Role of vascular Kinin B-1 and B-2 receptors in endothelial nitric oxide metabolism

ContentslistsavailableatScienceDirect

Peptides

jo u rn al h om epa g e:w w w . e l s e v i e r . c o m / l o c a t e / p e p t i d e s

Role

of

vascular

Kinin

B

1

and

B

2

receptors

in

endothelial

nitric

oxide

metabolism

Rodrigo

A.

Loiola

_,

_Felipe

_C.G.

_Reis

_,

_Elisa

_M.

_Kawamoto

_,

_Cristoforo

_Scavone

_,

Dulcinéia

S.

Abdalla

_,

_Liliam

_Fernandes

,

João

Bosco

Pesquero

a_{DepartmentofBiophysics,FederalUniversityofSãoPaulo,SãoPaulo,SP,Brazil} b_{DepartmentofPharmacology,UniversityofSãoPaulo,SãoPaulo,SP,Brazil}

c_{DepartmentofClinicalAnalysisandToxicology,UniversityofSãoPaulo,SãoPaulo,SP,Brazil}

d_{DepartmentofBiologicalSciences,FederalUniversityofSãoPaulo,CampusDiadema,RuaProf.ArthurRiedel275,Diadema,SP09972-270,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received18April2011

Receivedinrevisedform8June2011

Accepted8June2011

Availableonline16June2011

Keywords:

B1receptor

B2receptor

Knockout Bradykinin

Vascularreactivity

Nitricoxide

a

b

s

t

r

a

c

t

KininB1andB2receptorsplayanessentialroleininflammatoryprocessandcardiovascularhomeostasis. Thepresentstudyinvestigatedthevascularreactivityandnitricoxide(NO)generationintheisolated mesentericarteriolarbedfromB1(B1−/−)andB2receptor(B2−/−)knockoutmice.Endothelial-dependent relaxationwassignificantlydecreasedinarteriolesfrombothB1−/−andB2−/−incomparisontowild type(WT)mice,withnodifferencesforendothelial-independentrelaxatingorvasoconstrictoragents. PlasmaticandvascularNOproductionweremarkedlyreducedinbothB1−/−andB2−/−.Incontrast, inthepresenceofl-arginine,Ca2+andco-factorsfortheenzyme,NOsynthaseactivitywashigherin homogenatesofmesentericvesselsofB1−/−andB2−/−.Thepresentstudydemonstratedthattargeted deletionofB1orB2receptorgeneinmiceinducesimportantalterationsinthevascularreactivityof resistancevesselsandNOmetabolism.Thesevereimpairmentintheendothelial-mediatedvasodilation accompaniedbydecreasedNObioavailability,despitetheaugmentedNOSactivity,stronglyindicatesan exacerbationofNOinactivationinB1−/−andB2−/−vessels.Thepresentdataprovidevaluableinformation inordertoclarifytherelevanceofkininreceptorsinregulatingvascularphysiologyandmaypointtonew approachesregardingitscorrelationwithendothelialdysfunction,oxidativestressandNOavailability.

©2011ElsevierInc.

1. Introduction

Thekallikrein–kininsystemplaysanimportantroleinseveral biological functions,including inflammation and cardiovascular homeostasis[7].Thediverserangeofeffectselicitedbykininsis mediatedbyactivationofGprotein-coupledreceptors,namedB1

andB2.Bradykinin(BK)isthenaturalagonistoftheB2 receptor,

anditsdegradationbycarboxypeptidasesgeneratestheB1receptor

agonist,des-Arg[9]-BK[34].WhereasB2 receptorsare

constitu-tivelyexpressedandmediatemostoftheknowneffectsassigned tokinins,B1receptorsareweaklydetectableunderphysiological

conditions,butrapidlyinducedbyinflammatorystimuli[7,23]. BothB1andB2receptorsactthroughG␣qtostimulate

phospho-lipaseC␤followedbyphosphoinositidehydrolysisandintracellular freeCa2+_{mobilization[19].TheresultingintracellularfreeCa}2+_is

theinitialstepin theactivationofnitricoxidesynthase(NOS), whichcatalyzesoxidationoftheterminalguanidinenitrogenof

l_{-arginine toform}l_{-citrulline andnitricoxide(NO) [32].Three}

NOSisoformshavebeendescribed:neuronalNOS(nNOSorNOS1),

∗Correspondingauthor.Tel.:+551133193300;fax:+551140436428.

E-mailaddresses:lfernandes@unifesp.br,liliamfernandes05@gmail.com

(L.Fernandes).

inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3). The iNOS isoform differs from nNOS and eNOS in that it is fullyactivein theabsence ofCa2+ _{[27]. TheNOS isoforms}

have similar enzymatic mechanisms and require presence of co-factorstetrahydrobiopterin(BH4), nicotinamide-adenine

din-ucleotide(NADPH),flavinadeninedinucleotide(FAD)andflavin mononucleotide(FMN)foritsproperfunction[25].Inthe vascula-ture,onceformedbyNOS,endothelialNOdiffusesintothesmooth muscleandactivatessolubleguanylatecyclasethatcatalyzesthe formationof3′_,5′_{-cyclicguanosinemonophosphate(cGMP),} result-inginsmoothmusclerelaxationandthereforevasodilation[13].

In the last recent years, the development of genetically engineered mice lacking kinin receptors has allowed a better understandingof thephysiological and pathological role ofthe kallikrein–kininsysteminawiderangeofbiologicalevents[31]. Mice witha targeted deletion of thegene for the B1 receptor

(B1−/−)aredescribedtobehealthy,fertileandnormotensive,but

exhibit blunted responsesto bacteriallipopolysaccharide injec-tion and hypoalgesia [30]. Under physiological conditions, B2

receptorknockoutmice(B2−/−)presentnormaldevelopment[9],

renalhemodynamicsandsaltbalance[2,26,35].Nevertheless,data regardingtheeffectsofB2receptordeletiononbloodpressure

reg-ulationarecontroversial.Someauthorshavedemonstratedthat B2−/−arenormotensive[1–3,11,12,26,35,37,39]whileothergroups

0196-9781 ©2011ElsevierInc.

doi:10.1016/j.peptides.2011.06.010

Open access under the Elsevier OA license.

observedaslightbutsignificantincreaseinbloodpressurelevels [15,16,21,22].

Consideringthatboth B1 andB2 receptorsarelocatedinthe

endothelium and in vascular smooth muscle cells [7,19], and that resistance vessels are the most important sites for deter-miningperipheralvascularresistance[38],thepresentstudywas addressedtoinvestigatethevascularreactivityofmesenteric arte-riolesofB1−/− andB2−/−inresponsetoendothelium-dependent

and-independentagonists.Inparallel,plasmaNOlevels,vascular NOreleaseandNOSactivityinthemesentericvesselswerealso analyzedinordertoprovideinformationaboutNObioavailability inthesemicestrains.

2. Methods

2.1. Animals

C57Bl/6male knockoutB1 (B1−/−), B2 (B2−/−)and wildtype

(WT)mice,aged10–14weekswereobtainedfromthebreeding stockof Centro deDesenvolvimento deModelosExperimentais paraMedicinaeBiologia(CEDEME–UNIFESP).Micewerekeptina temperature-controlledroomona12hlight/daycycle,60% humid-ity,standardmicechowandwateradlibitum.InB1−/−andB2−/−,

theabsenceofthekininsreceptorswasshown byundetectable levelof mRNAencoding for theB1 or B2 receptor,respectively,

usingasemi-quantitativeRT-PCRtechnique.Allprocedureswere approvedandperformedinaccordancewiththeguidelinesofthe EthicsCommitteeoftheUNIFESP(protocolnumber0928/05), con-formedwiththeGuidefortheCareandUseofLaboratoryAnimals publishedbytheUSNationalInstitutesofHealth(NIHPublication No.85-23,revised1996).

2.2. Vascularreactivityintheperfusedmesentericvascularbed

Isolatedmesentericvascularbedswerepreparedaspreviously describedfortheratpreparation[24],withslightadaptationsfor themouse.Themesentericvascularbedwasperfusedwith Krebs-Henseleitsolution,pH7.4,37◦_{C,gassedwith95%O2and5%CO2,} ataconstantrateof2mL/minusingaperistalticpump.Vascular responseswereevaluatedbychanges intheperfusionpressure (mmHg)measuredbya dataacquisitionsystem(PowerLab8/S, ADInstrumentsPtyLtda,Australia).Toconfirmtheviabilityof tis-sues,preparationswereperfusedwithKCl(90mmol/L)addedto theKrebssolutionfor5min.After30minofstabilization, increas-ingdosesofnorepinephrine(NE)(5–100nmol),acetylcholine(ACh) (0.1–10nmol)andsodiumnitroprusside(SNP)(0.1–10nmol)were injectedinbolus,inavolumerangeof30–100␮L,witha3-min intervalbetweeneachdose.ForAChandSNPassays,preparations werepre-contractedwithNE(10␮mol/L)addedtoKrebssolution andvascularresponseswerecalculatedaspercentageof contrac-tioninducedbyNE.

2.3. MeasurementsofcirculatingNOlevels

TheplasmaNOlevelswereevaluatedbyNOderivativesnitrate and nitrite, as previously described [28]. Blood samples were collectedintoEDTA-coatedtubesandplasmawasimmediately sep-aratedbylow-speedcentrifugation(1500×g).Theconcentration ofnitrateinbloodwasdeterminedbychemiluminescence,elicited bythereactionofNOwithozoneafternitratereductionwithVCl3

saturatedsolutionin 1mol/LHCl, at90◦_{C,usinga NOanalyzer} (NOATM280_{SieversInstrumentsInc.,Boulder,CO,USA).Nitritewas}

determinedafterreductionwith1%KClsolutioninglacialacetic acidtoconvertnitritetoNO.

2.4. BasalNOproductioninmesentericarterioles

BasalNOinmesentericarterioleswasdeterminedbyusinga fluorescentcellpermeabledyeforNO,4,5diaminofluorescein diac-etate(DAF-2DA,Alexis,USA),aspreviouslydescribed[10].Once insidethecell,DAF-2DAishydrolyzedbycytosolicesterasesthus releasingDAF-2.ThereactionbetweenDAF-2andNOyieldsthe cor-respondingbrightgreen-fluorescenttriazolofluoresceins(DAF-2T). Themesentericarteriolesweredissected, immersedinmedium for cryosectioningand cut into10␮mthicksections(Leica CM 1850 cryostat,Leica Instruments,Germany). In order to stimu-lateNOSactivationandprovideoptimallevelsofsubstrate,slices werepre-incubatedwithphosphatebuffer(PB)solution contain-ingCaCl2(0.45␮mol/L)andl_{-arginine(100␮mol/L)during30min}

at37◦_{C.Sliceswerewashed,incubatedwithPBcontainingDAF-2} DA (10␮mol/L) for 30min at 37◦_{C and observed on a} micro-scope(Axiovert100M– CarlZeissSMT,Germany)equippedwith fluorescein filter(excitationat488nmand measuringemission at515nm).Fluorescenceemittedin responsetoNOproduction wasquantifiedthroughopticdensitometry(arbitraryunits,a.u.) using theAxioVision 4.8. digital imagesanalysissoftware (Carl Zeiss).Thesemi-quantitativeanalysisofbasalNOproductionwas determined,atleast,inthreeslicesfromeachanimal.Significant auto-fluorescencewasdiscardedbyexperimentsperformedinthe absenceofDAF-2DA.

2.5. DetectionofNOSactivityinthemesentericvascularbed

NOS activity was measured by the biochemical conversion of l-[3H] arginine tol-[3H]citrulline according tothe method

describedbyReesetal.[33].Mesentericvesselsweredissected, washed,homogenizedinice-coldbufferandstoredat−80◦_C.On thedayofassay,homogenateswereincubated(37◦_C/60min)in abuffercontainingFMNandFAD4␮mol/L,calmodulin10␮g/mL, Ca2+_{1.25mmol/L,NADPH1mmol/L,l-arginine120nmol/L,l-[3H]}

arginine 50nmol/L (NEN Life Science Products, USA) and BH4

10␮mol/L.For the determination ofiNOS activity,experiments wereperformedintheabsenceofCa2+_{.Reactionwasterminated}

by the addition of cation-exchange resin (Dowex 50WX8-400) to remove the excess of substrate. Theresin wasleft to settle for 30min atroom temperature,thesupernatant was carefully removed in vials withscintillation liquid and the radioactivity tol-[3H]citrullinewasquantified. Resultswere normalizedby

proteinconcentrationandNOsynthaseactivitywasexpressedas pmol/mgmin.

2.6. Drugsandreagents

NE,AChandSNPwereacquiredfromSigmaChemicalCo.(St. Louis,MO).Exceptwhendescribed,allotherdrugsandreagents werepurchasedfromMerck,Sharp&Döhme(WhitehouseStation, NJ).

2.7. Statisticalanalysis

ComparisonsweremadebyANOVAfollowedbyTukey–Kramer test.Valueswerereportedasmean±standarderrorofmean(SEM). StatisticalsignificancewassetasP<0.05.

3. Results

3.1. VascularreactivitytoNE,AChandSNP

After30minofstabilization,basalperfusionpressurein mesen-tericvascularbedfromB2−/−(48±1.8mmHg;n=8;P<0.05)was

Perfusion Pressure (mmHg)

0 50 100 150 200

A

B

4

2 6 8 10 12 14

Time (minutes)

NE

5 nm

ol

NE

10 nm

o

l

NE

50 nm

o

l

N

E

10

0 nmol

0 50 100 150

B1 -/-WT

NE (nmols)

B2

-/-5 10 50 100

increase in perfusion pressure

(mmHg)

Fig.1.(A)Representativerecordingofperfusionpressurealterationelicitedbyin

bolusinjectionofNEinthemiceisolatedmesentericarteriolarbed.Arrowsindicate

drugapplication.(B)BarsgraphshowsthevasoconstrictoreffectofNE(determined

asincreaseinperfusionpressure)onisolatedmesentericarteriolarbedofWT,B1−/−

andB2−/−.Resultsaremean±S.E.M.,n=5–7foreachgroup.

andB1−/−(41±1.0mmHg;n=8)preparations.Injectionof

vaso-constrictorNEonisolatedvascularpreparationselicitedrapidand dose-relatedconstrictionthatincreasedtoasinglepeakandthen declinedtobasalperfusionpressure,usuallywithin2min(Fig.1A). NEinjectionpromotedsimilarresponsesinallvascular prepara-tionsfromWT,B1−/−andB2−/−,asdemonstratedinFig.1B.

Theendothelialfunctionofmesentericarterioleswasassessed throughtheeffectofACh(anendothelium-dependentrelaxating agent)andSNP(anendothelium-independentrelaxatingagent)in pre-contractedvessels(NE10␮mol/L).Inallexperiments,ACh pro-ducedasignificantdose-dependentreductioninperfusionpressure (atthedosesof0.1,1and10nmols).AsshowninFig.2,vascular responsetoAChwasmarkedlyreducedinB1−/−andB2−/−

prepa-rationswhencomparedtoWTresponses,foralltesteddoses.In allgroups,SNPinjectionelicitedaconsistentdecreaseinperfusion pressure(about60%ofcontractioninducedbyNEperfusionatthe doseof10nmols).Nosignificantdifferencesweredetectedamong strainsforalltesteddosesofSNP(Fig.3).

3.2. PlasmaticNOlevels

SincetheNOmetabolitesreflecttheoverallNOproductionin theorganism,wedeterminedtheplasmanitrite/nitrate concentra-tioninbloodsamplesobtainedfromWT,B1−/−andB2−/−mice.A

significantdecreaseincirculatingNOlevelswasdetectedinboth B1−/−andB2−/−whencomparedtoWTsamples.Dataareshown

inFig.4.

0 5 10 15 20

WT

B1

-/-*

*

*

0.1 1 10

ACh (nmol)

B2

-/-*

*

*

decrease in perfusion pressure (%NE)

Fig.2. BarsgraphshowstherelaxatingeffectofAChinisolatedmesentericarteriolar

bedofWT,B1−/−andB2−/−.Responsesareexpressedas%ofcontractioninducedby

NE(10␮mol/L).Resultsaremean±S.E.M.,n=5–7foreachgroup.*P<0.05vsWT.

0 20 40 60 80

B1 -/-WT

10 1

0.1

SNP (nmol)

B2

-/-decrease in perfusion pressure (%NE)

Fig.3. BarsgraphshowstherelaxatingeffectofSNPinisolatedmesentericarteriolar

bedofWT,B1−/−andB2−/−.Responsesareexpressedas%ofcontractioninducedby

NE(10␮mol/L).Resultsaremean±S.E.M.,n=6–7foreachgroup.

3.3. BasalNOproductioninmesentericarterioles

VascularNOproductionwasassessedinmesentericarterioles sectionsincubatedwithDAF-2DA,asensitivefluorescentindicator fordetectionofNO.ImagesareshowninFig.5A.Thefluorescence

WT B₁-/- B₂

-/-0 50 100 150 200

*

*

NO plasma levels [

μ

M ]

Fig.4.BarsgraphshowstheNOplasmalevelsofWT,B1−/−andB2−/−.Resultsare

Fig.5.(A)RepresentativeimagesoffluorescenceinmesentericarteriolesslicesfromWT,B1−/−andB2−/−afterincubationwithDAF-2DA(10␮mol/L),afluorescentcell

permeabledyeforNO(200×magnification).(B)BarsgraphshowsthefluorescenceemittedafterincubationwithDAF-2DAandrepresentthebasalNOproduction.Fluorescence

wasquantifiedbyopticdensitometry(arbitraryunits,a.u.).Resultsaremean±S.E.M.,n=5foreachgroup.*P<0.05vsWT.

intensityofDAF-2DAwassignificantlydiminishedinvesselsfrom B1−/−andB2−/−whencomparedtoWTsamples,indicatingthat

basalNOproductionwasdecreasedinmesentericarteriolesfrom bothstrains(Fig.5B).

3.4. NOSactivityinmesentericvessels

TheNOSactivitywasassessedinhomogenatesofmesenteric vesselsbybiochemicalconversionofl_{-[3H]arginineto}l_-[3H]

cit-rullineinpresenceofsubstrateandco-factors.Surprisingly,instead oftheexpectedreduction,totalNOSactivity(Ca2+_{-dependent)was}

enhancedinmesentericvesselsfromB1−/−andB2−/−when

com-paredwithWTsamples,asrepresentedinFig.6.TheincreaseofNOS activityinvesselsfromB1−/−andB2−/−probablyisattributedto

increaseinactivityofeNOSornNOS,sinceexperimentsperformed inabsenceofCa2+_{todetermineiNOSactivity(Ca}2+_{-independent)}

showedsimilarresultsamongstrains.

0 1 2 3 4 5

*

B1 -/-WT

(-) Ca2+ (+) Ca2+

B2

-/-*

NOS activity (pmol/mg.min)

Fig.6.BarsgraphshowstheNOSactivityinpresenceorabsenceofCa2+_to

evalu-atetotalNOSactivityandinducibleNOSactivity,respectively,inhomogenatesof

mesentericvesselsfromWT,B1−/−andB2−/−.Resultsaremean±S.E.M.,n=4–5for

eachgroup.*P<0.05vsWT.

4. Discussion

TheadventofpotentandselectiveB1andB2receptor

antago-nistshaspermittedtoassesstheroleofkininsinseveralbiological systems;however,receptorantagonistsarenotdevoidof unspeci-ficity. The recent development of genetically engineered mice lackingthekininB1andB2receptorhasallowedtheopportunityto

investigatethephysiologicalroleofthekallikrein–kininsystemin absenceofpharmacologicalinterventions.Byanalyzingtheeffect ofvasoactiveagentsinmesentericarteriolesandmeasuring cir-culatingandtissueNOproduction,wefindseveralevidencesthat targeteddeletionofkininB1orB2receptorimpairs

endothelium-mediatedvasodilationbyreducingNObioavailability.

Firstly,we observedthat B2−/− arterioles exhibitincrease in

basalperfusionpressureincomparisontoWTandB1−/−.Although

mostof thestudieshave reportedthatB2−/− are normotensive

[1–3,11,12,26,35,37,39],thesemiceappeartoexhibitexaggerated responsestohypertensivestimuli[3,11,12,15,20,21].Thus,even withoutanessentialroleinbloodpressureregulation,B2

recep-torisclearlyrelatedtomodulationofvasculartonusandcontrolof regionalbloodflowtotheorgans.

ConsideringthatvasodilationinducedbyAChisdirectly depen-dentonendothelialNOrelease[17]andthatrelaxatingeffectof SNPisattributedtodirectNOdeliveryonthesmoothmuscle[8], ourresultsdemonstrateasevereimpairmentintheendothelialNO –dependentvasodilationinmesentericarteriolesfrombothB1−/−

andB2−/−.Thisfindingisinagreementwithpreviousdatashowing

thatthevasodepressorresponsetoinjectionofAChwasshiftedto therightinB2−/−[2].Inthepresentstudy,wedemonstratedforthe

firsttimethatimpairedvascularresponsetoAChisalsopresentin theB1−/−mice.

Contrastingin partwithourresults,a preservedresponseto AChin B2−/−mesentericvesselshasbeenpreviouslyrelatedby

pre-contractingagents,Krebscompositionandenzymaticblockers orotherinhibitorsaddedtotheperfusion. Inthepresentstudy, flowvelocity waschosenonthebasis of itsability toinducea sustainedandsub-maximalvasoconstrictiontoNE(10␮mol/L),in theabsenceofotherdrugs.Vasodilatoragentsweretestedin arte-riolessubmittedtoanapproximate80mmHgincreaseinperfusion pressure,and,inthis case,ACh(10nmol)evoked avasodilation around 12mmHg, confirming that preparations were able to dilate.

In order to confirm whether a possible impairment in NO bioavailability in B1−/− and B2−/− couldbe responsible for the

reducedAChresponse,weanalyzedplasmaticNOlevelsand vas-cularNO generationin both strains. Asexpected, we observed a significant reduction on circulating NO levels and basal NO releaseinmesentericarteriolesfromB1−/−andB2−/−.Similarly,

studies have described lower nitrite/nitrate plasma levels [18] and reduced renal nitrite excretion [35] in B2−/− when

com-paredtoWT mice.Moreover,induced hypertension bychronic NOsynthesisinhibitionis lesspronouncedinB2−/− when

com-paredtoWTresponses[20].Therefore,B2receptordeletionmay

severelyinterfere withNO bioavailability. Our data show that, besidesB2,B1receptorsarealsoinvolvedinbasalandstimulated

NOmetabolism.

Reduction in NO levels can occur through several potential mechanisms,suchasreducedNOSenzymaticactivityorincreased NOinactivation [29]. Consideringthat thebioavailability of NO is largely dependenton NOS, we analyzed theNOS activity in mesentericvesselsbybiochemicalconversionofl-[3H]arginine

tol-[3H]citrulline inpresenceofsubstrateand co-factors.

Sur-prisingly, instead of the expected reduction, total NOSactivity (Ca2+_{-dependent) waselevatedin homogenatesof vesselsfrom}

B1−/−andB2−/−.Theseresultsarepartiallyinagreementwith

Bar-bosaetal.[4],thatobservedadecreaseinrelaxatingeffectofSNP instomachfundusfromB1−/−,despiteincreaseiniNOSactivity

andcGMPlevels.Thesefindingsindicatethat,atleastinpresence ofsupplementationwithexogenoussubstrateandco-factors,NOS frombothB1−/−andB2−/−isfunctional.

Thepresentdatadonotgivesupportforexplainingthe contrast-ingresultsaboutdecreasedNOlevelsaccompaniedbyenhanced NOSactivity in kinin knockout mice. One possible mechanism responsibleforthiscouldbethefactthatuncouplingofNOSinduces NOS-derivedproductionofsuperoxideanionandhydrogen perox-ide[14,36].Inthiscase,reducedNObioavailabilityinB1−/− and

B2−/−couldberelatedtoincreaseinvascularoxidativestress

asso-ciatedwithelevatedsuperoxideanionproductionandconsequent NOinactivation.Infact,superoxideanionrapidlyinactivatesNOto formthehighlyreactiveintermediateperoxynitrite,which repre-sentsamajorpotentialpathwayofNOreactivityanddegradation [5,36].Nevertheless,thegenerationofreactiveoxygenspeciesin B1−/−andB2−/−micehasnotyetbeenconsistentlyanalyzedand

furtherstudieswillberequiredtotestthishypothesis.

5. Conclusion

Inconclusion, thepresent studydemonstrated that targeted deletion of B1 or B2 receptor gene in mice induces important

alterationsinthevascularreactivityofresistancevesselsandNO metabolism.Thesevereimpairmentintheendothelial-mediated vasodilationaccompaniedbydecreasedNObioavailability,despite theaugmentedNOSactivity,stronglyindicatesanexacerbationof NOinactivationinB1−/−andB2−/−.Thepresentdataprovide

valu-ableinformationinordertoclarifytherelevanceofkininreceptors inregulatingvascularphysiologyandmaypointtonewapproaches regardingitscorrelationwithendothelialdysfunction,oxidative stressandNOavailability.

Sourcesoffunding

SupportedbygrantsfromFundac¸ãodeAmparoàPesquisado EstadodeSãoPaulo(FAPESP2007/59039-2,2008/06676-8), Con-selhoNacionaldeDesenvolvimentoCientíficoeTecnológico(CNPq) andCoordenac¸ãodeAperfeic¸oamentodePessoaldeNívelSuperior (CAPES).

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