积分法_一种药理学中分析电压依赖性瞬时外向钾电流的方法_英文_

Integration:amethodforevaluatingvoltage2dependenttransientoutwardpotassiumcurrentsinpharmacologyDONGDe2Li1,SUNZhi2Jie2,JIAOJun2Dong1,YUEPeng1,WANGQing2Hui1,FANGZhi2Wei1,YANGBao2Feng13(1.DepartmentofPharmacology,HarbinMedicalUniversity,Bio2pharmaceuticalKeyLaboratoryofHeilongjiangProvince,Harbin150086,China;2.SchoolofMaterialScienceandEngineering,HarbinInstituteofTechnology,Harbin150001,China)Abstract:AIMToevaluatetheintegrationmethodforanalysisofvoltage2dependentCa2+2independenttransientoutwardK+currents(Ito)inpharmacology.METHODSTheinactivationphasesofItowerebestfittedbythesumoftwoorthreeexponentialsequations.Theareaundertherawcurrentcurves(AUC)wasobtainedbytheintegrationofexponentialequations.TheAUCnormalizedtothecellcapacitancerepresentedthenetK+chargeflowduringanydepolarizeddurationandwasastheindexforcompar2ison.Calcineurinoverexpressiontransgenic(TG)miceshoweddownregulationofIto.Thesedataweretestedbytheintegrationmethod.RESULTSAUCobtainedfromthreeortwoexponentialsfittingswascalculatedas:AUC=A11+A22+A33+A0t-A11e-t/1-A22e-t/2-A33e-t/3orAUC=A11+A22+A0t-A11e-t/1-A22e-t/2.The50%and90%actionpo2tentialduration(APD50,APD90)inventricularmyocytesofmiceareabout10msand30ms,respectively.AUCat10ms(AUC50,AUCof50%APD)and30ms(AUC90,AUCof90%APD)inleftventriclecardiomyocytesofwildtype(WT)andTGmicewerenormalizedtothecellcapacitance.ThenormalizedAUC50andAUC90ofWTgroupweresignificantlymorethanthoseofTGgroup,whichwasconsistenttotheprolongationofAPDinTGmiceandthepreviouspublishedresults(downregulationofcomponentsofItoinTGmice).CONCLUSIONTheintegrationmethodwasanidealwayforanalysisoftran2sientoutwardK+currentsinpharmacology.Keywords:electrophysiology;potassiumchannels;Receiveddate:2004206201Accepteddate:2004210215Foundationitem:TheprojectsupportedbyNaturalScienceFoundationofHeilongjiangProvince(D0246);andNationalNaturalScienceFoundationofChina(30271599)Biography:DONGDe2Li(1970-),male,nativeofQiqihar,HeilongjiangProvince,associateprofessor,PhD,mainresearchfieldiscardiovascularpharmacologyandionchannelpharmacology.3Correspondingauthor.E2mail:[e-mail]:862451286671354Fax:[phone]voltage2dependenttransientoutwardpotassiumcurrents;integration;exponentialfittingCLCnumber:R965Documentcode:AArticleID:100023002(2005)0120064206Thevoltage2dependentCa2+2independenttransientoutwardK+current(Ito)hasbeenrecordedincardiacmyocytesfrommanyspecies[1-3].Itoplaysakeyroleindeterminingthedurationofactionpotential(APD)incardiaccells.Sincethepatch2clamptechniqueshadbeeninvented,themountingdatawerereportedaboutthepropertiesandphysiologicalsignificanceofItointheheart.SeveralwayshavebeenappliedfortheanalysisofIto:Itoisdividedintotwocom2ponents,ItandIss.Issisthesteadystatecompo2nent,ItisobtainedbysubtractingIssfromthepeakoutwardcurrent[4-6].ForthekineticschangeinItoisnotexactlyexpressedbytheabovemethod,anewanalyticwaywasdevelopedbyXu,etal[7]usingtwoorthreeexponentialsfitting.Accordingtotheirstudies,Itoisseparatedintofourcomponentswithdifferentinactivationcon2stants.Theseparationwasevidencedfurtherbythepharmacologicalwayintheirpaper.ThemethodwasalsoadoptedfortheItoanalysisinourpreviousstudy[8].AlthoughtheexponentialfittingprovidesmoreinformationaboutIto,therearestillsomelimitations:forexample,theobtainingofdifferentcomponentsisdependentonthemathe2maticswaysartificially.AnidealanalyticwayshouldreflectthenetchargeflowduringanAPD46ChinJPharmacolToxicol20052;2005Feb;19(1):64-6919(1):64-69oraholdingpotentialduration.ThemiceexpressedItoabundantlyintheheartandcardiomyocytesareexcellentsamplesforItoanalysis.Calcineurinisacalcium2activatedphosphatasethatdephosphorylatesacytosolicpro2teinnamedasnuclearfactorsofactivatedTcells(NF2AT3)whichtranslocatesintothenucleustointeractwiththetranscriptionfactorGATA24thusactivatingarangeofgenesinvolvedincardiachy2pertrophy[9].Comparingwiththewildtype(WT)mice,calcineurinoverexpressiontransgenic(TG)miceshowedcardiachypertrophyanddownregula2tionofK+currents[8].Theintegrationmethodwastestedbyourpublisheddataanditshowedmanyadvantagescomparedwithpreviousmeth2ods.TheaimofthepresentstudywastoevaluatetheintegrationmethodforItoanalysis.1MATERIALSANDMETHODS1.1ChemicalsandanimalsAllchemicalswereSigmaproducts.FVBWTmiceandcalcineurinoverexpressionTGmice,maleandfemale,22-26g,werekindlygiftedbyJefferyD.Molkentin(DivisionofMolec2ularCardiovascularBiology,DepartmentofPedi2atrics,ChildrensHospitalMedicalCenter,Cincinnati,USA).1.2SingleventricularmyocyteisolationHeartswereperfusedretrogradelywithTy2rodessolution,containing1mmolL-1CaCl2,pH7.4,37,for2min,thenperfusedwithaCa2+2freeTyrodesfor5min,andfinally,withaCa2+2freeTyrodessolutioncontaining25kUL-1ofYakultcollagenaseforWTand35kUL-1forTGmicefor10min.Thefreewalloftheleftventriclewasseparated.Theleftventricularfree2wallwascutandtituratedfor2minandthenincubatedinhigh2K+storagesolutionconsistingof(mmolL-1):KCl25,KH2PO410,MgCl23.0,potassiumglutamate70,EGTA0.5,BAPTA2AM1,22bis(22aminophenoxy)ethane2N,N,N,N2tetraaceticacidtetrakis(acetoxymethylester)0.01,glucose10,HEPES10,taurine20,andKOH(pH