olizes arachidonic acid (AA) chiefly by a cytochrome. P450 monooxygenase ...... ducts have been reported to generate 5,6-epoxyeicosatrienoic acid and 11,12-.
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Val. 266, No. 19, Issue of July 5, PP. 12306-12312,1991 Printed in U.S.A .
(e) 1991 by The American Society for Biochemistry and Molecular Biology, Inc
Structural Identification of CytochromeP450-dependent Arachidonate Metabolites Formed by Rabbit Medullary Thick Ascending Limb Cells* (Received for publication, March 26, 1991)
Mairead A. Carroll$$, Angelo Salan, Catherine E. DunnS, John C. McGiffS, and RobertC. Murphyn From the $Department of Pharmacology, New York Medical College, Valhalla, New York 10595 and TDepartment of Pediatrics, National Jewish Centerfor Immunology and Respiratory Medicine, Denver, Colorado 80206
The medullary thick ascending limbof Henle’s loop Arachidonic acid (AA)’ can be metabolized via the cyto(mTALH) contributes importantly to the regulation of chrome P450 monooxygenase pathway to yield biologically extracellular fluid volumeand composition and metab- active metabolites. When cells of the medullary thick ascendolizes arachidonic acid (AA) chiefly by a cytochrome ing limb of Henle’s loop (mTALH), which are invested with P450 monooxygenase pathway. Rabbit mTALH cells, high cytochrome P450 monooxygenase activity andhave negwhen incubated with radiolabeled [14C]AA,form prod- ligible cyclooxygenase activity ( l ) , are incubated with radioucts that segregate into two peaks designatedP1 and labeled [1-14C]AA,they form two principal peaks that have Pn based on their reverse-phase high pressure liquid been designatedP1/PI and P p / P I I based on their HPLC retenchromatography retention times. We have now defin- tiontimes (2, 3). P1 and P2 havecharacteristic biological itively identified their chemical structures. mTALH activities and can be differentiated by their principal actions cells, isolated from the rabbit outer medulla, were ho- on blood vessels relative to their effects on Na’-K+-ATPase mogenized and incubated with [14C]AAin the presence activity. Thus,theprimaryaction of P1 is relaxation of ofNADPH. The AA metabolites in P1 and Pz were isolated blood vessels, whereas Pp exerts a predominantly identified by gas chromatographic-mass spectrometricNa’-K+-ATPase inhibitory effect (3). These AA metabolites methods, including fast atom bombardment, negative may play a role in integrated renal function as their formation ion electron capture, and electron ionization.All mass can be altered by peptide hormones, such asvasopressin and spectrometric data, the lack of UV chromophores, and calcitonin (3),experimental andgenetic forms of hypertension comparisons with authentic standards were consistent (2,4), andmodification of dietary potassium(5).The mTALH with P1 containing two principal components: 19-hy- has one of the highest concentrations of Na+-K’-ATPase droxy-5,8,11,14 eicosatetraenoic acid (19-HETE)and found in mammalian tissue (6), reflecting the importance of 20 - hydroxy - 5,8,11,14 - eicosatetraenoic acid (20this nephron segment in the regulation of extracellular fluid HETE), Pzcontained primarily 1,20-eicosa-5,8,11,14- volume. It is, therefore, crucial to further our understanding tetraenedioic acid (20-COOH-AA). The biological of the regulation of the renal circulation and extracellular properties of P1 and Pz were compared with thoseof fluid volume to identify structurally theAA metabolites aristhe authentic standards of 19- and 20-HETE and 20- ing from the mTALH. This will enable us to study the full COOH-AA. P1dose dependently relaxed precontracted range of the biological activities of these novel AA products mesenteric arterial rings, as did authentic (19s)and and to determine their contribution to normal renal function (19R)-HETE, whereasSO-HETE relaxed at lower and as well as to diseases of the kidney. contracted at higher concentrations. As P1contained a We now report on the definitive structural analyses of PI mixture of 19- and 20-HETE, eachof these AA metab- and Ppby mass spectrometry and have compared the biologolites presumably contributed to the vascular relaxa- ical activities of P1 and P1 with their principal constituent tion producedby P1. Neither Pz nor 20-COOH-AA AA metabolites, 19- and 20-hydroxyeicosatetraenoic acids exhibited vasoactivity, but each demonstrated a simi- (19- and20-HETE)and 1,20-eicosatetraenedioicacid(20lar potency in inhibiting rabbit medullary Na+-K+COOH-AA), respectively. ATPaseactivity. As previouslyreported, Pz was a more potent inhibitor of Na+-K+-ATPase thanP1. The EXPERIMENTALPROCEDURES lesser inhibitory activityof PI presumably reflects the Materials-Cell isolation mediawere obtained fromGIBCO; bovine presence of similar amounts of 19-HETE, the least serumalbuminfrom Sigma; AA from NuChekPreps(Elysian, active metabolite, and 20-HETE, which resembles 20- MN), and [l-”C]AA (56 mCi/mmol) from Amersham Corp. COOH-AA in its capacity to inhibit Na+-K+-ATPase. [5,6,8,9,11,12,14,15-zH8]AAwas a gift fromDr.HowardSprecher, Thus, thebiological activity of the less polar peak, PI, Ohio StateUniversity. Synthetic 19- and 20-HETEwere synthesized can be accounted for by 19- and 20-HETE, and that of by Dr. J. R. Falck, University of Texas, Southwest Medical Center. 20-COOH-AA was biosynthesized from 20-HETE (1 N M ) incubated P z , by 20-COOH-AA. with alcohol dehydrogenase (1p ~Sigma) , and ratliver mitochondria (10,000 X g pellet) as a source of aldehyde dehydrogenase for 1 h at
* This research was supported in part by National Institutes of HealthGrants GM41026, RR01152, HL34300, and HL25394 and American Heart Association Grant 90-075G. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked“advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact. To whom correspondence should be addressed.
The abbreviations used are: AA, arachidonic acid; mTALH, medullary thick ascending limb of Henle’s loop; 19-HETE, 19-hydroxy5,8,11,14-eicosatetraenoicacid; 20-HETE, 20-hydroxy-5,8,11,14-eicosatetraenoic acid; 20-COOH-AA, 1,20-eicosa-5,8,11,14-tetraenedioic acid; RP-HPLC, reverse-phase high pressure liquid chromatography; FAB, fast atom bombardment; GC/MS, gas chromatography/ mass spectrometry; PFB, pentafluorobenzyl; EI, electron ionization; ECL, equivalent chain length.
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Metabolism Arachidonate Renomedullary 22 "C in phosphate buffer with M NAD. The 20-COOH-AA was extracted and purified by RP-HPLC (System A). Bis(trimethylsily1)trifluoroacetamide was obtained from Supelco. Diazald, which was used to prepare an ethereal solutionof diazomethane, 5% rhodium on alumina, pentafluorobenzyl bromide, and N,N-diisopropylethylamine were obtained from Aldrich. All solvents used were HPLC grade. mTALH Cell Isolation-The isolation, separation, and identification of rabbit mTALH cells have been described (1, 3). A total of 30 male New Zealand White rabbits (2.5-3.0 kg) were used in this study. The rabbits (two per day) were anesthetized with xylazine, ketamine hydrochloride, and acepromazine maleate (13:60:0.06 mg/kg) given intramuscularly. Kidneys were perfused in situ through the renal artery with 0.9% saline to remove blood elements. The inner stripe of the outermedulla was excised, cut into small pieces, and trypsinized for 20 min a t room temperature in trypsin-EDTA plus 0.1% bovine serum albumin. The pieces were mechanically disrupted in RPMI 1640 culture media plus excess bovine serum albumin (1.0%) to stop trypsinization. After filtration througha 30 p~ nylon mesh, thesingle cell suspension of outer medullary cells was separated into several fractions by centrifugal elutriation (1) a t 2,000 rpm and 20 "C. Two fractions were collected; outer medullary cells, depleted of mTALH cells, were eluted a t a flow rate of 9.5 ml/min, andcells derivedmainly from mTALH were eluted at a flow rate of 19 ml/min. Greater than 90% of the cells were shown to be viable as determined by trypan blue exclusion, electron microscopic appearance, andmetabolic activity. Various histochemical, biochemical, and morphological criteria have previously shown the mTALHcell fraction to consistof approximately 80-85% mTALH cells, the remainingcells being derived from proximaltubulesand collecting ducts (1). Identification of the mTALH cell fraction was based on the following criteria: positive staining for Tamm-Horsfall protein, a specific marker for mTALH cells, enriched Na'-K'-ATPase activity, and low alkaline phosphatase activity, a marker for proximal tubule cells. A total of 6.75 X loH mTALH cells were isolated in this study from 30 male New Zealand White rabbits. Incubation Conditions-Following determination of mTALH cell yield (approximately 2 X 10'/two kidneys), a broken cell preparation was prepared by freeze thawing in distilled water. mTALH cell (3 X 10"; 100 pg of protein) homogenates were incubated with AA in the presence of NADPH (1 mM) and indomethacin (10 y ~ in) 1 ml of phosphate-buffered saline for 30 min at 37 "C in a shaking water bath. In order to identify radioactive AA metabolites, three experimental protocols were used: addition to mTALH homogenatesof (A) radiolabeled ["CIAA (7 PM);(B) a combination of ["CIAA (10.5 p ~ containing deuterated AA (6.7 p ~ and ) unlabeled AA (6.7 p ~ ) (C) ; ["CIAA (3.5 p M ) and unlabeled AA (20 yM). Followinga30-min incubation, the reaction was stopped by addition of 2 volumesof ethyl acetateand acidification to pH 4.0 using 8.8% formic acid. The organic phase was evaporated and the extract resuspended in methano1:water (1:l)and sealed under argon. As a quality control procedure, an aliquot of sample (1/25) obtained from each protocol was separated by RP-HPLC (System A; see below) prior to processing for GC-MS analysis. To confirm the identity of PI and Pp in terms of theirchromatographicelutiontimesand biological activities, AA metabolites were prepared using protocol C. This sample, prepared from 39 X 10' mTALH cells, was divided equally. The sample for bioassay was separated on RP-HPLC, the fractions collected (0.5 ml), and an aliquot (1/20) taken for scintillation counting.The amountof PI and P, was calculated based ona specific activity of 1yg = 70,000 cpm. The remaining sample was separated by RP-HPLC into PI and P, and then injected with authentic standards to compare elution times. HPLC Separation of Radioactive AA Metabolites-Samples were resuspended in acetonitrile and separated aon yBondapak CIR column using a linear solvent gradient of 1.25%/min ranging from water:acetonitrile (1:1, containing 0.1% acetic acid) to 100% acetonitrile (containingO.l% acetic acid) at a flow rate of 1ml/min (System A). The elution profile of the AA metabolites was monitored by radioactivity using the on-line radioactive detector (Radiomatic Instruments & Chemical Co., Inc., Tampa, FL), and the radioactivity was counted using a liquid scintillation counter. Identification of mTALH-AA Metabolites by Mass SpectrometyThe HPLC-purified PI and P, metabolites were taken to dryness under reduced pressure, redissolved in 1 ml of 30% methanol/water and rechromatographed on a second RP-HPLC. The elution system used (System B) was a linear gradient from methanol/water (0.02% acetic acid, adjusted to pH with NH,OH) to 100% methanol over 30
12307 min a t a flow rate of 1ml/min. Fractions were collected every minute, and an aliquot (1%)was taken for radioactivity determination. Separate fractions of PI and P, were then taken to dryness and resuspended in 80% methanol containing 2% glycerol for fast atom bombardment (FAB) mass spectrometricanalysis. Continuous flow FAB was performed on a VG-7070E mass spectrometer using methanol/ water/glycerol (40:lO:l) as themobile phase with a flow rate of 10 pl/ min. Samples were dissolved in thismobile phase and desorbed from the probe tip with a beam of xenon atoms from a saddle field atom gun operated at 2 mA, 7 kV acceleration potential. The mass spectrometer was scanned for negative ions between 50 and 600 daltons. Samples analyzedby GC/MS were converted to either methyl ester trimethylsilyl ethers or pentafluorobenzyl ester trimethylsilyl ethers prior to analysis (see below). Furthermore, a portion of each sample was reduced to determine the number of double bonds in PI and P, by catalytic hydrogenationof the samples as previously described (7). Briefly, the samples were dissolved in 0.5 ml of 50% methanol/H20, and 5-10 mg of 5% rhodium on alumina was added and hydrogen bubbled into the suspension for 20 min. The suspension was then treatedwith 10-20 p1 of 1 N NaOHandthen centrifuged. The supernate was acidified to pH 4.0 with concentrated HC1 and extracted with CH,Cl,. The organic layer was taken to dryness and derivatized for gas chromatographic separation. Pentafluorobenzyl (PFB) esters were madebylyophilization of each HPLC fraction to dryness followed by treatment with50 yl of a 10% solution of pentafluorobenzyl bromide in acetonitrile and 50 pl of a 10% solution of N,N-diisopropylethylamine in acetonitrile.After evaporation of these reactants, free hydroxyl groups were trimethylsilylated using bis(trimethylsily1)trifluoroacetamide(50 pl), with 50 yl of acetonitrile a t 60 "C for 30 min. Negative ion electron capture mass spectrometry was performed on a Nermag RlOlO mass spectrometer(Delsi-Nermag,Paris)operatedin negativeionelectron capture mode using methane as moderating gas at 0.5-Torr source pressure. Samples analyzed by electron ionization (EI) mass spectrometry were first methylated with ethereal diazomethanefor 5 min in methanol,takentodryness,andthenreactedwith bis(trimethy1sily1)trifluoroacetamide in acetonitrile as described above. EI-GC/ MS was carried out on a Hewlett-Packard 5970 mass spectrometer using an ionizing electron beam seta t 70 eV. Gas chromatographywas carried outusing aDB-1 (J&WScientific, Rancho Cordova, CA) capillary column (10 X 0.25-mm inner diameter, 0.2-ym film thickness) directly interfaced to eachmass spectrometer. The column was linearly programmed from 100 to 300 "C at ) 15 "C/min, and helium was employed as carrier gas. Identical gas chromatography conditions were employed for the pentafluorobenzyl ester trimethylsilyl ethersas for the methyl ester trimethylsilyl ether derivatives. Fatty acid equivalent chain length was determined using a mixture of either fatty acid PFB esters or fattyacid methyl esters between 19 and 25 carbons. The retention time for each of the fatty acid derivatives was plotted uersus the fatty acyl carbon length anda linear regression used to calculate fatty acid equivalent chain length for eluting P1 and Pz derivatives. Synthetic standards of 20-HETE and 20-COOH-AA were analyzed using identical mass spectrometric conditions as described above for PI and P,. Bioassay of mTALH-AA Metabolites-The determination of biological activity (vasoactivity andeffects on Na+-K+-ATPase activity) was performed in a blind fashion, and absolute amounts (ng/ml) of endogenous products and authentic standards were used. The AA metabolites were tested on precontracted rabbit mesenteric artery rings. The mesenteric artery was carefully excised, cleaned of connective tissue, and cut into 2-4-mm rings. The rings were bathed in Krebs-Henseleit solution and oxygenated with 95% 0, and 5% CO, a t 37 "C a t a tension of 1.5 g for 90 min prior to administration of samples. The tension was increased to 3-4 g by precontracting the arterial rings withphenylephrine M ) . Changesintension were monitored isometrically. The functional integrity of the endothelium was assessed by a relaxation response to acetylcholine (5 X lo-' M ) . The biological activity of P, and Pp(50 ng to 1 yg/ml) was compared with that of 20-HETE, 19-HETE ( S and R stereoisomers), and 20COOH-AA. The results were calculated as percent change of the phenylephrine-induced constriction. The effect ofAA metabolitesonNa+-K'-ATPaseactivity was determined using Na'-K'-ATPase purified from rabbit renal outer medulla based on the method of Jorgensen (6). Briefly, the inner stripe of the outer medulla obtained from four rabbit kidneys was homogenized (10 ml/g of tissue) in an ice-cold solution of 0.25 M sucrose and 30 mM histidine.Afterseveral centrifugation steps, a
12308
Metabolism Arachidonate Renomedullary
microsomal pellet was obtained following centrifugation a t 48,000 X g for 30 min. Na+-K'-ATPase was purified by incubating the micro-
somal fraction for 45 min in a solution containing 0.5 mg/ml sodium dodecyl sulfate, 2 mM EDTA, 50 mM imidazole, and 3 mM ATP at 20 "C. The sample was purified by a discontinuous density gradient consisting of sucrose (29.4, 15, and 10%) in 25 mM histidine and 1 mM EDTA (pH 7.5), followed by centrifugation a t 60,000 rpm for 90 min. The pellet was resuspended in 2 ml of 25 mM imidazole and 1 mM EDTA (pH7.5), aliquoted, and stored -70 at "C. This preparation was inhibited with ouabain (lo-" M) by 90 & 7%. The assay media contained 1.0-2.0 pg of protein, 2.0 mM MgC12, 60.0 mM NaC1, 5.0 mM KCl, 0.1 mM EDTA, 92.0 mM Tris-HC1 buffer, 1.0 mM ATP. Na+-K+-ATPaseactivity was assessed in the presence and absence of ouabain (lo-'' M) and the effects of 50-500 ng/ml PI, P,, PO-HETE, 19-HETE ( S and R stereoisomers), and 20-COOH-AA were tested. Each tube was brought to a final volume of 0.5 ml with histidine (30 mM). Incubations were performed for 15 mina t 37 "C. The inorganic phosphate liberated was assayed by the method of Lanzetta et al. (8). The enzymatic reaction was stopped by addition of 800 pl of a solution containing ammonium molybdate (4.2%), malachite green (0.045%), and sterox (Perkin-Elmer Cetus Instruments). After mixing, 100 p1 of 34% citrate solution was added, and the final solution was then read a t 660 nm in a Perkin-Elmer spectrophotometer. Results are expressed as percent inhibitionof total Na+-K+-ATPaseactivity. Statistics-Comparisons among groups were made by analysis of variance followed by a paired t test. Data are expressed as means & S.E., and p c0.05 was considered significant.
mophores inherent in the structures of these arachidonate metabolites. The two active components were separately collected and repurified on a second RP-HPLC system prior to mass spectrometric analysis. Structural Characterization of PI
Negative ion FAB mass spectrometry of PI (Fig. 2A) revealed only one major ion at m / z 319 aside from the glycerol matrix ions typically observed when glycerol is used as the FAB matrix. Since negative ion FAB mass spectrometry of eicosanoids yields abundant carboxylate anions ( 9 ) ,this material likely had a molecular mass of 320 atomic mass units. Considering the molecular mass ofAA (304 atomicmass units), metabolite PI was consistent with incorporation of a single atom of oxygen into theAA structure. Gaschromatography-massspectrometricanalysis of the trimethylsilyl ether PFB ester using electron capturechemical ionization conditions revealed two separable components in this HPLC fractionof approximately equal abundant ionsat m/z 391 (Fig. 3, inset).The massof this ion would correspond to the carboxylate anion typically observed for PFB esters and a trimethylsilyl esterof a monohydroxy arachidonic acid. The earliest eluting peak ((Pia, 21.7 equivalent chain length (ECL)) had a negative ion mass spectrum (Fig. 3 A ) similar, RESULTS but not identical with thenegative ion mass spectrum of the secondisobaric component (Plb, 22.3 ECL) (Fig. 3 B ) , the Production of PI and P2 by m T A L H Homogenates latter of which had a slightly less abundant ion at m / z 301 Incubation of radiolabeled AA with rabbit mTALH cell corresponding t o loss of trimethylsilanol (M - 90). The abunhomogenates in the presence of NADPH (1 mM) and indo- dance of ions m / z 393 and 303 relative to m / z 391 and 301, methacin (10 p ~ resulted ) in the formationof several metab- respectively, was approximately 25%, indicating thepresence olites more polar than AA (Fig. 1). As described in previous of I4C isotope in these metabolites. This isotopic abundance , AA metabolites, is substantially higher than expected for the natural abuninvestigations with this system ( 2 , 3 ) those designated PI (17.5 minretentiontime)and Pp (16 min dance of carbon, hydrogen, oxygen, and silicon atoms (theoretention time), had characteristic biological activities that retical 7.0%). The mass spectrum of synthetic 20-HETE as differentiated them; uiz. PI was more potent than Pp in its the PFB ester, trimethylsilyl ether using electroncapture ability to relax precontracted isolated blood vessels, whereas negativeion massspectrometry is shownin Fig. 3C(22.3 P, was a more potent inhibitor of Na'-K'-ATPase activity ECL). than P1.Those AA products thatsegregated on RP-HPLC as Catalytic reduction of PI (as a mixture of Pla and Plb) P1and Pzwere the major radioactive components observed in followed by derivatization as above resultedin two major this incubation system. Several other minor AA metabolites could be separated but with slightly different RP-HPLC characteristics. The effluent of the HPLC shown in Fig. 1 was also monitored for elution of UV-absorbing material with a photodiode array detector. None of the radioactive metabolites and, in particular, neither PI nor Pz absorbed UV light above 210 nm, suggesting the lack of any conjugated chro150
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FIG. 1. Reverse-phase HPLC separation of mTALH radioactive AA metabolites. Separation was performed following incubation of AA with rabbit mTALH cell homogenates in the presence of NADPH (1mM) and indomethacin(10 FM). The major metabolites of AA (20:4) indicated as P, and P.) were collected from the HPLC effluent for subsequent analysis.
FIG. 2. FAB mass spectrometry of P I and Pz.A , negative ions were obtained by FABmassspectrometry of PI (Fig. 1) obtained under continuousflow conditions. A backgroundspectrum taken prior to the analysis of fraction PI was subtracted to remove those ions arising from the glycerol matrix. The abundant ion a t 319 is designated asa molecular anion (M - H), typically observed for carboxylic acids analyzedby FAB mass spectrometry. Thissuggests that PI has a molecular weight of 320. B, negative ions were obtained by FAB mass spectrometrv of P2 as described above. The abundant ion at m/z ( M - H) suggests a molecular weight of 334 for PB.
Metabolism Arachidonate Renomedullary
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trimethylsilyl ether methyl ester yielded a single compound (23.4 ECL) with a mass spectrum identical (Fig. 4C) with that obtained from metabolite Plb. Finally, co-injection of 20 nCi of P1 (as a mixture of Pla and Plb)with 3 pg of synthetic 20-HETE resulted in perfect coelution of the biological metabolite (PI radioactivity) with the synthetic material (monitored at 205 nm). All mass spectrometric, spectroscopic, and chromatographic data are consistentwith the structureof 20HETE for metabolite Plb, assuming that the position and geometry of the double bonds were unaltered. Thus,the mTALH cell homogenate produced both 19-HETE and 20HETE as the HPLC peak identified as PI. Structural Characterization of P,
Analysis of Pp by direct FAB mass spectrometry using a glycerol matrix resulted in the production of an abundantion at m/z 333 that corresponded to anadditional 28 atomic mass units to the arachidonate structure for this metabolite (Fig. 2B). Derivatization of metabolite P p as the PFB ester trimethylsilyl ether was somewhat more difficult than that experienced for metabolite P1.Yet a negative ion electron cap30 1 319 ture mass spectrum (Fig. 5A,26.3 ECL) was obtained that 0 . . . . , . . . , . . . . . . . . . , . ! . , . . . . , . . . . ~ . . _ . , revealed essentially a single ion at m/z 513 (M-PFB). There 100 150 250 200 300 350 400 450 500 was no obvious ion corresponding to a loss of trimethylsilanol. M/Z Catalytic hydrogenation of Pz resulted in a mass spectrum of FIG. 3. Negative chemical ionization mass spectra of PI. the PFB ester with a single abundant ion at m/z 521 (data Inset, capillary GC-MS analysis of P, using negative ion chemical ionization conditions following derivatization of P1 as the pentafluo- not shown) that would correspond to thepresence of 4-olefin robenzyl ( P F B ) ester trimethylsilyl ether (2"s).Two components bonds in P,. The difference in mass between the FAB mass eluted from the gas chromatographic column with abundant ions at spectrometry ( m / z 333) and thenegative ion electron capture m/z 391 as illustrated a t 10.39 and 10.64 min. A, mass spectrum of mass spectrometry for the PFB ester derivative of P, (m/z the earliest eluting component in the GC-MS analysis of PI. This 513) corresponded to 180 atomic mass units or the replacecomponent is designated Pla. B , mass spectrum of the latest eluting ment of one additional carboxylic acid proton with CH2CsFs. component is designated as P,b. C, mass spectrum of synthetic 20hydroxy-5,8,11,14-eicosatetraenoic acid as the pentafluorobenzyl es- This supported the hypothesis of two carboxylic acid moieties ter trimethylsilyl ether using identical GC-MS conditions for the in P, corresponding to oxidation of the C-20 methyl to COOH, analysis of Pla and P,b. The retention time of this synthetic com- an addition of 28 atomic mass units to that of AA. Electron pound was 10.64 min, identical withthat shown for Plb (panel B ) . ionization mass spectrometry (Fig. 5B) of the methyl ester of catalytically reduced P2 (22.4 ECL) had the most abundant components (22.5 and 23.0 ECL, respectively). Both compo- ion at m/z 98, typical for long chain dicarboxylic acid methyl nents had similar but not identical mass spectra and each esters (11).The ion observed at m/z 339 (M-OCH,) suggested shifted 8 atomic mass units higher than the unreduced me- a molecular mass of 370 daltons for the saturated dimethyl tabolites, confirming the presence of 4-olefin bonds remaining ester, which corresponded to 342 daltons for reduced Ps and in the structure. The major difference in the mass spectral 334 daltons for native Pz. A sample of 20-COOH-AAwas behavior for these two compounds was related to the ion prepared from synthetic 20-HETE by incubating with alcohol corresponding to theloss of trimethylsilanol from the carbox- dehydrogenase, rat liver mitochondria, and NAD+ in buffer. The semisynthetic 20-COOH-AA was found to be poorly ylate anion (M - 181-90). Gas chromatography-massspectrometricanalysis of the soluble inacetonitrile andthus slowly converted tothe reduced metabolite, derivatized as the trimethylsilyl ether bis(pentafluorobenzy1) ester. This derivative had an identical methyl ester, using E1 likewise revealed two major compo- mass spectrum and gas chromatography retention time (data nents with quite different mass spectrometric behavior. The not shown) to that of derivatized P,. The E1 mass spectrum first component (Fig. 4A, 22.8 ECL) had the most abundant of the dimethyl esterof reduced, semisynthetic 20-COOH-AA ion at m/z 117 (CH3(Me3SiO)CH+)and significant high mass shown in Fig. 5C is identical with that of reduced methylated 370 (M-CH,CHO), P2 (Fig. 5B). Finally, co-injection of 10 nCi of P, with 2.5 pg ions at m/z 367 (M-CH,-CH,OH), m/z and m/z 399 (M-CH,). The appearance of the ion at m/z 370 of 20-COOH-AA on RP-HPLC resulted in coelution of radioactive labeled and unlabeled product (measured at 205 nm). was consistent with the previously reported rearrangement process observed for trimethylsilyl derivatives of saturated The mass spectrometric, spectroscopic, and chromatographic hydroxy fatty acid methyl esters (10). All mass spectrometric data,as well as comparison with semisynthetic material, data and the lack of a UV chromophore in metabolite Pla support structural characterization of Pz as 20-COOH-AA, were consistent with a structure of 19-HETE. The absolute assuming that the position and geometry of all double bonds configuration of the hydroxy group at (2-19 is unknown, and were unaltered. although no data are available, the geometry of the olefin Biological Characteristics of 2O-HETE, (19R)-and (19s)bonds is assumed to be cis, since no biochemical substitution HETE, and 20-COOH-AA Compared with Native P, and P2 reactions occurred near these olefin groups. The second component (Plb), analyzed as the reduced meVascular Actiuity-Rabbit mesenteric arterial rings, pretabolite by EI-GC/MS (Fig. 4B, 23.4 ECL), had abundant contractedwithphenylephrine M), relaxed dose dependions at m/z 103 (Me3SiO-CHt), 367 (M-CH30H), and 399 ently by 20-80% in response to the addition of 10-9-10"j M (M-CH,). Synthetic 20-HETEanalyzed by EI-GC/MS as the acetylcholine, indicating the presence of functional endothe-
12310
FIG. 4. Positive electron impact spectra of PI. A , mass spectrum of reduced Pla as a methyl ester trimethylsilyl ( M e , T M S ) ether derivative using electron ionization conditions (70eV) andGC-MS. B , massspectrum of reduced metabolite P,b following derivatization as a methyl ester trimethylsilyl ether derivative usingelectron ionization (70 eV) and GC-MS. C, mass spectrum of reduced synthetic 20-hydroxyeicosatetraenoic acid as a methyl ester trimethylsilyl ether derivativeusing electron ionization (70 eV).
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FIG. 5. Mass spectra of Pz. A , Negative ion mass spectrum of P, derivatized asthe pentafluorobenzyl ( P F B ) esterobtained using electron capture conditions during GC-MS analysis. B, positive ion mass spectrum of reduced P, as the methyl ester( M e ) using electron ionization(70eV)duringGC-MS analysis. C , positiveion mass spectrum of the methyl ester of semisynthetic 20-carboxyeicosanoic acid using electron ionization (70 eV).
lium. As shown in Fig. 6, addition of PI over a concentration range of 0.05-1.0 pg/ml resulted in relaxation of mesenteric arterial rings, achieving a relaxation of 67 10% a t 1 pg/ml (3.1 X M), the highest concentrationtested.Theauthentic standards of those AA metabolites identified as major components of PI had variable effects on mesenteric arterial rings. 20-HETE resulted in relaxation at low concentrations
*
and biphasicresponses at higher concentrations (Fig. 6A). 0.05 (1.6 X M ) and 0.1 pg/ml (3.1 X M ) 20-HETE caused relaxation of 4 f 1 and 15 f 4%, whereashigher M ) and 0.5 pg/ml (1.6 X amounts (0.2 pg/ml (6.3 X M)) produced relaxation of 18 f 5 and 4 f 2%, respectively, followed by contraction of vascular rings. 1.0 pg/ml (3.1 X M ) resulted in contraction only. Boththe S and R stereoisomers of 19-HETE relaxed mesenteric rings; the reM ) was 37 f 6% with (19R)laxation at 1 pg/ml(3.1 X HETE and 35 f 9% with (19s)-HETE (Fig. 6). Neither P2 nor 20-COOH-AA, 0.05 to 1pg/ml(l.3 X M to 2.6 X M), exhibited vascular activity. HPLC solvent control and vehicle control (100 pl of Krebs-Henseleit solution) relaxed mesenteric rings by less than 3%. Na+-K+-ATPaseActiuity-Both PI and P2 and 20-HETE and 20-COOH-AA dose dependently inhibited rabbit renal medulla Na+-K+-ATPase (Fig. 7). (19R)- and (19s)-HETE had minimal activity, inhibiting Na+-K+-ATPase by 12 f 4 and 7 f 4% a t 0.5 pg/ml (1.6 X M ) , the highest amount tested. Vehicle and solvent controls had nodetectable effects of Na+-K+-ATPaseactivity. Pzwas 10-fold morepotent than P1 ( p < 0.05), confirming our previous studies on therelative Na+-K+-ATPase inhibitory activityof mTALH cell AA metabolites (2, 3); 20-HETE and 20-COOH-AA also inhibited Na+-K+-ATPase activity, demonstrating a potency similar to that of P2.The estimated IC5ovalues of the ouabain-inhibitM for 20-HETE, ableNa+-K+-ATPase (90%) were 5.1 X >1.5 X M for PI, and 2.9 X and 2.1 X M for 20COOH-AA and Pz,respectively. In addition toP1 and Pp,seven minor peakswere separated by HPLC. These peaks could not be identified structurally because of insufficient material. The peaks lacked vasoactivity and were without effect on Na+-K+-ATPase activity. However, the amounts purified (50-100 ng) may have been insufficient for detection of biological activity in ourassays. DISCUSSION
The purpose of this study was to identify definitively the principal mTALH cell P450-AA metabolites and to compare
Renomedullary Arachidonate Metabolism
12311
the biological properties of native Pl and P2 with those of authentic AA metabolites identified as principal components of P1 and P2.The AA metabolites generatedby mTALH cells were separated by RP-HPLC and segregated primarily into two peaks (PI and P2). The principal components in each 0.2 0 5 I O W 0.I 0.1 0 5 peak were structurally characterized. Based on mass spectroPE PE 5.10% 2.0-HETE 5110”M PI metric analysisof derivatized compounds of these AA metabolites, anw-oxidation product of AA, 20-HETE, was found in 1 the less polar peak (Pl), as was an w-1 oxidation product of AA, 19-HETE. The more polarpeak (P2)contained predominantly 20-COOH-AA. There were lesser quantities of at least J 1 om02 I O P P three other AA metabolites that did notsegregate with those nl nq AA products comprising either Pl or P2(Fig. 1)and could not be identified by GC-MS because of insufficient quantities. Proximal convoluted tubules and collecting ducts have been reported to generate 5,6-epoxyeicosatrienoic acid and 11,12dihydroxyeicosatrienoic acid, respectively (12, 13).Therefore, these tubular segments, which represent less than 20% of cells collected by the isolationprocedure, presumably did not contribute to the principal AA products identified in P1 and P2 (1,3). However, the presence of larger numbers of proximal and collecting tubular cells in the mTALHcell fraction in the initial study may have accounted for the preliminary identification of 5,6-epoxyeicosatrienoic acid and 11,12-dihydrox.,_, yeicosatrienoic acidas significant components of mTALH cell -80 cytochrome P450-AA metabolism (3). IO” 10-6 10-5 It has been known for several years that cells within the CONCENTRATION (M) kidney contain the enzymatic systems capable of oxidizing FIG. 6. Vasoactivity of mTALH cell-AA metabolites. A , de- fatty acids, as well as prostaglandinsinto w- and (w-1)picts representative bioassay traces of the response of rabbit mesenhydroxylated products (14, 15). Renal cortical homogenates M), tericarterial rings, preconstricted with phenylephrine (PE; and microsomes supplemented with NADPH metabolize AA to the mTALH cell-AA metabolite, PI,and authenticstandards, (19s)-and (19R)-HETE and20-HETE. B, dose-response curves were to 19-HETE, 20-HETE,20-COOH-AA, and several other AA generated tothe mTALH cell-AA metabolite, PI,and authentic products. Formation of these products is mediated by cytostandards, 19- and 20-HETE. Percent relaxation of mesenteric arte- chromeP450-linkedrenal monooxygenase (15). The enzyrial rings was calculated as percent change from the phenylephrinematic capability to metabolize AA t o 20-COOH-AA was induced constriction. The results shown represent the means & S.E. higher in low speed supernates than inhigh speed particulate of five separate experiments (*, p < 0.05 for authentic standards, compared with PI). Neither P, nor 20-COOH-AAwas vasoactive fractions, suggesting the involvement of soluble enzymes that (results not shown). catalyze oxidation of the terminal methyl group toa carboxylic acid moiety. The metabolism of more complex eicosanoids, such as leukotrienes Bq and C4, also involves a cytochrome P450 monooxygenase, yielding 20-hydroxy metabolites (16). The participation of alcoholdehydrogenase and aldehyde dehydrogenase may mediate the furthermetabolism A of these w-hydroxyeicosanoids into 20-COOH metabolites 1WSWETE (17). These NAD-dependentenzymes may also be involved in the metabolism of 20-HETE to20-COOH-AA in the mTALH T cells. Studies based on rat liver microsomes have indicated that formationof w- and w-1-oxidation products, aswell as wP1 COOH metabolites of fatty acids, involves cytochrome P450 monooxygenasesinducibleby phenobarbital.For example, the formation of w-1-oxidized products of palmitic acid and formation of 16-COOH-palmitic acid were substantially enhanced in phenobarbital-treated rats (18) as compared with controls. I We compared the biological properties of endogenous AA products associated with PI and P2to those of the authentic 10” 10-6 10-5 10-7 10-6 10-5 standards of their major components: 20-HETE, 19-HETE, CONCENTRATION(M) CONCENTRATION(Y) and 20-COOH-AA. We defined the biological effects of these FIG. 7. Effect of mTALH cell-AA metabolites on rabbit re- authentic standards on vascular tone and Na+-K+-ATPase nomedullary Na+-K+-ATPase activity. Total Na+-K+-ATPase activity, which are biological properties that we have used to activity was determined from the maximum inhibition produced by distinguish P1 from Pz (2, 3).There was good agreement M), as described under “Experimental Procedures.” ouabain between the biological properties of the natural AA products Results depicted are the means k S.E. of five separate dose-response contained in each peak and those of authentic standards of curves. Poand 20-COOH-AAdid not differ in their Na’-K+-ATPase inhibitory activity ( A ) , whereas 19- and 20-HETE differed signifi- these AA metabolites. For example, the effect of 20-COOHcantly from PI (*, p < 0.05) in their capacity to inhibit Na+-K+- AA on vascular tone of preconstricted mesenteric arterial ATPase ( E ) . rings corresponded to that of P2 in that neither Ps nor the
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12312
Metabolism Arachidonate Renomedullary
endogenous AA products provided additional evidence for the standard affected vascular tone (2,3). On the other hand, the of the major AA products material identified in PI dose dependently relaxed mesenteric correctness of the structural analysis artery rings as did authentic (19s)- and (19R)-HETE (Fig. generated by rabbit mTALH. Pz and its major component, 6). Although (19s)- and (19R)-HETE have been reported to 20-COOH-AA, were devoid of vasoactivity and were the most increase the basal toneof rat aortic rings (19), in the present potent inhibitors of Na+-K+-ATPase.PI contained two prinstudy both (19s)- and (19R)-HETE exhibited the capacitycipal to components,each of which was vasoactive and together of 20-HETE and 19-HETE) relax precontracted mesenteric artery rings. Thus, the pres- (assuming similar concentrations ence of either theR or S isomer of 19-HETE inPI could have could account for the vasoactivity of PI and the lesser Na'contributed to the vasodilatory properties of P1. Addition of K+-ATPase inhibitory capacity when compared to P2. The 20-HETE resulted in relaxationat lower, and contraction at identification of these principal products of AA metabolism by mTALH is therequisite first step in defininga physiologhigher, concentrations. As PI contained a mixture of20HETE and 19-HETE, both of these AA metabolites could ical role for these AA metabolites (23). have contributed to the vascular relaxation produced by P1. REFERENCES Based upon the abundance of the carboxylate anions atrnlz 1. Ferreri, N. R., Schwartzman, M., Ibraham, N. G., Chander, P. 391 (Fig. 3, inset) following separation of the trimethylsilyl N., and McGiff, J. C. (1984) J. Phurmacol. Exp. Ther. 231, ether pentafluorobenzyl ester derivatives of 19-HETE and20441-448 HETE during the GC-MS analysis, these two metabolites 2. Carroll, M. A., Schwartzman, M., Baba, M., Miller, M. J. S., and achieved comparable concentrations in the fraction analyzed McGiff, J. C. (1988) Am. J. Physiol. 255, F151-F157 (Pl).Some caution should be exercised in the interpretation 3. Schwartzman, M., Ferreri, N. R., Carroll, M. A,, Songu-Mize, E., and McGiff, J. C. (1985) Nature 314, 620-622 of these results as there could havebeen loss of either of these 4. Sacerdoti, D., Abraham, N. G., McGiff, J. C., and Schwartzman, two components in the HPLC purification step(s) that would M. L. (1988) Biochem. Pharmacol. 37,521-527 alterthe relative abundance of these two componentsin 5. Carroll, M. A., Louzan, M., and McGiff, J. C. (1990) Am. J. subsequent fractions. P h y ~ i ~258, l . F1084-F1087 6. Jorgensen, P. L. (1974) Methods Enzymol. 32, 277-290 The differential potency of the authentic AA standards 7. Balazy,M., and Murphy, R. C. (1986) Anal. Chem. 58, 1098relative to their capacity to inhibit renal Na'-K+-ATPase 1101 activity is in accord with our previous reports that Pz is a 8. Lanzetta, P. L., Alverez, L. J., Reinach, P. S., and Candia, 0. A. more potent inhibitor that P1 (2, 3). 20-COOH-AA, the prin(1979) Anal. Biochem. 100, 95-97 cipal component of Pz, dose dependently inhibited Na+-K+- 9. Zirrolli, J . A,, Davoli, E., Bettazzoli, L., Adams, J., Gross, M. L., and Murphy, R. C. (1991) J. Am. SOC. Mass Spectrom. 1,325ATPase activity. Thus, P2 and 20-COOH-AA showed corre333 spondence of biological activity in termsof inhibition of Na+K+-ATPase activity andlack of vasoactivity. This similarity 10. VanRollins, M., and Murphy, R. C. (1984) J . Lipid Res. 25,507517 of biological activity for Pz and 20-COOH-AA has been con- 11. Ryhage, R., and Stenhagen, E. (1959) Ark. Kemi 14, 497 firmed using a morefunctional indicatorof transport activity, 12. Douglas, J. C., Romero, M., and Hopfer, U. (1990) Kidney Int. 38, S43-S47 i.e. oxygen consumption, uiz. Pzand 20-COOH-AA were equipotent in inhibiting oxygen consumption of cultured mTALH 13. Hirt,D. L., Capdevila, J., Falck, J. R.,Breyer, M. D., and Jacobson, H. R. (1989) J . Clin. Znuest. 84, 1805-1812 cells (20). 14. Navarro, J., Piccolo, D. E., and Kupfer, D. (1978) Arch. Biochem. The lesser Na+-K+-ATPase inhibitory capacity of PI when Biophys. 191, 125-133 compared with P2presumably reflects the presenceof similar 15. Oliw, E. H., Lawson, J. A., Brash, A. R., and Oates, J. A. (1981) amounts of 19-HETE and 20-HETE in this peak as the 19J. Bid. Chem. 256,9924-9931 HETEs had neglible ability to inhibit Na+-K+-ATPase activ- 16. Soberman, R. J., Sutyak, J. P., Okita, R. T., Wendelborn, D. F., Roberts, L. J., 11, and Austen, K. F.(1988) J . Biol. Chem. 263, ity. (19s)-HETE has also been reported to stimulate Na'7996-8002 K+-ATPase activity (21). However, in that study, Na+-K'17. Baumert, T., Huber, M., Mayer, D., and Keppler, D. (1989) Eur. ATPase was purified fromratrenal corticalmicrosomes, J. Biochem. 182, 223-229 whereas in the present study rabbit medullary microsomes 18. Bjorkhem, I., and Danielsson, H. (1970) Eur. J . Biochem. 17, 450-459 were used as thesource for Na+-K+-ATPase. Thus, the presW. C., Falck, J. R., Yadagiri, P.,and ence of either (19s)- or (19R)-HETE in PI would offset the 19. Escalante,B.,Sessa, Schwartzman, M. L. (1989) J. Pharmacol. Exp. Ther. 248, Na+-K+-ATPase inhibitory capacity of 20-HETE. Additional 229-232 evidence for the functionalsignificance of the capacityof 20- 20. Escalante, B., Erlij, D., Falck, J. R., andMcGiff, J. C. (1990) Adu. HETE to affect renal transport mechanisms has also been Prostaglandin Thromboxane Leukotriene Res. 2 1, 209-212 obtained, uiz. 20-HETE, when infused into the rat kidney 21. Escalante, B., Falck, J. R., Yadagiri, P., Sun, L., and LaniadoSchwartzman,M. (1988) Biochem. Biophys. Res. Commun. .~ produceda natriuresis (22); and when addedtoisolated 152,1269-1273 mTALH cells, 20-HETE, as noted above for 20-COOH-AA, 22. Takahashi. K.. CaDdevila. J.. Karara, A., Falck, J . R., Jacobson, inhibited oxygen consumption (23). H. R., and Badr,-K. F. (1990) Am. J. F%ysiol.'258, F781-F789 In conclusion, correspondence of the biological properties 23. Escalante, B., Erlij, D., Falck, J. R., and McGiff, J. C. (1991) Science 251, 799-802 of the authentic standards of AA products with those of the
