Evidence that kinin B-2 receptor expression is upregulated by endothelial overexpression of B-1 receptors

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Peptides

jo u r n al h om ep ag 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

Evidence

that

kinin

B

2

receptor

expression

is

upregulated

by

endothelial

overexpression

of

B

1

receptors

Eliete

S. Rodrigues

a

_,

_Rafael

_F.

_Silva

a

_,

_Renan

_P.

_Martin

a

_,

_Suzana

_M.

_Oliveira

a

_,

_Clovis

_R.

_Nakaie

a

_,

Regiane

A. Sabatini

b

_,

_Vanessa

_F.

_Merino

c

_,

_João

_B.

_Pesquero

a

_,

_Michael

_Bader

a

_,

_Suma

_I.

_Shimuta

a,∗

a_Department_of_Biophysics,_Federal_University_of_São_Paulo,_São_Paulo_04023-062,_Brazil

b_Department_of_Nephrology,_Federal_University_of_São_Paulo,_São_Paulo_04023-062,_Brazil

c_Oncology,_Johns_Hopkins_University_School_of_Medicine,_Baltimore,_USA

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3May2011 Receivedinrevisedform 28December2012 Accepted2January2013 Availableonline8January2013

Keywords:

AngiotensinII Bradykinin des-Arg9_-bradykinin Kininreceptors ACE

a

b

s

t

r

a

c

t

Bradykinin(BK)anddes-Arg9_-bradykinin_(DBK)_of_{kallikrein-kinin}_system_exert_its_effects_mediated_by

theB2(B2R)andB1(B1R)receptors,respectively.ItwasalreadyshownthatthedeletionofkininB1R

orofB2Rinducesupregulationoftheremainingreceptorsubtype[10,12,16,28,36].Howeverstudieson

overexpressionofB1RorB2Rintransgenicanimalshavesupportedtheimportanceoftheoverexpressed

receptorbuttheexpressionofanotherreceptorsubtypehasnotbeendetermined[17,19,33].Previous

studydescribedamarkedvasodilatationandincreasedsusceptibilitytoendotoxicshockwhichwas

asso-ciatedwithincreasedmortalityinresponsetoDBKinthoracicaortafromtransgenicratoverexpressing

thekininB1R(TGR(Tie2B1))exclusivelyintheendothelium.Inanotherstudy,miceoverexpressingB1Rin

multipletissueswereshowntopresenthighsusceptibilitytoinflammationandto

lipopolysaccharide-inducedendotoxicshock.ThereforetheroleofB2Rwasinvestigatedinthethoracicaortaisolatedfrom

TGR(Tie2B1)ratsoverexpressingtheB1Rexclusivelyinthevascularendothelium.Ourfindingsprovided

evidenceforhighlyincreasedexpressionleveloftheB2Rinthetransgenicrats.Itwasreportedthatunder

endotoxicshock,theseratsexhibitedexaggeratedhypotension,bradycardiaandmortality.Itcanbe

sug-gestedthatthehighmortalityduringthepathogenesisofendotoxicshockprovokedinthetransgenic

TGR(Tie2B1)ratscouldbeduetotheenhancedexpressionofB2Rassociatedwiththeoverexpressionof

theB1R.

1. Introduction

Kininsarepotentinflammatorymediatorsandinduce contrac-tionandrelaxationinseveralvascularandnon-vascularsmooth muscles[4,24].Thekininsbelongtothekallikrein-kininsystem (KKS)involvedintherenalandcardiovascularregulation[4,13]. Bradykinin(BK:Arg1_-Pro2_-Pro3_-Gly4_-Phe5_-Ser6_-Pro7_-Phe8_-Arg9_), isanonapeptidehormonewhichmediatestheactionofthe con-stitutivelyexpressedkininB2 receptor(B2R).Ontheotherhand, thekininB1receptor(B1R)isaninducedreceptoranditseffectis mediatedbydes-Arg9_-BK_(DBK),_a_1–8_fragment_of_BK_[25]_._The expressionlevel of B1Ris verylow inhealthy tissues but high in inflammatory conditions or aftertime-dependent incubation

[13,15,25].

Geneticallyengineeredanimalshavebeeninbredtoallowa bet-terunderstandingofthefunctionofkininsandtherole ofeach subtypeofreceptors.Thereforeseveraltransgenicanimalshave

∗Correspondingauthor.Tel.:+551155724583;fax:+551155715780.

E-mailaddresses:sshimuta@unifesp.br,sshimuta@gmail.com(S.I.Shimuta).

beencreated,suchasmicedeficientinthekininB1R[21,28],in thekininB2R[5,9,10,12]andalsoinbothkininB1RandB2R[8],as wellasmiceoverexpressingtheB1R[17,19]ortheB2R[34].Ithas beenshownthatthelackofkininreceptorsmayaffectthe reactiv-ityofthevascularsmoothmusclepreparationsorcausechanges intheexpressionlevelofsomereceptorsofkallikreinkininsystem (KKS)andreninangiotensinsystem(RAS)inthesametissue[26].

Across-talkbetweentheRASandKKSisbasedontheeffect ofangiotensinI(AngI)convertingenzyme(ACE),whichis respon-sible for the cleavage of AngI into the potent vasoconstrictor angiotensinII(AngII)andofthevasodilatorBKintonon-active pep-tidefragments[4,6,37].TheACEenzymeis mostlyexpressedin theendothelialvascularsmoothmuscle,mainlyinthepulmonary arteries[4].

Severalstudieshavedemonstratedinteractionsbetweenkinin receptors and AngII type 1 receptor (AT1R). Therefore it was reportedthatspontaneousinteractionofB1RandB2Rincreasesthe abilityofB1RbutnotofB2Rtobestimulatedbyitsagonist[11]; het-erodimerizationbetweenB2RandAT1Rcausesincreasedactivation ofGproteinsignalingtriggeredbyAT1RbutnotbyB2R[1];AngII mayregulatetheexpressionofB2RmRNA[32],thatB2Rgeneisa

http://dx.doi.org/10.1016/j.peptides.2013.01.002

Open access under the Elsevier OA license.

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andKKSand theevidence forexpressionofAngII AT1Rprotein andmRNAinendothelialcells[18,22,23,31,35]providerationale forstudyingtheinteractionsbetweenAngIIandBKreceptorsin additiontotheassessmentaboutvascularreactivityofthekinin aswellastheexpressionlevelofB2Rintheaortaisolatedfrom transgenic(TGR(Tie2B1))rats.

2. Materialandmethods

2.1. Animals

Experimentswerecarriedoutusing300–350gSprague-Dawley ratsascontrol(WT)andoverexpressingB1R(TGR(Tie2B1)),[17] fromthe“CentrodeDesenvolvimentodeModelosExperimentais” (CEDEME)oftheUniversidadeFederaldeSãoPaulo(UNIFESP).The animalsweremaintainedonstandard ratchowat21–23◦_C _and

kepton12hlight:12hdarkcycleandallowedadlibitumaccess tofoodandwater.Theprotocolsusedinthisstudywerein accor-dancewithcurrentguidelinesforthecareoflaboratoryanimals andethicalguidelinesforinvestigationsapprovedbytheAnimal CareCommitteeofUNIFESP.

2.2. Isometricrelaxationandcontractionrecordings

Thoracicaortawere isolatedfrom rat, clearedof connective tissueand mounted asringpreparations into5ml organbaths. The ringsof aortawere bathed in carboxygenated (95% O2/5% CO2), and modified Krebs-Ringer solution: 144mM NaCl, 5mM KCl,1.1mMMgSO4,25mMNaHCO3,1.1mMNaH2PO4,1.25mM CaCland5.5mMglucoseat37◦_C _(pH_7.4)._Resting _tension_was

maintainedat 0.5gand thetissues wereleft to equilibrate for 90min,withfrequentchangingofbathingsolution.Thetissue via-bilitywasassessedwithaprimingdoseof80mMKCland1␮M norepinephrine(NE),asdescribedpreviouslyby[30].Followinga 90minwashoutandrecoveryperiod,changesintensionproduced bythestimulantsweremeasuredwithanisometrictransducer TRI201(Panlabs.l.,Cornella,Barcelona,Spain)throughan ampli-fierPowerlab4/30andsoftwareLabchartProV7(ADInstruments, ColoradoSprings,CO,USA).

Cumulative concentration–response curves were

con-structed for BK applying increasing concentrations (0.1nM to 1␮M) of the agonist. On the other hand non-cumulative concentration–responsecurveswereobtainedfor AngIandAng IIto avoid desensitization, as described previously by [3]. The presenceoftheendotheliuminthethoracicaortawasconfirmed pharmacologically by the acetylcholine-induced relaxing effect onaorticringspre-contractedwithNE1␮M.Somecurveswere obtainedinthepresenceandabsenceoflisinopril(1␮M),anACE inhibitor,pre-incubatedfor20minandinthepresenceofR-715, specificantagonistofB1R,sincethetissuewasisolatedfromthe animal.TheeffectofspecificblockersofB2R,HOE-140(1␮M),the nitricoxidesynthaseinhibitor,L-NAME(1mM)andthe cyclooxy-genaseinhibitor,indomethacin(1␮M)pre-incubatedfor20min weretestedonthemaximalresponseinducedbyBK.Curve-fitting analyses(GraphPad-Prismsoftware, SanDiego, California, USA) wereusedtodeterminetheapparentaffinityofagonistsinterms ofpD2,which isthenegativelogarithm oftheconcentrationof agonistthatproduces50%ofthemaximaleffect)andthemaximal effect(Emax)wascalculatedinrelationtotheeffectinducedby 1␮MNE,whichwasconsidered100%.

isolatedusingTRIzol®_reagent_(Invitrogen,_Carlsbad,_CA,_USA)._After purification,thepresenceofintactRNAwasverifiedonan ethid-iumbromide-stainedagarosegel.ThetotalRNAwassubmittedto reversetranscriptioninthepresenceof2.5ng/␮Lofrandom hex-anucleotidesand2.5␮Mofoligo(dT)20,200␮MofdNTP,10mMof MgCl2 and2unitsofSuperScriptTMIIIFirst-Strandreverse trans-criptase(Invitrogen,Carlsbad,CA,USA).

TodeterminetheexpressionlevelsofkininB2andAT1 recep-tors,andACE,real-timePCRwasperformedusing5␮lofsamples containing1:10dilutedcDNA.Eachreactionwascarriedoutwith 10␮LofTaqManUniversalPCRMasterMix2×(Applied Biosys-tems,FosterCity,CA,USA),1␮Lofeachpairofspecificprimersand aprobelinkedwithaTAMRAdyeandaFAMquencher.Theused primers were for B2R (reverse primer 5′ -CACCACGCGGCACAG-3′_, _forward _primer ₅′_{-ATCACCATCGCCAATAACTTCGA-3}′ _and

probe 5′_{-6FAM-CACCTCTCCGAACAGC-TAMRA-3}′_), _for _ACE

(reverse primer 5′_{-CCTGCTGTGGTTCCAGGTACA-3}′_, _forward

primer 5′_{-AACACGGCTCGTGCAGAAG-3}′ _and _probe, ₅′

-6FAM-CCTCCCAGAGTCCAGTCGCGTCA-TAMRA-3′₎ _and _for _AT

1 receptor (reverse primer 5′_{-CAGTGTCCACGATGTCAGAAATTTT-3}′_,

for-ward primer 5′_{-ACTTTCCTGGATGTGCTGATTCAG-3}′ _and _probe

5′_{-6FAM-CTGGGCGTCATCCAT-TAMRA-3}′₎_and_beta-actin

endoge-nouscontrol (reverseprimer 5′_{-GCCTGGATGGCTACGTACATG-3}′_,

forward primer 5′_{-GGCCAACCGTGAAAAGATGAC-3}′ _and _probe

5′_{-6FAM-CAGATCATGTTTGAGACCTT-TAMRA-3}′₎_and_Mili-Q_water

(MiliporeCorporation)to20␮L.

Thereal-timePCRswereperformedwithanABIPRISM®₇₀₀₀ sequencedetectionsystem(Applied)andcycleconditionswere: 50◦_C_for₂_min,₉₅◦_C_for₁₀_min,_followed_by₅₀_cycles_of₉₅◦_C_for

15s(meltingstep),60◦_C_for₁_min_{(anneal/extend}_step)._Increases

intheamountofreporterdyefluorescenceduringthe50 ampli-ficationcyclesweremonitoredusingSequenceDetectorsoftware (SDSversion1.6,AppliedBiosystems).Quantificationofthetarget amountwasperformedbymeasuringthethresholdcycle,CT,which isdefinedasthefractionalcyclenumberatwhichthefluorescence encountersafixedthreshold.Anormalizedvaluetoevaluatethe mRNAexpressionwascalculatedasthedifferenceinthethreshold cycle:theCTvaluesofreceptorminusCTofinternalstandard(␤ -actin),resultinginCT.SinceitisuncommontouseCTparameter asarelativeexpressionparameterduetothislogarithmic char-acteristic,the2−C_T _parameter_was_used_to_express_the_relative

geneexpressiondata[14].Finaldataareexpressedastheratioof thefold-changeinthetargetgeneinthetransgenicratoverthe fold-changeinthetargetgeneofcontrolrat.

2.4. ACEactivity

Thoracicaortaisolatedfromratwerequicklyharvested,rinsed, blotted,frozedandhomogenizedinTris–HClbuffer,pH7.0, con-taining50mMNaClandTween20,0.2%.Subsequently,thesamples werecentrifugedat1000g for10minand thesupernatant was frozenat−20◦_C._The_protein_contents_of_the_samples_were

mea-suredbythemethodofBradfordusingbovineserumalbuminas standard.TheACEactivitywasdeterminedusing Abz-FRK(Dnp)P-OH (Abz=ortho-amino benzoic acid; Dnp=ethylenediamine) as substratesfollowing themethodology previously described [7]. Theincreaseinthefluorescencewascontinuouslymeasuredina HitachiF-7000fluorimetersetatem=420nmandex=320nm andtheassayscarriedoutin96-wellplates(finalvolumeineach well=0.2mL).TheevaluationofthoracicaortaACEactivitywas per-formedat37◦_C_in_0.1_M_Tris–HCl_buffer,_pH_7.0,_containing_0.05_M

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Fig.1. EffectoftheoverexpressionofB1receptorsontherelaxationinducedbyBK inrataorta.Cumulativeconcentration–responsecurvesforbradykinin(BK)were determinedinthoracicaorticringsisolatedfromWTandtransgenicrat overex-pressingtheB1receptorspecificallyinthevascularendothelium(TGR(Tie2B1)).The isometricrelaxingresponseswerecalculatedinrelationtotheincreasedtonusofthe aortainducedby1␮Mnorepinephrine(NE),considered100%.Dataareexpressed asmeans±SDof5experiments.*SignificantlydifferentfromcontrolWTanimals (P<0.05).

wewant tosuppress(10␮ME64,1␮Mpepstatin,1mM PMSF, 100␮MTLCK,and100␮MTPCK).Beforestartingthereactionbythe additionof10␮MofAbz-FRK(Dnp)P-OH,thetissueshomogenates werepreincubatedfor5minintheassaybuffer,at37◦_C._To_define

thespecificityforACE,theassayswerealsoperformedinthe pres-enceofthecocktailofinhibitorsplus1␮Mofthelisinopril.The slope wasconverted into nMof substrate hydrolyzed/min.The measurementswereperformed intriplicateand theresultsare expressedasmeans±SD.

2.5. Drugsandprimers

BK,AngI,AngII,lisinopril,L-NAME,indomethacinandHOE-140 werepurchasedfromSigmaChemicalCo.(Dorset,U.K).R-715was agiftfromD.Regoli,UniversitédeSherbrooke,Quebec,Canada.

Concentratedsolutionsofpeptidesandotheragentswere pre-pared in water and kept at 20◦_C _until _they _were _used. _The

stocksolutionswereseriallydilutedwithKrebs–Ringersolution. OligonucleotideprimerandfluorogenicprobesetsforTaqmanTM Real-TimePCRweredesignedforkininreceptorsandbeta-actin usingAssays-by-DesignService(AppliedBiosystems).

2.6. Statisticalanalysis

Valuesareexpressedasmeans±SDand(n).TheStudentt-test wasusedtodeterminethestatisticaldifferences,withthelevelof significancesetasP<0.05.

3. Results

3.1. EffectofkininBKontheaorticringsisolatedfromwildtype andtransgenicratsoverexpressingtheB1R

RecordingsoftherelaxanteffectinducedbyBKwereobtainedby cumulativeincreasingconcentrationsoftheagonistintothoracic aorticpreparationsisolatedfromWTandratoverexpressingthe B1Rspecificallyintheendothelium,TGR(Tie2B1).Non-cumulative concentration–response curves induced by BK were not differ-entfromthecumulative concentrationcurves.Fig.1showsthe concentration-dependentrelaxationtoBKintheaorticrings iso-latedfromWTandTGR(Tie2B1)rats.Themaximalresponses(%) were21±2(4)forWTand50±5(5)forTGR(Tie2B1)rats.ThepD2 (-logEC50,concentrationoftheagonistthatinduces50%ofthe max-imalresponse)valueswere8.0±0.3(4)forWTand8.1±0.3(5)for TGR(Tie2B1).

Fig.2.EffectofspecifickininB1receptorantagonist,R-715,ontherelaxantresponse inducedbybradykinin(BK)inratthoracicaorta.EffectofR-715(1␮M)onBK inducedresponseinaortaisolatedfrom(A)WTand(B)transgenicratwith endothe-lialoverexpressionofkininB1receptor(TGR(Tie2B1)).Theresponseswerecalculated inrelationtotheeffectinducedby1␮Mnorepinephrine(NE),whichwasconsidered 100%.Dataaremeans±SDof3experiments.

3.2. Effectofkininreceptorantagonistsontherelaxantresponses inducedbyBKinthoracicaortaisolatedfromTGR(Tie2B1)rats

Toevaluatewhethertheenhancedrelaxantresponsesinduced byBKwerepartlyduetotheactivationofB1R,theringsofthoracic aortaisolatedfromFig.2A,WTandFig.2B,ratoverexpressingthe B1Rspecificallyinthevascularendothelium(TGR(Tie2B1))were preincubatedwith1␮MofR-715,specificinhibitorofB1R.Ascan beseeninFig.2,concentration–responsecurvesforBKintherat thoracicaortaweresimilarbetweenWTandTGR(Tie2B1).ThepD2 valuesforBKinthepresenceofantagonistwere7.8±0.1(3)forWT and7.8±0.2(3)forTGR(Tie2B1), whereasinpreparations with-outthepresenceoftheantagonistwere8.0±0.3(4)forWTand 8.1±0.3(5)forTGR(Tie2B1).Themaximalresponse(%)toBKin thepresenceof1␮MR-715was21±1(3)forWTand50±3(3)for TGR(Tie2B1)andinnon-treatedpreparationsthevalueswere21±2 (4)forWTand50±5(5)forTGR(Tie2B1).Ontheotherhandwhen 1␮MHOE-140waspre-incubated,BK(100nM)inducedresponse

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Fig.4. EffectofL-NAMEandindomethacinonbradykinininducedresponseinrat aorta.Relaxantresponsesinducedbybradykinin(BK,1␮M)inthepresenceorin theabsenceof1␮ML-NAMEor1␮Mindomethacin,pre-incubatedfor20minin ringsofthoracicaortaisolatedfromWTratandtransgenicratoverexpressingthe B1receptorexclusivelyintheendothelium(TGR(Tie2B1)).Therelaxantresponses werecalculatedinrelationtotheeffectinducedby1␮Mnorepinephrine(NE).The datashownarethemean±S.D.of3experiments.*Significantlydifferent(P<0.05) fromthecontrol(withoutinhibitors)and#_different_from_WT.

wastotallyinhibitedinrataortaisolatedfromWTandTGR(Tie2B1) asshowninFig.3.

3.3. EffectofL-NAMEandindomethacinonthe bradykinin-inducedrelaxation

Toverify iftheBK-inducedrelaxation wasmediated byNO, theinhibitor ofNOsynthaseactivitywastested.Pre-incubation with1mML-NAMEfor20mincompletelyblockedthemaximal relaxationinducedbyBKinthoracicringswithendothelium-intact isolatedfromWTratandTGR(Tie2B1).Ontheotherhand,asshown inFig.4,theresponsesinducedbyBKinbothpreparationswere not blocked by pre-incubation for 20min withcyclooxygenase inhibitorindomethacin(1␮M).

3.4. PharmacologicaldeterminationofACEactivityinthoracic ringsaortaisolatedfromtransgenicratsoverexpressingtheB1R

ThefindingthatthereactivitytoBKwasenhancedinthe trans-genickininB1Rknockoutmice[20]andthatACEactivitycanbe influencedbyB2RandB1R[2,27],ledustotesttheresponsiveness ofthethoracicaortatoAngIandtoBKinthepresenceoflisinoprilto evaluateapossiblechangeintheACEactivityinTGR(Tie2B1)rats.

Therole of ACEwastested ontherelaxingresponsestoBK usinglisinopril(1␮M)pre-incubatedfor30min.Underthis con-dition,thecurvesconcentration–responsestoBKwereobtainedin thethoracicaortaofWTandTGR(Tie2B1)rats.Fig.5showsthat thesigmoidaldoseresponsecurvesweresimilarinboth prepara-tions(WT,Fig.5AandTGR(Tie2B1),Fig.5B),incomparisontothose obtainedintheabsenceoftheinhibitor,indicatingnosignificant differenceinthesensitivityofBKtoACEactivityintheTGR(Tie2B1) rats.Moreover,todeterminetheactivityofACEinTGR(Tie2B1) rats, onthe conversion of AngI to AngII, contractile responses induced by AngI pre-incubated for 30minwith lisinopril were tested.Concentration–responsecurveswereobtainedincubating non-cumulativeconcentrationsofAngItoavoiddesensitization.In thepresenceofACEinhibitortherewassimilarinhibitionofthe responsesthroughoutalltestedconcentrationsoftheagonistin bothstrainsoftherats(WT,5CandTGR(Tie2B1),5D).ThepD2 val-uesexpressingthepotencyandthemaximalresponse(Emax)values arepresentedinTable1.

3.5. DeterminationofACEactivitywithAbz-FRK(Dnp)P-OH hydrolysis

TheACEactivitywasalsodeterminedusingaselective fluores-cencesubstrateassayforACEwithAbz-FRK(Dnp)P-OHassubstrate.

Fig.5. PharmacologicaldeterminationofACEactivityinthoracicaorta. Cumu-lative concentration–relaxant responsecurves for bradykinin(BK)in thoracic aorta isolated from (A), WT and (B), from transgenic rat overexpressing theB1 receptorspecificallyintheendothelium(TGR(Tie2B1)).Non-cumulative concentration–contractileresponsecurvesforangiotensinI(AngI)in(C),WTand in(D),TGR(Tie2B1)rats.Thecurvesweredeterminedinthepresenceorabsence ofACEinhibitorlisinopril(1␮M),pre-incubatedfor20min.Theisometricrelaxant responsestoBKandcontractileresponsestoAngIareexpressedaspercentageof theeffectof1␮Mnorepinephrine(NE),considered100%.Dataaremeans±SDof3 experiments.*SignificantlydifferentfromthecontrolWTanimals(P<0.05).

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Table1

EffectoflisinoprilontheapparentaffinityandmaximalresponsesinducedbyBKandAngIinratthoracicaorta.

BK BK+lisinopril AngI AngI+lisinopril

pD2 Emax pD2 Emax pD2 Emax pD2 Emax

WT 8.0±0.3(4) 21±2(4) 8.6±0.2(3) 45±3(3)* _7.9

±0.3(4) 41±8(4) 7.8±0.2(3) 13±0.8(3)*

TGR(Tie2B1) 8.1±0.3(5) 50±5(5)# _8.7

±0.4(3) 58±5(3)* _8.0

±0.3(4) 41±3(4) 7.8±0.3(3) 14±1(3)*

BKinducedrelaxantresponsesandcontractileresponsesinducedbyAngIinthepresenceorintheabsenceofACEinhibitorlisinopril(1␮M)inthoracicaortaisolatedfrom WTratandtransgenicratoverexpressingtheB1receptorspecificallyintheendothelium(TGR(Tie2B1)).ThepD2values(negativelogarithmoftheagonistconcentration(M) thatinduces50%ofthemaximalresponse)wereextractedfromtheconcentration–responsesigmoidlogisticcurves.Themaximalresponse(Emax)wascalculatedinrelation to1␮Mofnorepinephrine,whichwasconsidered100%.Dataaremeans±S.D.and(n)numberofexperiments.

*_Different₍_P_<_0.05)_from_BK_or_Ang_I_(without_inhibitor).

#_Different_from_WT.

Fig.6.FluorescenceassayofACEactivity.ACEactivityassaywasperformedusing aselectivefluorescentsubstrateforACE,Abz-FRK(Dnp)P-OH(ortho-aminobenzoic acid-FRK-(2,4-dinitrophenyl)P-OH)inthethoracicaortaisolatedfromWTratand transgenicratoverexpressingtheB1receptorspecificallyinthevascular endothe-lium(TGR)Tie2B1)).Dataaremeans±SDof3experiments.

Fig.7.RelativegeneexpressionofkininB2receptor,ACEandangiotensinIItypeI receptor.TheexpressionlevelsofB2receptor,ACEandangiotensinIItype1(AT1 receptor)weredeterminedinthoracicaortaisolatedfromwild-type(WT)ratand fromendothelialoverexpressedkininB1receptor(TGR(Tie2B1)).ThevaluesofmRNA expressionlevelswerequantifiedbyreal-timePCR.Thevaluesoftherelativegene expressionwerecalculatedas2−CT_parameter,_which_is_obtained_by_subtracting

theCT(thresholdcycle)ofgenetargetfromtheCTofinternalstandard.Finaldata areexpressedastheratiooffoldchangeofcDNAinthetransgenicrat(TGR(Tie2B1)) targetgeneversusthefoldchangeofcDNAincontrol.Thedataaremeans±S.D.of 3–5experiments.*Significantlydifferentfromthecontrolvalue(P<0.05).

3.6. ExpressionlevelofkininB2receptorsandofACE

TheexpressionlevelsofB2RweredeterminedbyrealtimePCR relativequantification,sincethemaximumeffectinducedbyBK inthetransgenicTGR(Tie2B1)ratswashigherthanintheWTrats. Furthermore,expressionlevelofACEwasevaluated.Fig.7shows theresultsaboutthelevelsoftheirexpression,which was cal-culatedbyfold-upchangeofthetransgenicratoverthecontrol group.TheexpressionlevelofB2Rincreasedaboutthreefoldsin theTGR(Tie2B1)ratwhereasthatofACEmRNAexpressionwasnot significantlydifferentfromthecontrolWTrats.

3.7. VascularreactivitytoangiotensinIIandexpressionlevelof AT1receptorinaortaisolatedfromTGR(Tie2B1)rat

Responsivenessof thethoracic aortic ringsto angiotensinII (AngII) induced contractile response was assessed to evaluate

Fig.8. EffectofendothelialoverexpressionofkininB1receptorsonthecontraction inducedbyAngIIinrataorta.Non-cumulativeconcentration–responsecurvesfor angiotensinII(AngII)weredeterminedinthethoracicaortaisolatedfromWTrat andtransgenicratoverexpressingtheB1receptorspecificallyintheendothelium (TGR(Tie2B1)).Theisometriccontractileresponsesareexpressedaspercentageof theeffectof1␮Mnorepinephrine(NE),considered100%.Dataaremeans±SDof5 experiments.*SignificantlydifferentfromthecontrolWTanimals(P<0.05).

any cross-talking betweenkinin and AT1 receptors under con-ditions where the expression level of B2R was shown to be increasedinTGR(Tie2B1)rat.Theconcentration-responsescurves wereobtainedusingnon-cumulativemannerforstimulationsto avoiddesensitization.Thedatashowthatthevascularreactivity toAngII(Fig.8)intheaorticringsfromTGR(Tie2B1)ratswasnot alteredwhencomparedtothatofWTrat.Themaximalresponse values(%)were31±4(4)forWTand30±2(5)forTGR(Tie2B1) andthepD2valueswere7.8±0.2(4)forWTand7.9±0.2(5)for TGR(Tie2B1).Inaddition,thedeterminationofAT1receptormRNA expressionrevealed thatin aortaoverexpressingtheB1 and B2 receptors,therewasnosignificantdifferencefromthecontrolWT rats(Fig.7).

4. Discussionandconclusion

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pawand edema induced by carrageenan and high susceptibil-ity toendotoxicshock induced by lipopolysaccharide[19]. The presentstudy showedthat B2Rwassurprisingly overexpressed intheendotheliumofthoracic aortafromTGR(Tie2B1)rat.This findingwasunexpectedsinceadownregulationshouldoccuras acounterregulatorymechanismforoverexpressionofB1R.Ithas beenreportedthatthelackofonekininreceptoriscompensated bytheup-regulationoftheothersubtype,asshowninthecaseof deletionofB2R[10,12,36]andofB1R[16,28].

In another study [28], lipopolysaccharide treatment caused enhancedB2RmRNAwhichwasfurtherincreasedinB1KOmice with increased mortality. Although some studies have been reportedaboutoverexpressionofB1R[17,19]orB2R[33]assessing theimportanceoftheoverexpressedreceptor,theexpressionof theotherremainingreceptorsubtypehasnotbeendetermined.

TheenhancedB2RmRNAexpressioninTGR(Tie2B1)ratwas cor-relatedwiththeincreasedresponsivenessofrataortatoitsagonist BK.ThefindingthattheabilityofACEtoconvertAngItoAngIIwas notreducedneithertheACEmRNAwasaltered,providedevidence thattheincreaseintheBKreactivitywasnotmodulatedbyACE activityduetothehighexpressionoftheB2R.Thisconclusioncould notconfirmaneffectofACE/kininB2RinteractionmodulatingACE activityaspreviouslydescribed[20,27].

Itisnoteworthythatwasfoundnoevidenceforincreased activa-tionofAT1RsincethevascularreactivitytoAngIIwasmaintainedin theaortaisolatedfrom(TGR(Tie2B1))rats.Thereforethehypothesis thataspontaneousheterodimerizationofAngIIandBKreceptors couldtriggertheAT1Ractivationwasnotconfirmedincontrastto thatpreviouslyreported[1].

Inconclusion,transgenicratsoverexpressingkininB1R exclu-sively in the endothelium of TGR(Tie2B1) rats were shown to overexpressthekininB2Randtocauseincreasedresponsiveness toBK. It was reported that after lipopolysaccharidetreatment, thesetransgenicratspresentedamorepronouncedhypotensive responseandmarkedbradycardiaassociatedwithincreased mor-talitywhencomparedtonon-transgeniccontrolrats[17].Itwas alreadyreportedthatB1RandB2Rwereupregulatedbyendotoxins andthatB2RmRNAwasfurtherincreasedinB1KOduringtheacute phaseofendotoxinshockinvolvingincreasedmortality[28]. There-forethemechanismbywhichB2RmRNAexpressionisincreased inratsoverexpressingkininB1Rneedsfurtherinvestigation.Our findingsupportsanimportantroleofB1andB2receptorsduring thepathogenesisofendotoxicshock.Fromthisstudyitcanbe sug-gestedthatoverexpressionandincreasedactivationofkininB2R couldbeinvolvedinthehighmortalityduringthepathogenesisof endotoxicshock,whereinB1Rexpressionishighlyinduced.

Acknowledgements

This study was supported by grants from São Paulo State ResearchFoundation(FAPESP):FAPESPN◦_{2009/08336-2;}_FAPESP

N◦_{2010/05255-9)}_and_by_the_Brazilian_National_Research_Council

(CNPqN◦_{300247/2010-9).}

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