病理学与病理生理学课件

eNOSandCardiovasculardiseases

YongJiAtherosclerosisResearchCenterNanJingMedicalUniversity2008-11-20...eNOSandCardiovasculardiseas1SUMMARYTheregulationofeNOSeNOS-derivedNOandatherosclerosisRecentachievement...SUMMARYTheregulationofeNOS2TheregulationofeNOS...TheregulationofeNOS...3L-arginine

NO+L-citrullineeNOScalmodulin(CaM)tetrahydrobiopterin(H4B)flavinmononucleotideFADNADPH...L-arginineNO+L-citr4RegulationofeNOSexpression

RegulationofeNOSmRNAstability

eNOSlevelsinendothelialcellscanalsobeefficientlyregulatedbychangesineNOSmRNAstability.

Hypoxiaandcytokines,suchasTNF,alsodownregulateeNOSmRNAlevelsbydecreasingthehalf-lifeofeNOSmRNAfrom48hunderbasalconditionsto3h....RegulationofeNOSexpression

5......6IntracellularTranslocation

FunctionaleNOScanbedetectedinatleastthreemembranecompartments,theplasmamembrane,plasmalemmalcaveolae,andtheGolgiapparatus.However,thedisruptionoftheGolgiapparatusdoesnotaffecttheagonist-induced,endothelium-dependent,andNO-mediatedrelaxationofporcinecoronaryarteries,suggestingthattheGolgieNOSpoolcontributeslittletotheregulationofvasculartone....IntracellularTranslocation

Fu7TwogroupshavereportedthateNOScanalsobedetectedinthenucleusofendothelialcellsandbrownadipocytes.AlthoughthenuclearlocalizationofeNOSmayfacilitatetheinteractionofNOwithtranscriptionfactors,theconditionsassociatedwithalocalizationofeNOSanditsexactroleinthiscellcompartmentremaintobedetermined....Twogroupshavereportedthat8eNOSsignalingcomplexSignaltransductionmoleculesproteinsColocalizationEnzymeactivaionNOproductionDownstreameffectorpathway...eNOSsignalingcomplexSignalt9Ca2+andeNOSActivityCa2+-dependentactivationofeNOS

CaMbindingtotheCaM-bindingmotifisthoughttodisplaceanadjacentautoinhibitorylooponeNOS(andnNOS),thusfacilitatingNADPH-dependentelectronfluxfromthereductasedomainoftheproteintotheoxygenasedomain.(acetylcholine,bradykinin,histamine)Ca2+-independentactivationofeNOS

fluidshearstress

ActivationofPI3KActivationoftheserinekinasesAktActivationofproteinkinasesAP-eNOSeNOS...Ca2+andeNOSActivityCa2+-dep10eNOSPhosphorylationSer1177.

WhenSer1177isphosphorylated,thefluxofelectronsthroughthereductasedomainand,asaconsequence,alsoNOproductionareincreasedtwo-tothreefoldabovebasallevels.Thr495.

Thisresidueisconstitutivelyphosphorylatedinalloftheendothelialcellsinvestigatedtodateandisanegativeregulatorysite,phosphorylationisassociatedwithadecreaseinenzymeactivity....eNOSPhosphorylationSer1177..11OtherPhosphorylationSites

Ser633Ser615Ser114Tyr83PhosphorylationeNOSactivity...OtherPhosphorylationSitesS12eNOS-derivedNOandatherosclerosis...eNOS-derivedNOandatheroscl13Atherosclerosis...Atherosclerosis...14......15TheNobelPrizeinPhysiologyorMedicine1998RobertF.Furchgott

LouisJ.Ignarro

FeridMurad

FortheirdiscoveriesconcerningnitricoxideasasignallingmoleculeinthecardiovascularsystemDiscoveryofNO...TheNobelPrizeinPhysiology16UncoupledeNOSeNOSuncoupling:electronsflowingfromthereductasedomaintothehemearedivertedtomolecularoxygenratherthantothesubstrateL-arginine,therebyresultinginproductionofsuperoxideinsteadofNO....UncoupledeNOSeNOSuncouplin17

Thesignaltransductionpathways

...Thesignaltransductionpathw18

PromotevasorelaxationPreventplateletaggregationandleukocytesadhesionInhibitvascularpermeabilityInhibitatherogenesisL-arginineL-citrullineeNOSO2

NADPHNONADPEffectofeNOS-DerivedNOonatherosclerosis

InhibitvascularSMCmigration,proliferation...PromotevasorelaxationPrevent19Recentachievement...Recentachievement...20

ToinvestigatethemechanismbywhichthisassociationbetweeneNOSandβ-actincanregulateeNOSactivity....Toinvestigatethemechanis21β-actincoimmunoprecipitateswitheNOSinplatelets.JiY,etal.ProcNatlAcad

SciUSA2007;104:8839-8844.

...β-actincoimmunoprecipitatesw22eNOSbindstotheglobular,butnotthefilamentous,formofβ-actininplatelets....eNOSbindstotheglobular,bu23Polymerizationstatusofβ-actinregulateseNOSactivityandNObioactivityinplatelets....Polymerizationstatusofβ-act24Polymerizationstatusofβ-actinhasnochangeinintracellularCa2+orserine-1177phosphorylationofeNOS....Polymerizationstatusofβ-act25Hsp90colocalizeswitheNOSand

β-actin.β-Actin,Hsp90,andeNOSwereeachdetected(A–C,respectively)byImmunoGoldlabelingwithelectronmicroscopy.Triplelabelingshowsmultipleareas(arrowed)whereβ-actin(5-nmgoldparticles),eNOS(10-nmgoldparticles),andHsp90(15-nmgoldparticles)colocalize(D).g,plateletgranule....Hsp90colocalizeswitheNOSan26Globularβ-actinformsaternarycomplexwitheNOSandHsp90.eNOSbindingtomonomericβ-actinwasconcentration-dependent,andreversible.(B)PreincubationofeNOSwithHsp90increasedthedegreeofbindingtoimmobilizedmonomericβ-actin,by2.8±1.2-fold,andthisbindingbecameirreversible....Globularβ-actinformsaterna27ConclusionHere,wedemonstrateinhumanplateletsthatthepolymerizationstateofβ-actincruciallyregulatestheactivationstateofeNOS,andhenceNOformation,throughalteringitsbindingofHsp90....ConclusionHere,wedemonstrat28ArticlesinrecentyearsJiY,FerracciG,FerroA,etal.beta-Actinregulatesplateletnitricoxidesynthase3activitythroughinteractionwithheatshockprotein90.ProcNatlAcad

SciUSA2007;104:8839-8844ChenL,LiuY,JiY,etal.17Beta-oestradiolpartiallyattenuatestheinhibitionofnitricoxidesynthase-3byadvancedglycationend-productsinhumanplatelets.ClinExpPharmacolPhysiol2007Oct;34(10):972-8.QueenLR,JiY,FerroA,etal.Mechanismsunderlying{beta}2-adrenoceptor-mediatednitricoxidegenerationbyhumanumbilicalveinendothelialcells.JPhysiol2006;576(pt2):585-94JiY,DiaoJ,HanY,etal.Pyridoxinepreventsdysfunctionofendothelialcellnitricoxideproductioninresponsetolow-densitylipoprotein.Atherosclerosis2006;188(1):84-94BiaoXu,YongJi,KangYao,etal.Inhibitionofhumanendothelialcellnitricoxidesynthesisbyadvancedglycationend-productsbutnotglucose:relevancetodiabetes.ClinSci2005;109:439-46....ArticlesinrecentyearsJiY,29ArticlesinrecentyearsJiY,HanY,DiaoJ,etal.Inhibitionofendothelialnitricoxidegenerationbylow-densitylipoproteinispartiallypreventedbyL-arginineandL-ascorbate.Atherosclerosis2004;176:345-53.FerroA,CoashM,JiY,etal.Nitricoxide-dependent{beta}2-adrenergicdilatationofrataortaismediatedthroughac