- Email: [email protected]
Biosynthesis of paf-acether XIV. Paf-acether output in murine peritoneal macrophages is regulated by the level of acetylhydrolase
Vol.
162,
No.
July
14, 1989
BIOCHEMICAL
1, 1989
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
475-482
BIS OFPAF-ACEIHER XIV. PAF-m wrHYrINr4i?mNE v-w IS~BYTHEIEVELOF~L?GEl
Rhi
Palmantier, Jacques
lNERMU.200, Received
May
25,
hradherent
I-hssina Etra
Maiza,
Ninio
Universit& Paris-S& 32 medes 92140 Clamart, France
Cam&s,
1989
SWM&RY Faf-acether(paf) 24
&me DiLia&, Benveniste and
and
synthesiswasprwicuslyshowntobeinpaimdin Bacillus
~in-activated
mrine
peritoneal
macx@ages as axpared toresidentmacmphages.Wereportherethatthe induction of acetylhydrolasewasxxsponsible forthedemmsed Pf ovltrxrt in 24~adhere& nmx+qes. The kinetic analysis of the enzymes derived frun 2 W,24hr-iudBooactivatedadherentmacrq&ges ClItlfmmplaSna r-weal& that the s for paf was similar whateverthe sourceofthe acetylhydmlase whereas the V was five-fold in 24 hraillzllrd ma-. The ace!$ftqdmlase activitywas -+ Cd -ir&pde& and was not inhibited by addition of alkyl-acyl(longc3Eain)-glycem@sphocMlinesuggestingthattheenzymewasnotapho@mlipaseA2. m1989AcademicPress, Inc.
Paf-acether (paf) metabolic pathway y& the ~latioq/acetylation me&mismhasbeenshcwntobeinstnrmentdlinseverdlcelltypes (mviewed in 1 and 2). It involves phoqholipase A2 (PIA2) hydrolysis of alkyl-acyl-glycero-#z5phocholine (alkyl-acyl-GFC) frm menWane -lipids into alkyl*lX (lyso paf) and its mbseqmnt acetylation by an l-O+lkyl-GEC, aa&yl-CoA : acetyltransferase (EC 2.3.1.67). The acetyltransferase level is responsible for the regulation of paf bonemnuw+erivedmast bioqmt%esisinleulmq&s ardplatelets,muse cells, a2ld mrine maczqhages (mviexedin2). Pafdegradationwas first attrituted to an Acid-Labile Factor described in human sera (3, 4), and later to an l+alkyl-2-acetyl-GFC : acetylhydrolase (EC 3.1.1.48) present in cytosolic fractions of varicms rat tissues (5)‘ in humn xra (6), in plasma and platelets frcxn several species, and inhumnmutmphils, en%thelial cells (7-10). HuEan or rat plasma eosincphils and I
A part of this wxk hasbeenpresentedasapreliminary report The Federation of American Societies Annual Meeti.ng of Experimental Biology (at Las Vegas, NV, USA, May 1-5, 1988) (17).
72nd
475
atthe for
0006-291X/89 $1.50 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
162,
No.
1, 1989
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
mthofthfmare acetylhydmlase is baud to lipoproteins (4-12). acid-labile, Ca2+-kkper&nt and can hydmlyse onlyshortdknfatty acids at m-2 position of glycerol (6). Acetylhydrolase acts as an inpmtantmdulatingfactminpafcoltprt arr?l thus can ~~lpafconcentrationinblood.Indeed,hi~~levelof ac!etylhydrolase was foury3 in plasm frum hypertensive ratsthanfmgn nom&znsive ones (13) and in plasma frcan patients with is&em& cerebmvasculardiseaseascmparedtocontrolsubjects(14). Moreover, acetylhydrolase can be released fm platelets during aqqqation (15). 'Ihe lm level ofpafbiosynthesis inmrineBooactivat&mmq&qes (EDS-MB) or macmFhagesmaintained24hoursinculture(24hr-m))wasnot attributed to a defect in the anabolic pathway (16-18). Thus, it was postulated that in these cells,theregulationcouldbegwernedbythe level ofacetylhydrolase.Webrink~~idencethatinmrrine~~~ maintained in ailture for24 hrbutnot i.nBcoadivated~, the ahanced catabolism of paf v&acetylhydrolaseinAucticmismsponsible for the low a&put of the mdiator.
Ma-cfes
Macr@lages k~ere obtained as described in (16) and plated for 2 or 24 hr. Tim hour-adherent activat&*were&tainedfrmtioeinjectedwith Bacillus CalmetteGu&rin (wx;) (In&it& l&steurProdukicn,Mames-IaCogEtte,France) forapericdoflto2mek(~,16). Identification and ouantitation of oaf metabolites FJaf metabolism by lmr' peritomlmacro@ageswasdekrmimd ' (l-O-[ 3 locizdeql-2-acetyl-, 179 [hoctadecyl-Pf U.K.) or [2H]acetyl paf (l-l-2Amersham). Labeled paf fmlll5to5OOnM, (0.01 to 0.05 wi) was a&d to adhere& cells in 500 /,A1ml.tllr.X lI&im an3 inwbaW for 5 to 120 min at 37'C. F&actions wzre stqq& by adding ethanol (80% final wnwntration). Lipidswereextracted frmsupematants and cell mnolayers by omrnight incubation at 4'C. After centrifqation (1500 x g, 20 min, 4°C) the ethanolic supernatiwerecollectedand dried uMer an air stream at 40'C and kept at -20'C prior to the high pressure liquid chmmtogramy (HPLC) analysis.Unspecificpafmetabolian (less than 5%) imatal conditicms, except thatthe wem itzukated for lhrat37’Cwith zymyzan (200 w/ml, Sigm &em. Co, St Icuis, MO, USA) before addition of [ Hloctadecyl-paf. Lipid extracts were resolved by HPLC (Mkroporasil aAmm,Waters,Milford,~,USA), diCWO33XlE~ /mfk?mml/k&er (60:50:5, v/v) as mabile phase. -ing Fkactions were collected atlmin internals, dried, dissolved inethanol and mixed with 0CS (Amershm Int.) prior to scintill.aticmcumting. S@kganyelin ti @oqAatidylcholinefmnSigmand Olabeleapafand wereusedas-. lyso paf (laaetyl-wfPa' used,thereleaseof[H]acetatewasdeterminedas~~below. 476
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AcetYlhvdrolase assay The acetylhydmlaseactivitywasmasured inosllsonica~frau2hr-, 24 ~and~~~g~andinrmrinepl~.Plasaawas~after centrifugation of mouse blood (800 x g, 15 min) collected usirq EDI!A (2.5 mM final) as anticoagulant. Then platelets ~remvedbyacklitional centrifugation (1500 x g, 15 min, 4'C). Protein content was determined in oell lysates and in plasm using umy's m&hod (19). 5-15 pg protein) or plasm diluted 1:lOO M==@=w 1Y=* (50 a, were added to 440 1.I1ofabufferpH8.0containirqinmM:NaCl l37,KCl 2.6, HEIFS 4.2, EDTA 2 orCaC1210.After 3 min psdmubatim at 37'C, the ma&ion was initiated with 10 ~1 of [ Hlacetyl-paf (0.01 Gi, 2.5 to 50 /AM final cwwntration). Incubationswereperformed forlominat 37'C lnilessstated~iseandstappedinanicebathwithsfaultaneous addition of 0.4 ml acetic acid (5%) $jolution confining [ C]acetate (6000 m) as an internal sitar&d. [ H]acetyl-paf was extracted in oyganic phase by adding 2 (1:1, v/v). Muctures were osntrifqed 10 min) to facilitate #mse separation. The anmnt of Iacetate in the aqueous@mseresulting fxnn the acetylhydrolase assay was determined byliquidscintillation assolvent. Emlkspreparedusirqheat*~ using ACS (Amemham Ilk.) enzyme (100°C for 10 min) never exceed& 5% of test& sanples and were subtracted prior to calailati$ns of -f"y" activities. The results are expm in nml x min x nq14 protein after correction for extractionlosses calculatedusingthe [ C]acetate internal star&ud.
Metatmlismofoaf in intactmacrmhacfes Incubation of [3H]cctadeql-paf with intact macrqhages for 1 hr at 37°C resulted in the fomtion of two prcducts corresponding to lyso paf and alkyl-acyl-GFC (Table I). In 2hr-andEKX+macmphages,onlyl8to 38 % of paf washydmly&,whereas in 24 hr-m-, 69 to 90 % of PafmAabolisminvarimsmacmPhagepoFPrlations
TableI.
w.
M-wMf=
Fadioactivitydistributiana
N' Faf
Lyso Pf
I
2hr-Ma 24 h-W
82.0 55.5 27.0
18.0 37.5 69.0
0.0 7.0 4.0
70.0 61.0 6.0
27.0 32.0 90.0
3.0 7.0 4.0
II 2hr-m 24 h-N
BOG- ad24 hr-xe~gesjnalbaw forlhrat 37'C with [3H]octade@-paf (0.01 jCi, 100 H4). Lipids were pcteflanaseparateausingHpI13. values are IreaIls of duplicate detankations with variations belaw 10 %.
2 *,
477
Vol. 162, No. 1, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the added [3H]ocbdecyl-paf was reamred as lysopafwithinlhr. In of mamqhages, only a small part frun 0 to 7 % of all types [3H]octa&cyl-paf was mstabolised into alkyl-acyl%FC. !UE initial furtherstudicdin2hr-and24 velccity of paf clfzqradation was The release of [3H]acetate was hr-ma~es using [3H]acetyl-paf. pmportional with timeupto15ard5minfor2 hr-&24h~~~cn@ages respectively andrea&~&theplateauat15minonlyfor24hrmaczu@ges. mtion 500 nM ; The hydrolysis of paf was still unsaturatedat conoentmtionsbeingstimulatorytointactmamqhages (20). In t.w m@= separate experiments the initial ve.locitycxkulated inthepzesenceof 500 rM ofpafandthelinearportion ofthetime-ccxlrsecurvewas :13 D 189 pm1 x ny-' pmt. for2hrar~I24hr-mcmphagesmspectively. of mf acetvlhvdmlase activitv in muritle lMcroDhage lvsatesand&SsIna In the folluwing experiments, acetylhydrolaseactivitywasdetemined in suspension ofsonicatedunstimlatdmacmphagesbythemeasurpmerrtof free [3H]acetate released frcan 10 /JM [3H]acetyl-paf. Zmstylhydrolase asaX@arEdtolAetimeof activity inalltypesofmacrqhageswaslinear incubation thmugh at least 10 min (Fig. 1) and to protein a8xmkraticm at least up to 15 I.rg xml-l (datanotshcm). Pafhydmlysiswasthen studid as a fun&ion of paf concmtmtion. [3H]acetatereleasefrm [3H]aoetyl-paf reached a plateau intheprfzsenm of25jAMpafin2hP,
Characteristics
01
20
40
60
Time (min)
02
-0.0s
0
0.06
0.16
0.24
0.32
14s1
Time-course of [3H]acetyl-paf Lysates fran 2 m?aa were inabted at 37.C with /AM). At the i.rdicaM times, quantifiedbsdesczrikedinMaterialsandEWhcds.
hydmlysis
by
mine
.
Rekase
of
[3H]acetati frcm lysates incubated
nwns of duplicate detmnimtims (with DpXsedativeof3to6experimnb(statistical~~~isgivenin Table II). 478
2 hrfor variations
(0.01 pCI,
10
(0 ), 24 h?? ( A) ad 10 min with defined below
10%) andare
0.40
BIOCHEMICAL
Vol. 162, No. 1, 1989
Table II.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Ximticcm&antsofacety~ydrolaseinmur~ nla~aKl.inplasma
2 lx--m
14.8 _+ 3.2 17.8 _+ 2.2 13.6 f 1.4 19.3 _+3.8
(6)
(3) 24 lx--M3 (3) Plasm (3) -
3.8 5.1 21.9
17.6
for acetylhy3rolase were ca1aUatedfx-m %3x dcuble reciprccal plots shown in Fig. 2 and 4 arxl f sm. In parentheses, nmber of represerrts experiments. and
24 hr-
[email protected]~,theLineweaver EUrk plot of the data gave qpamntsimilarK,valuesforpaf (Fig. 2 and Table II). lQn?hsmm acetylhydrolase fran 2 hr- andED+maM exhibiteda maparable level of activity. Incontmst, amarkedly inmmsedV.was cbsemed in 24 h-==PwF lysatEeasczCqamdto2hr-and (Table II). After zymsan stimlation the --@age lysates acetylhydmlase kinetic constants remained unchatq&lforallmcr@qe ~ati~studiedas~tounstinailatedones(data~~). the deacetylation activity in mrine plasma. The N-c% we characterised aaetylhydmlase activity was linear witithetime of i.mubtionuptu15 machby a plateau at 60 min and with protein azmoentitimatleast m, up to
50 pg x ml-l
(datz
not
shm).%andV--determinedas
described for macmphage lysates (Fig. abtainedusing~~gelysatesandplasnraweresimilar.
3).
We fourd
that
I$, values
[3H]aoetate release in the preserce of he PlaSmi F’aure 3 iitaebd. for 10 min with defined cmcmtrations of % [ ]acetyl-paf (0.01 /.tci, 5 to 50 @%).Inset : Lbeweaver-Eurk plot of the data. Fesulte are means of duplicatedetenninationswithvariationSbelCw1O% ad am representativeof3separate~~~(statisticalanalysisis given in Table II). 479
BIOCHEMICAL
Vol. 162, No. 1, 1989 Wle
XII.
Acetylhydmlaee
AND BIOPHYSICAL RESEARCH COMMUNICATIONS inhibition
ad
specificity
J-F=-
IxUitionsa 2hr-m
EmA(2 w ELWA (2 I@¶) + FMSF (2 IrM) ca2+ (10 rn) Ca2+ (10 @I) + EMSF (2 Ml) Alky1-=Y1(10 Pa Aucyl-acyl~ (20 Pw Alkyl-acylGFC (40 #f)
2.1_+ 0.8 1.9 2.2 -+ 0.9 1.4 2.1_+ 0.8 2.0 _+ 0.8 1.9 _+ 0.9
24 hr-m 8.8 7.4 7.2 7.3 7.7 7.5 7.0
PlB 6.9 -+ 0.1 5.5 -+ 0.6 6.5 5.5 6.7 6.6 6.3
_+ 2.7 _+ 2.2 f 1.8 _+ 2.4 -+ 2.1
a Added capcads were disolved in the acetylhydrol buffer& pdmubdted 5 min at 37°C with the enzyme prior to T[ ]acetyl-paf eupplaaentation for 10 min. seeMaterialsaniMeI3udssecticmfor
details.
starting
solution
of RGF (1M indimthylsulfoqde)
dissolved by eonication inthebuffer; alkyl-acylGFC ethanol)waedimctlyacUedtotheaesay. Results am expressed in rim1 acetate releaeed pmt. ardaremem3~smof3exFxxi.menk,exceptwhereSmare cm.i~,therefllltsbeingmeansof2experiments.
(2nMin80 x
was %
minB1xqq
In order to detenninewhethertheacetylhydrolaseactivitiee~ in nurine macrqhages and plasma were different fmnthatoftypical ccqmmk known to influence theactivityofPLA2~or purz, various of acetylhydmlase, wereaddedtothe inahtionmedium. Resulteobbimd after addition of EUTA, ca2+, phenylmethylsulfonyl fluoride (RBF) and alkyl-acyl-GFC are shcmn in Table III. The presence of Ca2+ ami RBF did not mdifytheacetylhydmlaseactivity.AdditionofdLkyl-acyl~ (10 to 40 /AM) to the assay did not diminish significantly the yield of acetylhydrolase reaction, supporting the specificity of the latter enzym for short chain fatty acid esterified to the m-2 position of glycem&m@lipids. ThesereeultsalsosuggestthatpIAzdidnot contributetopafhydrolysis inmacrophag~andinplasma. DISUJSSICN
Paf is degmded to variaxs degmss by different cell types (5-10). m wa shcw that while intact 2 badherent resident or Booactivated macrqhagesdegradedpaftoscmeextent, thisdegradationwasgreatly e&axed when cells wem cultured for 24 hr. Interestingly, only a mall portion of lyso paf was rmcylated with long chain fattyacidinto alkyl-acyl-GE by Imxuphages as qpce& to human (21) and rakbit IleubqWls (22) kalt in good agreement withthemsultsabtainedwiti 480
Vol.
162,
No.
1, 1989
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
human eMio#elial cells (9). Haever a Ca2+-w PIA was descrw in macrcphages (23) it semsunlikelythatthejxmasedpaf hydmlysis in 24 hr-xnacrophag~ was duetothatpartiaAarenzym.The induction of an acetylhydrolase that is ca2+-iMeqmk& and not inhibited by alkyl-acyl(long chain)-GFc explains certainly betterthe obsemed pheruanenon. ?hekineticanalysisofacetylhydrolasederived fmn 24 hr-, andBaimacm@a~indi~tedthatin24hr-macqhages, 2 hr-, of the reaction was inueamAabout5timsasmaparedto2 the %ax hr-wt mxrophages and EXG-macrophages.Hmever, the specificityof the acetylhydrolase remained similar asjudgfxIbysimilarY,valuesfor These data i~@ythatmstprobablytklevelofacetylhydmlaseis paf. kreasedduring24hradhermce but not its affinity for paf. This incrase is likely duetothedenmosynthesisoftheenzymbecau.seof the relatively long period of induction. In
omtmst
to acetyltransfemse that is inm~~+& after zymean we fou%l no variation in acetylhydrolase activity after mlenge (16) I zymxan-induced macraphage stinulation. The latter result ard the fact that acetylhydrolase activity was not increased in BOG-activated ma~ges idkate that induction of acetylhydmlaseisnotadirect comegum of [email protected] impair& paf formation by ECG-activatedmacrophagesremainsstill ancpenquestion Inlightof ourresults, thisdefectcannotbereadilyexplair&by (16). the erhncd catabolism ofpafbythesecells.Hmever,~~that the deacetylatioqkeacylation reaction leading to alkyl-acyl+XC form&ion flmn exogenously added [3H]octadecyl-paf was slighty increased in Eloana~ges as ccsnparedto 2 hr-adherentmcrqAages. Finally, it seems likelythatacetylhydmlaseactivity inmae may represent one of the mechanisms that controlspafoutqxt.Sucha self-controling mechanism could operate in vivo. Inked, prolonged adherence of macrophages to biological surfaces suchasadjacentcell mendaranes (e.g. in infkmmkory lesions) and to bloodvessels, could conlxibute to a reduction of paf availability anrl thus of its effects.
We would sdl@ZS.
1iJce to
thankMr.
Gilles
E?essou for the HPLC analysis
Rzsarch 1. Snydm, F. (1985) in Medicinal ed. Jchn Wiley a.txI Sons, Inc. pp. 107-140. 481
Rewievs, Vol.
of (xv
5. F. Snyder,
Vol. 162, No. 1, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2. Ninio, E. (1987) in New Horizons in Platelet-Activati Factor Reseaxh, C.M. Whlcw and L.M. Ice, &Is. John Wiley and Sons, Inc. up. 27-35. 3. Pinckard R.N., Farr, R.S. and Hanahan, D.J. (1979) J. Imnunol. 123, 1847-185;. 4. Fax-r, R.S., Co& C.P., Wardlow, M.L. ti Joqensen, R (1980) Clin. limmol. Immopdthol. 15, 318-330. 5. Blank, M.L., Lee, T-c., Fitzgerald, V. and Snyder, F (1981) J. Biol. #em. 256, 175-178. 6. Farr, R-S., Wa.mbw, M.L., Cox, C.P., Merq, K.E. ard Greene, D.E. (1983) Fed. Prcc. 42, 3120-3122. 7.&e, 'p-c., Malone, B., Wassermn, S.I., Fitzgerald, V. and Snyder, F (1982) Biochem. Biqhys. Res. Ccmmun.105, 1303-1308. 8. Kramer, R.M., Patton, G.M., Pritzker, C.R. and Deykin, D. (1984) J. Biol. C&m 259, 13316-13320. 9. Blank, M.L., Spector, A-A, Kxiuce, T.L., Lee T-c. ard Snyder, F. (1986). Biokim. Biophys. Acb 876, 373-378. 10. Hirafuji, M., bkncia-Huerta, J.M. and Benveniste, J. (1987) Bicchim. Biqchys. kta 330, 359-369. 11. Stafforini, D-M., McIntyre, T-M., Carter, M-E., and Presmtt, S.M. (1987) J. Biol. Chem. 262, 4215-4222. 12. Pritchard, P.H. (1987) Biohem. J. 246, 791-794. 13. Blank, M-L., Hall, M-N., Cress, E.A. and Snyder, F. (1983) Biochem. Biqhys. Res. Ccmnm. 113, 666-671. 14. Satoh, K., Imaizumi, T., Kawamura, Y., Yoshida, H., Tabmatsu, S., and Mizono, S. (1988). Prostaglandins 35, 685-698. 15. !iksuki, Y., Miwa, M., Harada, M., and Matxxmdo, M. (1988). Eur. J. Biochem. 1, 117-120. 16. Roubin, R., Ihlicust, A., Haye-Legran& I., Ninio, E. and Bemmkte I J. (1986) J. Immnol. 136, 1796-1802. 17. Palmantier, R., hlioust, A., Benveniste, J. and Ninio, E. (1988) FASEBJ. 2, Al739 (abstr.) 18. biliouSt, A., Viviex, E., Meslier, N., Rmbin, R., Haye-Legrand, I., and Benveniste, Biochem. J. (inpress). 19. Lmry, O-H., Rosebrough, N-J., Farr, A.L. and Randall, R.J. (1951) J. Biol. C&m. 193, 265-275. 20. Harhnq, H-P., FSwnham, M-J., Winkelnmn, J., Ehglbeqer, W. and Hadding, U. (1983) Int. J. scol. 5, 115-121. 21. Hilton, F-H., O'Flaherky, J-T., Marshall Ellis, J., Smr&en, C.L. and Wykle, R.L. (1983) J. Biol. Chn. 258, 7268-7271. 22. Muddler, H-W., O'Flaherty, J-T. and Wykle, R.L. (1982) Lipids 17, 72-77. 23. FX+mson, N., Bechrdite, S., Wang, P., Wiate, M. and Elsbach, P. (1973) Bicchim. Biophys. Acta 296, 365-373.
482
162,
No.
July
14, 1989
BIOCHEMICAL
1, 1989
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
475-482
BIS OFPAF-ACEIHER XIV. PAF-m wrHYrINr4i?mNE v-w IS~BYTHEIEVELOF~L?GEl
Rhi
Palmantier, Jacques
lNERMU.200, Received
May
25,
hradherent
I-hssina Etra
Maiza,
Ninio
Universit& Paris-S& 32 medes 92140 Clamart, France
Cam&s,
1989
SWM&RY Faf-acether(paf) 24
&me DiLia&, Benveniste and
and
synthesiswasprwicuslyshowntobeinpaimdin Bacillus
~in-activated
mrine
peritoneal
macx@ages as axpared toresidentmacmphages.Wereportherethatthe induction of acetylhydrolasewasxxsponsible forthedemmsed Pf ovltrxrt in 24~adhere& nmx+qes. The kinetic analysis of the enzymes derived frun 2 W,24hr-iudBooactivatedadherentmacrq&ges ClItlfmmplaSna r-weal& that the s for paf was similar whateverthe sourceofthe acetylhydmlase whereas the V was five-fold in 24 hraillzllrd ma-. The ace!$ftqdmlase activitywas -+ Cd -ir&pde& and was not inhibited by addition of alkyl-acyl(longc3Eain)-glycem@sphocMlinesuggestingthattheenzymewasnotapho@mlipaseA2. m1989AcademicPress, Inc.
Paf-acether (paf) metabolic pathway y& the ~latioq/acetylation me&mismhasbeenshcwntobeinstnrmentdlinseverdlcelltypes (mviewed in 1 and 2). It involves phoqholipase A2 (PIA2) hydrolysis of alkyl-acyl-glycero-#z5phocholine (alkyl-acyl-GFC) frm menWane -lipids into alkyl*lX (lyso paf) and its mbseqmnt acetylation by an l-O+lkyl-GEC, aa&yl-CoA : acetyltransferase (EC 2.3.1.67). The acetyltransferase level is responsible for the regulation of paf bonemnuw+erivedmast bioqmt%esisinleulmq&s ardplatelets,muse cells, a2ld mrine maczqhages (mviexedin2). Pafdegradationwas first attrituted to an Acid-Labile Factor described in human sera (3, 4), and later to an l+alkyl-2-acetyl-GFC : acetylhydrolase (EC 3.1.1.48) present in cytosolic fractions of varicms rat tissues (5)‘ in humn xra (6), in plasma and platelets frcxn several species, and inhumnmutmphils, en%thelial cells (7-10). HuEan or rat plasma eosincphils and I
A part of this wxk hasbeenpresentedasapreliminary report The Federation of American Societies Annual Meeti.ng of Experimental Biology (at Las Vegas, NV, USA, May 1-5, 1988) (17).
72nd
475
atthe for
0006-291X/89 $1.50 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
162,
No.
1, 1989
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
mthofthfmare acetylhydmlase is baud to lipoproteins (4-12). acid-labile, Ca2+-kkper&nt and can hydmlyse onlyshortdknfatty acids at m-2 position of glycerol (6). Acetylhydrolase acts as an inpmtantmdulatingfactminpafcoltprt arr?l thus can ~~lpafconcentrationinblood.Indeed,hi~~levelof ac!etylhydrolase was foury3 in plasm frum hypertensive ratsthanfmgn nom&znsive ones (13) and in plasma frcan patients with is&em& cerebmvasculardiseaseascmparedtocontrolsubjects(14). Moreover, acetylhydrolase can be released fm platelets during aqqqation (15). 'Ihe lm level ofpafbiosynthesis inmrineBooactivat&mmq&qes (EDS-MB) or macmFhagesmaintained24hoursinculture(24hr-m))wasnot attributed to a defect in the anabolic pathway (16-18). Thus, it was postulated that in these cells,theregulationcouldbegwernedbythe level ofacetylhydrolase.Webrink~~idencethatinmrrine~~~ maintained in ailture for24 hrbutnot i.nBcoadivated~, the ahanced catabolism of paf v&acetylhydrolaseinAucticmismsponsible for the low a&put of the mdiator.
Ma-cfes
Macr@lages k~ere obtained as described in (16) and plated for 2 or 24 hr. Tim hour-adherent activat&*were&tainedfrmtioeinjectedwith Bacillus CalmetteGu&rin (wx;) (In&it& l&steurProdukicn,Mames-IaCogEtte,France) forapericdoflto2mek(~,16). Identification and ouantitation of oaf metabolites FJaf metabolism by lmr' peritomlmacro@ageswasdekrmimd ' (l-O-[ 3 locizdeql-2-acetyl-, 179 [hoctadecyl-Pf U.K.) or [2H]acetyl paf (l-l-2Amersham). Labeled paf fmlll5to5OOnM, (0.01 to 0.05 wi) was a&d to adhere& cells in 500 /,A1ml.tllr.X lI&im an3 inwbaW for 5 to 120 min at 37'C. F&actions wzre stqq& by adding ethanol (80% final wnwntration). Lipidswereextracted frmsupematants and cell mnolayers by omrnight incubation at 4'C. After centrifqation (1500 x g, 20 min, 4°C) the ethanolic supernatiwerecollectedand dried uMer an air stream at 40'C and kept at -20'C prior to the high pressure liquid chmmtogramy (HPLC) analysis.Unspecificpafmetabolian (less than 5%) imatal conditicms, except thatthe wem itzukated for lhrat37’Cwith zymyzan (200 w/ml, Sigm &em. Co, St Icuis, MO, USA) before addition of [ Hloctadecyl-paf. Lipid extracts were resolved by HPLC (Mkroporasil aAmm,Waters,Milford,~,USA), diCWO33XlE~ /mfk?mml/k&er (60:50:5, v/v) as mabile phase. -ing Fkactions were collected atlmin internals, dried, dissolved inethanol and mixed with 0CS (Amershm Int.) prior to scintill.aticmcumting. S@kganyelin ti @oqAatidylcholinefmnSigmand Olabeleapafand wereusedas-. lyso paf (laaetyl-wfPa' used,thereleaseof[H]acetatewasdeterminedas~~below. 476
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AcetYlhvdrolase assay The acetylhydmlaseactivitywasmasured inosllsonica~frau2hr-, 24 ~and~~~g~andinrmrinepl~.Plasaawas~after centrifugation of mouse blood (800 x g, 15 min) collected usirq EDI!A (2.5 mM final) as anticoagulant. Then platelets ~remvedbyacklitional centrifugation (1500 x g, 15 min, 4'C). Protein content was determined in oell lysates and in plasm using umy's m&hod (19). 5-15 pg protein) or plasm diluted 1:lOO M==@=w 1Y=* (50 a, were added to 440 1.I1ofabufferpH8.0containirqinmM:NaCl l37,KCl 2.6, HEIFS 4.2, EDTA 2 orCaC1210.After 3 min psdmubatim at 37'C, the ma&ion was initiated with 10 ~1 of [ Hlacetyl-paf (0.01 Gi, 2.5 to 50 /AM final cwwntration). Incubationswereperformed forlominat 37'C lnilessstated~iseandstappedinanicebathwithsfaultaneous addition of 0.4 ml acetic acid (5%) $jolution confining [ C]acetate (6000 m) as an internal sitar&d. [ H]acetyl-paf was extracted in oyganic phase by adding 2 (1:1, v/v). Muctures were osntrifqed 10 min) to facilitate #mse separation. The anmnt of Iacetate in the aqueous@mseresulting fxnn the acetylhydrolase assay was determined byliquidscintillation assolvent. Emlkspreparedusirqheat*~ using ACS (Amemham Ilk.) enzyme (100°C for 10 min) never exceed& 5% of test& sanples and were subtracted prior to calailati$ns of -f"y" activities. The results are expm in nml x min x nq14 protein after correction for extractionlosses calculatedusingthe [ C]acetate internal star&ud.
Metatmlismofoaf in intactmacrmhacfes Incubation of [3H]cctadeql-paf with intact macrqhages for 1 hr at 37°C resulted in the fomtion of two prcducts corresponding to lyso paf and alkyl-acyl-GFC (Table I). In 2hr-andEKX+macmphages,onlyl8to 38 % of paf washydmly&,whereas in 24 hr-m-, 69 to 90 % of PafmAabolisminvarimsmacmPhagepoFPrlations
TableI.
w.
M-wMf=
Fadioactivitydistributiana
N' Faf
Lyso Pf
I
2hr-Ma 24 h-W
82.0 55.5 27.0
18.0 37.5 69.0
0.0 7.0 4.0
70.0 61.0 6.0
27.0 32.0 90.0
3.0 7.0 4.0
II 2hr-m 24 h-N
BOG- ad24 hr-xe~gesjnalbaw forlhrat 37'C with [3H]octade@-paf (0.01 jCi, 100 H4). Lipids were pcteflanaseparateausingHpI13. values are IreaIls of duplicate detankations with variations belaw 10 %.
2 *,
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the added [3H]ocbdecyl-paf was reamred as lysopafwithinlhr. In of mamqhages, only a small part frun 0 to 7 % of all types [3H]octa&cyl-paf was mstabolised into alkyl-acyl%FC. !UE initial furtherstudicdin2hr-and24 velccity of paf clfzqradation was The release of [3H]acetate was hr-ma~es using [3H]acetyl-paf. pmportional with timeupto15ard5minfor2 hr-&24h~~~cn@ages respectively andrea&~&theplateauat15minonlyfor24hrmaczu@ges. mtion 500 nM ; The hydrolysis of paf was still unsaturatedat conoentmtionsbeingstimulatorytointactmamqhages (20). In t.w m@= separate experiments the initial ve.locitycxkulated inthepzesenceof 500 rM ofpafandthelinearportion ofthetime-ccxlrsecurvewas :13 D 189 pm1 x ny-' pmt. for2hrar~I24hr-mcmphagesmspectively. of mf acetvlhvdmlase activitv in muritle lMcroDhage lvsatesand&SsIna In the folluwing experiments, acetylhydrolaseactivitywasdetemined in suspension ofsonicatedunstimlatdmacmphagesbythemeasurpmerrtof free [3H]acetate released frcan 10 /JM [3H]acetyl-paf. Zmstylhydrolase asaX@arEdtolAetimeof activity inalltypesofmacrqhageswaslinear incubation thmugh at least 10 min (Fig. 1) and to protein a8xmkraticm at least up to 15 I.rg xml-l (datanotshcm). Pafhydmlysiswasthen studid as a fun&ion of paf concmtmtion. [3H]acetatereleasefrm [3H]aoetyl-paf reached a plateau intheprfzsenm of25jAMpafin2hP,
Characteristics
01
20
40
60
Time (min)
02
-0.0s
0
0.06
0.16
0.24
0.32
14s1
Time-course of [3H]acetyl-paf Lysates fran 2 m?aa were inabted at 37.C with /AM). At the i.rdicaM times, quantifiedbsdesczrikedinMaterialsandEWhcds.
hydmlysis
by
mine
.
Rekase
of
[3H]acetati frcm lysates incubated
nwns of duplicate detmnimtims (with DpXsedativeof3to6experimnb(statistical~~~isgivenin Table II). 478
2 hrfor variations
(0.01 pCI,
10
(0 ), 24 h?? ( A) ad 10 min with defined below
10%) andare
0.40
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Table II.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Ximticcm&antsofacety~ydrolaseinmur~ nla~aKl.inplasma
2 lx--m
14.8 _+ 3.2 17.8 _+ 2.2 13.6 f 1.4 19.3 _+3.8
(6)
(3) 24 lx--M3 (3) Plasm (3) -
3.8 5.1 21.9
17.6
for acetylhy3rolase were ca1aUatedfx-m %3x dcuble reciprccal plots shown in Fig. 2 and 4 arxl f sm. In parentheses, nmber of represerrts experiments. and
24 hr-
[email protected]~,theLineweaver EUrk plot of the data gave qpamntsimilarK,valuesforpaf (Fig. 2 and Table II). lQn?hsmm acetylhydrolase fran 2 hr- andED+maM exhibiteda maparable level of activity. Incontmst, amarkedly inmmsedV.was cbsemed in 24 h-==PwF lysatEeasczCqamdto2hr-and (Table II). After zymsan stimlation the --@age lysates acetylhydmlase kinetic constants remained unchatq&lforallmcr@qe ~ati~studiedas~tounstinailatedones(data~~). the deacetylation activity in mrine plasma. The N-c% we characterised aaetylhydmlase activity was linear witithetime of i.mubtionuptu15 machby a plateau at 60 min and with protein azmoentitimatleast m, up to
50 pg x ml-l
(datz
not
shm).%andV--determinedas
described for macmphage lysates (Fig. abtainedusing~~gelysatesandplasnraweresimilar.
3).
We fourd
that
I$, values
[3H]aoetate release in the preserce of he PlaSmi F’aure 3 iitaebd. for 10 min with defined cmcmtrations of % [ ]acetyl-paf (0.01 /.tci, 5 to 50 @%).Inset : Lbeweaver-Eurk plot of the data. Fesulte are means of duplicatedetenninationswithvariationSbelCw1O% ad am representativeof3separate~~~(statisticalanalysisis given in Table II). 479
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XII.
Acetylhydmlaee
AND BIOPHYSICAL RESEARCH COMMUNICATIONS inhibition
ad
specificity
J-F=-
IxUitionsa 2hr-m
EmA(2 w ELWA (2 I@¶) + FMSF (2 IrM) ca2+ (10 rn) Ca2+ (10 @I) + EMSF (2 Ml) Alky1-=Y1(10 Pa Aucyl-acyl~ (20 Pw Alkyl-acylGFC (40 #f)
2.1_+ 0.8 1.9 2.2 -+ 0.9 1.4 2.1_+ 0.8 2.0 _+ 0.8 1.9 _+ 0.9
24 hr-m 8.8 7.4 7.2 7.3 7.7 7.5 7.0
PlB 6.9 -+ 0.1 5.5 -+ 0.6 6.5 5.5 6.7 6.6 6.3
_+ 2.7 _+ 2.2 f 1.8 _+ 2.4 -+ 2.1
a Added capcads were disolved in the acetylhydrol buffer& pdmubdted 5 min at 37°C with the enzyme prior to T[ ]acetyl-paf eupplaaentation for 10 min. seeMaterialsaniMeI3udssecticmfor
details.
starting
solution
of RGF (1M indimthylsulfoqde)
dissolved by eonication inthebuffer; alkyl-acylGFC ethanol)waedimctlyacUedtotheaesay. Results am expressed in rim1 acetate releaeed pmt. ardaremem3~smof3exFxxi.menk,exceptwhereSmare cm.i~,therefllltsbeingmeansof2experiments.
(2nMin80 x
was %
minB1xqq
In order to detenninewhethertheacetylhydrolaseactivitiee~ in nurine macrqhages and plasma were different fmnthatoftypical ccqmmk known to influence theactivityofPLA2~or purz, various of acetylhydmlase, wereaddedtothe inahtionmedium. Resulteobbimd after addition of EUTA, ca2+, phenylmethylsulfonyl fluoride (RBF) and alkyl-acyl-GFC are shcmn in Table III. The presence of Ca2+ ami RBF did not mdifytheacetylhydmlaseactivity.AdditionofdLkyl-acyl~ (10 to 40 /AM) to the assay did not diminish significantly the yield of acetylhydrolase reaction, supporting the specificity of the latter enzym for short chain fatty acid esterified to the m-2 position of glycem&m@lipids. ThesereeultsalsosuggestthatpIAzdidnot contributetopafhydrolysis inmacrophag~andinplasma. DISUJSSICN
Paf is degmded to variaxs degmss by different cell types (5-10). m wa shcw that while intact 2 badherent resident or Booactivated macrqhagesdegradedpaftoscmeextent, thisdegradationwasgreatly e&axed when cells wem cultured for 24 hr. Interestingly, only a mall portion of lyso paf was rmcylated with long chain fattyacidinto alkyl-acyl-GE by Imxuphages as qpce& to human (21) and rakbit IleubqWls (22) kalt in good agreement withthemsultsabtainedwiti 480
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human eMio#elial cells (9). Haever a Ca2+-w PIA was descrw in macrcphages (23) it semsunlikelythatthejxmasedpaf hydmlysis in 24 hr-xnacrophag~ was duetothatpartiaAarenzym.The induction of an acetylhydrolase that is ca2+-iMeqmk& and not inhibited by alkyl-acyl(long chain)-GFc explains certainly betterthe obsemed pheruanenon. ?hekineticanalysisofacetylhydrolasederived fmn 24 hr-, andBaimacm@a~indi~tedthatin24hr-macqhages, 2 hr-, of the reaction was inueamAabout5timsasmaparedto2 the %ax hr-wt mxrophages and EXG-macrophages.Hmever, the specificityof the acetylhydrolase remained similar asjudgfxIbysimilarY,valuesfor These data i~@ythatmstprobablytklevelofacetylhydmlaseis paf. kreasedduring24hradhermce but not its affinity for paf. This incrase is likely duetothedenmosynthesisoftheenzymbecau.seof the relatively long period of induction. In
omtmst
to acetyltransfemse that is inm~~+& after zymean we fou%l no variation in acetylhydrolase activity after mlenge (16) I zymxan-induced macraphage stinulation. The latter result ard the fact that acetylhydrolase activity was not increased in BOG-activated ma~ges idkate that induction of acetylhydmlaseisnotadirect comegum of [email protected] impair& paf formation by ECG-activatedmacrophagesremainsstill ancpenquestion Inlightof ourresults, thisdefectcannotbereadilyexplair&by (16). the erhncd catabolism ofpafbythesecells.Hmever,~~that the deacetylatioqkeacylation reaction leading to alkyl-acyl+XC form&ion flmn exogenously added [3H]octadecyl-paf was slighty increased in Eloana~ges as ccsnparedto 2 hr-adherentmcrqAages. Finally, it seems likelythatacetylhydmlaseactivity inmae may represent one of the mechanisms that controlspafoutqxt.Sucha self-controling mechanism could operate in vivo. Inked, prolonged adherence of macrophages to biological surfaces suchasadjacentcell mendaranes (e.g. in infkmmkory lesions) and to bloodvessels, could conlxibute to a reduction of paf availability anrl thus of its effects.
We would sdl@ZS.
1iJce to
thankMr.
Gilles
E?essou for the HPLC analysis
Rzsarch 1. Snydm, F. (1985) in Medicinal ed. Jchn Wiley a.txI Sons, Inc. pp. 107-140. 481
Rewievs, Vol.
of (xv
5. F. Snyder,
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AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2. Ninio, E. (1987) in New Horizons in Platelet-Activati Factor Reseaxh, C.M. Whlcw and L.M. Ice, &Is. John Wiley and Sons, Inc. up. 27-35. 3. Pinckard R.N., Farr, R.S. and Hanahan, D.J. (1979) J. Imnunol. 123, 1847-185;. 4. Fax-r, R.S., Co& C.P., Wardlow, M.L. ti Joqensen, R (1980) Clin. limmol. Immopdthol. 15, 318-330. 5. Blank, M.L., Lee, T-c., Fitzgerald, V. and Snyder, F (1981) J. Biol. #em. 256, 175-178. 6. Farr, R-S., Wa.mbw, M.L., Cox, C.P., Merq, K.E. ard Greene, D.E. (1983) Fed. Prcc. 42, 3120-3122. 7.&e, 'p-c., Malone, B., Wassermn, S.I., Fitzgerald, V. and Snyder, F (1982) Biochem. Biqhys. Res. Ccmmun.105, 1303-1308. 8. Kramer, R.M., Patton, G.M., Pritzker, C.R. and Deykin, D. (1984) J. Biol. C&m 259, 13316-13320. 9. Blank, M.L., Spector, A-A, Kxiuce, T.L., Lee T-c. ard Snyder, F. (1986). Biokim. Biophys. Acb 876, 373-378. 10. Hirafuji, M., bkncia-Huerta, J.M. and Benveniste, J. (1987) Bicchim. Biqchys. kta 330, 359-369. 11. Stafforini, D-M., McIntyre, T-M., Carter, M-E., and Presmtt, S.M. (1987) J. Biol. Chem. 262, 4215-4222. 12. Pritchard, P.H. (1987) Biohem. J. 246, 791-794. 13. Blank, M-L., Hall, M-N., Cress, E.A. and Snyder, F. (1983) Biochem. Biqhys. Res. Ccmnm. 113, 666-671. 14. Satoh, K., Imaizumi, T., Kawamura, Y., Yoshida, H., Tabmatsu, S., and Mizono, S. (1988). Prostaglandins 35, 685-698. 15. !iksuki, Y., Miwa, M., Harada, M., and Matxxmdo, M. (1988). Eur. J. Biochem. 1, 117-120. 16. Roubin, R., Ihlicust, A., Haye-Legran& I., Ninio, E. and Bemmkte I J. (1986) J. Immnol. 136, 1796-1802. 17. Palmantier, R., hlioust, A., Benveniste, J. and Ninio, E. (1988) FASEBJ. 2, Al739 (abstr.) 18. biliouSt, A., Viviex, E., Meslier, N., Rmbin, R., Haye-Legrand, I., and Benveniste, Biochem. J. (inpress). 19. Lmry, O-H., Rosebrough, N-J., Farr, A.L. and Randall, R.J. (1951) J. Biol. C&m. 193, 265-275. 20. Harhnq, H-P., FSwnham, M-J., Winkelnmn, J., Ehglbeqer, W. and Hadding, U. (1983) Int. J. scol. 5, 115-121. 21. Hilton, F-H., O'Flaherky, J-T., Marshall Ellis, J., Smr&en, C.L. and Wykle, R.L. (1983) J. Biol. Chn. 258, 7268-7271. 22. Muddler, H-W., O'Flaherty, J-T. and Wykle, R.L. (1982) Lipids 17, 72-77. 23. FX+mson, N., Bechrdite, S., Wang, P., Wiate, M. and Elsbach, P. (1973) Bicchim. Biophys. Acta 296, 365-373.
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