Cocaine administration increases angiotensin I-converting enzyme (ACE) expression and activity in the rat striatum and frontal cortex

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ContentslistsavailableatSciVerseScienceDirect

Neuroscience

Letters

jo u r n al h om epa ge :w w w . e l s e v i e r . c o m / l o c a t e / n e u l e t

Cocaine

administration

increases

angiotensin

I-converting

enzyme

(ACE)

expression

and

activity

in

the

rat

striatum

and

frontal

cortex

Bruna

Visniauskas

a

_,

_Juliana

_C.

_Perry

a

_,

_Vitor

_Oliveira

b

_,

_Fernanda

_M.

_Dalio

b

_,

_Monica

_L.

_Andersen

a

_,

Sergio

Tufik

a

_,

_Jair

_R.

_Chagas

a,c,∗

a_Departamento_de_{Psicobiologia,}_Universidade_Federal_de_São_Paulo_(UNIFESP),_Rua_Napoleão_de_Barros,_925,_04024-002,_São_Paulo,_SP,_Brazil b_Departamento_de_Biofísica,_Universidade_Federal_de_São_Paulo_(UNIFESP),_Rua_Pedro_de_Toledo,_669,_04039-032,_São_Paulo,_SP,_Brazil c_Departamento_de_Ciências_da_Saúde,_Universidade_Federal_de_São_Paulo_(UNIFESP),_Rua_Ana_Costa,_95,_11060-001,_Santos,_SP,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14April2011

Receivedinrevisedform

21September2011

Accepted20October2011

Keywords:

AngiotensinI-convertingenzyme(ACE)

AngiotensinII(AngII)

Centralnervoussystem

Cocaine

Frontalcortex

Striatum

a

b

s

t

r

a

c

t

SomecentraleffectsofcocaineadministrationseemtoberelatedtoangiotensinII(AngII)orits

metabo-lites.Nonetheless,itisstillanopenquestionwhetherornotthelevelsofangiotensinI-convertingenzyme

(ACE),themainAngIIgeneratingenzyme,aremodifiedbycocaineadministration.Toevaluatetheeffect

ofacuteandsubchroniccocaineadministrationonACEactivityandmRNAexpression,maleratswere

randomlyassignedtosalineorcocainegroup.Acuteandsubchroniccocaineadministrationinduceda

significantincreaseinACEactivityandmRNAexpressioninthefrontalcortexandstriatumbutnotinthe

hippocampus.TheseresultssuggestthatsomeoftheAngIIrelatedeffectsofcocaineuponthecentral

nervoussystemcanbemediatedbychangesontheexpressionandactivityofACEinthestriatumand

frontalcortex.

1. Introduction

Cocaineisoneofthemostcommonlyabusedillicitdrugs[18]. In2007,theestimatedworldwide totalnumberofpeople,aged 15–64yearsold,whousedcocainewas15.6–20.8million(0.4–0.5% ofthoseinthisagegroup)[40].

It is well-documented that systemic acute cocaine adminis-tration induces autonomic responses mediated by the central nucleusoftheamygdala(CeA)[39].Periphericinjectionofcocaine (7mg/kg)inducessignificantalterationsinanumberofbehaviors, suchasstereotyped,locomotor,rearingandanxiety-likebehaviors

[5].Inaddition,ithasbeenshownthatrepeatedadministrationof cocaineatasimilardoserange(10mg/kgfor5days)caninduce locomotorsensitizationinrodents[15],ananimalmodelof addic-tion[21,37].SakooriandMurphyusinga7.5mg/kgdoseshowed cocaine-associatedplacepreference[34].Moreover,Reicheletal. demonstrated theeffectsof low dosesofcocaine (5–7.5mg/kg) inamodifiedvariantoftheplaceconditioningtask,referredasa

∗Correspondingauthorat:DepartamentodePsicobiologia,UniversidadeFederal

deSãoPaulo,RuaNapoleãodeBarros,925,04024-002,SãoPaulo,SP,Brazil.

Tel.:+551121490155x189;fax:+551121490155.

E-mailaddress:jchagas1@gmail.com(J.R.Chagas).

‘reference–conditioning’procedurethatcomparedtwopotentially rewardingstimuli(highvs.lowdose)[31].Manyoftheseactivities arerelatedtodopaminere-uptakeblockade.However,thespecific molecularmechanismsinvolvedorwhetherthisactivationisdue tothedirecteffects ofcocaineondopaminergicneuronsand/or actionsonotherneuromodulatorysystemsarestill understudy

[11].

Dopamine release in thestriatum of the ratcan be directly affectedby angiotensinII (AngII) [13,28]or throughan Ang II metabolitelikeangiotensinIV(AngIV)[8,9,38].AngII,increases corticotropin releasing factor (CRF) release [1] an effect also induced by cocaine withdrawal [12]. Of note, angiotensin I-convertingenzyme(ACE,EC3.4.15.1),themainAngIIgenerating enzyme,isanessentialpartoftherenin-angiotensinsystem(RAS) presentinthebrain.Arecentreviewemphasizestheimportance andpossibleimplicationsofthecompleteRASinthebrain,where itactstoregulateanumberofphysiologicalprocesses(e.g. cardio-vascularmaintenance,memory,fluidintake,energybalance)[3]. AngiotensinI-convertingenzymeinhibitors(ACEi)areligandsthat formacomplexwithZn2+_at_the_active_site_of_ACE._ACEi_are_effective

inreducingbloodpressureinhypertensiveindividualsastheyblock theconversionofangiotensinItoAngIIandreducethe degrada-tionofbradykinin.AspointedoutbyMargolinetal.ACEiareable toincreasedopaminereleaseinthestriatum,aneffectprobably

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506 (2012) 84–88 85

mediatedbytheopioidsystem.AstheyindirectlycouldblockCRF releaseanddirectlyblockAngIIproduction,ACEihavebeen sug-gestedtobeusedinthetreatmentofcocaineabuse[27].Moreover, itwasobservedthatchronicadministrationofACEiincreasedthe turnoverofdopamineinthestriatumofrats[20].

Itis stillan openquestionwhetherornot thelevelsofACE aremodifiedbycocaineadministration,potentiallyleadingtoan increase in the generation of Ang II and its metabolites. In an attempttoanswerthisquestion,followingacuteandsubchronic cocaineadministrationinrats,weevaluatedACEactivityandmRNA expressionindifferentareas,inthecentralnervoussystem,known tohaveincreasedextracellulardopamineunderAngIIeffectorto belinkedtomotoractivity.

2. Materialsandmethods

MaleWistarrats(3monthsofage)wereobtainedfromCEDEME – Universidade Federal de São Paulo (UNIFESP). The animals werehousedinaroommaintainedat22◦_C_in_12:12_h_light–dark

cycle(lightsonat07:00a.m.andoffat19:00h)insidestandard polypropylenecages.Allproceduresusedinthepresentstudy com-pliedwiththeGuidefortheCareandUseofLaboratoryAnimals

[19].TheexperimentalprotocolwasapprovedbytheEthical Com-mitteeofUNIFESP(#1001/09).

Cocainehydrochloride(SigmaChemicalCo.,St.Louis,MO,USA) wasfreshlydissolvedinsalinesolution.Intraperitoneal(ip) injec-tionsweregivenataconstantvolumeof1mL/kg.Naivesubjects wererandomlyassignedto2treatmentgroups,salineandcocaine administration.A single dose of cocaine 7mg/kg or salinewas administeredintraperitoneallytotheratsat7:30a.m.only1day (acute)orduring5days(subchronic)atthesametime.Sixhours afterthesingledose(acute)or6hafterthefifthdose adminis-tration(subchronic),theanimalsweredecapitatedwithminimum discomfort.Twoadditionalgroups(withdrawalandtherespective control)receivedipdailyinjectionsofcocaine(7mg/kg)orsaline for5 consecutivedays andwere euthanized24hafterthefifth administration.Thedoseof7mg/kgwaschosenonthebasis of previousstudies[4,5].

Afterdecapitationthefrontalcortex,striatumandhippocampus wererapidlydissected forprotein ortotal RNA extraction (Tri-zol,InvitrogenLifeTechnologies,Carlsbad,CA,USA).Forprotein extraction,tissueswerehomogenizedinabuffercontaining50mM Tris–HCl,pH7.4,NaCl100mMand0.1%Triton.Homogenateswere centrifugedat1000gfor15minandthesupernatantwasfrozenat −20◦_C._The_protein_contents_of_the_samples_were_measured_by_the

Bradfordmethod[7]usingbovineserumalbuminasastandard. ACEactivityontissueextractswasdeterminedusingtheFRET peptideAbz-FRK(Dnp)P-OH(10␮M)aspreviouslydescribed[10]. ThereactionswerecontinuouslyfollowedinaGeminiXS fluorime-ter(MolecularDevicesCompany,Sunnyvale,CA,USA)measuring thefluorescenceatex=320nmandem=420nm.Measurements

wereperformedinduplicateandACEactivityvalueswerereported asnanomolarofsubstratehydrolyzedperminutepermilligramof protein(nMmin−1_mg−1_)._In_separate_control_experiments,_cocaine

concentrationsupto1mMwereaddedtoincubationinorderto verifythepossibilityofadirectactionofcocaineonenzymeactivity. ACE, ␤-actin and GAPDH mRNA expression in tissue were assessed by SYBR Green real-time PCR using 10ng of total cDNA, SYBR Green Universal Master Mix (Applied Biosys-tems, Carlsbad, CA, USA), and the following set of primers independently: ACE (5′_{CGGTTTTCATGAGGCTATTGG3}′ _and

5′_{TCGTAGCCACTGCCCTCACT3}′ _GenBank _accession _number

NM012544), ␤-actin (5′_{AGGCCAACCGTGAAAAGATG3}′ _and

5′_{CCAGAGGCATACAGGGACAAC3}′ _GenBank _accession _number

NM031144.2) and GAPDH (5′_{TGCCCCCATGTTTGTGATG3}′ _and

5′_{GCTGACAATCTTGAGGGAGTTGT3}′ _GenBank _accession _number

NM017008) [26]. ACE, ␤-actin and GAPDH mRNA expressions wereobtainedfromthecyclethreshold(Ct)associatedwiththe exponentialgrowthofthePCRproducts.Quantitativevaluesfor ACEmRNAexpressionwereobtainedbytheparameter2−Ct_,_in

whichCtrepresentsthesubtractionofthe␤-actinortheGAPDH CtvaluesfromtheACECtvalues.

Studentˇıs-ttestforindependentsamplesbygroupwasusedto analyzethedata.Valuesareexpressedasthe±standarderrorof mean(SEM).p<0.05wasconsideredstatisticallysignificant.

3. Results

Fig.1showsthatacutecocainetreatmentsignificantlyincreased ACEgeneexpressionintheratcortex(t:−2.99;df:12;p<0.01) andstriatum(t:−4.48;df:12;p<0.001)comparedtosaline treat-ment.SimilaralterationswereseeninACEmRNAaftersubchronic cocainetreatment(frontalcortext:−2,36;df:10;p<0.04and stria-tumt:−3.24;df:10;p<0.009).Thereweresignificantdifferences amonggroupsinACEexpressionrelativetoendogencontrols␤ -actinorGAPDH(datanotshown).ACEactivityvaluesareshownin

Fig.2.Acuteandsubchronicadministrationinducedasignificant increaseinACEactivityinthefrontalcortex(acutet:−10.24;df: 10;p<0.001andsubchronict:−4.69,df:11;p<0.001/Fig.2Aand D)andstriatum(acutet:−3.80;df:11;p<0.003andsubchronict: −3.53;df:11;p<0.005/Fig.2BandE).Inaseparatecontrol,there wasnoeffectofcocainealone,addedupto1mMintheincubation medium,onACEactivity(datanotshown).However,no signifi-cantdifferencewasobservedinACEactivityafter24hwithdrawal ofrepeatedcocaineadministration(frontalcortext:0.91;df:12; p=0.37;striatumt:0.11;df:13;p=0.91andhippocampust:−1.08; df:12;p=0.3;Fig.2G–I).Inaseparatecontrol,therewasnoeffect ofcocainealone,addedupto1mMintheincubationmedium,on ACEactivity(datanotshown).

Nosignificantchanges wereobtainedeitherfor ACEactivity (acutet: −1.54;df: 11;p=0.15 and subchronict: 0.85;df:11; p=0.41)ormRNA(acutet:−1.82;df:12;p=0.09andsubchronict: −1.78;df:10;p=0.10)intissueextractsfromthehippocampus.

4. Discussion

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86 506 (2012) 84–88

0,0 0,5 1,0 1,5 2,0 2,5

cocaine saline

A

C

E

m

R

N

A

e

x

pr

e

s

ion

0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0

cocaine saline

AC

E

m

R

N

A

e

x

p

re

s

io

n

*

ACUTE

SUBCHRONIC

*

Frontal cortex Striatum Hippocampus

Fig.1. PlotrepresentationofACEmRNArelativeamountafter(A)acuteandsubchronic(B)cocaine(7mg/kg,i.p.)administrationinthefrontalcortexrelativeto␤-actin(A)

*_p_<_0.02_and_(B)*_p_<_0.04,_striatum_relative_to_␤_-actin_(A)*_p_<_0.001_and_(B)*_p_<_0.007_and_hippocampus_relative_to_␤_-actin_(A)*_p₌_0.09_and_(B)*_p₌_0.19._Values_are_expressed

asthemean±standarderrorofmean(SEM).

Bothcocaineregimens(acuteandrepeated)producedthesame levelofresponsenotonlyattheACEactivitybutalsoatthemRNA expressioninthestriatumandinthefrontalcortex.Inthecaseof therepeatedregimen,itcanalsobeinterpretedasaresponsetothe lastcocaineinjectionandnottotherepeatedtreatment.Inlinewith thelatterpossibility,whenanothertimepointfurtherawayfrom theinjection(i.e.24h)wasconsideredinordertomeasure estab-lishedlongtermeffects,noeffectsonACEactivitywasobserved ineitherbrainarea.Both,striatumandthefrontalcortex,seemto berelatedtothebehavioralsensitizationphenomenonandtodrug addiction[17,23,25];Giventhat thedoserangeand durationof cocainerepeatedadministrationusedhereareeffectiveat produc-ingbehavioralsensitization[15],onewouldexpectthattheeffects ofrepeatedcocaineonACEactivityandmRNAexpressionwould alsosensitizeafterrepeatedadministration.Thusitisimportant tohighlightherethatanypossibleimplicationofthefindings pre-sentedincocainebehavioraleffectswouldneedfurtherbehavioral experimentstobecorroborated.

Althoughnotyetdemonstratedinnervoustissues,beyondits abilitytoformAngIIandprocessmanyactivepeptides,ACEalso actsasasignalingmoleculebyitselfatthecellmembrane, trig-geringintracellularphosphorylationevents[14]andcalciumentry

[16].Theserecentfindingsindicatethat,potentially,anincrease inACEexpressioncouldnotonlyaffectAngIIformation,activating angiotensinreceptorsbutalsoincreaseapossiblesignalingthrough ACEitself.

Invivo,ACEisabletohydrolyzebradykininand substanceP

[36].Opioidpeptides,corticotrophin[33,35],hemopressin[32]and amyloid␤-protein[29]werealsoshowntobeinvitroACE sub-strates.Opioidpeptides,likeendorphinsandenkephalins,areACE substrateswhosedynamicsareprobablyalteredbychangesinACE activitywithconsequencesinpainperception[2].Moreover, mod-ificationsonACEactivitycanpotentiallyresultinalterationsinthe metabolismoftheseneuropeptidesand invivocouldberelated tococaine addiction.ACEinhibitors have beensuggestedto be usedfortreatingcocaineaddictionsincetheywereableto stim-ulatethereleaseofdopamineintheCNS[20].Furtherstudiesalso pointtootherofACEinhibitorsintheCNSthatmayhavepotential applicabilityforthetreatmentofcocaineaddiction[24,27].

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506 (2012) 84–88 87

Fig.2. PlotrepresentationforACEspecificproteolyticactivityinthecontrolandintheacutecocainetreatedanimalsin:(A)frontalcortex(*_p_<_0.001),_(B)_striatum₍*_p_<_0.003)

and(C)hippocampus(p=0.15),subchroniccocainetreatedanimalsin:(D)frontalcortex(*_p_<_0.001),_(E)_striatum₍*_p_<_0.005)_and_(F)_hippocampus₍_p₌_0.41)_and_after₂₄_h

withdrawalofrepeatedcocainetreated:(G)frontalcortex(p=0.37),(H)striatum(p=0.91)and(I)hippocampus(p=0.30).Valuesareexpressedasthemean±standarderror

ofmean(SEM).

Acknowledgements

WearegratefultoDr.RobertoFrussaFilhoforhisvaluable sug-gestions.ThisworkwassupportedbygrantsfromAssociac¸ãoFundo deIncentivoàPesquisa(AFIP),Fundac¸ãodeAmparoàPesquisa –FAPESP(#98/14303-3),ConselhoNacionaldeDesenvolvimento Científico e Tecnológico – CNPq (#558924/2008-5 to JCP) and (#481104/2004-6toJRC).

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