血管紧缩素II 信号的稀释再配对eNOS 和禁止Nonendothelial 氮化物活动在糖尿病老鼠Word文件下载.docx
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Abbreviations:
AngII,angiotensinII;
AT1R,AngIIreceptortype1;
CMH,methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine;
DETC,diethyldithiocarbamicacid;
DHE,dihydroethidium;
DHFR,dihydrofolatereductase;
eNOS,endothelialnitricoxidesynthase;
ESR,electronspinresonance;
H4B,tetrahydrobiopterin;
L-NAME,N-nitro-L-argininemethylesterhydrochloride;
NOX,NAD（P）Hoxidase;
ROS,reactiveoxygenspecies;
STZ,streptozotocin;
VSMC,vascularsmoothmusclecell
ABSTRACT
TOP
ABSTRACT
RESULTS
DISCUSSION
RESEARCHDESIGNANDMETHODS
REFERENCES
AngiotensinII（AngII）levelsareincreasedinpatientswithdiabetes,butmechanismsunderlyingitscontributiontodiabeticvasculardiseasesareincompletelyunderstood.Werecentlyreportedthatinaorticendothelialcells,AngIIinducesendothelialnitricoxidesynthase（eNOS）uncouplingtoproducesuperoxide（O2·
–）ratherthannitricoxide（NO·
）,uponlossofthetetrahydrobiopterin（H4B）salvageenzymedihydrofolatereductase（DHFR）.Here,wefoundthatstreptozotocin-induceddiabeticmicehadamarkedincreaseinaorticO2·
–production,whichwasinhibitedbyN-nitro-L-argininemethylesterhydrochloride,indicatinguncouplingofeNOS.AngIIreceptortype1blockercandesartanorACEinhibitorcaptoprilmarkedlyattenuatedeNOS-derivedO2·
–andhydrogenperoxideproductionwhileaugmentingNO·
bioavailabilityindiabeticaortas,implicatingrecouplingofeNOS.O2·
–andNO·
productionwerecharacteristicallyandquantitativelymeasuredbyelectronspinresonance.DHFRexpressionwasdecreasedindiabeticaortasbutsignificantlyrestoredbycandesartanorcaptopril.EitheralsoimprovedvascularH4Bcontentandendothelium-dependentvasorelaxationindiabetes.Rac1-dependentNAD（P）Hoxidase（NOX）activitywasmorethandoubledintheendothelium-denudeddiabeticaortasbutwasattenuatedbycandesartanorcaptopril,indicatingthatNOXremainsactiveinnonendothelialvasculartissues,althoughuncoupledeNOSisresponsibleforendothelialproductionofO2·
–.ThesedatademonstrateanovelroleofAngIIindiabeticuncouplingofeNOSandthatAngII–targetedtherapyimprovesendothelialfunctionviathenovelmechanismofrecouplingeNOS.DualeffectivenessonuncoupledeNOSandNOXmayexplainthehighefficacyofAngIIantagonistsinrestoringendothelialfunction.
Productionofreactiveoxygenspecies（ROS）isincreasedinbothtype1andtype2diabetes,contributingnotonlypartiallytoetiologyofdiabetes（1–4）,butalsosignificantlytodiabeticaccelerationofvasculardiseases（2,4–9）.OneimportantmechanismforROS-inducedvasculardamageisoxidativedegradationofnitricoxide（NO·
）.ThisleadstoendothelialdysfunctionthatischaracterizedbyalossinNO·
-dependentvasodilatation（6,9）.Endothelium-dependentvasodilatationisimpairedindiabetes,andthiscanbesignificantlyreversedbysuperoxidedismutaseortheantioxidantvitaminC（10–12）.Indiabeticcoronaries,ROSalsohinderendothelium-dependenthyperpolarizingfactor–mediatedvasodilatation（13）.Whatremainstobefullyelucidatedistheenzyme（s）responsibleforproductionofROSindiabetes.RecentstudiesdemonstratethatvascularNAD（P）Hoxidase（NOX）servesasthepredominantsourceofROSinthediseasedbloodvessels（9,14）.Uncoupledendothelialnitricoxidesynthase（eNOS）,however,couldbeanotherimportantsourceofROS（seebelow）inthediabeticendothelium（15）,likelydownstreamofNOX（14,16）.MitochondriaasROSresourceshavebeeninvestigatedinnonvascularcellsindiabetes.RecentstudiessuggestthattheremightbecrosstalksbetweendifferentenzymaticsourcesofROS,resultinginROS-dependentself-propagation（16）.
ByproducingNO·
toinactivatesuperoxide（O2·
–）anditsderivatives,eNOSnormallyfunctionsasapotentantioxidantenzymeinthevasculature.Togetherwithotheranti-inflammatoryandantithromboticpropertiesofNO·
thisantioxidantroleofeNOSisessentialforprotectionagainstendothelialdysfunction.Underpathologicalconditions,however,eNOScantransformintoapro-oxidant,generatingO2·
–ratherthanNO·
.This"
uncoupling"
ofeNOSoccursnotonlyin"
traditional"
vasculardiseasessuchashypertensionandatherosclerosis（14,17–19）,butalsoindiabeticbloodvessels（15）.ThenextobviousquestionisthenwhatisthecommonmechanismthatinducesuncouplingofeNOSindifferentvasculardiseases?
AngiotensinII（AngII）isacirculatingvasoconstrictivehormonewhoseproductionisoftenelevatedinpatientswithhypertensionandhypercholesterolemia.InfusionofAngIIinapolipoproteinE–deficientorLDLreceptor–deficientmicedramaticallyacceleratedatherogenesis（20–22）.Ofimportance,productionofAngIIisalsoincreasedinpatientswithdiabetes,particularlythosewithhypertensionandrenaldysfunction（23,24）.Itisgenerallybelievedthathyperglycemiaintheearlystagesofdiabetesinduceshyperfiltrationandnatriuresis,andthisactivatesAngIIsynthesisasacompensatorymechanismtomaintainnormalbloodpressure（25,26）.HighglucosealsopotentlyupregulatesexpressionoftheAngIIreceptortype1（AT1R）（27）.ThisresponsecouldsensitizevascularcellstoAngII.ItisthusrationaltohypothesizethatAngIImaymediatecommonpathologicalmechanismsinvolvedinthedevelopmentofdifferentvasculardiseases.OnepossibletargetcouldbeeNOS.
Werecentlyreportedthatinculturedaorticendothelialcells,AngIIinducesNOX-dependentuncouplingofeNOS（19）.Dihydrofolatereductase（DHFR）isthekeyenzymeresponsibleforsalvationoftheeNOScofactortetrahydrobiopterin（H4B）fromitsinactive,oxidizedform.NormalendothelialDHFRisrequiredforbasalendothelialH4BandNO·
bioavailability（19）.AngII,however,caninduceDHFRdeficiencyviahydrogenperoxide（H2O2）–dependentmechanisms,resultinginalossofH4Band,consequently,theuncouplingofeNOS.Inkeepingwiththis,scavengingH2O2oroverexpressingDHFRwaseffectiveinrestoringNO·
productionfromeNOSwhileeliminatingeNOS-derivedO2·
–production（19）.InviewofthepotentialsignificanceofAngIIindiabeticvasculardisease,inthecurrentstudy,weinvestigatedwhetherAngIIisinvolvedindiabeticuncouplingofeNOSinvivo,andwhetherattenuationofAngIIsignalingwithAT1RblockercandesartanorACEinhibitorcaptopriliseffectiveinrecouplingeNOStoimproveendothelialfunction.Tissue-specific,differentialcontributiontototaldiabeticvascularO2·
–productionofuncoupledeNOSandNOXwasalsoexamined.
RESULTS
Hyperglycemiainstreptozotocin-induceddiabeticmice.
Diabeticmicewerecreatedbystreptozotocin（STZ）administrationasdescribedindetailinRESEARCHDESIGNANDMETHODS.Ondayofharvest（7th–8thdayafterinitialSTZinjection,samehereafter）,bloodglucosewaselevatedto452.0±
15.1mg/dlindiabeticmiceversus148.4±
3.2mg/dlintheC57BL6controls（Fig.1）.Candesartan（100mg·
kg–1·
day–1,samehereafter）orcaptopril（100mg·
day–1,samehereafter）treatmentsinceday4hadnosignificanteffectonSTZinductionofhyperglycemia（522.5±
14.6or478.3±
14.6mg/dl,respectively;
Fig.1）.Fortheentirestudyperiod,miceweremonitoredandfoundcomfortablewithoutanysignsofseverelossinbodyweight.
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FIG.1.Bloodglucoselevels.Ondayofharvest,bloodwascollectedfromtailveinforglucoseanalysisforeachmouse,usingOneTouchglucosemonitorandmatchingstripes.P<
0.05vs.control,ANOVA,n=36.
UncouplingofeNOSindiabetes.
DiabeticaortasshowedamarkedincreaseintotalROSproduction,detectedbydihydroethidium（DHE）fluorescence（Fig.2A）.WhereasN-nitro-L-argininemethylesterhydrochloride（L-NAME）（100µ
mol/l;
inhibitorofNO·
synthase）increasedROSproductioninthecontrolsbecauseofscavengingofNO·
fromcoupledeNOS,italmostcompletelyattenuatedROSinthediabeticmice,stronglysuggestinguncouplingofeNOS（Fig.2A）.Aorticproductionofsuperoxideanion（O2·
–）wasalsodetectedspecificallyusingelectronspinresonance（ESR）andacell-permeableO2·
–-specificspintrapmethoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine（CMH）（fordetails,seeRESEARCHDESIGNANDMETHODS）.AsshownbyrepresentativeESRspectrainFig.2BandquantitativedatainFig.2CandD,aorticO2·
–productionwasmorethandoubledindiabeticmice（3.3±
1.6vs.7.0±
2.6nmol/lpermin/mgwetwtaortaforcontrolvs.diabeticmice）,andthisresponsewasattenuatedtonearbaselinebyL-NAME.L-NAMEdidnotpotentiatedetectableO2·
–incontrolmice,likelybecauseoftherapiddegradationofO2·
–intootherspecies,becauseL-NAMEdidenhancetotalROSproductiondetectedbyDHE.Takentogether,bydirectlymeasuringL-NAME–sensitiveaorticROSingeneralandO2·
–productionspecificallyandquantitatively,thesedataclearlydemonstrateth
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