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CHARACTERIZATION OF THE CHIRALITY OF THE MONOHYDROXYEICOSATETRAENOIC ACIDS PRODUCED BY RAT BASOPHILIC LEUKEMIA CELLS Alan N. Baer*, Patrick B. Costello,and Floyd A. Green Division of Rheumatology,Departmentof Medicine, State University of New York at Buffalo and the VeteransAdministration Medical Center, Buffalo, New York 14215 Received
April
16,
1990
Incubation of tat basophilicleukemiacellswith exogenousarachidonicacid andpermeabihzii concentrationsof ethanolresultedin the production of 5-, 12-, and 15hydroqeicosatetraenoicacids. With chiral phasehigh performanceliquid chromatography,it was demonstrated that the 5hydroxyeicosatetraenoicacid had strict (S) stereospecificitywhile contrary to expectation, the 12-and the 15-hydroxyeicosatetraenoicacidswere non-tacemicmixtures of the stereoisomerswith the s/R ratios averaging 8.6 and 2.2, respectively. If the strict (S) stereospecificityof mammalianlipoxygenasesholdstrue, theseresultssuggestthat the 15-and 12hydroxyeicosatetraenoicacidsmay be derived from non-lipowgenasesources.Examination of the chimlity of the oxygenaseproductsof unsatumtedfatty acidsmay be of value in defming the enzymeswhich areactivated in vivo in pathologicalstates. 0 1990Academic Press,Inc.
Rat basophilicleukemia(RBL- 1) cellsmay be activated by an IgE-dependentpathway or by the calcium ionophore,A23 187, resultingin the releaseof amchidonicacid and in the secretion of histamine(1). The subsequentmetabolismof arachidonicacid in thesecellsproceedsprimarily via a 5-lipoxygenase,yielding 5-HETE and the leukotrienesB4 (LTB4), C4, and D4 (2,3,4), and via cyclooxygenase, yielding the prostaglandinsD2, Ez, andF2a (5,6). Arachidonate 5lipoxygenasehasbeenpurified from RBL cellsand its N-terminal aminoacid sequencehasbeen found to be homologouswith other mammalianlipoxygenases(7,8). The production of 12HETE by a calcium-dependentenzyme, presumedto be a lipoxygenase,hasalsobeendescribedin RBL- 1 cells(9,lO). The chirality of theseproductshasnot beenpreviously reported. The HETEs have a variety of biologic activities, including regulationof cellular lipoxygenaseand cyclooxygenaseactivity (11,12), mediationof chemotaxis(13), and promotion of cell growth and differentiation (14,15). In mammaliancells,the HETEs are derived primarily *To whom correspondenceshouldbe addressed. ABBREVIATIONS HETE (hydroxyeicosatetmenoicacid); HODD (hydroxyoctadecadienoicacid); LTB4 (leukotrieneB4); RBL- 1 cells(rat basophilicleukemiacells); HPLC (high performanceliquid chromatography);LC (liquid chromatography);W (ultraviolet); HEPES (N-2-hydroxyethylpipemzine-N’-Zethanesulfonicacid). 0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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from lipoxygenasepathways. However, there is someevidencefor the participation of other enzymatic pathways, including that of cyclooxygenaseand cytochrome P450, in the production of the HETEs. Analysis of the chirality of the oxygenaseproductsin different cells is a necessaryfirst step towardsunderstandingthe natureof the enzyme action aswell asthe transcellularmetabolismand the regulatory propertiesof their products. Furthermore,the chimlity of theseproductscanbe looked upon asenzyme-imprinted“tracks” which canhelp to recognizethe cellular enzyme sources of mono-HETEs producedin vivo during pathologicalprocesses.Such observationsmay help to define the role of in vivo activation of oxygenasesin pathogenesis. METHODS Cells, Incubations. and SamnlePrenaration. RBL- 1 cellswere purchasedfrom the American Type TissueCollection, Rockville, MD (CRL 1378). The cellswere cultured in minimumessentialmedium(Eagle)with 10%fetal calf serumandmaintainedin an atmosphereof 5% CO2 in air at 37oC. The cells were harvestedby scrapingthe flaskswith a rubber policeman, and then washedthree times. The cellswere routinely suspended in Medium 199with 40 mM, pH 7.6, HEPES buffer, at a concentrationof 30-40 x 106celldml. In someexperiments,the incubation mediumwasHank’s balancedsalt solutionwith 40 mM HEPES buffer (pH 7.6). Production of HETEs by the RBL- 1 cellswasroutinely achievedby incubationwith exogenousarachidonicacid (300 PM) in ethanol(0.8M). The incubationwasperformed in a 370 water bath for varying amountsof time, rangingfrom 15 to 120minutes. The reaction was terminatedby freezing the reactionvial in an acetone-dryice bath. The samplewaslyophilized and then treatedby the anhydrousmethanolicNaOH methodof Rates(16) to quantitatively tmnsesterifyall esterifiedfatty acidsand their derivatives to their respectivemethyl esterswhile preserving free fatty acidsand their derivatives intact. A singlemodification was madein order to partition non-estetifiedfatty acidsand their hydtoxylated derivatives into the organiclayer: after the addition of water to separatethe phases,100~1of 2 M ammoniumformate buffer, pH 3.2, was added. After washingwith methanol/water(10:9) saturatedwith chloroform, the lower phasewas reconstitutedinto a mobile phaseconsistingof methanol:waterzcetic acid, 80:20:0.1. The average recovery of fatty acidswith this extraction procedurewas 65%. Reversed-Phase High PerformanceLiouid Chromatoaranhv. Reversed-phase HPLC was performed isocratically on a Hewlett-Packard 1090liquid chromatographusingHewlett-Packard ODS-Hypersil columns, 20 cm in length x 4.6 mm at a flow rate of 0.4 ml/min. This instrumentis equippedwith a diode array spectrophotometerand a computer for on-line display and storageof UV spectra,and thusprecisemeasurementof the hmaxof eachpeakcan be performed after each run is completed. The LC tunswere monitoredat a wavelength of 236 nm, but recall was availableat 270 nm for detectionof the leukotrienes. StereochemicalAnalvses. Free hydroxylated fatty acidscollected during the LC runswere methylatedwith etherealdiazomethaneand rechromatographed on the sameLC columns(delayed retentiontime of approximately 15min). The methylatedfatty acidswere collected during the LC run, and then rechromatographedon two 25 cm Bakerbondchiral phaseLC columnsin series (dinitrobenzoylphenyl glycine coupledionically over aminopropyl residues;J.T. Baker Research Products). The mobile phasemixture for this straight phaseHPLC consistedof hexane:isopropanol,1000:15. Mixing experimentswith synthetic standardswere carried out for eachHETE to confirm exact retention timesin the chiral phaseHPLC studies. In the figures showingchromatographicresults,the ordinatescaleis given in milliabsorptionunits and all chromatogramsare reproduceddirectly from the LC computerplots. RESULTS When RBL- 1 cellswere incubatedwith exogenousarachidonicacid anda permeabiliziig concentrationof ethanol (17), 15HETE, 12HETE, and 5HETE were produced(Figure 1). In 333
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Fire 1. Reversed-phase HPLCof theatachidonicacidmetaholites of RBL-1 cells(34 x 1OVml;5 ml) incubatedwith arachidonic acid(300JAM)for 2 hours.Compounds A, B, C, D, E co-chromatogtaphed with 12-epi,&tram+-LTB4,LTB4, 15-BBTE,12-HFTE,and5-HBTE, respectively.The insetshowstheh&g UV spectrain mobilephaseof the5 compounds. The wavelengthmaximumof LTB4 wasslightlyhighercompared to itsisomer.
mostsuchincubations,5-HETE was the predominantmono-hydroxylated fatty acid. Leukotriene B4, and its diastereoisomers,6-transLTB4 and 6-ttans, 12-epiLTB4 eluted prior to the monoHETEs. The peptido leukotrieneswere not resolvedwith theseLC conditions. Production of the HETEs wasobservedat arachidonicacid concentrationsaslow as20 PM, and becamemaximal at concentrationsof 300-600uM. The stereospecificityof the free HETEs producedby the RBL- I cellswasanalyzed using chit-alphaseHPLC columnsunder straight phaseconditions. For theseanalyses,the free HETE
I
SR=l.4
14’ 12 108’ z
6. 4.
30
48 Time (mid
2:
se Time (min.)
Firrum2. Representative straightphasechitalHPLCchromatogtams of themethylesterof 15-HBTE.Theouterchromatogram isthatof thematerialderivatixedfrompeakC in Fire 1. Thechromatogram shownin theinsetisthatof thesamematerialto whichmcemic15-HETE methylestersyntheticstandard hasheenadded.Thes/R ratiosof theoriginalmaterialandthatof theoriginalmaterialto whichtacemic15-HBTEmethylesterhasheenaddedareshown. 334
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30
18
40
98
Time bin3
10
20 Time
I
36 (min.)
40
S0
j%mue3. Representative straightphasechirslHPLCchmmatograms of themethylesterof 12-HETE.Thecuterchromatogram is thatof thederivatizedmaterialfrompeakD in Ei 1. Thechromatogram shownin theinsetis thatof thesamematerialto whichracemic12-HETE methylestersyntheticstandard hasbeenadded.Thes/R ratioof theoriginalmixtureof stereoisomers decreased aftertheadditionof thetacemicstandard.
waspooled from severaldifferent incubations,methylatedwith etherealdiazomethane,purified with reversed-phase chromatography,and then rechromatographedon chiral phasecolumns. An exampleof an LC tun of 15-HETE methyl esteris shownin Figure 2. Mixing experimentswere performedasillustratedin Figure 2 to confirm the identity of the compoundandto differentiate the stereoisomers.The 15-HETE colkcted from the RDL- 1 cellswasa non-racemicmixture of the S and R stereoisomers,with the WR ratio ranging from 1.4 to 4.4 (mean+ SEM, 2.2 It 0.4, n=7). The 12-HETE wasalso a non-mcemicmixture of stereoisomers with the s/R ratio being 8.6 f 1.7
Wavelength
10
20
30
40 50 Time (min.)
(nd
60
70
60
Firmre 4. Representative straight phase chiral HPLC chromatogram of the methyl ester of S-HETE. The S-HETE was generated by incubation of RBL- 1 cells (40 x 1OVml) with amchidonic acid (300 PM), then esterified, and re-chromatogmphed on RP-HPLC prior to the chiral phase HPLC shown here. The S-HETE methyl ester eluted as a single peak and was shown to be the (S) stereoisomer. The inset shows the b & UV spectrum in mobile phase of the 5HETE methyl cater.
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106 30-
0.5
262
. i:
400.v\ 225
238
235
WaVelength
la-
16
RATIOS
1.3
60
2
. II0
S/R
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245
kd
30 Time (min.)
40
s
J
60
Fiaure5. Representative straightphasechimlHPLCchromatogtam of themethylestersof 1IHODD and9-HODD. The l3-HODDand9-HODDweregenerated duringincubations of RRL-1cehswith linoleic acid(300a) andthenesterified.There was separation of thetwo compounds; 13&R)-HODD methylesterselutingin thischromatogram at 50-53minand9(&R)HODDmethylesterselutiagat 59-62min. Theinsetshowstheitr &u W spectra(overlaid)of 13(S)-HODD,9(S)-HODD,and9(R)-HODDmethylesters.
(mm f SW, n=6) (Figure 3). In eachof 5 samplesstudied, 5-HETE had strict Sstereospecificity(Figure 4). Additional experimentswere performedin order to evaluatethe mechanismby which a non-racemicmixture of 15HETE stereoisomers wasproduced. The REtL-1 cellswere incubated with exogenouslinoleic acid to confirm the presenceof a o-6 oxygenase. When run on reversedphaseHPLC, the productsof this incubation includeda peak eluting at 22.5 min with a Amaxat 234 nm. This peak had the retentiontime of authentic 13-HODDBHODD standards.When the compoundsin this peakwere methylated, and then run on chiral phaseLC, peakswhich comigratedwith 13(S)-HODD, 13(R)-HODD, 9(S)-HODD, and 9(R)-HODD were observed(Figure 5). The s/R ratio of 13-HODD was 3.6 f 0.4 and that of 9-HODD was 0.55 * 0.01 (mean* SEM, n=2). When incubationsof amchidonicor linoleic acid were performedwith boiled RBL- 1 cells, smallpeaksat the retentiontimesof 15HETE and 13BHODD were observed. When thesepeaks were collected from severalsuchincubations,methylatedin vitro with diazomethaneand run on chiral phaseLC, racemicmixtures of 15HETE, 13-HODD, and 9-HODD were observed. The production of 15-HETE amountedto 33 + 10%(meanf SEM, n=8) and that of 13/9-HODD amountedto 44-56% (2 experiments)of that observedin parallel incubationswith live cells. After incubationof RBL-1 cellswith arachidonicacid in the presenceof either indomethacin,7 PM, or SKF 525A, 100 pM (a cytochrome P450inhibitor) (18), the s/R ratio of the 15HETE producedwas comparableto that of the 15-HFTE producedin the absenceof drug. DISCUSSION In the current studies,it was demonstratedthat RBL- 1 cells produceboth 15HETE and 12-HETE, in addition to 5HETE, when incubatedwith exogenousarachidonicacid. The 336
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production of 15HETE by RBL- 1 cells had not beenpreviously reported. The formation of severalpossibleHETEs other than U-IETE wasobservedby Ford-Hutchinsongl d (4), but these productswere not characterizedfurther. The analysisof the stereospecificityof HETE production by RBL- 1 cellsprovides important information asto the enzymatic origin of thesecompounds.Mammalian lipoxygenases have beenshownthus far to have strict S-stereospecificity- asexemplified by 12(S)-HETE derived from humanplatelets(19) and murineeosinophils(20); 1S(S)-HETE derived from rabbit reticulocytes (21), rat mononuclearcells(22), and humanplatelets(23), eosinophils(24), and keratinocytes(25); 13(S)-HODD from humanleukocytes(26); and S(S)-HETE derived from humanneutrophils(27). Recently, 12(R)-HETE or a non-tacemicmixture of the 12HETE stereoisomers hasbeenisolatedfrom mammaliantissues,including bovine comealepithelium (28), psoriatic skin scales(29,30), and humanepithelium (3 1). Cytochrome P450 monooxygenasesare thought to be responsiblefor the 12HETE production in someof thesetissues. In the presentstudy, the 5HETE derived from RBL- 1 cellshad strict (S) stereospecificity, compatiblewith its known origin from a 5-lipoxygenase. The 12HETE wasnot enantiopure,with a s/R ratio of approximately 9. This smallamount of the R isomermay be compatiblewith lipoxygenaseaction. The moststriking finding wasthe consistentpresenceof a non-mcemic mixture of 15-HETE stereoisomers with a S/R ratio that averaged2.2. Incubationsof the RBL-1 cellswith linoleic acid alsoresultedin non-racemicmixtures of the w-6 oxygenaseproducts, 13HODD and 9-HODD. There are severalreasonsto arguethat thesenon-racemicstereoisomermixtures were derived from enzymic sourcesand not simply from a combinationof autooxidation and the action of a w-6 lipoxygenase. First, production of a mixture of stereoisomers with a SR ratio of 2.2 by the combinationof a lipoxygenasewith strict (S) stereospecificityandautooxidation would imply that the formation of the HETE by autooxidationexceededthat by enzymic sourceswhere in fact autooxidation is likely to be inhibited in the presenceof intact cells. Second,the linoleic acid productsof the w-6 oxygenaseconstituteda mixture of 13-HODD and 9-HODD stereoisomers that would not be expectedwith a lipoxygenase. In particular, the predominanceof the 9(R)-HODD stereoisomerarguesagainstan oxygenasewith strict (S) stereospecificity. Third, the s/R ratios of the 15-HETE were found to be within a relatively narrow range,an observationunlikely to occur with autooxidation. The nature of the o-6 oxygenasein RBL-1 cellsneedsto be defined further. The resultsof the current study would not be expectedif this enzyme were a lipoxygenasesimilar to those describedin other mammaliansystems. Both cytochrome P-450 monooxygenasesand cyclooxygenasedemonstrateo-6 regiospecificity andhave beenfound to yield stereoisomeric mixtures of the monohydroxylated derivatives of arachidonicand linoleic acid (32,33).
ACKNOWLEDGMENTS The authorsgratefully acknowledgethe technicalassistanceof Norine Yuskiw. This work was supportedby the VeteransAdministration, the WesternNew York Chapterof the Arthritis Foundation, the Julesand Gwen Knapp CharitableFoundation, and NM Grant HL 24009. 337
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REFERENCES
1. 3: 4. 5. 6. 7. 8. 9. :t 12. 13. ::: 16. 17. 18. ii: 21. 22. 23. 24. 25. 26.
McGivney, A., Morita, Y., Crews, F.T., Hirata, F., Axelrod, J., and Siraganian, R.P. (198 1) Arch. B&hem. Biophys. 212,572-580. Morris, H.R., and Taylor, G.W. (1980) Prostaglandins 19, 185-200. Jakschik, B.A., Harper, T., and Murphy, R.C. (1982) J. Biol. Chem. 257, 5346-5349. Ford-Hutchinson, A.W., Piper, P.J., and Samhoun,M.N. (1982) Br. J. Pharmacol. 76, 215-220. Jakschik, B.A., Lee, L.H., Shuffer, G., and Parker, C.W. (1978) Prostaglandins 16, 733-748. Levine, L., Morgan, R.A., Lewis, RA., Austen, K.F., Clark, D.A., Marfat, A., and Corey, E.J. (1981) Proc. Natl. Acad. Sci. USA 78, 7692-7696. Hogaboom, G.K., Cook, M., Newton, J.F., Varrichio, A., Shorr, R.G.L., Sarau, H.M., and Crooke, ST. (1986) Mol. Pharmacol. 30,510-519. Sisal, E., Grunberger, D., Craik, C.S., Caughey, G.H., and Nadel, J.A. (1988) J. Biol. Chem. 263,5328-5332. Jakschik, B.A., Sun, F.F., Lee, L-H., and Steinhoff, M.M. (1980) Biochem. Biophys. Res. Comm. 95, 103-110. Hamasaki,Y., and Tai, H-H. (1984) Biochim. Biophys. Acta 793, 393-398. Vanderhoek, J.Y., Bryant, R.W., and Bailey, J.M. (1980) J. Biol. Chem. 255, 1006410066. Setty, B.N.Y., and Stuart, M.J. (1986) J. Clin. Invest. 77, 202-211. Goetzl, E.J., Weller, P.F., and Sun, F.F. (1980) J. Immunol. 124, 926-933. Setty, B.N.Y., Graeber, J.E., and Stuart, M.J. (1987) J. Biol. Chem. 262, 17613-17622. Rapoport, S.M., and Schewe,T. (1986) Biochim. Biophys. Acta 864,471-495. Kates,M. (1972) Laboratory Techniquesin Biochemistry andMolecular Biology. Vol. 3, Part II, p. 558, North-Holland, Amsterdam. Green, F.A., and Claesson,H-E. (1986) Biochem. Biophys. Res. Comm. 140:782-788. Boeynaems,J.M., Demolle, D., and Van Coevorden, A. (1986) Prostaglandins32,145149. Hamberg, M., and Samuelsson,B. (1974) Proc. Natl. Acad. Sci. USA 71,3400-3404. Turk, J., Rand, T.H., Maas, R.L., Lawson, J.A., Brash, A.R., Roberts, L.J., Colley, D.G., and Oates,J.A. (1983) Biochim. Biophys. Acta 750, 78-90. Bryant, R.W., Bailey, J.M., Schewe, T., and Rapoport, S.M. (1982) J. Biol. Chem. 257,6050-6055. Maas, R.L., Turk, J., Oatcs, J-A., and Brash, A.R. (1982) J. Biol. Chem. 257, 70567067. Wong, P.Y.-K., Westlund, P., Hamberg, M., Granstriim, E., Chao, P.H.-W., and Samuelsson,B. (1985) J. Biol. Chem. 260,9162-9165. Turk, J., Maas, R.L., Brash, A.R., Roberts, L.J.11, and Oates, J.A. (1982) J. Biol. Chem. 257, 7068-7076. Green, F.A. (1989) Biochem. Biophys. Res. Comm. 160,545-55 1. Reinaud, O., Delaforge, M., Boucher, J.L., Rocchiccioli, F., and Mansuy, D. (1989) Biochem. Biophys. Res.Comm. 161,883-89 1. Borgeat, P., and Samuelsson,B. (1979) Proc. Natl. Acad. Sci. USA 76:2148-2152. Schwartzman, M.L., Balazy, M., Masferrer, J., Abraham, N.G., McGiff, J.C., and Murphy, R.C. (1987) Proc. Natl. Acad. Sci. USA 84,8125-8129. Woollard, P.M. (1986) Biochem. Biophys. Res. Comm. 136, 169-176. Baer, A.N., Costello, P.B., and Green, F-A. (1990) J. Lipid Res. 31, 125-130. Holtzman, M.J., Turk, J., and Pentland, A. (1989) J. Clin. Invest. 84, 1446-1453. Hamberg, M., and Samuelsson,B. (1980) B&him. Biophys. Acta 617,545-547. Capdevila, J., Yadagiri, P., Manna, S., and Falck, J.R. (1986) B&hem. Biophys. Res. Comm. 141,1007-1011.
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CHARACTERIZATION OF THE CHIRALITY OF THE MONOHYDROXYEICOSATETRAENOIC ACIDS PRODUCED BY RAT BASOPHILIC LEUKEMIA CELLS Alan N. Baer*, Patrick B. Costello,and Floyd A. Green Division of Rheumatology,Departmentof Medicine, State University of New York at Buffalo and the VeteransAdministration Medical Center, Buffalo, New York 14215 Received
April
16,
1990
Incubation of tat basophilicleukemiacellswith exogenousarachidonicacid andpermeabihzii concentrationsof ethanolresultedin the production of 5-, 12-, and 15hydroqeicosatetraenoicacids. With chiral phasehigh performanceliquid chromatography,it was demonstrated that the 5hydroxyeicosatetraenoicacid had strict (S) stereospecificitywhile contrary to expectation, the 12-and the 15-hydroxyeicosatetraenoicacidswere non-tacemicmixtures of the stereoisomerswith the s/R ratios averaging 8.6 and 2.2, respectively. If the strict (S) stereospecificityof mammalianlipoxygenasesholdstrue, theseresultssuggestthat the 15-and 12hydroxyeicosatetraenoicacidsmay be derived from non-lipowgenasesources.Examination of the chimlity of the oxygenaseproductsof unsatumtedfatty acidsmay be of value in defming the enzymeswhich areactivated in vivo in pathologicalstates. 0 1990Academic Press,Inc.
Rat basophilicleukemia(RBL- 1) cellsmay be activated by an IgE-dependentpathway or by the calcium ionophore,A23 187, resultingin the releaseof amchidonicacid and in the secretion of histamine(1). The subsequentmetabolismof arachidonicacid in thesecellsproceedsprimarily via a 5-lipoxygenase,yielding 5-HETE and the leukotrienesB4 (LTB4), C4, and D4 (2,3,4), and via cyclooxygenase, yielding the prostaglandinsD2, Ez, andF2a (5,6). Arachidonate 5lipoxygenasehasbeenpurified from RBL cellsand its N-terminal aminoacid sequencehasbeen found to be homologouswith other mammalianlipoxygenases(7,8). The production of 12HETE by a calcium-dependentenzyme, presumedto be a lipoxygenase,hasalsobeendescribedin RBL- 1 cells(9,lO). The chirality of theseproductshasnot beenpreviously reported. The HETEs have a variety of biologic activities, including regulationof cellular lipoxygenaseand cyclooxygenaseactivity (11,12), mediationof chemotaxis(13), and promotion of cell growth and differentiation (14,15). In mammaliancells,the HETEs are derived primarily *To whom correspondenceshouldbe addressed. ABBREVIATIONS HETE (hydroxyeicosatetmenoicacid); HODD (hydroxyoctadecadienoicacid); LTB4 (leukotrieneB4); RBL- 1 cells(rat basophilicleukemiacells); HPLC (high performanceliquid chromatography);LC (liquid chromatography);W (ultraviolet); HEPES (N-2-hydroxyethylpipemzine-N’-Zethanesulfonicacid). 0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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from lipoxygenasepathways. However, there is someevidencefor the participation of other enzymatic pathways, including that of cyclooxygenaseand cytochrome P450, in the production of the HETEs. Analysis of the chirality of the oxygenaseproductsin different cells is a necessaryfirst step towardsunderstandingthe natureof the enzyme action aswell asthe transcellularmetabolismand the regulatory propertiesof their products. Furthermore,the chimlity of theseproductscanbe looked upon asenzyme-imprinted“tracks” which canhelp to recognizethe cellular enzyme sources of mono-HETEs producedin vivo during pathologicalprocesses.Such observationsmay help to define the role of in vivo activation of oxygenasesin pathogenesis. METHODS Cells, Incubations. and SamnlePrenaration. RBL- 1 cellswere purchasedfrom the American Type TissueCollection, Rockville, MD (CRL 1378). The cellswere cultured in minimumessentialmedium(Eagle)with 10%fetal calf serumandmaintainedin an atmosphereof 5% CO2 in air at 37oC. The cells were harvestedby scrapingthe flaskswith a rubber policeman, and then washedthree times. The cellswere routinely suspended in Medium 199with 40 mM, pH 7.6, HEPES buffer, at a concentrationof 30-40 x 106celldml. In someexperiments,the incubation mediumwasHank’s balancedsalt solutionwith 40 mM HEPES buffer (pH 7.6). Production of HETEs by the RBL- 1 cellswasroutinely achievedby incubationwith exogenousarachidonicacid (300 PM) in ethanol(0.8M). The incubationwasperformed in a 370 water bath for varying amountsof time, rangingfrom 15 to 120minutes. The reaction was terminatedby freezing the reactionvial in an acetone-dryice bath. The samplewaslyophilized and then treatedby the anhydrousmethanolicNaOH methodof Rates(16) to quantitatively tmnsesterifyall esterifiedfatty acidsand their derivatives to their respectivemethyl esterswhile preserving free fatty acidsand their derivatives intact. A singlemodification was madein order to partition non-estetifiedfatty acidsand their hydtoxylated derivatives into the organiclayer: after the addition of water to separatethe phases,100~1of 2 M ammoniumformate buffer, pH 3.2, was added. After washingwith methanol/water(10:9) saturatedwith chloroform, the lower phasewas reconstitutedinto a mobile phaseconsistingof methanol:waterzcetic acid, 80:20:0.1. The average recovery of fatty acidswith this extraction procedurewas 65%. Reversed-Phase High PerformanceLiouid Chromatoaranhv. Reversed-phase HPLC was performed isocratically on a Hewlett-Packard 1090liquid chromatographusingHewlett-Packard ODS-Hypersil columns, 20 cm in length x 4.6 mm at a flow rate of 0.4 ml/min. This instrumentis equippedwith a diode array spectrophotometerand a computer for on-line display and storageof UV spectra,and thusprecisemeasurementof the hmaxof eachpeakcan be performed after each run is completed. The LC tunswere monitoredat a wavelength of 236 nm, but recall was availableat 270 nm for detectionof the leukotrienes. StereochemicalAnalvses. Free hydroxylated fatty acidscollected during the LC runswere methylatedwith etherealdiazomethaneand rechromatographed on the sameLC columns(delayed retentiontime of approximately 15min). The methylatedfatty acidswere collected during the LC run, and then rechromatographedon two 25 cm Bakerbondchiral phaseLC columnsin series (dinitrobenzoylphenyl glycine coupledionically over aminopropyl residues;J.T. Baker Research Products). The mobile phasemixture for this straight phaseHPLC consistedof hexane:isopropanol,1000:15. Mixing experimentswith synthetic standardswere carried out for eachHETE to confirm exact retention timesin the chiral phaseHPLC studies. In the figures showingchromatographicresults,the ordinatescaleis given in milliabsorptionunits and all chromatogramsare reproduceddirectly from the LC computerplots. RESULTS When RBL- 1 cellswere incubatedwith exogenousarachidonicacid anda permeabiliziig concentrationof ethanol (17), 15HETE, 12HETE, and 5HETE were produced(Figure 1). In 333
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Fire 1. Reversed-phase HPLCof theatachidonicacidmetaholites of RBL-1 cells(34 x 1OVml;5 ml) incubatedwith arachidonic acid(300JAM)for 2 hours.Compounds A, B, C, D, E co-chromatogtaphed with 12-epi,&tram+-LTB4,LTB4, 15-BBTE,12-HFTE,and5-HBTE, respectively.The insetshowstheh&g UV spectrain mobilephaseof the5 compounds. The wavelengthmaximumof LTB4 wasslightlyhighercompared to itsisomer.
mostsuchincubations,5-HETE was the predominantmono-hydroxylated fatty acid. Leukotriene B4, and its diastereoisomers,6-transLTB4 and 6-ttans, 12-epiLTB4 eluted prior to the monoHETEs. The peptido leukotrieneswere not resolvedwith theseLC conditions. Production of the HETEs wasobservedat arachidonicacid concentrationsaslow as20 PM, and becamemaximal at concentrationsof 300-600uM. The stereospecificityof the free HETEs producedby the RBL- I cellswasanalyzed using chit-alphaseHPLC columnsunder straight phaseconditions. For theseanalyses,the free HETE
I
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Firrum2. Representative straightphasechitalHPLCchromatogtams of themethylesterof 15-HBTE.Theouterchromatogram isthatof thematerialderivatixedfrompeakC in Fire 1. Thechromatogram shownin theinsetisthatof thesamematerialto whichmcemic15-HETE methylestersyntheticstandard hasheenadded.Thes/R ratiosof theoriginalmaterialandthatof theoriginalmaterialto whichtacemic15-HBTEmethylesterhasheenaddedareshown. 334
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30
18
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20 Time
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S0
j%mue3. Representative straightphasechirslHPLCchmmatograms of themethylesterof 12-HETE.Thecuterchromatogram is thatof thederivatizedmaterialfrompeakD in Ei 1. Thechromatogram shownin theinsetis thatof thesamematerialto whichracemic12-HETE methylestersyntheticstandard hasbeenadded.Thes/R ratioof theoriginalmixtureof stereoisomers decreased aftertheadditionof thetacemicstandard.
waspooled from severaldifferent incubations,methylatedwith etherealdiazomethane,purified with reversed-phase chromatography,and then rechromatographedon chiral phasecolumns. An exampleof an LC tun of 15-HETE methyl esteris shownin Figure 2. Mixing experimentswere performedasillustratedin Figure 2 to confirm the identity of the compoundandto differentiate the stereoisomers.The 15-HETE colkcted from the RDL- 1 cellswasa non-racemicmixture of the S and R stereoisomers,with the WR ratio ranging from 1.4 to 4.4 (mean+ SEM, 2.2 It 0.4, n=7). The 12-HETE wasalso a non-mcemicmixture of stereoisomers with the s/R ratio being 8.6 f 1.7
Wavelength
10
20
30
40 50 Time (min.)
(nd
60
70
60
Firmre 4. Representative straight phase chiral HPLC chromatogram of the methyl ester of S-HETE. The S-HETE was generated by incubation of RBL- 1 cells (40 x 1OVml) with amchidonic acid (300 PM), then esterified, and re-chromatogmphed on RP-HPLC prior to the chiral phase HPLC shown here. The S-HETE methyl ester eluted as a single peak and was shown to be the (S) stereoisomer. The inset shows the b & UV spectrum in mobile phase of the 5HETE methyl cater.
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. i:
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Fiaure5. Representative straightphasechimlHPLCchromatogtam of themethylestersof 1IHODD and9-HODD. The l3-HODDand9-HODDweregenerated duringincubations of RRL-1cehswith linoleic acid(300a) andthenesterified.There was separation of thetwo compounds; 13&R)-HODD methylesterselutingin thischromatogram at 50-53minand9(&R)HODDmethylesterselutiagat 59-62min. Theinsetshowstheitr &u W spectra(overlaid)of 13(S)-HODD,9(S)-HODD,and9(R)-HODDmethylesters.
(mm f SW, n=6) (Figure 3). In eachof 5 samplesstudied, 5-HETE had strict Sstereospecificity(Figure 4). Additional experimentswere performedin order to evaluatethe mechanismby which a non-racemicmixture of 15HETE stereoisomers wasproduced. The REtL-1 cellswere incubated with exogenouslinoleic acid to confirm the presenceof a o-6 oxygenase. When run on reversedphaseHPLC, the productsof this incubation includeda peak eluting at 22.5 min with a Amaxat 234 nm. This peak had the retentiontime of authentic 13-HODDBHODD standards.When the compoundsin this peakwere methylated, and then run on chiral phaseLC, peakswhich comigratedwith 13(S)-HODD, 13(R)-HODD, 9(S)-HODD, and 9(R)-HODD were observed(Figure 5). The s/R ratio of 13-HODD was 3.6 f 0.4 and that of 9-HODD was 0.55 * 0.01 (mean* SEM, n=2). When incubationsof amchidonicor linoleic acid were performedwith boiled RBL- 1 cells, smallpeaksat the retentiontimesof 15HETE and 13BHODD were observed. When thesepeaks were collected from severalsuchincubations,methylatedin vitro with diazomethaneand run on chiral phaseLC, racemicmixtures of 15HETE, 13-HODD, and 9-HODD were observed. The production of 15-HETE amountedto 33 + 10%(meanf SEM, n=8) and that of 13/9-HODD amountedto 44-56% (2 experiments)of that observedin parallel incubationswith live cells. After incubationof RBL-1 cellswith arachidonicacid in the presenceof either indomethacin,7 PM, or SKF 525A, 100 pM (a cytochrome P450inhibitor) (18), the s/R ratio of the 15HETE producedwas comparableto that of the 15-HFTE producedin the absenceof drug. DISCUSSION In the current studies,it was demonstratedthat RBL- 1 cells produceboth 15HETE and 12-HETE, in addition to 5HETE, when incubatedwith exogenousarachidonicacid. The 336
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production of 15HETE by RBL- 1 cells had not beenpreviously reported. The formation of severalpossibleHETEs other than U-IETE wasobservedby Ford-Hutchinsongl d (4), but these productswere not characterizedfurther. The analysisof the stereospecificityof HETE production by RBL- 1 cellsprovides important information asto the enzymatic origin of thesecompounds.Mammalian lipoxygenases have beenshownthus far to have strict S-stereospecificity- asexemplified by 12(S)-HETE derived from humanplatelets(19) and murineeosinophils(20); 1S(S)-HETE derived from rabbit reticulocytes (21), rat mononuclearcells(22), and humanplatelets(23), eosinophils(24), and keratinocytes(25); 13(S)-HODD from humanleukocytes(26); and S(S)-HETE derived from humanneutrophils(27). Recently, 12(R)-HETE or a non-tacemicmixture of the 12HETE stereoisomers hasbeenisolatedfrom mammaliantissues,including bovine comealepithelium (28), psoriatic skin scales(29,30), and humanepithelium (3 1). Cytochrome P450 monooxygenasesare thought to be responsiblefor the 12HETE production in someof thesetissues. In the presentstudy, the 5HETE derived from RBL- 1 cellshad strict (S) stereospecificity, compatiblewith its known origin from a 5-lipoxygenase. The 12HETE wasnot enantiopure,with a s/R ratio of approximately 9. This smallamount of the R isomermay be compatiblewith lipoxygenaseaction. The moststriking finding wasthe consistentpresenceof a non-mcemic mixture of 15-HETE stereoisomers with a S/R ratio that averaged2.2. Incubationsof the RBL-1 cellswith linoleic acid alsoresultedin non-racemicmixtures of the w-6 oxygenaseproducts, 13HODD and 9-HODD. There are severalreasonsto arguethat thesenon-racemicstereoisomermixtures were derived from enzymic sourcesand not simply from a combinationof autooxidation and the action of a w-6 lipoxygenase. First, production of a mixture of stereoisomers with a SR ratio of 2.2 by the combinationof a lipoxygenasewith strict (S) stereospecificityandautooxidation would imply that the formation of the HETE by autooxidationexceededthat by enzymic sourceswhere in fact autooxidation is likely to be inhibited in the presenceof intact cells. Second,the linoleic acid productsof the w-6 oxygenaseconstituteda mixture of 13-HODD and 9-HODD stereoisomers that would not be expectedwith a lipoxygenase. In particular, the predominanceof the 9(R)-HODD stereoisomerarguesagainstan oxygenasewith strict (S) stereospecificity. Third, the s/R ratios of the 15-HETE were found to be within a relatively narrow range,an observationunlikely to occur with autooxidation. The nature of the o-6 oxygenasein RBL-1 cellsneedsto be defined further. The resultsof the current study would not be expectedif this enzyme were a lipoxygenasesimilar to those describedin other mammaliansystems. Both cytochrome P-450 monooxygenasesand cyclooxygenasedemonstrateo-6 regiospecificity andhave beenfound to yield stereoisomeric mixtures of the monohydroxylated derivatives of arachidonicand linoleic acid (32,33).
ACKNOWLEDGMENTS The authorsgratefully acknowledgethe technicalassistanceof Norine Yuskiw. This work was supportedby the VeteransAdministration, the WesternNew York Chapterof the Arthritis Foundation, the Julesand Gwen Knapp CharitableFoundation, and NM Grant HL 24009. 337
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1. 3: 4. 5. 6. 7. 8. 9. :t 12. 13. ::: 16. 17. 18. ii: 21. 22. 23. 24. 25. 26.
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