Vol. 171, No. 3, 1990 September 28, 1990

BIOCHEMICAL

RECEPTOR-MEDIATED AND

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 944-948

ACTION

OF HEPOXILIN

ARACHIDONIC

ACID FROM

A3 RELEASES HUMAN

DIACYLGLYCEROL

NEUTROPHILS

SantoshNigam*, Sim Nodes*, Gunter Cichon*, Elias J. Corey** and Cecil R. Pace-As&k+1 *Departmentof GynaecologicalEndocrinology, Kiinikum Steglitz, Free University of Berlin, D-l 000 Berlin 45, FRG **Department of Chemistry, Harvard University, Cambridge,Mass. 02138 +ResearchInstitute, Hospital for Sick Children, Toronto M5G 1X8, Canada Received

August

2, 1990

We have previously shownthat hepoxilin A3 increasesthe intracellular concentrationof Ca+2 in humanneutrophils. Herein we addressthe initial events of hepoxilin action on the neutrophil which precedethe rise in intracellular calcium. We showthat hepoxilin A3 at 10-1000nM concentrationsreleasesfrom [ 1-14C]-arachidonicacid labeledneutrophilsdiacylglycerol and unesterifiedarachidonicacid in a time andconcentrationdependentfashion. The releaseof arachidonicacid and diacyglycerol are receptor-mediatedeventswhich are blocked by pertussis toxin. This data showsthat hepoxilin A3 stimulatesphospholipases C and A2 in the cell which may be involved in the rise in cytosolic calcium. Thus, hepoxilins may representa hitherto unrecognisedclassof cellular mediators. 01990 Academic Press, Inc. Much interesthasrecently beendirected to the 5lipoxygenase pathway of arachidonicacid metabolismasproducts derived from this pathway, e.g. the leukotrienesandlipoxins, have biologicalpropertieswhich have implicatedthem aspotential mediatorsof inflammation and hypersensitivity reactions(l-4). Although the 12-lipoxygenasepathway is abundantin cells, little attention hasbeendirected to its products. We have previously shownthat within the 12lipoxygenasepathway, the primary intermediate,12-HPETE, is metabolizedinto the hepoxilins (A3 and Bg), hydroxy epoxide metabolitesformed through the catalytic action of ferrihemeproteins

(5-7). In fact it hasbeendemonstratedthat the actionsof 1ZHPETE are mediatedthrough the formation of the hepoxilins asthe latter productsreleaseinsulin from pancreaticislets(7,8), facilitate the transportof calcium acrossmembranes(9), andraiseintracellularcalcium in human neutrophils(10). Hepoxilins alsohave synaptic effects on the Aplysia motor neurons(11) and on mammalianhippocampalCA1 neurons(13). Hepoxilins have beenimplicated in the openingof Stype K+ channelsin the Aplysia by hematin-treated1ZHPETE in inside-outpatchesof the Aplysia neurons(12). Hepoxilin A3 is further transformedinto a biologically active glutathioneconjugate (14,lS). 1 To whom correspondenceshouldbe addressed. Abbreviations used: HxA3, hepoxilin A3, 8-hydroxy-11,12-epoxyeicosa-5Z, lOE, 14Ztrienoic acid; HxB3, hepoxilin B-j, lo-hydroxy-11,12-epoxyeicosa-5Z, 8Z, 14Ztrienoic acid; TLC = thin layer chromatography;PBS = phosphatebuffered saline;DAG, sn-1,2-diacylglycerol. 0006-291X/90 Copyright

All rinhts

$1.50

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

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MATERIALS

AND

METHODS

Materials: [ 1-14C]Arachidonic acid (spec.act. 52 mCi/mmol) and [$2P]-ATP (spec.act. 425,000 cpm/nmol) were purchasedfrom NEN (Dreieich, FRG). TLC silica gel-60 plateswere from Merck (Darmstadt,FRG); pertussistoxin was from Sigma (Munchen, FRG); FMLP, BSA and phospholipidTLC standards,Octyl-P-D-glucoside, cardiolipin, dithiothreitol and imidazole were from Sigma (Munchen, FRG). Diacyl glycerol kinasefrom E. Coli waspurchasedfrom Calbiochem,FRG. HxA3 was usedasthe methyl esterand a stock solution was madeup in DMSO (&g/@) and aliquotswere further diluted with buffer to the appropriateconcentrations(lo1000 nM). Isolation of human neutrophils: Peripheral blood was drawn into heparinized tubes from healthy donorsby venipuncture. Neutrophilswere isolatedby Ficoll-Hypaque gradient centrifugation (LSM, OrganonTeknica Co., Durham, NC) followed by dextran sedimentation. Red cells were removed by hypotonic lysis followed by centrifugation. Isolated cells were suspendedin Dulbecco’sphosphatebuffered saline(PBS) containing both CaC12(0.6 mM) and MgC12 (1.0 mM), pH 7.4. These suspensions contained 98 + 1 % PMNs asdeterminedby light microscopy and were viable (98 + 1%) astestedby trypan blue dye exclusion. Labelling of cells: PMNs (30 x l@/ml) were incubated with 0.25 PCi of [l-14C] arachidonicacid for 20 min at 37cC. After washingtwice with Hanks balancedsalt solution containing BSA (0.25%) without CaC12to remove unesterifiedfatty acid, the cells were suspended in PBS (5 x 106cells/ml). 74 + 11 % (n=9) of arachidonicacid wasincorporated into lipids. The labeledcells showed98 i 1 % exclusion of trypan blue (n=9). Release of free arachidonic acid: [l-14C] arachidonic acid-labeled PMNs (5 x lo6 cells/ml PBS) were incubatedat 37OCwith HxA3 methyl esterin PBS (10-1000nM) for various times (5, 10,30,60,120 and 300 set). Incubationswere terminatedat specific times by addition of 3.5 ml of chloroform/methanol (2:5, v/v) and extracted. Sampleswere acidified to pH 3.5 by the addition of 1N HCl andthe organic phasewas separatedby addingchloroform (1 ml) and water (1 ml). The organic phasewas separatedby centrifugation and washedwith 1 ml water. The organic phasewastaken to drynessin vacua andthe residuewas suspendedin chloroform/methanol(50 ~1; 2/5, v/v) and spottedon heatactivated silica gel-60 TLC plates. The plateswere developedwith petroleumether/ diethyl ether/ acetic acid (50/50/l, v/v). Lipids were visualized by iodine staining,and the bandmigrating asauthenticstandard(unesterified arachidonicacid) was scrapedand suspendedinto Instagelliquid scintillation fluid (Pharmacia, Freiburg, FRG). The amountof radiolabelwasquantified by scintillation counting. Release of diacylglycerol: 5 x 106neutrophils (unlabeled) in 0.5 ml PBS were equilibratedat 37OCfor 5 min. The reaction was startedby simultaneousaddition of 0.4 ml of hepoxilin (final cont. 0- 1 @f) and 0.1 ml FMLP (final cont. 100 nM) for various periodsof time. The reaction was stoppedwith 3.5 ml of chloroform/methanol(2:5, v/v) and the lipids were extracted asdescribedabove. DAG was assayedasreported elsewhere(16,17). Briefly, extracted lipids were dissolved in 20 ~1of a solution containing 7.5% octyl-P-D-glucoside and5 mM cardiolipin and mixed with 50 yl of a solution containing 100 mM imidazole, 100 mM NaCl, 25 mM MgC12 ,2 mM EGTA, 0.5 mg/ml Glycerol kinase (spec.act. 10~mol/min/mg protein) at pH 7.0, 10 pl of 20 mM dithiothreitol and 10 ~1 of 10 mM [Y-~~P]-ATP. Following incubation for 10 min at 25OC,the reaction mixture wasextracted asdescribedabove. The lipid residuewas reconstitutedin 5% methanolin chloroform andan aliquot was spottedon a silica gel-60 TLC plate and developedin chlorofornnmethanol:acetic acid (65:15:5, v/v/v). The radioactive spot correspondingto phosphatidicacid was scraped,suspended in scintillation fluid and countedfor radioactivity. The amountof DAG wascalculatedfrom the amountof [32P] phosphatidicacid producedand the specific activity of [$2P]-ATP used. RESULTS

AND

DISCUSSION

Incubation of radiolabelledarachidonicacid with PMNs resultsin its active incorporation (74 + 11 %, n=9) into lipid storesof which 27 + 2 % (n=9) wasfound esterified in phospholipids 945

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(18). Treatment of these labeled cells with HxA3 (100 r&l) resulted in a rapid release of diacylglycerol (starting within 10 set up to 300 sec)$ig. 1A) and unesterified arachidonic acid (Fig. 1B). The time-dependent release of both diacylglycerol and archidonic acid was blocked by preincubating the cells with pertussis toxin (2 pg/ml). The time course of release of diacylglycerol and arachidonic acid is worthy of comment. The lipoxin-induced release appears biphasic while that for HxA3 appears to lack the initial rapid phase but retains the second phase of release (see below). Whether the second phase of response of lipoxin-induced release is mediated via the formation of the hepoxilins is the subject of further investigation. In separate experiments, the concentration dependency of HxA3 on the release of arachidonic acid from activated PMNs was investigated. The PMNs were activated by prior challenge with the chemotactic peptide, Fh4LP, followed by HxA3. As shown in Fig. lC, HxA3 potentiates the release of arachidonic acid by

DAG RELEASED (%

increase

over

FMLP

(CPM

value)

1‘l-“C&AA change 300

RELEASED over

control) HxA3

B

+ pertussis

(100

toxin Y

I 01030

120 60

iis

I

300

180

020

3.

60

[I-‘?Z]-AA (CPM

120 TIME (set)

TIME (set)

change

RELEASED over

control)

- FMLP

HxA3

CONCENTRATION

(nM)

Figure 1. HxA34nduced (100 nM) time-dependentappearanceof A) diacylglycerol (DAG) measured after conversion into [y-32P]-labeledphosphatidic acid (see Methods) and B) [l14C]-labeledfree arachidonic acid from human PMNs (5 x 106 cells/ml PBS) prelabeled in their lipid stores with [l-14C] arachidonic acid (20 min at 37oC). Stock solutions of hepoxilin A3 were made up in DMSO (ll.rg/pl stock solution) and aliquots were addedin PBS. In other experiments, the cells were pretreated with pextussis toxin (2 pg/ml) for 90 min at 37OC and challenged with hepoxilin A3. Results shown are the mean + SE of 3 separate experiments. C). Effect of HxA3 at various concentrations on the release of free [l14C] arachidonic acid from PMNs prelabcled with [l-i4C] arachidonic acid in their lipid stores. Diacylglycerol and arachidonic acid were quantified by TLC (see Methods for details). The inhibitory effect of pertussis toxin (2 pg/ml) on the HxAg-induced release of both products is also shown.

300

nM)

BIOCHEMICAL

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FMLP (100 nM) at a threshoIdconcentrationof 10 - 100nM. This responseto HxA3 is similar in potency (thresholdIO-*- 10-YM) to that recently reportedfor the lipoxins Lx& and B4 (18) The potentiation by HxA3 is approximately 2-fold at 100 nM concentrationof HxA3 and about 5-fold at the highestconcentrationused(1000nM). At all concentrationsused,the effect of HYxA3was blocked by pertussistoxin (2 pg/ml). The synergistic action of the hepoxilins with proinflammatory mediatorssuggeststhat the hepoxilins may serveto amplify or sustainthe inflammatory response. The releaseof both diacylglycerol aswell asunesterifiedarachidonicacid by HxA3 suggests that HxA3 is capableof stimulatingphospholipases C or D and A2 respectively. The lipid substrate for thesephospholipases appearsto be primarily phosphatidylcholine rather than phosphatidyl inositol asrecently shownfor the lipoxins (18). This evidence stemsfrom two additional setsof experiments. First, when total inositol phosphates(IP) were measured(data not shown), release of Ip (only lo- 13%of control) occuredat high concentrationsof hepoxilin (5 J&I) suggestingthat the phospholipidsubstratefor hepoxilin-mediatedphospholipaseis not a phosphatidyl inositol pool and that DAG must be derived from a separatephospholipidpool. The possibility of the releaseof DAG from phosphatidicacid after phospholipaseD hydrolysis of phosphatidylcholine cannot be excluded from theseexperiments. Second,preliminary evidence (not shown)from phospholipid analysissuggeststhat the arachidonicacid releasedby phospholipaseA2 activation by hepoxilin is derived from a phosphatidylcholine pool. Thus, both DAG and aracbidonicacid may result from the action of phospholipases C andA2 on phosphatidyl choline. Since an appreciableamountof IP wasnot releasedafter hepoxilin stimulation,we suggestthat hepoxilin activatescalcium mobilization directly from the endoplasmicreticulum. We have previously shownthat the

HxA Pertussis Toxin

3

(-) t G-Protein

Plase e

C DAG

PC -b

+ choline-phos

PC = Phosphatidyl choline PI = Phosphatidyl inositol PIP2 = PI-bis phosphate IP3 = lnositol triphosphate DAG = 1.2.diacylglycerol PK = Protein kinase Plase = Phospholipase LOX = Lipoxygenase AA = Arachidonic acid LPC = LysoPC

Figure2. Schematic representation describing thestepsby whichhepoxilinA3 mayraise intracellularcalciumin thehumanneutrophil. 947

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hepoxilin-inducedrise in cytosolic calcium appearsto be derived both through influx from the extracellular mediumaswell asthrough the mobilization from intracellular stores(10). Although the actionsof the hepoxilins reportedhereinare receptormediated,we cannot determinefrom these experimentswhether hepoxilin action is initiated via a receptoron the plasmamembraneor on the endoplasmicreticulum. The previously observedcellular actionsof hepoxilins in other systems e.g. in the releaseof insulin from pancreaticislets(7) andin the modulation of neurotransmission in the mammalian(11) and molluscanbrain (13) may result from a similar activation of phospholipases and calcium mobilization asshownherein (Fig 2). Subsequentcalcium dependent activation of phospholipaseA2 may result in the further synthesisof hepoxilins which may serveto amplify or prolong the initial signal. Thesefindings suggestthat HxA3 (andpossiblyother hepoxilins) may be involved in phospholipidmetabolismand remodellingin humanPMNs. Inhibition of the actionsof HxA3 on phospholipidmetabolismreportedin this paperandof the increasein intracellular calcium by pertussistoxin reported earlier (IO) suggeststhat HxA3 may act through a receptor activating a Gprotein. Whether a specific hepoxilin-type receptor is presentin the neutrophil requiresfurther experimentation. ACKNOWLEDGMENTS

This study was madepossibleby grants to S.N. (AICR, UK), E.J.C. (NII-I) and C.R.P-A mw. REFERENCES

1. 32: 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Borgeat, P., Hamberg, M., and Samuelsson,B. (1976) J. Biol. Chem. 251, 7816-7820. Samuelsson,B. (1985) Adv. Prostagl.Thromb. Leukotriene Res. 15, l-9. Serhan, C.N., Hamberg, M., and Samuelsson,B. (1984) Biochem. Biophys. Res. Commun. 118, 943-949. Serhan, C.N., Hamberg, M., and Samuelsson,B. (1984) Proc. Natl. Acad. Sci. (USA) 81, 5335-5339. Pace-Asciak, C.R., Granstrom, E., and Samuelsson,B. (1983) J. Biol. Chem. 258,68356840. Pace-Asciak, C.R. (1984) J. Biol. Chem. 259, 8332-8337. Pace-Asciak, C.R. , and Martin, J.M. (1984) Prostagl. Leukotriene and Med. 16, 173-180. Pace-Asciak, C.R., Martin, J.M., Corey, E.J., and Su, W.G. (1985) Biochem. Biophys. Res. Commun. 128, 942-946. Derewlany, L.O., Pace-Asciak, C.R., and Radde, I.C. (1984) Can. J. Physiol. Pharmacol. 62, 1466-1469. Dho, S., Grinstein, S., Corey, E.J., Su, W.G., and Pace-Asciak, C.R. (1990) Biochem. J. 266, 63-68. Piomelli, D., Shapiro, E., Zipkin, R., Schwartz, J.H., and Feinmark, S.J. (1989) Proc. Natl. Acad. Sci. (USA) 86, 1721-1725. Belardetti, F., Campbell, W.B., Falck, J.R., Demontis, G., and Roslowsky, M. (1989) Neuron 3, 497-505. Carlen, P.L., Gurevich, N., Wu, P.H., Su, W.G., Corey, E.J., and Pace-Asciak, C.R., (1989) Brain Res. 497, 171-176. Pace-Asciak, C.R., Laneuville, O., Chang, M., Reddy, CC., Su, W.G., and Corey, E.J. (1989) Biochem. Biophys. Res. Commun. 163, 1230-1234. Pace-Asciak, CR., Laneuville, O., Su, W.G., Corey, E.J., Gurevich, N., Wu, P.H., and Carlen, P.L. (1990) Proc. Natl. Acad. Sci. (USA) 87, 3037-3041. Preiss, J., Loomis, C.R., Bishop, W.R., Stein, R., Niedel, J.E., and Bell, R.M. (1986) J. Biol. Chem. 261, 8597-8600. Nigam, S., and Muller, S. (1989) Free Rad. Res. Commun. 7, 171-178. Nigam, S, Fiore, S., Luscinskas,F.W., and Serhan, C.N. (1990) J. Cell Physiol. 143, 5 12-523.

Receptor-mediated action of hepoxilin A3 releases diacylglycerol and arachidonic acid from human neutrophils.

We have previously shown that hepoxilin A3 increases the intracellular concentration of Ca+2 in human neutrophils. Herein we address the initial event...
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