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CELLULAR AND MOLECULAR
Department of Pharmacology, New York Medical College, Valhalla, New York (J.-S.W., F.Z., M.J., B.A.Z., N.G.A., A.N., M.L.-S.); and Department of Physiology, Medical College of Georgia, Augusta, Georgia (M.-H.W.)
Received May 4, 2004; accepted July 21, 2004.
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Abstract |
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-hydroxylation to 20-HETE at rates of 0.85 ± 0.29 and 0.27 ± 0.04 nmol/107 cells/h, respectively. Transfection of interlobar arteries with either plasmid yielded EGFP immunofluorescence that was localized to the intima, media, and adventitia. Arteries transfected with pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2 showed increased vasoreactivity displaying EC50 to phenylephrine of 0.24 ± 0.07 and 0.11 ± 0.03 µM, respectively, as compared with arteries transfected with pIRES2-EGFP (1.11 ± 0.21 µM; n = 6, p < 0.05). The increased vasoreactivity to phenylephrine was inhibited by N-methylsulfonyl-12,12-dibromododec-11-enamide, an inhibitor of CYP4A-catalyzed reactions, suggesting that a product of CYP4A1 and CYP4A2 catalytic activity contributed to the increased constrictor responsiveness. Removal of the endothelium did not prevent the sensitization to phenylephrine in vessels transfected with the plasmid containing the CYP4A1 cDNA, suggesting that the CYP4A product responsible for the sensitizing effect, presumably 20-HETE, is not of endothelial cell origin.
-hydroxylation metabolite of arachidonic acid, is a major eicosanoid in the renal microvasculature (Ma et al., 1993
; Wang et al., 1998b). When added to isolated preparations of renal interlobular and afferent arterioles, 20-HETE causes vasoconstriction (Imig et al., 1996). In isolated interlobar arteries, increased synthesis of 20-HETE or addition of 20-HETE increased responsiveness to constrictor agonists such as phenylephrine (Kaide et al., 2003). Recent studies indicated that 20-HETE promotes Ca2+ entry by depolarizing smooth muscle cells secondary to blockade of KCa channels and by increasing conductance of L-type Ca2+ channels (Imig et al., 1996; Zou et al., 1996; Gebremedhin et al., 1998). These findings suggest that 20-HETE serves as an intracellular regulator of ion channel activity rather than a direct vasoconstrictor. Studies demonstrating the effect of 20-HETE on signaling proteins including protein kinase C and mitogen-activated protein kinase (Nowicki et al., 1997; Muthalif et al., 1998; Sun et al., 1999) also reinforce the notion that 20-HETE acts as an intracellular messenger. Moreover, the identification of structurally related analogs of 20-HETE that antagonize the constrictor activity of 20-HETE in renal arterioles implicated the existence of a 20-HETE's specific surface or intracellular binding site/receptor (Alonso-Galicia et al., 1999). Studies that characterize the bioactivity of 20-HETE must take into consideration that it is a lipid molecule. As such, exogenously added 20-HETE may reach targets different from those affected by the endogenously generated 20-HETE since its interaction with the cell membrane is multifaceted; it readily enters the cell, can be esterified to phospholipids, or interact with a surface protein. Consequently, producing 20-HETE within the cell/tissue may shed light on its role as a cellular effector molecule.
A viable approach to increasing 20-HETE in a given cell or tissue is overexpression of P450 proteins that catalyze its formation by gene transfer techniques. CYP4A proteins including CYP4A1, CYP4A2/4A3, and CYP4A8 have been extensively characterized as the arachidonic acid -hydroxylases in the rat kidney. We have shown that CYP4A1 and CYP4A2/4A3 are major CYP4A proteins expressed in the rat renal microcirculation and both contribute significantly to vascular synthesis of 20-HETE (Wang et al., 1998b). Accordingly, we constructed two bicistronic vectors, pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2, in which coexpression of EGFP allows assessment of transfection efficiency and detection of transfected CYP4A proteins. In this study, we demonstrate the coexpression of EGFP and CYP4A proteins in COS-1 cells, in A7r5 vascular smooth muscle cells, and in microdissected rat interlobar arteries. We further show that these vectors efficiently drive the expression of CYP4A1 and CYP4A2 by increasing protein levels and 20-HETE synthesis. Furthermore, interlobar arteries transfected with pIRES2-EGFP-4A1 or pIRES2-EGFP-4A2 demonstrated increased constrictor responsiveness to phenylephrine, presumably via increased CYP4A1 and CYP4A2 catalytic activity. The ability to monitor P450 expression by following EGFP immunofluorescence provides a useful tool for evaluating the consequences of increasing endogenous levels of 20-HETE, an important modulator of vascular function.
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Materials and Methods |
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), was released by digestion with EcoRI and BamHI, purified, and then ligated into the pIRES2-EGFP vector (BD Biosciences Clontech, Palo Alto, CA), a 5.3-kilobase vector that contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus between the multiple cloning site and the EGFP coding region. CYP4A2 cDNA (1720 base pairs), originally subcloned into the EcoR I site of PCR II vector, was released by digestion with EcoRI, purified, and then ligated into pIRES2-EGFP vector. Both the CYP4A (cloned into the MCS) and the EGFP are translated separately from a single bicistronic mRNA expressed under the control of the CMV promoter. The orientation of the cloned CYP4A1 and CYP4A2 was verified by restriction analysis, and its identity was confirmed by polymerase chain reaction analysis with isoform-specific primers. Cell Culture and Transfection. A7r5 and COS-1 cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (DMEM) that was supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, and 100 µg/ml streptomycin. Cultures were maintained in a humidified atmosphere of 5% CO2 in air at 37°C. The medium was changed every 2 days, and cells were passaged with Trypsin-EDTA at least once a week. Cells were grown in T-75 flasks to about 50% confluence. The cells were incubated with a mixture containing 10 µg of plasmid CYP4A2-pIRES2-EGFP or CYP4A1-pIRES2-EGFP and 10 µl of Lipofectamine reagent (Invitrogen, Carlsbad, CA) for 6 h in 2 ml of Opti-MEMI medium at 37°C, to which 10 ml of DMEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 10 µg/ml streptomycin was added, and then the cells were incubated for an additional 36 h. For immunofluorescent staining, cells were grown on two-chamber glass slides to 40 to 50% confluence. Transfection was performed by adding 1 µg of plasmid and 3 µl of Lipofectamine reagent in 200 µl Opti-MEMI medium for 6 h at 37°C after, which 1 ml of DMEM medium was added, and cells were incubated for an additional 36 h at 37°C. Cells were washed in PBS, fixed with 90% ethanol for 10 min at room temperature, and rinsed with PBS three times. The fixed cells were then permeabilized with a blocking solution consisting of 2% bovine serum albumin and 0.05% Tween 20 in PBS for 1 h. After washing with PBS three times, the cells were incubated with goat anti-rat CYP4A1 antibody (1:100; BD Gentest, Woburn, MA), which cross-reacts with all CYP4A proteins, and with mouse anti-EGFP monoclonal antibody (1:100; JL-8, Invitrogen) in blocking solution for 1 h at room temperature. After further washing with PBS, slides were incubated for 1 h at room temperature with a secondary antibody labeled with Alexa Fluor 594-conjugated rabbit anti-goat IgG (Molecular Probes, Eugene, OR) for CYP4A (1:1000) and a secondary antibody labeled with Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes) for EGFP (1:300). Control experiments included omission of primary antibodies and addition of nonimmune rabbit and mouse serum for CYP4A and EGFP, respectively.
Microdissection and Transfection of Renal Interlobular Arteries. Sprague-Dawley rats were anesthetized with pentobarbital sodium (50 mg/kg b.wt.). Kidneys were excised, and interlobar arteries were microdissected and freed from cortical and connective tissue under a microscope as previously described (Kaide et al., 2003). Isolated rat interlobar arteries (10 vessels/dish) were incubated with a mixture containing 30 µg of CYP4A2-pIRES2-EGFP or CYP4A1-pIRES2-EGFP vector and 30 µl of Lipofectamine Reagent for 6 h in 200 µl of Opti-MEMI medium in a humidified atmosphere of 5% CO2 in air at 37°C, after which 1 ml of DMEM with 10% 
-Serum was added and incubation proceeded for an additional 18 h. Arteries were fixed in 4% formaldehyde in phosphate-buffered saline, dehydrated in ethanol, penetrated by xylene, embedded in OCT (Sakura Finetek Europe, Zoeterwoude, the Netherlands), and sectioned 5 µm thick. Deparaffinized and rehydrated specimens were stained with anti-EGFP antibodies (1:100) and anti-CYP4A antibodies (1:100) followed by secondary antibodies as described above. Control experiments included omission of primary antibodies and addition of nonimmune rabbit and mouse serum for CYP4A and EGFP, respectively. Immunostaining was visualized by confocal microscopy.
Western Blot Analysis. A7r5 or COS-1 cells (T-175; 50% confluent) were transfected with a mixture containing 25 µg of CYP4A1 or CYP4A2 recombinant plasmids and 25 µl of Lipofectamine as described above. Cells were harvested using cell lysis buffer (0.1 M potassium phosphate buffer, 0.25 M sucrose, 1 mM EDTA, 0.1% NP-40, 0.1 mM phenylmethylsulfonyl fluoride, and Sigma protease inhibitor cocktail 1:1000, pH 7.4). The lysates were centrifuged at 4000g for 10 min, and the protein concentration in supernatant was measured using the BioRad assay. Microdissected interlobar arteries were snap-frozen and homogenized in 10 mM potassium phosphate buffer, pH 7.2, containing 25 mM sucrose, 0.1% NP-40, 1 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride. The homogenized arteries were centrifuged at 4000g for 15 min, and the supernatant was used for Western blot analysis. Proteins (10–20 µg) were separated by SDS/polyacrylamide gel electrophoresis at 25 mA and 4°C for 18 to 20 h and were transferred electrophoretically to a nitrocellulose membrane. Immunoblotting was performed as described previously (Marji et al., 2002) using the following primary and secondary antibodies: goat anti-rat CYP4A1 polyclonal antibody (1:1000; Gentest), mouse anti-EGFP monoclonal antibody (1:1000; Invitrogen), rabbit anti-goat IgG (1:2000), and goat anti-mouse IgG (1:1000). Immunoreactive proteins were detected using the ECL Plus detection system (Amersham Biosciences Inc. (Piscataway, NJ) according to the manufacturer's instructions. The anti-rat CYP4A1 polyclonal antibody cross-reacts with all CYP4A isoforms, i.e., CYP4A2, CYP4A3, and CYP4A8 (Nguyen et al., 1999).
Measurement of Enzyme Activity. Control and transfected cells were washed three times with DMEM and incubated with [1-14C]lauric acid or [1-14C]arachidonic acid (2 µCi) in 5 ml of Optimedia for 2 to 6 h at 37°C. Aliquots from the culture medium (2 ml) were taken at 2, 4, and 6 h after addition of the radiolabeled substrates. In some experiments, microsomes were prepared by centrifugation. Briefly, cells were scraped and homogenized in 1 ml of 0.25 M sucrose containing 1 mM EDTA. The homogenate was centrifuged at 1000g for 5 min and the supernatant collected and further centrifuged at 100,000g for 60 min. The 100,000g pellet was resuspended in 50 mM potassium phosphate buffer, pH 7.5, containing 0.2 mM EDTA and 20% glycerol and stored at –80°C until used. The 100,000g pellet (100–300 µg of protein) were incubated with [1-14C]arachidonic acid or [1-14C]lauric acid (0.4 µCi, 7 nmol) and NADPH (1 mM) in 0.3 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, and the metabolites were extracted with ethyl acetate and separated by HPLC as previously described (Marji et al., 2002).
Measurements of 20-HETE Levels. Interlobar arteries were transfected with the various plasmids as described above and incubated for an additional 24 h. The medium was collected, and [2H2]20-HETE (0.5 ng/ml) was added as an internal standard. 20-HETE was isolated by ethyl acetate extraction and HPLC separation and was further derivatized to the pentafluorobenzyl ester, trimethylsilyl ether. Negative chemical ionization-gas chromatography/mass spectrometry was performed on an HP6890 mass spectrometer (Hewlett Packard, Palo Alto, CA) interfaced with a capillary gas chromatographic column (HP-5MS, 30 m x 0.25 mm x 0.25 µm; Agilent Technologies, Palo Alto, CA). Single ions were monitored: m/z 391 and 393 for the derivatized 20-HETE and [2H2]20-HETE, respectively. 20-HETE levels were quantified as described previously (Marji et al., 2002).
Measurement of Isometric Tension in Vascular Rings. Interlobar arteries (
230 µm, internal diameter) were cut into ring segments (2 mm). Rings were mounted on wires in the chambers of a multivessel myograph (JP Trading, Aarhus, Denmark) filled with Krebs' buffer (37°C) gassed with 95% O2/5% CO2. After 30 to 60 min of equilibration, the vessels were set to an internal circumference equivalent to 90% of that which they would have in vitro when relaxed under a transmural pressure of 80 mm Hg. Isometric tension was monitored continuously before and after experimental interventions, and constrictor response to 80 mM KCl was determined in each vessel at the beginning of the experiment. Subsequently, a cumulative concentration-response curve to phenylephrine (10–9-5 x 10–5 M) was constructed. Phenylephrine-induced increase of isometric tension is expressed as the percentage of the increase in tension produced by 80 mM KCl. In some experiments, the response to phenylephrine was measured in vessels pretreated with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; 30 µM), a selective inhibitor of arachidonic acid -hydroxylation (Wang et al., 1998a). The endothelium was removed by passing through a human hair (Buus et al., 2000). Endothelium removal was further confirmed by the lack of endothelium-dependent relaxation to acetylcholine (1 µM) in phenylephrine (1 µM)-preconstricted arteries.
Data Analysis. Data are expressed as mean ± S.E.M. Concentration-response data derived from each vessel were fitted separately to a logistic function by nonlinear regression. The maximum asymptote of the curve and the concentration of agonist producing 50% of the maximal response (EC50) were calculated using commercially available software (Prism 2.01; GraphPad Software Inc., San Diego, CA). Concentration-response data were analyzed by a two-way analysis of variance followed by a Duncan multiple range test. Other data were analyzed by a Student's t test for paired or unpaired observations as appropriate. The null hypothesis was rejected at p < 0.05.
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Results |
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; Nguyen et al., 1999). The rate of 20-HETE production in cells expressing CYP4A1 was 0.85 ± 0.29 nmol/107 cells/h. Cells transfected with CYP4A2 metabolized arachidonic acid to 19/20-HETE and EET at a rate of 0.27 ± 0.04 and 0.07 ± 0.03 nmol/107 cells/h, respectively. Cells treated with the control vector (pIRES2-EGFP) did not metabolize arachidonic acid (Fig. 3C).
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Similar results were obtained when lauric acid, a known substrate of CYP4A proteins, was used as the substrate. As seen in Fig. 4, CYP4A1-transfected cells metabolized lauric acid primarily to the 12-OH-lauric acid at a rate of 1.66 ± 0.41 nmol/107 cells/h (Fig. 4A) as did CYP4A2-transfected cells, which also exhibited significant -1 hydroxylation of lauric acid (Fig. 4B). CYP4A2-transfected cells metabolized lauric acid to 12-OH-lauric acid at a rate of 0.76 ± 0.32 nmol/107 cells/h. Cells that were treated with the control vector (pIRES2-EGFP) did not metabolize lauric acid (Fig. 4C). Using confocal immunofluorescence histocytochemistry techniques, we examined the colocalization of EGFP and CYP4A in interlobar arteries transfected in vitro with pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2. As seen in Fig. 5, EGFP and CYP4A immunofluorescence in pIRES2-EGFP-4A1- and pIRES2-EGFP-4A2-transfected arteries was apparent in all vascular layers including the intima, media, and adventitia. Merging the two images shows that these two proteins are colocalized within the transfected arteries (Figs. 5, C and F). Western blot analysis was further performed to document an increase in the levels of CYP4A immunoreactive proteins following transfection of interlobar arteries with these plasmids. Transfection with pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2 increased CYP4A immunoreactive proteins to levels higher than the control cultured arteries or arteries cultured with the control plasmid, pIRES2-EGFP (Fig. 6, A and B). In addition, the amount of 20-HETE in the culture medium of vessels transfected with pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2 was 1.5- and 1.7-fold higher, respectively, than the amount in the medium of vessels transfected with the control plasmid, pIRES2-EGFP (Fig. 6C). To further evaluate whether the bicistronic vectors described in this study are useful for functional studies, we examined vascular reactivity to phenylephrine in interlobar arteries transfected with pIRES2-EGFP-4A1, pIRES2-EGFP-4A2, and pIRES2-EGFP. As seen in Fig. 7A, the concentration-response curves to phenylephrine in arteries transfected with pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2 were shifted to the left, resulting in EC50 values that are, respectively, 20 and 10% of the EC50 in arteries transfected with the control vector, pIRES2-EGFP. The maximal response to phenylephrine was not different between arteries transfected with pIRES2-EGFP-4A1, pIRES2-EGFP-4A2, or pIRES2-EGFP (Fig. 7A). Pretreatment with DDMS, a selective inhibitor of CYP4A-catalyzed arachidonic acid -hydroxylation (Wang et al., 1998a), increased the EC50 to phenylephrine in arteries transfected with the control pIRES2-EGFP from 1.11 ± 0.21 to 3.25 ± 0.75 µM, further substantiating previous reports that a product of CYP4A enzymes contribute to constrictor responsiveness in these arteries (Kaide et al., 2003). Likewise, pretreatment with DDMS caused a rightward shift in the concentration-response curves to phenylephrine in pIRES2-EGFP-4A1- and pIRES2-EGFP-4A2-transfected arteries, increasing the EC50 by about 20-fold to values not different from the EC50 of vessels transfected with the control vector pIRES2-EGFP and pretreated with DDMS. DDMS did not affect the maximal response to phenylephrine in any of these vessels (Fig. 7B).
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Since immunostaining showed the presence of immunore-active protein in the endothelial layer, we examined whether removal of the endothelium altered vascular response to phenylephrine. The results depicted in Fig. 8 indicate that removal of the endothelium sensitized vessels transfected with either pIRES2-EGFP or pIRES2-EGFP-4A1 to phenylephrine, resulting in a 2-fold reduction of the EC50 values relative to corresponding values in vessels with endothelium. Removal of the endothelium did not interfere with the sensitizing effect of CYP4A1 transfection. The contractile response to 80 mM KCl was not affected by removal of the endothelium (viz., 1.88 ± 0.19 versus 1.50 ± 0.26 mN/mm in intact and denuded pIRES2-EGFP-transfected arteries and 1.18 ± 0.10 versus 0.94 ± 0.10 mN/mm in intact and denuded pIRES2-EGFP-4A1-transfected arteries).
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Discussion |
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; Paine, 1991; Lin et al., 1995). Thus, transfection of P450 genes provides a useful tool for functional analysis of metabolic pathways. Chen et al. (1999) demonstrated successful stable transfection of the renal epithelial cell line, LLCPKc14, with an active 14(S),15(R)-arachidonate epoxygenase of bacterial origin containing a P450 domain fused to a CPR domain. These authors further demonstrated that the 14(S),15(R)-EET functions as an intracellular second messenger in response to EGF. Endothelial cells transfected with CYP2C epoxygenases were instrumental in elucidating the role of EETs because EDHF and cellular mechanisms underlie their effect on cell proliferation and angiogenesis (Fisslthaler et al., 1999; Fleming et al., 2001; Michaelis et al., 2003). The use of bicistronic vectors in this study provides the means to estimate transfection efficiency and to trace and localize the expression of the transfected CYP4A within the tissue. The results show that both GFP and CYP4A proteins are coexpressed in cultured cells and in freshly isolated and cultured renal interlobar arteries. The increased expression of CYP4A1 or CYP4A2 using these bicistronic vectors was associated with increased -hydroxylation of lauric acid, the preferred substrates of CYP4A enzymes, and arachidonic acid, as well as with increased constrictor responsiveness to phenylephrine.
In COS-1 and A7r5 cells, transfection efficiency was about 95% as estimated by GFP fluorescence. These cells do not contain measurable CYP4A1 or CYP4A2 proteins, nor do they metabolize arachidonic acid to 20-HETE. Transfection of the bicistronic plasmids yielded expression of both proteins as measured by confocal immunofluorescence cytochemistry and by Western blotting. The transfected CYP4A1 and CYP4A2 exhibited their characteristic activity as -hydroxylases of lauric and arachidonic acids. Cells transfected with the CYP4A1-containing plasmid metabolized arachidonic acid to 20-HETE at the higher rate than cells transfected with the CYP4A2-containing plasmid, consistent with the finding that CYP4A1 is the most efficient arachidonic acid -hydroxylase among the CYP4A isoforms (Okita and Okita, 2001). Cells transfected with the CYP4A2-containing plasmid also produced 11,12-EET, a property previously documented for the recombinant Sf9-expressed CYP4A2 protein in a reconstituted system (Wang et al., 1996; Nguyen et al., 1999).
Interlobar arteries are an important site of 20-HETE synthesis and function. These arteries (200-µm diameter) produce a significant amount of 20-HETE (Marji et al., 2002). Moreover, constrictor responsiveness to phenylephrine is attenuated in the presence of CYP4A inhibitors, such as DDMS, and is greatly magnified in the presence of exogenously added 20-HETE (Kaide et al., 2003), thus implicating 20-HETE as an endogenous modulator of constrictor responses The results of this study clearly showed that the levels of immunoreactive CYP4A protein in these arteries can be increased as a result of transfection with expression plasmids containing the CYP4A1 or CYP4A2 cDNA. In examining the consequences of increased expression on vascular reactivity in renal interlobar arteries, we showed that both CYP4A1- and CYP4A2-transfected arteries exhibited a marked increase in their sensitivity to phenylephrine. Moreover, the ability of DDMS, a selective inhibitor of CYP4A-catalyzed reactions, to offset this increase suggests that a CYP4A product(s) contributes to the increased sensitivity to phenylephrine displayed by CYP4A1 and CYP4A2 transfected arteries. Indeed, interlobar arteries transfected with plasmids expressing CYP4A1 and CYP4A2 displayed a greater catalytic activity than arteries transfected with the control plasmid as evidenced by their ability to produce significantly more 20-HETE. Hence, the increased sensitivity to phenylephrine following overexpression of CYP4A1 or CYP4A2 could be attributed to increased levels of 20-HETE, the major arachidonate metabolite of CYP4A1 and CYP4A2 that is known to sensitize renal vascular smooth muscle to constrictor agonists (Alonso-Galicia et al., 1999; Zhang et al., 2001; Kaide et al., 2003). On the other hand, 20-HETE may not be the sole mediator of CYP4A-increased vascular reactivity to phenylephrine in arteries transfected with CYP4A1 or CYP4A2. It is well known that the CYP4A substrate specificity encompasses short- and long-chain fatty acids and their derivatives (Okita and Okita, 2001). Accordingly, we cannot exclude the possibility that increased CYP4A expression promotes vascular production of oxygenated products derived from fatty acids other than arachidonic acid. Neither can we exclude the possibility that such products facilitate constrictor responsiveness to phenylephrine. An additional possibility is suggested by observations that EETs are excellent substrates for CYP4A1 and CYP4A2 (Cowart et al., 2002). Hence, since EETs are putative endothelium-derived hyperpolarizing factors (Campbell and Harder, 1999), it is conceivable that the increased vascular reactivity to phenylephrine in vessels overexpressing CYP4A is, in part, the result of increased /-1 hydroxylation of EETs with attendant reduction of the EETs bioactivity. Although vessels transfected with CYP4A1 and CYP4A2 expressed and produced comparable levels of immunoreactive CYP4A proteins and 20-HETE, respectively, CYP4A2-transfected vessels showed greater sensitivity to phenylephrine than CYP4A1-transfected vessels. This difference may be explained by differences in their catalytic activity and/or their ability to degrade EETs as described above.
The present study demonstrates significant levels of CYP4A immunoreactivity in the endothelial layer of the renal interlobular arteries transfected with pIRES2-EGFP-4A1 or pIRES2-EGFP-4A2. However, removal of the endothelium did not prevent the sensitization to phenylephrine in vessels transfected with the plasmid containing the CYP4A1 cDNA. This observation implies that the CYP4A product responsible for sensitization to phenylephrine in vessels transfected with CYP4A-containing plasmids is not of endothelial cell origin. Endothelium removal increased the sensitivity to phenylephrine in vessels transfected with either pIERS2-EGFP-4A1 or the control plasmid, pIERS2-EGFP. It is likely that endothelial denudation excludes factors such as NO, prostaglandin I2, EETs, and lipoxygenase-derived eicosanoids (Li et al., 1997; Campbell and Harder, 1999; Campbell et al., 2003) that counteract vasoconstrictor mechanisms.
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Footnotes |
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ABBREVIATIONS: 20-HETE, 20-hydroxyeicosatetraenoic acid; P450, cytochrome P450; EGFP, enhanced green fluorescence protein; IRES, internal ribosome entry site; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; HPLC, high-performance liquid chromatography; DDMS, N-methylsulfonyl-12,12-dibromododec-11-enamide; EET, epoxyeicosaterienoic acid.
Address correspondence to: Dr. Michal Laniado-Schwartzman, Department of Pharmacology, New York Medical College, Valhalla, NY 10595. E-mail: michal_schwartzman{at}nymc.edu
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, p < 0.05 from corresponding vessels not treated with DDMS.
, p < 0.05 from corresponding vessels with the endothelium.