ABSTRACTS FROM AOes JOURNALS

fBUy acids were evaluated from 90 10 170 bar at 40 and 6O'C. The com-plex mixture of anhydrous milk. fat {AMF} was evaluated from 100-310bar at 4O'C. TIle viscosicie5 of tbe methylatN fauy acids saturated withSC~ decreased between 5 and 10 limes wben the pressure increasedfrom 1 10 80 bar, followed by II further decrease by. faclor of2 10 3 ""henthe pressure was i~ased from 80 10 180 bar. "The viscosities of the fallyacids lII1dAMF saturated with SC-CO:z had viscosity reduction similar tothe methylated fauy acids between I and 80 W, but they decreased muchless between 80 and 350 bar. AI constant pressure, the viscosity of theIany acids and AMF decreased with increasing temperature. whereas thevlscoshy of the methylated fan), acids increased wlth increasing tempera-lure. The lipid/SC-C02 mixtures were Newtonian. and their viscositieswere best inlerpreled by using the mass ccocemreucn of dissolved SC-~ in lhc lipids and !he pure component Viscosities.IJAQCS 68, 912-921 (1991)]

Structural In\'Wlllations of IJ' TriacylglycerolJi: An X-R.y Dlffrac-lion .nd Mkl'05COpk Study of Twinned ~. Cryst.ls

Inhibitory Effect or Boldine on Fish 011 Oxidation

Alfonso \'.lenzuelaQ, Susana Nietoi', Broce K. casselsb .nd Hernan5peiskyDQUnidad de Bioquimic. Fannacologlca y Lipidos. INTA. Universidad deChile, Santialo, OIile and boepanamento de Quimlca, Facu1tad de Cien-cias, Universidad de OIUe, Samialo, Chile

Paul J.M.W.L. 8irker, Sijmen de Jong, Eli C. Roijers and Ton C. vanS.,,,Unllever Rescan:h Laboratorium Vlurdingen, Olivier van Noortlaan 120.3133 AT Vlaardingen. 11Ie Netherlands

To reveal the ItnlClure of ~- triaeylglycemls in detail, LML (CI2CI4CIVwas pumled by. ZDne-melling procedure. and twinned crystall of W Sta-ble LML were obtained from. melt, P- LML cf}'llallius in the tnOIIIXlin-inc space group 0, with eight molecules in the unit cell. A po ....der X-raydiffraction sludy of solid compounds of I: I mixtures of selected triacyl-glycerols led to the conc:lusion thattne triecylglycerol motecutes in the p'modification have 112 c!Ulir-confonmuion (i.~.,the fally acid ch.ins onglycerol positiorn I and 2 are adjacent. with the chain on the 3-positiOflforming the back rest of the chBir). Packing studies and the positiOl1$ oftwo-fold lUeS and two-fold screw lUes in the unit cell require that themoleeules are ~nt at the glycerol site. The fauy aeid chains make anangie of 2Y with the 1000gaxis of the unit cell. Electron micrograplu andprecession phoIognphs indicate that the twinning results from the stack-ing of. lerge number of thin crystalline platelets in two distinct orienta-tions.IJ~OCS68, 895-906 (I99I)]

Dc·Oiling Contaminated Bleaching Clay by Hlgh·Pressure Extrael!on

C. W.ldmann lind R. EggersTcchnische Universitlt HambuIB-HarbuIB. Arbehsbereich Verfahreosleeh-nik II. HambuIB, Germany

The disposal and re-use of spent bleaching clay from the vegetable oilprocessing industry is a problem of growing imporlllllCe.

Although today the only practical w.y of mnov.1 of !be spent materi-.1 is disposal, extraction with organic solvents is II well-known method ofde-oiling conuuninated bleaching clay. In our investigations we comparethe extractability of IWO different types of bleaching clays with CO;z as asolvent. All experiments were carried out with a high-pressure extractionplant. The extraction and separation eonditions, temperature and pressure.as well as the ~ mass flow. were v.ned during the experimems, Theaim of our invesligalions was II complete separariee of tbe oil frum theadsorbent. The lanel'"&bould lhen be re-used U bleaching clay. The oil andlhe bleaching clay were analyzed and tested. respectively. The relIultsshow that oil of good qup]jty can be recovered and bleaching clpy still hasiIU activity approximately:5O% of fresh clay.IJ~OCS68, 922-930 (1991)]

Polymorphic Behavior of High-Melting Glycerides from H)"dI"ogt'Rllt-td c.noIa. Oil

V. D'Souza, L, deMan and J.M. deManDepartment of Food Science, University of Guelph, Ontario N IG 2WI.Canada

Canola oil was hydrogenated with I commercial niekel c:ataly$t It 175'C.nd 15 psi hydrogen pressure. Samples were taken during the reaction5tarting at 15 min and thereafter at ten-minute intervals. The reaction wasstopped after tWO hours. The high-melting glycerides (HMG) wereobtained by fraclional crystallization at IYC with acelOne as $Givent. TheHMGs were anal~ for f.ny aeid and triglyceride composition by gllSliquid chromatography and trans was determined by infrared spec-troseopy, In the first 45 min of hydrogenation of canola oil, the 18:0 fattyacid increased 81 a low nile while the trans terry acid content increased atII much faster rate. The 16:0 end 18:0 content of the HMG was highestand trans contenl the lowest during Ihe period in ....rueh the uigiyceOdecomposition Wat the most diverse, The S4-carbon uig1~ride content ofthe HMG increased from 64.. to 78 .. during the , .....0 bouTS ofhydrogena·lion. The short spacings for the HMG showed Ihe presence of ~ crysllllsas well as several intermediate forms. The number of short-spacingsincreased with hydrogenation lime. 11Ie differential scanning calorimetry(DSC) melling profile of the HMG sho .....ed one broad peak between 20and 3O"C and two peaks IlrOlInd 6Q·C.nd above. Crystallization tempera-tures of the HMG were in the range of 4O-45'C.IJ~OCS68,907-911 (1991)]

O:lIid.lin InteractiOns 01' Choleslfrol with Triacylgl)'cerols

S.K. Kim .nd W. W. NawarDepartment of Food Science. University of Masskhu5ells. Amherst. MA01003

Ttlecylgtycerols (TGs) acceterered the decomposhlon of cholesterol at130·C. Addition of stemric and linoleic acids also accelerated cholesteroldeeompositiOfl and produeed ch.raeteristic chcleaterol oxide profiles,qu.litatively differenl from those pradocm in the presence of TGs. Milkfal .,;celerated eoolesterol decomposition at 130'C and praduced a choles-terol oxide pattern similar to that arising from the addition of pure TG.Not only did TGs affect cholesterol oxidation. but cholesterol influencedthe de<:omposilon of TG$. Addition of eholesterol accelerated the destruc-tion of TGs It the beginning of helting while protecting them later. Asimilar pattern, ie., acceleration followed by proteetion, could .Iso beseen when triacylglycerols were heated in the presence of olhcr triaeyl-glycerols. The results of this work demonstrate that the stability of lipidcomponents in complex mixtures is influenced by interactions amongthese componenl5 and/or their decomposition prodUCI5. Such intenctiOlUdo not ~Iy shift. te., aecekrau: or delay. the oxidation rate. they mayeisc modify the shape of the oxidation curve ilSelf.'J~OCS68, 931-934 (1991)]

ViJeosities 01' Fatty Adds Ind Meth)'lated Fatty Adds SatUTlled withSupercritkaJ Carbon Diodde

P. Kashulinesl', 5.S.H. RI:tvIlQ, P. Harriottb and J.A. Zollwtllb(llnstitute of Food Science, Cornell University. Ithaca. New Yorl!: 14853and bSchool of Chemical Engineering. Cornell University, Ithaca. NewYork: 14853

1lIe visrositlea or several types of lipids wunlled with supercritical car-bon dioxide (SC-C0:2) were measured with I high pressure c.pilllU)' vis-C"",,,ler_ Oteje JlCidand Iinoleie aeid were evalulted from 85 to 350 bar at40 and 6O'C. The more SC-~.soluble methylated derivatives of these

INFORM, Vol. 3, no. 1 (January 1992)

119

120

ABSTRACTS FROM AOCS JOURNALS

The anrioxidatlve effect of boldine. an alkaloid Utracted from Peumusboldus Mol. (bolda). was assayed 011the spontaneous and on the metal-induced oxidation of fish oil. The inhibitory effect of boldine was com-pared LOthose of dl-a tocopherol, the navonoid qucn:clin III1d the synthet-ic antioxidants bulyllllcd hydroxytolucne and butyl,ted hydroxyanisffie.Boldine. in all auays. showed. good antilnidative effect. which wascompanble 10 that of quercetin and even bener than thai of dl-a toco-pherol and the synthetic antioxidants. Additive effects were observedwhen mixtures of boldine and quercetin Of dl-a tocopherol were assayed.The present study supports tile poIenlial use of boldine as a novel naturalantioxidant for Ilsh oil.IJAOCS68. 935-937 (l99I)J

Prooxidalhe lind Antioxidati\'c Efftd5 or Phospbolipid~ on Milk Fa'

z..Y. Chen and W.W. NawarDepartment of Food Science, Univer,;il)' of MUSIIChusclls. Amherst. MA01003

The effects of dipalmitoylphosphatidylelhanolaminc (OPE) and dipalmi-toylphosphatidyJcholine (OPC) on milk fat oxidation were examined atSO·C and 9YC under various conditions by monitoring oxygen uptakeand fany acid composition. OPE 5lmngly inhibited milk fat oxidation boIhat SO·C and 9YC in the absence of water. OPC was less effective thanOPE. In aqueous systems. the reverse was observed. OPE II(:celeraledmilk (II oxidation at both SO·C and 9YC. OPC acceltnlled tile oxidationIt SO'C. but inhibited ir at 9S"C. 11tc: free amino group in OPE may beresponsible for its inhibiting effect in the dry system. The accereraungactivity of OPE in the aqueous system is probably due 10 the formatioo ofa more dispersed strucfure with bener oxygen accessibility.(JAOCS 68. 938-940 (\991)]

Antioxidath·e Acth·ity Measurement in Lipid Peroxldation Systemswith Malonaldehyde and 4-Hydroxy Nonenal

H. Tamura and T. ShibamotoDepartment of Environmental Toxieology. University of California.Davis. CA 95616

Amioxidative II(:tivities of vitamin E. ethylenediamine tetntaeetic aeid(EDTA). ferolic lICid. and butylated hydroxy toluene (8H1) were mooi.lend with lipid peroxidiltion 5yJlems. Formation of malonaldehyde (MA)and 4-hydroxy noncnal (4-HN) frum elhyllinoleate or ratliver microsomeoxidized by FeC12{H202 or ADP/FeS04 was measured by gas chro-matography. Over 90% of MA and 4-HN formation was inhibited by 100mmolJ\.. of Vitamin E. A synthetic antioxidant. 8HT. showed the strongcstanlioxidative Ictivity. followed by thai of vitamin E. whereas EOTAaccelerated formation of MA and 4-HN in the ethyllinoleate/FeCI2/H2~system. Vitamin E did IlOl svppress lipid peroxidation significilIltly whenmicrosome WIS oxidized by AOP/FcS04' EDTA inhibited oxidation ofmicrosome by AOPJFeS04 considerably: in OOIItrast, it did not in the caseof oxidation by FeCl2/H2~'[;AOCS68. 941-943 (1991)]

The Effeel of Dry Htat on the Bioavailability of Iron in Soy)<lour

s.s. Jonnalagadda. P. Sabhaf"-.I. C.A. Prau and W. BarbeauDepattment of Human Nutrition and Foods. Virginia Polytechnic Instituteand Stale University. Blacksburg. Virginia 24061

8iOllvaiiability of iron in $l)y noor was invesligau;,d by the HemoglobinRegentTlltion Emc:iency (HRE) procedure in 50 1h~_momh..,ld Spngue-Dawley TlU$. Rau weighing 250 ± 7 g and with a mean hemoglobin levelof 12.9 gldl were mndomly ISsigned to one of five treatment groups:baseline: (8L), unheated soy flour (UII). soy flour heated at 225'F fOf"either 10 min (H 10), 30 min (1-130).or 120 min (HI20). The animals WCM

fed diets (46 ppm iron) coouoining the soy no..r (or 21 days. HREs of UH.IHO. H30. HilO dku were t7.6. 16.8, t7.7 and 16.8%. rnpo:c:tively.Apparent iron ad$l)rp!ion from the UH. H 10. HlO. H 110 diets were 17.6.

16.8.17.7 and 16.8%. respectively. Apparent iron adsorption from theUH. H 10, H3O. H20 was 94.1. 94.3. 93.9 and 94.3%. respectively. Serumiron was significantly lower (p<.()()I) and tOI:11iron binding capacity wassignifteantly higher (p<.OOJ) in rats fed the HllO diet. Iron concentrationsin the liver. spleen. bean and kidney were signifICantly InwCT in rats fedU30 or HIM dicl5. These: results suggest ttw prolonged heating of soynoormay reduce iron bioavailability and resuu in deplelion of iron stores.IJAOCS68. 944-948 (1991)1

InterlMbont!ory Comparison of Soybean Protein and Oil Determina·"OM

Randy A. Hartwig and Charles R. Hurburgh, Jr.Department of Agricultural Engineering. Iowa State University. Ames.Iowa 5001 I

Iowa SUlte University coordinated an interlaboi1ltory comparison study ofKjeldahl protein and ether oil extl1lCtion methods. Blind duplicates of 10clean. single-variety sobyean samples were sent 10 30 laboratoriesgrouped in 3 calegories of 10 ellCh in public (goyernment and university).commm:ial and processor facilities. Five of the commercial laboratorieswere AOCS-cenified.

Standard deviations among labomtory means acl"OSlall samples were3.87 and 1.82 percenlBge points (dry basi5) for protein and oil. respeceve-ly (0.48 and 0.27. mspectively. for the AOCS-a:nlfled labontorics). Theaverage differences between blind duplicates of I sample were 0.71 per-centage points for protein and 0.87 percentage points for oil (0.28 Dnd0.45. respectively. for the certified laboratories). Average standard devia-tions across jubcrarcries on an individual sample were 2.37 and 1.71 per-cenlage poinlli for protein and oil. respectively (1.81 and 0.99. respective-ly. for the «"if ted labontories).[JAOCS 68.949-955 (1991)1

Nutrition of Rice Bran 011 In Relation to Its Purification

S. Sarkar and O.K. 8hallacharyyaDepartment of Chemical Technology. UniYCl"liity Colleges of Science &.Technology. Calcutta University. Caiculla· 700 009. India

A ccsnparatlve nutritive sludy was made 10 show that the extent of purifi-ClItion rnarl:;edly influences the nutritive chane-teristic5 of rice bran oil.The coefTtdent of digestibility was 93.8'1. when rice bran oil that ]wibeen purified by degumming. deacidifying. bleaching and deodorizingwas fed to ruts: whereas it WQS 94.8% when extremely pure rice bran oil,which was echieved by including an additional dewaxing step, was used.Rice bran oil withoul deodorization. but purified by other treatmeras.showed a 96.2% coeffICient of digestibility. which is somewhat lower thanthat o( groundnut oil. However. after a feeding experiment over threemonths. the highly purified rice. bnm oil showed bener results than theother two purified samples of rice bran oil. and was sometimes better thangroundnut oil in temu of total lipid. triglyceride and cspecially for chole$-terot COlItent in strom. Hver and hean tissues.IJAOCS68. 956-962 {199l)1

GC-MS Characterizalion (Chemical Ionization Mnd Electron ImpaclModes) or the Metbyl Esters and OUloUne Derlvath·es of Cycio-propcnoid Fatty Acids

Volker SpitzerLehrstuhl fUr Lebensrnlnelwissenschaft und Lebeesmiuelchemle.Rheinische Friedrich Wilhelms-UniyersiIDt. 0-5300 Bonn I

The C1-{CH4) mus spectrll of the methyl eslera and lhe EI ma.u spectraof the oxazoline: derivDtivC$ of three cyclopropenoid fatty acids (malvaJic.sterculic and a-hydroxy·sterculic acid) from the seed oil of PuclriruaqUlltica were found to be useful for stnJctu~ elucidation of such com-pounds. Funhcrmore. some hithcno unknown minor fatty acids wereidentified and the nuc;l.,.r magnetic; re$Ona.nce dDtl of the oil and o-hydroxyslcrculic acid methyl ester art presenled.

INFORM, Vol. 3, no. 1 (Jonuary 1992)

121

[JAOCS68. 96l-969 (1991)1Enlymlllic Methylation of CanDia Oil Deodorizer Distillate

Suresb Ramllmurthi, Prakash R. Bhirud lind Alan R. McCurdyDepartment of Applied Microbiology and Food Science, University ofSaskatchewan, SasI;:au;Kln, Saskatche ....an. Canada S7N OWO

Methylation of canota oil deodorizer distillate catalyzed by II nonspecificlipase was investigated. lbe conversion of fauy acids to methyl esters hasbeen optimU,ed by using II statistical design. Up 10 96.5% conversion ofrany acids 10 their methyl CS!e1'Slias been achieved without the aid of vac·uurn or any water-removing agelll. The effecl$ of tempeTalure, I1Itio of thereactanlS (methanol: fany acids in the deodorizer distillale) and mzymecoroeenr.tation on the equilibrium eonvenion were studied. The tempera-ture and 1111;0 of the reactants showed II significant effect on the conver-sion of fauy acids to methyl esters and tile)' exhibited II strong interactiveeffect. Enzyme cencentrarien in the range of 2.7% 10 4.3% did nOI show IIsignificant dfecl on the equilibrium conversion of fatly Kids. Greaterthan 95% conversion of Ierty acids to methyl esters was achieved at rem-perarures around SO'C and ilt a ratio of the n:ac:tanl5 between 1_8and 2.0.TIx: inhibitory effect of hydrophilic methanol on the enzyme .etivity waslargely reduced by working at the lower temperatere range (around SO'C).IJAOCS68. 970-975 (199I)J

L..auric: Add-Containing 1iiglyttrides in ~ or UIfI~lfu1.tJrWI (II/i_/(m'U:tI NUll, (LaUr1lCtae)

T. Rcynolds. J.V. Dring and C. HughesJodrcU Laboratory. Royal Botanic Gardens, Kcw. Richmond, Surrey.United Kingdom

A white crystalline malerial isolllled from the seeds of UmMllularia cali-forflica conlIisted mainly of the milled lriglyttride. dilaurocaprin, withsmaller amounts ofttilaurin and dicaprolaurin and a IIlICe of triclprin. TIx:yield of crude oil from I tree growing al Royal Botanic Gardens. Kew,was considerably leu than that reported previously for trees growing inCalifornia.IJAOCS68. 976Jn7 (1991)1

NO\'cl Fally Acid5ln Adnw TdraUlnlll1l Seed Oil

C,D. Daulailibad. V,A. Desai. K.M. Hosamanl and A.M. JamkhundJDepartment of Chemisuy. Kamatak Uni~ersity. Dharwad 580 003. India

A:illW UIrOCIlfl/M Lam. belonging to the Sal~adoraceae plant family.was found to contain ricinoleic .cid (9.8") and I:yclopropenoid fany.eilis (9.6") along whh normal fauy.eKa..IJAOCS68. 978-979 (1991))

AllIIlysis or Neutral Lipids and Glycerolysi5 ProducU from Olin Oilby Liquid Chromatography

Baokang Yllng and Jyhplng ChenCenter for Dairy Research. Department of Food Science. Unlversuy ofWisconsill-Madison. Mooison. WI 53706

A simple method was developed to separate model trigly«ridcs. 1.3-digly«rides. 1.2--digly«rides, monoglycerilb and free fany .eids andproductS generated in enzymatic glycerolysis of oIi~e oil by high-perfor-mance liquid chromatography with a laser light-scattering detector. Theseparation was carried OUtwith II silica column at 3O'C. A gnldiellt elutionwith mobile phue A (hexane/o;hlorofonnjformic acid) and mobile phaseB (henne/acetone/chloroform) can separate the compounds lme distinctpeaks in leu than 20 min.IJAOCS 68, 980-982 (1991)1

The' En-C'd orTe'mperatu~ on the Induction Time' or a Stablliud Oil

Greg ReynhoutKalscc.lrtc .. Kalamazoo, Michigan 49005

Soybean oil was fonilied with the antioxidants BHT (butylated hydroxy-toluene). BHA (butylated hydrollyanisole), TBHQ (tertiary butylhydro-quinone). rosemary extract (HerbaloxiIJ SelSoning) and tocopherol.Induction times were dctennined against a control on each s.ample ill aMctrohm Rancimat over a temperature range of W'C to ISO·C. A lineareffect of data was obtained when the log of induction time was plottedagainst temperature. TIx: Me!rohm R.aocimal was found 10 be capable ofdetcnnining induction times within the range of 0.510 10 hr.fJAOCS68. 983-984 (1991)]

Studies on thC' SriC'ct1~lty or EntylMS In"otvC'd in PlatOO-Activating""actor FOf"mation in StimulatC'd Cdls

H. van den 8osc:hD, A. Sturkb, J.W. ten Caleb and AJ. AarsmanQ

vcenue for Biomcmbrnnes and Lipid EIl~ymology, Uni~ersily of Utrecht,Padull18.lln 8. 3584 CH Utrecht, and bDcpanmeni of Hematology. Divl-sion of Hemostasis and Thrombosis. Academic Medical Centre. 1105 AZAmsterdam. TIx: Newrlands

The present studies were undertaken to obtain further insighl into theselectivities of the eru.ymes, Le .• phospholipase A2 and .cetyhnnsferasc,involved ill platelet-activating factor (PAF) production upon stimUlationof human polymorphonuclear leukocytel (PMN) and platelets. Afterappropriate sWnulliion of the «lis in the presence of ,3H]acetaJe the totalPAF and analogs. te.. l·alkyl-2-accryl-. l-alkenyl-2-aceryl-. and l-acyl-2-acetyl-glyeero-3-phosphocholine were isolated by high performance liq-uid chromatography. The isoJllIed mixture was wbjeelCd 10 treatment withphospholipase A I to differentiate acetate illCOI"pOI1ltioninto l-etber linkedand l-ester linked species. The ratio of ecetate incorporation into t-etherlinked end l-ester !.inkcd '15 l-ester linked PAP ererogs amounted to 13.8± 1.0 and 1.3 ± 0.1 for PMN and platelets, respecuvety, When comparedto the ratio of l-etber linked and l-ester linked species in the diradylglyc-erophosphol;:ltoline precUC$Of1in each cell type. Le.. 1.13 for PMN and0.22 for platelets. these data suggested a pronounced selectivity for thephospholipase A2 andIOf .eetyltransfense in the process of PAF produc-tion. When the experiments were repeated with «lis that had been pre-!reated with phcnylmelhancsulfOllylfluoride (PMSF) to block the acetyl-hydrolase. the most dramatic effCO::I5were observed on acetllte lncorpont-tion into l-acyl-2-.cetyl-slycero-3-phosphocholine. which increasedmuch more than that into 1-a1k(en)yl-2·acetyl-glyccro-3-phosphocholine.Under these conditions. the ratio of acetate incorporation into t-etherlinked V$ l-ester linked PAF analogs became 1.4 ± 0.2 and 0.11 ± 0.02 forPMN and platelets. respectively, These ~alueillre ~el)' close 10 the l-etberlinked vs l-ester linked species ill 100 diradylglycerophosphocbcline pre-cursors for PAF in the respecuve «II type. Tbese data suggested that theselectivities of phospholipase A2 and/or lItetyl transferase for ether-linkedspecies. as observed in ROR-PMSF treated «lis, are only appill:llt andcaused by rapid degntdation of the I-.eyl analog either berore or lfieracetylation. In line with tlti, inle~ion. we demonstrated thai 1'lCyl-2-Ketyl.(iPC can be de.eylatcd to water-SOluble acetyl-GPC and OPC bysonicated PMN and platelets and that Ihis deacylaticn is completelyblocked in sonicatCli from PMSF-pretreatcd cells. In addition. evidence ispresented which indicates that the en"tyme responsible for dcllCylationmay be. Iysophospholipase.[Lipid$ Z6. 967-973 (1991)1

INFORM. Vol. 3, no. 1 (January 1992)

122

GPC. This and other properties, such as pH Ikpcndrnoe and !henna] SIa-bility. iMicale tha1 ral Inin may have two distil1C1 enzymes for the syn-thesis of PAF and ether choline phospholipids. The amnily of ueseenzymes (0( CDPeholine is similar to thaI reponed for other tiSSlleS, theKm being 421UJl and 55 11m with alkylacetylgly«rol and dioclanoylgJyc:·eml as lipid substrates. ~speclively. The Vmax values were 3.0 and 2.2nmol/mg prot/min fot PAF and diocumoyl-GPC, respectively. In addition.it was shown Ihallhe microsomal (melion of tal brain ccmains an acelyl-transferase wllich can convert lywPAF 10 PAF. Since it has been reportedpreviously lhal brain nssue possesses phoI;pholipase A2 aclivity 1hal canhydrolyze alkylacy]-GPC to I)'$OPAF. we conclude thaI brain tissue has allenzymic activities for the synthesis of PAF by the Krernodeling pathway",111e role of the 1'1'0 routes of PAF biosYlithesis in nervous tissue remains10 be established.)lipids 26, 9&6-991 (1991»)

ABSTRACTS FROM AOCS JOURNALS

Regulation of the Biosynthesis or Plateltl·Acth-aling Factor in Ah'to-lar ~bcl"'Ophages

Tak.ayukl Sugiurll, Ayak.o Ojimll·UchlYllma, VIISUO MaSU1.llwII,Masamichi Fujita, Yasuhilo Nakagawa and Kebo WakuFaculty of Pharmaceurkal Sciences. Teikyo Uniyenity, Kanagawl 199·01. Japan

Activities of enzymes which metabolite IY$Oplltelet·activating factor(lysoPAF) and plaltlet·activhting focto!" (PAF) were studicd in rabbit etve-olar mocrophage lysates. Substantial ecetyltraasferese activity was notedin the presence of 100 11M ecetyl-ceenzyme A (CoA), and this IICtivitywas increased in A23187-5timulated cell tysare. On the other hand, in theabsence of ellogeroolu acetyl-CoA, lysoPAF was mainly acylated througha transacylation pathwlY rathcr than by acetyltransferase in both controland A23187·stimulated cell lysates. We confirmed thai the intracellularconcentntion of ICCtyl-CoA is relatively low. The observations suggestthat the lI1InSaCylation l)'5ltm may play an tqUllly important !"Ok in theregulation of the IVlillbility of lysoPAF in intact cells. IntracellullrlywPAF was also maintained II rdltively low levels. Inle~tingly. 1llf1Camounts of PAF were produced even in unstimullted cells upon Idditionof an excess of exogenous lysoPAF. suggesting that generation of an ade-quate amount of lysoPAF within cells may be sufficient to trigger PAPsynthesis in this type of cells.jUpids 26. 974-978 (1991)1

Platelet-Activating )-"actor Acetylhydrolase Activity In Human Tlssuesand Blood Cells

Diana M. StafTorini, Stephen M. Preseou, Guy A. Zimmerman andThomas M. Mclnl}"rtThc Nora Eccles Harri$Of1 Cardiovascular Research and Training Instituteand the Departments oflmemal Medicine and Biochemistry. University ofUtah School of Medicine, Sail Lake Cily. Utah 84112

Human tissues, blood cells, and plasma have enzymes that catalyze thehydrolysis of PAF (1-O-llkyl-2-acetyl-.m·glycero-3-phosphocholinc). Theactivities are no! due to phospholipascs A2 thai hydrolyze long chain acylgroups at the $11-2position of glyeemphospholipids. 5i~ they are calci-um-independent and are specific for hydrolysis of short chain acyl groups..We examined the biochemical properties of the$e PAF acelylhydrolueatlivities (EC 3.1.1.47) in homogenates of human liver and spleen, inwhite blood cells (neutrophils and monocytes), and in erythrocyte5. Thedata suggest thai tbe plasma end lmracellular PAF acetylhydrolase activi-ties pre likely due 10 different proteins. Second, the intracellular PAFacetylhydrolase activities in liver and spleen share several biochemicalfeatures that differentiate them from the activities in blood cells. Third,the activities in rnonoeylu and neutrophils have pmpenia that diffen:nti-ate them from the activity present in human erythrocytes. Finally, the ery-throcyte activiry has unique properties !hal place it in I separate categoryof short chain aeylhydrolases. In conclusion. there is • family of distinctenzylTlC$thai can be identified 115 PAF acetylhydrohues based on their ear-cium-independence and specificity for a sOOn residue at the sll-2 positionof phospholipids.[Upith 26. 979-JJ85 (1991)]

Properties of PAF-SynthesiJ.ing Phospboeholinttransferase and Evi-dence for LysoPAF Aretyltransferase Activity In Rat Brain

Glanfranreseo Goracci and [rmelinda FranrescangeliDipartimento di MedicinD Sperimentale e Scienze Blochlmicbe. Ueiversi-ta di Perugia. 06100 Perugia, Italy

Several reportS have indicated thai platelet·actiy"ing factor (PAF) mayplaya role in the physiopathology of nervous tissue. We previoosly havedemonstrated the preseece. in tbe microsomal fractions of 1111broin, of aphosphochoJioctrnnsfera$C which is able to synthesize PAF by the dl' 110''(1pathway. The presence of dithiOlhreitol in the medium increases the rateof PAF biosynthesis, whereas it inhibits the synthesis of long-chain alky-lacyl- and diacyl·glyeerophosphocholines (OPC). including dioctanoyl-

The Metabolism of' I-Acyl-PAF In Rabbit P1atdets And Ill! ~ibleInteraction With PA)-'

Laeusseine Touqul, Clystenes Soares Silvil and Bernardo Borio! var-gamgUnite de Pharmacologic Cellulaire, Unite Asscciee INSERM/PasteurU.2lIS.institut Pasteur. 75015 Paris. France

The metabolism of 1·.,;:yl·2-acetyl·slt·glycero-l·phosphocholinc (t-ecyt-PAF). a nalUnlly occuning analogue of platelet !lCtiyaling factor (PAF).WIS lnvesttgeted in rabbit pteteree. Our studies showed that l-Icyl-j3H] PAF (I·pal mil0yl.2 -acet yl-slI-gl ycero-3-phospho[ N-me! hy/.3 H]-choline) was converted by pletelets into phOSPho!i'!r.1I3H]Choline([3H]PC) in a lime-dependent fashion. The Iorrnaticn of I HJPC occurredat a rote similar to that observed when lyso-(3H]PC (palmuoyl-sa-glyc-ero-3-phospho/N-melh,·/_3H)eholine) was used as substrate. In addition, •limc-dependcnt inereue in the level of wlter-soluble ndioactiyity wa!i

observed during the incubalion of plltelets with either l-acYl-~H]PAF orlyso-,3H]PC. Thio! increase was parallel 10 the formation of j H]PC andwas 001 observed in lhe presence of jl4c)PAF (I-octadecyl-2-acetyl-slt-glyccm-3-phospho[N mt'lhy/_l4C)-chQJine). Analysis by thin·layer chrc-matography showed that the soluble radioactivity was mainly associatedwith glycempho5phoeholinc (OPC). On the othc:T hand. the preincubationof platelets with phcnylmethylsulfonyl fluoride. an inhibitor of lhe acetyl-hydrolase, reduced the hydrol)'llb of !·.,;:yl_,3HIPAF to ,3H}GPC with aoonoomitant accumulation of radioactivity in I-acyl-PAF. These findingssuggest !hat l-acyl·PAP is convened into PC through deacetyltuion-rcac:y-lation with lysoPC as an obligatory intermediate. The findings also indi-care that the ly.oPC resulting from I-DcyI·I'AF is eith~r reacylated tophosphatidylcholine (PC) or hydrolyzed to OPC by lysophcsphcllpase.Finally. we showed that the stimulation of platelets with PAF led \0 atime- and conecnll1ltion-dcpendcnt trcreese in the convenion of l-ecyl-jlH]PAF to ,3H]PC. The stimulatory effect of PAF was not observedwhen platelets were lysed before incubation, sllggCllting thaI PAFenhances the metabolism of l-acyl-PAF. probably by accelerating itstnlnSlocation through the plasma membrane.jUp;th 26, 992-996 (1991)]

Plateltt-Aclivatln& Fllctor (PAF) Stimulates the LysoPAF Acetyl-transferase In LtllkOC}"It-Rich Plasmll: U. in PAF Anl.'&Oo~ Stud-

'"Thomas W. Doebber, Margarel S. WU, Anthony Mauriello and AlfredAlbertsDepartment of Biochemical Regulation, Merck Sharp & Dohme ResearchLebcratcries, Rahway. New Jersey 07065-0900

Addition of platelet·activating faclor (PAF; I-O-alkyl·2·acetyl-slt-glyc-ero-3-ph05phocholine) to leukocyte- rich plasma from seYeral speciesrescuee in the rapld and pronounced activation of lhe PAF biOlynlhetieenzyme acetyl·CoA: 1-Q-alkyl-s/t-glycero-3·phosphocho\ine aceryhrans-Ierese (EC 2.3.1.67). Activation of acetyltransfera5C by PAF occurred injeukccyte-rich plasma from human. ehimpanzee. rhesus monkey. and dog.The neutrophil was indicated to be the major oelJular 501U'ceof the actio

INFORM, Vol. 3. no. 1 (January 1992)

vtl.b1e eeetyltransferase in leukocyte-rich plasma. The induction ofauryltransferue was substantial wilh 10 nM PAF, and maximal al 1()-305eCOnds.. Measurable lCeIyltransferase activation was sig.nirlClJlllly greaterwhen the PAF-xlivaled cells wen. separated from the plasma by centrifu-gation before the ecetyltransferase assay. This may be due in pan to theremoval of the PAF-specific ecetylhydmlase present in plasma which cancleave the acetyl group from PAF. Measuring PAF activation of acetyl-transferase in leakccyte-nch plasma can be useful to detennine the poten-cy of PAF antagonists with neutrophils in plasma compam:l to isolatedneutrophile in aqueous buffer, IU1dU IU1u: vtvo assay to detcnnine theefficacy and plasma eoneentraticn equivalents of anlagonislS administeredto whole animalS. 11K: PAF anUlgonist 1,...-659,989was shown to be 3-5times more poten! in inhibiting PAF induction of acetyltransferase in tsc-IDted human neulrophils than in human leukocyte- rich plasma, with ICSOvalues of 10 nM Ind 40 nM, respectively. In the e¥ vim assay. oraladministrll.tion of !he PAF antagonist L-667.131 10 clop resulted in very,ub!ilantial inhibition of PAF induction of aoelyitmuferase in the leuko-cyte·rich plasma, Utilizing the t¥ \'i\'O assay. oral administration of Img/kg L·6S9,989 to rats was found to resuf in plasm. concentrationequivalents of approximately 200-300 nM L--6S9,989. Our findings offer1new approach for characlelUing the in \'itro and in I·i.·o efficacy of PAPreceptor anUlgonists and demonslnte that PAF m.y be able to activateneulrophils in the blood in ""1'0, further enhancing PAF synthesis.IUpids 26. 997-1003 (1991)]

Kinetic Studies or Human and Rat Neutrophll LysoPAF Acetyltrans·rerase Using LysoPAF and DansyllysoPAF as Sub'111Ites

Peter W. Schindleri' and Ewa Nlnmbof'tuuma fofschung, Hoechsl AG. 6230 Frantfun 80. Germany. and biN.SERM U200, Universite Paris-Sud, 92140 Oamart, France

Enzyme kinetic studies of IY50PAF aceryhransferase from microsomalpreparationll of human and mt neutrophils were carried out using IY50PAFor dansyllysoPAF u wbslfate. With the human enzyme. incomple!e con-version of the wbstm!e lnte !he product was observed 11 37"C with bothsub!ilrlltes.. 1be KC:tyltransferase was inactivated 11 3rC in the absence ofsubstra!e with I half·life of 7.5 min. However. the initial me of productformation under Inc assay conditions was linear up to 10 min. Bothenzymes were optimally active at 40 j1M cOIlCentnuion whh either sub-Slnue. bul enzyme activity Wa5 inhibited at higher substrate levels. At aconstant sub!iltate c:orK:entmtion (40 j1M), the Km (11M) and Vmax (nmolproduct/min/ml protein) values for the human eceryltransferase, withrespect to acetyl·eoA were 132 and 23.1 respectively, with IY50PAF assubstrate. and lOS and 26.7, respectively. when damyllY50PAF was used.The Km and V max values for the rat enzyme were lOS and 6.5, respec-tively. with lysoPAF as substr&te, and 120 and 5.4. respectively. whendlnsyllysoPAF W85 used. Under our standard conditions, lysoPAFrequired I mg of BSA per mL in the assay, wben:as full activiry of bothCIll)'lllC$ was seen with dan5yllysoPAP even in the absence of BSA. Theresults dJow that dansyllysoPAF can replace lysoPAF in the assay withoutany signirJCant changes in kinetic paramC:!ers.IUpid$26.1004-1010(1991)]

The Elfed. or Inhibitors of Platelet Allrtgatton on the Metaboli~m ofPl8le1et·Acth1ltlna Factor (PAf1ln Washed. Rlbbit PI8telet$

C. O'NeiltG. AJ. Ammltll', R. Korthb, S. F1emint' and X. weuss0Human Reproduction Unit. Royal North Shol1l Hospital of Sydney. St.Leonerds, 2065. NSW Australia IU1dbJnsenn U200, untvesue Paris, Sud,F·92I40 Clamatt. France

The rabbit platelet metabollzea platelet·activating factor (PAF) imracellu-lady. PAF is dcacetylaled to produce IY50PAF which, in turn, can be ecy-lated to produce 1.o.alkyl.2.acyl,slI·llycero-3.pllosphocholine (alkylacylGPC). Some PAF receptor antagonists have been shown to inhibit thismetabolic convenion. In the present study we examined whether the PAFreceptor anllgoni't$ SRI 63·44\ and WEB 2086 would inhibh themeUlbolism of PAF by intact rabbit platelets. In addilion. we examinedwhether iloprmt. a 51able analogue of prmtagllndin 12 (PG12). Ind I

potent inhibitor of platelet aclivalion induced by • range of agonists.would also inhibit PAP metabolism. We found that SRI 63-441 and WEB2086 caused an almost complete inhibition of the c:onvemon of PAF toalkylacyl GPC. Ilopmst caused up 10 1 SO% inhibition of PAF meUlbolismcompared to antagonist·free controls. Iioprost (and PGIz) is thought toinhibit platelet response by elevation of cAMP, while rcceptCITantagonistsact by blocking PAF binding to Iu receptor. Since iloprost caused partialinhibition of PAF metabolism, the resuus of this study suggest thai inhibi·tion of PAF melllbolism does not occur solely due to competitive inhibl-tion of PAP binding to its receptor.IUpids 16, 1011-1014 (1991)]

The Hormonal Rrgulation or PI8Ielet·Actiyallng "'actor Acelylhydro-1ase AcI;"ity In Plasma

Shuichi Miyaura, Nonci Makl, William Byrd aDd John M. JohnstonTbe Depanmc:ntJ of Biochemistry. Ob5teuics..Qynecology and the CecilH. & Ida Green Center for Reproductive Biology Sciences. The Univenl·ty of Texas Southwestern Medical Center, Dallas. Texas 75235·9038

We have previously reported that certain fetal lissues including the lunaand kidney have an incrusai platelet·activating factor {PAf} content andenzymatic mechanism for ilS elevated biosymhesiJ durilli the latter sta~sof pregnancy. In COIItnst. in the maternal plasma compartment of both therabbit and human. a decreased capacity to inactivate PAF has beendemonstrated. The PAF ecetylhydmlese in the fetnl plusma is also sup-pressed. The present study was undertaken to determine the mechanism(~)involved in the regulation of PAF acetylhydmlase. The l7a.ethynyICStl1l·diol was administered (intraperiloocal/i.p.12.5 m&lkg body wt 5 days) tofemale and male ralS. The plasma PAF ICClylhydroluc activiry decreasedS·fold. A decrease was observed when. concentl1ltion of the estrogen aslow as 50 j1gJkg was employed. The injection of deltamethasone (i.p .. 1.3mg/kg body WI,S days) to male and female rllJl resulted in a 3-foldIncrease in the plasma PAF acetylhydrolase activity. 11K: activity returnedto the values prior to hormone ~atment 4 clays af!er cessation of treat·menr, Testosterone and progesterone were wilhout effect on plasmaacetylhydrollSC: activity. The change in PAF acetylhydrolase activitycaused by ulrogen and the gluooconicoid was rt:nected by a change inthe activity in the HDL fraction and not due to the presence of an inhibitoror activator in the plasma of the normooe- treated animals. Human serumobtained from a group of women. in which the 171)-estradiol concentra-tion WlIli elevated in preparation for an ill I'i,ro fertilization procedure.showed an inverse rt:latiornhlp between the pl8lima estrogen concentra·tion and the PAF ICCtylhydrolase activity. It is suggested that estrogen isresponsible for the regulation of PAF acetylhydrolase and the decrease Inthe plasma PAF lICetylhydrolase during the laller stages of pregnancy inboth the maternal and fetal plasma caused by tnc hyperestrogcnlc statethat occurs during this period. The observed i/lCreallC in PAF eceryjhydro-lase by dexamethasone may account for, in pan, the known anti·innam·malory propenies of this stCTOidby decreasing the concentration of thispolent aulaCOid.IUpids 26,1015-1020 (1991))

Recent Advances in Our Understanding or the Biochemical tmerec-tions Between Platelel·Actiutina Factor and Arathidonic Add

floyd H. Chilton, Man:: Cluul Ind Massimo Triggianl11K: Johns HopkillS University S<:bool of Medicine, 11K: Johns HopkillSAsthma and Allergy Center. Baltimore, Maryland 21224

In the last few yean. it has become increasingly apparent that the bio-chemistry of PAF (platelet·activating fllCter) and that of arachidonic acidan: interTelated in a number of innammatory cells. Experiments presentedhere further point out that arachidonic acid plays I crucial role in thecatabolism and biosynthesis of PAF. In addition. they suggest that thesame phospholipid molecular species may serve as a source for botharachidonic acid Ind l·alkyl·2-lyso-sn-gJycero-3·phosphocholine durinilcell activation. Finally. !hey reveal thai there m.y be common regul.IOT)'mechanisms for the hiOlynthesis of PAF and arachidonic acid ITIelabo!ite$.Taken toaether. nuetes examining ttle relationship between PAF Ind

INFORM. Vol. 3. no. 1 (Jonuol'( 1992)

123

124

ABSTRACTS FROM AOCS JOURNALS

anclIidonk .:id &uggesl it may be dilflClll! to consider the bioc1'lmlisuyofPAF without considering arachidonic acid metabolism and vice VCf$II.

(Lipids 16. 1021-1027 (1991))

Inositol Phospholipid Turnever In PAF Trall5membrane SignallingShh'endra D. ShuklaDepartment of Pharmacology. School of Medicine. University of Mis-$OIIri. Columbia. Missouri 65212

In a variel)' of tells and tissues, platelet activoting faclOr (PAF) stimulatesphospholipase C catalyzed breakdown of phosphoinositides. This resultsin the generation of \be second mencngers. il1051101 trisphosphate anddiglyceride. This proce$!I occurs independently of utnccllular Ca2., Anumber of PAF Slrucrural analogues, tttqM~ antagonists and drugs navebeen utilized to phannaeologically probe the actiyatk!n of phospholiPQeC. PAF stlmulauon orthe phosphoinosilide turnover was shown to be sen-shive \0 pertussis toxin in some systems, btu not in ethers. The involve-men! of guanine nucleotide binding protein(s) and tyrosine kinase(s) inIhl$ process have also been postulated. These developments give newinsighlS into PAF·receptor function al ~ molecular level. and also pro-vide leads toward5 I beUeT undeBlanding of ~ cellular responses to PAF.{lipids 26.102&-1033 (199I)J

Transmembrane Signalling And Par·Acelher Biosynthesis

Eon NinKi and Francine JolyInstilUI National de: la SlUltt et de: la Recherche M&licaJ U200. 92140 aa·mart, France

EXpression of Iyso paf-acether (lY50 paO:acetyl·CoA acetyltransfcreseand its activation above basal levels by specific agonists controls the roteof par biosynthesis in proinllammetery cells. AcetyltransJerase activalionin these eens is due to the rapid postranslational modification of anina<:tive precursor by phosphorylation. most probably catalyzed by •cAMP·dependent kinase. However. the possibility exists that acakium/calmodulin-dependent kinase can be implicated as well. Unlikemurine cultured mast cells. human neutrophils fomt paf when stimulatedwith phorbol myristate acetate (PMA) or diacylglycerol. In both celltypes, acetyltnn,ferase is lCIivated by PMA. Controveny exists as towhether PMA activates ce remodeling pathway, t.e. the activation ofphospholipase A2 and acetyltnnsferale. or the dr 110\'0 route throughCDPcholine cholinepbospborransferase action on alkylacetylglyceroJ.There is some indication thai PKC might regulate paf biosynthesis. Theimplication of a GTP-regulated protein has also been postulated in signaltransduction leading to par formation in endothelial cells. neutrophils, lindmast cells. The topography of paf formation is discussed in light of thesubcellular distribution of acetyhransferase in neutrophils and Krebs IIeeus,(lipids 26.1034--1037 (1991)1

PAF EfTtclll on Transmt'mhl'1llM Signaling Pathways in Rat KuprrerCells

Chandrashekbar R. Gandbi and Mt'rlt S. Ol5onDepanlntnt of Biochemistry. Univenity of Texas Health Science Centerat San Antonio. San Amonio. 'rexes 78284

Platelet activating factor (PAF) was found to stimulate the metabolism ofinositol phospholipids via deacylation and phospholipase C in Kupffercells. !be resident macrophagCll in liver. PAF-induced phospboinosilide:Intlabolisrn occurred in two ~. Within 5CCOI1dJi lner stimulation. inthe absence of cxtrlCellulaf ea++-. platclet aaivating factor caused thepbospbodiester bydrolysis of phosphalidylillO$itol 4.~-biphosphate andphos;phatidylinOlitol 4-phosphate wilh the release of inosilol 1.4.~-trisphosphate and inosilol 1.4-bispllosphate. This WI! followed by anutrocellular Ca- -<kprncknt release of g1yoerophospboioositol. inositolmonophosphatCll and illO$;lol bisphosphllCll. Various Ca--mobilizingagonisu failed to evokc hydrolysis of phospboinositidc:s. Plllcict .,.ivat.ing Iector also stimulated the synthelislnd release of prostagllndins from

INFORM. Vol. 3. no. 1 (Jonuory 1992)

these cells. Platelet activating faclor-stimulated phosphodiestermelabolism of phosphoinositidc:5 and prostaglandin synthe:si5 was inhibit-ed by treatment with pertussis toxin and cholera toxin. Pc:T1l1ssistoxin alsoinhibited platelet Dctivating factor-induced gtycercpnosphclneshotrelease. Cholera toxin. in ccrurasr, stimulated platelet activating Iector-induced glycerophosphoinositol release and prostaglandin synthesis andsynergistically stimulated the effect of platelet activating factor on Iheseprocesses. The results suggest that plalelet activlling faclor-inducedInttlbolism in the KupfTer cells occurs via specinc receplors and may bemediated through the lCIivation of different G-proteins.llipids 26, 103&-1043 (1991)1

Platelet·Actlvlt;nl FlKtor May Pat1k:lpalt' In Signal TransductionProces.ws in Rabbit I..t'ukocykS

AlaSlair G. Stewart and Trudl HllrrisDepartment of PIIysiology, University of Melbourne. Victoria, Australia

The bactcrial chemotactic peptide, Icrmyl-methionyl-leucyl-phenylala-ninc (fMLP). induces the genention of platelet-activating factor (PAF).the mobiliution of anchidonic acid and genention of Juperoxide anion(~l in nbblt poI.ymorphonu<:leaf leukocytes (PMNs). The PM receptorantagoniSlS. WEB 2086 (IO-HX)~) and CV 6209 (1-10 11M). reducedthe mobiliUltion of amchidonic acid and the ~- genention in response tofMLP but 001 that in n:sponse to A23187. PreU'Catment of PMN, with !bephoSpholipase A2 inhibitor. chloroquine. or the serine protease inhibitor.tosyl-phc:nylalanine chloromethyl ketone. reduced the fMLP-51imulatedgeneration of PAF and also reduced the gmera1ion of ~-. The respiralOfyburs.t induced by a submaximal concentration of pborbol myristate acetatewas not affected by these compounds. These data are consistent with thesuggestion that endogenous PAF mlly contribute to the signal transductioncascade initiated by fMLP.[lipids 26.1044-1049 (1991)1

Inhibition by Ihe PAr Antagonist WEB 2086 of PAr IndUm:llnositol-1,4.5-trispbosphale Production in Human PlaleleU

1-:W. Birke and H.A. ElI5ingerBoehringer Ingelheim KG. Department of Biochemistry. D-6S07 Ingel·helm. Germany

Platelet-activating factor (PAF) ectlvates human platelet! by binding 10 aputative PAF receptor which evokes the rapid formation of in05itol-I.4,~-trisphosphate (lp) by phospholipase C mediated phO!lphatidylinositol·4.5·bispllosphale (PIP2) lIydrolysis. Stimulation of 13H]inosi!01-labeledhuman platelets by PM (I nM-1 11M) resulted in a conccntnllion-<kpen-de:nt increase of intncellular IP3' 1P2 and inositolmonophosphote (JPI)'IPI IeveJ$ increued up to ttu=-rold upon muimum stimulation by 100nM PAf. The EC50 cuncenllation for PM was 1.2 ± 0.3 n.ld. Addition ofthe hetnlU'piooic PAF antagonist. WEB 2086, inhibited PAF stimulatedhydrolysis of P1P2 in a dose-dependent manner. WEB 20116 (100 11M)blocked inositol·1.4.5-trisphosphate formation down to baseline levels(ICSO=33 ± 12 11M WEB 2086). In tllrombin and ADP stimulatedplatelets. inositol phosphate (lP) generation was not influenced by WEB2086. It is concluded that WEB 2086 selectively antagonizes PAF-induced inrn:aseJ in IP and does not interfere directly with imracellulersignal transduction. Instead. WEB 2086. whicll has been shown to bindspecifically and with high affinity (Kj IS nM) to human platelets. acts as acompetitive antagonist at the PAF receptor level.[lipids 26.1050-1053 (199I)J

Phorbol Ester Stimulates PAF Syntbcsis ria the Actlutlon of' ProteinKi~ C In Rat Leukocytrs

MllSIIhiko Hayashi, Yoltsuke lmal and Sacbiko Oh-lshlDepartment of Pharmacology. School of Pharmaceutical Sciences,Ki1aHto Univers.ity. Tokyo 108. Japan

When rat pleunl mononuclear leukocytes were stimulated with 1 11M

phorbol myristate acetate (PMA). platelet·actiyating factor (PAF}·liteactivity was lIe!CC'led in the supernatant and the cellular frKlion$ of theincubatioo mlamre, 11$ measured by nbbil ptatelel agg.regltion. CI@,AFactivity peated Ill: 30 min in both ftactiolu. Awylttanderase activity inthe rnicmsomal fraction of the stimulated cells also il1CR:ased rapidly ands~ a peat al \0 min. A protein ki~ C inhibilor. staurosponne. andan inhibilor of phospholipase A2. p·bromophenaeylbromide. inhibitedstimulated PAF formation in both fractions. SlIIurosporine abo inhlbitedPMA induced acelyltrnnsfeTllSC lICtiyily. 'The dllia luggUI that PMA stim-ulates PAF synthesis by the remodeling patllway in nu pleurp\ cellsthrough acliYation of both phospholipase A2 and eceeyhransferase. andthat the acetyhransferase. in 111m,may be actlyated through activation ofprotein kinase C.[UpidsZ6. \054-1059(1991»)

Tumor Nec:rosis Factor Relnsc by Human !\.1onoc:ytes Stimulaledwith Piateitt-Adivaling Factor

N.M. Ruts, J.K. R05C'and F.H. ValoneDepartment of Medicine, Vetel1Uls Adminiltnllion Medical Center. SanFmncisco. CA 94121

Plalelet-lICtiyating factor (PAF) is a low molecular weight phospholipidwhich enhances human monocyte cytotollicity for IUmor cells. In the cur-rent studies. the eapacity of PAF to stimulate release of tumor necrosisfactor u (lNFa) by human monoc)'les was U$CSsed. PAF induced maxi-mal TNFa synthesis 2-3 hr after monoc)'le stimulation as assessed by dotblotting of cell-associated TNFQ using polyclonll anti-TNF anlibody.Maximal net release of TNFa occum:d 5-16 hr aner monocyte stimula-tion.. as assessed by a specifIC EUSA. Dose-response studies revealed thata maximal two- to three-fold Increase in release of TNFa occurs ItI()...IOO pM PAF. LysoPAF and the opIicll isomer of PAF did not stimu-late release of TNFo., suggesting that $!imulation is mcdillled by 5pCCiflCPAF receplOrS. Scau:hanl analysis of [3H)PAF binding to rnDfIlX:)'le mem-bnwcs revealed 651 :t 495 binding sitcslmonocyte with a Kd of 4.7 ± 4.2X 1O-lllM. PAF is I structurally unique ac1ivator of monocytCll whoseinteractions with TNFa Ind other eytokinel mlY be critical to hostdefCfl5Cagainst tumors.(Upids 26. 1060-1064 (1991))

Guinea Pig Bone Marrow Cells Treated wllh Platelet·Adivallng "'IIC'

tor Generate Factor(s) Which A"ecl$ Their DNA Syothui5 andMicrobicidal Aclivlly

Ichiro Kudo, Toshiyuki Kala. Hidetoshi Hayuhl, Ryohel Yanoshlla,Kolchi Ikil.llwa, Hlroko Uda lind Keilo InoueFaculty of Pharmaceutical Sciences. University of Tokyo. Hongo.Bunkyo-ku. Tokyo 113. Japan

We have preyiously reponed that platelet-lCtiyating facio.- (PAf) inducesproliferation and microbicidal activity of guinetl pig bone IIUIITOW cells. Inthe present study. we have foond that the conditioned medium of PAF- ornonmetabo1izable PAF 19onist-treated guil1el pig bone marrow cells lug-menll:d DNA synthesis and induced microbicidal ac1iyity of bone marrowcells. A PAF specifIC &nllgoni$!. CV-6209. inhibited generation of theacti~ oooditioncd medium by PAF. Addition of the PAF &ntagoni$! onlypanially suppressed tbe augmentative cffec:t of the ICtive conditionedmedium OIl DNA synthesis: lhis is CQIl.SJsu,ntwith the fact that. because ofthe rapid breakdown. no appreciable amount of PAF remained in the eco-ditioned medium ofPAF-treated cells. Although rnDU$C bone marrow cellsdid IlOl respoed to PAF unlitc guinetl pig cells. their DNA synthesis WIIS

significantly enhanced by the cooditioncd medium of PAF-Irtated guineapig hone marrow cells. Thus. tome newly gentl1lted f3Ctor{s) distinctfrom the originally inoculated PM seemed to mociulmte the bioactions ofPM on bone marrow cells. An appreciable amount of PAF was prodllCedby cakium ionophore-ltClted guinel pig bone mlllTOw cells. Tbese find-ings indicate that PAF synthesized in Buinca pig bone marrow cellsinduces generation in the cells of some factor(l) which affects prolifera-tion or microbicidalllCtivity.[Upids 26. 1065-1070 (1991)]

["ec:1 of Plalelet_Activating Factor 00 Tumor rseeresrs Factor-Inducecl Sapcroxide Generalion from Humao Neulrophils. PossibleIn,-oh-ement of G Proteins

Pierre Braquet4l', David Hosfordfll• Philipt KoIlzb, Jeln Guilbaudband Moniqut Paulltr1_BraquelbUlnstitute Henri Beaufour. 92350 Lc Plcssil-Robins.on and bClinicalRC!;CIIl'ChUnit Bum Center. 92141 Claman, FI'llfICC

The effect of platelct-activating factor (PAP) and of tWO specific PAFantagonists on tumor necrosis fIlCH)!'(TNF) induced superoxide produc-tion by human polymorphonuclear neutrophils (PMN) was examined.PAF alone (0.1 pM to 0.1 nM) failed to evoke sepercxlde produclion:however. when PAF was added for 10 min to cells upon prior incubationwith 10 nglmL TNF f"" 50 min. superoxide production was significantlyenhanced as compared to th~t induced by TNF alone. MlIllimum amplifl-cation (+]0%) was obtained with 10 pM PAF: however. the effect WII$completely abolished by two structurally unrelated PAF MtagonislS. BN52021 and BN 52111. 'The antagonists a1w decreased by 25~the super-oxide production elicited sclely by TNF, implicating the involvement oferoogenccs PAF in this process. PrcltCatmCnt of the PMN with either per-tussis 0.- cholcra toxin anenuaied the PAF amplified 5uperollidc preduc-tion in TNF stimulated cells. suggesting thlll 0 pmteill$ sensitive 10 thesetoxins may be involved in the mechanisms controlling amplification.(Upids 26.1071-1075 (199I)J

Protein KInase C is nOI Involved In Ihe Desensllizalion of PlaltlelActivating Faclor Receptor in Rabbit Pllteleb

L«-Young CbauInstitute of Biomedicil Sciences. Acadcmil Sinica. Nankang. Taipei11529. Taiwan, Republic ofOJina

Rabbit platelets pretreated with platelet ICtiv.ting factor (PAF) becamerefractory to further 5timulatioo by PAF. 'The effect was specifIC fo.- PAF.In this study. the a1tcratioo in the specifIC agonist binding to PAF ~plorin platelets following dC!iensilizalion WIIS inywigall:d. As KVealcd by theScatchard analysis of radioligand binding dati. the Iffinity for specificPAF binding to dcscru;itizcd platelet membranes WII$substantilily low.ered as compared with thl! to control platclet membranes. Ouaninenucleotide triphosphate. which Wll$ shown to decrease the affinity of spe-cific PM binding to platelet membranes. had less effect on the PAF bind-ing affinity to tile desensitized preparation. In plotelets pretreated withphorool 12-myristatc-13-acetatc. Ihe binding affinity of PAF receptorremained unaltered. Pretreatment of platolets with 1-(5-isoquinolinc-sulphonyl)-2-mclhylpipcrll;dne. a protein klnase C Inhibitor. or neomycin.an inhibitor of the polyphosphoinositide breakdown. failed to prevent theredudion of specific PAF binding affinity following subsequent exposure10 PAF. These results suggest that the agonist.induced ~nsitizlltion ofPAF receptor in rabbit platelets is independent of aclivation of proteinkinase C.\Upids 26.1076-1079 (1991)]

A Unique Pool of Free Arachidonllt Stn·u as Subslrlle ror bolhC,·clooxygcBa.'it and LipoJr)·geBa.'it In Pllteitb

••~ Chevy. Claude woIr aod OdUe ColardURA CNRS 1283. Departernent de Biochimie. Uniyersit~ Paris VI. 75571Paris. France

Stimulation of plltelelS induces a rapid release of arnchidonate from spe-cifIC phospholipids and subsequent remodeling of arachidonnle-contaJn.ing phospholipids. This process is accompanied by transfonnatioo ofreleased arachidonate by cycloollygenll5C and lipoltygcnasc enzymes. Wcaddressed the qucstioo of whether tbe cyclooxygenll5C and the lipoltyge-nase products odginaied from the same arnchidonDte-containing phosp/lo-lipids. [14C]Arachidonate prelabeled platelets were stimulated by throm-bin or by ionophorc A 23187. We monitored the CyclOOllygenase pathwayby following 12-hydrolly-5.8.IO-htpladecatricooic acid [12(S}-IUIT) for-mation and the lipoltygena$e pathway by following 12-hydrolly-

INFORM. VOl. 3. no. 1 (Jonuory 1992)

125

126

ABSTRACTS FROM Aces JOURNALS

S.8.10.l4-eicoSlIC!l1IC:ootc flt"id (12(S}-HETE] fonnalion and l'om~specinc activities. 'The data showed !hal the $alJIe pool of released arachi-donale can be utilized by either cyclooaygenase or by llpoxygenase.Indeed. the spccilic activity of both producu was identical when bothenzymes were acting. Since cyclOOllygenase was rapidly deactivatedwhile lipoxygenase cootinued to be active. the specific activity of 12(S)-HETE became lower lhan the specific aclivity of 12{S)-HHT when largeamounts of 12(S)·HETE were synthesized. Based on comparison of spe-cific activity between phospholipids and oxygenated products, me poolsof erachldonete-containing phospholipidS involved in the synthesis ofoxygenated products are dependent on the amount of aracbidonatereleased.[Lipids 26, 108G-l085 (199l)]

Blotransformlltlon or Alkylglyterols in Pt"nl Cell Cullures: Produc·lion of Platelet At'livlling Faclor and Olher Biologk:ally Acti..-e EtberUplds

Heimul K. Mangold, Shashank S. Aple. lind Nikolaus WeberFederaJ. Center for Lipid ReJeaJt:h. lnMitute for Biochemistry and Tech-nology. H_P.Klufmann-lnstiTute. D-44OO MOnster. Germany

Plant «lis in CIIlture Ill! caplble of inoorporatina exogenous I-O-alkyl-sII-glycerols into varioos neutral and ionic eeer lipids. 1-O-Alkyl-2-acyl-SII-glyccro-3-phosphocholines. the major class of compounds thusformed. are used for 1M preparation of platelet activlting factor (PAF) inhigJ\ yields. Similarly. the prochiral 2-0-alkyl-SII-glycerols are trans-formed to chiral 2-O-alkyl gly«ropho!;pholipids from which compoundscan be obtained that exhibit anTiviral activity in pllUlt and lUIima] cells.Rcaction of I·O·alkyl·2-acyJ-slI-glyccro--3-phosphocholines with phos-pholipase 0 in the presence of ethanolamine leads 10 1-O-alkyJ-2-acyl-sll-glycero--3-phosphocthanolamines. whkh serve IS statting material for thepreparation of I-O-alkyl-2-acyJ-slI-gJycero-3-phospho-(N-acyl)ethanolamines. compounds known 10 have antitumor activity.IUpids 26.1086-1092 (1991)]

Par_Acelher in Human Skin

\'vrs Oenboltl• Laurence Mkhelb, Jacques Benvenillle'l, Alain MfY-bfckl.', \'ol~ne ThomasD lind Louis DuberlTelb"INSERM V.200. 92140 Clamert, blNSERM U,312. 92010 ceen. aooc:t..VMH Recherche 92704 Colombes. France

Paf is a phospholipid mediator present in human skin which inducesinnammalory events. such as neutrophil infiltration and increased vascu-lar permeability. Recent ciAlI suggest that cutaneous cells. $UChas fibrob-luu and keratinocyt«. produce par and that par is Il!leased during allergiccutaneous ~acTions. It is tempting 10 speculate that par may contribute to!.be development in various 6kin disorden with acute and chronic skininnammation. ~f antagonisl5 may help in bringing answers to thishypothesis and may orrer new prospectS for the ~Itmen! of CUWleOtl5inllammawry dillUSC£.IUpids 26.1093-1094 (l99I)}

Chfmical Synlhf5iJ and Ph)'sloloiical Activity or Sulronium Ana-logues or Plalelel Activating Factor

Morris Katestl, Gforge A, Adamsb, Merle L. Blankt and FredSnyderC"Department of Biochemistry. University of Onawa, Ottawa, OntarioKIN 984. "univenlty of Ottawa Hean hutlillte. Onawa Civic Hospital.Onawa. Ontario K I Y 4E9. Clnooa and tMedical and Health SciencesDivision. Oak Ridge Allocialed Univenliies. Oak Ridge. Tennessee37831

Phosphatidylsulrocholine (PSC). the sulfonium analogue of phosphat idyl-

choline (PC). occun lWul1l11y in !iOIM diatOllU_ TIle replacement of !.be-N(CH3>J group by a .5(0I3h reslllu in an increase in the polar headgroup size in PSC relative to that of PC. coruistent wiTh the observedincrease in permeability of PSC billyers towards Urc:L It was of interest 10see whether replacement of the -N(CH3)3grouP in platelet&etivlting fac-tor (PAF) by an -~(CI"3h group leads 10 any change in platelet aggrega_tion or other physiological activity. Synthesis of the sulfonium analogueof PAF was carried out by suitable modifications of known procedures.The PAF-sulfonium analogue was found to have almost the same plateletaggregating activity as PAr itself. in the eoocenrreaon mnge 1-20 jJ.M.bur a much lower activity in the mnge O.ot-ljlM. The analogue had littleor no effect on the platclet aggregation aClivity of PAF when added in theconcentration range 0.01-1 jJ.M and had about half the hypotensive activi-ty of PAF towards hypertensive COF male rats. The ,utfonium analogue.however. wu much more cytotollic to HL-60 cells than PAF itself. in theconcenlrllion range 0-15 jlM: replacemenl of !he acetate group by II ben-loyl group increased the cytotoxicity 10 the level \"Ifthat of the methoxyanalogue of PAF. Thus, replacement of the -N{CH3» group by I~{CH3h group in the polar head group regKln of PM miulu in a rela-Lively small cltan&e in iu platelet a~gatKln activity and a decn:asc in iuhypolerU;ive activity. butgrellily iJICfUSCSiu antitumor activity,IUpids 26.1095-1101 (1991)]

Errect of Plale~-Acll\'aling Factor on Lipoprotein Upase and BloodUpids

Keiji Mimura, Susumu \'uh",., \'oshlo Mor!, Knuya Okada,Masal05hi Mune, Osarnu Nlshlkawli. Aklra Hiblno. M!yah!koSonobe, Tduya GOIO lind HinlShi NornoloThird Departmc:nt of Internal Medicine. Wakayama Medical University.Waklyama 640. Japan

We investigated the errecl of platelet-activlting factor (PAF) and of thePAF specific anllgonist CV-6209 on pluma lipid metabolism. and partic-ularly on post-heparin plasma lipolytic activily in male Wistar 11115.Lipoprotcin lipase (LPL) acTivity wu enhanced by intravenous injectionof PAF before intravenous injection of heparin when !.be PAF dose waslow (0.2 Ilgl1cg). PAF activated hepatic trilcylglycerol lipase (HTOL)acnvtry dose-dependently. Plasma triacylglycerols (TG) significantlydecreased with the activation of LPL and/or HTGL. Plasma total choles-terol (fC) and pho!;pholipld (PL) level. decreased at a low dose of PAF(0.2 Ilg/kg). but increased when higher doses were uscd. The PAF entago-nist CV-6209 partially reversed the PAF induced effects on HTGL. TC

"'" PLIUpids 26.1102-1107 (l991)J

Errect of Plalflel_Acllvating Factor on Cortl5ol and Cortiooslfroneseereuce by Pfrfu5e(l Dog Adrenal

Tadaomi A.ika ...aa, Taeko Hir~, 115uro Mll5umotoa, ToshikoMoritaWll"', TosIt!o Sh!ma<taG. \'urni MineA'. \'oshik! Tsujimotn" andYosbiro TsujibDepartmenu or QPhysioiogy and ~iltric: •• Nigasaki University Schoolof Medicine. Napsili 852. Japan

Administration of platelet-activating faclor (PAF) to perfused adll!nllincreased conisol and conicoslerone secretion. With hexadecyt PAF(C 16PAF; I-O-hexldecyl-2-acetyl_SII_glycero- 3-phosphocholine). theincrease- was significant It I nM and maximal at 10 nM. TI!e Il!spOI'I5CItO10 nM octedecyl PAF (CI8PAF; I-O-OClldccyl-2-acctyl-sn-glycero_3_phosphocholillC) were one fourth of those to 10 nM C 16PAF. The additionof CU;PAF 10 dispersed adrenal cells significantly increased cortisol andccntccsrercne production al 0.1 nM and 10 nM. respectively. Cl6PAFW8!l about loOO time. more potent than histamine on a molar buis inrespect to cortisol response In both perfused adrenal and dispersed adrenalcells. The results suggest Ihat PAF induces conlsot release from dogadrenal.IUpids 26,1108-1111 (1991)]

INFORM. Vol. 3. no. I (January 1992)

127

Tandem Mass Spedromelry of Negali~e Ions From Choline Phospho-lipid Mol~ular Species Related to Platelet Activating Factor

J.A. ZlrroIli. K.L. Clay lind R.C. MurphyDepanment of Pediatrics, National Jewish Center for Immunology andRespiratory Medidne, Denver. CoIOl'1ldo 80206

Fest atom bombardment mass spectrometry of choline phospholipids pro-duces negative ions characteristic of the intact molecule and tandem massspectrometry of collision-induced oeccmposuon of M-15 anions charac-terizes both the identity and substituent position of radyl groups. Certaincholine phospholipid molecular species which may be of special interestin the generation of platelet activating factor contain II highly unsaturatedIany IIcyl substituent 8.1sn-2 and an ether rad)'l group at se-t: othercholine phospholipid molecular species which contain esterified arachi-donic acid 8CCof interest as potential sources of arechldcnate foreicosanoid biosyrnhesis. Collisional activated decomposition of l-hexade-canoyl-2-arachidollOyl-lll-glycero-phosphocholine produce abundant car-bollylate anions at mh 303 (arochidonatc) and mil 255 (hcndecanoau:) ina ratio of 3: I, diagnostic for the .l"n-2arachidonoyl position. The etheranalog, I-O-hendecyl-2-arachidonoyl glyceropllosphocho\inc. producesonly one collision-induced dissociation ion at mlz 303 and no product ionscorresponding to the ether substituent at sn-!. MOlecular weight informa-tion from the M-15 ion combined with the CID generated carboxylateanions completely characterize these importanl phospholipids.

Precursor ion studies of M-15 anions from glycerophosphochcllnelipidll indicate that this ion is derived directly from a unique adduct ionfonned by attachment of the molecular species to a matrix alkollide ion.neutralizing the positive charge of the quaternary choline nitrogen.Decomposition of this adduct ion yields a methylated matrix moleculeand the nominal M-15 ion.[Lipids 26, 1112-1116 (1991)]

Determination of P\atelet·Acth·ating Factor by a ChemiluminescenceMethod and Its Application to Stimulated Guinea Pig Neutrophils

Yukio Hasegawa, Elko Kunow, Junko Shindou and Hidetaka YukiDepartment of Clinical Chemistry, School of Pharmaceutical Sciences.Toho University, Chiba 274, Japan

A simple, rapid and sensitive chemiluminescence method has been devet-oped to measure pletelee-actlvaung factor (PAF). Hydrogen peroxide gen-erated from PAF. upon phospholipase D cleavage, by choline encase isdetermined as chemiluminescence by a lumlnol-microperoxidese system.The detection limit of PAF by this method is 5 prnolaube. The method isreproducible with a 5.5% coefficient of variation at 10 pmol of PAF (n =5). Lipids were extracted from guinea pig neutrophils after stimulationwith cytochpJasin B and N-fonnyl-methionyl-leucyl-phenylalpnine, andPAF was lseleied by high-performance liquid chromatography and deter-mined by chemiluminescence measurements. The amount of PAF detect-ed was 96.1 ± 39.7 (mean ± SO, n = 7) pmovl08 cells. This highly sensi-tive method could be useful for the determlnation of PAF generated underpathophysiological conditions.[Lipids26.1117-1121 {l99i)J

Specific Binding of Antibodies to Platelet-Activating Factor (PAF) asDemonstrated by Thin-Layer ChromatographyfImmunostalning

Ken Karasawa, Noriko Satoh, Toshio Hongo, Yasuhito Nakagawa,Morio Setaka and Shoshlehi NojimaDepartment of Membnme Biology, Faculty of Pharmaceutical Science,Telkyo University. Sagamiko. Kanagewa 199-01. Japan

The specificity of rabbit antibodies produced by injection of 1-0-(15' -car-bollypentade<:yl)-2-N N -dimethylcarbamoyl-slI-glycero-3-pltosphocholinebovine serum albumin (BSA) conjugates was examined by a thin-layerchromatography (TLC)/immunostaining method. Phosphatidylcholtne(PC), Iysophosphatidylcholine (ly$OPC). Iyso platelet-activating factor(IY$OPAF), phosphatldylethanolamlne (PE), phosphatidylglycerol (PG).phosphalidylserine (PS). sphingomyelin (SM). phosphulidylil105itol (PI).

phosphatidic acid (PA) and cardiolipin (CL) were nOi immunostalned.Among several synthetic PAF-related compounds, the anlibodiC$ onlybound to PAF agonists which have the activity to induce washed rabbitplatelet aggregation. The results suggest that the birKIing sites of the anti-bodies on the PAF molecule are the acetyl group at the sn-2 position andthe choline moiety at the sn-3 position of glycerol, both of which areessential for exerting the biological function of PAF and for binding to thePAF receptors located on cellular membranes.[Lipids 26.1122-1125 (1991»)

Radioimmunoassay for Platelet-Activating Factor

Ken Karasawe, Noriko Satoh, Toshio Hongo, Yasuhito Nakagawa,Morio Setaka and Shoshichi NojimaDepartment of Membrane Biology, Faculty of Pharmaceutical Science,Teikyo University, 5agamiko. Kanagawa 199-01. Japan

A radioimmunoassay (RIA) f(ll""measurement of plalelel-activating Iector(PAF) was developed. At a final antiserum dilution of 1:640. the lowestdetection limit of PAF was 0.1 pmol (50 pg). The standard curve obtainedwas suitable for measurement of PAF in amounts ranging fmm 0.1 pmolto 39 pmol. The emiserurn showed high specificity. Cross-reaction forlysoPAF. lysophosphatidylcholine and long-chain phosphatidylcholineswas very low (less than 0.025%). I-Palmitoyl-2-acetyl-lll-glycero-3-phos-phccholine cress-reacted slightly (6.25%). PAF exogenously added tomacrophage suspensions was quantitDlively determined by RIA after sol-vent extraction and high-performance liquid chromatographie separation.RJA was also used to estimate PAF formation after stimulation of rabbitalvCQl1II"macrophages in suspension with calcium ionophore 06,23187.jLipid.r 26, 1126-1129 (1991)1

Synthesis or a PAF Immunogen and Production or PAr·Specific Anti-bodies

Mary A, SmaIQ, Brian A, Baldol'.b and John W. RedmolldcQKolling Institute of Medical Research, Royal North Shore Hospital, 51.I.eonards. N.5.W. 2()65. bDcpartment of Medicine. University of Sydney,Sydney, N.S.W. 2006 and cSchool of Chemistry, Mucquarie University.North Ryde. N.S.W. 21 13. Austmliu

Platelet activating factor (PAFj. a naturally occurring phospholipid withmany potent physiologicul and pharmacological activities. is implicated tIS

a mediator of many diseases. An immunoassay for PAF would greatlyimprove quamneucn. and hence PAF-specifie antibodies were required.Chemically-reactive analogs of PAF, containing an aldehyde group at theend of the I·O-alkyl chain (hellyl or dodecyl), were synthesized fromreadily available materials, During the multi-step l)'l1thetic procedure. thealdehyde group wus protected as an acetal. which was converted by mildacidic hydrolysis to the aldehyde immediately prior to protein coupling.lllese analogs wen: coupled to methylated bovine serum albumin and thoeresultant conjugates were injected into rabbits. Antibodies to PAF weredetected using a solid phase radioimmunoassay based on Protein A-Sephercse. The dodecyl PAF conjugate proved to be the more immuno-genic conjugate with more than half of the rabbits producing significantlevels of antibodie$ (at lellSt a la-fold increase in radioactive uptake overpre-immune levels). Results from solid phase irnrnuTlOlUsays employingnitrocellulose discs impregnated with PAE lysoPAF, lecithin. lysolecithinand 2-O-methyl-lysoPAF indicated that the antibodies recognized onlyPAE PAF-specific antibodies were isolated by affinilY chromatographyusing II column of PAF-poly(tysine) conjugated to carbcxy-acrlvated poly-acrylamide. The antibodies may be employed in a sensitive and specificimmunoassay for PAF and for many other studies involving PAF,(Lipid526,1130-1135(1991)1

A Specific, Sensitive and High-Capacity Immunoassay for PAF

Brian A. BaldlP.b, Mary A. SmalQ and Alistair C. McCas.ki!lcQKol1ing Institute of Med;cnl Research. Royal North Shore Hospital (IfSydney. St. Lconards. N.S.W., 2065. bDepunmenl of Medicine, Univers;-

INFORM, Vol. 3. no. 1 (January 1992)

128

ABSTRACTS FROM AOCS JOURNALS

I)' of Sydney_ Sydney. 2006 and C-SilcnUI Labonlories. Victoria. 3122.Australia

A specific radioimmunoassay for platelet-activating factor (PAf) sensitivein the range 10-1000 pg (O.02-2 pmoles) has been developed, Detailedquaruitative hapten inhibition $.Iudies showed specificity for the ~Iylgroup II C-2 of PAF•• requiremern for the etber linkage al Col and sometolerance for lubstituent! OIl the choline nitrogen. No signiilCalll cress-reactivity was found wlth ph05phalidylchoJine and Iysopbosphalidyl-choline or with lysoPAF.ILipids 26. 1136-1139 (1991)1

QlllInlilation by Radioimmunoassay or PAF in Human Saliva

Sue J. Coone"', Mary A. SmaJ6 and Brian A. BalikJIZ,bOKomng lostitute of Medical Rescan:h. Royal North Shore Hospital. 51.Leooards, N.S. W. 2065 and bDepartnent of Medicine. UniveBily of Syd-ney. N.S.W.o 2006. Auslnllia

Approximately 75~ of the PAF present in saliva is recovered on cxll'lC-lion of whole saliva (0.8 vol) wilh chloroformJrnelhanoi/waler (2:2:1,v/V/V). PAF levels. detennined by our rttCmly developed TlIdioimmullOllS-say. in saliva exlrat:ts ranged from 05-21 nglmL wilh S9% bet ....een 2-6ng/mL. Tllese figures. for apparently heallhy subjects. are higher than pre-viously reponed levels obtained by platelet assays. 11Je validity of ourradioimmunoassay lUUils wu checked by isolating and quanliulling thePAF fraction from whole $Iliv&. In addition. when we examined our sali-va sample$ by platelet aggregation. low jevels of PAF. comparable wilhthe values found in the literature. were detected. Investigations revealedthe presence of a substance(s) which inhibited PAF-induced plateletIggregation but Which did not affect the radioimmunoassay.[Upids 26.1140-1143 (1991)1

Inhibltor(s) of Plateitt·AC1iuting Factor (PA.') In Human Saliva

Mary A. Sma!D and Brian A. Oaldoa,baKolling Institute of Medical Research. Royal North Shore Hospital. SI.l..eonartls. N.S.W. 2065. and bDepanment of Medicine. University ofSydney. N.S.W. 2006. Australia

QuantiUllion of platelet-activating lector (PAF) in human saliva samplesby radioimmuooassay indicated there was. at times. sumcient PAF presentto aggregate platelets. However. in certain samples. we observed lillie orno aggregation. and furthermore, these samples ....ere found to inhibitaggregation induced by PAF (200 pg). Chsornatographic fractionation ofpooled saliv. incre~d the PAF octivity 4·fold. and the. observed inhibito-ry ecilvhy WIISfound to co-migrote with the fauy acids. 1l1e inhibitoryfraction was found to be active against platelet aggregation induced hyarachidonic acid (3.4 nmole) as well as PAF (2.5 pg). but not thrombin(20 mtr). Tbese results indicate the existence of a PAF inhibitor in saliva.which may uplain why potentially toxic levels of PAF can occur in the&aliva of nonna1. healthy individuals. These findings also highlight animponunt advantage of the. radioimmunoassay over platelet aggregationfor the quantitation of PAF in. at le15t. some biological Iluids,[Upids26.1144-1141(l99ll1

L459.989: A Usdul Probe in tbe Detection or Multiple Conforma·lional StatH or PAf-' Receptors

Sao_Bao HQng and My·Hanh LamMcn:k Sharp &; Dohme RClCarch Lllboratories. Depanmem of Biochemi-cal Rcgulation. Rahw.y. NJ 0"7<lM..0900

L-6S9.989 il II poIent. speclfic and competitive platelet-activating factorQ'AF) rttCplOf antagonisL The 2.5-tritium labeled L-659-989. similar to[3HIPAF. specifICally binds to rabbit platelet membr.mes with an C(juilib-rium di5SOCiition constant (KD) of 1.60 (±a.20) nM in 10 mM MgCI2'

However. guanosine S'-triphosphate (GTP) and several cations affect thespecific binding of [3HIPAF and of ~3HIL-659,989 to rabbit plateletmcmbrune5 in diffell'nt way •. K+. Mg -. Ca2+ and Mn2+ porennaie thesr,:cifie binding of both ligands. Na+ and u- inhibit the specific[ H)pAF binding. but enhance the binding of [3H)l=659,989: GTPreduces the fHIPAF bindin~ but his no effect on the binding of (3HIL.6S9.989. Ni + inhibi15 the I H1L-659.989 binding. bul has no effect 011

the binding of 13U1PAF. In the presence of ISO mM NaCI. [3H]t...-659.989exhibits identical KO and detectable binding sitts (BtrUU:) valllC$ as thosein the presence of 10 mM MgCI2. while KD and Bmu values of,3H)PAFan: dramatically reduced in the ~nce of 150 mM NaCi com-pared 10 those in 10 mM MgCl2' These results SUggest the existence ofmultiple eonfonnaliona1 stales of the PAF specific receptor and that PAFand L-659.989 bind differently 10 those statcs.ln the ~or 150 mMNaCi and I mM GTP. rttCptors appear to exist in a single coofonnationalstate with an equilibrium diuociation conSWit (KO) of 0.93 J1M fOf PAFas derived from the Schild plot. In isolated rabbit plate\e15 P!'-'treatcd with10 j1M ETH 227. a Na+-specific ionophore. the detectable [3H)PAF bind-ing sites ctroP from 260 10 100 binding sites per platelet. but the bindinglites for ,3H1t...-659.989 remain roughly the same. The Na+ binding siteswhich modulate the conformation of PAF receptoI"$ an: !bcll'fore proteCtedfrom extracellular Na+ until ionophorc is added. and ~ probably Ioaledon the cytoplasmic side of the plasma membrane.IUpids 26.1148-1153 (1991»)

Cheminl and Biochemical Cbaracterizalion or Lignan Analogs asNovel PAF gecepror AnlagonislS

T.Y. ShenOepal'tment of Chemistry. University of Virginia. Charlottesville. Virginia22901

Various derivatives and isosteres of nco1ignans of the 2.5-diaryl tetrahy-drofu11ln type have been synthesized as antagonists of platelet-activatingfaelor (PAF). A detailed analysis of their structure-activity relationship(SAR) has revealed a clear preference for an asymmetrical moi«ularcon-figuration with a high degree of stereo and chiral specificity associatedwith greater potency. TlIe frans-2S.5S enantiomers are generally 10-200umes more potent in \';fro than !heir COITe$ponding cis or frans-2R.5R iso-mers. A similar stereochemical preference is indicated by the recentlyreported PAF anlagonist MK-287 which has undefKOIle ciinical evalua-tion. An l\Zido derivative L-662,025 has been charecterized as a photola-bile irreversible anUlgOl1istof PAF for the lnvestlgatlon of solubilized andpal'tiaJly purined PAF binding proteins from cell membranes. The biologi-cal justification for concomitant inhibition of both PAF reCeptOr and 5-Hpoxygenase in innammation is well recognized. The feasibility of devel_oping such dual-functional pgents has been demonstrated by a group ofdithlolane analogs ofneolignans and several derivatives of futoenone.IUpids 26. 11j4...11~(l991)1

Thieno-trinolo-I.4-dlauplnes as Antagonists or Piatelel·Aclivating.'acta.": Pruc:nt StlitUll

Jorge Casall;·SlenulBoehringer In~lheim KG. 6507 Ingelheim lUll Rhein. Germany

1lIe platelct-activaling faclor, 1-O-alkyl-2-acetyl-slI-glycero-3-phospho-choline (PAF) antagonistic !lCtivity of thienotriazolodiazepines his recent-ly been described. The lead compound in Ihis series was brotizolam.which also exhibits sedative and hypnotic effects. 8y combirwion of bro-tizolam with the benzodlazeplne receptor antagonist RO 15·1788. PAFantagonistic and central nervou.s sylttm (CNS) activities could be segre_gated. SysttmDlic structure variation has led 10 pOlenl and selective PAFanta&Ql\ists without eNS effects. WEB 2086 and its analogues WEB 2170and STY 2108 are ~nlative examples of this AlUCtunaJ.type and haveshown a high potency and selectivity in PAF-induced and PAF-dependentin ";fro tests and in Cllperimcntal models. Studies in healthy volunteershave demon..wated potent pharmaoological activity and good safety and10lel1ll1CCof oral. intravenous or inhaled WEB 2086 in man. These agellts

INFORM. Vol. 3. no. 1 (January 1992)

should therefore prove useful for the further elucidation of the pathophys-iological role of PAF and provide an oppor1unity for the!3P<'"tic applica.tioru; in diseases in which the: involvement ofPAF has been implicated.[Upid~26, 1157-1161 (1991)]

PAt-"Receptor Structure; A Hypothesis

J.-J. GodfroidQ, G. OJ''eb, J. LamoUe-Brasseur'», J.-P. BauQ and F.HeymansDQLaboratoire de Pharmacechimie Moleculaire, Universite Paris 7, Paris.Prance and bCemre d'Ingenierie des rroretoes. lostitut de Chlrrtie. Li~ge-Scrt-Tllman. Belgium

Different hypotheses of the structure of platelet-activating factor (PAF)receptor based on structure-activity relationships of agonisls and antago-nists are reviewed. For an agonistic effect. strong hydrophobic interac-tions and an elber fuoction ~ required in position-I of the: glycerol back-bcoe. chain length limitations and steric hindrnno:::edemand a small groupin posilion-2. The unusual structural properties of non-PAF-like amago-ntsts required 3-D electrostatic potential calculations. This method applied10 seven potent antagonists suggests a strong "Cacbe-oreilles" (ear-muff)effect. i.~..two strong etecceoeganve wells (isocontour at -10 KClIl/mole)are located HI ISO" to each other and at a relatively constant diSlIlIlCe. Ini-tial consideration of the: "Cacbe-oreilles" effect implied the: structure of abipolarized cylinder of 10-12 A diameter for the receptor. However. veryrecent studies with agonists and antagonists suggest tnat the receptor mayin fact be a multi-polarized cylinder.[Lipids 26. 1l62-1166 (1991)]

PAt-" Receptor And "Cachf:-Oreille!i" Errect. Simple PAt-"Antagonists

J. Lemcne-Bresseure. to: Heymansb, G. DiveD. A. L.amourib, J.-P.Baub, C. Redeuilhb, D. Hosrordt', P. Braquett' and J.-J. GodrroldbaCenlre d'Ingenterie des ProI6rn:s.institut dc Chimie, Liege-Sart-Tllman,Belgium. bLaboraloire de Pttarmacochlmle Moleculaire. Universite Paris7. Paris. France and <lnstinn Henri aeauroor.Le Plessis Robinson. FI1IIlCe

Nine simple and structurally nexible PAF antagonists were synthesizedand their inhibitory effects on PAF induced platelet aggregation weremeasured. Compounds with PAF antagonistic activity exhibited a nega-tive electrostatic potential generated by two trimethoxyphenyl groups(isocontour at -10 KcaVmole) at various distances between the negativeclouds. The optimal distance between the negative clouds generating the"cecbe-oreiues'' system for exhibiting potent PAF amagoni5tic activity isestimated to be 11-13 A.ln the flexible molecules studied. the dispersionof the electronic distribution is not necessarily favorable for anti-PAFactivity. llie data support the simple bipo1ari~ed rnoo:kt for the PAF ~ep-tor thot hal; been proposed by the authors.[Lipids26, 1167-1171 (1991)1

Discovery and Preliminary Pharmacology of Sch 31370. a DualAntagonist or PAF and Histamine

M. Motllsim BillahQ. Robert W. EganD, Ashit K. Gllngul,·b. MichaelJ. Greenb, William Krt'utnerD, John J. Piwill5kib, Mar-vin I. SiegelQ,t-'rank J. Vmanib and Jesse K. WongbDepartments of bChemislry and GAliergy and lnflarnmation BiologySchering-Plcugh Research, Schering-Plough Corporation. Bloomfield.New Jersey 07003

From a series of amide W13.logsof the histamine HI ant.agonist. RUtadine.II potent, omlly active. dual platelet-activating factor (PAF) and histamineantagonist. Sch 37370, namely l-acetyl-4·(8-chloro·5.6-dihydro-lIH-benzo-[5.6]cyclohcpto[ 1.2-b]pyridin-ll-ylidine)piperidine. was discov-ered. Sch 37370 selectively inhibits PAF-induced aggregation of humunplatelets in "il'O (IC50 '" 0.6 IJ.M). and in """0 inhibits PAF- and his·

tamine-induced bronchospasm in guinea pigs with ED.50 values of 6.0 and2.4 mglkg p.o .. respe<:lively. Seh 37370 is expected to be IIlOfe efficaciousthan single mediator antagonists in allergic diseases. such at asthma.[Lipids 26. 1172-1174 (1991)]

PAF Inhibitory Activity of Dlketepiperazjnes: Structure-ActivityRelaticnshlps

Norihiko Shimaz.aki, Icbiro Shima, Mll$llnori Okamoto. Keizo ¥oshi.da, Keiji Hemml and Masashl HasblmotoExploratory Research Laboratories. Fujisawa Pharmaceutical Co .. Ltd .•Tsukuba. lbaraki, Japan

FR900452. a natural product isolated from the culture broth of Strepto-myus phQto[QcitllS No. 7739. was found to inhibit PAF-induced rabbitplatelet aggregation with an ICSO on.7 x 1O-7M. FR900452. l-methyl-3·[ 1-]5-metbylthiomethyl-6-oxo-3·(2·oxo- Lcycjopemen-t-yltdenej-z-pipenlZinyl]ethyl]-2-indolinc. hat an oxocydopentylidern: group incorp::!-rated as a vinylogous amide in a diketopiperazine skeleton. This uniquestructure led us to synthesize diketopiperazfne derivatives. 3·arylalkyJ-6-substituted-piperazine-2,5-diones. Their observed PAF inhibitory activitysuggest that the D-D coeflgeration of diketopiperazine is an imponantfactor for anti·PAF aclivity and rhm the hydrophobic aromatic portionmay playa specirlc role in the binding of the diketopiperuzinc 10 the PAFreceptor,[lipids 26. I17~1178 (1991)]

Pharmacological Properties of YM461, a New Orally Active Platelet-Activating Factor Antagonist

Toshimitsu Yamada, Muoetoshi Saito. Toshiyasu Mase, Hirt.mu Mara,Hitoshi Nagack&, Ki}'oshi Murase and Kenicbi TomiokaMedicinal Research I..alxJr.Ilories. Yamnnouo;hi f'barmaa:utical Co .. Ltd ..Tsukuba.Jbarakl 305. Japan

The antagonistic effect of YM461 [1-(3-phenyJpropyl)-4-[2-{3-pyridyl)lhi-azolidin-a-ylcarbonyllpiperazlne fumarate] against platelet-activating fac-lor (PAF) was examined io several/I! ";11'0 and ill ";''0 systems. We foundthat YM461 inhibited 13PAF! binding to rabbit platelet membranes with apKj value of 8.90. YM461 inhibited PAF induced rabbit and humanplatelet aggregation with pA2 valUe! of 7.52 and 7.29. respectively; the:slopes of the Schild plots were 1.07 and 1.01. respectively. However.YM46L Bt 10-4 did not affect rabbit and human platelet aggregationinduced by ADP. collagen. arachidonic acid or epinenphrtne. YM461inhibited PAF induced death in mice with an EDSO (50% effective dose)value of 0.35 mg!kg p.o. YM461 at doses above 0.3 mg!kg ;.~. inhibitedPAF induced hypotension in rats. YM461 showed a dose-dependent inhi-bition of PAF induced hemoconcentration in rats with EDSO values of 0.15and 0.21 mg!kg p.Q.. respectively, at 0.5 and I hr after oral administration.TIle anti-PAF effect of YM461 persisted more than 6 hr after 3 mglkg p.o.in rats. YM461 inhibited the brcnchoccnstriction induced by PAF with anEDSO value of 1.2 mg.l1cgp.o. in anesthetized guinea pigs. Furthermore.the compound ot doses above 3 mglkg p.o. significantly inhibited antigen-induced anaphylactic asthma in conscious guinea pigs pretreated withmepyramine and propanolol. These results indicate that YM461 is a selec-tive. potent and orully active PAt-"antagonist.[lipids 26. 1179-1183 (I99I)]

Bioactions of 5-Hydroxyicosatetraenoatl' and its Interaction withPlatl'let_Aclivllting Factor

Adriano G. Rossi and Joseph T. O'FlahertyOepanment of Medicine. SC(;tion of Infectious Discasel>. Wake ForestUniversity Medical Center. Winston-Salem. NC 27103

In a variety of stimulated cells. platelet-activating factor (PAF) andnumerous arnchidonate derivatives are co-products that form as a conse-quence of ~epIOl'·mediated phospholipid mobilization. These lipid eo-produt'ts produce 0 plethora of biological effects in 0 wide: variety of ceu

INFORM. Vol. 3. no. 1 (January 1992)

130

ABSTRACTS FROM AOCS JOURNALS

SYSICIlU.Furthermore, they often have a fascinating. although less widelyappreciated. imerecticn. S-HETE. at submicromolar ccocenuaucns. exensrelatively few dlrect bioaclions. II doell, ho_ver, poIently (16--160 oM)mse c:ytosolic free eak:ium [C.2+h and augment PAF-induced responsesin human polymorphonuclear neutrophils (PMN) by as much as 100- 10\OOO-foid. 5-HETE IICt5 on PMN by • slnIClUraily spec::irlc:. stereospecificand pertussis lO11in-inllibitable mechanism. In addition. PMN exposed to5·HETE exhlblt homologous but noI heterologous desensitization. 'Thesefindings suggest that 5-HETE. like PAF. may bind to its own specificplasmalemm.al receptors 10 exen its unique set of bioactions. However.funher investigation is required to dClIIOfL'ltrate any punitive 5-HET£receptors. Other potenti.1 mechanisms of S-HETE-induccd bioaclionstogether with the po6Sible effects of 5·HETE on PAF transduction mecha-nisms an: also discussed.IUpids 26. 1184-1188 (1991)]

Platdel-Acllntlnl Factor (PAF) Receptor Antagonists InbibitArtlchldonic Add-Induced Platelet Aggregation In Rabbit WboleBlood

Alai ... Jean Ammlt and Chris O'NeillHuman Reprodoction Unit, ROyl1 Nonh S~ Hospital of Sydney, SI.1..eonan1s. 206S, N.S.W .. Australia

Tbe potency of several platdet-lC'Iivating factor (PAF) receptor antago-nists was measured by ooSC'rving their inhibitory erreeu against PAFinduced platelet aggregation. Their SC'lectivity was assessed by monitoringtheir effect on platelet aggregation induced by anchidonic acid (AA) andadenosine: diphospllJ,lC: (ADP). The antagonisls inhibiled platelet aggrega·tion induced II the PM ECSO (0.023 jJm) with the following rani;: OI'dcrofpotency; WEB 2086 :> WEB 2170 :> SRI 64-412 > SRI 63-675 :> BN52021> kadsurellO!lC: > SRI 63-441 > alprazolam. While the antagonistshad no inhibitory effect II the ECSO for ADP (10 jJm), they did not inhibitplatelet aggregatioo Induced at the ECSO for AA (55 jJm). However. therewas eonsidenable variability in the 'lope of the inhibitory response and therelative potency of each anllgonist against PM induced plllClel aggrega-tion as compared to AA induced platelet aggregation. The antagonist 1CSO(Jim) against PAF and AA were as follows, whh those that showed 5ignifi-clntly different (p:> 0.01) slopes indicated by an esterick: SRI 63-441·(3.8. 15.1); SRI 63-675 (1.4. 36.2): SRI 64-412 (0.5. 10.5): BN 52021·(2.4, 58.9): hdsllrenone· (2.8, 28.3): alpl"llUllam· (10. 25): WEB 2086(0.055.0.220). and WEB 2170 (0.107, 0.534). Therefore. in rabbit wboleblood Inc anlagonists were potent. although not completely selective.inhibitOfl of PAF induced platelet aggregation. Tbese resull!i ~ggest thatthe mode of action of PAF and AA Induced platelet aggregation mayshQre some common features. However. since the slope of the inhibitoryresponse against PAF and AA for some entagonists differed, mechanisticdifferences in their action appear to exist.(Lipids 26.1189-1192(1991)]

Biological Response of Guinea Pig Peritoneal Macrophages toPlatelet·Activatlng.'actor

Hldeloshi Haya5hlG, Ichiro Kudo", Shoshichi Nojimab and Keizo1_"QFllCUltyof I'twmaceutical Sclencet, 'The Univenily of Tokyo. Hongo,Bunkyo-ku, Tokyo 113, and bf"aculty of Phann~utical Sciences. TcikyoUnlvershy, Sagamikomachi. 1'Iukui-gun. Kanagawa 199-01, Japan

We investigated the effects of platelel-lC'Iivating faclor (PAF) 00 guineapig peritoocal macrophagC:$. SpecifIC and high-affinity binding sites forPM were detected on guinea pia peritoneal m~gcs. ScaIchard anal-Ylis 00 PAF bindina revealed high affinity binding sites (7.9 x I04JaIJ)with a dissociation constant of 2.3 x 10-10 M. When treated wilhto'9_10'!i M PAF. guinea pig peritoneal macrophages released hydrogenperoxide into the medium in a time-de:pcndent manner. 'The release reac-lion upon 5timulation with 1O-!i M PAF reached a plateau within 30 minand the extent of rejeese was twice as high as that when stimulaIcd by N-formyl-L-methionyl-lcllCyl-L-phenylaianinc (tMLP: 2 J.lM}-treated cells.

Neither lysoPAF nor the PAF enanuomer was effective. PAF·jnducedH2~ release was inhibited specincaUy by PAF antagonists, suggestingthat PAF activated macrophagC:$ through binding 10 specific sites. Lysoso-mal ensyme (N-lICC:tyl·~D-gl\ICosaminidase) was released from guineapig pcriKInCal macrophages upon treatment with 10'5 M PM for 60 min.Guinea pig peritoneal macropbaSts were ~ated with PM for 8 ItT and theconditioned medium was examined for cylokines. The medium exhibitedcytocidDlactivity .gainst mouse fibrobl8.'i1 L929 cells [tumor necrosis fac-tor (rNF) activity], and this activity was compamble to thai detected aftertreauncnt of cells with the bacterial lipopolysaccharide (LPS). Further-more, the same conditioned medium also ~ coIooy-stimulating fac-tor (CSF) activity. Oencl1ltion of these C)1OItines was Sicn:ospcciflC. Ourfindill8s suucst that PAF is a unique macrophage activator thDl ~nti-eres both respiratory buut/ly.osomal enzyme release (early-phaseresponse) and monokine production/glucose consumption (late-phaseresponse).(Lipids 26. 1193-1199 (1991)]

Production of Plalelet-Activatlng Factor by Human No.-modensc andHypodensc Eo5Inophlis

Ayako Ojima-Ue:.hlyamaG, Vasuo Ma.suz..awa". Takayuki Sugiurtl",Keizo Waku", likeshi Fukudab and Sobei Makin06°Faculty of Phannaccutical Sciences. Teikyo University. KanagaWJI 199-01. Japan, and bootkyo University, School of Medicine. Tochigi, 321-02.Japan

Nonnodcnse cosinophils and neutrophils from nonnal dooon producedcoosiderable amounts of platc:!et-lcIivlting factor (PAF) when stimulatedwith ionopbore A23187. PAF produced by eoslnophils appeared to bedegraded more ~pidly thin PAF fonned by neutroptnts. suggesting Ihigher activi1y of PAF-dcgrading enzyme in cosinophils. Substantial pro-portions of PAF newly fonncd by both eosinophils and neutrophils wereshown to be cell-associated. By comparison. hypodensc eosincphilsobtained from I patient with idiopathic hypcrcosinophilic syndrome pro-duced an extremely la.ge amount of PAF and released mIlCh of it into tncincubation medium. The accelerated fonnation of PAF in hypodensceosinophils may be related 10 various cardiovascular complications asso-ciated wilh h~pereosinophilic syndrome.[Upitlt 26. 1200-1203 (1991)]

Immunoregulalory Funcllons or Pa'-Acethtr. VI. Dual Effecl onHuman 8 Cell Prollferalion

Corinne Leprinceo• Eric Vlvlerb• Dominique Tretun'", PierreGalanaud", Jacques Benvenlsleb, Volande Richardlf and VolheThomasb°Llbontoire d'lmmunopathologie et d'lmmnunologie Viralc. InslilutNational de 10 Samt er de la Recherche Mtdicale, U131, and buboratoired'lmmunopharmacologie de l'Allergie et de l'{nflarnmation. lnsrltutNational de la Santt er de la Recherche Medlcale. U200, Universitt Paris-SUd. 9214() Claman. France

'The role of pat-accther (pal), I phospholipid cytoir:inc. in the modulatin ofhuman B cell function was investigated. Pal. from I x 10-5 M to 10-6 M.decreased B cell proliferation Induced by both phorbol myristare lICCIate(PMA) and anti-lgM antibodies (anti-lgM Ab). By contrast. I x 10-7 M toI II. 10'9 M par enhanced PMA triggered, but not anti-lgM triggered Bcell proIife~tion. B cell prolifel1ltion was modulated hctween 24 and 72hr of cuhure indicating that the effect of pat did not me:n:ly reflect I lIIiflin prolifel1ltKln kinetics. IntC:fW:ingly. pal aI$O enhanced the spontaneousproliferation of a Burkitt Iymphoma-dcrivcd B cell line. Raji. which lug-gcsu that paf can dim:lly act on B cells. n.c modulatory effect of paf onperipheral blood B cells was independent of PMA concentration, yet theeffect of Raji cells was dependent upon cell density. 1lIc data suggC:5t thaIpat is • ~nt modulator of B c.ell function. and may be involved in thecontrol ofhumonl immune response.[Lipids 26.1204-1208 (1991)]

INFORM. Vol. 3, no. 1 (January 1992)

131

Intra«lIular Cal...Concentration and "202 Production In MousePeritoneal Macrophages Are Stimulated by Platelet Activating Factor

Masayuki Sasaki, Katutaka MaeYlima lind Takehiko WatanabeDepartment of Phamlllcology I. Toboku University School of Medicine.Sendai 980, Miyagi.lapan

The intracellular calcium concentration UCa2+Ji) in mouse peritonealmacrepbages cultured on a covergress was measured in the superfusionsystem using fum-l as a nuorescenl calcium probe and plalelet a<:livatingfactor (pAF) as a stimulant In the presence of extracellular Ca2+, 10-7 MPAF sharply increased ICa2+]i from a basal level of 90 oM 10 340 AM.'IlIm:afier, the {Ca2+11 level gradually decreased in two phases. in an ini-tial rapid phase and a subsequent slow phase of decrease. The calciumresponse was dependem on PAF concentration. In !he absence of exrracel-lular Ca2+, a single sharp peak was observed, suggesting IWOdifferentmodes of Ca2+ movement, one from intracellular stores and the otherfrom the extracenufar medium. A simple. sensitive fluorometrk assaysystem was developed for measuring H202 in the scpertusare ofmacrophages after stimulation by use of immobilized peroxidase and 3-{p-hydroxyphenyl)propionk acid as a Iluorogernc substrate. With thisstysrem. as liule as 2 pmol of H2~ could be measured. PAF (I [lM)increased H2~~lroductioo in peritoneal macrophages in the presence ofelltraceliular Ca +ibut H2~ production was not observed in tile absenceof'extracellular Ca +.{Upids 26, 1209-1213 (1991)]

Ptatdet-Activating Fador Stimulates Receptor-Mediated Formationof Reecnv e Oxygen Inltrmediales in Human Monocytes

M.G. Pustynnikuv, N.V. Porodenko, O.V, Makarova, A.V. Kozyukov,E. Vu. Moskaleva, A.A. Sokolovsky and E.S. SeverinResearch Center of Molecular Diagncstlcs. USSR Ministry of Health.113149 Moscow, USSR

Stimulation of production of reactive oxygen Intermediates (ROI) wasuamined in human peripheral blood monocyres by lurninol-dependentchemiluminescence. The dose-response curve characterizing the depen-dence of ROI production on the concentmtion of platelet-activating factor(PAr! showed that stimulation occurred within a concentration range of 2x ]()-9M to 5 II [~M. Transformation of the dose-response curve to an&die-Hofstee plOi indicated that the process is characterized by twO Kmvalues. The Km value com:sponding to the high-affinity branch of thecurve is 1.3 ± 0.14 nM. In the same cells. tlie dissociation conSlani for the13H]PAF/receptor complex was determined. The Kd value was 0.8 ± 0.1nM. which agreed quite well with the high-affinity Km value obtained inthe Eadie-Hofstee plot. The dat.a indicate thot stimulation of ROI genera-tion is mediated thmugh PAF binding at specific receptor sites at nanomo-lar PAF ccecennarjcns. Along with the specific receptor-mediated ROI&eneration, a nonspecific effect of PAF at a high concentration wasdemoostnlted.{Lipid! 26,1214-1217 (1991)]

Cakium Channel BIOf,:kade lnl)ibit$ Platelet Activating Factor Pro-duction by Human Umbilical Vein Endothelial Cells

J.P. Tolins, A. Melemed, D. Suldner, K.s. Gustafson and G.M. Verttl-

"""University of Minnesota Schoo! of Medicine and Veterans AdministrationMcdical Center, Minneapolis. Minnesota :554[7

An increase in intracellular calcium level is an important signal in the reg-ulation of cellular responses under normal and pathological conditiOfl5.Because two key enzymes in the synthetic pathway of platelet IICtivatingfactor (PAF). phospholipase A2 and aceryltransferase. are calcium depen-dent, we hypothesized that calcium channel blockade may inhibit agonist-induced PAF synthesis. Primary cultures of humQn umbilical veinendothelial cells {Eq, pre-incubated with 13H]pcetate. were e~posed 10thrombin (:5 U/mL) and PAF production .....as quantitated by iocOl"pOmtionof radiolabel into the EC lipid fraction co-migrating with exogenous PAF

in thin-layer chronlBtogr&phy. The effect of ~-incubation .....hh calciumchannel blockers (verapamil. dihiazem. 10-4 M) or buffer was deter-mined. Results (triplicate experiments. - P<fI.O:5 vs buffer, t P<O.05 vsthrombin) demonstrate that pre-incubation with calcium channel blockermarkedly inhibits thrombin-induced PAF production (verapamil:buffer273 ± 122. thrombin 10.735 ± 1524-, thrombin + verapamll 178 ± 91 tcpmIplate: diltiaz.em:buffer 1097 ± 581. thrombin 15,283 ± 2661-, throm-bin + diltiazem 280 ± 56 t c~late). The effect of diltiazem was dose-dependent (% inhibition: 10- M.46%; 10-5 M, 60%; 10-4 M 98%). en.tiazem also inhibited bradykinin (l0-8 M) induced PAF s~nthesis. In cal-cium-free medium or in the presence of LaCI3 (l0- M), the PAFresponse of EC to thrombin was blunted (buffer 582 ± 360, thrombin5394 ± 1069, thrombin + calcium free medium 1055 ± 57!. thrombin +LaCI3 1271 ± :58 cpm/plate). We oorx:lude that calcium channel blocken;prevent agonist-induced PAF synthesis. possibly by preventing cellularcalcium influx and activation of PAF synthetic enzymes. We speculatethat this mechanism may underlie, at least in pan. the beneficial effect ofcalcium channel blockade under various pathological conditions.{Upids26,1218-1222(199I)]

The Errect or EicosapentHnok Acid Consumption on Human Neu-trophil Chemiluminescence

Philip J. Thompsona, Neil L.A. MissoD, Marlon Passarellia and Mar-tin J. PhlllipsbIl])epanment of Medicine, University of Western Austnllia and boepan-ment of Respiratory Medicine. Sir Oiarles Gainlner Hosphal. Ned[ands.W.A. 6009, Austnllia

The effect of eicosapentaenoic acid (EPA) on the inflammatory potentialof neutrophils was investigated by supplementing the diets of 12 $ubjectswith 2.16 g of EPA or 12 g of olive oil per day for 4 weeks in a doublehlind crossover study. Neutrophil function as assessed by luminolenhanced chemiluminescence responses to platelet-activating faciO!"(PAF)and fonnyl-methionyl-leucyl-phenylalanine (FMLP) was significantlyreduced after EPA but not after olive oil consumption in the subjects .....hoconsumed EPA first. In contrast. EPA had no significant effect on neu-trophil chemiluminescence in the subjects who consumed olive oil first.Dietary supplementation with EPA inhibits neutrophil responses toinflammatory mediators such as PAF while other fally acids appear \0modify the effects of EPA.[Upjds 26. [223-1226 (1991)]

Role of Platelet-Activating Factor CPA!') in Superoxide Production byHuman Polymorphonuclear Leukocytes

Shujl TakahllShi, Toshika:zu Voshikawa, Vuji Naito, Toru Tanigawa,Norimasa Yoshida and Motoharu KondoFin;t Department of Medicine, Kyoto Prefectural Universlry of Medicine.Kyoto 602, Japan

The effect of platelet-activating factor (PAF) on supcroooe production byhuman polymorphonuclear leukocytes (PMN) was studied. Cypridinaluciferin analog (CLA) dependent chemiluminescence was used to detectsuperoitide anion radicals. PM induced supeecxide generation in humanPMN in a dose-dependent manner. Preincuba1ion .....hh a small amount ofPAF (5 it 10-9 M) enhanced PMN superoxlee release induced by variousstimuli. such as phorbo[ myristate acetate (PMA). opsonized zymosan(Ol), calcium ionophore (A23187) and N-formyl-methionyl-Ieucyl-phenylalanine (FMLP). The PAF antagonist, CV-6209. inhibited superox-ide production induced by PAF. bU1 not that induced by other stimuli.These findings would indicate thai PAF may play an important role atinflammatory reaction sites and that CV-6209 may inhibit excessiveinflammatory reaction.[Upids26,1227-123O(1991)]

IliA Immune Aggregates Stimulate Platelet_Activating Factor andSuperollde Anion Production by Human Neutrophils. "Comparisonwith IgG Aggregates

INFORM. Vol. 3. no. 1 (January 1992)

132

ABSTRACTS FROM AOCS JOURNALS

I'aloma Hernando, Jesus Egldo, Rosario de Nicoli!; and Eva Gon:tiiluDepanmem of Nephrology. Fundaci6n Jimtnez Diu. UniveraidadAUI6noma-CSIC. Madrid. Spain

We have examined the pos5ibilily thai human polymorpbonUl:Iear cellsexposed to ISA immune complexes can mediate the production ofplarelet-ectiveting factor (PAF) and oxygen radiClI5. We found thaihuman [gA and IgG immune aggregates stimulated. to a similar extent.PAF and Of production by human polymorphonuclear eens (PMN) in aconcenlnnion UId lime dependent manner, The PAF.rnal was llIJEe1yasso-ciated with eeus. was shown to be identical 10 synthetic PAF. as deter-mined by pbysicQChemical. chmmatographic and mzynuuic assay. Fur-thcnnorc. d, 110''0 synthesis of PAP by PMN was shown to occur byincorporation of radioactive precursors. such as 13HlaceUIe. TIle additionof normal human serum 10 PMN incubated with JgG aggregates resultedin a significant amount of PAF rorm~tion which wes not observed withIgA aggregates. By contrast. no change was seen in PMN ~~ with eitheraggregates. The p~incubation of PMN with cytechalasln B. an inhibitorof p/ulgocytosb. did not affect PAF alld ~~ production by both agg~-gatcs. The results suggest that the inleruction of PMN with the IgA com-plexes in blood vessel walls of dirre~nttissues can reJluit in tile release oflipid mediators. such as PAF and oXYl!en radicals that could contribute tothe inflammatory response.[Upids 26. 1231-1235 (1991)J

Platelet·AC1i.-ating Fador and PoI}"unsatu~led Fauy Acids in Cere-b~1 Ischemia or Con\'ulsions: Int ... CC'lIuln PAF.Binding Siles andActiulkIn ofa Fo.<WJunlAP-1 TrallJO"lplk>nal Signaling System

Nicolllll G. BazanD,b,d, Slephen P. ~ulnloD. Pierre Braquelil!, ThomllllPanetta" and ViClor L. MarchesdUb;dllI>epartmenl$ of Biochemistry and Molecular Biology. bophthalmology,and £'Surgery, dNeul1l!iciencc Ccmer. Louisiana State. University MedicalCenter School of Medicine, New Orleans. Louisiana 70112 and ~tilUlHenri Beaufour, Le Plessis Robill5Of\, France

Platejet-ecrlvating factor (PAl') Is a lipid mediator formed in the earlyresponse of the central nervous system to ischemia or convulsi0n5. F~polyunsaturated fatty acids and arachidonie and docosahc:uenoic acidsare accumulated along with PAF. Antal!onino; of PAF have been found toimprove ce~bral blood flow and panially block the rise in fR:Cfally acids,an effect that may arise by way of Inhibition of PAF receptOr'S or stimula-tion of the reacylation of tree fany acids released upon insult. Three imra-cellular PAF-binding sites hove been identified in rut cerebral cortes.These very high-affinity binding sites ore inhibited by PAF antagonists.with certain antagonists exhibiting specificity for a particular binding site.This specifICity indicates heterogeneity in these billding sites. Ischemia orstimulation also leads to pmlooocogene tmnscriplional activation. Here.we discuss studies with cells in culture snowing that PAF promoces tran·scriptional activDtion of immediate-early genes. PAF activates the tmn-scription of the immediate-early genee los and jun, whose gene productsan: regulutOl'l of the transcription of other genes. Trnnscriplion of los isalso activated by convulsion or ischemia in the central nervous system.The aaivalion of these genes by PAF can be inhibited by PAF an13gonistJ.and is ~ntJy accomplished by wly of an AP-I tnnscription ~gulato-ry sequence in the promoter ~gjon of the~t Jenl'.S. Studies with dele-tion muWlts show that PAF can also exen its activating p~rties by wayof cyclic adcnosine-rY-monophO$phate-(cAMP) and Ca +.~sponsiveelements. and suggest that PAF is involved in an Intereonoected networkof cell signaling that may coordinate soorHenn and long-tenn responsesof cells to stimulus and injury.IUpids 26.1236-1242 (I99I)J

Calcium Ion MQbilizalion in Neuronal Cellllinduced by PAF

Elizabeth Kornecki" and Yigal U. Ehrlichb0Department of Anatomy and Cell Biology. Suite University of New YorkHealth Science Center. Brooklyn. New York 11203 and the bCSIIIBRCenter for Developmental Neurosciences. College of Staten Island, CityUniversity of New York. Staten Island. New Yorl; 10301

INFO~M. Vol. 3. no. I (January 1992)

We have reponed previously that platelet·activating factor (PAFj inteructswith the neuronal cell line NGI08-15 (neuroblastoma X glioma hybrid)and the pheochromocytoma cell line. PC12. PAF acts on these: cells byraising levels of inuxcUular free calcium ions. In the ~t repert, weexl£nd these studies.. PAP induced the vC$icular release of adenosine 5".triphosphate (ATP) from PCI2 eells in I dose-dependent manner. 'ThePAF-induced ATP release was inhibited by the PAF antagonists. CV-J988and CV-6209. and the calcium antagonist pn:nylamine. The relevance ofthe interaction of PAP with neuronal cells was investigated further byusing brain synaplosomal preparations and primary conical and neostri-atal cells. NDI"IOIYlOlarccocentrauces of PAF induced calcium transients inacquorin-loaded synaptO$Olllai p~par1Ition5. and cortical and neostriatalcells were sensitive to the action of PAF. The possible physiological andpathophysiological rolcs of PAF in brain functioo are discussed.ILipidsZ6.124J...1246(I99I)J

Role or Platdet·Actlvating FacIOI' In Aggnogalion or Leukocyles andPlatdels in Cerebrallso::hemia

S. Uchiyama, M. YamllUlki and S. MaruyamaDepartment of Neurology. Neurological Institute. Tokyo Women's Medi·cnl College. Tokyo 162. Japan

Leukocyte and platelet agg~gation stimulated with rormyl-methionyl-leueyl-phenylalanine (FMLP) wu measured in 32 patients with cerebralischemia and in 15 controls. usin, I whole blood eggregometer. Theincreases in impedance and the rcductiom in leu..lr::ocyleand platelet countswere signiflCMtly grelller in stroke patients than in controls. AggregationWIlS inhibited by orul ticlopidine, but not by 0l'Il1aspirin. Thc effects wereclearly counteracted by platelet-activating factor (PAl') antagoni$l5, andcounteracted in part by I 5-lipo,;ygenue inhibitor. The results suggest thatplatelets tend to be activated by PAF and leukou-itnes liberated fromhypenggregable leukocytes in patients with iaehemic SU'Oke.IUpids 26,1247-1249 (1991)1

Cardiovascular Etrccl5 or Plalelet·AcllvlIUng Factor

Robert E. Gold~eln, Giora Z. FeutrSlein, Linda M. Bntdley. JosephJ. Siambouly. F~ndsco R.M. Laurlndo and Nancy J. Oa\'enportDiviliion of Cardiology. Department of Medicine. and the Division ofNeurobiology, Unifonned Services Uni~el'llity of the Health Sciences,Bethesda. Maryland 20814-4799

Sudden release of platelet·activating rector (PAl') inlO the circulation cancause hypotensioo. tachycardia. and circulatory collapse. To funller UW11-

tee this re:sponse. we perfonned detailed studies of cardiovascular func-tion after PAF adminlstratlcn to young domestic pigs Ind newbornpiglets. Our results indicate thPI circulatory dysfunction after PAP ~neclSsevere coostriction of pulmonary resistance vessels and eoesequera ecueright ventricular Iallure. Ahhough PAF-induced coronary Brtery constric-tion and contractile dep~S$ion may be complicating problems. Icft ven-tricular underpcrfusion and dysfunction after PAF a~ mainly the result ofsystemic arterial hypolmsion and diminished left ventricular filling. Tbead~erse hemodynamic effects of PAF are aec;ompanied by substantial~ka5eof thromboxane A2 (T,;AV. lltese effectS are mimicked by theTxA2 agonist U-466l9 and panially blocked by specifIC and nonspecifICinhibitors of TxA2 synthesis (OKY-046 and indomethacin). Even rTIQI'e

potent blockade of PAF action is exerted by tile T,;A2 receptor blocker.SQ 29.548. Taken together. these findings indicate that severe pulmonaryvascular constriction and hemodynamic collapse soon afler intravenousPAF-induced TltA2 rejease.

Tachyphytuif 10 PAF innuence has been observed in siudies ofie.ukoeyte and plateie.t function. We hypothesized that tachyphyluis toPAF might also occur in our studies of coru;lrictor responses in pulmonaryvessels. Recently. we have examined the capacity of PAF to produce sus-tained pulmonary vesecenstrictlon in openci1ested. anesthetized newbompigie.ts. Infusions sufficient to produce 100% increase in mean pulmonaryanery pressure after J min showed no lou of effICacy .....hen sustained for30 min. 1lIe same wu true for infu$iom of U-46619. Thus, tile pulmonaryvasoconstrictor innuence: of PAF or U-46619 is not readily diminiShed by

tachyphyluis. These findings flvor the viewpoint that PAF or TxA2release during innammatory proces5e1i could hive prolonged adverseaclions on pulmonary and systemic circulations. .ILipid: 26. 12S0--1256 (1991)1

P1ate~t·Actinting Factor in ~rdio"lUaIlIIr Stress Situatiom

Reu,"en Rablnovici. 11an.U YUI!' and Giora fo'l!'uersieinCardiovuculllT Phannacology. SmithKlinc Beecnem Laboratories, Kingof Pruasia, PA 19406-0939

Since the ellK:Klation of its chl!'mical Sll\ICttJre two ~ ago, plltelet-activating facto!" (PAF) has ~ u an important mediato!" of variouscardiovascular stress situations. Most notably. PAF was implicated as akey factor in the septic shock syndrome, blUed on the similarities betweenendotOJ(in end PAF biological effects. tbe elevation of circulating Ill1dus-sue levels of PAF during endotoxemia, and the protective effect of PAFIll1taSoni5iS in the sepuc slOte. In IIddition. IICcumulating dalO suggest theinvolvement of PAF in the pathophysiological processes associated withischemia, hemorrhage and treuma, where PAf exerts its effects dirc:c:tly oncells Dnd blood elements or indirectly through interactions wilh othermediaton such as cytokines and prostaglandins. Nevertheless, the relativecontribution of PAF to the pathophysiological processes in endorcxemie isstill unknown and should await further investigations. The primary aimsof this chapter are: to delineate the erreeu of PM on the cardiovascularsymm. to summarize the cialll which sug&eSt the involvement of PAF inStn:ss situltioos of the cardiovascular system, and to identify areas wherefuture experiment.al efforts should be foeu$C!(i.ILipidJ 26.1257-1263 (199I)J

Studies on tM R'* of P1atHet-Acti ..atin& Factor in Blood PressureRegulation

Katsuhlko SakaguchiD, Shl&eto MorimotoD, Fuminorl MasuglD,Shulchl Saekl'l', T05hio O&iharaD, Koujl Vama.dab and Is»o VamalsubUOcpartmCnt of Geriatric Medicine. O5a1:a University Medical School.Fukushima·leu. Osaka 553 and brsukuba Research Laboratories. Eisa;Co., Ud., Tsukllb.a.lbanllr.i 300-26, Japan

Circulating levels of I·O·hexadecyl-2·acetyl-sn·glycero·3·phospho-choline (CI6PAF) in human subjects were measured by gas chremarogra-phy/m\lSS spectrometry using negative ion chemical ionization. The !DC1ll1(t S.D.) circlilDling CI6PAF levels in patients with essential hypertension(18.1 ± 5.3 pg/mL. n=16) were I'lOI significantly different from those inncrmcrenstve subjects (17.2 t 7.2 pg/mL. n.. 14). During a salt balancestudy, high sal! intake (20 s/day)significantly increased the circulatinglevel of CI6PAF, and changes io circulating CI6PAF significantly andpositively correlated with changes in mean arterial blood pressure (r-O.47.p<O.05). Changes in C 16PAF also correlate with changes in creatinineclearance (r-O.55, p<O.05), but did I'lOI COrTelate with changC$ in plasma$Odium concentmion. plasma chloride concenlration and plasma volume.An intravenous injection of 50 Ilg of human atnal natriuretic peptide(hANP) decreased circulatin& ClijPAF levels from 20.0 ± 2.7 to 13.9 ±2.4 pg/mL of blood (0=10. p<O.OI) in healthy subjects. Tbe data appear toindiclle that ClijPAF levels are changed by salt lntake-indeced mildincrease in blood pressure, and that hANP may be an ~IlOUS fllCtocwhich lowen circulating C 1ijPAF.IUpidJ26.1264-1268(I99I)]

Intravascular Release of a Platelet-Activating Factor·Llk!!' Lipid(PAF·LL) Induced by Cigarette Smokln&

Tada-atsu Im.aizumiDepartment of Pathologic Physiology, Institute of Neurological txseases,Hirosaki Unlveriry School of Medicine, Hlresaki, 036, Japan

To lWCSS the role of plateleHlctivating factOf (PAF) in smoking·inducedvascular injury. the effect of cigarette smoking on PAF-lite lipid (PM·LL) in plasma was studied. The subjects wen: 12 young smokers (22 ± 1.3

years old). 13 young non·smokers (22 t 2.1 years old). 14 older smoken(59 ± 9.6 yean old), and II older non·smokers (60 ± g.7 years old).Lipids were eXlfacled from 5 mL of plasma and then were separated bythin-layer chromatography. The fraclion with the same migration asauthentic PM was recO\lerc:d and tested for the ability to Iggregate humanpolynKll"phonuclellT neutmphils. The IIC:tivity WL'i identified as PAF·LLbcaluse it WlIS inactiv~ by phospholipase A2. and because the effectson PMN were: blocked by CV·39S8. I oompetitive antagonist of the PAFreceptor. PAF·LL was detected in plasma from 3 young non:smokel$(23~), 5 young smokCT$ (42~). 3 older non·smokers (27%) and II oldersmoken (79%). The incidence ortbe detection of plasma PAF·LL in oldersmokers was significantly higher than those in young non-smokers(p<O.OI) Of in older eco-smoeers (p<O.OS). In young smokers. the acuteeffect of smoking was also studied. Plasma PAF-LL was detected in III ofthe 12 subjects immediately aner smoking a cigarette, in sharp contrast tothe incidence of 42% before smoking. Plasma ~.thr(lmboglobulin washighest in older smokers, and it increased significantly after smoking aci&arette in young smokers. Smoking is essoctered with increased produc-tion of PAF or a similar lipid. which may play an important role in lhepathogeocsis of smoking-induced vascular diseases.IUpids 26. 1269-1273 (1991)1

Platcift-Activatin& FaclQr (PAlo') Induces Contraction or Saponin·Skinned Smooth Muscle orCoronary Artery

A.1. SoIol'ifY" and P.8raquelltDA.A. Bogomcletz Instilute of Physiology. Kiev-24. 252024. Ukraine.U.S.S.R., and bH. Beaufeur lnatitute, F·92350. Le Plessis Robinson.F_

The: effC:cl of platelc1-lCIivating fact« (PAF) on the dcvekJpment of iso-metric tension by saponin-skinned coronary artery was studied. PAFcaused two types of conlrnctions of coronary smooth muscle cells (SMC):(I) rapid. transient (phasic) contractions of SMC wen: induced by inOliitoi1,4,5-trisphosphate dependent Ca2+ n:lease from san:oplasmic reticulum(SR): (ii) slow sustained (tonic) oontnctions were induced by ~ase inCa2+ ·sensitivity of the con!Taclile apparatus of SMC by proIein kinase Cactivation. "The presera results suppon the hypothesis Ibat, in SMC ofcoronary artery, PAF rettpton arc: located not only on the plasma memobrane. but also on the SR membrunes.Il.ipids 16. 1274-1276 (1991))

RQle f(lf"Platelet-ActiVlllting FaC10r In Asthma

K.F. Chung and PJ. BarnesDepartment of Thoracic Medicine, National Heart & Lung Institute andRoyal Brompton National Hearl & Lung Hospital. London SW3 6LY,United Kingdom

Recent studies of the effects of platelet·activating factor (PAF) on humanand animal airways would support I putative role for this lipid mediator inII1Ithma.PAF can induce many aspects of the clinical and pathological fea-tures seen in asthmalic airways such as airway oedema. eosinophil eccu-mulation in the aiTWllYwall, and broor:hial hyperresponsiveness. PAF haspoIent ICIivity as a chemotactic agent and as an lICtivator of eosinophil!.which are prominent cells in asthmatic airways. through \be activation orspecirtc surface receptors. Thc: intenction between PAF and eoIiinophiismay be crucial in the pathogenesis of bronchial hyperrc:sponsivencsi inasthma. A role for PAF in asthma can now be studied using the recentlydeveloped anlOgonists of \be PAF receptor.IUpids 26. 12n-I279(I99I))

The Contribution or Platf!et·Actlvaling Factor to Allergen·lnduel!'(!I-Alilnophillnliltration and Bronchll/ll Hyperresponsivenes5

C.P. PageDepartment of Phannacology. KinlJ'. College. University of London.London SW3 6LX. United Kingdom

lNFOr<M. Vol. 3. no. I (Jonuary 1992)

133

134

ABSTRACTS FROM AOCS JOURNALS

Plalelet-a::tivllilll fKror (PAf) is .n ether-linked phospholipid hiving arange of biological properties of relevance 10 our understanding of thepathogenesis of bronchial asthma and related allergic diseases. III panicu-lar the ability of PAF to induce eosinophilllCtivation and recruitment hasreeeivcd considerable attention. Since in uperimenlli animals PAF andallergcn-induced eosinophil infillnllion are dependent upon platelet Kli."Ilion, it is suggested IIuII platelet activation may be an important compo-nent of the allergic process. The ability of PAF antagonists to inhibit vari-ous aspects of the allergic response has been demonstrated in a number ofanimal models bul not all of them have been extended inlO man.[lipidt 26,1280-1282 (1991)]

Pialcici Aclivatlng Factor-Induced Pulmonary Accumulation orUIJndium.Oxlne labelled Neutrophilsln Anl!'ilhtllzed Guinea Pigs

UI'5UIa Hultklist·lk:ngt5500, Gary P. AlKkl'5OR. Maddrioe Kings andJohn MOrieyPreclinical Research. Sandol AG, Basel, SwllUTland

'The thoracic accumulation of neutrophils labelled with 111lndium-oxineIn response to infusion of platelet activlting faclOr (PAF. 18 nflkglmin X5 min, tv.) was studied using an automated isotope monitoring system(AIMS plus) in anesthetized guinea,pigs. Loss of tell lI5$IXialed ndioac-t;vlty ill vuro was len than l % over 4 hr. Labelled neutrophlls maintainedtheir functional capacily (oxidative response to the cell stimulants N-fonnyl-L-mcthionine-L-leucine·L-phenylalanine Ind phorbol myristateacetate) and >95% viability (ethidium bromide/acridine orange Slain) jll";fro. Total thoracic ndioactiYily increased significantly from baseline inresponse 10 PAF with. slight tachyphylwr.ia in the n.eulrophU-actumula-tion after a repeat PAF infusion. The higheSI ulios of radlolabel(dssue,lblood) were found in the spleen s- liver> lung.[UpUb26.12gJ..-1286(199I)]

Platdd Actinting Fador Induc:ed R~piTlltory Mucn>al Damalle

Ken·lchi Hisamalsu, Telsuya Ganbo. Tsutemu Nakaz.awa and YO!hI·hiko MurakamiDepartment of Otorhinolaryngology. Yamanashi College of Medicine.Tamaho-cho. Nakakoma-gun. Yamanashi 409-38, Japan

Human sinus mucosal specimens from eight normal individuals wereespesed 10 plalelel activating factor (PAF) at concentrations ranging from10~ M to ](rll M in I humidified CO:2 chamber at 37·C. The mllCOSlllsurface of the specimenl was recorded on video tape$ and magnified2,500 times on a 19·;nch television (TV) monitor. Cililll)' activity of eachciliated cell was photoelectrically measured on the TV monitor in realtime. PAF induced mucosal damage which resulted in a coarse profileincluding cilioswis and exfoliation of epithelial cells. The length of theincubation period in which the initial coarse profile occurred on themueosal surface inversely correlated wilh the concenl(ation of exposedPAF ranging from 10-6 M 10 10-10 M with r. 0.712 (p<2 x 10-4). BoIhthe control medium and 10-8 M lysoPAF snowed 110 effect on ciliaryactivity or mucosal surface alteration even after 24 Iv of exposure. Signif-ical11ciliary inhibition was nou:d aner 6 hr of exposure to PAF at c;:oncen-uati0n5 of 10-8 M and 10-10 M (p<O.05). After 10 Iv of exposure, siJllifi-canl ciliary inhibition (p<O.OI) was llOIed at all a:JIlCCntnlliolls. Inhibitioncccurred in a time- and dose-dependent manner. 'The length of the intuba-lion period in which initial cilioswis cccened and the level of PAF con-centration showed an inverse torrelation whb r. 0.918 (p<I0-6). Theseresults indicate that PAP is cytotoxic to human respimtOTY muoosa.IUplth26.1287-1291 (1991)1

Platelet-Activating Factor Det~ted In BronchOlilyeolaT Lavage Fluidsfrom lin Asthmatic I'atlent

Taugio Horila, H!loshi Okazaki", Minoru Kino", YohnosukeKoo.yll5bi", KiyOl'ibl Satoucbib and Kunihiko SaltobuDepanmcnt of Pediatrics and boepattment of Medical Olemistry, Kan·58i Medical University, Moriguchi, Owka, 570, Japan

It m::ently has been recognized that plateiet-activating factor (PAF) maybe a mediator of asthma exacerbation. We had the opportunity 10 analyubronchoalvcolar lavage fluids from an asthmatic infant. whiCh were char-acterized by neutrophil infill(atioo. Tbe patient's lungs were washed onthree occasions with ... line during asthmatic attaCks. PAP was found ineach case on the basis of its ability 10 cause the immediate aggregation ofwashed mbbil platelets. The PAF detected was eqeivalem 10 1-1.4 pmol ofI"O.hexadecyl-2-acetyl,slI-glyccro-3-phosphochollne, three quarters ofwhich were recovered in cell-associated rcrm. By contrast, we did notdetect PAF in brcnchcalvecter exudates from patlems with laryngcals~nosis or with respiratory distress syndrome. LysoPAP, the dim::t precur-sor as well as initial metabolite of PAP. WI5 also analyzed after being con-verted to PAF by acetylation. There was a wide vanation in the amount oflysoPAF present in individual patients. suggesting that lys.oPAF levelscannot. be taken as an indicator for the presence of PAF.[Upids26,lm-I296(1991)1

Platelet-Activating .-actor (PAF) In Alltrgic Diseases: Inhibitor,Errects or Antl·Alterlllc Drugs, Ketotiren and Three KampoMedicines on PA"- Production

Tsuoeyo6hi Nakamu .... , Motoakl Kurlyama", Keiko (ShihaTller, YukloMlitsumuTllb lind Tl"rumlWl MiYllmotolruDepartment of Internal Medicine and PhY5itai Therapy, School ofMedicine, University of Tokyo. 7·3·1. Hongo. Bunkye-ku. Tokyo andbMalSumura Clinic and Research Institute for Asthma and ImmunologicalDiseases. 5-52-2. Higa.shinippori. Arakawa-ku. Tokyo.lapan

Platelet-activating factor (PAF) is considered an imponant mediator Inallergic and inflammatory reactions. To further investigate the role of PAFin allergic diseases, we examined the jll vttro and ex vivo effect of anti-allergic drugs on PAF production by human neutrophils. Ketotlfen andthltt Kampa medicines, which an: widely used in the treatment nf alle'licd~ in Japan, inhibited PAF prodUt:tion by normal human neutrophilldose-depcndcntly. wherell5 disodium chromoglycate (OSCG) and tranitastdid not have any inhibitory effect. KetOlifcn also supprused ex ..i..o PAFproduction by neutrophill from hellthy subjects. PAF productionOecreased from 40.5 t 5.0 units to 21.6 t 4.7 units al\er on] administm-lion of2 mg of kelOlifcn per day for one week. We also measured the CQII-

centntion 0( lysoPAF In serum from patients with Japanese cedaT pom·1I05is during the pollen season and followed the effect of ketOlifen onlys.oPAF jeve!s in the serum from these patients. We found that the con-centration of lysoPAP. I precursor and degmdation product of PAF, inpatients with cedar pollinosis (87.g t g.7 units) was signifltantly higherthan in healthy subjects (54.7 ± 7.7 units). After two weeks, lysoPAF lev.ets decreased significantly (82.6 units 10 41.3 units) only in the grouptreated with nasal aerosol and ketotlfen.These results suggest that PAFmay playa role in allergic diseases and that the clinical effect of antt-allergic drugs may atleast partially be due to their anti-PAF action.[Upids 26. 1297-1300 (1991)]

Errect or the Selective PAF Antagonist SM·I0661 on an AsthmaticModel. I. Effect on Passive Anaphylactic Bronehoconstrictlon InGuinn Pigs

Mll5llko Uchida, NorIakl lmanishi, T05hlnari SUgasaWli and Shigeall.l

"<>n><>I<aResearch Laboratories, Sumitomo Pharmaceuticals Co., LId .. Osaka 554,Japan

The effect of SM-](166t, I selective antagonist 0( platelet-activating fac-tor (PAF), on passive anaphylactic bronchocOftSlriction was examined inguinea pigs. A challenge of ovalbumin to passively sensitized guinea pigsinduced bronchoconstriction, which peaked at 4 min. When SM-I0661WII5administered intravenously 2 min before ovalbumin challenge, bron-choconslriction WIJ inhibited dose·dependently with an ID.'iO of 68mg/kg. In guinea pigs pR'treated with U j.L&lkgmepyramine which il asuboplimaI dose. anti sen-induced bronchoconstriction peaked at 4-6 min.btu was inhibited by SM-I0661 with an 10.50 of21 mglkg. When guineapigs were pretreated intravenously with 2.5 mglkg mepyramine, I mglkg

.

INFORM. \otlI. 3. no. I (Jonuay 1992)

135

indomethacin and 0.01 msJl:a: propranolol, the antigen-induced bron-cboconslridion peaked 1.6 min. SM-I0661 inhibi~!be ~ with an1050 of 45 mgllr:J. Hbwnine- and kukooienc 04-induced bronchocon·nncnces were unaffected by up to 100 mg/kg SM-I0661. Ovalbuminchallenge of minced lunp from passively Knsitiud guinea pigs uiggc~the release of Icukooienes and histamine. SM-I0661 had no effeel 011theantigen- induced reJcue of peptide kukotriclIelI or histamine up to 10-4M. These m;ults indicate !hal SM-I066\ may be a userut eottc investl-gate the role ofPAF in antigen- induced anaphylactic bronchoconstriclioo.[Upids 26. \301-1304 (1991)]

Effect of the Selective PAF Antagonist SM·I0661 on an AsthmaticModd. 2. E"eel Or Anllgcn-lndu«1i Dual Asthmatic: Response andInfiltration of LeukOCYltlI Inlo Airways in Actively Sensitiled Con.scious Guine. Pigs

Toshinari Sugasawa. NoMak! hnanishi and Shiguki MoruokaResearch Laboratories, Sumilomo ptuumacculitais Co .. Lul .• Osaka 554.Jo,.,The effect of a selective platelet-activating factO!"(PAP) n:ceplO!" antago-nbt. SM-I0661. on antigen-induced dual asthmatic response and leuko-cyte infiltration into the Ilrways of actively sensitiw:l coescioes guineapigs was investigated. The animals were pretreated with mepyraminemaleate (10 mg/kg Lp.) and then challenged with ovalbumin. Tbe inhala-tion of ovalbumin cau$cd an immediate decline in specific airway conduc-tance (sGaw) which peaked at 5 min after the challenge. sGaw graduallyreturned to the baseline 6 hr after the challenge. After the carly as!hmalicresponse (EAR). a I«Ond phase change. a late asthmatic response (UR)in sOaw peaking II 17-20 hr was observed. When 1'1>wtv disodium cro-moglycale was inhaled for 2 min on twe occasions. EAR was not affected,but LAR was Jignifinntly inhibited. Oral administration of 50 mg/k:gfenote:roI (30 min bef~ challenge) signiflCUltly inhibited EAR. but hidno signifICant effect on LAR. SM-I0661 administered orally 1 hr beforethe challenge inhibited EAR dose-dependently (S()<;l, inhibitory dose(1050>; 59 mg/k.g, but was even IJIOfe cffective against the LAR ODSO>;13--16 mgi\g). When 30 mYkg of SM-I0661 was administered onlly 6hr after ovalbumin challenge. LAR was completely inhibited. 11Ie numberof 100aicells. macrophaaes and eosinophils in bronchoalveolar lavage flu.ids rose signiOcantly 17 hr after antigen challenge compared 10 thatobserved afler saline challenge. 1l1e number of neutrophils and lympho-cytes also increased in response to the ovalbumin challenge. but not sig-nificemly, SM-l0661 (30 mgtkg) administcred orally I hr before the chal-lenge significantly Inhibited the increase in LOIalcells, macrophages andeosinophils. By comparison, when 30 mg/kg SM-I0661 was administeredorally 6 hr after the challenge. these increases were not significantlyinhibited. These results suggest that PAP is involved in EAR and LAR.and IliBt SM-I0661 may be a usefultheropcutic agent for the treatment orallergic asthma.[Upids 26,1305--1309 (1991»)

The Role of Platelet·Actlvating Factor (PAF) in E"perimenlalGlomerular Ill,jury

A, Ortl~ M, Gome:t-Cbiarri. J.L Ltrma, E. GoIuaJtz and J. EgMioDepartmenl of Nepllrology. Fundaci6n Jimtnel Diaz, 28040 Madrid.S,.mPlatelet·activatinl factor (PAF) is I potent autacoid thaI panicipates ininnammation Ind other pilhophysiological precesses. In this review wedeal with recent evidence suggesting thll PAF is a mediator that isreleased early during glomcndar injury. PAF can be synthesized in theglomerulus by inmtrating imrinsic glomertJllIT cells. Normal glomeruliproduce PAP upon stimulation. and glomerular PAF synthesis is increasedin a variety of expenmenul glorncrulopathics. The local infusion of PAFinto the renal artery or isolated blood-free kidneys induces proteinuria.PAF aUrlCl1 and Ictivatel inflammalory celb. Glomerular mel.lngill.endothelial and epithelial cells are also \ariets for PAF. TIterapy with spe-cific PAF rcttplOf antagonists has prevented or reduced proteinuria andimproved glomerol ... inn.mmation in seve~ eJl.perimenlal models of

proIifemive glomerulonephritis and minimal change ncphms:is. However,the bmcflCiai effect of administration of PAF antJgonists once proteinuriais ful1y developed has been minim.l. PAF m.y also playa role in therecruitment of innammatory intemiti.1 cells.[Upids 16,1310-1315 (1991)]

Tbe Effects of R-75,J17 on AntiglomcruJar Basement MembraneGlomerulonephritis In Rats

Masaakl Miyamoto", Hlroyukl Koike", Toshlo SadaQ, VasuteruIljlmab, Junichlro Fukushllel' and NoMo NakamuraI':QBiolOlical Research Laboratories. bNew Lead Research Laboratoriesand 'Fennentation Research Laboratories. Sankyo Cc., Led., Tokyo 140.J.""Platelet-attivatina ractor (PAA is a potent innammatory mediator whichis released by various inflammatory cells and produced by cenain tissues.including the kidney. PAF has been shown to increase glomerular perme-ability to protein and to decrease glomcrolar filtration rate (GFR) by con-uacting mcsangium. On the basis of these observations. it has been SUI-pected that PAF may playa role as medillor of Ilomcro\ar damage inglomerular nephritis, To examine this possibility. we studied the effects ofa specific PAF antagonist, R·75,317. on the development of an experi-mental model of anri-gtcmerular b.sement membrane (anti-GBM)Ilomcrulonc:phritil. Glomcrolonephritis WIS initiated by injecting rabbitanti;tat GBM serom into rots. PTOteinuria gradually developed after seruminjection. plateaued al week. 2, and rem.ined It the high level of week. 2throughout the experimental period (6 wk.). Chronic treatment wilh R-75.317 (10 mglki/day i.p.) tended 10 delay the onset of proteinurea andsignificantly accelerated the ~overy phase. Creatinine cleaJ'llOCe (O;:r)fell to 40'1> It week 3, R·75.317 treatment completely prevented thisdecline of Ccr. Hi51ological changes in !his model (glomerular hypertro-phy. proliferation or meungial matrix and interstitial fibrosis) were .Isoameliorated by the R-7~.317 treatment. 1l1e results suggest that PAF mayplaya role in the development of glomerulonephritis and thai PAP antago-nists could be used in the treatment of human renal disease.(Upids 26, 1316--1319 (1991»)

Nephrotoxicity or Cyclosporine: The Role of Platelet·ActiYlting Fac·tor and Thromboxlnt

Oscar F. PniO dos SantosD. Mirlan A, 8olma, Elvino J.G. 8arrosG,Eduardo Plrot'l.kyb, Pierre 8raquetb and Nestor Seho.-oQNeghrology Division, Escoln PQulistP de Medictna, S10 Paulo. Braziland lnsthut Henri Beaufour. Les Ulls, France

Cyclosporine «(;sA). an lmmuncsuppresslve ageru, is polentially eephrc-10xic. We had previously observed that acute administration of CsA toMunich-Wisw ntIS induced a decrease in single nephron glomerolar fil-tration rate. due to I decline in glomerular plasma flow, and in theIlomerular ultrafiltnttion coefficient_ Moreover, these alterations wereprevented when an antlgonist of platelet-activating factor (PAF) wasadminimred, In the present study we examined whether the protectiveeffect of the PAF blocker in (:$A nephrot:Ollicity could have been mediat-ed by thromboune (fxA2)' Our daa show that the PAF effects were notmediated by TxA2. ,inee administration of dazmegrel, a thrombounesynthetase inhibitor, did not ameliorate the acute renal failure caused byCsA. ThU5, PAF appcan 10 be a di~t mediator of acute CsA nephn:ItoJl.i-city, while TJl.A2 is not signifiCantly involved in this process.[Upids 26.1320-1323 (1991)]

Errecl of Plalelel-Actlvatlng Faclor Antagonist BN 52M3 on theNephrotoxicity or Clsplatin

Oscar F. Pavia dos SantosO. Mirlan A. 801""', F.Jrino J.G. Barros",Eduardo PITOI:r.kyb,Plel'l"f! Br1lquetb and Nestor Sch0r4uNephrolOiY Division. EscoIJ PaulislI de Medicina, SAo Paulo. SP, Bruiland DJn5litut Henri Beaufcer, Les Uiis, France

INFORM. Vol. 3. no. 1 (January 199'2)

136

ABSTRACTS FROM AOCS JOURNALS

Cisplatin (DDP) is an effective anticancer agem lllnt has been successfullyapplied against various solid tumors. However, DDP commonly causesnephrotoxicity. We observed Ihal DDP led 10 ~ignilicam ahcratlons inrenal microcirculation when administered to Munich·Wistilr rats. wlth aconcomitant decrease in single nephron glomerular Illtrarlon rate due LOreduction in glomerular plasma now and \nI.n~.pil1al')' hydraulic prcs~uredifference. BN 52063. a platelet-activating faciO!"antagonist. caused astriking change in acute renal failure induced by DDP leading lowardsIIOmIaliuuion of all parameters of renal function. 1be results suggest WIBN 5206J could be used as I ~I drug IOcontrol DDP nephl'Oloxicily.(Upids 26. 1324--1328 (1991)]

Effects of PA.- Antagonises Oft Renal Vascular Escape and Tac:hyphy-taxis in Perfused Rabbit KidMy

M.G. Ferrcln.Q, P. Braquetb and M.e. Fonlll'l~lIUnidade de Pesquisas Clfmcas. Centro de Cicncias de Saudc. lfnlversi-dade Fcdeml do Ceani. Pcrtateza, Brazil, and hlr'l.~tllut Henri Beeufcur, F-92350. Le Plessis Robinson. Frunce

Renal vascular escape is a physlotogjcal phenomenon of adaptation tlimoccurs in vascular smooth muscle. II has been described in many prepare-nons subjected to electrical stimulation or treated with vasoactive agents.such H norepinephrine. angiotensin and vasopressin. We have recentlydemonstrated that a naturally ocurring ginkgolide (BN 52021), which is aPAF antagonist, was able to block non:pinephrine-induced escape in per-fused rabbit kidney. In the pruent wort other PAF antagonislJ. such 115the ginkgolidcs BN 52022 and BN 52024. and the synthetic oompounds48740 RP and WEB 2086. were tested, Their enecu on renal vascularescape, perfusion pressure and tachyphyluis were evaluated. They IIIwere shown 10 block the escape. Among lhe ginkgolides. BN 52024 isgenenrJly recognized as one of the weaker PAF antagonists. However. inspite or this, BN 52024 was able to signiflCalllly and simultaneously blockrenal vascular escape and tachyphyluis in perfused rubbit kidney infusedwith norrpinephrine.[lipids 26, 1329-1332 (1991)[

~DCr uf' Platl!'let-Acth'ating Factor in Pyuria In HumaM

I(hiro Ihda4l, Makoto Oda4l, Muneki Sahkura41 and KojiroYasunagablIThe I):l!artment of Internal Medicine, Saiseikai teec Hospital. Osaki551 and ~e First Department of Internal Medicine, Kansai MedicalUniversity. Moriguchl Osaka 570, Japan

Tlie relationship between the occurrence of platelet-activating factor(PAF) and ncutrophlls in urine from patients with urinal')' InlCt infectionwas examined, PAF was detected in human pyuria. when leukocyte levelsreached at least 300 cell$/J1L (n=45), but 001 in normal urine (n=12). Theamount of PAF found in pyuria. measured by platelet aggrt'gation assay,was 0.01 to 13.3 pmol/mL A erose OOrTClation was seen between theamount of PAF ~t and the number of urinary leukocytes (p < 0.01.r=O.70). The leukocytes in pyuria consisted almoM mtirely of neutrophils(96 ± 4'),. mean ± S.D.). Our findings suggest 1M the OIX'IITe~ of PAFis associated with the accumulation of eeutrophils in urine.[Upids16,1333-1335(199I)1

Signtncan« of Platelet-Activating FaClor In Muenleric Ischemia·Reperfusion

Ja .... Hlep", Pierre Braquelb and Tibor MOlU~Deparunems of uPathophysioiogy and 'Tmumatology, Semmelweis Uni-versity Medical School. 1445 Budapest, Hungary. and blnstitut HenriBeeufour, 92350 Le Plessis Robinson. France

Reperfusion of the ischemic mescnterium is frequently followed by acutecirculatory eollapse. This review focuses on the possible role of platelet-IICtivating ractor (PAF) in ischemia-induced damhlle. It provides evidenceIhat (i) PAF concentrations are elevated in the mesenterie circulation fol-

INFORM, Vol. 3. no. I (January 1992)

lowing tcmporary ischemia; (ii) administration of exogenous PAF into lhesuperior mesenteric vein mimies many events observed during repetfu-sion: and (iii) pretreatment of tile experimentaillnimab with specific PAFreceptor antagonisl5 prevent the circulatory collapse. These findings sag-gest that PAF may play an important role in the development of circulato-ry collapse caused by mesenteric lscbemta-repetfuslon.[lipids 16. 1336-1339 (1991»)

Hypoxia, PAF, and Ncavtizing Enterocolitb

Mithad S. Caplan, Xiao-Ming Sun and Wd HsuehDepastmcnlS of Ptdiatrics and Pathology, Children's Memorial Hospital,Northwestern Univer.;ity Medical School, Chicago, Illinois 60614

Necrolizing enterocolitis (NEe) is an important neonatal disease with ahlgh mortality rate. The pathophysiology is unclear but epidemiologicstudies suggest thai hypoxia and infection are important risk (IICtOI'$.Inthis review we discuss the effect of liypoda and plmelet-ectivating foctor(PAF) on intestinal blood flow and imesunul necrosis. and implicate PAFas an importa.nt medialor in hypoxia-induced intestinal injury, Finally weprovide evidence that PAF may be important in neonatal NEe.[Lipids 26. 1340-1343 (l99I)[

Antagonism of PIalelet-AClivaling Factor In lsoialeel Rat CcMon: Pus-§ible MKhanism

Akira Tokumura. Nobuyukl Yube. MOlonor' Terao and HlroakiTsukataniFaculty of Pharmaeeutieal Sciences, The University of Tokushima.Tolushima 770, Japan

The eon tractions of three different regions of rat colon in response toplalelel-acliv8ting factor (PAF) _re oompan:d, The ascending ooIon wasfound to be the mo:st I1:$ponsive. The slow contraction or lhe ~inllcolon induced by PAF was dqxOOent on external Ca2+. CV-3988, • .utuc-tural analog of PAF. slowly induced irreversible inhibition of PAF-induced eontnlClion, whereas FR-9()()452, whleli is structurally unrelatedto PAF, caused rapid revertible inhibition of PAF-induced COIltnlClion. Noinhibitory effects of CV-3988 were cbserved when the Strip Willi washedwith Tyrode's solution COIltaining I'), bovine serum albumin (BSA). Theresults suggest that PAF and CV-3988 penetrate slowly into the outer halfof the lipid bilayer of plasma membrane!; of cells in isolated rat colon, andthen rapidly diffuse laterally to associate firmly with specific bindingsites.ILipids 26, 1344-1346 (1991»)

Molecular Heterogeneity or Platelet-"cti~atlng Fador (PAl') in RatGlandular Stomach Drtermlned by Gas Chl"OmalographyfMlH5 Spee-trometry, PAF Molecular Species Changes upon Wlter-Immef$ion"....Junko Sugatani4l, K81.uyo Fujimurab, MISIO Miwat, KlyoshiSaloucbj41 and Kunihiko Saito"°Department of Medical ~mistry and b-rbe Third Depanmc:nt of lmer-nat Medicine. Kansai Medical School, Moriguehi. Ouka 570, and '1:le-partment of Biochemistry. School of Pharmaceutical Science, Universityof Shizuoka. Shizuoka 422, Japan

The molecular heterogeneily of 1-alkyl-2-llCety1-sn-glycero-).phospho-choline (alkylaeelyl-GPC) and l-acyl-2,acetyl-sn-glycero-3-phospho-choline (acylacelyl-GPC) in noonal nil glandular Itom...:h was Itudied bygas ehromatogmphytmass speetTOlnelry (OCtp.1S) and tandem mD$5 spec-trometry, The percentage eompositions of the molecular speeies of 1-a1kyi-2'&eetyl-GPC and l-acyl-2·acetyl-GPC in the antrum were. respec-tively. I -alkyl [16:0 (34'),) and 18:0 (66'),)I-2-acetyl-GPC and I-acyl116:0 (60%), 18:0 (14'),) and 18;1 (26'1.)[-2-acetyl-GPC. The alkyl ehainoomposition of l-atkyl-2-acetyl-GPC was quite differenl from that of 1-alkyl-2·acyl-GPC in both the antrum and corpus, demonslraling a higlidegree of selectivilY of alkyl chain utilil.lltion in PAF biosynthesis. 11Ie

amoon! of 1-ar.:;yl-2·acetyl-GPC was much greater than that of l-alkyt·2·IlCelyt-GPC. 'The molecular heterogeneity of t-a1kyl-2·acetyl--GPC and 1-acyt-2-lICetyt--GPC in the corpus was similar to that in the antrum, waicr-immer.;ioo SIJe$S affected not only the amount of l-alkyl-2-acelyl-GPCand l-acyl-2-acetyl-GPC. but also their molecular heterogeneity in theantrum and corpus. wnerees the amount of l-hexadecyl-2-acetyl-GPCand l-acyl [16:0. 18:0 and 18:1I-2-acetyt-GPC decreased markedly (toless than one-fifth) in the antrum after such stress for I hr. the amount ofl-cctadecyl-Z-acetyl-Gl'C increased markedly (up to 4-fold) in the corpusand severe lesions were observed after stress for 7 hr. The changes may beassociated with the pathogcnicity of gastric ulcers.[Lipids 26,1347-1353 {199I}j

The Effect of CV-3988 and CV-6209 on the Acute Gastric Erosions ofRalll Due to Water-ImmersX>n and Restraint Stress

Makolo Nogamia, Yoshto Hoshihara<l. Kunihiko Tsubura<l. TakashiYamamot<fl, Masafumi Tabuchi<l, Terumasa MiyamotcP and JunjlShigabaDepan:ment of Medicine and Physical Therapy, and bDepan:mem ofPathology, Faculty of Medicine, University of Tokyo. 7-3-1. Hongo.Bunkyc-ku. Tokyo. 113. Japan

CV-3988 and CV-6209 inhibited gastric erosions in rats due to water-immersion and restraint stress in a dose-dependent manner. The aboveinhibitory effects of CV-3988 were observed in the presence ofindomethacin. which may indicate that the inhibition is not prostaglandindependent. The studies indicate that platelet-activating factor may heinvolved in the formarlcn of erosions in rats under water-immersion andrestraint stress.[Lipid£ 26.1354-1355 {199I)1

Platelet-Activating Factor May Mediate Dexamethasone-InducedGastric Damage in the Rat

Janos Filep'l. Ferenc Hermanb and Pierre BraquetCDepartments ofapathophysiology and hJ>harmacodynamics, SemmelwelsUniversity Medical SCOOOI.1445 Budapest, Hungary IlJId CJnsitut HenriBeaufour. 92350 Le Plessis Robinson. France

The possible role of platelet-activating factor (PAF) in dexamethasone-induced gastric mucosal damage was studied in rats. PAF was measuredby a platelet aggregation assay. The identity of the PAF-like productrecovered from gastric tissues was ascertained by thin-layer chromatogra-phy and high-pressure liquid chromatography. Low levels of PAF weredetected in the normal nil stomach. while in dexamethasone-treated ani-mals PAF levels were significantly higher. Pretreatment of the animalswith BN 52021, a specific PAF receptor antagonist. significantly auenuat-ed dexamethasone-Induced mucosal injury. These findings suggest thatPAF may be a mediator of mucosal damage induced by glucoconicoids.[Lipid£ 26.1356-1358 (1991)J

My~lectric Intestinal Disturbances in Escherichia coli EndotoxicShock in Rats. In\,oh'ement of PJatelet-Acti\'ating Factor

Laurent Ponsll, Marie·Thirest Droy-Lefaixb, Pierre Braquetb andLionel Bueno"°Oepan:ment of Pharmacology INRA. 31931 'toutouse CMex, and"IPSEN{lHB Laboratories, 92350 Le Plessis-Robinson. France

Administration of BN 52021 (50 mg/k8 i.v.). a specific antagonist ofplatelet-activating factor (PAF). significantly reduced .lI).eintestinal myo-electric disturbances induced by E, coli endotoxin injection (50 J.IgIl:gi.I'.)by 62%. Thus, PAF may by involved in the intestinal motor alterationsobserved in endorcxtc shock. When given in combination withindomethacin (10 mg/kg i.p.). BN 52021 inhibited endotoxic shockintestinal disturbances. Indomethacin alone also reduced PAF induced (25~g i-p.) disruption of migrating myoelectric complexes. Endetoxinsmay act on intestinal motility "ia release of endogenous PAF and

prostaglandins, the effects of PAF being mediated through the release ofprostaglandins.[Lipids26. 1359-1361 {199!)1

Platelet-Activating Factor and Granulocyte-Mediated O!(;dati\'l~Stress. Strategy for in vivo Oxyradical Visualization

Makoto Suematsu lind Mesabaru TsuchiyaDcpanmeru of Internal Mcdicine. School of Medicine, Keio University.Tokyo 160. Japan

Platelet-activating factor (PAF) and leukotrienc B4 are potent lipid media-tors of endotbelium-granulocyte interactiOfl which results in granulocyte·dependent increased microvascular perrneabilhy, The specific mechanismby which PAF induces granulocyte-mediated endothelial injury has nOtbeen fully investigated. Digital imaging p/KM.onicintensified microscopyhas revealed that PAF effectively induces granulocyte-mediated cxidanvestress on microvascuillf beds. 'The method has made it possible to visual-ize lurninol-dependent photonic burst released from PAF-treatedmicrovascular beds in the rat mesentery. The pllotooic activili« clearlycorrespoeoed to the localization of sticking granulocytes in post-capillaryvenujes. By contrast, no significant chemilumigenic response could bedetected in the leukotriene B4-induced' activation of endothelium-granulo-cyte interaction in vivo: The present Ilndings suggest that oxygen radicalsare flO{ prerequisites for granulocyte adhesion in the microcirculation andprovide evidence for a dissociation of in I';"Q granulocyte functionbetween !cukOlDctic and oxidative activation in Ieukcmene B4-inducedmicrovascular changes.[Lipids 26. 1362-1368 (1991)1

In\'oh'ement of Phltciet.Aelivating .'aelor (PAlo') in Septic Shock andPriming as Indicated by the Effect of Hetrazepinoic PM Anlagonlsls

Hubert O. HeuerDepanment of Pharmacology, Boehringer Ingclheim KG. 0-6501. lngel-heim/Rhein. Gennany

Pharmacological data obtairted with betrazepincic platelet-activating fac-tor (PAF) antagOflist.s. such as apafant (WEB 2086) and bcpafan\ (WEB2170). indicate a role for PAF in septic shock end in the priming process.The effect of PAF antagonists in different models of shock states favors arole for PAF in endotoxin associated lethality. activation of inflammatoryblood cells with release of mediators. cardiovascular failure and increasedvascular penneability. and in the development of shock organs and organfailure. The priming process (e.g .. by endotoxin or tumor necrosis factor)towards an increased susceptibility towards minute amounts of PAF has tobe taken into account when considering the pathophysiological signifi-cance of PAF under in 1'11'0conditions IlJId in septic shock.[Lipids 26,1369-1373 (1991)1

Effect of the Hetrazepinoic Platelet-Actlvatfng Factor- AntagonistBepafant (WEB 2110) in Models of Acnve and Passive Anaphytuis inMitt and Guinea Pigs

H.O. HeuerDepartment of Pharmacology. Boehringer lngelheim KG, W-6501Ingel-heimJRhein. Gennany

The selective hetrazcpincic platelet-activating factor (PAF) antagonistWEB 2170 (Bepafaru) was used to study the pathophysiological role ofPAF in several models of anaphylaxis in mice and guinea pigs. In active-ly sensitized mice. the PAF antagonist WEB 2170 (I.O-IO mg/k.g p.o.)protected mice from anaphylactic death in a dose-dependent manner whenthe anaphylactic response was potentiated by the beta-receptor antagonistpropranolol. When active anaphylaxis in guinea pigs was induced intr~-venously by 100 mg/k.g ovalbumin (OA) in the presence of small doses ofthe antihistamine mepyr~mine. additional treatment with oral or intra-venous WEB 2170 protected the guinea pigs from anaphylactic death.Also. the remaining anaphylactic bronchocoostriction and blood pressure

INFORM. VOl. 3. no. 1 (January 1992)

137

138

ABSTRACTS FROM AOCS JOURNALS

changes (including anaphylactic hypotension) were auenuared. Whenguinea pigs were passively sensitized wilh a heterologous antibody "ill thetracheal mute and then challenged by ovalbumin (100 mglkg i.v.) 24 hrafter sensitization in the presence of 0.003 mglkg i.v. mepyraminc. addi-tional treatment with tracheal WEB 2170 at 0.1-1 mglkg protected theguinea pigs dose-dependently not only from anaphylactic death but elsefrom a funher decrease of respiratory flow and changes of blood pressure.Increased levels of PAP-like IICtivity (20-50 IIg PAF{whoJe tung) weredetected in lungs removed from antigen-challenged animals. The resultssuggest a causative role for PAF in active and passive anaphylaxis.[Lipids16,1374-138O(l99I)1

Platekt-Actiuting Factor Type Activity in Plasma rrom Patients withSepticemia and Other Diseases

Hubert O. HcuerO, Harald Dariusb• Helmut F. Lohmannc• JuergenMeyerf', Manuela Schierenbc!rgb and Norbc!rt TreesebQDepanment of Pharmacology. Boehringer Ingelheim KG, 6507 Ingel-helm, bUniversity Clinic of Johannes Gutenberg University Maim" 6500MailU. and "Human Phannacology Center. Boehringer Ingelheim KG.6507 lngetheim. Gennany

The purpose of the present 5tudy was to determine whether increased lev-els of platelet-activating factor (PAF) type activity can be detected in plas-ma from patients with septicemia and other diseases. A level of PAFbelow 0.5 nglrnL of plasma was considered normal. We found that plasmafrom a patient with adverse enephylecioidic reaction to meavenccs anal-getics contained 2.1 ng PAF/mL. In seven patients with septicemia.including urosepsis, endocarditis and peritonitis, and with positive bloodculture, increased plasma PM levels (1-20 ng PAFhnL) were observed.Other patients with clinical indications of septicemia had negative bloodcultures and/or increased levels of C-reactive protein (CRP). Yet, in theplasma from these patients. no Increased PAF levels were detected underthe assay conditions used. Two patients with allergic asthma, requiringtreatment with steroids, had no measurable plasma PAF. In the plasmafrom a patient with idiopathic thrombocytopenic purpura (ITP) only an-eedogenoes'' inhibitor of PAF induced platelet aggregation was initiallyobserved. In spite of this. the palient responded to treatment with the PAFantagonist WEB 2086 with a dramatic increase in platelet count(Lohmann et QI .• Lanett ii, 1147. 1988). Thereafter, also increased PAFlevels (3.3 ng PAFhnL) were detected in plasma. although some "endoge-nous" inhibitor of PAF was still present. In conclusion. increased PAF lev-els in plasma from patients support a role of PAF in certain human diseasestates, such as in anaphylactoid reaction, sepsis and septic shock. Thetype. relevance and specificity of endogenous inhibitors of PM deservefurther study.[Lipids 26.1381-1385 (1991)]

Dirrerenllal Errcrt of a PAF Antagonist CV-3988 on Active and Pas-sive Anaphylactic Shock in Various Mouse Strains

Akinori Arimura and Minoru HaradaShionogi Resean::h Laboratories, Shionogi & Co .• Ltd., Osaka 553, Japan

To define the role of platelet-activating factor (PAF) in anaphylactic shock.in the mouse, the suppressive effect of CV-3988. a PAF antagonist. onactive and passive anaphylactic shock. was studied. Various mouse strainstreated or not treated with Bordnella pertussis (8. per/llssis) were used.We found that the effect of CV-3988 on anaphylactic shock. in the micethai were actively sensitized with bovine serum albumin plus 8. pertussisdiffered mark.edly according to mouse strain. CV-3988 suppressed theanaphylactic shock in C3H/He and CBNJN mice at a low dose of 3mg/kg, whereas antagonists to other mediators such as histamine. sere-ronln. thromboune A2 and leukcrrienes did not show a suppressiveeffect. This suggests thai PM play! a major role in anaphylactic shock. inthese strains. On the other hand. CV-3988 did not suppress aclive anaphy-lactic shock. in cataract Shionogi (CTS). NOD and DS stmins even at ahigh dose of 30 mg/kg. which could be interpreted to suggest that PAF isnot active in these strains. However, this possibility was ruled out basedon the similar results obtained in passive anaphylactic shock and PAF-

INFORM, Vol. 3, no, 1 (Jonuory 1992)

induced shock in these mice. Passive anaphylactic shock in as micemediated by IgGl antibody was markedly suppressed by CV-3988 but notat all by antagonists to other mediators. Furthermore, the suppressiveaction of CV-3988 against passive anaphylactic shock. and PM-inducedshock. was greatly attenuated when the mice were pretreated with 8. per-mssts. From these results, the conclusion can be drawn that PAF is themain mediator of active and passive anaphylactic shock. in the mouse ingeneral, even though the effect of CV-3988 differs depending on themouse stmin and on whether or not 8.pertussis treatment is used.[Lipids26.1386-1390(l99I)[

Erred or a Selective PAF Antagonist SM-l0661 ((±}-cis-3,5-Dimethyl-2-(3-pyridyl)thia:wlidin-4-one HCI) on Experimental DisseminatedIntravascular Coagulation (DIC)

Noriak! lmanlshl, Yoshihiro Komuro and Shigeakl MorookaResearch Labomtories, Sumitcmo Pharmaceuticals Co., Ltd., Osak.a, 554Japan

Intravenous infusion of endotoxin (0.25 mglkglhr for 4 he) was shown toinduce disseminated intravascular coagulation (Ole) in rats. which result-ed in hypofibrinogenemia. prolongation of prothrombin (PT) and partialthromboplastin time (PIT). thrombocytopenia, and elevated levels of fib-rinogen/fibrin degradation products (FOP). Oral administration (100mg/kg) of the selective PM antagonist, SM-I0661 ({±}-cis-3.S-dimethyl-2-(3-pyridyl)thiazolidin-4-one HC!). counteracted the changes caused bythe endotoxin. Intravenous infusion of SM-I0661 (6 mglk.g bolus 2 minbefore endotOlCin infusiCH1+ 6 mg/kglhr for 4 hr infusion) also counteract-ed Die. When suboptimal doses of gebexaie mesllare, a synthetic proteaseinhibitor (3 mg/kg i.p.). and SM-I0661 (2 mglkg bolus + 2 mglk.glhr for 4hr infusion) were administered concomitantly, hematological parametersimproved. The results suggest that PAF may pl8Y a role in the pathogene-sis of DlC, and that together with the results already reported for otherPAF IIl1tagonists, SM-I0661 may be useful in the treatment of DlC.[Lipids 26,1391-1395 (1991)1

Effecl or PAF·Acether Antagonists on Acnve Anaphylactic MousePaw Edema

Claudia Zuany Amorima, Maria das Gra\!8s Muller de 01inira Hen-riquest', VI~ian Baseches wega. Renato sergio Balii.(1Cordeinfl, andB. Boris VarganlgbQFund~lo Oswaldo Cruz, IOC, Departmento de Fisiologia e Farmaced-inamica. CEP 20040. Rio de Janeiro, Brazil, and bUnit~ de Phermaco!o-gie Ce!lulnire. Unit~ Assoctee lnstitut Pasteur INSERM-U.285, 75015Paris, France

A new model of active anaphylactic reaction in mice was developed. Theedemaiogenic reaction appeared 5 min after the intraplamar injection ofovalbumin. peaked at 30 min after the antigenic challenge, and decreasedthereafter. Using the non_steroidal. anti-inflammatory ageruaindomethacin and aspirin. we found that cyclooxygenase products do notparticipate in the reaction. In contrast, vasoactive amines appear to beinvolved. because meclizine and methysergide reduced the edema. Dex-amethasorte. BW753e. LY 171883 and WEB 2170 effectively interferedwith the edesnarogenic reaction. which suggests that lipid mediators suchas teukomenes and PAF playa role in the active anaphylactic response.[Lipids 26. 1396-1399 (1991)1

Pooling of Blood in Pestlschemlc Shock Is Modulated by Platelet-Acti~aling Factor (pAF)

V.F. Saga<:ho, A. V. Dmitrie,·aQ and P. BraquelbQA.A. 80gomolets Institute of Physiology. Kiev. USSR and blns.tllltHenri 8eaufour, Le Plessis-Robinson. France

In experiments on dogs. i.v. administration of platelet-activating factor(PAF) (500 nglkg) was shown to induce hypotension which. apart fromdecreased myocardial contractility. was characterized by blood pooling in

veins (82.6 ± 6.8 mUkg). This was ecccmpamed by restriction of venousreturn to !he heart and reduction of cardiac output (CO). During post-ischemic shock the cardia- and hemodynamic disturbances were similar tothose induced by Lv. administration of PAF. In the postischemic shockmodel. preliminary blockage for PAF receptors wilb the PAF receptorantagooist BN 52021 (6 mgJkg. i.~.)significantly decreased the amountof blood pooled in shock from )8.7 ± 5 to 18.) ± 2 mL/kg (p<O.OI).Simultaneously, the reduction of CO and blood pressure, induced byreperfusion of the continuously inschemized tissues of a rear timb, wasless significant in pretreated vs. the nontreated group. The data suggestIbat PAF may be involved in postischemic blood pooling and that PAFantagonists could be used to correct pcstisehemtc cardto- and hemo-dynamic disturbances.[Lipids 16. 1400-140) (l99I)]

Induction of Platelet- Activating Factor In Mice by IntravenousAdministration of a Neutral Fraction or Bakers' Yeast Mannan

Takeshl Mlkamja. Ken Fukushla, Mikl ishitani", Kouki Ishitani",Shigeo Suwkib and Masuko Su:r.ukiQQDepanment of Microbiology and bSecond Department of HygienicChemistry, Tohoku College of Phannacy. Sendai 981. Japan

A neutral subtraction of mannan of bakers' yeast (WNM) was found toshow a lethal effect in mice when administered intravenously. Symptomscaused by intravenous (Lv.) administration of WNM resembled thoseresulting from the administration of platelet-activating factor (pAf). CV.)988 and ONQ-..624O, selective PAF antagonists, prevented hypotensionand death caused by !he administration of WNM or PAF. A jl.-adrtllOcep-tor agonist was shown to prevent death caused by WNM, whereaspropranolol increased the lethal activity ofWNM.lntravenous administra-tion of WNM into mice produced PAF in gall bladder Iluid which wasdetennined by platelet aggregation assay. The findings indicate thar WNMis able to induce PAF in mice and thaI the resultant PAF may participate inthe WNM-induced lethal activity observed in mice.[Lipids 16. 1404--1407 (1991»)

InVOlvement of Platelet-Activating Factor in Zymosan-Induced RatPleurisy

Yohsuke Imai, Masahiko Hayashi and Sachiko Oh-ishiDepartment of Pharmacology, School of Phannaceutical Sciences.Kitasato University, Tokyo lOS, Japan

The role of platelet-activating factor (pAF) in innammatory reactions wasstudied in zymosan-Induced flit pleurisy. Pleurisy was induced by injec-tion of a 2% :r.ymosan suspension into the pleural cavity of rats. The timecourse of plural exudate accumulation, the exudation rate. and exudateleukocyte numbers were followed then for 96 hr. Peak pleural exudateaccumulation was about 3 mL II 24 hr, whereas the exudaticn rateincreased biphasically with peaks at 0.5 hr and 5 hr. The migration ofleukocytes into the pleural cavity increased wilb time up to 48 hr. Thepolymorphonuclear leukocytes were the dominant white celts in the exu-date between 5 and 16 hr. but mononuclear leukocytes started to outnum-ber them around 24 hr. Pretreatment with cyproheptadine (5 mglkg). aninhibitor of both histamine and serotonin, significantly sUPJ'R'ssed pleuralnuid eccumulauon and the exudation rate at 0.5 hr. The PAF antagonistCV-6209 (l mgJkgJ significantly suppressed pleural fluid accumulationand the exudation rate at both 0.5 and 5 hr. At either time point, thepanuneters were not suppressed by indomethacin. We detected PAF ecuv-ity in the high-perfOOllance liquid chromatography (HPLC) fraction (witha retention time cormsponding to Ibat of authentic PAF) of the exudates at0.5 hr. 5 hr, and 16 hr using an aggregation bioassay with washed rabbitplatelets. The results suggest that in zymosan-induced rat pleurisy. his-tamine and/or serotonin an: the main mediators of exudation Bt 0.5 hr andthat PAF may be partly responsible for exudation at 0.5 hr and later at 5 hr\0 t6 hr.[Upids 26, t408-14!1 (1991)]

Hexadecylpbosphocholine: Preclinical and the First Clinical Resultsor a New Antitumor Drug

Clemens Unger"'" aod Hansjol'J EiblbQDivision of Hematology/Oncol~. Department of Internal Medicine,University Hospital G6l1ingen and Max-Planck-Institute for BiophysicalChemistry. D-l4OO G6ttingen, Federal Republic ofGennany

Dose-response studies on cytOioxic alkyl lysophospholipids wilb variouschemical structures revealed that a long alkyl chain and a polar group areessential for antitumor activity. The combination of both the long alkylchain and a phosphocholine group thus results in alkyl phosphocholines.Preclinical studies wilb heudecylphosphocholine (He-pc) as a represen-tative compound indicate distinct antineoplastic scuvuy on leukemia cellsof human origin. He-PC is highly effective in inhibiting the growth ofchemically induced rat mammary carcinomas. Even more striking is thefact that a high percentage of the tumors regressed completely. In a clini-cal phase I trial on breast cancer patients with local recurrences. topicallyapplied He-PC resulted in regression of skin metastases. A phase II trialfor topical treatment and a phase I trial for orally applied He-PC havebeen initiated 10 further evaluate me antitumoral activity of this new com-pound.[Lipids 16, 1412--1417(1991)]

Comparison or Selective Cytotoxidty or Alkyl Lysophospholipids

W.R. Vogler", A.C. OlsonQ, S. Okamoto", M. Shoji", R.L. Raynor"'",J.F. Kuo", WE. BerdetD, H. Eiblb, J. Hajdue and H. NomuradQEmory University School of Medicine. Atlanta. Georgia )0)22, i1Max-Planck-Institut rtir Biophysikalische Chemic, 3400 GOttingen. Gennany.cCalifomia State University, Northridge. California 91330 and UrakedaChemicallndustries, Ltd .• Osaka 532, Japan

Alkyllysophospholipids have been shown to be cytotoxic to a number ofneoplastic tissues. One. ET-18-OCH), has been used to selectively purgeleukemic ceus from mixtures with normal marrow progenitor cells. illvuro and in l'ivo. We have measured the.5O% inhibitory (lCSO) effect of aseries of ether lipids (EL) on leukemic cells (HL60. KS62, Daudi. KG-I,KG-Ia) and nonnal marrow progenitor cel1s. Cel1s were incubated withvarying concentrations of EL for 4 hr and assayed for viability.[)H[thymidine incorporation and cjcnogenicity in semi-solid media. Theeffect on protein kinase C (PKC) activity was assayed for each compound.Compounds tested included three glycerophosphocholine analogs-ET·IS-OCH3. ET-16-NHCOCH). and BM 41.440. In addition. a lipoidalamine. CP 46665, an ethylenegJycolpllospholipid. AEPL. and four singlechain alkylphosphochcllne analogs, HePC2' HePC3' HePC4 and HePC6were also tested. During the period of incubation. the cells remainedviable (>70%) as judged by trypan blue dye exclusion. The glycemphos-phocho1ines were the most active and showed the highest therapeuticIndex. The lipoidal amine was active, buttoxic 10 normal marrow progen-itor cells. The ethyleneglycolphospholipid was active against HL60, butnot against the other cel1lines. The single chain alkylphosphocholineanalogs were less active. All of !he compounds inhibited PKC IICtivity:however. the glycerophnsphocholines were the most inhibitory.rUpids 26. 1418-142) (1991))

Stereospecific Synthesis or Antitumor Active Thioether PAF Analogs

Su~h K. Bhatia aod Joseph HajduDepartment of Chemistry. California Slate University. Northridge.Northridge. California 91))0

A novel srereospectnc synthesis of antitumor active thicether analogs ofplatelet-activating faclor (PAF) is reported. The synthesis is based upon: i)the use of D-serine to provide the chiral center for the construction of theoptically active phospholipid molecule; ii) development of the 511-1-thioalkyt fUllCtion via thioaa:tate displacement of methanesutfonate-acti_vated primary hydroxy group followed by alkylation of the sII_I_thioJatefunction; and iii) introduction of Ihe phosphochollne moiety through the2·chloro-2-o:o;o-l.),2-diO)laphospholanc/trimelhylam ine sequence. The

INFORM. Vol. 3. no. I (January 1992)

139

140

ABSTRACTS FROM AOCS JOURNALS

entire scheme relies on the use of a single protecting group. The syntheticthiocther phospholipid 1-S-hcudeeyl-2-N-acetamidodeoxy-sn-glyC1:ro-3-phosphoclloline has been ~hown 10 be II polcnt antitumor active phospho-lipid, exhibiting tumor cytotoxicity against II lyrnphcblasroid lymphoma(Li-A) cell line and a mulignem histiocytic (DHL4) cell line of humanorigin at the same level of potency as ET-18-0Mc lind I-O-ocladecyl-2-N-acelamidodcoxy-.iIl-glyccro-3-phosphocholine. The synthetic methoddescribed has a great deal of Ilexibllity. providing a convenient generalmute 10 II wide range of rhicether PAF analogs.[Lipids16,1424--]43O(l99I)[

Antitumor Acti ...i.)' of IImufosine (8M 4J.44{I) in Ihe 3Lewls.LungCarcinoma Model

Dieter OJ. Herrmanna, Hans-George Opitzlf and Paul G. Munder!'oBochringer Mannheim GmbH. Department of Immunopharmacology,6800 Mannheim and bMax-Planck-lnstitute for Immunobiology, 7800Freiburg, Germany

llmofosine (l-hexadecylthio-2-metho~ymethyl-I.3-propanediol-phospho-cooline, BM 41,440) is a thioether phospoolipid with cytostatic/cytotoxicproperties. The antineoplastic oct;vity of this compound was investigatedin ..iro in the 3Lewis-lung call:inoma system. 3Lewis-lung IUmor-bearingC!i7B 1/6 mice were treated with 0.625 to 40 mg IImofosine/kg per dayp.o. either from days I to 9 or from days II to 28 after ltnrafoot-padtumor cell inoculation. llmofosine caused a significant dose-relatedresponse: on tumor growth and metesreses. expressed in terms of tumordiameter. tumor weight. sUf\'ival time and number of mctasrases-Itee ani-mals as compared 10 sham-treated and positive (cyclophosphamide) con-trols. The results suggest that direct cytostatic/cytOloxic effects, rutherthan immune-modulatory mechanisms. preferentially contribute to theantitumor activity of Ilmofosine;n ";"0.[Lipids 26. 1431-1436 (l99I»)

I--/>-D-Arablnofuranosylcyloslne Conjugates of Ether and ThfoetherPhospholipids. A N~ CIIl5S of Ara-C Prodrug ",ilh Irnpreved Antitu-mor Acli ..ity

Chung II Honga, Charles R. WestD, Ralph J. Bernackib, Cameron K.TebW: and Wolfgang E. BerdeldDepanments of °Neurosurgery. il£;o;perimental Therapeutics and ('Pedi-ames. Roswell Park Cancer Institute. Buffalo. New York 14263. and dOe_partment of Hematology and Oncology. Free University of Berlin. Berlin45. Germany

The I-~D-arabinofuranosylcytosine (era-C) conjugates I-O-alkyl(ether) and l_S_alkyl (thiocther) phospholipids, being analogues of era-CDP-m-I.2-0-dipalmitoylglycerol (I). showed significant antitumor

AOCS Mission StatemenlTo be a forum for the exchange of ideas. information and experienceamong those with a pmfessiOllll1 interest in the science and teehllOlogyof fats, oils and related substances in ways that promote personalexcellence and provide for II high standard of quality.

Dectaratlon by the Governing BoardThe American Oil Chemists' Society is organited for charitable. edu-caliOllal and scientific purposes. II does not have as its purpose thepromotion of any product. manufacturers. laboratory or business.Members of the Society. and employees of the Society do not and maynot speak for or 00 behalf of the Society without the e;t;preSKd per-mission of the Gov ....rning B03rd. This prohibition includes the use of

activity against LI210 and P388 leukemia in ";1"0.The more octive conju-gates include lhe !-O-alkyl analogues. arn-CDP-rur-l-O-Ilendccyl-2-0-palmitoylglycerol (2) and ara-CDP-Tar- I -O-octlldccyl-2-0-palmitoylglyc-erot (3), and the corresponding I-S-alkyl analogues. ara-CDP-ruc-I-S-he;t;adecyl-2-0-palmi!Oyl-l-thioglycerol (4) and IlTa-CDP-roc-I-S-octade-cyl-2-0-plllmiloyl-I-lhioglyccrol (5, Cytoros). The conjugates were for-mutated by sonication. in which the conjugates e;t;isted as discs (size0.01-0.04 ).4m). Among the conjugales of the three different phospho-lipids. tile I-S-alkyl analogues 4 and 5 displayed tbe strongest antitumoractivity against L 1210 leukemia in mice. followed by the \-O-alkyl (2 andJ) and the l-O-acyl (I) analogues. The I-S-alkyl analogue 5 was consid-erably more effecti ve than the I-O-acyl analogue I against myelomono-cytic WEHI-3B leukemia in mice. Conjugate 5 (Cyt()fl)li) showed a signif-icant therapeutic aclivity in mice with colon 26 carcinoma. M5076 sarco-ma. and C-IlOQ neuroblastoma. Furthermore. this agent inhibited livermetastases of M5076 sarcoma. Conjugates 3 and 5 also inhibited themetastasis of 3-Lewis lung eercinorna to the lungs of mice. Cytoros (5)and its analogues. with other ether and tbiocther phospholipids. appear tooffer increased therapeutic benefit to mice with tumors.[Lipids 26, 1437-1444 (1991)[

Induclion or Differentiation of Human Myeloid Leukemia HL-60Cells by No,'el Nonpho!'iphorus Alkyl Ether Lipids

Yo:shio HonmaD, Takashi Ka.sukabeD, Motoo HozumJU, Hiroshi Akl-molob and Hlroaki NomurabDDepanment of Chemotherapy. Sa.itama Cancer Center Research Institute.Sailama-362 and heentral Research Division. Takeda Chemical Ind. Lid ..Osaka-5J2. Japan

We synthesized a new series of ncnphospborus alkyl ether gtyceronpros.in which the 2-acetyl group of platelet-activating factor was replaced by apyrimidin-z-yl group and the 3-phosphocholinc portion by an w--(substi-tuted ammonio)ethoxyethyl side-chain including to-thiazollo-. imidasolio-and pyridinio groups with or without a carboxyl substituent. respectively(compound I-XI). Their effects on cell proliferation and differentiation ofhuman myeloid leukemia HL-60 cells were examined. Incubation of HL-60 cells with these cationic and zwiuerionic alkyl ether lipids inhibitedproliferation of HL-60 cells with 1CSO values ranging from \0 to 500nghnL. The cells were induced by the lipids to differentiate into morpho-logically and functionally mature granulocytes. Among the compoundswe tested. l-octadecyl-2-pyrimidinyl-3-13-(5-carbo;t; ylatepenyljlmldazo-lioethcxyethyllglycercl (compound I) was the moot effective in inducingdifferentiation of HL-60 cells. Compound [ showed on a molar basis, aninhibitory effcct on the leukemic cells over 50 limes greater than did 2-(2-dodecyloxycthcxyjcthyl 2-pyridinioetbyl phosphate, the antileukemicalkyl ether phospoolipid.[Lipids 26. 1445-1449 (1991}) •

the American Oil Chemists' Society logo or lencrbead when making asteremern of a technical or economic nature. Members of the Society,or employees of the Society speak only for themselves when givingopinion or making statemerns concerning technical matters and eco-nomic maners.

Use of AOCS Name and Logo~ A Individually or in combination. the d....sign of Ihe

~ name and logo of the American Oil Chemists' Soci-ety (AOCS) may nor be reproduced or used by any person ororganiza-tion other than lhe AOCS. its elected and appointed officers. sections.or committees. except by special permission of the AOCS GoverningBoard.

INFORM, VOl. 3, no. 1 (Jonuory 1992)