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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-adrenergicdilatationofrataortaismediatedthroughactivationofbothproteinkinaseAandAkt.BrJPharmacol2004;143:397-403.
LRQueen,YJi,FerroA,etal.Nitricoxidegenerationmediatedbyβ-adrenoceptorsisimpairedinplateletsfrompatientswithType2diabetesmellitus.Diabetologia2003;46:1474-82.JiY,HanY,DiaoJ,etal.Rabbitaorticendothelialdysfunctionbylow-densitylipoproteinisattenuatedbyL-arginine,L-ascorbateandpyridoxine.BrJPharmacol2003;140:1272-82.JiY,HuangY,FerroA,etal.Cardiaceffectsofamilorideandofenalaprilinthespontaneouslyhypertensiverat.JHypertens2003;21:1583-9.O’KanePD,QueenLR,
JiY,etal.Aspirinmodifiesnitricoxidesynthaseactivityinplatelets:effectsofacuteversuschronicaspirintreatment.CardiovascRes2003;59:152-9....ArticlesinrecentyearsJiY,30eNOSandCardiovasculardiseases
YongJiAtherosclerosisResearchCenterNanJingMedicalUniversity2008-11-20...eNOSandCardiovasculardiseas31SUMMARYTheregulationofeNOSeNOS-derivedNOandatherosclerosisRecentachievement...SUMMARYTheregulationofeNOS32TheregulationofeNOS...TheregulationofeNOS...33L-arginine
NO+L-citrullineeNOScalmodulin(CaM)tetrahydrobiopterin(H4B)flavinmononucleotideFADNADPH...L-arginineNO+L-citr34RegulationofeNOSexpression
RegulationofeNOSmRNAstability
eNOSlevelsinendothelialcellscanalsobeefficientlyregulatedbychangesineNOSmRNAstability.
Hypoxiaandcytokines,suchasTNF,alsodownregulateeNOSmRNAlevelsbydecreasingthehalf-lifeofeNOSmRNAfrom48hunderbasalconditionsto3h....RegulationofeNOSexpression
35......36IntracellularTranslocation
FunctionaleNOScanbedetectedinatleastthreemembranecompartments,theplasmamembrane,plasmalemmalcaveolae,andtheGolgiapparatus.However,thedisruptionoftheGolgiapparatusdoesnotaffecttheagonist-induced,endothelium-dependent,andNO-mediatedrelaxationofporcinecoronaryarteries,suggestingthattheGolgieNOSpoolcontributeslittletotheregulationofvasculartone....IntracellularTranslocation
Fu37TwogroupshavereportedthateNOScanalsobedetectedinthenucleusofendothelialcellsandbrownadipocytes.AlthoughthenuclearlocalizationofeNOSmayfacilitatetheinteractionofNOwithtranscriptionfactors,theconditionsassociatedwithalocalizationofeNOSanditsexactroleinthiscellcompartmentremaintobedetermined....Twogroupshavereportedthat38eNOSsignalingcomplexSignaltransductionmoleculesproteinsColocalizationEnzymeactivaionNOproductionDownstreameffectorpathway...eNOSsignalingcomplexSignalt39Ca2+andeNOSActivityCa2+-dependentactivationofeNOS
CaMbindingtotheCaM-bindingmotifisthoughttodisplaceanadjacentautoinhibitorylooponeNOS(andnNOS),thusfacilitatingNADPH-dependentelectronfluxfromthereductasedomainoftheproteintotheoxygenasedomain.(acetylcholine,bradykinin,histamine)Ca2+-independentactivationofeNOS
fluidshearstress
ActivationofPI3KActivationoftheserinekinasesAktActivationofproteinkinasesAP-eNOSeNOS...Ca2+andeNOSActivityCa2+-dep40eNOSPhosphorylationSer1177.
WhenSer1177isphosphorylated,thefluxofelectronsthroughthereductasedomainand,asaconsequence,alsoNOproductionareincreasedtwo-tothreefoldabovebasallevels.Thr495.
Thisresidueisconstitutivelyphosphorylatedinalloftheendothelialcellsinvestigatedtodateandisanegativeregulatorysite,phosphorylationisassociatedwithadecreaseinenzymeactivity....eNOSPhosphorylationSer1177..41OtherPhosphorylationSites
Ser633Ser615Ser114Tyr83PhosphorylationeNOSactivity...OtherPhosphorylationSitesS42eNOS-derivedNOandatherosclerosis...eNOS-derivedNOandatheroscl43Atherosclerosis...Atherosclerosis...44......45TheNobelPrizeinPhysiologyorMedicine1998RobertF.Furchgott
LouisJ.Ignarro
FeridMurad
FortheirdiscoveriesconcerningnitricoxideasasignallingmoleculeinthecardiovascularsystemDiscoveryofNO...TheNobelPrizeinPhysiology46UncoupledeNOSeNOSuncoupling:electronsflowingfromthereductasedomaintothehemearedivertedtomolecularoxygenratherthantothesubstrateL-arginine,therebyresultinginproductionofsuperoxideinsteadofNO....UncoupledeNOSeNOSuncouplin47
Thesignaltransductionpathways
...Thesignaltransductionpathw48
PromotevasorelaxationPreventplateletaggregationandleukocytesadhesionInhibitvascularpermeabilityInhibitatherogenesisL-arginineL-citrullineeNOSO2
NADPHNONADPEffectofeNOS-DerivedNOonatherosclerosis
InhibitvascularSMCmigration,proliferation...PromotevasorelaxationPrevent49Recentachievement...Recentachievement...50
ToinvestigatethemechanismbywhichthisassociationbetweeneNOSandβ-actincanregulateeNOSactivity....Toinvestigatethemechanis51β-actincoimmunoprecipitateswitheNOSinplatelets.JiY,etal.ProcNatlAcad
SciUSA2007;104:8839-8844.
...β-actincoimmunoprecipitatesw52eNOSbindstotheglobular,butnotthefilamentous,formofβ-actininplatelets....eNOSbindstotheglobular,bu53Polymerizationstatusofβ-actinregulateseNOSactivityandNObioactivityinplatelets....Polymerizationstatusofβ-act54Polymerizationstatusofβ-actinhasnochangeinintracellularCa2+orserine-1177phosphorylationofeNOS....Polymerizationstatusofβ-act55Hsp90colocalizeswitheNOSand
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....Hsp90colocalizeswitheNOSan56Globularβ-actinformsaternarycomplexwitheNOSandHsp90.eNOSbindingtomonomericβ-actinwasconcentration-dependent,andreversible.(B)PreincubationofeNOSwithHsp90increasedthedegreeofbindingtoimmobilizedmonomericβ-actin,by2.8±1.2-fold,andthisbindingbecameirreversible....Globularβ-actinformsaterna57ConclusionHere,wedemonstrateinhumanplateletsthatthepolymerizationstateofβ-actincruciallyregulatestheactivationstateofeNOS,andhenceNOformation,throughalteringitsbindingofHsp90....ConclusionHere,wedemonstrat58ArticlesinrecentyearsJiY,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-mediatednitricoxidegenerationbyhumanumbi
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