Rev. bras. farmacogn. vol.25 número4

w w w . s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

G-protein

activation

revealed

by

[

35

_S]-GTP␥S

_binding

_assay

_is

involved

on

the

antidepressant-like

effect

of

Hypericum

caprifoliatum

and

Hypericum

polyanthemum

cyclohexane

extracts

Alice

Fialho

Viana

_,

_Jean

_Costentin

_,

_Jean-Claude

_do

_Rego

_,

_Stela

_Maris

_Kuze

_Rates

a_{ProgramadePós-graduac¸ãoemCiênciasFarmacêuticas,UniversidadeFederaldoRioGrandedoSul,PortoAlegre,RS,Brazil}

b_{ServiceCommund’AnalyseComportementale,InstitutdeRechercheetd’InnovationBiomédicaleIRIB,FacultédeMédecineetPharmacie,UniversitédeRouen,RouenCedex,France} c_{CentreNationaldelaRechercheScientifique,France}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received30May2015 Accepted24June2015 Availableonline26July2015

Keywords: Hypericum

[35_{S]-GTP␥Sbindingassay}

UliginosinB Antidepressant Forcedswimmingtest Monoaminereceptors

a

b

s

t

r

a

c

t

Previousstudiesbyusdemonstratedtheantidepressant-likeandantinociceptiveeffectsoflipophilic extractsanddimericacyl-phloroglucinolsfromspeciesofthegenusHypericumnativetoSouthernBrazil. UliginosinBandHC1(anenrichedphloroglucinolfractionfromHypericumcaprifoliatum)areableto inhibitmonoaminesynaptosomaluptake withoutbinding tothemonoaminergic sitesonneuronal transporters,unlikeclassicalantidepressants.ThecurrentstudyaimedatinvestigatingtheactionofH. caprifoliatumCham.&Schltdl.andHypericumpolyanthemumKlotzschexReichardt,Hypericaceae, cyclo-hexaneextractsandtheirmaincomponent,HC1anduliginosinB,onGproteincoupledreceptorsbyusing the[35_{S]-guanosine-5}′_{-O-(3-thio)triphosphate([}35_{S]-GTP␥S)bindingassay,whichrevealstheGprotein}

activity.Theantidepressant-likeeffectofacute(oneorthreetreatmentswithin24h)andrepeated(five dayswithandwithoutathreedaywash-out)treatmentswiththecyclohexaneextractswasevaluated usingtheratforcedswimmingtest.The[35_S]-GTP

␥Sbindingtomonoaminesandopioidreceptors

stim-ulatedbyagonistswasperformedexvivoinbrainmembranesofratsacutelyorrepeatedlytreatedwith thecyclohexaneextracts.TheeffectofHC1andUliginosinBon[35_{S]-GTP␥Sbindingassaywas}

per-formedbydirectincubationwithbrainmembranesintheabsenceofagonists.Theirantidepressant-like effectwasevaluatedthroughthemiceforcedswimmingtest.Theextracts,HC1andUliginosinBshowed antidepressant-likeeffectintheforcedswimmingtest.Theacutetreatmentswithextractsincreasedthe [35_S]-GTP

␥Sbindingstimulatedbythemonoamines,whileafterfivedaysoftreatmentthe[35S]-GTP␥S

bindingwasreducedevenafterthreedaywash-out.TheseeffectsarenotduetoHC1orUliginosinB inter-actionwiththereceptors,sincedirectincubationwiththesephloroglucinolsdidnotaffect[35_S]-GTP␥S

bindingtomembranes.OurfindingsindicatethatH.caprifoliatumandH.polyanthemumextractsbring aboutadaptivechangesinmonoaminereceptors,whichreinforcestheirantidepressant-likeprofile.

©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

Naturalproductscanbeasourceofsubstanceswithinnovative mechanismsofaction(Rates,2001;Rollingeretal.,2006).In pre-viousworkswedemonstratedthatHypericumcaprifoliatumCham. &Schltdl.(HCP)andH.polyanthemumKlotzschexReichardt(POL), Hypericaceae,cyclohexaneextractshavepotentialantidepressant andantinociceptiveeffects(Vianaetal.,2003,2005;Steinetal.,

2012; Stolz et al.,2012, 2014a,b).H. caprifoliatumextracts and

itsenrichedphloroglucinolfraction(HC1)actonpre-synapseby

∗ Correspondingauthor.

E-mail:[email protected](S.M.K.Rates).

inhibitingmonoamineuptakebutdifferentlyfromantidepressants theydonotbindtothemonoaminesitesonneuronaltransporters

(Vianaetal.,2005).Furthermore,theantidepressantlike-effectin

theforced swimming test(FST) wasinhibited bydopamineD1

andD2receptorantagonists(Vianaetal.,2005).The

antinocicep-tiveeffectwasblockedbynaloxone(opioidreceptorantagonist)

(Vianaetal.,2003).POLanduliginosinBhadsimilareffects,

ulig-inosin B was able to inhibit synaptosomal monoamine uptake withoutbindingtotheirsitesonneuronaltransporters(Steinetal., 2012).Thepretreatmentwithdopaminereceptorantagonists,␣ -adrenoceptorantagonists,pCPA(inhibitorofserotoninsynthesis) andMK-801 (N-methyl-d-aspartic acidreceptorantagonist)

sig-nificantlypreventedtheHP4antidepressantandantinociceptive effects(Steinetal.,2012;Stolzetal.,2014a).Recently,Stolzetal.

http://dx.doi.org/10.1016/j.bjp.2015.06.008

SAL 0

HCP POL

Fig.1. EffectofT2(threetreatmentsduring24h),T3(twotreatmentsperdayduring fivedays)andT4(twotreatmentsperdayduringfivedaysfollowedbyathree dayswash-out)with90mg/kgofH.caprifoliatum(HCP),H.polyanthemum(POL)or saline(SAL1ml/kg)onratforcedswimmingtest.Dataarepresentedinmean±SEM (n=8rats/group).SignificantlydifferentvaluesweredetectedbyonewayANOVA followedbyposthocStudent–Newman–Keulstest:*p<0.05;**p<0.01;***p<0.001 ascomparedtotheirrespectivesaline(SAL)group.

(2014b)verifiedthatuliginosinBhassynergisticeffectwith

mor-phine, while with amitriptyline or clonidine the interaction is additive.Altogether,thesedataindicatethattheantidepressant and antinociceptive effects of the phloroglucinols from species ofHypericumnativetoSouthern Brazilinvolvetheactivationof severalneurotransmissionpathways,butdirectevidenceoftheir ability to activate G protein coupledreceptors (GPCR) are still lacking.

Theknowledgethat antidepressantagents affectGPCR

func-tion(Donati andRasenick, 2003; Avissar and Schereiber,2006;

Czysz et al., 2015) prompted us to investigate the effect of

H. caprifoliatum (HCP) and H. polyanthemum (POL) cyclohex-ane extracts on some GPCR implicated with the mode of actionofantidepressantandanalgesicdrugs byusingthe[35

S]-guanosine-5′_{-O-(3-thio)triphosphate([}35_{S]-GTP␥S)bindingassay,}

whichrevealstheGproteinactivity.Wetestedtheeffectofacute andrepeatedadministrationofHCP(H.caprifoliatum)andPOL(H. polyanthemum)cyclohexaneextractsonimmobilitytimeinFSTand onmonoamineandopioidreceptor-stimulated[35_S]-GTP␥S

bind-inginratbrainmembranes.Wehavealsoinvestigatedtheeffectthe phloroglucinolsderivatives,HC1anduliginosinBonmiceFSTand carriedoutthe[35_{S]-GTP␥Sbindingassayafterdirectincubationof}

thesecompoundswithbrainmembranes.

Materialsandmethods

Plantmaterialandpreparationoftheextract

TheaerialpartsofHypericumcaprifoliatumCham.andSchltdl., Hypericaceae,werecollectedin theregionof Viamão(29◦₅₇′_S; 50◦₅₉′_{W), and the ones from H. polyanthemum Klotzsch ex} Reichardt,Hypericaceae,werecollectedintheregionofCac¸apava doSul(30◦₅₄′_S;53◦₈₇′_{W).Thevoucherspecimensaredeposited} intheherbariumoftheFederalUniversityofRio GrandedoSul (ICN)(Bordignon,1400and 1429,respectively).Plant collection wasauthorizedby Conselho deGestão do PatrimônioGenético – CGEN and Instituto Brasileiro do Meio Ambiente – IBAMA – 003/2008P02000.001717/2008.60.

HypericumextractswerepreparedasdescribedbyVianaetal.

(2005)andSteinetal.(2012).Thephloroglucinolderivatives,HC1

fromH.caprifoliatumanduliginosinB(hereinnamedHP4)from H.polyanthemum wereisolatedbymeansof thinlayerand col-umnchromatographyonpreparativesilicagelGF254(Merck)using

0

SAL HC1 HP4

Fig.2.EffectofacutetreatmentwiththephloroglucinolsHC1(Hypericum caprifo-liatum)andHP4(H.polyanthemum)90mg/kgp.o.;orsaline(SAL10ml/kg)onmice forcedswimmingtest.Dataarepresentedinmean±SEM(n=10mice/group). Sig-nificantlydifferentvaluesweredetectedbyonewayANOVAfollowedbyposthoc Student–Newman–Keulstest:***p<0.001ascomparedtotheirrespectivesaline (SAL)group.

chloroform/hexane(3.5:1,v/v) aseluent (Nör etal.,2004;Stolz

etal.,2012).

Animals

AdultMaleSpragueDawleyrats,weighing(180–200g),were purchasedfromIFFA-CREDO/CharlesRiverLaboratories(Domaine desOncins,Saint-GermainsurL’Arbresle,France).AdultMaleCF1 mice(25–35g)fromFundac¸ãoEstadualdeProduc¸ãoePesquisaem Saúde(FEPPS-RS,Brazil)breedingcolonywereused.Theratswere housedbyfiveinMakroloncages(42cm×27.5cm×18cm);mice werehousedinplasticcages(17cm×28cm×13cm)ingroupsof eightmice.Allanimalswerekeptundera 12hlight/darkcycle (lightsonbetween7a.m.and7p.m.)inawellventilatedroom, atconstanttemperatureof22◦_±1◦_{C,withfreeaccesstostandard} certified rodent diet and tapwater. All the behavioral experi-mentswere performedaccordingto guidelinesof TheNational ResearchEthicalCommittee(publishedbyNationalHeathCouncil –MS,1998),whichareincompliancewiththeEuropean Commu-nitiesCouncilDirectiveof24November1986(86/609/EEC).The behavioralexperimentswereapprovedbyNationalCommission ofResearch Ethics –Brazil(projectapproval number 01-188at 07/26/2001).

[35_{S]-GTPSbindingassay}

Ratsweresubmittedtofourdifferenttreatmentregimenswith POLorHCP90mg/kg,p.o.:T1–onetreatmentbyoralgavage1h beforetheassays;T2–threetreatmentswithin24h(23,5and 1hbeforethesacrifice);T3–twotreatmentsperdayduringfive days;T4–twotreatmentsperdayduringfivedaysfollowedbya threedaywash-outbeforetheassays.Theextractsweredissolved inwaterwith5%polissorbate80toaconcentrationof90mg/ml, thevolumeofadministrationwas1ml/kg.

Onehourafterthelastadministration(T1,T2andT3)orthree dayslater (T4) theanimalswerekilledby decapitation.Frontal cortex, striata, hypothalamus and thalamus were removed and homogenizedin20volof0.32Msucrose.Thesamebrainstructures wereremovedfromnon-treatedratstobeusedascontrolandfor directincubationwithHC1orHP4(10−10_–10−6_M).Crude

–8 0 20 40 60 80 100

–7 –6

Log [DA] (M)

% stim

ulation

A

–5 –4 –3 –8

0 20 40 60 80 100

–7 –6

Log [5-HT] (M)

Saline HCP POL

% stim

ulation

C

–5 –4 –3

–8 0 20 40 60 80 100

–7 –6

Log [NA] (M)

% stim

ulation

B

–5 –4 –3 –9

0 20 40 60 80 100

–8 –7

Log [DAMGO] (M)

% stim

ulation

D

–6 –5 –4

Fig.3. EffectofT1(singleadministration)withHCP,POL(90mg/kg,p.o.)orsalineonagonist-stimulated[35_S]-GTP

␥Sbindingmeasuredexvivo.Membranespreparedfrom

ratstriatum(A),hypothalamus(B),frontalcortex(C)andthalamus(D)wereincubatedwithincreasingdosesofdopamine(A),noradrenaline(B),serotonin(C)orDAMGO(D) inthepresenceof0.1nM[35_{S]-GTP␥Sand10␮MGDP.DataarepresentedaspercentageofbindingstimulationoverGDPbasalstimulation(absenceofagonist).Mean±SEM}

fromfourseparatedexperimentscarriedoutinduplicate.

two centrifugationswerecombinedand centrifuged(17,000×g for30min).TheresultingpelletwasthensuspendedinTrisbuffer (50mMTris/HCl,100mMNaCl,5mMMgCl2,and1mMEDTA,pH

7.4),sonicated,andcentrifuged(50,000×gfor10min).Thefinal proteinconcentrationwasmeasuredbythemethodofLowryetal.

(1951).

[35_{S]-GTP␥Sbindingassayswereperformedasfollows.}

Mem-branes(15–20␮g/100␮l)wereincubatedintheassaybuffer(Tris bufferaddedwith1mMdithiothreitol(DTT)and0.1% metabisul-phite) with 10␮M GDP (Sigma–Aldrich Co.), 10−4–10−8M of dopamine,noradrenaline,serotoninorDAMGO ([D-Ala2_,

N-Me-Phe4_,Gly5_{-ol]-Enkephalin)inthepresenceof0.1nM[}35_S]-GTP␥S

(1250Ci/mmol;Perkin-Elmer,Courtaboeuf,France)inatotal vol-ume of 1ml. Basal binding was assessed in the absence of monoaminesorDAMGOandpresenceofGDP(guanosine-5′_-O -(3-thio)diphosphate),andthenon-specificbindingwasassessedin thepresenceof10␮MGTP␥S(Sigma–Aldrich).Inparallel, incuba-tionswithHC1orHP4intheabsenceofmonoaminesorDAMGO weremade.Theentiremixturewasincubatedat25◦_Cfor2hand filteredthroughWhatmanGF/Bglassfiberfilters,whichhadbeen pre-soakedfor2hinTrisbuffer,andwashedthreetimeswith4ml ofice-coldTrisbuffer,usingaMilliporeSamplingManifold (Biller-ica,MA,USA).BoundradioactivitywasdeterminedinTri-Carb2100 TRliquidscintillationcounter(Packard)afterovernightextraction ofthefiltersin4mlofUltimaGoldscintillationfluid(Perkin-Elmer). Fourindependentexperimentsforeachassaywerecarriedoutin duplicate.

Forcedswimmingtest

AnotherbatchofratsweresubmittedtotreatmentsT2,T3and T4andevaluatedintheFST.Inbrief,beforethebeginningofthe treatment,ratsweresubmittedtoswimmingfor15minin30cm deepwaterwithtemperaturebetween23±2◦_C.Theambient tem-peraturewasapproximately22◦_{C.Attheendof theswimming} exposition,theanimalswereremovedfromthewaterandgently dried.Onehourafterthelasttreatment(T2andT3)orthreedays aftertreatmentdiscontinuation(T4),theanimalsweresubmitted toasecondswimmingexposure(5min),andtheirimmobilitytime wasmeasured.Inordertoevaluatethephloroglucinolsderivatives, HC1andHP4,antidepressant-likeeffect,wetestedthematthe sin-gledoseof90mg/kg(p.o.)onmiceFSTaccordingtoPorsoltetal.

(1977,1978)andCenturiãoetal.(2014).

Statisticalanalysis

The data were evaluated using one or two-way analysis of variance(ANOVA)followedbyStudent–Newman–Keulstestor Stu-dent’st-testdependingontheexperimentaldesign.p-Valuesless than0.05wereconsideredasstatisticallysignificant.

Thepercentstimulationof[35_S]-GTP

␥Sbindingwascalculated accordingtothefollowingformula:(S−B)/B×100%,whereSisthe stimulatedleveland Bisthebasallevel of[35_{S]-GTP␥Sbinding}

–8 0

–7 –6

Log [DA] (M)

–5 –4 –3 –8

0

–7 –6

Log [5-HT] (M)

Saline HCP POL

–5 –4 –3

–8 0 20 60

40 100

80 120 140 160

–7 –6

Log [NA] (M)

% stim

ulation

B

–5 –4 –3 –9

0 20 40 60 80 100

–8 –7

Log [DAMGO] (M)

% stim

ulation

D

–6 –5 –4

Fig.4.EffectofT2(threetreatmentsduring24h(23,5and1hbeforeassay)withHCP,POL(90mg/kg,p.o.)orsalineonagonist-stimulated[35_S]-GTP

␥Sbindingmeasuredex vivo.Membranespreparedfromratstriatum(A),hypothalamus(B),frontalcortex(C)andthalamus(D)wereincubatedwithincreasingdosesofdopamine(A),noradrenaline (B),serotonin(C)orDAMGO(D)inthepresenceof0.1nM[35_{S]-GTP␥Sand10␮MGDP.DataarepresentedaspercentageofbindingstimulationoverGDPbasalstimulation}

(absenceofagonist).Mean±SEMfromfourseparatedexperimentscarriedoutinduplicate.

Results

Forcedswimmingtest

All treatment regimens with HCP [one-way ANOVA: F(5,53)=17.46;p<0.05]andPOL[one-wayANOVA:F(5,54)=4.89; p<0.05] significantly reduced immobility time. The five day treatments(twiceaday)werenotmoreeffectivethantheclassical Porsolt protocol (T2). The antiimmobility effect remained even after three days wash-out (T4) although less pronounced than thoseobservedinT2andT5treatmenttwiceaday(T3)(Fig.1). Even though there are no statistically significant differences betweenPOL and HCP treatments, it can beobserved that the POLeffectsareweakerthanthoseofHCP(Fig.1).BothHC1and HP4wereeffective inreducing miceimmobilitytime[one-way ANOVA:F(2,27)=12.36;p<0.001](Figs.1and2).

[35_{S]-GTPSbindingassay}

Exvivoexperiments

Thedifferenttreatmentregimensinducedcontrastingresults. TreatmentsT1andT2withHCPandPOLincreasedthemaximal effect(Emax)of[35S]-GTP␥Sbindinginducedbyallmonoamines

(Figs. 3 and 4)and did not affect DAMGO curvefeatures. The

monoaminepotency(EC50)toinduce[35S]-GTP␥Sbindingwasalso increased.

Repeatedtreatment(T3and T4)withHCPor POLinduced a reductionintheEmaxandEC50of[35S]-GTP␥Sbindingstimulatedby

monoamines(Figs.5and6).Thisreductionwassignificantforthe threemonoamineswhenmembraneswerepreparedonehourafter thelasttreatment(T3)andevenfollowingathreedaywash-out (T4).DAMGO-stimulated curveswerenot affectedbythe treat-ments.

Invitroexperiments

Inorder toverify whethertheeffects observedexvivowere relatedtodirectphloroglucinolsactiononreceptorfunctionality, weevaluatedthe[35_{S]-GTP␥SbindingstimulatedbyHC1andHP4}

intheabsenceofagonists.HC1andHP4werenotabletostimulate thebindingof[35_{S]-GTP␥StoanyoftheG␣coupledtoreceptorsin}

membranespreparedfromnon-treatedanimals(datanotshown) (Fig.7).

Discussion

–8 0

20 40 60 80 100

–7 –6

Log [DA] (M)

% stim

ulation

A

–5 –4 –3 –8

0 20 40 60 80 100

–7 –6

Log [5-HT] (M)

Saline HCP POL

% stim

ulation

C

–5 –4 –3

–8 0

20 40 60 80 100

–7 –6

Log [NA] (M)

% stim

ulation

B

–5 –4 –3 –9

0 20 40 60 80 100

–8 –7

Log [DAMGO] (M)

% stim

ulation

D

–6 –5 –4

Fig.5.EffectofT3(twotreatmentsperdayduringfivedays)withHCP,POL(90mg/kg,p.o.)orsalineonagonist-stimulated[35_S]-GTP

␥Sbindingmeasuredexvivo.Membranes

preparedfromratstriatum(A),hypothalamus(B),frontalcortex(C)andthalamus(D)wereincubatedwithincreasingdosesofdopamine(A),noradrenaline(B),serotonin (C)orDAMGO(D)inthepresenceof0.1nM[35_{S]-GTP␥Sand10␮MGDP.DataarepresentedaspercentageofbindingstimulationoverGDPbasalstimulation(absenceof}

agonist).Mean±SEMfromfourseparatedexperimentscarriedoutinduplicate.

classicalantidepressantsiscoherentwiththeirchemicalstructure; thesemoleculesdonothavenitrogenatomintheirstructure,which isacorefeatureforbindingtoneuronalmonoaminestransporters

(Appelletal.,2004).However,untilthismomentthemolecular

mechanismsbywhichextractsofHypericum speciesexerttheir antidepressant-likeeffectsremainsunclear.

Itisaconsensusamongthefieldofpsychopharmacologythat theincreaseofmonoaminelevelsinthesynapticclefcausedby uptakeinhibitionisonlythefirstfromaseriesofneurochemical modificationsinducedbyantidepressant.Thisincreaseevidently willleadtopostsynapticmodificationsatdifferentlevelsofthe signaling cascades (Schreiber and Avissar, 2003; Millan, 2006;

Stahl,2008).Since antidepressant agents affect GPCRs function

(DonatiandRasenick,2003;AvissarandSchereiber,2006;Czysz

et al., 2015) we decidedto investigatethe effect of H. caprifo-liatum (HCP) and H. polyanthemum (POL) cyclohexane extracts onmonoaminergicreceptorsresponseinratbrainbymeasuring agonist-induced[35_{S]-GTP␥Sbinding.}

Acuteandchronictreatmentswiththeextractsshowed oppo-siteresultsregardingagonist-induced[35_{S]-GTP␥Sbinding:while}

acutetreatments(T1andT2)increasedit,repeatedtreatments(T3 andT4)diminishedit.The[35_{S]-GTP␥Sbindingincreaseindicates}

proteinGactivationandcouldberelatedtoreceptorsresponse tothesuddenincreaseofmonoamineatthesynapticcleft(Lohse etal., 2008).The[35_{S]-GTP␥Sbindingreduction after5days of}

treatment(T3andT4)mayrevealaregulatorymechanisms result-ingfromthepersistentlyhighmonoamineslevelsatthesynaptic cleft(Hermans,2003;Stahl,2008),whichcouldalsoexplainthe

persistentantiimmobilityobservedintheFSTafter3daysof wash-out.Theseresultsareinagreementwithmanyexperimentalresults that show a reduction in [35_{S]-GTP␥S–G-proteincoupling after}

chronic treatmentwith several classes of antidepressant drugs and monoamine transporter blockers (Hensler, 2002; O’Connor

et al., 2005;Castro et al., 2008; Rossiet al., 2008;Vidal etal.,

2010).

Receptors,transportersandG-proteinsarenotquiescentplasma membraneproteins,manyevidencesupportthattheydynamically traffictoandfromplasmaticmembrane,bothconstitutivelyaswell asinresponse topsychostimulantexposure,receptoractivation andneuronalactivity(Melikian,2004;Chisarietal.,2007;Evans etal.,2010).TheGPCRdecreasedresponsivenessafterrepeated agonist treatmentcan occur through a variety of mechanisms, includingdesensitization,down-regulationand redistributionof cellsurfacereceptors(trafficking)(Ferguson,2001;Harrisonand

Traynor,2003;Hermans,2003;O’Connoretal.,2005).Theexact

mechanisms that causes neuronal changes following antide-pressanttreatments are yetunknown.Many studies showthat antidepressantsmaymodulatethedensityofmonoaminergic neu-rotransmitterreceptorsinbrains(GouldandManji,2002;Avissar

andSchereiber,2006).It appearsthatGTP-bindingpropertiesof

–8 0

–7 –6

Log [DA] (M)

–5 –4 –3 –8

0

–7 –6

Log [5-HT] (M)

Saline HCP POL

–5 –4 –3

–8 0 20 40 60 80 100

–7 –6

Log [NA] (M)

% stim

ulation

B

–5 –4 –3 –10 –9

0 20 40 60 80 100

–8 –7

Log [DAMGO] (M)

% stim

ulation

D

–6 –5 –4

Fig.6.EffectofT4(twotreatmentsperdayduringfivedaysfollowedbyathreedayswash-out)withHCP,POL(90mg/kg,p.o.)orsalineonagonist-stimulated[35_S]-GTP ␥S bindingmeasuredexvivo.Membranespreparedfromratstriatum(A),hypothalamus(B),frontalcortex(C)andthalamus(D)wereincubatedwithincreasingdosesof dopamine(A),noradrenaline(B),serotonin(C)orDAMGO(D)inthepresenceof0.1nM[35_S]-GTP

␥Sand10␮MGDP.Dataarepresentedaspercentageofbindingstimulation

overGDPbasalstimulation(absenceofagonist).Mean±SEMfromfourseparatedexperimentscarriedoutinduplicate.

G-protein(Hensler, 2002,2003;Pejchal etal.,2002; Gould and

Manji, 2002; Castro et al., 2008).Recently, Czysz et al. (2015)

showedthatantidepressants,likeS-citalopram,butnotthe inac-tiveisomerR-citalopram,inducetranslocationofthesubunitG␣ outoflipidrafts.

Regardingtheinvivo tests,theFSTis agood screeningtool, useful as a first approach for assessing antidepressant activity

(Petit-Demouliereetal.,2005).Atafirstglance,theactivityofthe

HypericumextractsandtheirmainphloroglucinolsonFSTseems nottobedirectlyrelatedtothe[35_{S]-GTP␥Sresults,sincedespite}

–9 0

20 40 60 80 100

0 20 40 60 80 100

Striata Hypothalamus Frontal cortex Thalamus –8

Log [HC1] g/ml

% stim

ulation

% stim

ulation

A

_B

–7 –6 –9 –8

Log [HP4] g/ml

–7 –6

Fig.7.Concentration–responseeffectofHC1(A)andHP(B)on[35_S]-GTP

␥Sbinding.ThedataarepresentedaspercentageofbindingstimulationoverGDPbasalstimulation.

beingeffectiveonFST,directincubationofmembraneswithHC1 anduliginosinBwithoutagonists stimulationdidnotinfluence [35_{S]-GTP␥Sbinding.Therefore,theeffectsobservedinthe[}35

S]-GTP␥SbindingaftertreatingratswithPOLandHCPcouldnotbe creditedtoanagonisteffectofuliginosinBorHC1on monoamin-ergicreceptors.Theseresultsarein agreementwithOzawaand

Rasenick(1989)andChenandRasenick(1995)whoobservedthat

antidepressantdrugsdidnothavedirecteffectonadenylylcyclase, aproteinmodulatedbyG␣subunit.Inaddition,antidepressants thatacutelyreduce immobilitytimein theFST,normallyshow theireffectsonGPCRonlyafter14–21daysoftreatment(Hensler,

2002;Pejchaletal.,2002;Shenetal.,2002;Castroetal.,2003).

Thus,itisreasonabletoconsiderthattheeffectsofacuteandthe 5daytreatmentscouldbeaconsequenceofmonoaminesincrease inthesynapticcleftfollowingtheuptakeinhibitioninducedbythe extracts,HC1anduliginosinB(Vianaetal.,2005;Steinetal.,2012). Ofnote,thelackofinfluenceover DAMGO-stimulated[35

S]-GTP␥Sbindingcurvessupportsthepropositionthattheextracts have a selective effect on monoaminergic system. This result alsocorroboratesaprevioushypothesisthattheextractspresent antinociceptiveactivityviaindirectactivation ofopioid system, sincethiseffectispreventedbynaloxone,buttheirphloroglucinols donotinhibit[3_{H]-naloxonebinding(Stolzetal.,2012).}

In conclusion, ourfindings indicate that,at doses for which antidepressant-likeactivityhasbeendemonstratedinanimal mod-els, H.caprifoliatum and H. polyanthemum extracts bring about adaptivechangesinmonoaminereceptors,whichreinforcestheir antidepressant-likeprofile.Thesechangesarenotduetoan ago-nistactionofHC1orHP4onmonoaminergicreceptors.Theyare likelytobearesultofmonoaminesincreaseinthesynapticcleft, inconsequenceofmonoaminesuptakeinhibition.Furtherstudies areneededtoelucidatewhichsignalingpathwaysareactivated bytheseextractsandcompounds,buttheresultssofarconfirm thatH.caprifoliatumandH.polyanthemumandtheirmaindimeric phloroglucinolderivativesconstitutepromisingagentstofindout newantidepressants.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Authorcontributions

AFV(PhDstudent)andJCR(ResearchEngineer)performedthe experiments.SMKRandJCcontributedonthetestsdesignandto criticalreadingofthemanuscript.Alltheauthorshavereadthe finalmanuscriptandapprovedthesubmission.

Acknowledgements

This work was supported by CAPES-COFECUB (418/03 and 656/09),FondsEuropéendeDéveloppementRégional(FEDER, Con-ventionN◦ _{35233)andINTERREG-TC2N.Wewouldliketothank} Prof.G.L.vonPoserand herstaffforprovidingtheextractsand phloroglucinolderivatives.

References

Appell, M., Berfield, J.L., Wang, L.C., Dunn III, W.J., Chen, N., Reith, M.E.A., 2004.Structure–activityrelationshipsforsubstraterecognitionbythehuman dopaminetransporter.Biochem.Pharmacol.67,293–302.

Avissar,S.,Schereiber,G.,2006.TheinvolvementofGproteinsandregulatorsof receptor-Gproteincouplinginthepathophysiology,diagnosisandtreatmentof mooddisorders.Clin.Chim.Acta366,37–47.

Castro,M.E.,Diaz,A.,DelOlmo,E.,Pazos,A.,2003.Chronicfluoxetineinduces oppo-sitechangesinGproteincouplingatpreandpostsynaptic5-HT1Areceptorsin ratbrain.Neuropharmacology44,93–101.

Castro, E.,Díaz,A.,Rodriguez-Gaztelumendi,A.,Del Olmo,E., Pazos,A.,2008.

WAY100635preventsthechangesinducedbyfluoxetineuponthe5-HT1A receptorfunctionality.Neuropharmacology55,1391–1396.

Centurião,F.B.,Braga,A.,Machado,F.R.,Tagliari,B.,Müller,L.G.,Kolling,J.,Poser,G., Wyse,A.T.,Rates,S.M.,2014.Studyofantidepressant-likeactivityofanenriched phloroglucinolfractionobtainedfromHypericumcaprifoliatum.Pharm.Biol.52, 105–110.

Chatterjee,S.S.,Nöldner,M.,Koch,E.,Erdelmeier,C.,1998.Antidepressantactivityof Hypericumperforatumandhyperforin:theneglectedpossibility. Pharmacopsy-chiatry31,7–15.

Chen,J.,Rasenick,M.M.,1995.ChronicantidepressanttreatmentfacilitatesGprotein activationofadenylylcyclasewithoutalteringGproteincontent.J.Pharmacol. Exp.Ther.275,509–517.

Chisari,M.,Saini,D.K.,Kalyanaraman,V.,Gautam,N.,2007.ShuttlingofGprotein subunitsbetweentheplasmamembraneandintracellularmembranes.J.Biol. Chem.282,24092–24098.

Czysz,A.H.,Schappi,J.M.,Rasenick,M.M.,2015.LateraldiffusionofG␣sintheplasma

membraneisdecreasedafterchronicbutnotacuteantidepressanttreatment: roleoflipidraftandnon-raftmembranemicrodomains. Neuropsychopharma-cology40,766–773.

Donati,R.J.,Rasenick,M.M.G.,2003.Proteinsignalingandthemolecularbasisof antidepressantaction.LifeSci.73,1–17.

Evans,B.A.,Sato,M.,Sarwar,M.,Hutchinson,D.S.,Summers,R.J.,2010. Ligand-directedsignallingat␣-adrenoceptors.Br.J.Pharmacol.159,1022–1038.

Ferguson,S.S.G.,2001.EvolvingconceptsinGprotein-coupledreceptor endocy-tosis:theroleinreceptordesensitizationandsignaling.Pharmacol.Rev.53, 1–24.

Gould,T.D.,Manji,H.K.,2002.Signalingnetworksinthepathophysiologyand treat-mentofmooddisorders.J.Psychosom.Res.53,687–697.

Harrison,C.,Traynor,J.R.,2003.The[35_{S]-GTP’Sbindingassay:approachesand}

appli-cationsinpharmacology.LifeSci.74,489–508.

Hensler,J.G.,2002.Differentialregulationof5-HT1Areceptor-Gproteininteractions

inbrainfollowingchronicantidepressantadministration. Neuropsychopharma-cology26,565–573.

Hensler,J.G.,2003.Regulationof5-HT1Areceptorfunctioninbrainfollowingagonist orantidepressantadministration.LifeSci.72,1665–1682.

Hermans,E.,2003.Biochemicalandpharmacologicalcontrolofthemultiplicityof couplingatG-protein-coupledreceptors.Pharmacol.Ther.99,25–44.

Lohse,M.J.,Hein,P.,Hoffmann,C.,Nikolaev,O.,Vilardaga,J-.P.,Bünemann,M.,2008.

KineticsofG-protein-coupledreceptorsignalsinintactcells.Br.J.Pharmacol. 153,124–132.

Lowry,O.H.,Rosenbrough,N.J.,Farr,A.L.,Randall,R.J.,1951.Proteinmeasurement withtheFolinphenolreagent.J.Biol.Chem.193,265–275.

Melikian, H.E., 2004. Neurotransmitter transporter trafficking: endocytosis recycling,andregulation.Pharmacol.Ther.104,17–27.

Millan,M.J.,2006.Multi-targetstrategiesfortheimprovedtreatmentof depres-sivestates:conceptualfoundationsandneuronalsubstrates,drugdiscoveryand therapeuticapplication.Pharmacol.Ther.110,135–370.

Müller,W.E.,Singer,A.,Wonnemann,M.,2001.Hyperforin-antidepressantactivity byanovelmechanismofaction.Pharmacopsychiatry34,98–102.

Nör,C.,Albring,D.,Ferraz,A.B.F.,Schripsema,J.,Pires,V.,Sonnet,P.,Guillaume,D., vonPoser,G.L.,2004.PhloroglucinolderivativesfromfourHypericumspecies belongingtotheTrigynobrathyssection.Biochem.Syst.Ecol.32,517–519.

O’Connor,K.A.,Porrino,L.J.,Davies,H.M.,Childers,S.R.,2005.Time-dependent changes in receptor/G-protein coupling in rat brain following chronic monoaminetransporterblockade.J.Pharmacol.Exp.Ther.313,510–517.

Ozawa,H.,Rasenick,M.M.,1989.CouplingofthestimulatoryGTP-bindingproteinGs toratsynapticmembraneadenylatecyclaseisenhancedsubsequenttochronic antidepressanttreatment.Mol.Pharmacol.36,803–808.

Pejchal,T.,Foley,M.A.,Kosofsky,B.E.,Waeber,C.,2002.Chronicfluoxetinetreatment selectivelyuncouplesraphe5-HT(1A)receptorsasmeasuredby[35_S]-GTP

␥S

autoradiography.Br.J.Pharmacol.135,1115–1122.

Petit-Demouliere, B., Chenu, F., Bourin, M., 2005. Forced swimming test in mice:areviewofantidepressantactivity. Psychopharmacology(Berl.)177, 245–255.

Porsolt,R.D.,Bertin,A.,Jalfre,M.,1977.Behavioraldespairinmice:aprimary screeningtestforantidepressants.Arch.Int.Pharmacodyn.Ther.229,327–336.

Porsolt,R.D.,Anton,G.,Blavet,N.,Jalfre,M.,1978.Behaviouraldespairinrats:anew modelsensitivetoantidepressanttreatments.Eur.J.Pharmacol.47,379–391.

Rates,S.M.K.,2001.Plantsassourceofdrugs.Toxicon39,603–613.

Rollinger,J.M.,Langer,T.,Stuppner,H.,2006.Strategiesforefficientleadstructure discoveryfromnaturalproducts.Curr.Med.Chem.13,1491–1507.

Rossi,D.V.,Burke,T.F.,Hensler,J.G.,2008.Differentialregulationofserotonin-1A receptor-stimulated[35_S]-GTP

␥Sbindinginthedorsalraphenucleusby

citalo-pramandescitalopram.Eur.J.Pharmacol.583,103–107.

Schreiber,G.,Avissar,S.,2003.ApplicationofG-proteinsinthemoleculardiagnosis ofpsychiatricdisorders.Expert.Rev.Mol.Diagn.3,69–80.

Shen,C.,Li,H.,Meller,E.,2002.Repeatedtreatmentwithantidepressants differen-tiallyalters5-HT1Aagonist-stimulated[35_S]-GTP

␥Sbindinginratbrainregions.

Neuropharmacology42,1031–1038.

Stahl,S.M.,2008.EssentialPsychopharmacology:NeuroscientificBasisandPractical Applications.CambridgeUniversityPress,Cambridge.