer analysis no sequence homology with CYP4A2 or any known CYP sequences. The specificity and potency of these ODNs on CYP4A2 protein levels and CYP4A2-derived arachidonic acid omega -hydroxylation were assessed in the Sf9 cells expressing CYP4A2. As seen in Fig. 2A, 20-HETE synthesis in Sf9 cells treated with 0.1 µM 4A2-antisense ODN was inhibited by 72%. At high concentrations of 4A2-antisense ODN (1 and 10 µM), arachidonate omega -hydroxylase activity was decreased to ~20% of control. The 4A2-scrambled ODN had little effect on CYP4A2-catalyzed 20-HETE synthesis at 0.1 and 1 µM. However, at high concentration (10 µM) the specificity disappeared; 4A2-scrambled ODN caused ~50% inhibition of 20-HETE synthesis.


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Fig. 2.   Effect of cytochrome P-450 4A2 (CYP4A2; A) or CYP4A1 (B)-antisense oligonucleotides (ODNs) on 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis in Sf9 cells expressing either CYP4A2 or CYP4A1 proteins. Sf9 cells were infected with either CYP4A2 or CYP4A1 recombinant viruses. Concentrations from 0.1 to 30 µM of liposome-encapsulated 4A2-antisense ODN (4A2-AS), 4A2-scrambled ODN (4A2-S), 4A1-antisense ODN (4A1-AS), or 4A1-scrambled ODN (4A1-S) were added into culture dishes at 3, 24, 48, and 72 h after infection. Twenty-four hours later, the last treatment cells were harvested, and membranes were prepared for analyses. Arachidonic acid metabolism was measured as described in METHODS. Results are expressed as percent of control and are the means of 3 separate determinations.

With respect to CYP4A1, two different lengths of antisense ODNs were examined for their potency and specificity in the baculovirus-Sf9 cell system. Both were targeted to the ATG initiation codon of the CYP4A1 cDNA; one contained 18 nucleotides and the other 21 nucleotides. The results depicted in Fig. 2B indicate that both antisense ODNs inhibited arachidonate omega -hydroxylation in a concentration-dependent manner. However, the 21-mer antisense ODN was more potent, inhibiting 20-HETE synthesis by 75% at 10 µM. The 4A1-scrambled ODN had little effect on 20-HETE synthesis, inhibiting it by 20% at 10 µM (Fig. 2B). Additional control experiments revealed no effect of the liposome vehicle on 20-HETE synthesis. Moreover, treatment of cells expressing CYP4A1 protein with 4A2-AS or of cells expressing CYP4A2 with 4A1-AS did not affect 20-HETE synthesis (data not shown).

The specific antisense ODNs were used for evaluating whether selective inhibition of CYP4A-derived 20-HETE synthesis can be achieved in vivo. The dosage of the ODN preparations (phosphorothioate derivatives mixed with liposomes for increased efficiency of delivery and uptake) used for the in vivo studies was arbitrarily chosen; it was primarily based on literature research and partially on our in vitro screening. Figure 3 contrasts the effects of in vivo treatment of 4A2- and 4A1-antisense ODNs on 20-HETE synthesis by microvessels and proximal tubules homogenates. We used the 21-base antisense ODN (4A1-AS) for the in vivo experiments. Rats were treated with 4A2- or 4A1-antisense ODNs for 5 days at 167 nmol · kg-1 · day-1. Both 4A2- and 4A1-antisense ODNs significantly decreased 20-HETE synthesis in microvessel homogenates by 40 and 50%, respectively. Proximal tubular synthesis of 20-HETE was markedly inhibited by 50% in rats treated with 4A1-antisense ODN but was unaffected in rats treated with 4A2-antisense ODN. In two additional sets of experiments, rats were treated with both antisense preparations (each ODN preparation at 100 nmol/kg) following the same protocol. The combined administration of both antisense preparations resulted in a significant 75% inhibition of renal microvessel 20-HETE synthesis. Additional analysis revealed that 20-HETE synthesis was also inhibited in liver microsomes by ~50% in rats treated with the CYP4A antisense preparations. For example, 4A2-antisense ODN reduced 20-HETE synthesis from 134 ± 4 to 67 ± 1 pmol · mg-1 · min-1, whereas the 4A2-scrambled ODN reduced it to 108 ± 6 pmol · mg-1 · min-1. Western blot analysis further confirmed that the decreased synthesis of 20-HETE was associated with a decrease in the levels of CYP4A-immunoreactive proteins (Fig. 4). Treatment with 4A2-antisense ODN did not affect renal cytochrome P-450 (c) reductase (78 U/mg in control vs. 76 U/mg in 4A2-AS-treated rats) or CYP2E1-dependent p-nitrophenol hydroxylase activity (0.35 nmol · min-1 · mg-1 in control vs. 0.32 nmol · min-1 · mg-1 in 4A2-AS-treated rats).


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Fig. 3.   Effect of systemic administration of CYP4A1 and CYP4A2-antisense ODNs on proximal tubular (A) and renal microvessel (B) 20-HETE synthesis. 4A1-antisense ODN (4A1-AS), 4A2-antisense ODN (4A2-AS), or vehicle (liposomes without ODN) were administered into femoral vein of 7-wk-old rats (167 nmol · kg body wt-1 · day-1 for 5 days). Proximal tubules and renal microvessels were isolated, and homogenates were prepared for arachidonic acid metabolism. Results are means ± SE; n = 4 per group. * P < 0.05.


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Fig. 4.   Effect of the combined administration of CYP4A1 and CYP4A2-antisense ODNs on CYP4A protein expression in renal microvessels. ODNs (4A1- and 4A2-antisense ODNs, each at a dose of 100 nmol · kg body wt-1 · day-1) or vehicle was administered into the femoral vein of 7-wk-old rats as described in METHODS. Renal microvessel homogenate (30 µg protein) from vehicle treatment (lanes 1 and 2) and from ODNs treatment (lanes 3 and 4), 1 µg Sf9-expressed CYP4A1 (lane 5), and 5 µg Sf9-expressed CYP4A2 (lane 6) were subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblot analysis with anti-4A IgG as described under METHODS.

The finding that renal microvessel synthesis of 20-HETE is inhibited by 4A1-AS conflicts with reports indicating that CYP4A1 mRNA and protein could not be detected in the renal vasculature (12). Accordingly, we set out to investigate whether CYP4A1 is present in the renal microvessel by performing RT-PCR with CYP4A-specific primers using microdissected preglomerular renal microvessels freed from tubular, connective, and parenchymal tissues. As seen in Fig. 5A, ethidium bromide staining of the agarose gel demonstrated that all three CYP4A isoforms (4A1, 4A2, and 4A3) were amplified from reverse-transcribed whole kidney RNA (lanes 2, 5, and 8) as well as from their corresponding plasmid cDNAs (lanes 3, 6, and 9). Similarly, RNA isolated from renal microvessels revealed the presence of 317-bp and 321-bp products for CYP4A2 and CYP4A3, respectively (Fig. 5A, lanes 4 and 7). However, we did not detect a corresponding band (827 bp) for CYP4A1 (Fig. 5A, lane 1). Such a result could be explained by different amplification efficiency, since the CYP4A1-specific primers amplified a 825-bp fragment, whereas the CYP4A2- and CYP4A3-specific primers amplified much shorter fragments. We therefore performed additional nested PCR amplification using the first round RT-PCR mixture as the template. As shown in Fig. 5B (lanes 1-3), the expected 300-bp CYP4A1-specific fragment was present in renal microvessel preparation as well as in PCR mixtures from whole kidney and CYP4A1 plasmid. To confirm the authenticity of these PCR products, these fragments were transferred to nylon membranes and hybridized with each CYP4A cDNA probe. The results of Southern blot analysis indicated that CYP4A1 along with CYP4A2 and CYP4A3 are expressed in the renal microvessels (Fig. 5C). Western analysis using a large gel and Sf9 recombinant CYP4A membranes as standards revealed the presence of one major immunoreactive band with the elution profile of the recombinant CYP4A1 and another weak immunoreactive signal having the mobility of the recombinant CYP4A2. Also, addition of CYP4A1 and CYP4A2 recombinant proteins to the microvessel homogenates before SDS gel separation reinforced the intensity of the signal generated by CYP4A1 and CYP4A2 present in the renal microvessels (Fig. 6).


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Fig. 5.   RT-PCR analysis of renal microvessel mRNA with CYP4A isoform-specific primers. A: total RNA were extracted and amplified with CYP4A1-specific primers (825 bp, lanes 1-3), CYP4A2-specific primers (317 bp, lanes 4-6), or CYP4A3-specific primers (321 bp, lanes 7-9). PCR products were separated on an agarose gel and stained with ethidium bromide. Lanes 1, 4, and 7, RNA from single isolated branch of renal microvessel; lanes 2, 5, and 8, RNA from whole kidney; lanes 3, 6, and 9, corresponding CYP4A cDNA plasmids. B: first round CYP4A1 PCR products were amplified with CYP4A1 nested PCR primers (300 bp). Lane 1, RNA from single isolated branch of renal microvessel; lane 2, RNA from whole kidney; lane 3, CYP4A1 cDNA plasmid. C: Southern hybridization of 4A1 nested, 4A2, and 4A3 RT-PCR products with the corresponding CYP4A cDNA probes. Lane M, 1-kb DNA molecular weight marker.


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Fig. 6.   Western analysis of renal microvessel homogenates. Renal microvessel homogenate (20 and 80 µg of protein; MV-20 and MV-80, respectively) and the membrane fraction from Sf9 cells expressing CYP4A1 (0.05 µg; 0.02 pmol P-450), CYP4A2 (3 µg; 0.75 pmol P-450), and CYP4A3 (1 µg; 0.36 pmol P-450) proteins were subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblot analysis with goat anti-rat CYP4A1 antibody (1:1,000) as described in METHODS.

Of interest, measurement of blood pressure in the in vivo experiments revealed a significant reduction in blood pressure after treatment with either of the antisense preparations. Thus daily injection of 4A1-antisense ODN or 4A2-antisense ODNs for 5 days reduced the blood pressure by 17 ± 5 and 16 ± 4 mmHg, respectively (Table 1). When rats were treated with both antisense ODNs, blood pressure was reduced by 22 ± 6 mmHg. No significant changes were observed in rats treated with the vehicle control or in rats treated with the scrambled ODNs (Table 1).

                              
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Table 1.   Effect of antisense oligonucleotides on MABP in Sprague-Dawley rats


    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

In the present study, we evaluate the use of antisense ODNs for inhibition of specific CYP4A isoform expression and 20-HETE synthesis. One problem in assessing the specificity, potency, and efficacy of antisense ODNs against CYP-derived enzymatic activity is the lack of an in vitro system, e.g., cultured cells or cell line, expressing these activities. Many cells demonstrate a time-dependent rapid loss of CYP enzyme activity and expression following cultivation (24), which can be maintained, to some extent, with CYP inducers or cofactors (31). For example, proximal tubular epithelial cells, which are endowed with CYP enzyme activity in vivo, lose 20-HETE synthesizing capacity shortly after cultivation (18). We have used the Sf9 cells expressing CYP4A isoforms as an in vitro system to screen antisense ODNs for their specificity and efficacy in inhibiting isoform-specific expression and 20-HETE synthesis. To eliminate degradation, phosphorothioate-modified ODNs were synthesized, and to maximize uptake of ODNs, a liposomal mixture of the derivatized ODNs was administered (4, 19). Results of the in vitro studies in Sf9 cells revealed that CYP4A1- and CYP4A2/4A3-specific antisense ODNs have the ability to inhibit both protein expression and the corresponding catalytic activity, i.e., conversion of arachidonic acid to 20-HETE. These studies also yielded control ODNs that had identical base composition in a scrambled sequence but were devoid of biological activity.

The various ODNs were used for the subsequent in vivo studies where the dose, route, and duration of treatments were extrapolated in part from the in vitro experiments as well as from published studies with various antisense ODNs (4, 5, 32). We chose systemic administration, since it has been shown that phosphorothioate-modified ODNs administered intravenously accumulate mainly in the kidney and rapidly metabolize to shorter ODNs (4). Our studies clearly indicate that intravenous administration of antisense ODNs directed against CYP4A1 or CYP4A2/4A3 expression, given separately or combined, markedly inhibited 20-HETE synthesis in the renal preglomerular vessels.

The finding that the specific CYP4A1 antisense ODN preparation inhibited renal vascular 20-HETE synthesis in vivo is intriguing. There are several reports indicating that CYP4A1 expression in the rat male kidney is either undetectable or very low (25, 29). Moreover, CYP4A2 is believed to be the major CYP4A isoform expressed in the renal microvasculature of the male rat (11, 12). The results of the in vivo studies suggested that a significant portion of 20-HETE synthesis in the renal vasculature is CYP4A1 derived. It is possible that despite the clear specificity of the CYP4A1 antisense ODN in vitro (i.e., it did not affect CYP4A2-derived 20-HETE formation), in vivo this specificity was lost. However, RT-PCR and Western analyses provided substantial evidence to suggest that this vascular bed expresses, in addition to CYP4A2 and CYP4A3 mRNAs and proteins, CYP4A1 mRNA and CYP4A1-immunoreactive protein. Recent studies in our laboratory compared and contrasted the catalytic activities of the recombinant rat CYP4A isoforms. These studies clearly identified CYP4A1 as the low-Km arachidonic acid omega -hydroxylase; its capacity to form 20-HETE was 20 times greater than that of CYP4A2 or CYP4A3 recombinant proteins, namely, Km/Vmax of 928 vs. 72 and 22 min-1 for CYP4A1, CYP4A2, and CYP4A3, respectively (Nguyen and Laniado-Schwartzman, unpublished data). These findings suggest that, although present in lower amounts in the kidney, CYP4A1, with its high arachidonic acid omega -hydroxylation activity, may be a major catalyst for the formation of 20-HETE in the rat kidney.

The in vivo studies demonstrate that in rats treated with the CYP4A1 antisense ODNs, proximal tubular synthesis was inhibited, suggesting the significant contribution of CYP4A1 to proximal tubular 20-HETE synthesis. In contrast, treatment with CYP4A2 antisense ODN did not affect 20-HETE synthesis in the proximal tubules. Thus, despite the relatively high expression of CYP4A2 in the proximal tubules (9), CYP4A2 participation in 20-HETE production may be minimal. The relatively low catalytic turnover of arachidonic acid omega -hydroxylation by the recombinant CYP4A2 protein may explain the limited contribution of this isoform to proximal tubular 20-HETE production. On the other hand, one cannot exclude the possibility that the pharmacodynamic/pharmacokinetic properties of the CYP4A2 antisense ODN are different from the CYP4A1 antisense ODN, including differences in binding affinity to plasma proteins and susceptibility to degradation in the proximal tubules (4, 26). These differences may limit the efficacy of the former ODN in its ability to inhibit 20-HETE synthesis in the proximal tubules.

The present study also demonstrates that administration of antisense ODNs against the CYP4A isoforms that catalyzed 20-HETE synthesis (CYP4A1 and CYP4A2/4A3) is associated with a significant reduction in blood pressure. Several studies have reported that 20-HETE is a constrictor of renal and extrarenal arterioles (6, 8, 11, 20). The vasoconstrictor property of 20-HETE has been attributed to its ability to potently inhibit the opening of the large-conductance Ca2+-activated K+ channel in vascular smooth muscle cells (34). Hence, the vasodepressor effect of the CYP4A antisense ODNs may be due to attenuation of a vasoconstrictor mechanism mediated by endogenous 20-HETE at renal and extrarenal sites.

In summary, this study is the first to demonstrate the use of antisense ODNs for inhibition of CYP4A isoform-specific 20-HETE synthesis. It also implicates CYP4A1 as a major 20-HETE-forming enzyme. These antisense ODNs provide the specificity needed for evaluating the contribution of each CYP4A isoform to the endogenous production of 20-HETE and thereby can be used to examine the pathophysiological role of 20-HETE.


    ACKNOWLEDGEMENTS

This study was supported in part by National Heart, Lung, and Blood Institute Grant PO1-HL-34300 and by Fellowship Award 970104 from the American Heart Association, New York State Affiliate.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests: M. L. Schwartzman, Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595 (E-mail: michal_schwartzman{at}nymc.edu).

Received 16 July 1998; accepted in final form 8 October 1998.


    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Alterman, M. A., C. S. Chaurasia, P. Lu, J. P. Hardwick, and R. P. Hanzlik. 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[Medline].

2.   Altman, R. A., A. V. Orr, C. F. Lagenaur, N. P. Curthoys, and R. P. Hughey. Expression of rat renal gamma -glutamyltranspeptidase in LLC-PK1 cells as a model for apical targeting. Biochemistry 32: 3822-3828, 1993[Medline].

3.   Aoyama, T., J. P. Hardwick, S. Imaoka, Y. Funae, H. V. Gelboin, and F. J. Gonzalez. Clofibrate-inducible rat hepatic P450s IVA1 and IVA3 catalyze the omega - and (omega -1)-hydroxylation of fatty acids and the omega -hydroxylation of prostaglandins E1 and E2a. J. Lipid Res. 31: 1477-1482, 1990[Abstract].

4.   Crooke, S. T., M. J. Graham, J. E. Zuckerman, D. Brooks, B. S. Conklin, R. L. Cummins, M. J. Greig, C. J. Guinosso, D. Kornbrust, R. Manoharan, H. M. Sasmor, T. Schleich, K. L. Tivel, and R. H. Griffey. Pharmacokinetic properties of several novel oligonucleotide analogs in mice. J. Pharmacol. Exp. Ther. 277: 923-937, 1996[Abstract].

5.   Desjardins, J. P., and P. L. Iversen. Inhibition of the rat cytochrome P450 3A2 by an antisense phosphorothioate oligonucleotide in vivo. J. Pharmacol. Exp. Ther. 275: 1608-1613, 1996[Abstract].

6.   Escalante, B., K. Omata, W. Sessa, S.-G. Lee, J. R. Falck, and M. Laniado-Schwartzman. 20-hydroxyeicosatetraenoic acid is an endothelium-dependent vasoconstrictor in rabbit arteries. Eur. J. Pharmacol. 235: 1-7, 1993[Medline].

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

8.   Harder, D. R., D. Gebremedhin, J. Narayanan, C. Jefcote, J. R. Falck, W. B. Campbell, and R. Roman. Formation and action of a P-450 4A metabolite of arachidonic acid in cat cerebral microvessels. Am. J. Physiol. 266 (Heart Circ. Physiol. 35): H2098-H2107, 1994[Abstract/Free Full Text].

9.   Hardwick, J. P. CYP 4A subfamily: functional analysis by immunocytochemistry and in situ hybridization. Methods Enzymol. 206: 273-283, 1991[Medline].

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

11.   Imig, J. D., A.-P. Zou, D. E. Stec, D. R. Harder, J. R. Falck, and R. J. Roman. Formation and actions of 20-hydroxyeicosatetraenoic acid in rat renal arterioles. Am. J. Physiol. 270 (Regulatory Integrative Comp. Physiol. 39): R217-R227, 1996[Abstract/Free Full Text].

12.   Ito, O., M. Alonso-Galicia, K. A Hopp, and R. J. Roman. Localization of cytochrome P-450 4A isoforms along the rat nephron. Am. J. Physiol. 274 (Renal Physiol. 43): F395-F404, 1998[Abstract/Free Full Text].

13.   Iwai, N., and T. Inagami. Isolation of preferentially expressed genes in the kidneys of hypertensive rats. Hypertension 17: 161-169, 1991[Abstract].

14.   Kimura, S., N. Hanioka, E. Matsunaga, and F. J. Gonzalez. 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 (NY) 8: 503-516, 1989[Medline].

15.   Kimura, S., J. P. Hardwick, C. A. Kozak, and F. J. Gonzalez. 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 (NY) 8: 517-525, 1989[Medline].

16.   Kroetz, D. L., L. M. Huse, A. Thuresson, and M. P. Grillo. Developmentally regulated expression of the CYP4A genes in the spontaneously hypertensive rat kidney. Mol. Pharmacol. 52: 362-372, 1997[Abstract/Free Full Text].

17.   Laniado-Schwartzman, M., J.-L. Da Silva, F. Lin, M. Nishimura, and N. G. Abraham. Cytochrome P450 4A expression and arachidonic acid omega -hydroxylation in the kidney of the spontaneously hypertensive rat. Nephron 73: 652-663, 1996[Medline].

18.   Lin, F., A. Rios, J. R. Falck, Y. Belosludtsev, and M. Laniado-Schwartzman. 20-hydroxyeicosatetraenoic acid is formed in response to EGF and is a mitogen in rat proximal tubule. Am. J. Physiol. 269 (Renal Fluid Electrolyte Physiol. 38): F806-F816, 1995[Abstract/Free Full Text].

19.   Liu, Y., D. Liggitt, W. Zhong, G. Tu, K. Gaensler, and R. Debs. Cationic liposome-mediated intravenous gene delivery. J. Biol. Chem. 270: 24864-24870, 1995[Abstract/Free Full Text].

20.   Ma, Y.-H., D. Gebremedhin, M. Laniado-Schwartzman, J. R. Falck, J. E. Clark, B. S. S. Masters, D. R. Harder, and R. J. Roman. 20-hydroxyeicosatetraenoic acid is an endogenous vasoconstrictor of canine renal arcuate arteries. Circ. Res. 72: 126-136, 1993[Abstract].

21.   Omata, K., N. G. Abraham, B. Escalante, and M. Laniado-Schwartzman. Age-related changes in renal cytochrome P-450 arachidonic acid metabolism in spontaneously hypertensive rats. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F8-F16, 1992[Abstract/Free Full Text].

22.   Omata, K., N. G. Abraham, and M. Laniado-Schwartzman. Arachidonic acid omega /omega -1 hydroxylase along the nephron of the spontaneously hypertensive rat. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F592-F599, 1992.

23.   Omura, T., and R. Sato. The carbon monoxide binding pigment of liver microsomes. J. Biol. Chem. 239: 2370-2379, 1964[Free Full Text].

24.   Paine, A. J. The cytochrome P450 gene superfamily. Int. J. Exp. Pathol. 72: 349-363, 1991[Medline].

25.   Rice Okita, J., S. B. Johnson, P. J. Castle, S. C. Dezellem, and R. T. Okita. Improved separation and immunodetection of rat cytochrome P450 4A forms in liver and kidney. Drug Metab. Dispos. 25: 1008-1012, 1997[Abstract/Free Full Text].

26.   Sawai, K., R. I. Mahato, Y. Oka, Y. Takakura, and M. Hashida. Disposition of oligonucleotides in isolated perfused rat kidney: involvement of scavenger receptors in their renal uptake. J. Pharmacol. Exp. Ther. 279: 284-290, 1996[Abstract].

27.   Sharma, R. K., B. G. Lake, R. Kakowski, T. Bradshaw, D. Earnshaw, J. W. Dale, and G. G. Gibson. Differential induction of peroxisomal and microsomal fatty-acid-oxidizing enzymes by peroxisome proliferators in rat liver and kidney. Eur. J. Pharmacol. 184: 69-78, 1989.

28.   Stromstedt, M., S. I. Hayashi, P. G. Zaphiropoulos, and J. A. Gustafsson. Cloning and characterization of a novel member of the cytochrome P450 subfamily IVA in rat prostate. DNA (NY) 9: 567-577, 1990.

29.   Sundseth, S. S., and D. J. Waxman. 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].

30.   Tanaka, S., S. Imaoka, E. Kusunose, M. Kusunose, M. Maekawa, and Y. Funae. 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[Medline].

31.   Thurman, R. G., and F. C. Kauffman. Factors regulating drug metabolism in intact hepatocytes. Pharmacol. Rev. 31: 229-251, 1980[Medline].

32.   Tracewell, W., J. Desjardins, and P. Iversen. In vivo modulation of the rat cytochrome P450 1A1 by double-stranded phosphorothioate oligodeoxynucleotides. Toxicol. Appl. Pharmacol. 135: 179-184, 1995[Medline].

33.   Wang, M.-H., D. E. Stec, M. Balazy, V. Mastyugin, C. S. Yang, R. J. Roman, and M. Laniado-Schwartzman. 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[Medline].

34.   Zou, A.-P., J. T. Fleming, J. R. Falck, E. R. Jacobs, D. Gebremedhin, D. R. Harder, and R. J. Roman. 20-HETE is an endogenous inhibitor of the large-conductance Ca2+-activated K+ channel in renal arterioles. Am. J. Physiol. 270 (Regulatory Integrative Comp. Physiol. 39): R228-R237, 1996[Abstract/Free Full Text].

35.   Zou, A.-P., J. D. Imig, M. Kaldunski, P. R. Ortiz de Montellano, Z. Sui, and R. J. Roman. Inhibition of renal vascular 20-HETE production impairs autoregulation of renal blood flow. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F275-F282, 1994[Abstract/Free Full Text].

36.   Zou, A.-P., Y.-H. Ma, Z.-H. Sui, P. R. Ortiz de Montellano, J. E. Clark, B. S. Masters, and R. J. Roman. Effect of 17-octadecynoic acid, a suicide-substrate inhibitor of cytochrome P450 fatty acid omega -hydroxylase, on renal function. J. Pharmacol. Exp. Ther. 268: 474-481, 1994[Abstract].


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