ContentslistsavailableatSciVerseScienceDirect
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
Vascular
mechanisms
involved
in
angiotensin
II-induced
venoconstriction
in
hypertensive
rats
Rodrigo
Azevedo
Loiola
a,∗,
Liliam
Fernandes
b,
Rosângela
Eichler
a,
Rita
de
Cássia
Tostes
Passaglia
c,
Zuleica
Bruno
Fortes
a,
Maria
Helena
Catelli
de
Carvalho
aaDepartmentofPharmacology,InstituteofBiomedicalSciences,UniversityofSãoPaulo–Av.Prof.LineuPrestes,1524,SãoPaulo,SP05508-900,Brazil bDepartmentofBiologicalSciences,FederalUniversityofSãoPaulo–R.ArthurRiedel,275,Diadema,SP09972-270,Brazil
cDepartmentofPharmacology,FacultyofMedicineofRibeirãoPreto,UniversityofSãoPaulo–Av.Bandeirantes,3900,RibeirãoPreto,SP14049-900,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received22June2011
Receivedinrevisedform9September2011 Accepted10September2011
Availableonline16September2011
Keywords:
AngiotensinII Venoussystem Hypertension Angiotensinreceptors
a
b
s
t
r
a
c
t
ToinvestigatethevenoconstrictoreffectofangiotensinII(AngII)inspontaneouslyhypertensiverats
(SHR),weusedpreparationsofmesentericvenularbedsandthecircularmuscleoftheportalveins.
VesselsweretestedwithAngIIinthepresenceorabsenceoflosartan,PD123319,HOE140,L-NAME,
indomethacin,orcelecoxib.InthemesentericvenularbedofSHR,theeffectofAngII(0.1nmol)was
nearlyabolishedbylosartanandenhancedbyHOE140,indomethacin,andcelecoxib,whilePD123319
andL-NAMEhadnoeffect.Inportalveinpreparations,cumulative-concentrationresponsecurves(CCRC)
toAngII(0.1–100nmol/L)exhibitedalowermaximalresponse(Emax)inSHRcomparedtoWistarrats.AT1
receptorexpressionwassimilarinthetwostrains,whileAT2receptorlevelswerelowerinSHRportal
veinswhencomparedtoWistar.InSHRportalveins,losartanshiftedtheCCRCtoAngIItotheright,
whileindomethacinandHOE140increasedtheEmaxtoAngII.PD123319,celecoxib,andL-NAMEhad
noeffect.Takentogether,ourresultssuggestthatAngII-inducedvenoconstrictioninSHRismediated
byactivationofAT1receptorsandthiseffectmaybecounterbalancedbykininB2receptorandCOX
metabolites.Furthermore,ourdataindicatethattherearedifferentcellularandmolecularmechanisms
involvedintheregulationofvenoustonusofnormotensiveandhypertensiverats.Thesedifferences
probablyreflectdistinctfactorsthatinfluencearterialandvenousbedinhypertension.
©2011ElsevierInc.
1. Introduction
Thevenoussystemplaysanimportantroleincardiovascular homeostasissince it containsabout 65%of thetotal blood vol-ume[25].Thecapacitancepropertiesofthecardiovascularsystem areprimarilydeterminedbyveinsandvenules[24].Alterationsin venoustonusinducedbyhormones,peptidesordrugsinfluence directlythecardiacoutput,rightatrialpressure,and, therefore, cardiacperformance[32,37].
Increaseinvenoustonusandreductioninvenouscompliance
have beendescribed in various modelsof experimental
hyper-tension[10,16,27,30];however,dataarenotconclusiveandsome aspectsremaintobeelucidated.Moreover,methodological prob-lemsinvolvedinisolationofveinsandvenulescommitstudyof thisvascularbed.Inspiteofthis,isolatedportalveinandperfused mesentericvenularbedpreparationshavebeenusedin biologi-calresearchtoassesvenousfunctioninviewofthefactthatthese preparationsrespondtoavarietyofvasoactiveagents[32,37].Since
∗Correspondingauthor.Tel.:+551130917237;fax:+551130917237.
E-mailaddress:rodrigoazl@gmail.com(R.A.Loiola).
splanchnicvenousbedaccommodatesabout25%ofthetotalblood volume[32]andmesentericvascularbedcanbedestinationfor 10%ofcardiacoutput[37],investigationofvenousresponsesat thesevascular regionscouldyield important informationabout circulatoryfunctionandcontrolofbloodpressure.
Therenin-angiotensinsystem(RAS)isacoordinatedhormonal cascadeimportanttotheregulationofrenalsodiumexcretionand bloodpressure.AngiotensinII(AngII),themaineffectorpeptide ofRAS,bindstwomajorreceptors,AT1andAT2(AT1RandAT2R)
[38].ThevastmajorityofAngIIactionsoccurviatheAT1Rbinding, includingvasoconstriction,cellularproliferation,andactivationof thesympatheticnervoussystem[35].TheactionsofAngII medi-atedbyAT2Rarelesswellunderstood;however,itisknownthat AT2Rstimulationincludesvasodilation,inhibitionofcell prolifera-tionandmodulationofgrowthandremodelinginfetalvasculature
[3].
AngIIpromotesvasoconstrictioninisolatedmesentericvenules
[8,37]andportalveinpreparations[8,12,18,23]ofnormotensive rats; however,toour knowledge,the vascular effects ofAng II eitherinveinsorvenulesfromhypertensiveratshavenotbeen evaluated.Thus,theaimofthepresentstudywastoinvestigate theeffects of Ang II in themesenteric venularbed and in the
0196-9781 © 2011 Elsevier Inc. doi:10.1016/j.peptides.2011.09.011
Open access under the Elsevier OA license.
circularmuscleofportalveinsfromspontaneouslyhypertensive rats(SHR)byevaluatingtheparticipationofAT1RandAT2Ron AngIIresponse.Inaddition,weanalyzedtheroleof cyclooxyge-nase(COX)metabolites, nitricoxide(NO),and thekininB2Rin modulatingAngII-mediatedconstrictioninSHR.
2. Methods
2.1. Animals
MaleWistarandSHRsweighing200–300gwereobtainedfrom theInstituteofBiomedicalSciencesoftheUniversityofSãoPaulo (ICB-USP).Alloftheanimalexperimentswereconductedin accor-dancewiththeguidelinesoftheEthicCommitteeforResearchof ICB-USPandconformedtotheGuidefortheCareandUseof Lab-oratoryAnimalspublishedbytheUnitedStatesNationalInstitutes ofHealth(NIHpublicationNo.85-23,revised1996).Animalswere keptinatemperature-controlledroomona12hlight/12hdark cyclewith60%humidity,standardratchow,andwateradlibitum.
2.2. Mesentericvenularbedpreparation
Isolated perfusedmesenteric venularbed preparationswere performedaccordingtothemethodpreviouslydescribed[37].After ratswereanesthetizedwithchloralhydrate(450mg/kg,s.c.),a can-nula(PE50)wasinsertedretrogradely(1.0cm)intotheportalvein andthevascularmesentericbedwasdissectedoutatitsborder withtheintestine. Themesentericvenularbedwasperfusedat aconstantrateof2mL/minusingaperistalticpump(Miniplus3, Gilson,France)withKrebs-Henseleitsolution,pH7.4,at37◦Cin thepresenceof95%O2and5%CO2.Toconfirmtheviabilityof tis-sues,preparationswereexposedto90mmol/LKClfor5min.After 30minofwashingouttheKClwithKrebssolution,AngII(0.1nmol) wasadministeredinbolusinafinalvolumeof100Land vascu-larresponseswereevaluatedaschangesintheperfusionpressure (mmHg)(PowerLab4S;ADInstruments,Australia).
2.3. Portalveinpreparation
Isolatedportalveinringpreparationswereperformed accord-ingtothemethodpreviouslydescribed[2].Ratswereanesthetized withchloralhydrate(450mg/kg,s.c.),theportalveinwasexcised andconnectivetissuewasremoved.Ringsofportalveins(3–4mm length)weremountedunder0.5gofpassivetensioninanorgan bath(15mL)containingKrebs-Henseleitsolution,pH7.4,at37◦C with95%O2and5%CO2.Preparationswereallowedtoequilibrate for60min;duringthistime,thebathsolutionwaschangedevery 20min.Toconfirmtheviabilityoftissues,thepreparationswere exposedto90mmol/LKClfor5min.After30minofwashingout theKClwithnormalKrebssolution,acumulative-concentration responsecurve(CCRC)toAngII(0.1–100nmol/L)wasperformed andchangesinisometrictension(grams)wererecorded (Power-Lab4S,ADInstruments,Australia).CCRCwereanalyzedbyadata analysesprogram(Prism3,GraphPad)toevaluatetheEC50(the con-centrationofAngIIrequiredtoproduce50%maximumresponse) andmaximumresponse(Emax).Efficacyandsensitivityofportal veinringspreparationsinresponsetoAngIIwasdeterminedas EmaxandpEC50(−logEC50),respectively.
2.4. TreatmentsofMesentericvenousbedandportalveinring preparations
To investigate themechanisms involved in Ang II-mediated
contraction,preparationsof mesentericvenousbedsand portal veinringswereincubatedwithKrebs-Henseleitsolutioncontaining losartan(specificAT1Rantagonist,0.1mol/L),PD123319(specific
AT2Rantagonist, 0.1mol/L),HOE 140(specificB2Rantagonist, 20nmol/L) [13], indomethacin(COXinhibitor, 10mol/L), or L-NAME(inhibitorofNOsynthesis,10mol/L)30minbeforeAng IIinjection.Inaddition,a groupof SHRweretreatedwith cele-coxib (specific COX2 inhibitor, 10mg/kg) [20] administered by gavage3hbeforewerekilledandthemesentericvenularbedsand portalveinringswereprepared.Alltheconcentrationsof antag-onists/inhibitorsusedinexperimentswerebasedinpreliminary studiesperformedinourlaboratoryorintheliterature,when spec-ified.
2.5. RNAisolationandcDNAsynthesis
TotalRNAfromtheportalveinsofSHRandWistar ratswas extracted using Trizol reagent (Invitrogen, USA) in accordance withthemanufacturer’sprotocol.GenomicDNAwasdigestedwith DNaseIandfirst-strandcDNAwassynthesizedfrom2goftotal RNAinavolumeof20L,usingSuperScriptIIenzyme(Invitrogen); RNAseOut(Invitrogen)wasaddedtoprotectRNAduringthis pro-cess.Sampleswereincubatedat42◦Cfor50min,andthereaction wasstoppedbyheatingthesamplesat70◦Cfor15min.Samples werecooledat4◦Cfor10min.
2.6. Real-timePCR
Dilutedsamplesfromthereversetranscriptasereaction(1:10)
underwent real-time PCR amplification using Platinum SYBR
QPCR Supermix-UDG and specific primers for AT1R (forward,
CACTTTCCTGGATGTGCTGA;reverse,CCCAGAAAGCCGTAGAACAG;
141bp)andAT2R(forward,CTGCTGGGATTGCCTTAATGA;reverse,
AGCAGATGTTTTCTGATTCCAAAGT; 94bp). Gene expression of
GAPDH mRNA was used for normalization (forward,
GGTGCT-GAGTATGTCGTGGA;reverse,ACTGTGGTCATGAGCCCTTC;262bp).
Real-timePCRswereperformed,recorded,andanalyzedusingthe CorbettResearchsystem(CorbettLifeSciences,Australia).The con-ditionsforPCRwereasfollows:95◦Cfor2min,then40cyclesof 95◦Cfor15s,60◦Cfor1min,and72◦Cfor15s.Thespecificityofthe SYBRGreenassaywasconfirmedbymeltingpointanalysis. Expres-siondatawerecalculatedfromthecyclethreshold(Ct)valueusing theCt methodforquantification[22].Resultswereexpressed asfoldincreases.Allofthereagentsutilizedinthismethodwere purchasedfromInvitrogen(USA).
2.7. ImmunohistochemicalassayforAT1RandAT2R
Immunohistochemicalassayfordetectionofangiotensin
recep-tors in portal vein was realized according with the method
previously described [4]. The portal vein was fixed with 4%
paraformaldehyde(PFA)solutionfor6handimmersedinsucrose solution (30%) for 12h. After that, portal vein was embedded inmediumforcryosectioning,cutinto8mthicksectionswith
a Leica CM 1850 cryostat (Leica Instruments, Germany), and
placed onslides. The vessels were incubated with rabbit anti-goatAT1RorAT2Rantibody(IgG;SantaCruzBiotechnology,USA) at 1:25 and 1:10dilutions, respectively, at 4◦C for 18h. Slides
were washed with phosphate buffered saline (PBS) and
Perfu s ion pres sur e (mmHg)
0:0 0:5 1:4 2:3 4:1
An g I I minutes 3:2 25 A B 20 15 10 5 0 Con trol mol/L) μ Losa rtan (0.1 mo l/L) μ PD 1 23319 (0.1 HO E 14
0 (20 nm ol/L ) mol /L) μ L-N AME (10 mo l/L) μ Indom eth acin (10 Cel eco
xib (1 0m g/kg ) 0 5 10 15 20 25
*
*
*
*
Ang II (0.1 nmol)
in c re a s e i n p e rf u s io n pres sure ( m mH g )
Fig.1. (A)RepresentativerecordingofperfusionpressurealterationelicitedbyinbolusinjectionofAngIIintheratisolatedmesentericvenularbed.Thearrowindicates drugapplication.(B)EffectofdifferentdrugtreatmentsonAngII-inducedvenoconstriction.Bargraphshowingtheeffectof0.1nmolAngIIinisolatedmesentericvenular bedsfromSHR,expressedasanincreaseinperfusionpressure(mmHg).Resultsrepresentmean±SEM,n=5–8foreachgroup.*P<0.05versuscontrol.
determinedbyopticdensitometrywiththeKS-300imageprogram (Carl-Zeiss,Germany).
2.8. Drugsandreagents
AngIIwasfromBachem-CA;HOE140,PD123319,L-NAME,and indomethacinwerefromSigma–Aldrich;losartanwasfromMerck Sharp&DöhmeandcelecoxibwasfromPfizer.
2.9. Statisticalanalysis
ComparisonsweremadebyANOVAfollowedbyTukey-Kramer
testorStudent’st-testwhenappropriate.Valueswerereportedas
mean±standarderrorofmean(SEM).Statisticalsignificancewas setasP<0.05.
3. Results
3.1. EffectofAngIIonmesentericvenousbeds
Ang II injectioninduced a slight but consistent constriction in isolated mesenteric venules (Fig. 1A). No significant
differ-ences were observed between the responses of Wistar rats
(10.6±1.1mmHg;n=6)andSHR(10.6±1.3mmHg;n=8).
Basalperfusion pressure in mesenteric venousbed was not
modified by pre-incubation with different antagonists. In SHR
preparations, the constriction induced by Ang II was nearly
10 9.5 9 8.5 8 7.5 7 6.5
-log [Ang II] (mol/L)
0.2 0.4 0.6 0.8
A B
C
D
0 Tension (g )-10.0 -9.5 -9.0 -8.5 -8.0 -7.5
0.0 0.5 1.0 1.5 Wistar SHR
*
log [Ang II] (mol/L)
Increase in tension (g)
-10.0 -9.5 -9.0 -8.5 -8.0 -7.5 0.0
0.5 1.0 1.5
Control
Losartan (0.1μmol/L) PD 123319 (0.1μmol/L)
log [Ang II] (mol/L)
increase in tension (g)
#
-10.0 -9.5 -9.0 -8.5 -8.0 -7.5
0.0 0.5 1.0 1.5
Control
Indomethacin (10μmol/L)
*
HOE 140 (20 nmol/L)*
log [Ang II] (mol/L)
in cr eas e i n t e nsi o n (g )
abolished(P<0.05) byperfusionwithlosartan(0.8±0.2mmHg; n=7),whilePD123319andL-NAMEhadnoeffectatall.Incontrast, Ang II venoconstriction increased (P<0.05) after B2R blockade withHOE140(15.7±1.6mmHg;n=8),andalsoafterCOX inhi-bition with indomethacin (16.8±1.5mmHg, n=6) or celecoxib (18.8±1.4mmHg,n=5).TheresultsareshowninFig.1B.
3.2. EffectofAngIIonportalveinrings
Starting at 1nmol/L, Ang II contracted rings of portal vein
inaconcentration-dependent manner.TheEmaxwasreachedat
50nmol/L.Atconcentrationshigherthan100nmol/L,AngIIinduces rapiddesensitization(tachyphylaxis)inthispreparation(Fig.2A).
Fig.2Bshows the CCRCs toAng IIin both Wistar and SHR
portal vein preparations. The Emax to Ang II was significantly reduced(P<0.05)inSHR(0.62±0.09;n=6)comparedtoWistar rats(1.00±0.15;n=6).Nochangesweredetectedinresponseto KCl(Wistar:0.43±0.07g;n=7versusSHR:0.31±0.06g;n=8).
Pre-incubation of portal vein rings from SHR with
losar-tan shifted to the right the CCRC to Ang II [Control: pEC50:
8.62±0.05mol/L; n=6 versus Losartan: 7.95±0.06mol/L; n=4 (P<0.05)],whereasPD123319treatmenthadnoeffect(Fig.2C).
Pre-incubation with indomethacin and HOE 140 increased the
EmaxtoAngII[Control:0.57±0.09g,n=8versusIndomethacin: 1.21±0.14g,n=7andHOE140:1.01±0.08g,n=11(P<0.05)],as demonstratedinFig.2D.L-NAMEandcelecoxibdidnotalterthe AngIIresponse(datanotshown).
3.3. ExpressionofAT1andAT2receptorsinportalveins
To investigate a possible alteration in angiotensin receptor expressionbetweenSHRandWistarrats,wequantifiedthelevels ofAT1RandAT2RmRNAinsamplesfromportalveins.Theresults areshowninFig.3.
WhilenodifferencesweredetectedinAT1Rexpression,AT2R mRNAlevelsintheportalveinsamplesweresignificantlyreduced
0.0 0.5 1.0 1.5
Wistar SHR
*
AT1 AT2
re
c
e
ptor/
GA
PDH ra
tio
(a
rbitrar
y
u
n
its)
Fig.3.mRNAexpressionofangiotensinreceptors.BargraphshowingmRNA expres-sionofAT1RandAT2RinportalveinsfromWistarratsandSHR.Resultsfrom
real-timePCRrepresentmean±SEM,andwerenormalizedtoGAPDHmRNA expres-sion;n=5–7.*P<0.05versusWistarrats.
inSHR[0.34±0.13arbitraryunits(a.u.);n=7;P<0.05]compared toWistarrats(1.05±0.19a.u.;n=4)(Fig.3).
Immunohistochemical assays revealed similar results. Fig. 4
containsrepresentativeimagesofimmunohistochemicalstaining forAT1RandAT2RinSHRandWistarrats.AT1RandAT2Rwere present in the endothelium,vascular smooth muscle cells, and adventitiallayer.TherewasnodifferenceinAT1Rexpressionin SHRandWistar rats,whileAT2Rexpressionwasreducedinthe portalveinsofSHR(6.85±0.50a.u.;n=5;P<0.05)comparedto Wistarrats(9.08±0.25a.u.;n=5)(Fig.5).
4. Discussion
AngIIevokedaconsistentconstrictioninmesentericvenules and portal veinfromSHR. Inboth vascular preparations, losar-tanreducedornearlyabolishedtheAngII-mediatedconstriction,
while PD123319 did not modify this response. Ang II-induced
venoconstrictionwasmarkedlyincreasedbyindomethacin,while celecoxibwaseffectiveonlyinmesentericvenules.Whereas vascu-larresponsestoAngIIwereaugmentedbyHOE-140,L-NAMEhad noeffect.Byanalyzingourresults,wefoundthatAngII-induced
Fig.4.RepresentativeimageofimmunohistochemicalstainingofAT1RandAT2R400×magnification.AT1RstaininginportalveinfromWistarrat(A)andSHR(B).AT2R
0 2 4 6 8 10
SHR Wistar *
AT1 AT2
opt
ic d
e
ns
it
y
(a
rbit
rary u
n
its)
Fig.5.Proteinexpressionofangiotensinreceptors.Bargraphsshowing quantifica-tionofproteinexpressionofAT1RandAT2RinportalveinsofWistarratsandSHR,
expressedasarbitraryunits.Resultsrepresentmean±SEM,n=5foreachgroup. *P<0.05versusWistarrats.
constriction inmesentericvenules and portalveinfrom SHRis dependentofAT1RactivationandcounterbalancedbyCOX metabo-litesandkininB2R.
Severalaspectsofourresultsmaypointtoimportantdifferences
betweenthe venoussystem of normotensiveand hypertensive
rats.For instance, Ang II-induced constriction was significantly attenuatedinportalveinringsfromSHR.Besides,AngII-induced
venoconstrictionwasmediated byboth AT1Rand AT2R in
nor-motensiverats[8].Consideringthesefindings,wehypothesized thatdifferences betweenstrainscouldberelated tochanges in angiotensinreceptors expression.In fact, when AT1Rand AT2R wereevaluated,theAT2Rexpressionwassignificantlyreducedin portalveinfromSHR.Althoughseveralstudieshaveinvestigated theinfluenceofAT2Rinthevascularsystem,thefunctionalrole ofthissubtypeisnotcompletelyelucidated.Authorshave demon-stratedthatAT2Ractivationcaninducebothvasodilation[39]and vasoconstriction[34,40].Inthisregard,aconsistent vasoconstric-toreffectofAngIImediatedbyAT2Rinmesentericarteriolesof SHRhasbeendemonstrated[34,40].Moreover,AT2Ralso partici-patesofcontractileeffectofAngIIinportalveinpreparationsfrom normotensiverats[8,23].Probably,reductionofAT2Rlevelsin por-talveinfromSHRcanberesponsibleforthedecreasedresponse observedbyus.ThisresultcanindicatethatAT2Rplaysadistinct roleinthevasculatureofnormotensiveandhypertensiverats.
The basic hemodynamic disturbance in established
hyper-tensionis an elevation of total peripheralresistance, which is determinedmainlybyresistancevesselsfromarterialsystem.In fact,itiswellestablishedthathypertensivepatientshavesimilar valuesofcardiacoutputincomparisonwithnormotensivecontrols andtheelevatedbloodpressureismaintainedbyincreaseintotal peripheralresistance[16,26].Similarly,itwasdemonstratedthat cardiacoutputisnotalteredinSHR,agenerallyacceptedmodel forhumanessentialhypertension[31,36].Fromthispointofview, reducedAngIIresponseobservedinvenousfromSHRwouldnotbe influencingcardiacoutputcontrol.However,consideringthatAng II-inducedconstrictionis augmentedinarteriesfrom hyperten-siveanimals[6,11,13],reductionofAngIIresponsesinthevenous systemcouldberelatedtoacompensatorymechanism,avoiding exaggeratedincreaseinvenousreturnandalterationsincardiac output.
Thekalikrein–kininsystemplaysanimportantroleinthe main-tenanceof cardiovascularhomeostasis.In this regard,the kinin B2Rnullmicepresenthighsensitivitytohypertensivestimuli[1,5], impairmentofendothelium-dependentvasodilationanddecrease inNObioavailability [15].Moreover,studieshave indicatedthe existenceoffunctionalinteractionsbetweenangiotensinandkinin receptorsinvascularcells.Inthisrespect,Seyedetal.[29] demon-stratedthatAngII-mediatedvasodilationincoronaryvesselsfrom
dogs is dependent of B2R. This interaction was also observed
inarteriesfromnormotensive[9,19]andhypertensiverats[21].
ThepresentdatasuggestthatAngII-inducedconstrictionisalso counterbalancedbyB2Ractivationinvenulesandveinsfrom hyper-tensiverats.Therefore,thefinaleffectsresultedfromAngII,atleast onthesevascularbeds,shouldbeconsideredasacombinationof AT1Rsignalinginthepresenceofamodulatingactionelicitedby B2R.Furtherstudieswillrevealthephysiologicaland pathophysi-ologicalconsequencesofthisphenomenon.
WhereasCOXmetabolitesappear tocounterbalancetheAng
II-inducedvenoconstrictioninSHR,ourdatadonotsuggestthe par-ticipationofNOinthiseffect.Innormotensiverats,Fernandesetal.
[8]demonstratedthatNOcounteractstheAngII-induced venocon-striction,whileCOXmetaboliteswerenotinvolvedinthisresponse. Similarresultswereobservedinmesentericarteriolesfrom nor-motensiverats[19].IthasbeensuggestedthatalterationinNO metabolismisimplicatedinendothelialdysfunction,a common denominatorinessentialhypertension[7].Infact,severalvascular
bedsofSHRpresentimpairedendothelium-dependent
vasodila-tion[14,17,33].Inthisregard,increasedproductionofsuperoxide anioninvesselsofSHRhasbeenassociatedtoNOinactivationand elevationofthebloodpressure[28].Ourdatasuggestthat produc-tionofvasodilatoryeicosanoidsinvenousbedfromSHRrepresent analternativepathwaytoattenuatetheAngII-induced constric-tionatlowlevelsofNO.Moreover,COXmetabolitesprobablyare involvedinimpairmentofAngII-inducedconstrictioninportalvein
fromSHR.
Concluding,inSHR, theattenuationof Ang II-induced veno-constrictionbyCOXmetabolitesandB2Rmaybeinvolvedinthe localresponse toconserve thenormal cardiac outputin estab-lishedhypertension.Takentogether,ourdataindicatethatdifferent mechanismsareinvolvedintheregulationofvenoustonusof nor-motensiveandhypertensiverats.Thesedifferencesprobablyreflect distinctfactorsthatinfluencearterialandvenousbedin hyperten-sion.
Acknowledgments
Theauthors aregratefultoSonia Maria Rodrigues Leiteand MartaRodriguesdaSilvafromtheInstituteofBiomedicalSciences –USPfortechnicalassistance.
Financial support: This study was supported by grants the Fundac¸ãodeAmparoàPesquisadoEstadodeSãoPaulo(FAPESP), Coordenac¸ão de Aperfeic¸oamentode Pessoal de Nível Superior (CAPES),andPRONEX(CNPq).
References
[1]Alfie ME, YangXP, HessJF, Carretero OA.Salt-sensitive hypertensionin bradykinin B2 receptor knockout mice. Biochem Biophys Res Commun
1996;224:625–30.
[2]CamposAH,CalixtoJB.Mechanismsinvolvedinthecontractileresponsesof kininsinratportalveinrings:mediationbyB1andB2receptors.JPharmacol
ExpTher1994;268:902–9.
[3]CareyRM,PadiaSH.AngiotensinAT2receptors:controlofrenalsodium
excre-tionandbloodpressure.TrendsEndocrinolMetabol2008;19:84–7. [4]CeravoloGS,FernandesL,MunhozCD,FernandesDC,TostesRC,LaurindoFR,
etal.Hypertension2007;50:756–61.
[5]CervenkaL,Harrison-BernardLM,DippS,PrimroseG,ImigJD,El-DahrSS. Earlyonsetsalt-sensitivehypertensioninbradykininB2receptornullmice.
Hypertension1999;34:176–80.
[6] EndemannD,TouyzRM,LiJS,DengLY,SchiffrinEL.Alteredangiotensin II-inducedsmallarterycontractionduringthedevelopmentofhypertensionin spontaneouslyhypertensiverats.AmJHypertens1999;12:716–23. [7]FélétouM,VanhouttePM.Endothelialdysfunction:amultifaceteddisorder.
AmJPhysiol2006;291:H985–1002.
[8]Fernandes L, Loiola RA, Tostes RCA, Nigro D, Fortes ZB, Carvalho MHC. AngiotensinII-inducedvenoconstrictioninvolvesbothAT1andAT2receptors
andiscounterbalancedbynitricoxide.Peptides2005;26:2458–63. [9]FukadaSY,TirapelliCR,GodoyMA,OliveiraAM.Mechanismsunderlyingthe
endothelium-independentrelaxationinducedbyangiotensinIIinrataorta.J CardiovascPharmacol2005;45:136–43.
[11]GuidiE,HollenbergNK.Differentialpressorandrenalvascularreactivityto angiotensinIIspontaneouslyhypertensiveandWistar-Kyotorats. Hyperten-sion1987;9:591–7.
[12]HamonG,WorcelM.MechanismofactionofAngiotensinIIonexcitation con-tractioncouplingintheratportalvein.BrJPharmacol1982;75:425–32. [13]KostCK,JacksonEK.EnhancedrenalangiotensinIIsubtype1receptorresponses
inthespontaneouslyhypertensiverat.Hypertension1993;21:420–31. [14] LiJ,BukoskiRD.Endothelium-dependentrelaxationofhypertensiveresistance
arteriesisnotimpairedunderallconditions.CircRes1993;72:290–6. [15] LoiolaRA,ReisFC,KawamotoEM,ScavoneC,AbdallaDS,FernandesL,etal.Role
ofvascularkininB1andB2receptorsinendothelialnitricoxidemetabolism.
Peptides2011;32:1700–5.
[16]LondonGM,SafarME,SimonAC,AlexandreJM,LevensonJA,WeissYA.Total effectivecompliance,cardiacoutputandfluidvolumesinessential hyperten-sion.Circulation1978;57:995–1000.
[17] LüscherTF,DiederichD,WeberE,Vanhutte PM,Bühler FR. Endothelium-dependent responses in carotid and renal arteries ofnormotensive and hypertensiverats.Hypertension1988;11:573–8.
[18]MathisonR.Actionsofneurotransmittersandpeptidesonlongitudinaland circularmuscleoftheratportalvein.JPharmPharmacol1983;35:34–7. [19] MouraRS,ResendeAC,EmilianoAF,TanoT,RibeiroACM,CorreiaML,etal.The
roleofBradykininAT2andAngiotensin1-7receptorintheEDRF-dependent
vasodilatoreffectofAngiotensinIIontheisolatedmesentericvascularbedof therat.BrJPharmacol2004;141:860–6.
[20]MuscaraNM,VergnolleN,LovrenF,TriggleCR,ElliotSN,AsfahaA,etal. Selec-tivecyclo-oxygenaseinhibitionwithcelecoxibelevatesbloodpressureand promotesleukocyteadherence.BrJPharmacol2000;129:1423–30. [21]OgnibeneDT,OliveiraPR,CarvalhoLCM,CostaCA,EspinozaLA,CriddleDN,etal.
AngiotensinII-mediatedvasodilationisreducedinadultspontaneously hyper-tensiveratsdespiteenhancedexpressionofAT2receptors.ClinExpPharmacol
Physiol2009;36:12–9.
[22] PfafflMW.Anewmathematicalmodelforrelativequantificationinreal-time RT-PCR.NucleicAcidsRes2001;29:2002–7.
[23]PeletC,MironneauC,RakotoarisoaL,NeuillyG,AngiotensinII.receptor sub-typesandcontractileresponsesinportalveinsmoothmuscle.EurJPharmacol 1995;279:15–24.
[24]Rothe CF. Reflex control ofveins and vascular capacitance. Physiol Rev 1983;63:1281–342.
[25]RotheCF.Meancirculatoryfillingpressure:itsmeaningandmeasurement.J ApplPhysiol1993;74:499–509.
[26]SafarME,LondonGM,LovensonJA,SimonAC,ChauNP.Rapiddestraninfusion inessentialhypertension.Hypertension1979;1:615–23.
[27]SchobelHP, SchmiederRE,GatzkaCD,MesserliFH.Acentripetalshiftin intravascularvolumetriggerstheonsetofearlycardiacadaptationin hyper-tension.JHypertens1993;11:S94–5.
[28]SchnackenbergCG,WelchWJ,WilcoxCS.Normalizationofbloodpressureand renalvascularresistanceinSHRwithamembrane-permeablesuperoxide dis-mutasemimetic:roleofnitricoxide.Hypertension1998;32:59–64. [29]SeyedN,Xu X,NasjlettiA, HintzeTH. Coronary kiningeneration
medi-atesnitricoxidereleaseafterangiotensinreceptorstimulation.Hypertension 1995;26:164–70.
[30] SimonG.Alteredvenousfunctioninhypertensiverats.CircRes1976;38:412–8. [31]Smith TL, Hutchins PM. Central hemodynamics in the developmental stageofspontaneoushypertensionintheunanesthetizedrat.Hypertension 1979;1:508–17.
[32] Sutter MC. The mesenteric portal vein in research. Pharmacol Rev 1990;42:287–325.
[33] Tesfamariam B, Halpern W. Endothelium-dependent and endothelium-independent vasodilation in resistance arteries from hypertensive rats. Hypertension1988;11:440–4.
[34] TouyzRM,EndemannD,HeG,LiJS,SchiffrinEL.RoleofAT2receptorsin
AngiotensinII-stimulatedcontractionofsmallmesentericarteriesinyoung SHR.Hypertension1999;33:366–72.
[35]TouyzRM,SchiffrinEL.Signaltransductionmechanismsmediatingthe phys-iologicalandpathophysiologicalactionsofangiotensinIIinvascularsmooth musclecells.PharmacolRev2000;52:639–72.
[36]WalshGM,TsuchiyaM,CoxAC,TobiaAJ,FrohlichED.Alteredhemodynamic responsestoacutehypoxemiainspontaneouslyhypertensiverats.AmJPhysiol 1978;234:H275–9.
[37]WarnerTD.Simultaneousperfusionofratisolatedsuperiormesenteric arte-rialandvenousbeds:comparisonoftheirvasoconstrictorandvasodilator responsestoagonists.BrJPharmacol1990;99:427–33.
[38]WatanabeT,BarkerTA,BerkB,AngiotensinII.andendothelium:diversesignals andeffects.Hypertension2005;45:163–9.
[39]YayamaK,OkamotoH.AngiotensinII-inducedvasodilationviatype2receptor: roleofbradykininandnitricoxide.IntImmunopharmacol2008;8:312–8. [40]YouD, LoufraniL, BaronC,Levy BI,WiddopRE, Henrion D.Highblood
