INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

OUR STUDIES AND OTHERS clearly indicate that 20-hydroxyeicosatetraenoic acid (20-HETE), the -hydroxylation product of arachidonic acid, is a major metabolite in tubular and vascular structures of the renal cortex and outer medulla of the rat (17, 27). Numerous studies have demonstrated the potent biological activities of this eicosanoid and provided substantial evidence of an important role for 20-HETE in the regulation of renal vascular tone, tubular reabsorption, and the control of arterial pressure (23, 24, 40, 41). 20-HETE is also a prominent eicosanoid in other tissues, including the lung, and the cerebral microcirculation (5, 11, 25).

The -hydroxylation of fatty acids, including arachidonic acid, can be catalyzed by enzymes of the cytochrome P-450 (CYP) 4A family. In the rat, four isoforms have been identified: CYP4A1, -4A2, -4A3, and -4A8, and messages for all four have been identified in the kidney (19, 20, 33). These isoforms, although sharing 66-98% homology and a common, unique catalytic activity, i.e., hydroxylation of fatty acid at the -carbon, are localized to different renal structures and exposed to different regulatory mechanisms (20, 33). For example, whereas CYP4A2 is preferentially expressed in the outer medullary and thick ascending limb of Henle's loop region, CYP4A1, -4A3, and -4A8 are highly expressed in the proximal tubules (12, 18, 33). 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 (17). 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 (16, 19, 20, 34). To this end, the CYP4A2 gene is one of the few genes preferentially expressed in the kidney of the spontaneously hypertensive rat (14, 18), in which renal 20-HETE synthesis is high (26). Moreover, the CYP4A genotype has been reported to cosegregate with the development of salt-induced hypertension in F₂ populations derived from a cross of both spontaneously hypertensive rats (32) and Dahl S rats (31) with normotensive strains.

The renal expression of these isoforms is age dependent; CYP4A1 and CYP4A3 proteins are detectable in the fetus, and their levels gradually increase from newborn until ~9 wk of age and then decline to very low levels in adults. CYP4A2 protein is undetectable until 5 wk, but then the levels increase such that in adult male rats it is the major isoform detected in the kidney (21, 22). CYP4A8 mRNA levels are detected at 3 wk of age and thereafter follow a pattern of expression similar to that of CYP4A1 and CYP4A3 (21).

The role of each of these 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, 2, 10, 30, 35). Most of these studies made use of lauric acid as a substrate for examining the catalytic properties of these proteins, demonstrating mainly -hydroxylation and, to a lesser extent, -1-hydroxylation of the fatty acid. Previous studies in our laboratory with CYP4A2 expressed in the baculovirus Sf 9 cells indicated that, besides - and -1-hydroxylations, this isoform also catalyzes epoxidation of arachidonic acid at the 11,12 double bond (38). More recently we examined the selectivity of some newly synthesized metabolic inhibitors of arachidonic acid metabolism (36). These studies indicate the possibility that some of these compounds may be isoform specific. In the present studies, the four rat CYP4A cDNAs were expressed by means of the baculovirus-Sf 9 insect cell expression system, and recombinant membranes were used to compare and contrast catalytic properties and inhibitor selectivity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials. [1-¹⁴C]arachidonic acid (56 mCi/mmol), [1-¹⁴C]lauric acid (53 mCi/mmol) and [1-¹⁴C]linoleic acid (53 mCi/mmol) were purchased from Dupont-New England Nuclear (Boston, MA). Purified recombinant human NADPH-CYP oxidoreductase (specific activity, 58 µmol · min¹ · mg¹) was obtained from Oxford Biomedical Research (Oxford, MI). Purified human cytochrome b₅ (b₅; specific activity, 60 nmol/mg) was from Panvera (Madison, WI). Emugen E911 was obtained from KAO Atlas (Tokyo, Japan). The Sf 9 insect cells, the cationic liposome DNA transfection kit, and the pVL1393 expression vector were purchased from Invitrogen (San Diego, CA). 17-Octadecenoic acid (ODYA) was from Cayman Chemical (Ann Arbor, MI). The compounds 6-(2-propargyloxyphenyl)hexanoic acid (PPOH) and N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) were synthesized, and their structures were confirmed by spectral analysis as described by Falck et al. (9). Miconazole was obtained from Sigma (St. Louis, MO). Tissue culture and molecular biology reagents were purchased from GIBCO-BRL (Gaithersburg, MD). Enhanced chemifluorescent (ECF) substrate was from Amersham (Arlington Heights, IL). All organic solvents for HPLC were reagent grade.

Construction of CYP4A recombinant baculoviruses and protein expression. Recombinant CYP4A proteins were expressed in the baculovirus-Sf 9 insect cell system as described previously (38). The CYP4A cDNAs were kindly provided by Dr. Richard Roman (Medical College of Wisconsin). They were cloned from kidneys of Lewis-Wistar rats with RT-PCR and specific primers based on published sequences (19, 20, 33). The nucleotide sequences of the isolated clones were identical to the previously published sequences (Dr. Richard Roman, personal communication). CYP4A1 (2.1-kb EcoR I/BamH I fragment of CYP4A1-pBSK plasmid), CYP4A2 (1.7-kb Kpn I/Not I fragment of CYP4A2-pCRII plasmid), CYP4A3 (2.2-kb Kpn I/Not I fragment of CYP4A3-pCRII plasmid), and CYP4A8 (2-kb EcoR I fragment of CYP4A8-pCRII plasmid) were ligated into the Sma I site of the baculovirus expression vector pVL1393. Transfection of the CYP4A1-, CYP4A2-, CYP4A3-, and CYP4A8-recombinant vectors was performed individually by an AcMNPV linear transfection kit (Invitrogen). Sf 9 insect cells (GIBCO-BRL) were routinely maintained in complete Grace's medium containing 10% fetal bovine serum, 0.1% pluronic acid F-68, and 50 µg/ml antibiotic-antimycotic (GIBCO-BRL) in a 200-ml sterile glass spin flask at 27°C on an orbital shaker in a temperature-controlled incubator. Sf 9 cells (2 × 10⁶) were seeded into 60-mm dishes and transfected with 1 µg of AcMNPV DNA and 3 µg of CYP4A-pVL1393 construct via cationic liposome-mediated transfection. Transfected cells were incubated at 27°C overnight, after which the medium was removed, fresh medium was added, and the cells were incubated for 4-6 days. Identification of recombinant viruses was done initially by visualization of Occ-negative plaques and later by immunoblots with the anti-rat CYP4A1 polyclonal antibody and by P-450 spectral analysis. The recombinant viruses were amplified in Sf 9 cells. The virus stock was harvested 4 days postinfection, and virus titers were determined by plaque assay. Sf 9 cells (4 × 10⁶) were infected with either CYP4A1, CYP4A2, CYP4A3, or CYP4A8 recombinant viruses. Sf 9 cells infected with the wild-type viruses, and uninfected cells were grown in parallel under the same conditions for control incubations. Insect culture media were fortified with 5 µg/ml of hemin chloride at the time of infection. After 72 h, the cells were harvested, washed twice with PBS, and resuspended in sucrose buffer (50 mM KH₂PO₂, pH 7.4, 0.4 M sucrose). Cell lysates were prepared by brief sonication (4-5 bursts of 4-s duration), and membrane fraction was obtained by high-speed centrifugation (100,000 g) for 60 min. The membrane pellet was then resuspended in sucrose buffer and stored at 80°C. Protein concentration was measured by the method of Bradford (BioRad; Melville, NY). The concentration of CYP was determined by the CO-reduced difference spectral method of Omura and Sato (28) with an extinction coefficient _450-490nm = 91 mM¹ · cm¹.

Immunoblot analysis. Membrane proteins (1-5 µg) were separated by electrophoresis on an 8% SDS-polyacrylamide gel at 20 mA for 20 h. The proteins were transferred electrophoretically to a polyvinylidene difluoride membrane in a transfer buffer consisting of 25 mM Tris · HCl, 192 mM glycine, and 20% methanol (vol/vol). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) containing 10 mM Tris, 0.1% Tween 20, and 150 mM NaCl for 1.5 h and then washed three times with TBS. The membranes were incubated for 1 h with goat anti-rat CYP4A1 polyclonal antibody (1:1,000; Gentest, Woburn, MA) at room temperature, washed with TBS solution, and further incubated with 1:5,000 dilution of alkaline phosphatase-conjugated second antibody (Sigma) for 1 h. After being washed with TBS three times, the membranes were blotted dry and incubated with 1 ml of Vistra ECF substrate (Amersham). The membranes were air dried completely, and immunoreactive proteins were detected by the Storm PhosphoImager system (Molecular Dynamics; Sunnyvale, CA) and quantified by ImageQuant analysis.

Enzyme activity: lauric, linoleic, and arachidonic acid oxidations. Recombinant membranes (2.5-10 pmol P-450) were preincubated with and without various amounts of purified NADPH-CYP oxidoreductase and b₅ on ice for 10 min. [¹⁴C]arachidonic acid (0.4 µCi, 7 nmol), [¹⁴C]linoleic acid (0.4 µCi, 7 nmol), or [¹⁴C]lauric acid (0.4 µCi, 7 nmol) was added in a final volume of 0.15 ml incubation buffer (10 mM MgCl₂ and 100 mM KH₂PO₄, pH 7.2), and the reaction mixture was incubated at 37°C for 5 min. Reaction was started by the addition of 1 mM NADPH. The incubation was carried out for 30 min at 37°C. Control incubations consisted of uninfected Sf 9 cell membranes and recombinant membranes incubated without either NADPH or NADPH-CYP oxidoreductase. The reaction was terminated by acidification to pH 3.5-4.0 with 2 M formic acid, and the metabolites were extracted with ethyl acetate. The combined extracts were µm evaporated under nitrogen, and the residue was resuspended in 50 µl of methanol and injected onto the HPLC column. Reverse-phase HPLC was performed on a 5-µm octadecyl silane-Hypersil column (4.6 × 200 mm; Hewlett-Packard, Palo Alto, CA) with a linear gradient ranging from acetonitrile:water:acetic acid (50:50:1) 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. Activity was expressed as nanomoles per minute per nanomoles P-450 unless otherwise indicated.

Inhibitor studies. PPOH, DDMS, and 17-ODYA were diluted from ethanolic stock solutions 20-50 times with 100 mM KH₂PO₂ buffer, pH 7.5. Miconazole was dissolved in DMSO and further diluted 1:1,000 with distilled water. The diluted solution was then added to the incubation mixture containing recombinant CYP4A membranes, purified NADPH-CYP oxidoreductase and b₅ at a molar ratio of 1:14:4, NADPH (1 mM), and buffer (0.15 ml final volume). The mixtures were preincubated at 37°C for 10 min. [1-¹⁴C]arachidonic acid was then added, and incubation was carried out at 37°C for 30 min. Control incubations included the vehicle of the inhibitor. Extraction and HPLC analysis were performed as described in Enzyme activity: lauric, linoleic, and arachidonic acid oxidations.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Level of expression of CYP4A isoforms. Immunoblot analysis of membrane fractions prepared from Sf 9 insect cells infected with CYP4A recombinant baculovirus by means of the goat anti-rat CYP4A1 polyclonal antibody revealed the presence of distinct immunoreactive proteins (Fig. 1). Based on electrophoretic mobility, the estimated molecular mass of CYP4A1, CYP4A2, CYP4A3, and CYP4A8 were 50.2, 51.9, 53.3, and 52.4 kDa, respectively, and are in agreement with previous reports for the purified and recombinant CYP4A proteins (2, 15, 29). The CO-reduced difference spectrum of the expressed CYP4A isoforms exhibited a maximum absorbance at 450 nm as shown for CYP4A1 (Fig. 2). The total P-450 contents of CYP4A1-, CYP4A2-, CYP4A3-, and CYP4A8-expressed membranes were determined to be 0.34 ± 0.034, 0.22 ± 0.016, 0.25 ± 0.059, and 0.11 ± 0.006 nmol/mg protein, respectively.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Western immunoblot of baculovirus-Sf 9 cell-expressed cytochrome P-450 (CYP) 4A proteins. Membrane proteins from Sf 9 cells expressing CYP4A1 (0.1 µg, lane 4), CYP4A2 (5 µg, lane 5), CYP4A3 (1 µg, lane 6), and CYP4A8 (1 µg, lane 7), and microsomes from clofibrate-induced liver (5 µg, lane 1), male kidney cortex (5 and 10 µg, lanes 2 and 8, respectively), and female kidney cortex (10 and 20 µg, lanes 3 and 9, respectively) were separated on 8% SDS-polyacrylamide gel, transferred to polyvinylidene difluoride membrane, and blotted with goat anti-rat CYP4A1 polyclonal antibody. Immunoreactive proteins were detected by phosphoimaging methods using Vistra ECF substrate as described under EXPERIMENTAL PROCEDURES.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Representative CO-reduced difference spectrum of CYP4A1 expressed in baculovirus-Sf 9 insect cell system. After bubbling enzyme with CO, sample was divided into reference and sample cuvettes, and CO-reduced difference spectrum was measured after a few grains of Na2S2O4 were added to sample.

Fatty acid oxidation by CYP4A isoforms. To establish optimal conditions for the expression of CYP4A catalytic activity, the effects of varying amounts of NADPH-CYP oxidoreductase and b₅ on the arachidonic acid oxidation were studied. The results indicated that, as for CYP4A2 (38), a 14-fold excess of reductase and fourfold excess of b₅ are needed for maximal catalytic activity of all CYP4A isoforms (data not shown).

Figure 3 depicts representative HPLC traces of arachidonic acid metabolites formed in incubation of Sf 9 cells expressing the various CYP4A isoforms. CYP4A8 membranes showed no detectable catalytic activity. In contrast, CYP4A1, CYP4A2, and CYP4A3 readily metabolized arachidonic acid primarily via - and -1-hydroxylations to 20- and 19-HETEs (Fig. 3). However, CYP4A1-catalyzed - and -1-hydroxylations were six- to ninefold greater than that catalyzed by CYP4A2 and CYP4A3. In accordance with previous studies, CYP4A1 only metabolized arachidonic acid at the - and -1-carbons. On the other hand, CYP4A2 and CYP4A3, which exhibit >97% sequence homology, produced another active metabolite. This epoxide was found to coelute with the 11,12-epoxyeicosatrienoic acid (EET) standard on the HPLC, and its structural identity was verified by gas chromatography/l mass spectrometry analysis (38). The fact that the formation of 11,12-EET constituted an enzymatic activity and was not an intrinsic activity within the Sf 9 cells was further examined. 11,12-EET was not produced in incubations carried out under similar conditions with membranes from uninfected Sf 9 cells or with boiled membrane preparations from 4A2- or 4A3-expressing cells (Fig. 4).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Representative reversed-phased HPLC elution profiles showing arachidonic acid (AA) metabolism by baculovirus-Sf 9-expressed CYP4A isoforms. Cell membranes containing 10 pmol of expressed CYP4A1 (A), CYP4A2 (B), CYP4A3 (C), and CYP4A8 (D) were reconstituted with purified NADPH-CYP oxidoreductase (140 pmol) and cytochrome b5 (b5; 40 pmol) and incubated with 0.4 µCi (7 nmol) of [1-14C]arachidonic acid in presence of NADPH (1 mM). Reactions were carried out for 30 min at 37°C, and metabolites were extracted and separated by HPLC. HETE, hydroxyeicosatetraenoic acid; EET, epoxyeicosatrienoic acid; CPM, cycles per minute.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Reverse-phase HPLC of metabolites formed by native Sf 9 cells and Sf 9 cells expressing CYP4A2. CYP4A2 membranes (50 µg, 12 pmol P-450) or membranes from native Sf 9 cells (50 µg) were reconstituted with purified NADPH-CYP oxidoreductase (168 pmol) and b5 (48 pmol) and further incubated with 0.4 µCi of [1-14C]arachidonic acid (7 nmol) and NADPH (1 mM) in final volume of 150 µl for 30 min at 37°C. A: CYP4A2; B: Sf 9 cells; C: boiled CYP4A2.

Experiments were also carried out to compare the catalytic activities of the CYP4A proteins with various fatty acids. Lauric acid, although not physiologically significant, has been repeatedly used as an excellent substrate for all proteins of the CYP4A family and as an indication for enzyme activity. As shown in Table 1, all recombinant CYP4A proteins exhibited significant - and -1-hydroxylations of lauric acid; CYP4A1 had the highest specific activity of 36 nmol · min¹ · nmol¹ P-450, followed by CYP4A3, CYP4A2, and CYP4A8 with 10.1, 6.3, and 0.4 nmol · min¹ · nmol¹ P-450, respectively. Oxidation of linoleic acid, an unsaturated 18-carbon fatty acid (18:2, -6) was also examined. Similar to arachidonic acid oxidation, CYP4A1 only catalyzed oxidation of linoleic acid at the  and -1 positions, whereas CYP4A2 and CYP4A3 exhibited both hydroxylation and epoxidation activities. The relative ratios of linoleic acid -to--1-hydroxylations were determined to be 3:1, 3:1, and 5:1 for CYP4A1, CYP4A2, and CYP4A3, respectively. The linoleic acid epoxidation activity of CYP4A2 and CYP4A3 was about two times higher than - or -1-hydroxylations. The structural identity of the linoleic acid epoxide metabolite is yet to be determined. Arachidonic acid was found to be a slightly better substrate than linoleic acid for all isoforms. Moreover, the relative ratios of the  (20-HETE)- and -1 (19-HETE)-hydroxylated metabolites, namely, 12.6:1, 3.6:1, and 3.5:1 for CYP4A1, CYP4A2, and CYP4A3, respectively, indicated a relatively lower arachidonate -1-hydroxylation activity compared with linoleic acid. The results clearly indicate that 20-HETE is the major arachidonic acid metabolite of CYP4A1, amounting to more than 93% of metabolites formed, whereas it is only about 60% of all metabolites formed by CYP4A2- and CYP4A3-expressed membranes.

Initial velocity kinetic studies. Under optimal reconstitution conditions, CYP4A enzyme kinetic analyses were performed. Table 2 shows the Michaelis constant (K_m) and maximal velocity (V_max) values of CYP4A isoforms for arachidonic acid. Surprisingly, despite the high homology within the rat CYP4A family, the K_m values for CYP4A1-catalyzed 20-HETE synthesis were two and four times less than the K_m obtained for CYP4A2 and CYP4A3, respectively. Similarly, the V_max values for CYP4A1 catalytic activity were 7- and 10-fold higher than that found for CYP4A2 and CYP4A3, respectively. The catalytic efficiency, expressed as V_max/K_m, indicated that CYP4A1 metabolizes arachidonic acid most efficiently at the -carbon to yield 20-HETE, followed by CYP4A2, then by CYP4A3.

Effects of inhibitors on recombinant CYP4A-catalyzed arachidonate oxidation. Previous studies from our laboratory have demonstrated that PPOH, a terminal acetylenic compound, selectively inhibited microsomal arachidonic acid epoxidation. Given the unique characteristics between CYP4A1 and CYP4A2 or CYP4A3 (i.e., CYP4A1 only exhibited - and -1-hydroxylation), we examined the effect of PPOH on Sf 9-expressed recombinant CYP4A isoforms (Fig. 5). Addition of PPOH (1-50 µM) inhibited both CYP4A2- and CYP4A3-catalyzed arachidonate - and -1-hydroxylations and 11,12-epoxidation in a concentration-dependent manner, but not CYP4A1-mediated reaction (Table 3). 17-ODYA, a common acetylenic inhibitor of fatty acid -hydroxylation, was found to inhibit both - and -1-hydroxylations and epoxidation of arachidonic acid by Sf 9-expressed CYP4A isoforms (Table 3). However, differences in the sensitivity of these isoforms to 17-ODYA with regard to the two reactions were apparent. Thus the estimated IC₅₀ of 17-ODYA for CYP4A2-catalyzed 19- or 20-HETE formation was 4 µM, whereas that for CYP4A2-catalyzed 11,12-EET formation was 10 µM. On the other hand, DDMS, a specific inhibitor of microsomal arachidonic -hydroxylation (36), potently decreased - and -1-hydroxylations and epoxidation catalyzed by CYP4A1, CYP4A2, and CYP4A3 without differentiating between the two reactions and with similar IC₅₀ for all isoforms (Fig. 6, Table 3). In addition to these inhibitors, we also examined the effect of an imidazole derivative, miconazole, on arachidonic acid metabolism by recombinant CYP4A isoforms. As previously studied by others, imidazole-based compounds were shown to be potent and selective inhibitors of the arachidonic acid epoxygenase (6). Miconazole, at concentrations of up to 100 µM, had little effect (~20% inhibition) on 19- or 20-HETE and 11,12-EET formation by CYP4A2 and CYP4A3. However, CYP4A1-catalyzed -hydroxylation was inhibited by 20-65% in a concentration-dependent manner with an estimated IC₅₀ of 53 µM (Table 3).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Effect of 6-(2-propargyloxyphenyl)hexanoic acid (PPOH) on arachidonic acid - or -1-hydroxylase and epoxygenase activities of baculovirus-Sf 9-expressed CYP4A isoforms. PPOH was preincubated with reconstituted system containing cell membranes from expressed CYP4A isoforms (10 pmol P-450), NADPH-CYP oxidoreductase (140 pmol) and b5 (40 pmol), and NADPH (1mM) for 10 min. [1-14C]arachidonic acid (0.4 µCi, 7 nmol) was added, and reaction mixture was incubated for 30 min at 37°C. Arachidonic acid metabolites were extracted and separated by HPLC. Results are expressed as percentage of control and are means of three separate determinations; SE < 10%. Control values for CYP4A1, CYP4A2, and CYP4A3 - and -1-hydroxylations were 6.4, 1.25, and 0.66 nmol · min1 · nmol P-4501, respectively. Control values for CYP4A2 and CYP4A3 11,12-epoxidation were 0.27 and 0.17 nmol · min1 · nmol P-4501, respectively.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Effect of N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) on arachidonic acid - or -1-hydroxylase and epoxygenase activities in baculovirus-Sf 9-expressed CYP4A isoforms. DDMS at various concentrations was preincubated with NADPH-CYP oxidoreductase, b5, membranes containing expressed CYP4A isoforms, and NADPH for 10 min as described above. [1-14C]arachidonic acid (0.4 µCi, 7 nmol) was added, and reaction mixture was incubated for 30 min at 37°C. Arachidonic acid metabolites were extracted and separated by HPLC. Results are expressed as percentage of control and are means of three separate determinations; SE < 10%. Control values for CYP4A1, CYP4A2, and CYP4A3 - and -1-hydroxylations were 6.4, 1.25, and 0.66 nmol · min1 · nmol1 P-450, respectively. Control values for CYP4A2 and CYP4A3 11,12-epoxidation were 0.27 and 0.17 nmol · min1 · nmol1 P-450, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Despite the relatively high nucleotide and amino acid sequence homology and the common catalytic activity (hydroxylation of fatty acids and their derivatives at the -carbon) of the rat CYP4A isoforms, the current study demonstrates significant differences in their catalytic activity and inhibitor sensitivity. In the present study, the four rat CYP4A isoforms have been expressed in the baculovirus-Sf 9 insect cell expression system, and membranes expressing these proteins were used to characterize the catalytic properties. We found that although CYP4A1 catalyzed fatty acids, including lauric, linoleic, and arachidonic acids, exclusively at the - and -1-carbons, CYP4A2 and CYP4A3 catalyzed, in addition to - and -1-hydroxylations, epoxidation of the unsaturated fatty acids. Moreover, CYP4A1 was the most efficient arachidonic acid -hydroxylase, demonstrating 10- and 40-fold higher capacity to generate 20- and 19-HETEs than CYP4A2 and CYP4A3, respectively. Furthermore, CYP4A isoforms displayed different sensitivities to known inhibitors of CYP-derived fatty acid oxidation.

The epoxygenase activity of the CYP4A2-expressed Sf 9 cell membranes has been previously reported (38). Based on the high sequence homology between CYP4A2 and CYP4A3, 97% nucleotides and 96% amino acids (20), it was not surprising to find that CYP4A3 also acts as an epoxygenase. The epoxidation of arachidonic acid by CYP4A2 and CYP4A3-expressed membranes was at the 11,12 double bond. No other epoxides were detected. This activity was attributed to the expressed protein in each membrane preparation because membranes from native Sf 9 cells or cells infected with the wild-type virus were unable to catalyze the formation of 11,12-EET when incubated with arachidonic acid, NADPH-CYP oxidoreductase, b₅, and NADPH. Moreover, the specificity is further inferred from the inability of Sf 9 cells expressing CYP4A1 or CYP4A8 to catalyze the formation of 11,12-EET. These results are in contrast to a recent report by Helvig et al. (13) who demonstrated that native Sf 9 cells possessed intrinsic arachidonate epoxygenase activity, leading to the formation of all four epoxides. This discrepancy could be accounted for by differences in the Sf 9 batches and experimental conditions. Another important difference is that the sequence of CYP4A2 and CYP4A3 isoforms expressed by this group were not identical to the published sequence (19, 20). These differences may account for the lack or presence of epoxygenase activity. Another explanation for such differences may be the use of different preparations. Whereas in our studies, intact membranes from cells overexpressing specific CYP4A proteins were used for determination of catalytic activity, the study by Helvig et al. (13) used purified CYP proteins and measured catalytic activity in an in vitro reconstituted system composed of the CYP protein, the reductase, and phosphatidylcholine. Although the latter is the common method for measuring catalytic activity of specific CYP derived from a membrane containing many, one may argue that intact membranes expressing one CYP protein provide a better environment than that created by a reconstituted system. In all, differences in sequence, preparations, and batches of cells could contribute to the differences between the two studies.

The physiological implication of this dual catalytic activity may be of great importance. Several vascular beds, including the renal and the cerebral microcirculations, have been shown to express CYP4A2 and CYP4A3 proteins and to metabolize arachidonic acid to both 20-HETE and 11,12-EET (11, 17, 38). 11,12-EET exhibits biological activities opposite to those of 20-HETE; it vasodilates blood vessels and stimulates Ca²⁺-activated K-channels in vascular smooth muscle cells (11, 39). Hence the dual catalytic activity of these proteins may present a mechanism for balanced regulation of vascular tone. Whether this duality is expressed in vivo is unknown.

The catalytic activity of CYP4A1 has been extensively studied. This isoform is primarily expressed in the liver and is the isoform most susceptible to clofibrate and other peroxisomal proliferators (8). In the kidney, CYP4A1 expression is sex dependent; it is barely detectable in the male, whereas in the female this isoform is constitutively expressed (34). Studies with purified rat kidney CYP (2, 10, 30, 35) demonstrated that CYP4A1 is one of the most active isozymes, and it can hydroxylate laurate, palmitate, and arachidonate at the -carbon and, to a lesser extent, at the -1-carbon. Aoyama et al. (2) examined the catalytic activity of CYP4A1 by cDNA-directed expression with vaccinia virus and showed high specificity for -hydroxylation of lauric and palmitic acid; -1-hydroxylation amounted to only 5% of the -hydroxylation. The authors did not determine whether this recombinant protein could metabolize arachidonic acid. Recent studies by Alterman and colleagues (1, 7) demonstrated lauric acid -hydroxylation activity by an expressed rat CYP4A1-NADPH-CYP oxidoreductase fusion protein. Again, they did not report to what extent this fusion protein can catalyze the -hydroxylation of arachidonic acid. Our studies concur with these reports by restating the high regioselectivity of this isoform for - vs. -1-hydroxylations of fatty acids and, in particular, lauric acid. This regioselectivity extended to the 18- and 20-carbon fatty acids linoleic and arachidonic acid, respectively, albeit less efficiently. However, the relative -1-hydroxylation activity significantly increased. Indeed, studies that examined the active site structure and substrate specificity of the recombinant CYP4A1-NADPH-CYP oxidoreductase fusion protein demonstrated that regioselective -hydroxylation is indeed favorable for lauric acid, and increasing the chain length results in a small increase in -1-hydroxylation (3, 4). Of interest is the finding that arachidonic acid was a much better substrate than linoleic acid, further substantiating the notion that CYP4A1 is the low-K_m arachidonate -hydroxylase and thus by far the most efficient 20-HETE-synthesizing enzyme. This finding may have important implications. CYP4A1 catalytic properties imply that although it has low levels of expression in tissues such as the kidney, it may be the principal contributor of endogenous 20-HETE formation. To that end, we have recently reported that the renal microvessels also express CYP4A1 in addition to CYP4A2, and antisense oligonucleotides against CYP4A1 inhibit renal vascular 20-HETE synthesis (37).

CYP4A1 solely functioned as an -hydroxylase and showed no detectable capacity to carry on epoxidation of either arachidonic or linoleic acid. These findings may suggest differences in the active sites of these isoforms. Thus the active site of CYP4A2 and CYP4A3 may be wider and more flexible to allow the positioning of the 11,12 double bond within the catalytic center, whereas the active site of CYP4A1 may be much more rigid or narrow, permitting only the  end of the fatty acid to be in close proximity to the heme moiety of the catalytic domain. Recent studies by Alterman et al. (1) suggested that CYP4A1 has an elongated tube-shaped active site (14 Å in length) with a recognition site for polar groups, e.g., carboxyl, at its entrance and the (oxo)heme group at its terminus. It is possible that such an active site limits the catalytic activity to hydroxylation at the  side of the molecule. To date, no such analysis has been made for the active sites of CYP4A2 and CYP4A3.

The catalytic characterization singled out CYP4A1 and grouped CYP4A2 with CYP4A3 as similar enzymes. This was also reflected in the inhibitor studies. Both 17-ODYA and DDMS, which have been shown to inhibit arachidonic acid metabolism to 20-HETE in renal microsomes (36) and are generally accepted as inhibitors of fatty acid -hydroxylations, inhibit 20-HETE formation by all CYP4A isoforms with similar potency. The two so-called epoxygenase inhibitors, miconazole and PPOH, had contrasting effects. Although PPOH did not affect CYP4A1-catalyzed -hydroxylation, it did inhibit CYP4A2- and CYP4A3-mediated -hydroxylation and 11,12-epoxidation, suggesting that the ability of CYP4A2 and CYP4A3 to carry out epoxygenase reaction rendered them susceptible to this compound. PPOH is a 16-carbon fatty acid with a terminal acetylenic bond and a benzene group between carbon 7 and carbon 12; it resembles 17-ODYA in that it irreversibly inactivated the enzyme in a NADPH-dependent mechanism (36). However, its bulky carbon chain structure may contribute to its isoform specificity. These results argue that PPOH is not a distinct epoxygenase inhibitor, and its use may result in inhibition of 20-HETE synthesis. Moreover, the fact that only inhibition of arachidonate epoxygenase is detected in microsomes treated with PPOH (36) may suggest that perhaps the contribution of CYP4A2 and CYP4A3 to 20-HETE in that preparation is minimal. Miconazole, an imidazole-based inhibitor of CYP enzymes and a well-characterized epoxygenase inhibitor of microsomal activity (6), had the opposite effect; it did inhibit CYP4A1, albeit at much higher concentrations than microsomal epoxygenase activity (6), but it did not affect CYP4A2 and CYP4A3-mediated reactions at concentrations as high as 100 µM, suggesting differences between these CYP4A isoforms.

In summary, this is the first study to provide a complete comparison of the catalytic activity of the rat CYP4A isoforms with regard to arachidonic acid. This comparison denoted major differences that can provide the basis for developing isoform-specific inhibitors for better understanding the contribution of each to 20-HETE synthesis and thereby to the regulation of renal function and vascular tone.

Perspectives