Regulation of renal CYP4A expression and 20-HETE synthesis by nitric oxide in pr - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:42 |只看该作者 |倒序浏览 |打印

作者：Mong-Heng Wang, Jishi Wang, Hsin-Hsin Chang, Barbara A. Zand, Miao Jiang, Alberto Nasjletti, and Michal Laniado-Schwartzman作者单位：1 Department of Physiology, Medical College ofGeorgia, Augusta, Georgia 30912; and 2 Department ofPharmacology, New York Medical College, Valhalla, New York 10595 8 E- E+ z& X0 c+ ^) A/ [
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      【摘要】! s2 Y9 c% c: h; W. Q: p3 k
   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) hasbeen shown to bind to the heme moiety of the CYP4A2 protein and to inhibit20-HETE synthesis in renal arterioles of male rats. However, it is not knownwhether NO interacts with and affects the activity of CYP4A1 and CYP4A3, themajor renal CYP4A isoforms in female rats. Incubation of recombinant CYP4A1 and 4A3 proteins with sodium nitroprusside (SNP) shifted the absorbance at 440nm, indicating the formation of a ferric-nitrosyl-CYP4A complex. Theabsorbance for CYP4A3 was about twofold higher than that of CYP4A1. Incubationof SNP or peroxynitrite (PN; 0.01-1 mM) with CYP4A recombinant membranescaused a concentration-dependent inhibition of 20-HETE synthesis, with bothchemicals having a greater inhibitory effect on CYP4A3-catalyzed activity.Moreover, incubation of CYP4A1 and 4A3 proteins with PN (1 mM) resulted innitration of tyrosine residues in both proteins. In addition, PN and SNPinhibited 20-HETE synthesis in renal microvessels from female rats by 65 and59%, respectively. We previously showed that microvessel CYP4A1/CYP4A3expression and 20-HETE synthesis are decreased in late pregnancy. Therefore, we investigated whether such a decrease is dependent on NO, the synthesis ofwhich has been shown to increase in late pregnancy. Administration of N G -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, whichwas prevented by concurrent treatment with the CYP4A inhibitor 1-aminobenzotriazole (ABT). Urinary NO 2 /NO 3 excretiondecreased by 40 and 52% in L -NAME- and L -NAME  ABT-treated groups, respectively. Interestingly, renal microvessel 20-HETEsynthesis showed a marked increase following L -NAME treatment, andthis increase was diminished with coadministration of ABT. These results demonstrate that NO interacts with CYP4A proteins in a distinct manner and itinterferes with renal microvessel 20-HETE synthesis, which may play animportant role in the regulation of blood pressure and renal function duringpregnancy.
      【关键词】 cytochrome P A N G nitro L arginine methyl ester6 _, }% R3 `' h$ |4 O
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20- HYDROXYEICOSATETRAENOIC acid (20-HETE), the -hydroxylation product of arachidonic acid (AA), is a principaleicosanoid 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 ). Inthe 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 aswell as age and sex dependent( 9, 12, 13, 19, 20 ). For example, Sundseth andWaxman ( 37 ) demonstrated thathepatic and renal CYP4A1 and 4A3 expressions are similar in male and femalerats, whereas CYP4A2 expression is undetectable in female rats. Castration ofmale rats decreased the levels of CYP4A2, and treatment of castrated male ratswith testosterone reversed this decrease( 11 ). A recent study byNakagawa et al. ( 27 ) showedthat CYP4A8 expression is androgen sensitive. These sex-related differences inthe expression of hepatic and renal CYP4A proteins suggest that androgens andestrogens play an important role in the regulation of these isoforms.

Normal pregnancy in humans and rats is associated with increases inglomerular filtration rate and renal blood flow( 21 ) along with significantdecreases in arterial pressure and total peripheral resistance( 1, 17 ). 20-HETE possessesbiological effects that can potentially contribute to these physiologicalchanges during pregnancy. These biological effects include inhibition of iontransport along the nephron and vasoconstriction of renal arterioles( 24, 33 ). We demonstrated distinctupregulation 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 pregnancyare not known. We considered a role for nitric oxide (NO) in the regulation ofCYP4A expression and activity in renal microvessels during late pregnancybecause inhibition of NO synthesis increases 20-HETE synthesis( 31 ), NO donors decrease theproduction of 20-HETE in renal microvessels( 36 ), and NO productionincreases in pregnancy ( 1, 5 )./ r5 C3 v* ^& E+ E! F
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The present study was undertaken to explore possible biochemical mechanismsunderlying the effect of NO on CYP4A protein expression and activity and toexamine whether inhibition on NO synthesis alters renal vascular 20-HETEsynthesis in late pregnancy. We showed that NO readily binds to the hememoiety of the major CYP4A isoforms expressed in female rats, CYP4A1 andCYP4A3, with distinct isoform-specific affinity and that peroxynitrate increases tyrosine nitrosylation of these proteins. We also showed thatinhibition of NO synthesis during the third week of gestation leads to amarked increase in vascular 20-HETE synthesis. Furthermore, coadministrationof 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 vascular20-HETE synthesis may contribute to the regulation of blood pressure duringpregnancy.

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

Animals. All animals were purchased from Charles RiverLaboratories, Wilmington, MA. Experiments were conducted in male and female Sprague-Dawley rats (8 wk old), pregnant (timed pregnancy), and the same agecontrol female Sprague-Dawley rats. Experimental protocols were approved bythe Institutional Animal Care and Use Committee. Rats were maintained undercontrolled housing conditions of light and temperature and received standardlaboratory chow and water until used.1 D+ |; O; t* Z* F+ j" ^# e0 F3 v

Quantitative competitive RT-PCR for CYP4A2. The detailed procedure for the preparation of competitor DNA of CYP4A2 was described in themanufacturer's protocol (PanVera, Madison, WI). This method was designed togenerate CYP4A2 competitor (200 nucleotides) that is 117 nucleotides less thanthe target CYP4A2 cDNA. We used two sets of PCR to generate CYP4A2 competitorDNA. The first PCR primer set contained sequences that hybridize to a CYP4A2sequence and are flanked by DNA-specific sequences according to themanufacturer's protocol. The product of the first PCR set was purified andused as the template for the second PCR set. The second PCR primer setcontained sequences that hybridize to a CYP4A2 sequence and are flanked bySP 6 promoter-specific sequences. The product of the second PCR setwas purified and used to generate CYP4A2 competitor RNA by in vitrotranscription using a SP 6 RNA polymerase. The template was thendigested with DNase I, and the RNA was purified by phenol/chloroform/isoamyl alcohol method. The amount of the competitor RNA synthesized was quantified byspectrophotometry. Aliquots of total RNA (5 µg) from the kidneys of maleand female rats were prepared and a x 5 dilution series of competitor RNA(1,000; 200; 40; 8; 1.6; 0.32; 0.06 pg, respectively) was added into thesealiquots, and RT-PCR was performed. A reverse transcription reaction wasperformed 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 andvisualized by ethidium bromide staining. Gel pictures were scanned anddensitometry analysis was performed with Scion Image software using gray colorscale as a standard. The ratio of the density of the competitor RNA to theCYP4A2 RNA, plotted against the amount of the competitor RNA added to eachreaction, was used to estimate CYP4A2 mRNA levels as described( 30 ). The sequences of the primers used were as follows: CYP4A2 DNA: 5'-AGA TCC AAA GCC TTA TCA ATC GTA CGG TCA TCA TCT GAC AC-3' (forward primer), 5'-CAGCCT TGG TGT AGG ACC TTC ATT ACG CAT CGC TAT TAC-3' (backward primer);and SP 6 CYP4A2: 5'-ATT TAG GTG ACA CTA TAG AAT ACA GAT CCAAAG CCT TAT CAA TC-3' (forward primer), 5'-CAG CCT TGG TGT AGG ACCTTC ATT ACG CAT CGC TAT TAC-3' (backward primer).$ j% E8 f( @  x! c

Preparation of recombinant CYP4A membranes. CYP4A proteins were expressed using the baculovirus-Sf9 insect cell expression system as describedpreviously ( 28 ). CYP4Arecombinant Sf9 cell membranes were prepared after infection with therecombinant 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 lysatesas described ( 28 ). Themembrane pellets were resuspended in sucrose buffer (50 mM potassiumphosphate, pH 7.4, and 0.5 M sucrose) and stored at -80°C. Proteinconcentration 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 ).9 w$ S2 L4 x. ~! e2 w6 u7 m
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Effect of sodium nitroprusside and peroxynitrite on recombinant CYP4Acatalytic activity. The stability of the NO donor sodium nitroprusside(SNP) was examined with NO-sensitive litmus paper using Griess reagent [0.5 gof sulfanilamide plus 20 mg of N -(1-naphthyl)ethylenediaminedihydrochloride] dissolved in 10 ml of methanol( 29 ). In our preliminarystudy, SNP constantly released NO during a 10- to 20-min incubation (data notshown). Peroxynitrite (PN) stock was diluted in 0.3 M sodium hydroxide, andthe concentration was determined by the extinction coefficient of 1,670 M· cm - 1 · 302nm - 1 ( 3 ). SNP (0.01-1 mM) orPN (0.01-1 mM in 0.3 N NaOH) was added to mixture containing recombinantCYP4A1 or 4A3 membranes, purified NADPH-CYP oxidoreductase, and cytochrome b 5 at a molar ratio of 1:14:4. This mixture waspreincubated with NADPH (1 mM) in a final volume of 0.15 ml of buffer (10 mMMgCl 2 and 100 mM KH 2 PO 4, pH 7.2). Themixtures were preincubated at room temperature for 20 min.[1- 14 C]AA (0.4 µCi, 7 nmol) was then added, and incubation wascarried out at 37°C for 30 min. Control incubations included the vehicleof 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. Thefinal extract was evaporated under nitrogen, resuspended in 50 µl ofmethanol, and injected onto the HPLC column. Reverse-phase HPLC was performedon a 5-µm ODS-Hypersil column, 4.6 x 200 mm (Hewlett-Packard, PaloAlto, 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/minfor 30 min. The elution profile of the radioactive products was monitored by aflow detector (In/us System, Tampa, FL). The identity of 20-HETE was confirmedby its comigration with an authentic standard. Twenty-HETE formation wasestimated based on the specific activity of the added [1- 14 C]AA andwas expressed as nanomoles per minute per nanomoles of P -450.
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Nitration of tyrosine residues of soluble CYP4A proteins by PN. Sf9 cell membranes containing recombinant CYP4A1 or 4A3 were suspended in 2 mlof 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, proteaseinhibitor cocktail [aprotinin, leupeptin, pepstatin (1 mg/ml each)]}. Themixtures 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°Cfor 15 min. CYP content was determined by using the reduced CO-differencespectrum. Soluble CYP4A1 or 4A3 proteins (25 pmol) were incubated with PN (1mM) or vehicle control in a final volume of 1 ml immunoprecipitation buffer atroom temperature for 30 min. The reaction mixtures were incubated with proteinG-agarose/sepharose at 4°C for 10 min and spun down by centrifuge toreduce nonspecific binding. Anti-CYP4A antibody was added to the mixtures (10µg antibody/25 pmol CYP4A). The CYP4A/antibody mixtures were incubatedovernight at 4°C. The immunocomplex was captured with 100 µl of proteinG-agarose/sepharose by gently rocking for 2 h at 4°C. Theimmunoprecipitation product was collected by pulse centrifugation (5 s at14,000 rpm). The pellet was washed three times with PBS. The pellet was thenresuspended with 60 µl of sample buffer and boiled for 5 min. Theagarose/sepharose beads were collected by centrifugation, and SDS-PAGE wasperformed using the supernatant. Nitration of tyrosine residues of CYP4A proteins was determined by immunoblotting with anti-3-nitrotyrosine antibodies(Up-state Biotechnology).
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Protocol to evaluate the effect of inhibition of NO synthase and NOsynthase plus CYP4A on systolic arterial blood pressure, urinaryNO 2 /NO 3 excretion,and renal microvessal 20-HETE synthesis. Rats were placed in metaboliccages on the gestational day 13. On the gestational day 15,rats were treated with N G -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 aliterature search ( 15, 31 ). The dosage of ABT usedwas based on a previous study( 40 ). Pregnant rats in thecontrol group were treated with water. Systolic arterial blood pressure wasmeasured daily by tail-cuff sphygmography using a Natsume KN-210 apparatus (Peninsula Laboratories, Belmont, CA). Rats were warmed at 40°C for 10 minand allowed to rest quietly in a Lucite chamber before tail-cuffsphygmographgy; 10 pressure measurements were recorded for each rat, and theaverage systolic blood pressure was calculated. UrinaryNO 2 /NO 3 excretion was determined by a fluorometricmethod (Cayman, MI). After treatment, the rats were killed on day 21 of gestation and kidneys were removed for the preparation of microvessels tomeasure 20-HETE synthesis.. {# ]9 l& W' |- l+ `
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Measurement of 20-HETE synthesis in microvessels. Renalmicrovessels were isolated by microdissection and homogenates of tissue were prepared as described previously( 23 ). Homogenates ofmicrovessels (30 µg protein) were incubated with AA (20 µM) in 1 ml ofassay buffer containing 100 mM potassium buffer (pH 7.4), 10 mMMgCl 2, 1 mM NADPH, and 2 µM indomethacin for 60 min at 37°C.After incubation, [20,20- 2 H 2 ]20-HETE (1 ng) was added asan internal standard, and the reaction mixture was acidified to pH 4 with 1 Mformic acid. The mixture was extracted twice with 2 ml of ethyl acetate. Thefinal extract was subjected to reverse-phase HPLC. Fractions coeluting with the 20-HETE standard were collected, evaporated to dryness, and derivitized tothe pentafluorobenzyl bromide ester trimethylsilyl ether. 20-HETE wasquantitated by negative chemical ionization-gas chromatography/massspectrometry (NCI-GC/MS) by comparing the ratio of ion intensity (391:393) forderivatized 20-HETE vs. derivatized [20,20- 2 H 2 ]20-HETE( 39 ).
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Statistical analysis. Data are expressed as means ± SE. Alldata were analyzed by a one-way analysis of variance or the Student's t -test for unpaired samples. Statistical significance was set at P 3 M, `1 G% j. i& M, G* j

RESULTS5 r$ t. n) O- d" o* r: {- a

CYP4A isoform expression in the kidneys of female rats. To determine the major CYP4A isoforms expressed in the kidneys of female rats, weconducted Western blot analysis for renal cortical microsomes of female ratsalong with CYP4A standards and renal cortical microsomes of male rats. On thebasis of CYP4A protein mobility, the major isoforms expressed in the kidneysof female rats are CYP4A1 and 4A3, whereas the major isoforms in the male ratsare CYP4A2/4A8 and 4A3 ( Fig.1 A ). In addition, we also determined the expressionlevels of CYP4A2 by quantitative competitive RT-PCR in the kidneys of male and female rats. Figure 1 B demonstrates the profiles of RT-PCR products obtained by using a constantamount of total RNA from the kidneys of female and male rats and varyingamounts of the CYP4A2 competitor RNA. By using linear regression analysis between the ratio of amplified products of CYP4A2/competitor to theconcentration of the competitor, we estimated that there is about an 18-foldhigher expression level of CYP4A2 in the kidneys of males than that of females(2.7 x 10 - 16 vs. 0.15 x 10 - 16 mol of CYP4A2/µg of RNA in male and female rats, respectively). This finding is in agreement with the result ofWestern blot analysis ( Fig.1 A ). Taken together, CYP4A1 and 4A3 are the major CYP4Aisoforms expressed in the kidneys of female rats.
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Fig. 1. A : Western blot analysis of renal cortical microsomes of male andfemale rats along with cytochrome P -450 (CYP)4A isoform standards.Membrane proteins from Sf9 cells expressing CYP4A1 (0.1 µg), CYP4A2 (5µg), CYP4A3 (1 µg), CYP4A8 (1 µg), male kidney cortex (10 µg), andfemale 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 malerats in the presence of varying amounts of RNA competitor as described in MATERIALS AND METHODS. Lanes 1 through 7 aresamples in which 1,000; 200; 40; 8; 1.6; 0.32; and 0.06 pg of RNA competitorwere added, respectively. A single 317-bp band for CYP4A2 and a single 200-bpband for 4A2 competitor appeared in agarose gel for RT-PCR reaction.

Interaction between NO donor and baculovirus-expressed CYP4A isoformsin vitro. To examine whether NO binds to the major CYP4A proteinsexpressed in female rats, recombinant CYP4A1 and CYP4A3 were incubated withthe NO donor SNP (1 mM) at room temperature for 20 min. A representativevisible light absorption spectrum was shown in Fig. 2 A. Incubation ofCYP4A3 membranes with SNP increased absorption at 440 nm, indicating theformation of ferric-nitrosyl complexes at the CYP-heme binding site( 36 ). More interestingly, theabsorbance at 440-455 for CYP4A3 was about twofoldhigher than that of CYP4A1 (optical density of 0.01 for CYP4A3 vs. 0.0045 forCYP4A1). In other words, the binding affinity of NO to the heme moiety ofCYP4A3 was about twofold stronger than for CYP4A1. These results reveal asignificant difference in the binding characteristic of NO to the heme moietyof these two isoforms. Moreover, addition of SNP (0.01-1 mM) inhibitedboth CYP4A1- and CYP4A3-catalyzed AA -hydroxylation in aconcentration-dependent manner. At low concentrations, SNP had a greaterinhibitory effect on CYP4A3-catalyzed activity than on CYP4A1( Fig. 2 B ). Takentogether, these results suggest that the greater inhibitory effect of SNP onCYP4A3 may be due to greater binding affinity of NO for the heme moiety ofCYP4A3.$ s. m( l9 q3 a1 U
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Fig. 2. A : effect of sodium nitroprusside (SNP; 1 mM) on the visible lightspectrum of recombinant CYP4A1 and CYP4A3 membranes. Absorption spectra wereobtained after incubation of recombinant CYP4A1 or CYP4A3 membranes with SNPfor 20 min at 37°C. B : effect of SNP on arachidonic acid -hydroxylase activity in baculovirus-expressed CYP4A1 and CYP4A3.Control values for CYP4A1 and CYP4A3 -hydroxylation were 3 ±0.08 and 0.8 ± 0.02 nmol 20-hydroxyeicosatetraenoic acid (20-HETE)· min - 1 · nmol P -450 - 1. Results are means ± SE; n = 3; * P
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Effect of PN on recombinant CYP4A proteins. Incubation of PN (0.01-1 mM) with recombinant CYP4A1 and CYP4A3 membranes caused aconcentration-dependent inhibition of both CYP4A1- and CYP4A3-catalyzed20-HETE synthesis. PN had a greater inhibitory effect on CYP4A3-catalyzedactivity than CYP4A1 ( Fig.3 A ). To examine whether PN can modify tyrosine residuesof CYP4A isoforms, soluble preparations of recombinant CYP4A1 and CYP4A3 wereincubated with 1 mM PN at room temperature for 30 min. CYP4A proteins werethen isolated from the reaction mixtures by immunoprecipitation withCYP4A-specific antibody, and the nitration of tyrosine residues of CYP4Aproteins was determined by Western blot analysis with anti-3-nitrotyrosineantibody. As shown in Fig.3 B, a strong 3-nitrotyrosine-immunoreactive band wasobserved when CYP4A proteins were incubated with PN. These results suggestthat the nitration of tyrosine residues of CYP4A proteins by PN may contributeto PN inhibitory action on CYP4A-catalyzed 20-HETE formation.. `' B0 d5 Q: m
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Fig. 3. A : effect of peroxynitrite (PN; 0.01-1 mM) on arachidonicacid -hydroxylase activity of Sf9 recombinant CYP4A1 and CYP4A3membranes. Control values for CYP4A1 and CYP4A3 -hydroxylation activitywere 4 ± 0.1 and 0.7 ± 0.03 nmol 20-HETE ·min - 1 · nmol P -450 - 1. Results are means ± SE; n = 3; * P

Effect of SNP and PN on renal microvessel 20-HETE synthesis. Toexamine whether NO and PN have a similar effect on 20-HETE synthesis in renalmicrovessels isolated from female rats, homogenates were preincubated with SNP(1 mM), PN (1 mM), or vehicle control at room temperature for 30 min followedby incubation with AA and NADPH. 20-HETE synthesis was determined byNCI-GC/MS. As shown in Fig. 4,SNP and PN caused 59 and 65% inhibition of renal microvessel 20-HETEsynthesis, suggesting that NO and PN act as negative regulators of 20-HETEsynthesis in renal microvessels.
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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 wasdetermined by negative chemical ionization-gas chromatography/massspectrometry. Results are means ± SE; n = 4; * P
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Effect of NO synthase and NO synthase/CYP4A inhibition on systolic blood pressure, urinaryNO 2 /NO 3 excretion,and renal microvessel 20-HETE synthesis. L -NAME (0.25 mg/ml indrinking water), L -NAME (0.25 mg/ml in drinking water) plus ABT (25mg/kg ip), or vehicle control was administered for 6 days to pregnant ratsbeginning 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. UrinaryNO 2 /NO 3 excretion, an index for whole body production ofNO in pregnant rats and women( 5, 6 ), decreased by 40% ( P

Table 1. SBP urinary NO 2 /NO 3, excretion, and renalmicrovessel 20-HETE synthesis with L -NAME, L -NAME plusABT, or vehicle control treatment during pregnancy' D/ i, C" }. R) p

DISCUSSION; w$ c; g. b4 ]: E/ }$ `
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In a previous study, we demonstrated temporal changes in renal vascularCYP4A expression and 20-HETE synthesis during pregnancy in rats. CYP4A proteinlevels and the product of their catalytic activity on AA, 20-HETE, wereincreased during the first and second week of pregnancy but returned tocontrol levels during the third week of pregnancy. The exact mechanismsresponsible for these changes are unknown. We hypothesized that NO may constitute one of the factors regulating the production of 20-HETE in therenal microcirculation.2 Q3 L, K2 }. f& m+ C) m
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During pregnancy in rats, urinary excretion and plasma levels of nitrateare elevated and urinary excretion of cGMP is also increased. Chronictreatment with L -NAME reduces urinary nitrate excretion in pregnantrats ( 5 ). Moreover, the bindingof NO to hemoglobin is only detected in the blood of pregnant, but notnonpregnant, rats ( 5 ).Augmentation of NO production and increased expression levels of renalneuronal NO synthase (NOS) and inducible NOS have been observed during normalpregnancy ( 1 ), and chronicinhibition of NO synthesis by L -NAME during pregnancy resulted inpreeclampsia-like symptoms( 26, 41 ). These reports providedsubstantial evidence to implicate NO as a major contributor to theimplementation of a vasodilatory state in pregnancy( 18, 35 ). On the basis of thisinformation 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 thedecreased renal vascular 20-HETE synthesis during the third week of pregnancyand may contribute to the regulation of vascular tone and blood pressureduring pregnancy. In the present study, we showed that NO binds to the hememoiety of CYP4A1 and CYP4A3, the major isoforms expressed in female rats, andinhibits their catalytic activity. We also demonstrated that PN, which showselevated production of NO and superoxide ( 7, 22 ), causes nitrosylation oftyrosine residues on CYP4A1 and CYP4A3.

The CYP4A enzymes (CYP4A1, 4A2, 4A3, and 4A8) are considered to be themajor AA -hydroxylases in the rat kidney and thereby the primarycontributors of 20-HETE synthesis. With the use of quantitative RT/PCR andWestern blot analysis, we demonstrated that CYP4A1 and CYP4A3 are the majorCYP4A expressed in the kidneys of female rats. The expression levels of CYP4A2measured by quantitative RT/PCR were 18-fold lower in female rats compared with male rats. These results are in accordance with other reports documentinglower and even undetectable expression levels of CYP4A2 in the kidneys offemale rats ( 37 ) as well asreports demonstrating androgen-dependent expression of CYP4A2( 11 ). As for the CYP4A8expression, a recent study by Holla et al. ( 10 ) suggested that CYP4a12 (amurine homologue gene to CYP4A8) is a male-specific and androgen-regulatedenzyme and has very low expression in female kidneys. Nakagawa et al.( 27 ) showed that CYP4A8expression in the rat is androgen sensitive. CYP4A8 protein has a similarelectrophoretic mobility as CYP4A2( 28 ); the absence ofCYP4A2-immunoreactive protein in renal microsomes from female rats( Fig. 1 ) suggests lowexpression levels of CYP4A8. On the basis of these reports, our data, andprevious studies showing that the recombinant CYP4A1 and CYP4A3 proteins catalyze AA -hydroxylation to 20-HETE, it is likely that these isoformscontribute significantly to renal 20-HETE synthesis in female rats. However,we cannot rule out the possibility that other isoforms of the CYP4 gene familysuch as CYP4F proteins, CYP4F1, CYP4F4, CYP4F5, and CYP4F6, may be involved inthe renal production of 20-HETE in female rats( 4, 16 ). Kalsotra et al.( 14 ) showed significant levelsof expression of all CYP4F isoforms in kidneys of female rats and furtherdocumented an estrogen-sensitive expression of CYP4F1, CYP4F4, and CYP4F6. Thecatalytic activity of CYP4F isoforms toward AA and their ability to catalyzethe production of 20-HETE has not been fully examined. Further studies thatallow evaluation of the distinct contribution of each of the CYP4A and 4Fisoforms to 20-HETE synthesis are needed., }6 [: T( m. [5 u5 V2 x8 {
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NO inhibits heme-containing proteins such as CYP( 25 ). The mechanisms of theinteraction between NO and CYP proteins were described by Minamiyama et al.( 25 ), who demonstrated that NOcan interact with CYP in two ways: NO binds reversibly with the heme moietyand irreversibly with cysteine residues of CYP proteins. These NO-CYP adductsare enzymatically inactive in vitro. Moreover, Roberts et al.( 32 ) demonstrated that PN canmodify 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 studythe interaction between NO and CYP4A isoforms in renal tissues because renaltissues contain numerous CYP enzymes other than CYP4A isoforms.Baculovirus-expressed CYP4A isoforms provide a unique tool to study theinteraction between NO and individual CYP4A isoforms in vitro because there isa negligible level of CYP content in Sf9 insect cells( 39 ). Our results indicatedthat NO binds to the heme moiety of CYP4A1 and CYP4A3 with differentaffinities. The heme moiety of CYP enzymes is essential for the oxidationreaction. NO binding to the heme moiety can interfere with the electrontransport mechanisms of the oxidation reaction. It is possible that the ability of NO to inhibit CYP4A3-catalyzed 20-HETE synthesis to a greaterextent than that of CYP4A1 is due to the higher binding affinity of NO toCYP4A3. PN, a powerful oxidant, is derived from NO and superoxide. Because CYPisoforms can generate varying amounts of oxygen-derived free radicals such assuperoxide ion during the catalytic cycle of CYP enzymes( 8 ), it is possible thatsuperoxide ion generated from the CYP4A-catalyzed reaction can interact withNO and cause the formation of PN. We showed that PN inhibits 20-HETE synthesiscatalyzed by CYP4A1 and CYP4A3. The mechanisms underlying this inhibition arenot clear; however, the ability of PN to nitrosylate tyrosine residues ofCYP4A1 and CYP4A3 may constitute, at least in part, a mechanism of inhibition.However, additional studies are needed to demonstrate that tyrosinenitrosylation of CYP4A proteins occurs in vivo and that nitrosylated CYP4Aproteins are catalytically inactive.
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In contrast to normal pregnancy, preeclampsia is characterized by increasedarterial blood pressure, generalized vasoconstriction, increased systemicresistance, widespread vascular endothelial damage, decreased fetal growth,and proteinuria ( 21 ). The exact mechanisms that mediate preeclampsia are still unknown. Several reportshave suggested that NO may play an important role in its development( 2, 22 ). Moreover, two reportsdemonstrated that chronic inhibition of NO synthesis in late pregnancy in ratsresulted in signs similar to those of preeclampsia( 26, 31 ). That the increased renalmicrovessel production of 20-HETE following administration of L -NAME during the third week of pregnancy together with reportsthat NO inhibits 20-HETE synthesis and interferes with 20-HETE vasoconstrictoractivity in vivo ( 31, 36 ) suggest the contributionof 20-HETE to the implementation of renal vasoconstriction and increased bloodpressure ( Table 1 ) underconditions where NO production is suppressed. This notion is furthersubstantiated by data showing that coadministration of ABT, an inhibitor ofCYP4A activity, abolished the L -NAME-induced increase in bloodpressure in these rats ( Table1 ).

In summary, this study provides the first evidence to show that NO bindsdifferently to the heme of CYP4A isoforms and inhibits 20-HETE synthesis byrecombinant CYP4A proteins and renal microvessels in female rats. This studyalso shows that PN modifies tyrosine residues of CYP4A proteins and inhibitstheir catalytic activity. Additional data show that augmentation of renalmicrovessel 20-HETE synthesis after NOS inhibition is associated withincreased blood pressure and that this increase is negated by treatment with aCYP4A inhibitor. Hence, this study offers evidence that NO acts as a buffersystem to counteract 20-HETE-mediated vasoconstriction mechanisms duringpregnancy.

DISCLOSURES+ I6 A/ O1 j, ~/ P- O! W
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This study was supported by a National Institutes of Health (NIH) GrantPO1-HL34300 (to A. Nasjletti and M. Laniado-Schwartzman) and by NIH GrantR01-HL-70887 to M.-H. Wang.* r. K- G4 u" X7 n0 Z
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