【doc】培养牛脑微血管内皮细胞上血小板激活因子的...

【doc】培养牛脑微血管内皮细胞上血小板激活因子的... 培养牛脑微血管内皮细胞上血小板激活因 子的... 326?中国荮理AetaPharmacofogicaSinica1992Jill;13(4j amnesticeffeetsof【2chemicalsonpassiveavoidancere sponsesinmice:comparisonbetweenstepdownandstep throughtestsAetaPharmS/n【986;21】2—9 5PansYInfluencesofEntraventrIcu】arjnjectionof 6-hydroxydopamineonpassive-avoidanceresponse- acetyIcholine,andmuscarinicreceptors1nmousebrain ActaPharmaeo(Sinl99【12:344-7 6LowryoH.RosebroughNJ.FarrAL,RandallRJ ProteinmeasurementwiththeFolinpheno【reagentJ BiolCheJqa【951;193.26卜75 7CrawleyJNExploratorybehaviormodelofanxietyin miceNeuroscfBiobehavRev【985:9:37-44 8FileSEWhatcanbeteamedfromtheeffectsof benzodiazepinesonexploratorybehavior'?Ne~roscf BlobehaRevI985;9:45-54 9PierceyMF,VogelsangGD,FranklinSR,TangAH. Reversalofscopolamine—inducedamnesiaandalterations inenergymetabolismbythenootropicpiraeetam:impii— cationsregardingidentificationofbrainstructuresint volvedinconsolidationofmemorytracesBrainR I987;424:1-9 10MaquetP.DiveDSalmonE,SadzotB.FrancoG PoirrierR,alCerebra【glucoseutilizationduring sleep--wakecycleinmRndeterminedbypositronemission tomographyand【Fl}nuom一deoxy-D—glucose methodBrainResI990;513136-43 DiazdeIGuanteMA,Cruz-MoralesSE,Prado_Aleatd RATime-dependenteffectsofcholinergicblockadeof thestriatumOilmemoryNeuro$ciLett】99】122: 79—82 3,二 东茛菪碱对小鼠被动学习,探究行为及脑区毒 蕈碱受体的昼夜变化 潘思源 (北京中医北京100029,qi国) 提要来莨菪碱(Scoplip0.1,0.4mgkg实验于 07:00—:00,15:00—17:00和21:00—23:00进行.结果 显示.Scop对小鼠被动学习的抑制作用,及其增加探 究活动和减少排便的作用,以白天最明显.小鼠颞叶 皮层和海马部位的M受体数,白天多于夜晚而纹 状体M受体以,r=最少结果提示,Scop对小鼠学 习记忆和行为的影响,及其不同脑区的M受体,均 呈现一定的昼夜变化 =苯羟乙酸奎宁酯;记忆:探究 颞叶;纹状体i海马:昼夜节律 BIBLID:ISSN0253—9756中国药理ActaPharmacologicaSinicaI992Jul;13f41:326— 329 Specificbindingsitesofplateletactivatingfactorontheintactbovine cerebralmicrovascularendothelialceHsandantagonismofdrugs SUNDu-Xin,RUIYao—Cheng,ZENGGun—Qian,ZHUJu,SHENYou—An (DeparmentofPharmacology,SchoolofPharmacy,SecondMilitaryMedicalUniversity Shanghai200433,China) ABSTRAcT『JH]TriazolodiazepinefrH]WEB 20861_anantagonistofplateletactivatingfactor (PAF1rcceptor,wasstudiedasradioligandonintact cerebra1microvaseularendothelia1cellsCMEC). Theresultsshowedthatthebindingof [~H]triazolodiazepinereachedandmaintainedatan equilibriumafterl5—12Ominof1ncubationandthatit wassaturablewithincreasingconcentrationof Received1991Apt15Accepted1992May4 radioligandScatchardanalysisindicatedthatthere were2specificbindingsitesonCMEC,1ts. ,Kaanda2were313nmol'L,150 pmol/3×10cells,8396nmo1L,and1296 pmol/3x10cells,respectivelyThebindingof rH]triazolodiazepinetoCMECwfisdisplacedby C16-PAFand1,5is一(3.4一dimethoxypheny1)一 tetrahydro一(4研一pyran(SZ—I).whichICwere 043nmol_L,and0.125#mol?L一,respectively ThesedatasuggestedtheexistenceofPAFspecific bindingtesonCMEC 中国药理ActaPharmacofogicaSinica1992Jul;13【4)'327 KEYWORDSplateletactivatingfactor;vascular endothelium;drugreceptors PlateletactivatingfactorfPAF)isapo— tentphospholipidmediatorinvolvedinvarious inflammatory~t,respiratory(2},cardiovaseu- lar(. andrenaldisorders(4JPAFwassyn山e— sizedinbovinebrain,anditdose-depently constrictedthepialarteryofpiglets".PAF receptorantagonists,kadsurenoneand ginkgofides,dose-dependentlyenhancedthe recoveryaftercerebra1ischemia【75). However,thespecificbindingsitesof PAFonthecerebr0vascularsystemespecially 0nthecerebralmicrovascularendothelialcells (CMEC1havenotbeendescribedlnthe presentstudy,westudledthespecificbinding sitesofPAFonCMECwi山PAFreceptoran. tagonistrH]triazolodiazepine([sH]WEB2086) and山eantagonismofanev~compound l,5一bis一(3.4一dimethoxypheny1)一tetrahydro一 (4/t)一pyran(Sz—1)synthesizedinourDe- partment. .cn,o CHjO/\CH ) O .5一bs一(34一dimethoxypheny1)一 tetrahydro14一pyranfSZ一1) MATERIALSANDMETH0DS Minimumessentia1medium(MEM1(Gibco), trypsin(Sigma).rH]triazolodiazepine,triazolodia— zepine,andCl6一PAFweregiftedfromBoehringer Ingelheim,Germany. Cl_】tivationofhovi?cCMECCel】cultivation waspmparedbyamodificationofthemethod(i.In brief,thegraymatterofbovinebrainwascollecfed, rin~d4timeswithD—Hanksso】ution,andcut1nto smallpieces.Thenthegraymatterwashomogenized 20uDanddownstrokesinaglasshomogenizerwith MEM.Thehomogenatewasfilteredthrou曲anylon meshwithporesizeof74"mMicrovesse]swereco1. 【ectedbythenylonmesh+andwashedby centrifugationr800gfor8rain)with10m1MEM ThepelletwasresuspendedIn01%collagenaseand incubatedfor30minat37?.Attheendof incubation,thesuspensionwascentrifugated(800g r0r8rain1andthepelletresuspendedinMEMwith 20%retaIbovineselumandplatedontogelatin~coat. edtissuecultureflask.Thecultufeswemkept7dat 37?with5%CO,,fedwithfreshmediumevery2-3 dThecellsweresubculturedwith01‰trypsin BindingassavTheceilswerecollectedwith O1%trypsin,washedwithtris-HClbufrer(KCIl40 mlTIOlL,trisl0iYlmoI_L,BSA01%,pH 7.4,bycentrifugat;,on(800gf0r8mint,thepellet wereresuspendedint卜HCIburr6×l0 cells/rot)Theceilssuspension(3×10'cells1were addedtoatubecontainingrH】triazolodiazepine8 nmoL,withorwithout1O0o—foldunlabeled triazolodiazepine(fornonspecificbinding),incubated at25Cf0rI5min.Boundandfreeradioligandwere separatedbyrapid闻trationwithmicroporousnl_ teringfilmwhichwaspresoakedintris-HC1burror 24hThefilterswerewashed4timeswith2m】 tce-coldtris-HC】bufr(no0】%BSA1.The radioactivityofthefilterwasmeasuredbyscintillation with40%emciency RESU【JTS Thecellswerecharacterizedbvphase- contrastmicroscopy,transmissionelectron microscopy(TEM),scanningelectron microscopy(SEM),andfactorVIII-related antigenstaining.ThecelIsshowedthecharac- teristicsofendothelialcells,suchas cobblestonecoloniesunderthephase—contrast microscope.Tightjunctionsandpinocytic yesicleswerepresentunderTEM.Thestain. ingoffactorVIll-relatedanantigenwerepos. itive. KineticexperimentsThespecificbind. ingof[H]triazolodiazepine(8nmol?L)to CMECreachedrapidlyandmaintainedsteadi. 1yatanequilibriuminl5一l20minat25?. Thenonspecificbindingwasunchangeable duringl5一l20minandamountedtoonly 328?中国菇理ActaPharmacologicaSinica1992Jul;13(4) 50%ofthetotalbound(Fig1) 8 娶 Incubationtime,rain Fig1.[3HlTriazolodiazepinebindingtointact CMEC.Totalbound(0),nonspecificbomld (?),andspecificboundr×).=3,?S. SaturationstudvThebindingof [3HltriazolodiazepinetoCMECwasspecific andsaturablewiththeincreaseof rH]triazolodiazepineconcentrationr2-150 nluoI_L1.Thenonspecificbindingin— creasedJinearlywithradioligandconcentra- tion.Seatchardanalysisindicatedthatthere were2specificbindingsitesoffH]triazolo— diazepineonCMEC.Their1,, ?,andma2were313nmol.L,, 83,96nmoI_L,,1.50pmol/3×10cells, 0l0一"10-9l旷'10一 Corte~tration,mol?L-I Fig2.SaturationandScatchardanalysisof [JHltriazolodinzcpinespecificbindingtointactCMEC. =3,f?. and1296pmol/3×10cells,respectively (Fig2). CompetitionstudyThespecificityof F3HltriazolodiazepinebindingtoCMECwas establishedbydisplacingPH]triazolodiazepine 8nmoI_L—byC1^一PAFandSZ一1intom— petitionexperimentsCI6—.PAFf0.Ol一1O umol_L)andSZ-1rO.01一l0#mol.L) dose-dependentlyinhibitedthespeciticbind. ingof【jH]triazolodiazepinetocMEcwith lCnvaluesof0.44nmo1.L—and0l3 #mol_L,respectively(Fig31. Bound,pmo[/3×i0cc[Is Fig3.Displacementof[aHltriazolodinzepincbindingto intactCMECbyC16--PAFandSZ一1.=3,. DISCUSsloN Manyexperimentssuggestedthattheeel". ebra1vasculardisordersmi吐tberelatedtothe PAP.10)Thedatareportedheredescribed forthefirsttimethecharacterizationofPAF receptoronCMECusingrH]triazolodia— zepineThebindingof[3H]triazolodiazepine toCMECwasspecific.reversible.and saturable.Scatchardanalysisindieatedthat theenistenceof2specificbindingsiteson CMECisdifierentfromthatofothercells suchaSplatelets,neutrophilsexhibitingonly onekind0f山esites…'ThisSH卫gestedthat theimportanceofPAFinthe0erebr0vascuJar diseases.TheactionofPAFinthe cerebrovascularsystemmi曲tbemediated ? 叫] 8\鼍ja? 越?? 富一8—xn\目Ap】喜^l】=0lPtx0一 .Pu,0ulnu兰:.I!APa|oN暑皇墨^一 中国药理ActaYharmacotogicaSinica1992Jut;13(4)329 chroughthePAFreceptor. SZ-I,anewsyntheticcompound,not onlydisplacedtherH]triazolodiazepinebind— ingtoCMEC,butinhibitedtheaggregation ofwashedrabbitplateletsinducedbyPAFin vitro,thereleaseofC]arachidonicacidjn CMEC,andtheEvansbluestaininginrat braininducedbyPAFfdatanotshown)It suggestedthatSZ一1isanantagonistofPAF receptor. SpecificbindingsitesofPAFhavebeen foundinvariouscells.Butbindingstudies th『JHIPAFremainsomewhatdifncultbe. causeofthehighlevelofnonspecificbinding, theincor~sistentbiad2andugtakeofthe radioligand.Thus,wemadeuseofthePAF receptorantagonist【H]triazolodiazepineas radioligandtoinvestigatethePAFspecific bindingsitesontheintactbovineCMECfor 3PiperPJ,StewartAGAntagonismofvas0constctIon inducedbyplateletactivatingfactoringuiana—pig perfusedheartsbyselectiveplateletactivatingfactor receptorantagonists册JPharmaenll98790: 77l-83. 4CamussiGPotentialroleofplateletactivatingfactorin renalpathophysio[ogy.KidneyInt1986;29469—77 5KumarR.HarveySAK,KesterM.HanahaoDJ. 01sonMS.Productionandeffectsofplatelet—activating factorI12theratbrain.BiochimBiophysc1988, 963375—83 gArmsteadWM,PoureyrousM,MirroR.Lemer CW.Bus~aDW.PlateIetactivatingfactor:Apotent , constrictorofcerebralarteriolesinnewbornpigsCirc el988;62】一7 7KochanekpM,DutkaAJ,KumarooKK,Hallenbeck JMPlateletactivatingfactorreceptorblockadeen hancesrecoveryaftermultifocalbrain】schemiaL Scil987;412639-44 8SpinnewynB,BlavetN,ClostreF.BazanN,Braquet PInvolvementofplatelet-activatingfactor(PAP"1jn cerebraIpost-ischemicphasemMongoliangerOils. Prostaglandins】98734337-49 9Cai$otlMP,HaudenschildCCMicrovascular endotheliumandpericytes:Highyield?[owpassagecul- 54. tures/nVitroCDPBinl986;22344— 1OBraquetP.SpinnewynB,BIaverN,Matcbesell】V. Ro铀askaMBazanNG+PIateletactivatingfactoras amediator玎】cerebralIschemiaandrelateddisorders BiomedBJoehj.mActa1988,47:Sl95-2】8 l】UkenaD,DentG,BifkeFW.Roba'~tC.Svbrecht CW,BarnesPJRadioligandbindingofantagonistsof plateletactivatingfactortointacthumanplateletsFEBS Lett】988,228:285—9 l2Fg]desFi[epE.BraquetP.FilepJInhibitionbyBN 52021{ginkgolideBJofthebindingofrH]plateletacti 提要在体外培养的小牛脑微血管内皮细胞上,用 PAF受体拮抗剂[3Hitriazolodiazepine研究PAF受 俸结果明【3H]triazo~.odiazepine在该细胞上有两个 特异结台位点,其K.,2,且m1和丑毗分别为 313nmol.L,,8396nmolL,,1.50pm,M/3× 10cells,和1296pmol/3×10celts.而且 [3H]triazolodiazepine与内皮细胞的结台可按 Cifi-PAF和Sz一1所取代提示脑微血管内皮细胞上 有PAF特异结合部位. 关键词血小板澈话因子;血管内皮;药物受体 , ,垭腓?器 髓黎 删mna引淼 l;.,山曩一'薹恤一善要