Regulation of renal CYP4A expression and 20-HETE synthesis by nitric oxide in pregnant rats

Mong-Heng Wang,1 Jishi Wang,2 Hsin-Hsin Chang,1 Barbara A. Zand,2 Miao Jiang,2 Alberto Nasjletti,2 and Michal Laniado-Schwartzman2

1Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912; and 2Department of Pharmacology, New York Medical College, Valhalla, New York 10595

Submitted 14 February 2003 ; accepted in final form 4 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
20-Hydroxyeicosatetraenoic acid (20-HETE), which promotes renal vasoconstriction, is formed in the rat kidney primarily by cytochrome P-450 (CYP) 4A isoforms (4A1, 4A2, 4A3, 4A8). Nitric oxide (NO) has been shown to bind to the heme moiety of the CYP4A2 protein and to inhibit 20-HETE synthesis in renal arterioles of male rats. However, it is not known whether NO interacts with and affects the activity of CYP4A1 and CYP4A3, the major renal CYP4A isoforms in female rats. Incubation of recombinant CYP4A1 and 4A3 proteins with sodium nitroprusside (SNP) shifted the absorbance at 440 nm, indicating the formation of a ferric-nitrosyl-CYP4A complex. The absorbance for CYP4A3 was about twofold higher than that of CYP4A1. Incubation of SNP or peroxynitrite (PN; 0.01–1 mM) with CYP4A recombinant membranes caused a concentration-dependent inhibition of 20-HETE synthesis, with both chemicals having a greater inhibitory effect on CYP4A3-catalyzed activity. Moreover, incubation of CYP4A1 and 4A3 proteins with PN (1 mM) resulted in nitration of tyrosine residues in both proteins. In addition, PN and SNP inhibited 20-HETE synthesis in renal microvessels from female rats by 65 and 59%, respectively. We previously showed that microvessel CYP4A1/CYP4A3 expression and 20-HETE synthesis are decreased in late pregnancy. Therefore, we investigated whether such a decrease is dependent on NO, the synthesis of which has been shown to increase in late pregnancy. Administration of NG-nitro-L-arginine methyl ester (L-NAME) to pregnant rats for 6 days (days 15-20 of pregnancy) caused a significant increase in systolic blood pressure, which was prevented by concurrent treatment with the CYP4A inhibitor 1-aminobenzotriazole (ABT). Urinary NO2/NO3 excretion decreased by 40 and 52% in L-NAME- and L-NAME + ABT-treated groups, respectively. Interestingly, renal microvessel 20-HETE synthesis showed a marked increase following L-NAME treatment, and this increase was diminished with coadministration of ABT. These results demonstrate that NO interacts with CYP4A proteins in a distinct manner and it interferes with renal microvessel 20-HETE synthesis, which may play an important role in the regulation of blood pressure and renal function during pregnancy.

cytochrome P-450 4A; NG-nitro-L-arginine methyl ester


20-HYDROXYEICOSATETRAENOIC acid (20-HETE), the {omega}-hydroxylation product of arachidonic acid (AA), is a principal eicosanoid in vascular and tubular structures of the rat kidney whose synthesis is catalyzed primarily by isoforms of the cytochrome P-450 (CYP) 4A gene family (34). In the rat, four CYP4A isoforms, CYP4A1, 4A2, 4A3, and 4A8, have been identified. The renal expression of CYP4A isoforms has been shown to be tissue specific as well as age and sex dependent (9, 12, 13, 19, 20). For example, Sundseth and Waxman (37) demonstrated that hepatic and renal CYP4A1 and 4A3 expressions are similar in male and female rats, whereas CYP4A2 expression is undetectable in female rats. Castration of male rats decreased the levels of CYP4A2, and treatment of castrated male rats with testosterone reversed this decrease (11). A recent study by Nakagawa et al. (27) showed that CYP4A8 expression is androgen sensitive. These sex-related differences in the expression of hepatic and renal CYP4A proteins suggest that androgens and estrogens play an important role in the regulation of these isoforms.

Normal pregnancy in humans and rats is associated with increases in glomerular filtration rate and renal blood flow (21) along with significant decreases in arterial pressure and total peripheral resistance (1, 17). 20-HETE possesses biological effects that can potentially contribute to these physiological changes during pregnancy. These biological effects include inhibition of ion transport along the nephron and vasoconstriction of renal arterioles (24, 33). We demonstrated distinct upregulation of CYP4A expression and 20-HETE synthesis in renal microvessels from rats on days 6 and 12 of gestation, which returned to control levels at day 19 of gestation (40). The factors responsible for the reduction of CYP4A expression and 20-HETE synthesis in late pregnancy are not known. We considered a role for nitric oxide (NO) in the regulation of CYP4A expression and activity in renal microvessels during late pregnancy because inhibition of NO synthesis increases 20-HETE synthesis (31), NO donors decrease the production of 20-HETE in renal microvessels (36), and NO production increases in pregnancy (1, 5).

The present study was undertaken to explore possible biochemical mechanisms underlying the effect of NO on CYP4A protein expression and activity and to examine whether inhibition on NO synthesis alters renal vascular 20-HETE synthesis in late pregnancy. We showed that NO readily binds to the heme moiety of the major CYP4A isoforms expressed in female rats, CYP4A1 and CYP4A3, with distinct isoform-specific affinity and that peroxynitrate increases tyrosine nitrosylation of these proteins. We also showed that inhibition of NO synthesis during the third week of gestation leads to a marked increase in vascular 20-HETE synthesis. Furthermore, coadministration of 1-aminobenzotriazole (ABT), a CYP4A inhibitor, prevents this increase. These changes in vascular 20-HETE synthesis were associated with reciprocal changes in systolic blood pressure, suggesting that alteration in vascular 20-HETE synthesis may contribute to the regulation of blood pressure during pregnancy.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. [1-14C]AA (56 mCi/mmol) was obtained from DuPont-New England Nuclear (Boston, MA). Emulgen E911 was obtained from KAO Atlas (Tokyo, Japan). ABT was obtained from Aldrich Chemical (Milwaukee, WI). All solvents were HPLC grade.

Animals. All animals were purchased from Charles River Laboratories, Wilmington, MA. Experiments were conducted in male and female Sprague-Dawley rats (8 wk old), pregnant (timed pregnancy), and the same age control female Sprague-Dawley rats. Experimental protocols were approved by the Institutional Animal Care and Use Committee. Rats were maintained under controlled housing conditions of light and temperature and received standard laboratory chow and water until used.

Quantitative competitive RT-PCR for CYP4A2. The detailed procedure for the preparation of competitor DNA of CYP4A2 was described in the manufacturer's protocol (PanVera, Madison, WI). This method was designed to generate CYP4A2 competitor (200 nucleotides) that is 117 nucleotides less than the target CYP4A2 cDNA. We used two sets of PCR to generate CYP4A2 competitor DNA. The first PCR primer set contained sequences that hybridize to a CYP4A2 sequence and are flanked by {lambda}DNA-specific sequences according to the manufacturer's protocol. The product of the first PCR set was purified and used as the template for the second PCR set. The second PCR primer set contained sequences that hybridize to a CYP4A2 sequence and are flanked by SP6 promoter-specific sequences. The product of the second PCR set was purified and used to generate CYP4A2 competitor RNA by in vitro transcription using a SP6 RNA polymerase. The template was then digested with DNase I, and the RNA was purified by phenol/chloroform/isoamyl alcohol method. The amount of the competitor RNA synthesized was quantified by spectrophotometry. Aliquots of total RNA (5 µg) from the kidneys of male and female rats were prepared and a x5 dilution series of competitor RNA (1,000; 200; 40; 8; 1.6; 0.32; 0.06 pg, respectively) was added into these aliquots, and RT-PCR was performed. A reverse transcription reaction was performed using a first-strand cDNA synthesis kit (Pharmacia Biotech, Milwaukee, WI) as previously described (38). After RT-PCR, aliquots (10 µl) of PCR product were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining. Gel pictures were scanned and densitometry analysis was performed with Scion Image software using gray color scale as a standard. The ratio of the density of the competitor RNA to the CYP4A2 RNA, plotted against the amount of the competitor RNA added to each reaction, was used to estimate CYP4A2 mRNA levels as described (30). The sequences of the primers used were as follows: CYP4A2 + {lambda}DNA: 5'-AGA TCC AAA GCC TTA TCA ATC GTA CGG TCA TCA TCT GAC AC-3' (forward primer), 5'-CAG CCT TGG TGT AGG ACC TTC ATT ACG CAT CGC TAT TAC-3' (backward primer); and SP6 + CYP4A2: 5'-ATT TAG GTG ACA CTA TAG AAT ACA GAT CCA AAG CCT TAT CAA TC-3' (forward primer), 5'-CAG CCT TGG TGT AGG ACC TTC ATT ACG CAT CGC TAT TAC-3' (backward primer).

Preparation of recombinant CYP4A membranes. CYP4A proteins were expressed using the baculovirus-Sf9 insect cell expression system as described previously (28). 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 (28). 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). CYP content was calculated from the reduced CO-difference spectrum using an extinction coefficient of 91 mM (28).

Effect of sodium nitroprusside and peroxynitrite on recombinant CYP4A catalytic activity. The stability of the NO donor sodium nitroprusside (SNP) was examined with NO-sensitive litmus paper using Griess reagent [0.5 g of sulfanilamide plus 20 mg of N-(1-naphthyl)ethylenediamine dihydrochloride] dissolved in 10 ml of methanol (29). In our preliminary study, SNP constantly released NO during a 10- to 20-min incubation (data not shown). Peroxynitrite (PN) stock was diluted in 0.3 M sodium hydroxide, and the concentration was determined by the extinction coefficient of 1,670 M · cm1 · 302 nm1 (3). SNP (0.01–1 mM) or PN (0.01–1 mM in 0.3 N NaOH) was added to mixture containing recombinant CYP4A1 or 4A3 membranes, purified NADPH-CYP oxidoreductase, and cytochrome b5 at a molar ratio of 1:14:4. This mixture was preincubated with NADPH (1 mM) in a final volume of 0.15 ml of buffer (10 mM MgCl2 and 100 mM KH2PO4, pH 7.2). The mixtures were preincubated at room temperature for 20 min. [1-14C]AA (0.4 µCi, 7 nmol) was then added, and incubation was carried out at 37°C for 30 min. Control incubations included the vehicle of SNP or PN. 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 x 200 mm (Hewlett-Packard, Palo Alto, CA) using a linear gradient ranging from acetonitrile:water:acetic acid (50:50:0.1) to acetonitrile:acetic acid (100:0.1) at a flow rate of 1 ml/min for 30 min. The elution profile of the radioactive products was monitored by a flow detector (In/us System, Tampa, FL). The identity of 20-HETE was confirmed by its comigration with an authentic standard. Twenty-HETE formation was estimated based on the specific activity of the added [1-14C]AA and was expressed as nanomoles per minute per nanomoles of P-450.

Nitration of tyrosine residues of soluble CYP4A proteins by PN. Sf9 cell membranes containing recombinant CYP4A1 or 4A3 were suspended in 2 ml of ice-cold immunoprecipitation buffer {50 mM Tris · HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, protease inhibitor cocktail [aprotinin, leupeptin, pepstatin (1 mg/ml each)]}. The mixtures were incubated on an orbital shaker at 4°C for 15 min. Soluble CYP4A proteins were obtained by centrifuging at 14,000 g at 4°C for 15 min. CYP content was determined by using the reduced CO-difference spectrum. Soluble CYP4A1 or 4A3 proteins (25 pmol) were incubated with PN (1 mM) or vehicle control in a final volume of 1 ml immunoprecipitation buffer at room temperature for 30 min. The reaction mixtures were incubated with protein G-agarose/sepharose at 4°C for 10 min and spun down by centrifuge to reduce nonspecific binding. Anti-CYP4A antibody was added to the mixtures (10 µg antibody/25 pmol CYP4A). The CYP4A/antibody mixtures were incubated overnight at 4°C. The immunocomplex was captured with 100 µl of protein G-agarose/sepharose by gently rocking for 2 h at 4°C. The immunoprecipitation product was collected by pulse centrifugation (5 s at 14,000 rpm). The pellet was washed three times with PBS. The pellet was then resuspended with 60 µl of sample buffer and boiled for 5 min. The agarose/sepharose beads were collected by centrifugation, and SDS-PAGE was performed using the supernatant. Nitration of tyrosine residues of CYP4A proteins was determined by immunoblotting with anti-3-nitrotyrosine antibodies (Up-state Biotechnology).

Protocol to evaluate the effect of inhibition of NO synthase and NO synthase plus CYP4A on systolic arterial blood pressure, urinary NO2/NO3 excretion, and renal microvessal 20-HETE synthesis. Rats were placed in metabolic cages on the gestational day 13. On the gestational day 15, rats were treated with NG-nitro-L-arginine methyl ester (L-NAME; 0.25 mg/ml in drinking water) or L-NAME (0.25 mg/ml in drinking water) plus ABT (25 mg/kg ip) for 6 days (days 15 through 20 of pregnancy). The dosage of L-NAME treatment used for this study was based primarily on a literature search (15, 31). The dosage of ABT used was based on a previous study (40). Pregnant rats in the control group were treated with water. Systolic arterial blood pressure was measured daily by tail-cuff sphygmography using a Natsume KN-210 apparatus (Peninsula Laboratories, Belmont, CA). Rats were warmed at 40°C for 10 min and allowed to rest quietly in a Lucite chamber before tail-cuff sphygmographgy; 10 pressure measurements were recorded for each rat, and the average systolic blood pressure was calculated. Urinary NO2/NO3 excretion was determined by a fluorometric method (Cayman, MI). After treatment, the rats were killed on day 21 of gestation and kidneys were removed for the preparation of microvessels to measure 20-HETE synthesis.

Measurement of 20-HETE synthesis in microvessels. Renal microvessels were isolated by microdissection and homogenates of tissue were prepared as described previously (23). Homogenates of microvessels (30 µg protein) were incubated with AA (20 µM) in 1 ml of assay buffer containing 100 mM potassium buffer (pH 7.4), 10 mM MgCl2, 1 mM NADPH, and 2 µM indomethacin for 60 min at 37°C. After incubation, [20,20-2H2]20-HETE (1 ng) was added as an internal standard, and the reaction mixture was acidified to pH 4 with 1 M formic acid. The mixture was extracted twice with 2 ml of ethyl acetate. The final extract was subjected to reverse-phase HPLC. Fractions coeluting with the 20-HETE standard were collected, evaporated to dryness, and derivitized to the pentafluorobenzyl bromide ester trimethylsilyl ether. 20-HETE was quantitated by negative chemical ionization-gas chromatography/mass spectrometry (NCI-GC/MS) by comparing the ratio of ion intensity (391:393) for derivatized 20-HETE vs. derivatized [20,20-2H2]20-HETE (39).

Statistical analysis. Data are expressed as means ± SE. All data were analyzed by a one-way analysis of variance or the Student's t-test for unpaired samples. Statistical significance was set at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CYP4A isoform expression in the kidneys of female rats. To determine the major CYP4A isoforms expressed in the kidneys of female rats, we conducted Western blot analysis for renal cortical microsomes of female rats along with CYP4A standards and renal cortical microsomes of male rats. On the basis of CYP4A protein mobility, the major isoforms expressed in the kidneys of female rats are CYP4A1 and 4A3, whereas the major isoforms in the male rats are CYP4A2/4A8 and 4A3 (Fig. 1A). In addition, we also determined the expression levels of CYP4A2 by quantitative competitive RT-PCR in the kidneys of male and female rats. Figure 1B demonstrates the profiles of RT-PCR products obtained by using a constant amount of total RNA from the kidneys of female and male rats and varying amounts of the CYP4A2 competitor RNA. By using linear regression analysis between the ratio of amplified products of CYP4A2/competitor to the concentration of the competitor, we estimated that there is about an 18-fold higher expression level of CYP4A2 in the kidneys of males than that of females (2.7 x 1016 vs. 0.15 x 1016 mol of CYP4A2/µg of RNA in male and female rats, respectively). This finding is in agreement with the result of Western blot analysis (Fig. 1A). Taken together, CYP4A1 and 4A3 are the major CYP4A isoforms expressed in the kidneys of female rats.



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 1. A: Western blot analysis of renal cortical microsomes of male and female rats along with cytochrome P-450 (CYP)4A isoform standards. Membrane proteins from Sf9 cells expressing CYP4A1 (0.1 µg), CYP4A2 (5 µg), CYP4A3 (1 µg), CYP4A8 (1 µg), male kidney cortex (10 µg), and female kidney cortex (10 µg) were analyzed by immunoblot analysis. B: determination of CYP4A2 mRNA levels in the kidneys of female (a) and male (b) rats by quantitative competitive RT-PCR. RT-PCR was performed on total RNA (5 µg) from kidneys of female and male rats in the presence of varying amounts of RNA competitor as described in MATERIALS AND METHODS. Lanes 1 through 7 are samples in which 1,000; 200; 40; 8; 1.6; 0.32; and 0.06 pg of RNA competitor were added, respectively. A single 317-bp band for CYP4A2 and a single 200-bp band for 4A2 competitor appeared in agarose gel for RT-PCR reaction.

 

Interaction between NO donor and baculovirus-expressed CYP4A isoforms in vitro. To examine whether NO binds to the major CYP4A proteins expressed in female rats, recombinant CYP4A1 and CYP4A3 were incubated with the NO donor SNP (1 mM) at room temperature for 20 min. A representative visible light absorption spectrum was shown in Fig. 2A. Incubation of CYP4A3 membranes with SNP increased absorption at 440 nm, indicating the formation of ferric-nitrosyl complexes at the CYP-heme binding site (36). More interestingly, the absorbance at {lambda}440–455 for CYP4A3 was about twofold higher than that of CYP4A1 (optical density of 0.01 for CYP4A3 vs. 0.0045 for CYP4A1). In other words, the binding affinity of NO to the heme moiety of CYP4A3 was about twofold stronger than for CYP4A1. These results reveal a significant difference in the binding characteristic of NO to the heme moiety of these two isoforms. Moreover, addition of SNP (0.01–1 mM) inhibited both CYP4A1- and CYP4A3-catalyzed AA {omega}-hydroxylation in a concentration-dependent manner. At low concentrations, SNP had a greater inhibitory effect on CYP4A3-catalyzed activity than on CYP4A1 (Fig. 2B). Taken together, these results suggest that the greater inhibitory effect of SNP on CYP4A3 may be due to greater binding affinity of NO for the heme moiety of CYP4A3.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2. A: effect of sodium nitroprusside (SNP; 1 mM) on the visible light spectrum of recombinant CYP4A1 and CYP4A3 membranes. Absorption spectra were obtained after incubation of recombinant CYP4A1 or CYP4A3 membranes with SNP for 20 min at 37°C. B: effect of SNP on arachidonic acid {omega}-hydroxylase activity in baculovirus-expressed CYP4A1 and CYP4A3. Control values for CYP4A1 and CYP4A3 {omega}-hydroxylation were 3 ± 0.08 and 0.8 ± 0.02 nmol 20-hydroxyeicosatetraenoic acid (20-HETE) · min1 · nmol P-4501. Results are means ± SE; n = 3; *P < 0.05 from control.

 

Effect of PN on recombinant CYP4A proteins. Incubation of PN (0.01–1 mM) with recombinant CYP4A1 and CYP4A3 membranes caused a concentration-dependent inhibition of both CYP4A1- and CYP4A3-catalyzed 20-HETE synthesis. PN had a greater inhibitory effect on CYP4A3-catalyzed activity than CYP4A1 (Fig. 3A). To examine whether PN can modify tyrosine residues of CYP4A isoforms, soluble preparations of recombinant CYP4A1 and CYP4A3 were incubated with 1 mM PN at room temperature for 30 min. CYP4A proteins were then isolated from the reaction mixtures by immunoprecipitation with CYP4A-specific antibody, and the nitration of tyrosine residues of CYP4A proteins was determined by Western blot analysis with anti-3-nitrotyrosine antibody. As shown in Fig. 3B, a strong 3-nitrotyrosine-immunoreactive band was observed when CYP4A proteins were incubated with PN. These results suggest that the nitration of tyrosine residues of CYP4A proteins by PN may contribute to PN inhibitory action on CYP4A-catalyzed 20-HETE formation.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 3. A: effect of peroxynitrite (PN; 0.01–1 mM) on arachidonic acid {omega}-hydroxylase activity of Sf9 recombinant CYP4A1 and CYP4A3 membranes. Control values for CYP4A1 and CYP4A3 {omega}-hydroxylation activity were 4 ± 0.1 and 0.7 ± 0.03 nmol 20-HETE · min1 · nmol P-4501. Results are means ± SE; n = 3; *P < 0.05 from control. B: immunoblot of CYP4A proteins with anti-nitrotyrosine antibody. Soluble CYP4A1 or 4A3 (25 pmol) was treated with PN (1 mM) or the vehicle control, immunoprecipitated with anti-CYP4A1 IgG, and immunoblotted with anti-nitrotyrosine antibody as described in MATERIALS AND METHODS.

 

Effect of SNP and PN on renal microvessel 20-HETE synthesis. To examine whether NO and PN have a similar effect on 20-HETE synthesis in renal microvessels isolated from female rats, homogenates were preincubated with SNP (1 mM), PN (1 mM), or vehicle control at room temperature for 30 min followed by incubation with AA and NADPH. 20-HETE synthesis was determined by NCI-GC/MS. As shown in Fig. 4, SNP and PN caused 59 and 65% inhibition of renal microvessel 20-HETE synthesis, suggesting that NO and PN act as negative regulators of 20-HETE synthesis in renal microvessels.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 4. Effect of PN (1 mM) and SNP (1 mM) on renal microvessel 20-HETE synthesis. Renal microvessels were isolated from female rats. 20-HETE formation was determined by negative chemical ionization-gas chromatography/mass spectrometry. Results are means ± SE; n = 4; *P < 0.05 from control.

 

Effect of NO synthase and NO synthase/CYP4A inhibition on systolic blood pressure, urinary NO2/NO3 excretion, and renal microvessel 20-HETE synthesis. L-NAME (0.25 mg/ml in drinking water), L-NAME (0.25 mg/ml in drinking water) plus ABT (25 mg/kg ip), or vehicle control was administered for 6 days to pregnant rats beginning on day 15 of gestation. As seen in Table 1, systolic blood pressure in L-NAME-treated rats was significantly increased compared with pregnant control rats, whereas systolic blood pressure in L-NAME plus ABT-treated group remained unaffected. Urinary NO2/NO3 excretion, an index for whole body production of NO in pregnant rats and women (5, 6), decreased by 40% (P < 0.05) following L-NAME treatment (Table 1). Interestingly, renal microvessel 20-HETE synthesis increased threefold relative to control in the L-NAME-treated group but was unchanged in the L-NAME plus ABT-treated group (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1. SBP urinary NO2/NO3, excretion, and renal microvessel 20-HETE synthesis with L-NAME, L-NAME plus ABT, or vehicle control treatment during pregnancy

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In a previous study, we demonstrated temporal changes in renal vascular CYP4A expression and 20-HETE synthesis during pregnancy in rats. CYP4A protein levels and the product of their catalytic activity on AA, 20-HETE, were increased during the first and second week of pregnancy but returned to control levels during the third week of pregnancy. The exact mechanisms responsible for these changes are unknown. We hypothesized that NO may constitute one of the factors regulating the production of 20-HETE in the renal microcirculation.

During pregnancy in rats, urinary excretion and plasma levels of nitrate are elevated and urinary excretion of cGMP is also increased. Chronic treatment with L-NAME reduces urinary nitrate excretion in pregnant rats (5). Moreover, the binding of NO to hemoglobin is only detected in the blood of pregnant, but not nonpregnant, rats (5). Augmentation of NO production and increased expression levels of renal neuronal NO synthase (NOS) and inducible NOS have been observed during normal pregnancy (1), and chronic inhibition of NO synthesis by L-NAME during pregnancy resulted in preeclampsia-like symptoms (26, 41). These reports provided substantial evidence to implicate NO as a major contributor to the implementation of a vasodilatory state in pregnancy (18, 35). On the basis of this information and on data indicating that NO inhibits CYP4A expression and 20-HETE synthesis and action (31, 36), we postulated the existence of interactions between NO and CYP4A/20-HETE that may explain the decreased renal vascular 20-HETE synthesis during the third week of pregnancy and may contribute to the regulation of vascular tone and blood pressure during pregnancy. In the present study, we showed that NO binds to the heme moiety of CYP4A1 and CYP4A3, the major isoforms expressed in female rats, and inhibits their catalytic activity. We also demonstrated that PN, which shows elevated production of NO and superoxide (7, 22), causes nitrosylation of tyrosine residues on CYP4A1 and CYP4A3.

The CYP4A enzymes (CYP4A1, 4A2, 4A3, and 4A8) are considered to be the major AA {omega}-hydroxylases in the rat kidney and thereby the primary contributors of 20-HETE synthesis. With the use of quantitative RT/PCR and Western blot analysis, we demonstrated that CYP4A1 and CYP4A3 are the major CYP4A expressed in the kidneys of female rats. The expression levels of CYP4A2 measured by quantitative RT/PCR were 18-fold lower in female rats compared with male rats. These results are in accordance with other reports documenting lower and even undetectable expression levels of CYP4A2 in the kidneys of female rats (37) as well as reports demonstrating androgen-dependent expression of CYP4A2 (11). As for the CYP4A8 expression, a recent study by Holla et al. (10) suggested that CYP4a12 (a murine homologue gene to CYP4A8) is a male-specific and androgen-regulated enzyme and has very low expression in female kidneys. Nakagawa et al. (27) showed that CYP4A8 expression in the rat is androgen sensitive. CYP4A8 protein has a similar electrophoretic mobility as CYP4A2 (28); the absence of CYP4A2-immunoreactive protein in renal microsomes from female rats (Fig. 1) suggests low expression levels of CYP4A8. On the basis of these reports, our data, and previous studies showing that the recombinant CYP4A1 and CYP4A3 proteins catalyze AA {omega}-hydroxylation to 20-HETE, it is likely that these isoforms contribute significantly to renal 20-HETE synthesis in female rats. However, we cannot rule out the possibility that other isoforms of the CYP4 gene family such as CYP4F proteins, CYP4F1, CYP4F4, CYP4F5, and CYP4F6, may be involved in the renal production of 20-HETE in female rats (4, 16). Kalsotra et al. (14) showed significant levels of expression of all CYP4F isoforms in kidneys of female rats and further documented an estrogen-sensitive expression of CYP4F1, CYP4F4, and CYP4F6. The catalytic activity of CYP4F isoforms toward AA and their ability to catalyze the production of 20-HETE has not been fully examined. Further studies that allow evaluation of the distinct contribution of each of the CYP4A and 4F isoforms to 20-HETE synthesis are needed.

NO inhibits heme-containing proteins such as CYP (25). The mechanisms of the interaction between NO and CYP proteins were described by Minamiyama et al. (25), who demonstrated that NO can interact with CYP in two ways: NO binds reversibly with the heme moiety and irreversibly with cysteine residues of CYP proteins. These NO-CYP adducts are enzymatically inactive in vitro. Moreover, Roberts et al. (32) demonstrated that PN can modify tyrosine residues of CYP2B1 and inactivate CYP2B1-catalyzed reaction. It is likely that NO interacts with the major CYP4A isoforms in female rats, i.e., CYP4A1 and 4A3, in a similar manner. However, it is difficult to study the interaction between NO and CYP4A isoforms in renal tissues because renal tissues contain numerous CYP enzymes other than CYP4A isoforms. Baculovirus-expressed CYP4A isoforms provide a unique tool to study the interaction between NO and individual CYP4A isoforms in vitro because there is a negligible level of CYP content in Sf9 insect cells (39). Our results indicated that NO binds to the heme moiety of CYP4A1 and CYP4A3 with different affinities. The heme moiety of CYP enzymes is essential for the oxidation reaction. NO binding to the heme moiety can interfere with the electron transport mechanisms of the oxidation reaction. It is possible that the ability of NO to inhibit CYP4A3-catalyzed 20-HETE synthesis to a greater extent than that of CYP4A1 is due to the higher binding affinity of NO to CYP4A3. PN, a powerful oxidant, is derived from NO and superoxide. Because CYP isoforms can generate varying amounts of oxygen-derived free radicals such as superoxide ion during the catalytic cycle of CYP enzymes (8), it is possible that superoxide ion generated from the CYP4A-catalyzed reaction can interact with NO and cause the formation of PN. We showed that PN inhibits 20-HETE synthesis catalyzed by CYP4A1 and CYP4A3. The mechanisms underlying this inhibition are not clear; however, the ability of PN to nitrosylate tyrosine residues of CYP4A1 and CYP4A3 may constitute, at least in part, a mechanism of inhibition. However, additional studies are needed to demonstrate that tyrosine nitrosylation of CYP4A proteins occurs in vivo and that nitrosylated CYP4A proteins are catalytically inactive.

In contrast to normal pregnancy, preeclampsia is characterized by increased arterial blood pressure, generalized vasoconstriction, increased systemic resistance, widespread vascular endothelial damage, decreased fetal growth, and proteinuria (21). The exact mechanisms that mediate preeclampsia are still unknown. Several reports have suggested that NO may play an important role in its development (2, 22). Moreover, two reports demonstrated that chronic inhibition of NO synthesis in late pregnancy in rats resulted in signs similar to those of preeclampsia (26, 31). That the increased renal microvessel production of 20-HETE following administration of L-NAME during the third week of pregnancy together with reports that NO inhibits 20-HETE synthesis and interferes with 20-HETE vasoconstrictor activity in vivo (31, 36) suggest the contribution of 20-HETE to the implementation of renal vasoconstriction and increased blood pressure (Table 1) under conditions where NO production is suppressed. This notion is further substantiated by data showing that coadministration of ABT, an inhibitor of CYP4A activity, abolished the L-NAME-induced increase in blood pressure in these rats (Table 1).

In summary, this study provides the first evidence to show that NO binds differently to the heme of CYP4A isoforms and inhibits 20-HETE synthesis by recombinant CYP4A proteins and renal microvessels in female rats. This study also shows that PN modifies tyrosine residues of CYP4A proteins and inhibits their catalytic activity. Additional data show that augmentation of renal microvessel 20-HETE synthesis after NOS inhibition is associated with increased blood pressure and that this increase is negated by treatment with a CYP4A inhibitor. Hence, this study offers evidence that NO acts as a buffer system to counteract 20-HETE-mediated vasoconstriction mechanisms during pregnancy.


    DISCLOSURES
 
This study was supported by a National Institutes of Health (NIH) Grant PO1-HL34300 (to A. Nasjletti and M. Laniado-Schwartzman) and by NIH Grant R01-HL-70887 to M.-H. Wang.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M-H. Wang, Dept. of Physiology, Medical College of Georgia, Augusta, GA 30912 (E-mail: mwang{at}mail.mcg.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Alexander BT, Miller MT, Kassab S, Novak J, Reckelhoff JF, Kruckeberg WC, and Granger JP. Differential expression of renal nitric oxide synthase isoforms during pregnancy in rats. Hypertension 33: 435–439, 1999.[Abstract/Free Full Text]
  2. Baylis C, Beinder E, Suto T, and August P. Recent insights into the roles of nitric oxide and renin-angiotensin in the pathophysiology of preeclamptic pregnancy. Semin Nephrol 18: 208–230, 1998.[ISI][Medline]
  3. Beckman JS, Chen J, Ischiropoulos H, and Crow JP. Oxidative chemistry of peroxynitrite. Methods Enzymol 233: 229–240, 1994.[ISI][Medline]
  4. Chen L and Hardwick JP. Identification of a new P450 subfamily, CYP4F1, expressed in rat hepatic tumors. Arch Biochem Biophys 300: 18–23, 1993.[ISI][Medline]
  5. Conrad KP, Joffe GM, Kruszyna H, Kruszyna R, Rochelle LG, Smith RP, Chavez JE, and Mosher MD. Identification of increased nitric oxide biosynthesis during pregnancy in rats. FASEB J 7: 566–571, 1993.[Abstract/Free Full Text]
  6. Conrad KP, Kerchner LJ, and Mosher MD. Plasma and 24-h NO(x) and cGMP during normal pregnancy and preeclampsia in women on a reduced NO(x) diet. Am J Physiol Renal Physiol 277: F48–F57, 1999.[Abstract/Free Full Text]
  7. Daiber A, Herold S, Schoneich C, Namgaladze D, Peterson JA, and Ullrich V. Nitration and inactivation of cytochrome P450BM-3 by peroxynitrite. Stopped-flow measurements prove ferryl intermediates. Eur J Biochem 267: 6729–6739, 2000.[Abstract/Free Full Text]
  8. Fleming I. Cytochrome p450 and vascular homeostasis. Circ Res 89: 753–762, 2001.[Abstract/Free Full Text]
  9. Hardwick JP. CYP 4A subfamily: functional analysis by immunocytochemistry and in situ hybridization. Methods Enzymol 206: 273–283, 1991.[ISI][Medline]
  10. Holla VR, Adas F, Imig JD, Zhao X, Price E Jr, Olsen N, Kovacs WJ, Magnuson MA, Keeney DS, Breyer MD, Falck JR, Waterman MR, and Capdevila JH. Alterations in the regulation of androgen-sensitive Cyp 4a monooxygenases cause hypertension. Proc Natl Acad Sci USA 98: 5211–5216, 2001.[Abstract/Free Full Text]
  11. Imaoka S, Yamazoe Y, Kato R, and Funae Y. Hormonal regulation of rat cytochrome P450s by androgen and pituitary. Arch Biochem Biophys 299: 179–184, 1992.[ISI][Medline]
  12. Imig JD, Zou AP, Stec DE, Harder DR, Falck JR, and Roman RJ. Formation and actions of 20-hydroxyeicosatetraenoic acid in rat renal arterioles. Am J Physiol Regul Integr Comp Physiol 270: R217–R227, 1996.[Abstract/Free Full Text]
  13. Iwai N and Inagami T. Isolation of preferentially expressed genes in the kidneys of hypertensive rats. Hypertension 17: 161–169, 1991.[Abstract]
  14. Kalsotra A, Anakk S, Boehme CL, and Strobel HW. Sexual dimorphism and tissue specificity in the expression of CYP4F forms in Sprague-Dawley rats. Drug Metab Dispos 30: 1022–1028, 2002.[Abstract/Free Full Text]
  15. Kanashiro CA, Alexander BT, Granger JP, and Khalil RA. Ca2+-insensitive vascular protein kinase C during pregnancy and NOS inhibition. Hypertension 34: 924–930, 1999.[Abstract/Free Full Text]
  16. Kawashima H, Kusunose E, Thompson CM, and Strobel HW. Protein expression, characterization, and regulation of CYP4F4 and CYP4F5 cloned from rat brain. Arch Biochem Biophys 347: 148–154, 1997.[ISI][Medline]
  17. Khalil RA, Crews JK, Novak J, Kassab S, and Granger JP. Enhanced vascular reactivity during inhibition of nitric oxide synthesis in pregnant rats. Hypertension 31: 1065–1069, 1998.[Abstract/Free Full Text]
  18. Kone BC and Baylis C. Biosynthesis and homeostatic roles of nitric oxide in the normal kidney. Am J Physiol Renal Physiol 272: F561–F578, 1997.[Abstract/Free Full Text]
  19. Kroetz DL, Huse LM, Thuresson A, and Grillo MP. Developmentally regulated expression of the CYP4A genes in the spontaneously hypertensive rat kidney. Mol Pharmacol 52: 362–372, 1997.[Abstract/Free Full Text]
  20. Laniado Schwartzman M, Da Silva JL, Lin F, Nishimura M, and Abraham NG. Cytochrome P450 4A expression and arachidonic acid {omega}-hydroxylation in the kidney of the spontaneously hypertensive rat. Nephron 73: 652–663, 1996.[ISI][Medline]
  21. Lindheimer MD and Katz AI. Renal physiology and disease in pregnancy. In: The Kidney: Physiology and Pathophysiology, edited by Seldin DW and Giebisch G. New York: Raven, 1992, p. 3371–3431.
  22. Lowe DT. Nitric oxide dysfunction in the pathophysiology of preeclampsia. Nitric Oxide 4: 441–458, 2000.[ISI][Medline]
  23. Marji J, Wang HM, and Schwartzman M. Cytochrome P-450 4A isoform expression and 20-HETE synthesis in renal preglomerular arteries. Am J Physiol Renal Physiol 283: F60–F67, 2002.[Abstract/Free Full Text]
  24. McGiff JC and Quilley J. 20-HETE and the kidney: resolution of old problems and new beginnings. Am J Physiol Regul Integr Comp Physiol 277: R607–R623, 1999.[Abstract/Free Full Text]
  25. Minamiyama Y, Takemura S, Imaoka S, Funae Y, Tanimoto Y, and Inoue M. Irreversible inhibition of cytochrome P450 by nitric oxide. J Pharmacol Exp Ther 283: 1479–1485, 1997.[Abstract/Free Full Text]
  26. Molnar M, Suto T, Toth T, and Hertelendy F. Prolonged blockade of nitric oxide synthesis in gravid rats produces sustained hypertension, proteinuria, thrombocytopenia, and intra-uterine growth retardation. Am J Obstet Gynecol 170: 1458–1466, 1994.[ISI][Medline]
  27. Nakagawa K, Marji JS, Schwartzman ML, Waterman MR, and Capdevila JH. The androgen-mediated induction of the kidney arachidonate hydroxylases is associated with the development of hypertension. Am J Physiol Regul Integr Comp Physiol 284: R1055–R1062, 2003.[Abstract/Free Full Text]
  28. Nguyen X, Wang MH, Reddy KM, Falck JR, and Schwartzman ML. Kinetic profile of the rat CYP4A isoforms: arachidonic acid metabolism and isoform-specific inhibitors. Am J Physiol Regul Integr Comp Physiol 276: R1691–R1700, 1999.[Abstract/Free Full Text]
  29. Nims RW, Cook JC, Krishna MC, Christodoulou D, Poore CM, Miles AM, Grisham MB, and Wink DA. Colorimetric assays for nitric oxide and nitrogen oxide species formed from nitric oxide stock solutions and donor compounds. Methods Enzymol 268: 93–105, 1996.[ISI][Medline]
  30. Ogawa T, Linz W, Scholkens BA, and de Bold AJ. Variable renal atrial natriuretic factor gene expression in hypertension. Hypertension 33: 1342–1347, 1999.[Abstract/Free Full Text]
  31. Oyekan AO, Youseff T, Fulton D, Quilley J, and McGiff JC. Renal cytochrome P450 {omega}-hydroxylase and epoxygenase activity are differentially modified by nitric oxide and sodium chloride. J Clin Invest 104: 1131–1137, 1999.[Abstract/Free Full Text]
  32. Roberts ES, Lin H, Crowley JR, Vuletich JL, Osawa Y, and Hollenberg PF. Peroxynitrite-mediated nitration of tyrosine and inactivation of the catalytic activity of cytochrome P450 2B1. Chem Res Toxicol 11: 1067–1074, 1998.[ISI][Medline]
  33. Roman RJ. P-450 metabolites of arachidonic acid in the control of cardiovascular function. Physiol Rev 82: 131–185, 2002.[Abstract/Free Full Text]
  34. Simpson AE. The cytochrome P450 4 (CYP4) family. Gen Pharmacol 28: 351–359, 1997.[Medline]
  35. Sladek SM, Magness RR, and Conrad KP. Nitric oxide and pregnancy. Am J Physiol Regul Integr Comp Physiol 272: R441–R463, 1997.[Abstract/Free Full Text]
  36. Sun CW, Alonso-galicia M, Taheri MR, Falck JR, Harder DR, and Roman RJ. Nitric oxide-20-hydroxyeicosatetraenoic acid interaction in the regulation of K+ channel activity and vascular tone in renal arterioles. Circ Res 83: 1069–1079, 1998.[Abstract/Free Full Text]
  37. Sundseth SS and Waxman DJ. Sex-dependent expression and clofibrate inducibility of cytochrome P450 4A fatty acid {omega}-hydroxylases. Male specificity of liver and kidney CYP4A2 mRNA and tissue-specific regulation by growth hormone and testosterone. J Biol Chem 267: 3915–3921, 1992.[Abstract/Free Full Text]
  38. Wang MH, Guan H, Nguyen X, Zand BA, Nasjletti A, and Laniado-Schwartzman M. Contribution of cytochrome P-450 4A1 and 4A2 to vascular 20-hydroxyeicosatetraenoic acid synthesis in rat kidneys. Am J Physiol Renal Physiol 276: F246–F253, 1999.[Abstract/Free Full Text]
  39. Wang MH, Stec DE, Balazy M, Mastyugin V, Yang CS, Roman RJ, and Laniado Schwartzman M. Cloning, sequencing and cDNA-directed expression of the rat renal CYP4A2: arachidonic acid {omega}-hydroxylation and 11,12-epoxidation by CYP4A2 protein. Arch Biochem Biophys 336: 240–250, 1996.[ISI][Medline]
  40. Wang MH, Zand BA, Nasjletti A, and Laniado-Schwartzman M. Renal 20-hydroxyeicosatetraenoic acid synthesis during pregnancy. Am J Physiol Regul Integr Comp Physiol 282: R383–R389, 2002.[Abstract/Free Full Text]
  41. Yallampalli C and Garfield RE. Inhibition of nitric oxide synthesis in rats during pregnancy produces signs similar to those of preeclampsia. Am J Obstet Gynecol 169: 1316–1320, 1993.[ISI][Medline]