Characterization Cell Line Model of a for d -Serine Uptake Journal of Pharmaceutical and Biomedical Analysis

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Journal of Pharmaceutical and Biomedical Analysis

jou rn al h om e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j p b a

Characterization of a Cell Line Model for d -Serine Uptake

István Vincze, Péter P. Lakatos, Fruzsina Bagaméry, Tamás Tábi, Éva Szök ˝o

DepartmentofPharmacodynamics,SemmelweisUniversity,4Nagyváradtér,Budapest,H-1089,Hungary

a r t i c l e i n f o

Articlehistory:

Received31January2020

Receivedinrevisedform4May2020 Accepted6May2020

Availableonline13May2020

Keywords:

alanine-serine-cysteine-threonine transporters

capillaryelectrophoresis cellmodel

ratprimaryastrocyte d-serinetransport SH-SY5Ycellline

a b s t r a c t

d-Serineisanimportantco-agonistoftheN-methyl-d-aspartate(NMDA)receptorsinthebrainandits alteredactivitywasidentifiedinvariouspathologicalconditions.Modificationoftheextracellulard- serinelevelissuggestedtobeabletomodulatethereceptorfunction.Itstransportersmaythusserveas potentialdrugtargets.

Theaimofthisworkwastofindaneasilyavailablehumancelllinemodelappropriateforscreening moleculesaffectingd-serinetransporters.

Characteristicsofd-serinetransportintoSH-SY5Yhumanneuroblastomacelllinewerestudiedand comparedtothoseinculturedprimaryastrocytes.Uptakewasfollowedbymeasuringintracellulard- serineconcentrationbycapillaryelectrophoresiswithlaserinducedfluorescencedetectionmethod.

WefoundthatSH-SY5YcellsexpressfunctionalASCT-1andASCT-2neutralaminoacidtransporters andshowsimilard-serineuptakekineticstoculturedastrocytes.Neutralaminoacidsinhibitedd-serine uptakesimilarlyinbothcelltypes.Completeinhibitionwasachievedbyl-alanineandl-threoninealike, whilethetwo-stepinhibitioncurveoftrans-hydroxy-l-proline,aselectiveinhibitorofASCT-1supported thepresenceoffunctioningASCT-1andASCT-2transporters.Itshigheraffinitystepcorrespondingto inhibitionofASCT-1wasresponsibleforabout30%ofthetotald-serineuptake.

BasedonourresultshumanSH-SY5Ycelllineshowssimilaruptakecharacteristicstoprimaryastrocytes andthuscanserveasasuitablemodelsystemfortestingofcompoundsforinfluencingd-serineuptake intoastrocytes.

©2020TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/4.0/).

1. Introduction

Proper function of N-methyl-d-aspartate (NMDA) receptors of glutamate is crucial in neuronal development and plastic- ity, the molecular basis of several higher neuronal functions suchasmemoryformation,learning,etc.DysregulationofNMDA receptor activityleadsto numerouspathological conditions. Its

Abbreviations: Asc-1, alanine-serine-cysteine-1; ASCT-1, alanine-serine- cysteine-threonine-1;ASCT-2,alanine-serine-cysteine-threonine-2;CE-LIF,capil- laryelectrophoresislaserinducedfluorescence;DAAO,d-aminoacidoxidase;ECL, enhancedchemiluminescence;FBS,fetalbovineserum;HEPES,4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid; HPA-␤-CD, 6-monodeoxy-6-mono(3-hydroxy) propylamino-␤-CD; NBD-F, 4-fluoro-7-nitrobenzofurazan; NMDA,N-methyl-d- aspartate;RIPA,radioimmunoprecipitationassaybuffer;SR,serineracemase;TBSS, Trisbufferedsaltsolution;TBST,Tris-bufferedsalinecontaining0.1%Tween20;

t-Pro,trans-hydroxy-l-proline.

∗Correspondingauthor.

E-mailaddresses:vincze.istvan@pharma.semmelweis-univ.hu (I.Vincze),lakatos.peter@pharma.semmelweis-univ.hu (P.P.Lakatos),bagamery.fruzsina@pharma.semmelweis-univ.hu (F.Bagaméry),tabi.tamas@pharma.semmelweis-univ.hu(T.Tábi), szoko.eva@pharma.semmelweis-univ.hu(É.Szök ˝o).

hypofunction iswell establishede.g. in schizophrenia whileits over-activation resultsin excitotoxicity and consequential neu- rodegeneration[1].

NMDAreceptoractivationrequiresthebindingofaco-agonist in addition toglutamate that canbe eitherglycine or d-serine dependingonreceptorsubtypeanditscellularlocalizationinvar- iousbrainareas.D-Serineisregardedtobetheprimaryco-agonist onthesynapticNMDAreceptorsinthehippocampusandprefrontal cortex[2].Reducedd-serineconcentrationinbloodofschizophre- niapatientswasfirstreportedbyHashimotoetal.[3]andlater confirmedbyseveralresearchgroups(forreviewsee[4]).Further- more,highdosed-serinesupplementationeitheralone[5]orin adjuncttoantipsychoticdrugtherapy[6]wasshowntoimprove thesymptomsofthedisease.

Promising preclinicaland clinical datainitiated an intensive researchtobetterunderstandthesynthesis,releaseandelimina- tionofd-serineinthecentralnervoussystem.Serineracemase(SR) wasidentifiedastheuniquesourceofd-serinedenovosynthesis [7].SRexpressionwasfirstshowninastrocyteswhere d-serine was also found highly abundant,so originally it was regarded asagliotransmitter[8].However,recentlySRwasshown being

https://doi.org/10.1016/j.jpba.2020.113360

0731-7085/©2020TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).

predominantlyexpressedinneurons[9]and itsneuronspecific knockdownresultedinamoreconsiderablereductionofd-serine levelcomparedtoitsgliaspecificknockdown[10].Basedonthese resultsneuronsareregardednowastheprimarysiteofd-serine synthesis.Itsneuronalreleasewasdemonstratedtobeinducedby depolarization:however,itwasfoundcalciumindependentsug- gestinganon-vesicularmechanism[11].Alanine-serine-cysteine1 (Asc-1)transporterwasidentifiedtomediated-serinereleasefrom neurons[12].Asc-1isasodiumindependentsmallneutralamino acidexchangetransporterthatshowshighaffinitytod-serine[13].

Itsexpressionisrestrictedtoneuronsinthecentralnervoussystem andisonlyabundantinthepresynapticmembrane[14].

d-Serinewasalsoidentifiedtobesubstrateofalanine-serine- cysteine-threonine-1and-2(ASCT-1,ASCT-2),sodiumdependent hetero-exchangetransportersforsmallneutralaminoacids.Their expression was demonstrated in both astrocytes and neurons [15,16].Ribeiroandco-workersshowedfirstthatd-serineuptake intoastrocytesis mediatedby ASCTtype transportersand sug- gestedtheyplayanimportantroleintheregulationofextracellular leveloftheco-agonist[17].

Degradationofd-serineiscatalyzedbytheperoxysomalflavo- protein, d-amino acid oxidase (DAAO). The distribution of the enzymeinthebodyisuneven,itsexpressioninhigherbrainareas isinsignificantresultinginconsiderabled-serinelevel[18].

ThepossibilitiesforrestorationofthealteredNMDAreceptor activationinvariouspathologicalconditionsareextensivelystud- ied.Sincefinetuningofreceptoractivityisrequiredmodulationof theco-agonistlevelisapromisingapproach.Incase ofd-serine itssynthesizingand metabolizingenzymesaswellasits trans- portersmayserve aspotentialdrugtargets.Inhibitorsof DAAO wereusedconcomitantlywithd-serinesupplementationtoreduce itsrapidelimination.However,speciesdifferenceswereobserved suggestingdifferentroleofDAAOind-serinemetabolisminvar- iousanimals[19].Regulationofd-serinesynthesis bySRis not wellcharacterizedsofar,thusthewayofitspharmacologicalmod- ulationisnotclear.Asthetransportersplayamajorroleinthe regulationofextracellulard-serineconcentrationtheyseemtobe morepromisingdrugtargets.However,forstudyingtheeffectof drugcandidatesactingonthetransporters,asimplemodelsystem wouldberequired.Previouslymainlyprimaryastrocyteandneu- ronalcultureswereappliedforcharacterizationofthetransporters [17,20].Theirextensiveusefortestinghighnumberofmoleculesis difficultandraisesethicalissues.Celllinescouldbemoreappropri- ateforscreening.PreviouslyratC6gliomacelllinewascommonly usedfor studyingtransporters ofvarious amino acids,e.g. glu- tamine[21].d-Serinetransportintothesecellswasdemonstrated bySikkaetal[22].TheyshowedthatthecellsexpressSRandASCT-2 andintheabsenceofd-aminoacidoxidaseASCT-2isanimportant playerintheregulationofd-serinehomeostasis.ItsmRNAexpres- sionwasshowntobedependentond-serineavailability.Therole ofASCT-1inthetransport wasnotaddressed.Theotherwidely usedmodelforstudyingd-serinetransportareratMüllercellline (rMC-1)andmouseprimaryMüllercells.Thismodelwaschosen becaused-serineandSRweredetectedinMüllercells ofretina andastrocytesinvertebratesandd-serineincreasesNMDAinduced excitotoxicityinthesecells[23].Itstransportwascharacterizedby Dunetal[24]andwasfoundsodium-dependentandinhibitedby thesubstratesofASCTtransporters.Theyshowedthatthecellscon- tainedmRNAofbothASCT-1andASCT-2butbecauseglutamine, regardedspecificforASCT-2transporter,inhibitedd-serinetrans- porttheyfocusedonlyonASCT-2andclaimeditbeingimportantin theregulationofNMDAreceptoractivityoftheneighboringneu- rons.TheroleofASCT-1wasnotexplored.Fosteratal[20]werethe firstdemonstratingthatbothASCT-1andASCT-2contributestod- serinetransportintorathippocampalastrocytesandHEK293cells transfectedwithhumanASCT-1orASCT-2.Theydemonstratedthat

l-glutamineisapreferentialbutnotaspecificsubstrateofASCT-2 inthetransfectedcellsraisingthepossibilityofspeciesdifference in theaffinity of thesubstrates totheASCT transporters.They alsofounddifferingaffinityofsubstratestotransportersinastro- cytesandcellstransfectedwithhumanASCTtransporters.Thiswas explainedbythepossibledifferenceintransporterdensityonthe cellsurfaceduringtransfectionwhichcanbealimitationofusing transfectedcellsasamodel.

Regarding the suggested species difference, the goal of our presentstudywastofindahumancelllineexpressingASCT-1and ASCT-2toestablishamodelsystemforrapidscreeningofcom- poundsfor influencingd-serineuptake intoastrocytes.Wealso aimedatclarifyingthecontributionof ASCT-1transportertod- serineuptake.SH-SY5Yhumanneuroblastomacelllinewasthus studiedanditsd-serinetransportcharacteristicswascomparedto thoseofculturedprimaryastrocytes.

2. Materialsandmethods 2.1. Chemicals

d-Serine, l-alanine, l-threonine, l-glutamine, t-Pro, buffer components,trypsin,trypanblue,acetonitrile,l-cysteic-acid,acry- lamidewerepurchasedfromSigma-Aldrich(St.Louis,MO,USA) and cholinechloride wasfromAlfa Aesar (Haverhill,MS, USA).

Fluorescent reagent, 4-fluoro-7-nitrobenzofurazan (NBD-F) was providedbyTCI(Tokyo,Japan).6-Monodeoxy-6-mono(3-hydroxy) propylamino-␤-CD(HPA-␤-CD)waspurchasedfromCyclolabLtd.

(Budapest,Hungary).UltrapurewaterfromMilliQDirect8water purification system (Merck Millipore, Billerica, MA, USA) was appliedforallexperiments.

For preparationof cellculture medium,Dulbecco’s Modified EagleMedium/NutrientMixtureF-12(DMEM/F12)andfetalbovine serum(FBS)wereprovidedbyCorning(Tewksbury,MA,USA)and Biosera(Nuaille,France), respectivelyand stableglutamine was obtainedfromPanBiotech(Aidenbach,Germany).

For westernblot analysis Novex 4-12% polyacrylamide gels (Thermo Fisher Scientific, Waltham, MA, USA) were used.

ASCT-1andASCT-2primaryantibodiesweresuppliedbySigma- Aldrich and Cell Signaling (Danvers, MA, USA), respectively.

Horseradishperoxidaseconjugatedsecondaryanti-rabbitantibody andPierceenhancedchemiluminescence(ECL)detectionreagent fromThermoFisherScientificwereused.

2.2. Cellcultures

Wistarratpupsof1-3daysoldwerepurchasedfromToxi-Coop Ltd.(Budapest,Hungary)andusedforestablishmentofprimary astrocytecultureaccordingtotheprotocolofMechaetal.[25].

Animalexperiments wereapprovedbytheInstitutionalAnimal CareandUseCommitteeofSemmelweisUniversity(approvalnum- ber:PE/EA/850-2/2016).ProcedureswereinharmonywiththeEU CouncilDirectiveonlaboratoryanimals(86/609/EEC).Astrocytes wereusedafterreachingconfluencyofP1subculture.

SH-SY5YcellswereacquiredfromTheEuropeanCollectionof CellCultures(ECACC,Salisbury,UK)andmaintainedaccordingto therecommendationoftheprovider.

2.3. Analysisofcellulard-serineuptake

2.3.1. Time-andconcentration-dependenceoftheuptakeinto SH-SY5Ycells

Onthedayoftheexperimentcellsweretrypsinizedandsus- pendedinTrisbufferedsaltsolution(TBSS)atconcentrationof1 millioncellsin0.5mL.Cellsuspensionswereincubatedwith0,25, 50and200␮Mofd-serinefor0,15,30,60and120minutesat

37^◦C.Aftercompletionofincubationuptakewasterminatedby coolingthesamplesonicefollowedbycentrifugation(630g,4^◦C, 5min)andwashedtwicewithicecoldTBSS.Pelletedcellswere resuspendedin35␮Lmixtureofacetonitrile:water(2:1,v/v)and theprecipitatedproteinwasremovedbycentrifugation(3000g,4

◦C,20min).Supernatantwascollectedandstoredat−80^◦Cuntil analysis.

2.3.2. Timeandsodiumdependenceoftheuptake

Fortheanalysisofsodiumindependenttransportsodiumchlo- ridewas substitutedby cholinechloride. Samplescontaining 1 millioncellswereincubatedwith50 ␮Md-serinefor 0,15,30, 60 and 120 minutes at 37 ^◦C. After completion of incubation timeuptakewasterminatedandthesampleswereprocessedas describedinsection2.3.1.

2.3.3. Characterizationofd-serinetransportkinetics

Cellsuspensionswereincubatedinthepresenceofvariouscon- centrationsofd-serine(0-10000␮M)for15minat37^◦Ctostudy thetransportkinetics.Aftercompletionofincubationtimeuptake wasterminatedandthesampleswereprocessedasdescribedin section2.3.1.

2.3.4. Inhibitionofd-serineuptakebyneutralaminoacids

Cellswereincubatedwith25␮Md-serineinthepresenceof variousconcentrationsofl-alanine,l-threonine,l-glutamineandt- Pro,respectivelyfor15minat37^◦C.Aftercompletionofincubation timeuptakewasterminatedandthesampleswereprocessedas describedinsection2.3.1.

2.4. Determinationofintracellulard-serineconcentration

Capillaryelectrophoresiscoupledtolaserinducedfluorescence detection(CE-LIF)analysisofd-serinecontentofcellextractswas performedaccordingtoourpreviouslypublishedvalidatedmethod [26].Briefly,5␮Lsampleswerederivatizedbymixingwith5␮L ethanolicNBD-Fsolution(3mg/mL)and5␮Lboratebuffer(pH 8.5;20mM)containing5␮Ml-cysteicacidasinternalstandard andheatingfor20minat65^◦C.Preparedsampleswerestoredat

−20^◦CuntilCEanalysis.

AllmeasurementswerecarriedoutonaP/ACEMDQcapillary electrophoresissystemoperatedby32Karatsoftwareversion5.0 (BeckmanCoulter,Brea,CA,USA).LIFdetectorwithArgon-ionlaser sourcewasapplied.Excitationandemissionwavelengthswere488 and520nm,respectively.Separationtookplaceinfusedsilicacapil- lariesof75␮midand365␮mod(AgilentTechnologies,SantaClara, CA,USA).Beforeusecapillarieswerecoatedwithlinearpolyacry- lamide.Representativeelectropherogramsofd-serineseparation isshowninFig.1.

2.5. Westernblotanalysis

ExpressionofASCT-1andASCT-2inSH-SY5Ycelllinewasstud- iedbywesternblotanalysis.About5millioncellswereextracted by200␮Lradioimmunoprecipitationassaybuffer(RIPA,Thermo FisherScientific)andthesampleswereseparatedin4-12% gra- dientgelusingTris-glycine-EDTAbufferandtransferredtoPVDF membrane.Membraneswereblockedby5%non-fatdrymilkdis- solvedinTris-bufferedsalinecontaining0.1%Tween20(TBST)for1 hourandprobedwith1␮g/mLanti-ASCT-1or4␮g/mLanti-ASCT- 2antibodiesin 5%non-fat drymilkdissolvedinTBSTovernight at4^◦C.MembraneswerewashedthreetimeswithTBSTandthen incubatedwithhorseradishperoxidaseconjugatedsecondaryanti- rabbitantibodyfor1houratroomtemperature.ASCT-1andASCT-2 weredetectedonautoradiographyfilmsusingPierceECLreagent.

Fig.1.Electropherogramsdemonstratingdeterminationofintracellulard-serine contentofhumanSH-SY5Yneuroblastomacells.NumberedpeaksindicateNBD-F labelledglycine(1),d-serine(2)andl-serine(3).Intheinserttheelectropherograms ofsamples(A)afterincubationthecellswith25␮Md-serinefor15minand(B)after incubationthecellswith25␮Md-serinefor15mininthepresenceof3mMofl- alanineareshown.Separationconditions:50mMpH7HEPESbuffercontaining6 mMHPA-␤-CD;30/40cmx75␮midcoatedfused-silicacapillary,−24kV.Sample injection:3474Papressurefor5sec.

2.6. Dataevaluation

GraphPadPrism8forWindows(GraphPadSoftware,LaJolla, CA,USA)wasusedfordataanalysisandcurvefitting.Exponential plateaufittingscheme,fitlogIC50fittingmethodandMichaelis- Menten curves were applied for analysis of time dependence, inhibitionandkineticsofd-serineuptake,respectively.

Forestimationofintracellulard-serineconcentration6and7

␮mradiusofSH-SY5Ycellsandastrocyteswereused,respectively [27].

3. ResultsandDiscussion

d-Serineuptake characteristicsofSH-SY5Ycells wereevalu- atedandcomparedtothoseofprimaryastrocytes.Firstthecell linewasincubatedinthepresenceof25,50and200␮Md-serine, respectivelyfor upto120minutesanditsuptake wasassessed bymeasuringitsintracellularlevel.Thetransportwasfoundcon- centrationandtimedependent.Itwassaturatedafter30minutes incubationwhen200␮Md-serinewasusedassubstrate,whilein caseofitslowerconcentrationsonlyatendencyofsaturationwas seenafter2hours(Fig.2).

In sodium free buffer a very limited d-serine uptake was detectedindicatingoverwhelmingdominanceofsodiumdepen- denttransportmechanismintoSH-SY5Ycellsandastrocytesalike (Fig.3AandB).Sodiumindependentportionoftheuptakewasless than10%intoeachcelltypeprobablyduetoanon-specificprocess.

ASCTtransportersareknownresponsibleforsodiumdependent uptakeofd-serineintocells andwerereportedtobeexpressed onastrocytes[16].Thesefindingssuggestthattheundifferentiated SH-SY5Ycelllinemayshowsimilard-serinetransportcharacteris- ticstothosepreviouslyreportedforculturedastrocytes[20].

Toestablishthesimilarity, thekineticsofd-serinetransport intoSH-SY5Ycells andastrocyteswerecompared.Incubationof eachcelltypewithincreasingconcentrationsofd-serineshowed aproportionalelevationintheuptakerateaccordingtoMichaelis- Mentenkineticsandreachedplateauatsimilarconcentrationrange (Fig.4).EstimatedK_Mvaluesandmaximaluptakevelocity(v_max) werenotsignificantlydifferentindicatingcomparableaffinityand capacityofd-serinetransportinthesecelltypes(Table1).Wecan

Fig.2. Concentrationdependentd-serineuptakeintoSH-SY5Ycells.Intracellulard- serinecontentinSH-SY5Ycellswasdeterminedaftertheirincubationwithvarious concentrations(25,50,200␮M)ofd-serinefordifferentperiodsoftime(0,15,30, 60,120min).Dataareshownasmean±SEMofthreeexperiments(n=3).

Fig.3. Sodiumdependenceofd-serineuptakeintoSH-SY5Yandastrocytecells.

Uptakeofd-serineintoSH-SY5Ycells(A)andcorticalastrocytes(B)wasmeasured overaperiodof120minutesinsodiumcontainingorsodiumfreebuffers.d-Serine concentrationwas50␮M.Dataareshownasmean±SEMofthreeexperiments (n=3).

Fig.4. Kineticofd-serineuptakeintoSH-SY5Yandastrocytecells.Intracellulard- serineconcentrationmeasuredinSH-SY5Ycellsandratcorticalastrocytesinthe presenceofincreasingconcentrationsofd-serine.Dataareshownasmean±SEM ofthreeexperiments(n=3).Michaelis-Mentencurveswerefitted.

Table1

Kineticparametersofd-serineuptakeintoSH-SY5Yandastrocytecells.

SH-SY5Y astrocyte

mean 95%Cl mean 95%Cl p

Km(␮M) 2025 1081-3884 2545 1714-3847 0.5131

Vmax(␮M/10⁶cells/min) 135.0 108.4-172.0 121.9 105.8-142.8 0.4339

Fig.5.WesternblotanalysisofASCT-1andASCT-2inSH-SY5Ycells.SH-SY5Ycell lysate(40␮gprotein)wasanalyzedbywesternblotusing4-12%gradientgeland probedbyrabbitanti-humanASCT-1andrabbitanti-humanASCT-2antibody.ASCT- 1andASCT-2weredetectedatabout52andabout66kDa,respectively.

assumethatd-serineuptakeintoSH-SY5Ycellsishighlysimilar tothatofastrocytesthusitcanserveasmodeloftheastrocytic transportofthisd-aminoacid.

TofurtherconfirmtheroleofASCTtransportersinthed-serine uptakeintoSH-SY5Ycellstheirexpressionwasstudiedbywest- ernblotanalysis.Expressionofbothtransporterswasdetectedin SH-SY5Ycells.Themostintensivebandappearedatabout52kDa usinganti-humanASCT-1antibody(Fig.5),whichiscomparable tothepredicted55kDamolecularweightreportedearlierinante- riorcingulatecortex[28].Anti-humanASCT-2antibodydetected animmunoreactivebandat66kDa(Fig.5)whichisinlinewiththe previouslyreportedmolecularweightoftheglycosylatedprotein [15].

Fig.6.Inhibitionofd-serinetransportintoSH-SY5Ycellsandratcorticalastro- cytesbyl-alanineandl-threonine.l-Alanine(A)andl-threonine(B)concentration dependentlyinhibitedthed-serineuptake.One-stepinhibitioncurvewasobserved incaseofbothcelltypes.Inallexperimentsuptakeof25␮Md-serinewasstud- iedfor15min.Intracellulard-serineconcentrationintheabsenceofinhibitorwas regardedas100%uptake.Dataareshownasmean±SEMofthreeexperiments(n= 3).

Asourultimateintentionisstudyingdrugcandidatesacting byinhibitionofd-serineuptakeintoastrocyteswecomparedthe inhibitorsensitivityofthetransportersonthetwocelltypes.

Natural substrates of ASCT transporters, l-alanine and l- threonine concentration dependently inhibited d-serine uptake intobothSH-SY5Ycellsandastrocytesandalmostcompleteinhi- bitionwasachieved.ComparableIC50valuesindicatetheirsimilar inhibitoraffinitytothetransportersonthetwocelltypes(Fig.6A andB,Table2).Basedonliteraturedatatheseneutralaminoacids havesimilaraffinitytoASCT-1andASCT-2[20].Inlineofthisfind- ingweobservedaone-stepsigmoidalinhibitioncurve,notallowing thedistinctionofthetwotransportersandhavinginsightifboth ASCTtransportersareinvolvedind-serineuptakeoronlyoneof themisfunctioning.

Therelativecontributionoftheindividualtransporterstothed- serineuptakewasthusassessedbyusingtheirselectivesubstrates.

t-Prowaspreviously shownhavingconsiderably higheraffinity toASCT-1[29], whilel-glutamine wasreportedbeingaprefer- entialsubstrateofASCT-2 [30].Inhibitionofd-serineuptakeby

Table2

EstimatedIC50valuesofvariousneutralaminoacidinhibitorsond-serineuptake intoSH-SY5Yandastrocytecells.

SH-SY5Y astrocyte

mean 95%Cl mean 95%Cl p

L-AlaIC50(␮M) 174.4 40.21-772.4 177.7 66.21-470.4 0.982 L-ThrIC50(␮M) 157.9 50.52-521.8 158.1 74.47-335.5 0.998 L-GlnIC50(␮M) 97.47 53.12-182.5 67.95 26.12-206.3 0.515 t-ProIC50(␮M)* 8.218 1.98-26.5 3.975 0.16-19.7 0.522

*forASCT1

Fig.7.Inhibitionofd-serinetransportintoSH-SY5Ycellsandratcorticalastrocytes byt-Proandl-glutamine.T-Proresultedintwo-stepinhibitioncurveinbothcell types(A).One-stepinhibitioncurvewasobservedinthepresenceofl-glutaminein bothSH-SY5Ycellsandculturedastrocytes(B).Intracellulard-serineconcentration intheabsenceofinhibitorwasregardedas100%uptake.Dataareshownasmean

±SEMofthreeexperiments(n=3).

t-Proshowedatwo-stepcurveindicatingcontributionofbothASCT transporters(Fig.7A).TheIC50valuesofthehigheraffinitycompo- nentcorrespondingtotheinhibitionofASCT-1werecomparable inthetwocelltypes(Table2).Becauseoftheweakinhibitionof ASCT-2byt-Procompleteinhibitionoftheuptakewasnotreached inthestudiedconcentration-rangeandtheestimationofIC50val- uesofthesecondstepsuffersfromhigherror(datanotshown).

BasedontheheightofthefirststepthecontributionofASCT-1to d-serinetransportwasabout30%inbothcelltypes.Foralongtime,

theinvolvementofASCT-1ind-serineuptakewasneglected.How- ever,usingtransfectedcellsorculturedastrocytesitwasshown tobesubstrateofbothtransporters[12,20].Ourpresentresults furtherconfirmthatASCT-1 ispartiallyresponsibleford-serine uptakeintocultured astrocytesand similarresultwasfoundin SH-SY5Ycells.Usingl-glutaminehowever,asingle-stepinhibition curvewasdetectedcontrarytothefindingsofFoster etal[20], whoobservedslightselectivitytowardsASCT-2.Thecontradiction isprobablyexplainedbytherathersmalldifferenceintheaffinity ofl-glutaminetotheASCTtransporters(Fig.7B).

4. Conclusion

OurpresentresultsdemonstratethathumanSH-SY5Yneurob- lastomacellsexpressfunctionalASCT-1andASCT-2transporters and show comparable uptake kinetics of d-serine to cultured astrocytes.Assimilarinhibitioncharacteristicsoftheestablished competitive uptake inhibitor small neutral amino acids were observedin both celltypes, theundifferentiated SH-SY5Ycells seemtobeanappropriatemodelsystemforscreeningpotential inhibitorsubstances ofd-serineuptake intoastrocytes byASCT transporters.Theadvantages ofusingthis celllinearetheeasy availability of functioning human transporters, its inexpensive maintenanceandtheavoidanceofsacrificinglaboratoryanimals.

Authorstatement

EvaSzoko:Supervisionoftheproject,dataevaluation,prepara- tionofthepublication;IstvanVincze:Planningandperformingthe experiments,CEmeasurements,dataanalysis,preparationofthe draftofthemanuscriptandthefigures;PeterP.Lakatos:Perform- ingsomeexperiments,dataanalysis,preparation ofthefigures;

FruzsinaBagamery:Maintenanceofthecells,preparationofthe primaryculture,performingsomeoftheexperiments;TamasTabi:

Evaluationofexperimentaldata,preparationofthepublication

DeclarationofCompetingInterest

Theauthorsdeclarethattheyhavenoknowncompetingfinan- cialinterestsorpersonalrelationshipsthatcouldhaveappearedto influencetheworkreportedinthispaper.

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