信息编号11416501至11416550间共50条。
11416501:重症
多形红斑型
药疹的护理
重症多形红斑形药疹是重症药疹的一种，本病起病急剧，常有高热、头痛、全身不适等症状，皮疹广泛，有红斑、
丘疹、水疱、大疱、
血疱等，且伴有严重粘膜损害，如口腔、眼、外生殖器。部分患者还可有严重肝、肾等内脏损害，如抢救不及时，可因水、电解质紊乱、脏器受损严重以及合并感染导致死亡。我科1997年1月~2005年12月，收治了重症多形红斑型药疹13例，经过积极抢救治疗和精心细致的护理，提高了治愈率，现将护理体会报道如下...查看详细（3007字节）
11416502:Cocaine-andAmphetamine-RegulatedTranscriptIsLocalizedinPituitaryLactotropesandIsRegulatedduringLactation
ClaytonFoundationLaboratoriesforPeptideBiology,TheSalkInstituteforBiologicalStudies,LaJolla,California92037AbstractCocaine-andamphetamine-regulatedtranscript（CART）isahighlyexpressedpeptideimplicatedintheregulationoffeeding,rewardandreinforcement,andstress-relatedbehaviors.CARThasbeenlocalizedtodiscretecellpopulationsinthebrain,gut,adrenalgland,andpancreas.Incontrast,CART-producingcelltypesinthepituitaryglandremainilldefined.Inthepresentstudy,double-labelimmunohistochemistry,employingahigh-affinityantiserumwegeneratedagainstCART-（62C102）,wasusedtoidentifyCART-producingcellsinthepituitarygland.Intheanteriorpituitary,themajorityofCARTimmunoreactivity（-ir）waslocalizedinlactotropes;minorpopulationsofCART-ircellswereidentifiedassomatotropesandcorticotropes.Intheposteriorpituitary,CART-irextensivelycolocalizedwithoxytocin-containingfibers;incontrast,onlyafewvasopressinfiberscontainedCART-ir.Asexpected,CARTcolocalizedwithoxytocininmagnocellularneuronsofthesupraopticnucleus.Theeffectsofbromocriptine,apotentdopaminereceptoragonist,wereexaminedtodeterminewhetherCARTmRNAexpressionandproteinreleaseareregulatedinasimilarfashionasprolactin.Similartoprolactin,CARTmRNAexpressionandproteinreleaseweresignificantlydecreasedafterbromocriptinetreatmentofdispersedratanteriorpituitarycellsinculture.ToexploretheputativephysiologicalroleofpituitaryCART,wecomparedlevelsofCARTmRNAexpressioninlactatingandnonlactatingfemalerats.CARTmRNAlevelsweresignificantlyincreasedintheanteriorpituitaryandsupraopticnucleusoflactatingrats.Furthermore,levelsofCARTinthesystemiccirculationweresignificantlyelevatedattheonsetoflactation,peakedond10oflactationandreturnedtobaselinevalues10dafterpupswereweaned.ThecurrentstudydescribesthecellularlocalizationandregulationofCARTexpressionandproteinreleasefromtheratpituitarygland.ThesefindingssuggestaputativeroleforCARTinlactation.IntroductionCOCAINE-ANDAMPHETAMINE-regulatedtranscript（CART）wasoriginallyidentifiedbydifferentialdisplayPCRasanmRNAtranscriptthatwasup-regulatedinthestriatumofratsafteracuteadministrationofcocaineandamphetamine（1,2）.Sincethisinitialdiscovery,CARThasbeenimplicatedintheregulationoffeeding,rewardandreinforcement,andstress-relatedbehaviors（3,4,5,6,7）.CARTisahighlyexpressedpeptidethatiswidelydistributedthroughoutthecentralnervoussystem（CNS）,gut,pituitary,adrenal,andpancreas（5,8,9）.Inthebrain,CARTimmunoreactivity（-ir）hasbeenlocalizedincellbodies,fibers,andvaricositiesintheolfactorybulb,cortex,hypothalamus,mesencephalon,andbrainstem（10,11）.CART-irfibersareincloseoppositiontofenestratedcapillariesintheexternalzoneofthemedianeminence,andCARTproteinisfoundintheanteriorandposteriorpituitary（11）.Intheperiphery,CARTislocalizedinpancreaticislets（9）,entericneuronsinthegut（12）,andtheadrenalmedulla（11）.ThecellularlocalizationofCARThasbeenextensivelystudiedintheratCNS.Inthemedulla,CARTiscolocalizedwithadrenergicand-MSH-expressingneurons（13）.CARTisfoundinoxytocin（OT）-andvasopressin（AVP）-containingneuronsinthemagnocellularparaventricularnucleus（PVN）,TRH-containingcellsarefoundintheparvocellularPVN,andCART-ircellscolocalizewithproopiomelanocortininthearcuatenucleus（10,14,15）.CART-irhasalsobeenshowntocolocalizewithurocortin1incellsoftheEdingerWestphalnucleus（16）.Incontrast,thecellularlocalizationofpituitaryCARThasnotbeenclearlydefined.CARTmRNAisexpressedintheanteriorpituitary;however,CARTmRNAexpressionhasnotbeenreportedintheintermediateandposteriorpituitarygland（17）.Immunohistochemistry（IHC）studiesrevealedavarieddistributionofCARTintheanteriorpituitarygland.Koyluetal.（11）reportedthatthelocationandintensityofCART-irintheanteriorpituitaryvarieddependentontheantiserumused.Kuriyamaetal.（18）recentlydemonstratedthatCART-irislocalizedingonadotropes,whereasStanelyetal.（19）reportedthatCARTwasexpressedincorticotropes.Intheposteriorpituitary,CART-irislocalizedinahighdensityofvaricosefibers,andCART-irhasnotbeendetectedintheintermediatelobeofthepituitarygland（11）.InthepresentstudywegeneratedantiserumagainstCART-（62C102）toclarifytheanatomicaldistributionofCARTinthepituitarygland.AsensitiveRIAwasdeveloped,andreal-timePCRwasemployedtoidentifyfactorsthatregulateCARTmRNAtranscriptionandproteinreleasefromanteriorpituitarycells.Inaddition,wecomparedlevelsofCARTinthehypothalamus,pituitary,andsystemiccirculationoflactatingandnonlactatingratstoexplorethephysiologicalrolesofpituitaryCART.MaterialsandMethodsAnimalsandtissuepreparationAdultmaleandfemaleSpragueDawleyrats（HarlanSpragueDawley,Indianapolis,IN）,wereusedintheexperimentsdescribedinthisstudy.Animalsweremaintainedona12-hlight,12-hdarkcycle（lightsonat0600h）andprovidedratchow（Harlan-Teklad,Madison,WI）andwateradlibitum.Animalswereanesthetizedwithanoverdoseofchoralhydrate（1g/kgbodyweight,ip）andperfusedtranscardiallywith150mlsaline,followedby350ml4%paraformaldehydeinboratebuffer（pH9.5）.Afterperfusion,brainsandwholepituitarieswereremovedandpostfixedin25%sucroseinthesamefixativeat4Covernight.Tissueswerethenquicklyfrozenondryice,sectionedat25μmusingaslidingmicrotome,andstoredincryoprotectantatC20Cuntiluse.TheSalkInstituteanimaluseandcarecommitteeapprovedallproceduresdescribedinthisstudy.BloodsamplingBloodsampleswereobtainedfrompregnantandlactatingfemaleSpragueDawleyratsbytailbleed.Approximately0.3mlbloodwastakenfromeachanimalbetween0800C0900hond5,12,and18ofpregnancyandd2,10,17,and21oflactation.Controlbleedsweretaken10dbeforematingand10dafterweaningtodeterminebaselinelevelsofcirculatingCART.BloodsamplesweredrawnintochilledtubescontainingEDTAandcentrifuged,andplasmawasstoredatC20CuntilRIAanalysis.AntiserumandcontrolsAntiserumwasraisedinrabbitsimmunizedwithasyntheticpeptideencodingrat[Nle67]CART-（62C102）conjugatedtohuman-globulinsviabisdiazotizedbenzidineusingaprotocolpreviouslydescribedindetailforinhibinsubunits（20）.Inallexperiments,antiserumwasusedinanunpurifiedformattheindicateddilutions.Thespecificityofimmunostainingwasevaluatedusingprimaryantiserumpreabsorbedovernightat4Cwith0C300μMsyntheticratCART-（55C102）orCART-（62C102）beforeincubationwithbrainsections.CART-（55C102）andCART-（62C102）weresynthesizedinthislaboratorybysolidphasemethodologies（21）.RIATheanalog[Nle67]CART-（62C102）wasradiolabeledwith125IusingchloramineTandpurifiedbyHPLCwitha0.1%trifluoroaceticacid-acetonitrilesolventsystemforuseasatracerintheRIA.TheprocedurefortheCARTRIAwassimilartothatdescribedindetailforinhibinsubunits（20）.Briefly,rabbit6838anti-CART-（62C102）serumwasusedata1:150,000finaldilution,andsyntheticCART-（55C102）andCART-（62C102）wereusedasstandards.Rattissueswereacidextractedandpartiallypurifiedwithoctadecylsilicacartridgesaspreviouslydescribed（20）.Lyophilizedtissueextractswerereconstitutedinassaybuffer,pHwascheckedandadjustedifnecessary,andthreetosevendoselevelsweretested.Cellsecretionmediumwastestedwithoutdilutionatmultipledoses.Freetracerwasseparatedfromtracerboundtoantibodywiththeadditionofsheepantirabbit-globulinsand10%（wt/vol）polyethyleneglycol.TheminimumdetectabledoseandEC50forCART-（55C102）rangedfrom2C3and50C60pg/tube,respectively.Resultswerecalculatedusingalogit/logRIAdataprocessingprogramofFaden,Hutson,Munson,andRodbard（NationalInstituteofChildHealthandHumanDevelopment,ReproductiveResearchBranch,NationalInstitutesofHealth）.GelfiltrationchromatographyPooledmaleandfemaleratplasmawaspartiallypurifiedusingoctadecylsilicacartridgesaspreviouslydescribed（20）.ThepurifiedplasmawasappliedtoafastproteinliquidchromatographysystemequippedwithaSuperdex75HR10/30column（AmershamBiosciencesCorp.,Piscataway,NJ）.Thecolumnwaselutedwith30%acetonitrile/0.1%trifluoroaceticacidataflowrateof0.5ml/min.AliquotsoffractionswerelyophilizedandtestedintheCARTRIA.Inseparateruns,thecolumnwascalibratedusingvariouspeptideandproteinstandards.ImmunoblottingRatpituitaryglandswereacidextractedandpartiallypurifiedwithoctadecylsilicacartridgesaspreviouslydescribed（20）.Lyophilizedproteinswereresuspendedin1xsamplebuffer[50mMTris（pH6.8）,100mMdithiothreitol,2%sodiumdodecylsulfate,0.1%bromophenolblue,and10%glycerol].Sampleswereboiledfor5min,andproteinswereelectrophoresedona10%sodiumdodecylsulfate-polyacrylamidegel（InvitrogenLifeTechnologies,Inc.,Carlsbad,CA）.Electrophoresedproteinsweresubsequentlytransferredontonitrocellulosemembranesandthenprobedwithanti-CART-（62C102）（PBL6838）oranti-CART-（55C102）（PhoenixPharmaceuticals,Belmont,CA）overnightat4C.MembraneswerewashedinPBSwith0.05%Tween20andincubatedwithahorseradishperoxidase-conjugatedantirabbitIgG（AmershamBiosciences,LittleChalfont,UK）.ImmunoreactiveproteinswerevisualizedusingSuperSignalWestPicochemiluminescentsubstrate（PierceChemicalCo.,Rockford,IL）.IHCBrainandwholepituitarysectionswereincubatedinoneoracombinationofthefollowingantisera:rabbitanti-CART-（62C102）（1:10,000;PBL6838）;guineapiganti-PRL,-GH,-FSH,-TSH,and-ACTH（1:12,000;NationalInstitutesofHealth,Bethesda,MD）;guineapiganti-AVP（1:15,000;PeninsulaLaboratories,SanCarlos,CA）ormouseanti-OT（1:7,500;ChemiconInternational,Temecula,CA）inpotassiumPBS（KPBS）with0.4%TritonX-100at4Cfor48h.ThetissueswerethenrinsedinKPBSandincubatedinoneoracombinationofthefollowingsecondaryantibodies:biotinylateddonkeyantirabbitIgG（1:700;JacksonImmunoResearchLaboratories,WestGrove,PA）,fluorescein-conjugateddonkeyantirabbitIgG,rhodamineredX-conjugatedantimouseIgG,orCy3-conjugatedantiguineapigIgG（1:500;JacksonImmunoResearchLaboratories）inKPBSwith0.4%TritonX-100for1hatroomtemperature.Tissuesincubatedwithbiotinylatedsecondaryantibodiesweresubjectedto1-hincubationatroomtemperatureinavidin-biotincomplexsolution（VectastainABCElitekit,VectorLaboratories,Inc.,Burlingame,CA）.Theantibody-peroxidasecomplexwasvisualizedwith3,3-diaminobenzidine（0.2mg/ml）and3%H2O2（0.83μl/ml）in175mMsodiumacetatesolution.Whenstaininghadreachedanappropriateintensity,thetissuewasrinsedinKPBSandmountedongelatin-coatedglassslides.Slidesweredehydratedthroughgradedalcohols,clearedinxylenes,andcoverslippedwithDPXmountant（ElectronicMicroscopeScience,FortWashington,PA）.AdjacentseriesofbrainsectionswereprocessedforNisslstainingforreferencepurposes.RatanteriorpituitarycellculturesAnteriorpituitarycellsfrommaleSpragueDawleyratswerepreparedbydispersionwithcollagenaseaspreviouslydescribed（22）.Cultureswereplatedonpoly-L-lysine-coatedcoverslipsin24-wellcultureplatesforimmunocytochemicalanalysisorin48-wellplatesforproteinexpressionandreleasestudies.Cultureswereplatedatadensityof1.5x105cells/culturewellandwereallowedtorecoverfor72hincompletemedium[PitJulep（PJ）]supplementedwith2%fetalbovineserumandappropriategrowthfactors（22）.Beforeimmunocytochemicalanalysis,pituitarycellsinculturewererinsedwithKPBSandthenfixedfor20minwith4%paraformaldehydeatroomtemperature.CultureswerewashedinKPBSandprocessedforCART,GH,TSH,FSH,ACTH,andPRL-irfollowingtheproceduresdescribedaboveforbrainandpituitarysections.BeforetheexaminationofCARTexpressionandproteinrelease,pituitarycellswerewashedthreetimeswithPJand0.1%BSAandequilibratedfor1h.CellswerethenwashedwithPJand0.1%BSAandtreatedintriplicatewithbromocriptine（0.1C100nM）orforskolin（1μM）for3,6,and12hat37C.MediawerecollectedaftereachincubationtimepointformeasurementofsecretedCARTbyRIA.Cellswereharvested,andmRNAwasextractedusingTRIzolreagent（InvitrogenLifeTechnologies,Inc.）todetermineCARTmRNAlevels.InsituhybridizationAnimalswererapidlydecapitated,andwholebrainswereremovedandfrozenondryice.Coronalsections（20μm）werecutonacryostat,thaw-mountedontoglassslides,andstoredatC80Cuntiluse.AntisensecRNAprobesweretranscribedfromlinearizedcDNAtemplatescorrespondingtobp495C679oftheratCARTgene.[35S]UTP（PerkinElmerLifeSciences,Emeryville,CA）wasincorporatedintocRNAprobesduringtranscription,andthespecificactivityoftheprobeswasdeterminedtobeapproximately2x108dpm/μgcRNA.Thesaturatingconcentrationfortheprobesusedforassayswas0.3μg/ml/kb.Theprocedureusedforinsituhybridizationwasdescribedpreviously（23）.Briefly,brainsectionswerefixedin4%paraformaldehydeandtreatedwith0.25%aceticanhydridein0.1Mtriethanolamine（pH8.0）,followedbyarinsein2%standardsalinesulfate（SSC）,dehydratedthroughagradedseriesofalcohols,delipidatedinchloroform,rehydratedthroughasecondseriesofalcohols,andthenairdried.TheslideswerethenexposedtocRNAprobesovernightinhumidifiedchambersat55C.Afterincubation,theslideswerewashedinSSCofincreasingstringency,inribonuclease,andthenin0.1%SSCat63C;dehydratedthroughagradedseriesofalcohols;anddried.SlidesweredippedinNTB-2emulsion（EastmanKodakCo.,Rochester,NY）,exposedfrom7C14dat4C,anddeveloped.Afterdevelopment,theslideswerecounterstainedwithcresylviolet.TissueanalysisTissuesectionsandpituitarycultureswereinitiallyexaminedusinganE600lightmicroscope（Nikon,Tokyo,Japan）.ThecellularlocalizationofCARTwithpituitaryhormonesandhypothalamicneuropeptideswasdeterminedusingaFluoviewconfocalsystemonanIX70microscope（Olympus,Melville,NY）withthefollowinglaserexcitationlines:488nmforfluoresceinand568nmforrhodamineredXandCy3.Dichroic/emissionfiltersfordetectionwere500nmLP/515C565nmforfluorescein,and575nmLP/590nmLPforrhodamineredXandCy3.Thesefiltercombinationsresultedinverylowlevelsofcross-talkbetweenfluorochromesignals.TheextentofcolocalizationbetweenCARTandpituitaryhormoneswasascertainedfromsixseparatepituitaryculturesstainedforCARTandindividualpituitaryhormones.Thetotalnumbersofcellscontaining1）CART-ir,2）thepituitaryhormoneofinterest,and3）colocalizationofCART-irandaspecifichormoneweredeterminedforeachculture.TheresultsarepresentedasthepercentageofCART-ircellsthatcontainindividualpituitaryhormones.AutoradiogramsforCARTinsituhybridizationwerevisualizedunderdark-fieldilluminationusingaE600lightmicroscope（Nikon,Tokyo,Japan）andanalyzedwithImage-ProPlusimagingsoftware（MediaCybernetics,SanDiego,CA）.Integrateddensityvaluesofhybridizedneuronsofeachsideofthesupraopticnucleus（SON）andPVNweremeasuredinatleastfiveconsecutivesectionsforeachanimal.Nonlinearityofradioactivityintheemulsionwasevaluatedbycomparingdensityvalueswithacalibrationcurvecreatedfromautoradiogramsofknowndilutionsoftheradiolabeledprobesimmobilizedonglassslidesin2%gelatinfixedwith4%paraformaldehydeandexposedanddevelopedsimultaneouslywiththeinsituhybridizationautoradiograms.Allimageswerecapturedusingadigitalcamera（Photometrics,HuntingtonBeach,CA）andImage-ProPlusimagingsoftware（MediaCybernetics,SanDiego,CA）.TheimageswerecroppedandadjustedtobalancebrightnessandcontrastinAdobePhotoshop（version5.5;AdobeSystems,SanJose,CA）beforeimportintoCanvas（version6.0）forassemblyintoplates.RTandreal-timePCRanalysisCARTmRNAlevelsinanteriorpituitarycellswerequantifiedusingreal-timePCRanalysis.Afterdeoxyribonucleasetreatment,aconstantamountofRNA（1μg）wasaddedtoareversetranscriptasemixture（SuperScriptIIRNaseH-ReverseTranscriptase,InvitrogenLifeTechnologies,Inc.）.TotestforpossiblepseudogeneorgenomicDNAcontamination,eithertheRTenzymeorRNAwasomittedfromthereactiontube.LevelsofCARTexpressionweredeterminedwithprimersforCART（sense,5'-GTAAACGCATTCCGATCTATGAGA-3';antisense,5'-CCGATCCTGGCCCCTTT）andprimersforglyceraldehyde-3-phosphatedehydrogenase（GAPDH;sense,5'-GGAAGGGCTCATGACCACAGT-3';antisense,5'-CACAGTCTTCTGAGTGGCAGTGAT-3'）.ReactionmixturescontainedTaqManUniversalMasterMixwithSYBRGreen（Sigma-AldrichCorp.,St.Louis,MO）,80nMprimers,and1ngcDNAinafinalreactionvolumeof50μl.PCRswereperformedin96-wellplatesonanABIPRISM7900HT（AppliedBiosystems,FosterCity,CA）withsequencedetectionsystemsoftware.Foreachbiologicalsample,real-timePCRswereperformedinduplicate,andCARTexpressionwasnormalizedtoGAPDH.StatisticalanalysisStatisticalanalyseswereperformedusingunpairedStudent’sttestsandone-wayANOVAsasindicatedforeachstudy.TukeyanalysistestswereusedafterANOVAtomakecomparisonsbetweengroupsataparticulartimepointandbetweentimepointswithinaparticulargroup.DifferenceswereconsideredstatisticallysignificantatP<0.05.ResultsCART-irinratbrainandperipheraltissuesWegeneratedantiserumagainstasyntheticpeptideencodingratCART-（62C102）,whichallowedustodeterminethecellularlocalizationofCARTinthepituitarygland.TheaffinityandspecificityoftheantiserumwereexaminedbyIHC,immunoblotting,andsize-exclusionchromatography.Immunohistochemicalanalysisrevealedthatanti-CART-（62C102）stainedcellbodiesandfibersinmultipleregionsoftheratbrain,includingtheolfactorybulb,nucleusaccumbens,amygdala,thalamus,hypothalamus,andseveralnucleiinthemesencephalonandbrainstem.Preabsorptionofanti-CART-（62C102）with20μg/mlCART-（55C102）orCART-（62C102）completelyeliminatedCARTstainingintheSONandEdingerWestphalnucleus,demonstratingthespecificityofanti-CART-（62C102）（Fig.1）.ARIAwasdevelopedusingCART-（62C102）antiserumtodeterminethelevelsofCARTproteinintissuesandbiologicalfluids.TheCARTRIAishighlysensitive,withadetectionlimitof2C3pg/tube.AsshowninFig.2A,bothsyntheticCART-（62C102）（EC50,30pg/tube）andCART-（55C102）（EC50,50pg/tube）displacedtracerboundtoantibodyinasimilarmanner.CART-likeirwasmeasuredinacid-extractedrattissuespartiallypurifiedusingoctadecylsilicacartridges.ThehighestconcentrationsofCARTweredetectedintheposteriorpituitaryandhypothalamus,moderatelevelswerefoundinthecerebellumandpancreas,andlowlevelswereobservedintheheartandspleen.TheapproximateconcentrationsofCARTpermilligramofpartiallypurifiedtissueextractwereasfollows:2.8μg/mgposteriorpituitary,950ng/mghypothalamus,35ng/mgcerebellum,7ng/mgpancreas,0.7ng/mgheart,and0.08ng/mgspleen（Fig.2A）.Toexaminethespecificityofanti-CART-（62C102）,weemployedgelexclusionchromatographytodeterminethemolecularmassofCART-irproductsinratplasma.AsshowninFig.2B,anti-CART-（62C102）recognizedasingleimmunoreactivespeciesofapproximately5.8kDa.ThisisinagreementwiththepredictedmolecularmassofthatreportedforbiologicallyactiveCART-（55C102）.CellularlocalizationofCART-irinratanteriorpituitaryglandImmunoblotanalysiswasusedtoverifythatCARTpeptideswerepresentintheratpituitarygland.PartiallypurifiedproteinextractsfromwholeratpituitarieswereprobedwithantiserumwegeneratedagainstCART-（62C102）andcommerciallyavailableantiserumagainstCART-（55C102）usedbyotherresearchers（18）.Bothantiseradetectedbandsofapproximately5and8kDainpituitaryextractsandhadstrongimmunoreactivitywithsyntheticCART-（55C102）（Fig.3A）.IHCwasemployedtoexamineCARTproteindistributioninwholepituitarysectionsfromadultmalerats.ModeratelevelsofCART-irweredetectedthroughouttheanteriorpituitary.ThemajorityofCART-irwasdistributedaroundthelateraledgeoftheanteriorlobe（Fig.3B）.Preabsorptionofanti-CART-（62C102）with20μg/mlCART-（55C102）completelyeliminatedCARTstaininginthepituitary,confirmingthespecificityoftheantiserum（Fig.3D）.ThecellularlocalizationofCARTintheanteriorpituitarywasdeterminedbydouble-labelIHCusingrabbitanti-CART-（62C102）togetherwithantiseraraisedinguineapigsagainstpituitaryhormones（ACTH,FSH,GH,PRL,andTSH）indispersedratanteriorpituitarycellsinculture.ThemajorityofCART-irwaslocalizedinlactotropes,withminorpopulationsofCART-ircellsidentifiedassomatotropesandcorticotropes（Fig.4A）.AssummarizedinFig.4B,wedeterminedthat82%ofCART-immunopositivecellsalsocontainedPRL,10%ofcellscontainedGH,and5%ofCART-ircellsexpressedACTH.Lessthan1%ofCART-ircellscontainedTSHorFSH（Fig.4B）.ThepercentageofspecificanteriorpituitarycelltypesthatcontainedCART-irwasalsodetermined;thisrevealedthat25%oflactotropes,5%ofsomatotropes,and3%ofcorticotropescontainedCART-ir（Fig.4C）.ToensurethattheobservedcellularlocalizationofCART-irinculturedanteriorpituitarycellswasrepresentativeoftheintactpituitary,wedeterminedthecellularlocalizationofCART-irinwholepituitarysectionsanddispersedanteriorpituitarycellsfromfemalerats.Inallpreparations,similardistributionandlocalizationpatternswereobserved.DistributionofCART-irinposteriorpituitaryandhypothalamusStrongimmunostainingforCARTwasdetectedintightlypackedvaricosefibersthroughouttheposteriorlobeofthepituitary,andCART-irwasnotdetectedintheintermediatelobeofthepituitary（Fig.3C）.Double-labelIHCanalysisofwholepituitarysectionsrevealedthatalargepopulationofOTfiberscolocalizedCART-irintheposteriorpituitary（Fig.5A）.WenextexaminedthedistributionofCART-irinmagnocellularneuronsoftheSONandPVNthatcontributeaxonstotheposteriorpituitary.AlargenumberofCART-irneuronscolocalizedwithOTintheSON.Curiously,veryfewCART-ircellscolocalizedwithOTinthePVN（Fig.5A）.IncontrasttoOT,asmallnumberofAVPfiberscontainedCART-irintheposteriorpituitary（Fig.5B）.OnlyasmallpercentageofmagnocellularneuronsintheSONstainedforbothAVPandCART.CART-irwasnotdetectedinAVPneuronsinthemagnocellulardivisionofthePVN（Fig.5B）.Similardistributionandlocalizationpatternswereobservedinboththeposteriorpituitaryandhypothalamusofmaleandfemalerats.EffectsofbromocriptineonCARTmRNAexpressionandproteinreleasefromanteriorpituitaryTheeffectsofbromocriptine,apotentdopamine（DA）receptoragonist,onCARTmRNAexpressionandproteinreleasewereexaminedtodeterminewhetherCARTandPRLareregulatedbysimilarmechanismsintheanteriorpituitary.Anteriorpituitaryculturesweretreatedwithvaryingconcentrationsofbromocriptine（0.1C100nM）orforskolin（1μM）for3,6,and12hat37C.CARTmRNAexpressionlevelsweredeterminedbyquantitativereal-timePCRandnormalizedtothehousekeepinggeneGAPDH.Asignificantdose-dependentreductioninCARTmRNAexpressionwasobservedafter12-hincubationwithbromocriptine（Fig.6A）.NosignificantchangesinCARTmRNAexpressionwereobservedinculturesincubatedwithbromocriptinefor3and6h（datanotshown）.Asexpected,forskolin,anactivatorofadenylcyclase,significantlyincreasedCARTmRNAlevelsinanteriorpituitarycells（Fig.6A）.BromocriptinehadsignificanteffectsonCARTproteinreleasefromanteriorpituitarycultures.TheaccumulationofreleasedCARTwasreadilydetectableinculturemediumbyRIAanalysis.ThereleaseofCARTintotheculturemediumwassignificantlydecreasedafter3hofincubationwithbromocriptine.Specifically,theconcentrationofCARTwas200±18pg/mlforcontrols,77±10pg/mlfor0.1nMbromocriptine,67±6pg/mlfor10nMbromocriptine,and61±6pg/mlfor100nMbromocriptine（Fig.6B）.BromocriptinehadsimilareffectsonCARTreleaseafter6-and12-hincubationsofanteriorpituitarycellsinculture（datanotshown）.SimilartomRNAexpression,CARTproteinreleasewassignificantlyincreasedafterincubationwith1μMforskolin（Fig.6B）.RegulationofCARTduringlactationOurfindingsrevealedthatCARTiscolocalizedwithPRLandOT,importantregulatorsoflactation（24）,andsuggestedthatCARTmaypotentiallyfunctiontoregulatelactation.Toinvestigatethishypothesis,weexaminedtheeffectsoflactationonCARTmRNAexpressioninthehypothalamusandanteriorpituitary.ThelevelsofCARTmRNAexpressionintheSONandPVNoflactating（d1）andnonlactatingfemalerats（diestrouscontrols）weredeterminedbyinsituhybridization.CARTmRNAwaslocalizedinmagnocellularneuronsthroughouttheSONandinboththeparvocellularandmagnocellulardivisionsofthePVN（Fig.7A）.Densityvaluesweredeterminedbyimageanalysis,andlevelsofCARTmRNAwereincreased2-foldintheSONoflactatingrats（Fig.7B）.Interestingly,nosignificantchangesinCARTmRNAexpressionwereobservedineitherdivisionofthePVNinlactatingratscomparedwithnonlactatingcontrols（Fig.7B）.TheeffectsoflactationonCARTmRNAexpressionintheanteriorpituitaryweredeterminedbyquantitativereal-timePCR.LevelsofCARTmRNAwereincreasedbyapproximately5-foldintheanteriorpituitaryoflactatingratscomparedwithnonlactatingcontrols（Fig.7C）.SerumCARTwasmeasuredbyRIAduringpregnancyandlactationinratstodeterminewhethercirculatingCARTlevelscorrelatedwithchangesinmRNAexpressionintheSONandanteriorpituitary.Bloodwasseriallycollectedbytailbleedbetween0800C0900htoavoidconfoundswiththediurnalpatternofCARTinthecirculationofSpragueDawleyrats（25）.SerumCARTremainedconstantthroughoutpregnancy,withameanvalueof116±4pg/ml（Fig.7D）.ImmunoreactiveCARTlevelsweresignificantlyelevatedbyd2oflactation（162±14pg/ml）,andpeaklevelsweredetectedond10oflactation（255±14pg/ml）.LevelsofcirculatingCARTreturnedtobaselinevalues（150±11pg/ml）10dafterpupswereweaned（Fig.7D）.DiscussionTheanatomicaldistributionandspecificcelltypesthatexpressCARThavebeenextensivelystudiedintheratCNS（10,11,13,14,15,16）.FindingsfromanatomicalstudieshaveaidedinelucidationofthephysiologicalrolesofCARTintheCNS（3,6,7,26,27,28）.Incontrast,theanatomicallocalizationofCARTinthepituitaryglandremainsilldefined.ThecellularlocalizationofCARTinthepituitaryhasvariedinpublishedreports,andphysiologicalrolesforpituitaryCARThaveyettobedetermined（11,18,19）.Inthepresentstudy,antiserumwegeneratedagainstCART-（62C102）wasusedtoexaminethecellularlocalizationofCART.TheaffinityandspecificityofthisantiserumweredeterminedusingIHC,immunoblotting,andsize-exclusionchromatography.Analysisoftheratbrainrevealedthatanti-CART-（62C102）stainedmultipleregionspreviouslyreportedtocontainCARTmRNAandprotein（10,14,29,30,31）.Anti-CART-（62C102）andacommerciallyavailableantiserumagainstCART-（55C102）recognizedsimilarproteinsofapproximately5and8kDainpituitaryextracts.ThesefindingsareinagreementwithpreviousstudiesthatreportedthatCART-irpeptidesofasimilarsizearefoundinpituitarylysates（8,18）.Furthermore,anti-CART-（62C102）hadstrongimmunoreactivitywithasinglespeciesinratserumwithamolecularweightthatcorrespondstothebiologicallyactiveformofCART（1）.Thesefindingsdemonstratethattheanti-CART-（62C102）weproducedhashighaffinityandselectivityforendogenousratCART.AnRIAwasdevelopedtodeterminethelevelsofCARTproteinintissuesandbiologicalfluids.TheCARTRIAishighlysensitive,andbothsyntheticCART-（62C102）andCART-（55C102）displacedtracerboundtoantibodyinasimilarmanner.TherelativeconcentrationanddistributionofCARTproteinobservedintheCNSandperipheraltissuescorrelatedwellwithfindingsfrompreviousreports（1,8,19,32）.Intheanteriorpituitary,thevastmajorityCART-ircellscontainedPRL,andasmallpopulationexpressedGHandACTH.ThesefindingssupportpreviousstudiesthatdemonstratedthathighlevelsofCARTmRNAarefoundinGH3cells,aPRL-andGH-secretingcelllinederivedfromaratanteriorpituitarytumor（33,34,35）.OurresultsexpanduponfindingsbyStanleyetal.（19）,whoreportedthat28%ofCART-expressingcellscontainedACTH-ir,butdidnotidentifyotheranteriorpituitarycellsthatexpressedCARTmRNA.ItshouldbenotedthatweobservedasmallerpercentageofCART-ircellsexpressingACTH.DifferencesinthepercentageofanteriorpituitarycellsthatcolocalizedCARTandACTHcanbeattributedtothesensitivityofthedetectionmethodsusedinthesestudies.IHCwasusedtodetectCARTinourstudies,whereasStanleyetal.（19）usedinsituhybridizationtoexamineCARTdistributionintheanteriorpituitarygland.Incontrast,Kuriyamaetal.（18）reportedadivergentpatternofcellularlocalizationforCARTintheanteriorpituitary.TheyfoundthatCARTcolocalizedwithFSH-andLH-containingcells,butnotGH-,TSH-,ACTH-,orPRL-containingcells.ThediscrepancyinthepatternofcolocalizationobservedinourstudyandthereportfromKuriyamaetal.（18）maystemfromdifferentaffinitiesandepitoperecognitionsitesoftheparticularantibodiesusedineachstudy.Koyluetal.（11）alsoreportedmultipleantiserum-dependentstainingpatternsforCARTinthepituitarygland.AxonsofmagnocellularneuronslocatedintheSONandPVNformthesupraopticohypophysealtract,whichterminatesintheposteriorlobeofthepituitarygland（36）.Immunohistochemicalanalysisrevealedthatanti-CART-（62C102）stainedalargenumberofsupraopticohypophysealaxonfibersintheposteriorpituitarythatcontainedOT.IntheSON,CART-irneuronscolocalizedwithOT.InagreementwithLarsenetal.（37）,weobservedlittleCART-irinOTneuronsinthePVN,andCARTwasnotdetectedinAVPneurons.SimilarpatternsofcolocalizationforCARTinmagnocellularneuronshavepreviouslybeenreported（14,37）;however,wearethefirsttocomparethelocalizationofCARTwithOTandAVPinfibersoftheposteriorpituitary.AnatomicalstudiesrevealedthatthemajorityofanteriorpituitaryCARTwaslocalizedtolactotropes.PRLissecretedfrompituitarylactotropesinanepisodicmanner（38）underthetonicinhibitionofDAreleasedfromtuberoinfundibularneuronsinthehypothalamus（39,40）.DAbindsD2-typereceptorsonlactotropes,resultinginadecreaseinadenylatecyclaseactivityandasubsequentdecreaseinPRLmRNAtranscriptionandproteinrelease（41）.WeinvestigatedtheeffectsofDAreceptoractivationonCARTmRNAtranscriptionandproteinreleasetodeterminethephysiologicalsignificanceoftheobservedcellularlocalizationintheanteriorpituitary.AsignificantinhibitionofCARTmRNAexpressionandproteinreleasewasdetectedinanteriorpituitarycellsaftertreatmentwithbromocriptine.ThesefindingsdemonstratethatCARTandPRLmayberegulatedbysimilarmechanismsintheanteriorpituitary.Prolactinregulatesawidearrayofphysiologicalprocessesassociatedwithlactation（42）.Oxytocinisapowerfulgalactokinetichormonethatisessentialfortheprocessoflactation（43）.OurfindingsrevealthatCARTiscolocalizedwiththesepotentlactationregulatoryhormonesinthepituitaryglandandhypothalamus.Inaddition,invitrostudiesdemonstratethatCARTmRNAexpressionandproteinreleaseareregulatedsimilarlytoPRL.Toexpanduponthesefindings,weexaminedtheeffectsoflactationonCARTmRNAexpressionintheanteriorpituitary,SON,andPVN.LevelsofCARTmRNAweresignificantlyincreasedintheanteriorpituitaryandSONoflactatingratscomparedwithnonlactatingcontrols.ConsistentwiththelackofcolocalizationbetweenCARTandOT,nosignificantchangesinCARTmRNAexpressionwereobservedinthePVNoflactatingrats.Together,theseresultssuggestthatCARTmayplayaroleintheregulationoflactation.ProlactinsecretionislargelydependentuponlevelsofDA;however,PRLtranscriptionandproteinreleasearealsoregulatedbyfeedbacksignalsfromtargettissues,hypothalamicpeptides,andlocalfactorsinthepituitarygland（44）.TRH,OT,andvasoactiveintestinalpeptidehavebeenshowntostimulatePRLreleasefromanteriorpituitarycellsinvitro（45,46,47）.EndothelinadditionallyexertsbiphasiceffectsonPRL,bothinhibitingandstimulatingsecretioninvitro（48）.CARThasbeenshowntomodulatePRLreleasefromtheanteriorpituitary.Kuriyamaetal.（18）reportedthatCARTsuppressesPRLreleasefromanteriorpituitarycultures,andRaptisetal.（49）recentlyreportedthatCARTinhibitsTRH-inducedPRLsecretion.ThesestudiestogetherwithfindingsdetailedinthisreportsuggestthatpituitaryCARTmayfunctionasanautocrine/paracrinefactorthatregulatesthereleaseofPRLwhenlevelsofDAaresuppressed,suchasduringlactation.Theendocrinecontroloflactationisacomplexphysiologicalprocessrequiringtheproperregulationofmammogenesis,lactogenesis,galactopoiesis,andgalactokinesis（24）.Awidearrayofhormones,growthfactors,andproteinsareinvolvedintheregulationofmilkproductionandrelease.Multiplefactorsactascirculatinghormonesandparacrineautocrinefactorstostimulateandinhibitvariousphasesofmammaryglanddevelopmentandlactation（24,50）.Inadditiontothehypothalamusandpituitary,CARTispresentinthesystemiccirculationatphysiologicallevels（19,25）.WemeasuredCARTinserumfrompregnantandlactatingratstodeterminewhetherlevelsofcirculatingCARTcorrelatedwithchangesinmRNAexpressionintheanteriorpituitaryandhypothalamus.SerumlevelsofCARTremainedstablethroughoutpregnancy;however,significantincreasesincirculatingCARTweredetectedduringlactation.ThefindingspresentedinthisstudyimplicateCARTasanovelcandidatefactorinvolvedintheregulationoflactation.CARTmaypotentiallyactasanautocrine/paracrinefactorregulatingPRLreleasefromtheanteriorpituitarygland,oritcouldfunctionasacirculatinghormoneinvolvedintheprocessoflactation.Additionalstudies,includingtheidentificationofspecifictargettissuesandareceptorforCART,arerequiredtomoreclearlyelucidatethephysiologicalrolesofthisprotein.AcknowledgmentsWethankDr.JozsefGulyasforthesynthesisandpurificationofthepeptidesusedinthesestudies,Dr.LouiseBilezikjianforcriticalreadingofthemanuscript,andSandraGuerraforassistancewithpreparationofthismanuscript.FootnotesThisworkwassupportedbyNationalInstituteofDrugAbuseGrantDA-017550-02,NationalInstituteofDiabetesandDigestiveandKidneyDiseasesGrantP01-DK-26741,theRobertJ.Kleberg,Jr.,andHelenC.KlebergFoundation,andtheFoundationforResearch.W.W.V.isaseniorinvestigatorwiththeFoundationforResearch.S.M.S,J.M.V.,C.J.D.,R.E.F.,C.L.,andA.C.havenothingtodeclare.W.W.V.isacofounderandaboardmemberofNeurocrineBiosciences,Inc.,andAcceleronPharmaceuticals,Inc.FirstPublishedOnlineDecember8,2005Abbreviations:AVP,Vasopressin;CART,cocaine-andamphetamine-regulatedtranscript;CNS,centralnervoussystem;DA,dopamine;GAPDH,glyceraldehyde-3-phosphatedehydrogenase;IHC,immunohistochemistry;-ir,immunoreactivity,immunoreactive;KPBS,potassiumPBS;OT,oxytocin;PJ,PitJulep;PVN,paraventricularnucleus;SSC,standardsalinesulfate;SON,supraopticnucleus.AcceptedforpublicationNovember23,2005.ReferencesDouglassJ,McKinzieAA,CouceyroP1995PCRdifferentialdisplayidentifiesaratbrainmRNAthatistranscriptionallyregulatedbycocaineandamphetamine.JNeurosci15:2471C2481AdamsLD,GongW,VechiaSD,HunterRG,KuharMJ1999CART:fromgenetofunction.BrainRes848:137C140KristensenP,JudgeME,ThimL,RibelU,ChristjansenKN,WulffBS,ClausenJT,JensenPB,MadsenOD,VrangN,LarsenPJ,HastrupS1998HypothalamicCARTisanewanorecticpeptideregulatedbyleptin.Nature393:72C76LambertPD,CouceyroPR,McGirrKM,DallVechiaSE,SmithY,KuharMJ1998CARTpeptidesinthecentralcontroloffeedingandinteractionswithneuropeptideY.Synapse29:293C298KuharMJ,DallVechiaSE1999CARTpeptides:noveladdiction-andfeeding-relatedneuropeptides.TrendsNeurosci22:316C320KuharMJ,AdamsS,DominguezG,JaworskiJ,BalkanB2002CARTpeptides.Neuropeptides36:1C8SmithSM,VaughanJM,DonaldsonCJ,RivierJ,LiC,ChenA,ValeWW2004Cocaine-andamphetamine-regulatedtranscriptactivatesthehypothalamic-pituitary-adrenalaxisthroughacorticotropin-releasingfactorreceptor-dependentmechanism.Endocrinology145:5202C5209ThimL,KristensenP,NielsenPF,WulffBS,ClausenJT1999Tissue-specificprocessingofcocaine-andamphetamine-regulatedtranscriptpeptidesintherat.ProcNatlAcadSciUSA96:2722C2727WierupN,KuharM,NilssonBO,MulderH,EkbladE,SundlerF2004Cocaine-andamphetamine-regulatedtranscript（CART）isexpressedinseveralisletcelltypesduringratdevelopment.JHistochemCytochem52:169C177EliasCF,LeeCE,KellyJF,AhimaRS,KuharM,SaperCB,ElmquistJK2001CharacterizationofCARTneuronsintheratandhumanhypothalamus.JCompNeurol432:1C19KoyluEO,CouceyroPR,LambertPD,LingNC,DeSouzaEB,KuharMJ1997ImmunohistochemicallocalizationofnovelCARTpeptidesinrathypothalamus,pituitaryandadrenalgland.JNeuroendocrinol9:823C833EkbladE,KuharM,WierupN,SundlerF2003Cocaine-andamphetamine-regulatedtranscript:distributionandfunctioninratgastrointestinaltract.NeurogastroenterolMotil15:545C557WittmannGLZ,LechanRM,FeketeC2005Originofcocaine-andamphetamine-regulatedtranscript（CART）-containingaxonsinnervatinghypophysiotropiccorticotropin-releasinghormone（CRH）-synthesizingneuronsintherat.Endocrinology146:2985C2991VrangN,LarsenPJ,ClausenJT,KristensenP1999Neurochemicalcharacterizationofhypothalamiccocaine-amphetamine-regulatedtranscriptneurons.JNeurosci19:RC5FeketeC,MihalyE,LuoLG,KellyJ,ClausenJT,MaoQ,RandWM,MossLG,KuharM,EmersonCH,JacksonIM,LechanRM2000Associationofcocaine-andamphetamine-regulatedtranscript-immunoreactiveelementswiththyrotropin-releasinghormone-synthesizingneuronsinthehypothalamicparaventricularnucleusanditsroleintheregulationofthehypothalamic-pituitary-thyroidaxisduringfasting.JNeurosci20:9224C9234KoziczT2003Neuronscolocalizingurocortinandcocaineandamphetamine-regulatedtranscriptimmunoreactivitiesareinducedbyacutelipopolysaccharidestressintheEdinger-Westphalnucleusintherat.Neuroscience116:315C320MurphyKG,AbbottCR,MahmoudiM,HunterR,GardinerJV,RossiM,StanleySA,GhateiMA,KuharMJ,BloomSR2000Quantificationandsynthesisofcocaine-andamphetamine-regulatedtranscriptpeptide（79C102）-likeimmunoreactivityandmRNAinrattissues.JEndocrinol166:659C668KuriyamaG,TakekoshiS,TojoK,NakaiY,KuharMJ,OsamuraRY2004CARTPeptideintheratanteriorpituitaryglandislocalizedingonadotrophsandsuppressesprolactinsecretion.Endocrinology145:2542C2550StanleySA,MurphyKG,BewickGA,KongWM,Opacka-JuffryJ,GardinerJV,GhateiM,SmallCJ,BloomSR2004Regulationofratpituitarycocaine-andamphetamine-regulatedtranscript（CART）byCRHandglucocorticoids.AmJPhysiol287:E583CE590VaughanJM,RivierJ,CorriganAZ,McClintockR,CampenCA,JolleyD,VoglmayrJK,BardinCW,RivierC,ValeW1989Detectionandpurificationofinhibinusingantiserageneratedagainstsyntheticpeptidefragments.MethodsEnzymol168:588C617GulyasJ,RivierC,PerrinM,KoerberSC,SuttonS,CorriganA,LahrichiSL,CraigAG,ValeW,RivierJ1995Potent,structurallyconstrainedagonistsandcompetitiveantagonistsofcorticotropin-releasingfactor.ProcNatlAcadSciUSA92:10575C105759ValeW,VaughanJ,YamamotoG,BruhnT,DouglasC,DaltonD,RivierC,RivierJ1983Assayofcorticotropinreleasingfactor.MethodsEnzymol103:565C577KageyamaK,LiC,ValeWW2003Corticotropin-releasingfactorreceptortype2messengerribonucleicacidinratpituitary:localizationandregulationbyimmunechallenge,restraintstress,andglucocorticoids.Endocrinology144:1524C1532BuhimschiCS2004Endocrinologyoflactation.ObstetGynecolClinNorthAm31:963C979VicenticA,DominguezG,HunterRG,PhilpotK,WilsonM,KuharMJ2004Cocaine-andamphetamine-regulatedtranscriptpeptidelevelsinbloodexhibitadiurnalrhythm:regulationbyglucocorticoids.Endocrinology145:4119C4124VrangN,LarsenPJ,KristensenP,Tang-ChristensenM2000Centraladministrationofcocaine-amphetamine-regulatedtranscriptactivateshypothalamicneuroendocrineneuronsintherat.Endocrinology141:794C801StanleySA,SmallCJ,MurphyKG,RayesE,AbbottCR,SealLJ,MorganDG,SunterD,DakinCL,KimMS,HunterR,KuharM,GhateiMA,BloomSR2001Actionsofcocaine-andamphetamine-regulatedtranscript（CART）peptideonregulationofappetiteandhypothalamo-pituitaryaxesinvitroandinvivoinmalerats.BrainRes893:186C194VrangN,LarsenPJ,Tang-ChristensenM,LarsenLK,KristensenP2003Hypothalamiccocaine-amphetamineregulatedtranscript（CART）isregulatedbyglucocorticoids.BrainRes965:45C50Dallvechia-AdamsS,SmithY,KuharMJ2001CARTpeptide-immunoreactiveprojectionfromthenucleusaccumbenstargetssubstantianigraparsreticulataneuronsintherat.JCompNeurol434:29C39KoyluEO,CouceyroPR,LambertPD,KuharMJ1998Cocaine-andamphetamine-regulatedtranscriptpeptideimmunohistochemicallocalizationintheratbrain.JCompNeurol391:115C132FeketeC,WittmannG,LipositsZ,LechanRM2004Originofcocaine-andamphetamine-regulatedtranscript（CART）-immunoreactiveinnervationofthehypothalamicparaventricularnucleus.JCompNeurol469:340C350KuharMJ,YohoLL1999CARTpeptideanalysisbyWesternblotting.Synapse33:163C171BarrettP,MorrisMA,MoarKM,MercerJG,DavidsonJA,FindlayPA,AdamCL,MorganPJ2001ThedifferentialregulationofCARTgeneexpressioninapituitarycelllineandprimarycellculturesofovineparstuberaliscells.JNeuroendocrinol13:347C352DominguezG,LakatosA,KuharMJ2002Characterizationofthecocaine-andamphetamine-regulatedtranscript（CART）peptidegenepromoteranditsactivationbyacyclicAMP-dependentsignalingpathwayinGH3cells.JNeurochem80:885C893TashjianJrAH,YasumuraY,LevineL,SatoGH,ParkerML1968Establishmentofclonalstrainsofratpituitarytumorcellsthatsecretegrowthhormone.Endocrinology82:342C352VandesandeF,DierickxK1975Identificationofthevasopressinproducingandoftheoxytocinproducingneuronsinthehypothalamicmagnocellularneurosecretorysystemoftherat.CellTissueRes164:153C162LarsenPJ,SeierV,Fink-JensenA,HolstJJ,WarbergJ,VrangN2003Cocaine-andamphetamine-regulatedtranscriptispresentinhypothalamicneuroendocrineneuronesandisreleasedtothehypothalamic-pituitaryportalcircuit.JNeuroendocrinol15:219C226MeitesJ1977Neuroendocrinecontrolofprolactininexperimentalanimals.ClinEndocrinol（Oxf）6（Suppl）:9SC18SMacLeodRM,LehmeyerJE1974Studiesonthemechanismofthedopamine-mediatedinhibitionofprolactinsecretion.Endocrinology94:1077C1085RubergM,EnjalbertA,ArancibiaS,KordonC1981Regulationofprolactinsecretionatthepituitarylevel.ExpBrainRes（Suppl3）:182C199AlbertPR,NeveKA,BunzowJR,CivelliO1990Couplingofaclonedratdopamine-D2receptortoinhibitionofadenylylcyclaseandprolactinsecretion.JBiolChem265:2098C2104JaffeRB,YuenBH,KeyeJrWR,MidgleyJrAR1973Physiologicandpathologicprofilesofcirculatinghumanprolactin.AmJObstetGynecol117:757C773NishimoriK,YoungLJ,GuoQ,WangZ,InselTR,MatzukMM1996Oxytocinisrequiredfornursingbutisnotessentialforparturitionorreproductivebehavior.ProcNatlAcadSciUSA93:11699C11704SamsonWK,TaylorMM,BakerJR2003Prolactin-releasingpeptides.RegulPept114:1C5SamsonWK,SaidSI,SnyderG,McCannSM1980Invitrostimulationofprolactinreleasebyvasoactiveintestinalpeptide.Peptides1:325C332SamsonWK,LumpkinMD,McCannSM1986Evidenceforaphysiologicalroleforoxytocininthecontrolofprolactinsecretion.Endocrinology119:554C560LambertsSW,MacleodRM1990Regulationofprolactinsecretionatthelevelofthelactotroph.PhysiolRev70:279C318KanyicskaB,SellixMT,FreemanME2001Autocrineregulationofprolactinsecretionbyendothelins:apermissiveroleforestradiol.Endocrine16:133C137RaptisS,FeketeC,SarkarS,RandWM,EmersonCH,NagyGM,LechanRM2004Cocaine-andamphetamine-regulatedtranscriptco-containedinthyrotropin-releasinghormone（TRH）neuronsofthehypothalamicparaventricularnucleusmodulatesTRH-inducedprolactinsecretion.Endocrinology145:1695C1699LamoteI,MeyerE,Massart-LeenAM,BurvenichC2004Sexsteroidsandgrowthfactorsintheregulationofmammaryglandproliferation,differentiation,andinvolution.Steroids69:145C159...查看详细（47642字节）
11416503:12号吸痰管在
小儿先天性巨结肠灌肠中的应用
小儿先天性巨结肠发病率为1：5000，以男性多见，男女比例为4：1，临床表现：以便秘、腹胀常见，多需灌肠以帮助其排便，针对小儿灌肠，我科采用12号吸痰管代肛管灌肠，效果好，刺激小等优点。1用物准备12号吸痰管一根，在头部5cm处剪2~3个侧孔，50ml注射器、石蜡油、温生理盐水，温度计、卫生纸。2方法患儿取左侧卧位，50ml注射器抽取15ml石蜡油...查看详细（723字节）
11416504:EstradiolModulationofKainicAcid-InducedCalciumElevationinNeonatalHippocampalNeurons
DepartmentsofPhysiology（G.D.H.,L.L.B.,S.M.T.,M.M.M.）,Anesthesiology（L.L.B.）Psychiatry（S.M.T.,M.M.M.）andPrograminNeuroscience（S.M.T.,M.M.M.）,UniversityofMaryland,SchoolofMedicine,Baltimore,Maryland21201AbstractThedevelopinghippocampusofbothmalesandfemalesisexposedtohighlevelsofthegonadalsteroidestradiol.Theimpactofthisestradiolexposureondevelopinghippocampalneuronsisessentiallyunknown.Intherat,thenewbornhippocampusisrelativelyinsensitivetoexcitotoxicbraininjury,whichinadultsisassociatedwiththereleaseofaminoacids,inparticularglutamate,resultinginasignificantincreaseinintracellularcalciumandeventualcelldeath.Wehaveshownpreviouslyintheratthatadministrationoftheglutamateagonist,kainicacid（KA）,onthedayofbirthresultsinlimitedhippocampaldamage,whichisamelioratedbytreatmentwiththegonadalsteroid,estradiol.WenowshowthatKAinducesanincreaseinintracellularcalciumthroughL-typevoltage-sensitivecalciumchannelsearlyindevelopmentand,laterindevelopment,throughpolyamine-sensitive-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacidreceptorswithamodestincreasethroughN-methyl-D-aspartatereceptors.Pretreatmentwiththegonadalsteroid,estradiol,decreasesthepercentageofneuronsrespondingtoKAanddecreasesthepeakamplitudeofthecalciumtransientearlyindevelopmentbuthasnoeffectlaterindevelopment.Takentogether,thesedatasuggestthatthereisadevelopmentalshiftintherouteofKA-inducedintracellularcalciumandestradiolmodulatesKA-inducedintracellularcalciumtoatimerestrictedtoearlydevelopment,butwhetherthisisthebasisoftheneuroprotectiveeffectofestradiolremainstobedetermined.IntroductionDURINGTHEPERINATALperiod,thedevelopingbrainisexposedtohighlevelsofgonadalsteroids.Inthediencephalon,thisexposureisdistinctinmalesvs.females,inparticularneuronalaromatizedestradiol,whichisderivedfromandrogenprecursorsoriginatinginthemalestestis（1）.Functionally,estradiolmediatessexualdifferentiationintherodentbrain（2）,resultinginchangesinbehavior,volumetricdifferences,dimorphicsynapticpatterning（2,3）,andalterationsinneuronaldifferentiation（4）.Estradiolexertsbothtrophicandmaturationaleffectsthroughmodulationofexcitatoryaminoacids（5）andapoptosis（6,7）aswellasregulationoftrophicfactorssuchasbrain-derivedneurotrophicfactor（8）.EstradiolalsoactsdirectlyonN-methyl-D-aspartate（NMDA）receptorsandL-typevoltage-sensitivecalciumchannels（VSCCs）toregulatecalciuminflux（9,10）.Inthetelencephalon,includingthehippocampus,bothmalesandfemaleneuronsareexposedtohighlevelsofestradiol（11）,andthereisincreasingevidenceofdenovosteroidogenesisbyhippocampusneurons（12）.Thefunctionalimpactofelevatedestradiolonthedevelopinghippocampusislargelyunknown.Intheadult,thereisconsiderableinterestintheneuroprotectiveeffectsofestradiolafterischemicinjury（13,14,15,16,17）,andthehippocampusisparticularlysusceptibletothedeleteriouseffectsofhypoxiaasaconsequenceofexcitotoxicglutamate（18,19）.Thedevelopinghippocampusisalsosusceptibletoischemicinjury（20,21,22）,butthecellularmechanismsofdamagehavenotbeenwellcharacterized,inpart,becauseoftherelativelylowexpressionandfunctionalityofglutamatereceptorsearlyindevelopment（23,24,25）.Kainicacid（KA）isan-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid（AMPA）/kainatereceptoragonistthathasbeenwidelyusedtoinduceexcitotoxicbraininjuryinadults（26,27,28,29）byinducingincreasedintracellularcalciumandinternucleosomalDNAfragmentationandcellloss（30,31）.WerecentlyreportedthatKAadministrationcausessignificantdamagetothedentategyrusandmoderatedamagetotheCA2/3regionofthehippocampusinfemaleratpupsonthedayofbirth,whereasmalepupsareresistanttoKA-induceddamage.Damageinthefemaleisamelioratedbypretreatmentwiththegonadalsteroid,estradiol（32,33）.Thus,exploringtheimpactofestradioloncalciuminfluxinducedbyKAcouldprovideinsightintonormalbraindevelopmentaswellasapotentialmechanisticbasisofneuroprotectiveeffectsofestradiolinthedevelopingbrain.Usingthecalcium-sensitivedye,fura2-AM,andculturedhippocampalneurons,wereportherethatKAinducedanincreaseinintracellularcalciumthroughAMPAreceptor-mediateddepolarizationandsubsequentopeningofVSCCsaroundthedayofbirth.Approximately4dlater,incontrast,KAledtocalciuminfluxviapolyamine-sensitiveAMPAreceptorsandrecruitmentofNMDAreceptors.PretreatmentwithphysiologiclevelsofestradioldecreasedthepercentageofcellsrespondingtoKAanddecreasedthepeakamplitudeoftheKA-inducedcalciumtransientduringearlydevelopmentbuthadnoeffectontheKA-inducedintracellularcalciumresponseinmorematurehippocampalneurons.MaterialsandMethodsAnimalsSubjectswerefirstgenerationdescendantsofSpragueDawleyalbinoratsfromCharlesRiverLaboratory（Wilmington,MA）.FemaleratswerebredattheUniversityofMarylandSchoolofMedicine（Baltimore,MD）animalfacility.Animalswerehousedundera12-hlight,12-hdarkcyclewithunlimitedaccesstofoodandwater.AllanimalprocedureswereapprovedbytheUniversityofMarylandInstitutionalAnimalCareandUseCommittee.Numbersofanimalsusedwaskepttoaminimum,andprocedureswereperformedinamannerthatminimizedsuffering.HippocampalcellcultureAtimedpregnantSpragueDawleyfemalewaskilledbyCO2inhalationatgestationald18.Fetalhippocampiweredissociatedusingtrypsindigestionandtrituratedtogiveasinglecellsuspension.Thesuspensionwasincubatedinawaterbathfor15minat37Ctoallowthecellstosettle,andthesupernatantwasremoved.FivemillilitersofHanks’balancedsaltsolution+wasadded,andthecellswereallowedtosettlefor5min.Thisprocesswasrepeatedtwomoretimes.Cellsweredissociatedbypipettinganddeoxyribonucleasetreatment.Cellcountandviabilityweredeterminedusingahemacytometerandtrypanbluestainingbeforecellswereplatedatadensityof300,000cellspercoverslipon25-mmpoly-L-lysin-coatedcoverslips.Thesewerethenplacedin60-mmdishescontaining4mlplatingmedium（86mlMEM,10mlhorseserum,3mlfiltersterilized20%glucose,and1mlpyruvicacid,100mM）.Cellswereplacedina5%CO2incubatorfor4hat37C.Coverslipsweretransferredfromtheplatingmediatodishescontaining3mlNeurobasalmedia（1mlB-27supplement,1mlantibiotic/antimycotic100xliquid,and125μlL-glutaminetoavolumeof50mlwithNeurobasal）.Hippocampalneuronalculturesweretreatedwith1μMestradioldissolvedindimethylsulfoxide（DMSO）andaddedto3mlculturemediumtoachieveafinalconcentrationof1nM.Vehicle-treatedcontrolsreceivedthesamevolumeofDMSO.Thistreatmentresultsinaphysiologicconcentrationofapproximately200pg/mlestradiol（34）.Hippocampalcellculturesintendedforfluorescentimagingwereagaintreatedwithestradiolorvehicleondaysinvitro（DIV）2andDIV6.OnDIV3andDIV7,1mlmediumwasremovedfromeachculturedishandreplacedwith1mlfreshNeurobasalmediumcontainingeitherestradiolorvehicle.FluorescentimagingThedayculturesweregeneratedwasdesignatedasDIV0.Toevaluateintracellularcalciumresponsesdevelopmentally,daysinvitrowerecorrelatedtoestimatedpostnataldaysoflife（Fig.1）.Forexample,DIV4forneuronsculturedfromembryonicday18fetusesisconsideredroughlyequivalenttopostnatald0.OnDIV4,DIV6,DIV8,andDIV10,calciumimagingusingthecell-permeantfluorescentindicatorfura2-AMwasperformedonhippocampalcellcultures.Onthedayofimaging,hippocampalneuronswereincubatedwith3μlfura2-AMinDMSOwith20μlpluronicacidfor30min.Coverslipswerethentransferredtoatissuechamberonamicroscopestageandweresuperfusedfor30minwithaphysiologicsaltsolution[PSS;134mMNaCl,5mMKCl,1mMMgCl,3mMCaClsalts,and10mMHEPESin20%glucosesolution（pH7.4）]atroomtemperature.Superfusionremovesextracellulardyeandallowsforthedeesterificationofthefura2-AM.Atthebeginningofeveryseriesofexperiments,KClorvehiclecontrolwasadministered.AsharpriseinthecalciumtransientinducedbyKClindicatedthatthecellswerehealthyandcouldbeusedfordatacollection,andbaselinestabilityinresponsetovehiclecontrolindicatedreliabilityofthecellularresponse.AZeissAxiovert100invertedmicroscopewithilluminationbyaTilPhotonicsPolychromeIIMonochromatorwasused（AppliedScientificInstrumentation,Eugene,OR）,withexcitationmeasuredat340and380nmandemissionmeasuredat520nm.Imageswereobtainedusingacharge-coupleddevicevideocamera.ImageacquisitionandanalysiswasperformedusingtheMetamorph/Metafluor5.0ImagingSystem（UniversalImagingCorp.,Downingtown,PA）.Temperaturewasmaintainedbetween30and32Cthroughouttheexperiment.Calciumcalibrationwasperformedusingacalciumcalibrationbufferkit（Invitrogen,Carlsbad,CA）,andallanalysiswasperformedontherawfreecalciumconcentrationdata.BaselinecalciumfluorescenceratiowasobtainedwithPSSsuperperfusionfollowedby3-mininfusionsof10μMKA.KA,atthisconcentration,elicitedacalciumresponseinhippocampalcells,yetresultedinalowpercentageofcelldeath.PSSwasthensuperfusedfor4C5mintoallowforcompleteclearanceoftheKA.Receptorantagonistsat100μMconcentrations,including1）theNMDAreceptorantagonist,MK801;2）theAMPA/kainateantagonist,2,3-dihydroxy-6-nitro-7-sulfamoylbenzo（f）quinoxaline（NBQX）;3）theL-typeVSCCantagonist,diltiazem;and4）thespecificcalcium-permeableAMPAreceptorantagonist,1-naphtylacetylsperminetrihydrochloride（NAS）,werethensuperfusedfor4min.NBQX,diltiazem,andMK801wereobtainedfromSigma（St.Louis,MO）,andNASwasagiftfromAkiraIsokawa（DaicelChemicalIndustries,Ltd.,FortLee,NJ）.Afterthis,acombinationof10μMKAand100μMofeachantagonistorcombinationofantagonistswasappliedfor3min.Allantagonistswereadministeredatadose（100μM）determinedtosaturateallreceptorsandthatwas10-foldgreaterthantheconcentrationoftheagonist.Eachseriesofexperimentswithantagonistswasperformedonseparatecoverslipssothatcellswereexposedtoonlyoneantagonisttoeliminateapotentialconfoundduetotheorderofdrugapplication.Ratiometricmeasureswereobtainedevery5C30secthroughouttheexperimentalparadigm.Cellswereexcludedfromanalysisiftheirbaselineratiowasgreaterthan0.9,whichmightbeindicativeofanunhealthycell,iftheratiodidnotreturntobaselineaftereitherKAorantagonistperfusion,oriftherewasanindicationofcelldeathwithaprecipitousdropinratiotobelowbaseline.CellswereclassifiedasrespondersornonrespondersdependingonthesizeoftheKA-inducedriseinintracellularcalcium.Amorethan10%riseinintracellularcalciumhadbeendesignatedasthresholdinpriorimagingexperimentsbecauseanythingbelowthatwasdeemedunreliable（35）.Onlycellsrespondingwithamorethan10%riseinintracellularcalciumwereanalyzedfurthe
