Rev. bras. farmacogn. vol.26 número5

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w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Uliginosin

B,

a

natural

phloroglucinol

derivative

with

antidepressant-like

activity,

increases

Na

+

,K

+

-ATPase

activity

in

mice

cerebral

cortex

Ana

C. Stein

a

_,

_Liz

_G.

_Müller

a

_,

_Andréa

_G.K.

_Ferreira

b

_,

_Andressa

_Braga

a

_,

_Andresa

_H.

_Betti

a

_,

Fernanda

B. Centurião

a

_,

_Emilene

_B.

_Scherer

b

_,

_Janaína

_Kolling

b

_,

_Gilsane

_L.

_von

_Poser

a

_,

Angela

T.S.

Wyse

b

_,

_Stela

_M.K.

_Rates

a,∗

a_Programa_de_{Pós-graduac¸}_ão_em_Ciências_{Farmacêuticas,}_Universidade_Federal_do_Rio_Grande_do_Sul,_Porto_Alegre,_RS,_Brazil

b_Laboratório_de_{Neuroprotec¸}_ão_e_Doenc¸_as_{Metabólicas,}_Departamento_de_Bioquímica,_Universidade_Federal_do_Rio_Grande_do_Sul,_Porto_Alegre,_RS,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received23December2015 Accepted12April2016 Availableonline21June2016

Keywords:

UliginosinB

Antidepressant-likeactivity Na+_,K+_-ATPase

Veratrine

Voltage-gatedsodiumchannel

a

b

s

t

r

a

c

t

UliginosinB,aphloroglucinolisolatedfromHypericumpolyanthemumKlotzschexReichardt, Hyperi-caceae,hasantidepressant-likeeffectintheforcedswimmingtestinrodentsandinhibitsmonoamines neuronalreuptakewithoutbindingtotheirneuronalcarriers.StudiesshowedtheinvolvementofNa+_,K+

-ATPasebrainactivityindepressivedisorders,aswellasthedependenceofneuronalmonoaminetransport fromNa+_gradient_generated_by_Na+_,K+_-ATPase._This_study_aimed_at_evaluating_the_effect_of_uliginosin_B_on

Na+_,K+_-ATPase_activity_in_mice_cerebral_cortex_and_hippocampus₍₁_and₃_h_after_the_last_{administration)}

aswellastheinfluenceofveratrine,aNa+_channel_opener,_on_the_{antidepressant-like}_effect_of_uliginosin

B.Miceweretreated(p.o.)withuliginosinBsingle(10mg/kg)orrepeateddoses(10mg/kg/day,3days). Acuteadministrationreducedtheimmobilityintheforcedswimmingtestandtailsuspensiontestand increasedNa+_,K+_-ATPase_activity_in_cerebral_cortex₁_h_after_treating,_whereas_the_repeated_treatment

inducedtheantidepressant-likeeffectandincreasedtheNa+_,K+_-ATPase_activity_at_both_times_evaluated.

Nonetreatmentaffectedthehippocampusenzymeactivity.Veratrinepretreatmentpreventeduliginosin Bantidepressant-likeeffectintheforcedswimmingtest,suggestingtheinvolvementofNa+_balance

reg-ulationonthiseffect.Altogether,thesedataindicatethatuliginosinBreducesthemonoamineuptakeby alteringNa+_gradient.

Introduction

Basedonthewell-knownefficacyofHypericumperforatum(St. John’swortherb)forthetreatmentofmildtomoderatedepression

(Linde, 2009)ourgroup hasbeenstudying chemical and

phar-macologicalfeaturesofSouthBrazilianHypericumspecies(Daudt

etal.,2000;Ferrazetal.,2002;Viana,2007;Vianaetal.,2003,2005,

2006,2008;Steinetal.,2012).HypericumpolyanthemumKlotzsch

exReichardt,Hypericaceae,extractshaveshownantinociceptive

(Vianaetal.,2003;Haasetal.,2010)andantidepressant-likeeffects

inratsandmice(Steinetal.,2012).Itsmajorchemicalconstituents

∗ Correspondingauthor.

E-mail:stela.rates@ufrgs.br(S.M.Rates).

arethreebenzopyrans,namedHP1(6-isobutyryl-5,7-dimethoxy-2, 2-dimethyl-benzopyran),HP2 (hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran) and HP3 (5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran), and a phloroglucinol derivative,uliginosinB(VonPoseretal.,2006).UliginosinB(1) hasa dimericstructure consistingofphloroglucinolandfilicinic acidmoieties(Rochaetal.,1995;Nöretal.,2004;Duarteetal., 2014), and seems tobe responsible for theantidepressant-like effects observedin animalsbehavioral tests(Stein etal.,2012). Thiscompound showedantidepressant-like effectin theforced swimmingtestinmice,whichwaspreventedbytheimpairmentof themonoaminergicneurotransmissioninvivo;italsoinhibitedthe sinaptossomaluptakeofdopamine,noradrenalineandserotonin, butinterestinglyitdidnotinteractwiththeirrespectivesiteon neuronalcarriers(Steinetal.,2012).Thesefindingssuggestthat

http://dx.doi.org/10.1016/j.bjp.2016.04.005

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uliginosinBactsbyadistinctmechanismthantheclassical antide-pressantdrugs,whichactbyblockingmonoaminetransporters.

O

OH

OH HO

O

OH

1

Monoaminetransportersarelocatedintheplasmamembrane andaredrivenbytheelectrochemicalgradientofNa+_generated_by

Na+_,K+_-ATPase₍_Nelson_and_Lill,₁₉₉₄_)._It_has_been_shown_that_some

antidepressants,suchasamitriptyline,nortriptyline,imipramine anddesipramine,inhibittheNa+_,K+_-ATPase_activity₍_Carfagna_and

Muhoberac,1993;Sanganahallietal.,2000;ViolaandArnaiz,2007).

On theotherhand, dopamine,noradrenalineand serotoninare abletostimulatetheactivityof this enzyme(Violaand Arnaiz,

2007).

Na+_,K+_-ATPase_activity_is_essential_to_the_brain_normal_function,

sincetheflowofNa+_and_K+_ions_across_the_membrane_is_necessary

toneuronalexcitability,regulationofosmoticbalance,cellvolume andintracellulartransportofmoleculeslinkedtotheco-transport ofNa+_,_such_as_glucose,_amino_acids_and_{neurotransmitters}₍_Kaplan,

2002;Jorgensen etal.,2003).Severalexperimentalstudieshave

describedtheinvolvementofNa+_,K+_-ATPase_brain_activity_in_the

depressivedisorders (Zanattaet al.,2001; Gamaro etal., 2003;

Vasconcellosetal.,2005;Ackeretal.,2009).Maniaandbipolar

depressionhavebeenassociatedwithincreasedintracellularNa+

concentrations(LiandEl-Mallakh,2004).Ratsexposedtochronic variablestress(CVS)developeddespair-likeendophenotypesand haddecreasedhippocampalandamygdalarNa+_,K+_-ATPase

activ-ity (Crema et al., 2010).The antidepressant fluoxetine and the

moodstabilizerlithiumsimultaneouslypreventeddespairinduced by CVS and prevented the decrease in Na+_,K+_-ATPase _activity

(Vasconcellosetal.,2005).

The observation that several neuroactive drugs act on Na+

channelsalsoindicatestheimportanceofNa+_gradient _in_brain

disordersneurobiology.Sodiumchannelsarevoltage-dependent transmembraneproteinsresponsiblefortheincreaseof permeabil-itytosodium,whichinitiatesandpropagatestheactionpotential inexcitablecells(CestèleandCatterall,2000;Bourinetal.,2009). TheNa+_channels_are_targets_to_different_classes_of_drugs_such_as

anticonvulsants,localanestheticsandantiarrhythmics(Rasgdale etal.,1996).Someanticonvulsants(carbamazepine,lamotrigine, phenytoin,topiramate, valproatesodium) arealsousedtotreat bipolardisorder(Reinaresetal.,2012)andlamotriginehasbeen usedspeciallytotreatbipolardepression(Lengetal.,2013).

Inthiscontext,theaimofthepresentstudywastoevaluate theeffectoftheacuteandsub-acuteadministrationofuliginosin B on Na+_,K+_-ATPase _activity _in _mice _cerebral _cortex _and

hip-pocampusandtheinvolvementofsodiumchannelsinuliginosin Bantidepressant-likeeffect.

Materialsandmethods

Plantmaterial

HypericumpolyanthemumKlotzschexReichardt,Hypericaceae aerial parts were collected at Cac¸apavado Sul, in the state of Rio Grandedo Sul–Brazil(October, 2008).The voucher speci-mensweredepositedattheherbariumoftheFederalUniversity

ofRioGrandedoSul(ICNBordignon,3118Herbáriodo Departa-mentodeBotânica–InstitutodeBiociências–UFRGS).Theplant collectionwasauthorizedbyIBAMA(InstitutoBrasileirodoMeio AmbienteedosRecursosNaturaisRenováveis)(n◦_003/2008;

Pro-tocol02000.001717/2008–60).

Preparationofextract

H.polyanthemum lipophilic extractwas obtainedfromdried andpowderedplantmaterial(300g)extractedwithcyclohexane (plant/solventratio1:10,w/v)bystaticmacerationfor48h.The extractwasevaporatedtodrynessunderreducedpressureat45◦_C

toeliminatethesolvent,yieldingafreesolventextracttermedPOL (3.5%).Then,theextractwastreatedwithacetonetoremovethe waxes,accordingtoRochaetal.(1994),producing aninsoluble residue,whichwaseliminatedbyfilteringwithpaperfilter.

CharacterizationoftheextractbyHPLCanduliginosinBisolation

TheextractwasdissolvedinHPLCgrademethanol(2mg/ml), filtered(0.22␮mporesize,Merck)andanalyzedbyhigh

perfor-manceliquidchromatographyWaterHPLCsystem(Milford,MA, USA).UliginosinB(1)determinationwascarriedoutwithan iso-craticsolventcondition(95%CH3CN,5%H2O,0.01%TFA)througha

WatersNova-PackC18column(4␮m,3.9mm×150mm)adapted toaguardcolumnWatersNova-PackC1860A(3.9mm×20mm), flowrateof1mlmin−1_and_UV_detection_at₂₂₀_nm,_according_to

themethodpreviouslydescribed(Nunesetal.,2009).UliginosinB concentrationwasdeterminedas16%,isolatedbymeansof prepar-ativethinlayerandcolumnchromatographyandfinallyidentified by1_H–13_C_NMR_as_described_elsewhere₍_Rocha_et_al.,_1995;_Ferraz

etal.,2002;Nöretal.,2004).

Animals

BehavioralandbiochemicaltestswerecarriedoutwithmaleCF1 mice(25–30g)purchasedfromFundac¸ãoEstadualdeProduc¸ãoe PesquisaemSaúde,RS(Brazil).Theanimalswerehousedbyfive in plastic cages(17cm×28cm×13cm)and werekeptunder a 12hlight/darkcycle(lightsonat7a.m.)atconstanttemperature of23±1◦_C_with_free_access_to_standard_certified_rodent_diet_and

tapwater.AllexperimentalprotocolswereapprovedbyThe Ani-malCareLocalEthicalCommittee(CEUAUFRGS;Protocol18518), and performedaccording toBrazilian law(Brazil,2008), which areincompliancewiththeEuropeanCommunitiesCouncil Direc-tiveof24November1986(86/609/EEC)andInternationalGuiding PrinciplesforBiomedicalResearchInvolvingAnimals(Bankowski,

1985).

Experimentaldesign

Mice(n=8pergroup)weretreatedbygavage(10ml/kg)with uliginosinB(1)acutely(10mg/kg,p.o.)orsub-acutely(10mg/kg,

p.o.,onceaday,during3days),basedonpreviousresultsofour group(Vianaetal.,2008).Controlgroupswereperformedforboth treatmentregimens and theanimalswere treatedwithvehicle (saline+polisorbate802.0%).Differentgroupswereusedfor bio-chemicalandbehavioralexperiments(tailsuspensiontest,TST– andforcedswimmingtest,FST).

Time-courseof uliginosinBantidepressant-like effectonthe TST and FST was evaluated by testing independent groups of micewhich were acutelyor repeatedly treated withuliginosin B(10mg/kg,p.o.)orvehicle.Theanimalswereevaluatedinthe behavioral assays only once after treatment. Measurement of Na+_,K+_-ATPase _activity_was_performed_using _animals _that_were

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repeatedlytreated)wereeuthanizedbydecapitation1or3hafter receivingthelastdrugadministrationandbrainstructureswere removedimmediately.

Inanothersetofexperiments,weinvestigatedthepossible con-tributionofsodiumchannelstouliginosinBantidepressant-like effect.Weevaluatedtheinfluenceofthepre-treatmentwith ver-atrine,aNa+_channel_opener,_on_the_FST._Initially,_{dose-response}

experimentswereperformedintheFSTandlocomotoractivityin ordertodeterminethedoseofveratrinetobeused:veratrine(0.06, 0.125and0.5mg/kg,i.p.)wasadministered45minbeforethetest. Thesubeffectivedosewasdefinedasthedosenotabletoreduce immobilityintheFSTand withnoeffectonlocomotoractivity

(Centuriãoetal.,2014).TheassociationofveratrineanduliginosinB

consistedofthepretreatmentwithveratrine(0.06mg/kg)or vehi-cle,i.p.,45minbeforetheFST,plus uliginosinBorvehicle,p.o., 30minbeforethetest.

Assays

Tissuepreparation

Cerebralcortexandhippocampuswerehomogenizedin10 vol-umes(1:10, w/v) of0.32mMsucrose solutioncontaining5mM HEPESand1mMEDTA,pH7.5.Thehomogenateswerecentrifuged at 1000×g for 10min and thesupernatants wereremoved for Na+_,K+_-ATPase_activity_{determination.}

Na+_,K+_-ATPase_activity_assay

ThereactionmixtureforNa+_,K+_-ATPase_assay_contained₅_mM

MgCl2,80mMNaCl,20mMKCl,and40mMTris–HCl,pH7.4,ina

finalvolumeof200␮l.After10minofpre-incubationat37◦C,the

reactionwasinitiatedbytheadditionofATPtoafinalconcentration of3mM,andincubatedfor20min.Controlswerecarriedoutunder thesameconditionswiththeadditionof1mMouabain.Na+_,K+

-ATPaseactivitywascalculatedbythedifferencebetweenthetwo assaysaccordingtothemethoddescribedbyWyseetal.(2000). Releasedinorganicphosphate(Pi)wasmeasuredbythemethodof

Chametal.(1986).Specificactivityoftheenzymewasexpressed

asnmolPireleasedperminpermgofprotein.

Proteindetermination

ProteinconcentrationwasdeterminedbytheBradfordmethod

(1976)usingbovineserumalbuminasstandard.

Behavioralexperiments

Tailsuspensiontest(TST)

TheTSTwasconductedaccordingtoSteruetal.(1985)with minormodifications(Mülleretal.,2012).Micewereadaptedto thelaboratoryconditions1hbeforetheexperiment.Animalswere suspendedbytail60cmabovethefloorusingadhesivetape(1cm fromthetipoftheend)inadimlightroom.Immobilitytimewas recorded(inseconds)byablindtotreatmentobserverduring6min. Micewereconsideredimmobilewhen theyhungpassivelyand completelymotionless.

Forcedswimmingtest(FST)

TheFSTwascarriedoutaccordingtoPorsoltetal.(1978)with minormodificationsstandardizedandvalidatedinourlaboratory

(Vianaetal.,2005).Micewereadaptedtothelaboratoryconditions

1hbeforebeingexposedtotheFST.Theanimalswere individu-allyforcedtoswiminacylinderpool(10cmdiameter,13cmhigh, waterat22±1◦_C)_and_the_total_time_of_immobility_during₆_min

wasscored(inseconds).Immobilitytimewasrecordedwhenthe mouseremainedfloatingmotionlessor makingonlythe move-mentsnecessarytokeepitsheadabovewater.

Locomotoractivity

The spontaneous locomotor activity was performed in the open-field (Viana et al., 2005). Forty-five minutes after the administration, mice were placed in a transparent acrylic box measuring45cm×30cm×30cmwithadarkbottomdividedinto 24 equal quadrants. They were evaluated during 10min, after 5minhabituation.Thenumberofcrossingswasrecordedbyan observerblindedtotreatments.

Statisticalanalysis

Data wereexpressedasmean+SEMof themean.Data from biochemicalanalysiswereanalyzedbyStudent’sttest.Behavioral experimentsdatawereanalyzedbyone-wayortwo-wayANOVA followedbyStudent–Newman–Keuls.Differenceswereconsidered statisticallysignificantatp<0.05.Thestatisticalprocedureswere performedusing theSigmaStatsoftware 2.03 (JandelScientific Corporation).

Results

AcuteadministrationofuliginosinBincreasedNa+_,K+_-ATPase

activityincerebralcortex1haftertreatment[t(9)=2.447,p<0.05] (Fig.1A),butnotafter3h[t(8)=0.323,p=0.755](Fig.1B).Inthe hippocampus,Na+_,K+_-ATPase_activity_was_not_altered_at_both_times

studied:1h[t(9)=0.898,p=0.393](Fig.1C)and3h[t(8)=1.155,

p=0.281](Fig.1D).

Sub-acute administration of uliginosin B increased Na+_,K+

-ATPaseactivityincerebralcortex1hafterthelastadministration [t(10)=3.300,p<0.01](Fig.2A),aswellas3hafter[t(9)=2.518,

p<0.05](Fig.2B).Noalterationswereverifiedinthehippocampus atthetwodifferenttimes:1h[t(10)=1.494,p=0.166](Fig.2C)and 3h[t(8)=0.788,p=0.449](Fig.2D).

TheacuteadministrationofuliginosinB(10mg/kg,p.o.)inthe TSTandFSTareshowninFig.3.One-wayANOVAshoweda signifi-canteffectofuliginosinBadministrationintheTST[F(2,25)=4.411;

p<0.05]; posthoc analysis indicated a reduction onthe immo-bility time at 1h(p<0.05), but not at 3hafter administration (p=0.079) when compared to the control group (Fig. 3A).The sameeffectwasobservedwhenanimalsweresubmittedtotheFST [F(2,30)=11.136;p<0.001]:posthocanalysisindicatedasignificant decreaseintheimmobilitytime1hafteradministration(p=0.076), butnotafter3h(p>0.05)(Fig.3B).

The effect of sub-acute administration of uliginosin B (10mg/kg/day,p.o.)isshowninFig.3C.One-wayANOVArevealed a significanteffect of uliginosinB[F(2,21)=6.360; p<0.01] and

posthocanalysisindicatedasignificantanti-immobilityeffectat 1h(p<0.01)and3h(p<0.05)afterthelastadministrationwhen comparedtocontrolgroup.

Theanti-immobilityeffectofuliginosinB(10mg/kg,p.o.)was prevented by thepretreatment withveratrine (0.06mg/kg, i.p.) [two-way ANOVA: Fpre-treatment×treatment(1,31)=6,205, p=0.019]

(Fig.4).Theco-administrationof veratrineanduliginosinBhas noeffectwhencomparedtotheuliginosinBandvehicle(Tween), thusveratrineaffecttheantidepressant-likeeffectofuliginosinB inmouseFST.

Discussion

Inthepresentstudy,wedemonstratedthat uliginosinB, the mainphloroglucinolderivativefromH.polyanthemum,increases activity of Na+_,K+_-ATPase _in _mice _cerebral _cortex, _but _not _in

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120

100

80

60

40

20

0

Control Uliginosin B

1 hour

Na

+,K +-A

TP

ase activity

(nmol Pi/min, mg protein)

Acute treatment

Cortex

A

C

D

B

120

100

80

60

40

20

0

1 hour

Na

+,K +-A

TP

ase activity

Hippocampus

120

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0

3 hours

Na

+,K +-A

TP

ase activity

Hippocampus

120

100

80

60

40

20

0

3 hours

Na

+,K +-A

TP

ase activity

Cortex

Fig.1.EffectoftheacuteadministrationofuliginosinBonbrainNa+_,K+_-ATPase_activity._Mice_were_acutely_treated_with_uliginosin_B₍₁₀_mg/kg,_p.o_.)_and_euthanized_by

decapitation1or3hafterthelastadministration;immediatelycerebralcortexandhippocampuswereremovedtomeasureenzymeactivity.Studentttest;valuesare expressedasmean+SEM(n=5–6).DifferencefromControl*p<0.05.

120

100

80

60

40

20

0

1 hour

Na

+,K +-A

TP

ase activity

Cortex

A

120

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20

0

1 hour

Na

+,K +-A

TP

ase activity

Hippocampus

Sub acute treatment

C

120

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0

3 hours

Na

+,K +-A

TP

ase activity

Cortex

B

120

100

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60

40

20

0

3 hours

Na

+,K +-A

TP

ase activity

Hippocampus

D

Fig.2.Effectofthesub-acutetreatmentofuliginosinBonbrainNa+_,K+_-ATPase_activity._Mice_were_repeatedly_treated_with_uliginosin_B₍₁₀_mg/kg/day,₃_days,_p.o_.)_and

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250

200

150

100

50

0

Control

Immobility time (s)

1 hour

Uliginosin B (10 mg/kg, p.o)

TST

FST

3 hours

250

200

150

100

50

0

Control

Immobility time (s)

1 hour

Uliginosin B (10 mg/kg, p.o)

3 hours

250

C

B

A

200

150

100

50

0

Control

Immobility time (s)

1 hour

Uliginosin B (10 mg/kg/day, 3 days, p.o)

3 hours

Fig.3.Effectoftheacuteandsub-acutetreatmentofuliginosinB(10mg/kg,p.o.) ontheimmobilitytime.MicewereacutelytreatedwithuliginosinBandsubmitted totest,1or3hafteradministration:tailsuspensiontest(TST,panelA)andforced swimmingtest(FST,panelB);miceweretreatedfor3days,onceadayandsubmitted toFST1or3hafteradministration(FST,panelC).One-wayANOVAfollowedby Student–Newman–Keuls;valuesareexpressedasmean+SEM(n=8–10).Difference fromControl*p<0.05,**p<0.01.

FSTsuggestingthattheuliginosinBeffectis,atleastinpart,due to its action on Na+_,K+_-ATPase. _Furthermore, _the _pretreatment

withveratrine,aNa+_channel_opener,_prevented_the_uliginosin_B

antidepressant-likeeffect,reinforcingthattheactivityofuliginosin BinvolvestheregulationofNa+_balance.

Depressionis definedclinically asa pathological complexof psychological,neuroendocrineandsomaticsymptomsthatcannot be reproduced in animals. However, in mice, specific measur-ablebehaviors’canbeassayedsuchasFSTandTSTwhicharea

200 180 160 140 120 100 80 60 40 20 0

Tween

Immobility time (s)

Veratrine

@

Fig.4.Effectofthepretreatmentofmicewithveratrine(0.06,mg/kgi.p.)on theanti-immobilityeffectofuliginosinB(10mg/kg,p.o.)intheFST. Indepen-dentgroupsofmicewerepretreated(i.p.)withvehicle(saline+polisorbate80 2%=tween)andtreated(p.o.)withvehicle(Tween–Controlgroup)oruliginosin B(Tween–UliginosinBgroup);orpretreated(i.p.)withveratrineandtreated(p.o.) withvehicle(Veratrine–Controlgroup)oruliginosinB(Veratrine–UliginosinB group).Valuesexpressedasmean+SEM(n=8–10).Two-wayANOVAfollowedby Student–Newman–Keuls.*DifferencefromTween–UliginosinBversusTween– Control,p<0.05.@DifferencefromTween–UliginosinBversusVeratrine– Uligi-nosinB,p<0.05.

goodscreeningtoolswithgoodreliabilityandpredictivevalidity

(Petit-Demouliereetal.,2005;Cryanetal.,2005;Castagnéetal.,

2009).TheeffectofuliginosinBin reducingimmobilitytimein theTSTandFSTreinforcesourpreviousresultsthathavealready demonstrated theantidepressant-likeeffectof this phlorogluci-nol derivative(Steinet al.,2012).In addition,in this studywe movedonthepossiblemodeofactionofuliginosinBby study-ing its effecton theenzyme Na+_,K+_-ATPase. _Clinical ₍_Goldstein

et al., 2006, 2009; Tochigi et al., 2008) and preclinical studies

(Ackeretal.,2009;Gamaroetal.,2003;Vasconcellosetal.,2005;

Cremaetal.,2010;Kirshenbaumetal.,2011)reportedthatNa+_,K+

-ATPaseisdiminishedindepressivedisorders.Gamaroetal.(2003)

demonstrated thatthe Na+_,K+_-ATPase_activity_decreased _in _rats

hippocampus subjected to chronic stress model of depression, effectthatwasreversedbytherepeatedtreatmentwithfluoxetine andlithium(Vasconcellosetal.,2005),whereastricyclic antide-pressantsinhibitedtheenzymeactivity(Sanganahallietal.,2000). Although theanimalswere not subjectedto anydepression model, the resultsshowed that uliginosin B increased Na+_,K+

-ATPase activity in cerebral cortex about 18% 1h after the last administration,butitdidnotaltertheenzymeactivityafter3h. Inthesub-acutetreatment, uliginosinBwasabletoincreasein 20%theenzymeactivity1and3hafterthelastadministration, suggesting a longstandingeffectof therepeatedtreatment (for 3days).ThesefindingsareconsistentwithZanattaetal.(2001), whodemonstratedthatthechronicadministrationoffluoxetine (14days)increasestheenzymeactivity,factthatcancontributeto fluoxetinetherapeuticefficacy.

Ontheotherhand,noneoftheuliginosinBtreatmentregimens (acuteand sub-acute)havechanged theenzymeactivityin the hippocampus.Morphologicalandneurochemicalalterationshave beenreportedinthehippocampusofdepressedpatients(Sheline et al.,2003).Thus, thelackof theeffect ofuliginosin Bonthe Na+_,K+_-ATPase_activity_in_mice_hippocampus_may_be_due_the_fact

thattheanimalswerenotsubmittedtoanyexperimentalmodel ofchronicdepression.Thisfindingalsosuggestsaselectiveeffect ofuliginisonBoncorticalNa+_,K+_-ATPase,_which_could_be

particu-larlyrelevant,sincethisbrainstructureisinvolvedindepression neurobiology(Price andDrevets,2010)and postmortem studies have demonstratedalterations onNa+_,K+_-ATPase_activity_in _the

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thecortex,butnotinthehippocampus,ofanimalstreatedwith valepotriates.Wespeculatethatthisprofilecouldberelatedtothe irregulardistributionofNa+_,K+_-ATPase_isoenzymes._Three

␣

iso-formsareabundantinthebrain:␣1,expressedbyneuronsandglia; ␣2,predominantinglia;and␣3,neuronal(McGrailetal.,1991).

Tochigietal.(2008)studiedthegeneexpressionpatternin

post-mortembrainsofsubjectswithmajordepressionandfoundthat Na+_,K+_-ATPase_␣₃_gene_expression_is_decreased_in_prefrontal

cor-texofsubjectswithmajorandbipolardepression.Anotherstudy withNa+_,K+_-ATPase_␣₃_heterozygous_mice_showed_a_reduction_of

15%in neuronal Na+_,K+_-ATPase _activity._We _speculate _that _this

profilecouldbe related tothe irregulardistribution of Na+_,K+

-ATPaseisoenzymes.Three␣isoformsareabundantinthebrain: ␣1,expressedbyneuronsandglia;␣2,predominantinglia;and ␣3, neuronal (McGrail et al., 1991).Tochigi et al. (2008)

stud-iedthegeneexpressionpatterninpostmortembrainsofsubjects withmajor depression and found that Na+_,K+_-ATPase _␣₃ _gene

expressionisdecreasedinprefrontalcortexofsubjectswithmajor and bipolar depression. Another study withNa+_,K+_-ATPase

␣3

heterozygousmiceshowedareductionof15%inneuronalNa+_,K+

-ATPaseactivity.Furthermore,theseanimals werevulnerableto developincreased depression-like endophenotypesina chronic variablestressmodel(Kirshenbaumetal.,2011).Thesedata sug-gestthatNa+_,K+_-ATPase_␣₃ _in_cerebral_cortex_could_be_a _target

tonewantidepressantdrugsandtostudythepathophysiologyof depressivedisorders.

The pre-treatment with veratrine prevented the anti-immobility effect of uliginosin B in the FST, effect that is in linewiththeliterature,whichdemonstratedasimilarprofileto hyperbrasilolB (Centurião et al., 2014), hyperforin (Codagnone etal.,2007)andlamotrigine(Calabreseetal.,2008;Pricaetal.,

2008;Bourinetal.,2009).Arecentstudyfromourgroupshowed

thathyperbrasilolB,anatural dimericphloroglucinolderivative fromH.caprifoliatum,alsohaditsanti-immobilityeffectprevented byveratrine,anditwasabletoincreaseNa+_,K+_-ATPase_activity_in

micehippocampus,butnotcerebralcortex(Centuriãoetal.,2014). Hyperforin,aphloroglucinolderivativefromH.perforatum,which is an European species worldwide used for depression, seems to exert its antidepressant action by mechanisms dependent onNa+ _channels,_without_altering _the_activity_of_Na+_,K+_-ATPase

(Chatterjee et al., 1998a,b).In addition, hyperforin inhibitsthe

reuptake of monoamines without bindingto theirtransporters

(Wonnemann et al., 2000). Several authors demonstrated the

presenceofacarrier-mediatedmonoaminetransportmechanism, responsible for the entrance of dopamine, norepinephrine and serotonininthenerveterminal,accompaniedbyNa+_ions₍_Xhaard

etal.,2008;Kristensenetal.,2011)mechanismabolishedinthe

absenceofNa+ ₍_Krueger,_1990;_Gu_et_al.,_1994;_Pifl_et_al.,₁₉₉₇_).

Lamotrigine, in turn, is an anti-epiletic agent used at bipolar depression tomaintenance treatment.It acts bystabilizing the presynapticmembranethroughtheblockadeofvoltage-gatedNa+

channels(Codagnone etal.,2007).Vitezicet al.(2008) demon-stratedapartialprotectiveeffectoflamotrigineontheinhibition ofNa+_,K+_-ATPase_activity_induced_by_kainic_acid_in_rats_prefrontal

cortex and hippocampus. Also, Southam et al. (1998) demon-stratedinvitrothatlamotrigineinhibitsthereuptakeofserotonin, norepinephrine and dopamine, reinforcing the involvement of sodiumgradient(Xhaardetal.,2008).

ConsideringtheeffectofuliginosinBontheactivityofNa+_,K+

-ATPaseand onNa+ _channels_together _with_previous_studies _of

ourgroupdemonstratingthatuliginosinBinhibitsthereuptake ofmonoaminesinadifferentmannerfrommostantidepressants

(Steinetal.,2012)wecanspeculatethatuliginosinBreducesthe

monoamineuptakebyalteringNa+_gradient.

Inconclusion,thepresentstudyrepresentsonestepaheadinthe elucidationofthemechanismofactionoftheantidepressant-like

activityofuliginosinB,anaturalphloroglucinolderivative, suggest-ingthatinvolvementofuliginosinBinregulationofNa+_balance

mayoccurthroughincreasedofNa+_,K+_-ATPase_activity._The

reg-ulatorymechanisminvolvingNa+_may_be_regarded_an_important

property for antidepressant-like activity of this phloroglucinol derivative.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thattheallexperimentalprotocolswereapprovedbyTheAnimal CareLocalEthicalCommittee(CEUAUFRGS;Protocol18518),and performedaccordingtoBrazilianlaw(Brazil,2008),whicharein compliancewiththeEuropeanCommunitiesCouncilDirectiveof 24November1986(86/609/EEC)andInternationalGuiding Princi-plesforBiomedicalResearchInvolvingAnimals(Bankowski,1985). Alleffortsweremadetominimizeanimalsuffering,toreducethe numberofanimalsused,andtoutilizealternativestoinvivo tech-niques.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authors’contributions

ACSandLGMcontributedinrunningthelaboratorywork, chro-matographicanalysis,analysisofthedataanddraftedthepaper. AGKF,AB,AHB,FBC,EBS,JK,contributedtobiologicalstudies.GLVP contributedincollectingplantsampleandidentification,chemical analysesandcriticalreadingofthemanuscript.ATSWandSMKR plannedthestudy,supervisedthelaboratoryworkandcontributed tocriticalreadingofthemanuscript.Alltheauthorshavereadthe finalmanuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

ThisworkwassupportedbyCAPES-COFECUB[grantnumber 656/09],CNPq fellowshipsand Programa de Pós-graduac¸ãoem CiênciasFarmacêuticas(PPGCF-UFRGS).

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