absence of entourage: terpenoids commonly found in cannabisthe entourage concept applied to cannabis...
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Absenceofentourage:TerpenoidscommonlyfoundinCannabis
sativadonotmodulatethefunctionalactivityof∆9-THCat
humanCB1andCB2receptors
MarinaSantiago1,ShivaniSachdev1,JonathonCArnold2,3,IainSMcGregor2,4,andMarkConnor1
1DepartmentofBiomedicalSciences,MacquarieUniversity,NSW,Australia
2TheLambertInitiativeforCannabinoidTherapeutics,TheUniversityofSydney,NSW,Australia
3DisciplineofPharmacology,TheUniversityofSydney,NSW,Australia
4SchoolofPsychology,TheUniversityofSydney,NSW,Australia
Briefrunningtitle:THCandterpenoidactionsonCB1/CB2
Correspondingauthor:MarinaSantiago
Emailaddress:[email protected]
Keywords:phytocannabinoid,cannabinoidreceptor,terpenoid,entourageeffect,THC,signalling
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Footnotesandabbreviations:
SomeofthesedatawerepresentedatTheAustralasianSocietyofClinicalandExperimental
PharmacologistsandToxicologistsConferenceinMelbourne,Australia(2016).
β-Car β-caryophyllene
CBD cannabidiol
HBSS Hank'sbalancedsaltsolution
RFU relativefluorescenceunits
SEM standarderrorofthemean
SST somatostatin
∆9-THC ∆9-tetrahydrocannabinol
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Abstract
Introduction:CompoundspresentinCannabissativasuchasphytocannabinoidsandterpenoids,mayact
inconcerttoelicittherapeuticeffects.Cannabinoidssuchas∆9-tetrahydrocannabinol(∆9-THC)directly
activatecannabinoidreceptor1(CB1)andcannabinoidreceptor2(CB2),however,itisnotknownif
terpenoidspresentinCannabisalsoaffectcannabinoidreceptorsignalling.Therefore,weexamined6
commonterpenoidsalone,andincombinationwithcannabinoidreceptoragonists,onCB1andCB2
signallinginvitro.
MaterialsandMethods:PotassiumchannelactivityinAtT20FlpIncellstransfectedwithhumanCB1orCB2
receptorswasmeasuredinreal-timeusingFLIPR®membranepotentialdyeinaFlexStation3platereader.
Terpenoidsweretestedindividuallyandincombinationforperiodsupto30minutes.Endogenous
somatostatinreceptorsservedasacontrolfordirecteffectsofdrugsonpotassiumchannels.
Results:α-Pinene,β-pinene,β-caryophyllene,linalool,limoneneandβ-myrcene(upto30-100µM)didnot
changemembranepotentialinAtT20cellsexpressingCB1orCB2,oraffecttheresponsetoamaximally
effectiveconcentrationofthesyntheticcannabinoidCP55,940.Thepresenceofindividualora
combinationofterpenoidsdidnotaffectthehyperpolarizationproducedbyΔ9-THC(10µM):(CB1:control,
59±7%;withterpenoids(10µMeach)55±4%;CB2:Δ9-THC16±5%,withterpenoids(10µMeach)17±4%).
ToinvestigatepossibleeffectondesensitizationofCB1responses,allsixterpenoidswereaddedtogether
with∆9-THCandsignallingmeasuredcontinuouslyover30min.Terpenoidsdidnotaffectdesensitization,
after30minutesthecontrolhyperpolarizationrecoveredby63±6%,inthepresenceoftheterpenoids
recoverywas61±5%.
Discussion:NoneofthesixofthemostcommonterpenoidsinCannabisdirectlyactivatedCB1orCB2,or
modulatedthesignallingofthephytocannabinoidagonist∆9-THC.Theseresultssuggestthatifa
phytocannabinoid-terpenoidentourageeffectexists,itisnotattheCB1orCB2receptorlevel.Itremains
possiblethatterpenoidsactivateCB1andCB2signallingpathwaysthatdonotinvolvepotassiumchannels,
however,itseemsmorelikelythattheymayactatdifferentmoleculartarget(s)intheneuronalcircuits
importantforthebehaviouraleffectofCannabis.
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Introduction
AnenduringnotioninthemedicinalCannabisandcannabinoidfieldisthatofentourage:theideathatuse
ofthewholeplantmayexertsubstantiallygreatereffectsthanthesumofitsindividualparts.1Entourage
isusuallyconstruedasapositiveattribute,withtheassumptionthatsuperiortherapeuticactions,ora
morefavourable“high”,willbeobtainedfromconsumingthewholeCannabisplantratherthanindividual
componentssuchas∆9-tetrahydrocannabinol(THC).Somewhatsurprisingly,theevidenceforthiswidely
citednotionisrelativelysparse.
Cannabiscontainsalmost150phytocannabinoids,themostcommonofwhichareTHCandcannabidiol
(CBD),togetherwiththeiracidprecursorsTHCAandCBDA2.Cannabisalsocontainsalargenumberof
monoterpeneandsesquiterpenecompounds(togethercalledterpenoids),themostcommonofwhich
includeα-pinene,β-pinene,linalool,limoneneandβ-myrcene(monoterpenes)andβ-caryophylleneand
caryophylleneoxide(sesquiterpenes).3Terpenoidsarevolatilecompoundsthataresynthesisedalongside
phytocannabinoidsmainlyinthetrichomesofthecannabisplant,andprovidecannabiswithitsdistinctive
aromaandflavour.4Terpenoidsareoftenlostiftheextractionprocessinvolvesheating.5
Theentourageconceptappliedtocannabiscanencompassthepotentialforbothcannabinoid-
cannabinoidandcannabinoid-terpenoidinteractions.Withregardtotheformer,∆9-THC-CBDsynergyin
producinganalgesiawasreportedinananimalmodelofneuropathicpain6whileinhumans,CBDhasbeen
proposedtoamelioratesomeoftheadversepsychotomimeticandanxiogeniceffectsof∆9-THC.7,8This
claimiscontroversial,however,withanumberofcontraryfindings9,10CBDmaymodulate∆9-THCeffects
atthereceptorlevelactingasaCB1negativeallostericmodulator11,providingsomebiologicalplausibility
toamodulatoryinteraction.
Scientificevidenceforcannabinoid-terpenoidinteractionsisessentiallyabsentandmostlycomesfrom
websitesanddispensariesextollingthevirtuesofproprietaryCannabischemicalvarieties,or
chemovars.12,13However,someterpenoidsdohaveintrinsicpsychoactiveandphysiologicaleffects,and
modulatoryeffectson∆9-THCactionsisnotfarfetched.1,14Forexample,instudieswithlaboratory
animals,limonenedisplayedanxiolyticeffects,pineneincreasedgastrointestinalmotility,linaloolwas
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sedative,anticonvulsantandanxiolytic,whilemyrceneproducedsedation,analgesiaandmusclerelaxant
effects(summarisedinRussoandMarcu14).However,compellingevidenceforcannabinoid-terpenoid
interactionsorsynergydoesnotyetexist,anditisalsoworthnotingtherelativelylowconcentrationsof
terpenoidspresentinherbalcannabis.5,13,15,16
Withsomanybioactivecomponentspresentincannabis,thesystematic,granularelucidationofpossible
entourageeffectsposesasubstantialcombinatorialpuzzleandscientificchallenge.Asapreliminary
approachtoaddressingthischallenge,thepresentstudyexaminedwhethertheeffectsof∆9-THConits
cognatecannabinoidreceptors(CB1andCB2)wouldbemodifiedinthepresenceofterpenoidsthatare
commonlyfoundincannabis,eitheraloneorincombination.Thedemonstrationofsuchareceptor-level
entourageeffectmightleadtopredictionsregardingfunctionalcannabinoid-terpenoidinteractioneffects
thatcouldbetestedinvivo.
MaterialsandMethods
Cellculture
Experimentsusedmousewild-typeAtT20FlpIncells(AtT20-WT),orthesecellsstablytransfectedwith
humanCB1orCB2receptorswith3xN-terminushaemagglutinintags(AtT20-CB1andAtT20-CB2
respectively).17CellswerecultivatedinDulbecco’sModifiedEagle’sMedium(DMEM;Sigma-Aldrich)
supplementedwith10%foetalbovineserum(FBS;SIGMA/SAFC)and100Upenicillin/100µgstreptomycin
mL-1(Gibco).Selectionantibioticswere80µgmL-1Zeocin(Invivogen)forAtT20-WTor80µgmL-1
hygromycinBGold(Invivogen)fortransfectedcells.
Cellsweregrownin75mm2flasksat37°C/5%CO2andpassagedwhen80-90%confluent.Assayswere
carriedoutoncellsupto20passagesinculture.
PotassiumChannelActivityMeasurements
ChangesinmembranepotentialweremeasuredusingtheFLIPR®bluemembranepotentialdye
(MolecularDevices)inaFlexStation3,asoutlinedinKnapman2013.18Cellsfroma90-100%confluent
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75mm2flaskwereresuspendedinLeibovitz'sL-15Medium(Gibco)supplementedwith1%FBS,100U
penicillin/100µgstreptomycinmL-1andglucose(15mM)andplatedin96wellblackwalledclearbottom
microplates(Costar)inavolumeof90µLperwell.Cellswereincubatedovernightinhumidifiedambient
airat37°Cincubator.Membranepotentialdye,usedat50%ofthemanufacturerrecommended
concentration,wasresuspendedinHank'sBalancedSaltsolution(HBSS)ofcomposition(inmM):NaCl
145,HEPES22,Na2HPO40.338,NaHCO34.17,KH2PO40.441,MgSO40.407,MgCl20.493,CaCl21.26,
glucose5.55(pH7.4,osmolarity315±15).Dyewasloadedintoeachwell(90µLperwell)andequilibrated
at37°Cforatleast1hourpriortoassay.Fluorescencewasmeasuredevery2seconds(λexcitation=
530nm,λemission=565nm,λemissioncut-off=550nm).Assayswerecarriedoutat37°Canddrugs
wereautomaticallyaddedinvolumesof20µL.
DeterminingtheEffectsofTerpenoidsonAcuteHyperpolarization
Terpenoidswereaddedafteratleast60secondsofbaselinerecordingandincubatedfor5minutes
beforecannabinoid(CP55,940orΔ9-THC)addition.InAtT20-WTcells,somatostatin(SST)wasadded
insteadofcannabinoid.
DeterminingtheEffectsofTerpenoidsonSignallingDesensitization
HomologousdesensitizationwasmeasuredbysimultaneouslyaddingΔ9-THCwithterpenoidsafter120
secondsofbaselinerecording.Signallingdesensitizationwascalculatedaspercentagedecreasefrom
peakΔ9-THCresponseafter30minutesindrugs.SST(100nM)wasadded30minutesafterΔ9-THC
additiontoexaminethepotentialeffectsofprolongedcannabinoidreceptoractivationonnative
somatostatinreceptors(heterologousdesensitization).TheSSTresponsewascomparedbetweengroups
(withorwithoutterpenoids).
DrugDilution
Alldrugs(exceptSST)weremadeupinDMSOandstoredasfrozenstocksataconcentrationof10mM–
100mM.Terpenoidstocksolutionconcentrationswere100mM,withexceptionofβ-myrcene(30mM)
whichwasinsolubleat100mM.SSTwasdissolvedinwater.Freshaliquotswereusedeachday,withthe
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drugsdilutedinHBSScontaining0.1%bovineserumalbumin(Sigma-Aldrich)immediatelybeforethe
assay.ThefinalconcentrationofDMSOineachwellwas0.1to0.11%;thislimitedthemaximum
concentrationofterpenoidsabletobetested.Avehicle(HBSSplussolventalone)wellwasincludedin
eachcolumnofthe96wellplateandthechangesinfluorescenceproducedbyvehiclealonewere
subtractedbeforedeterminingthemaximumhyperpolarizationaftereachdrugexposure.
DrugsandReagents
Δ9-THCwasfromTHCPharm(Frankfurt,Germany).TerpenoidswerefromSigma-Aldrich;(+)-α-pinene,
(+)-β-pinene,(-)-β-caryophyllene,(+/-)-linalool,(R)-(+)-limoneneandβ-myrcene.Somatostatinwasfrom
AuspepandCP55,940fromCayman.Unlessotherwiseindicated,theotherchemicalsandreagentswere
fromSigma-Aldrich.
DataAnalysis
Eachexperimentwasindependentlyrepeatedatleast5times,with2technicalreplicatesineach
determination.Dataareexpressedasapercentagechangeinthefluorescencecomparedwiththepre-
drugbaseline(30sbeforedrugaddition),oraspercentageof1µMCP55,940response.Graphswere
plottedusingGraphpadPrism7.02,andscatterdotplotsshowmeanswithstandarderrorofthemean
(SEM).MeanswerecomparedusingunpairedStudent'st-testornomatchingone-wayANOVAfollowed
bycorrectionformultiplecomparisons(Dunnett);andnullhypothesiswasrejectedifp-valuewaslower
than0.05(p>0.05=notsignificant).
Results
TerpenoidsinAtT20-WTcells
Wefirstexaminedterpenoidactioninnon-transfectedAtT20cells.Weusedsomatostatin(100nM)asa
positivecontrolbecauseithyperpolarizesAtT20-WTcellsviaactivationofendogenousSSTreceptors(Fig.
1AandB).18,19Additionofα-pinene,β-pinene,β-caryophyllene,linalool,limonene(100μM)orβ-myrcene
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(30μM)didnotaffectthemembranepotentialofAtT20-WTcells(Fig.1C,opencircles).Thepresenceof
terpenoids(100μM/30μM)hadnoeffectonthesubsequentsomatostatinresponse(Fig.1C).
TerpenoidsinAtT20-CB1and-CB2cells
TheabsenceofaterpenoidresponseinAtT20-WTcellsenabledthestudyoftheireffectonmembrane
potentialinAtT20cellsexpressinghumanCB1orCB2.Weexaminedwhetherterpenoids(1nM–100μM,β-
myrcene300pM-30μM)hyperpolarisedcellsviathesereceptorsand,inparallel,whethertheyaffecteda
subsequentresponsetoamaximallyeffectiveconcentrationofCP55,940(1µM,Fig.2).17Asummaryof
thefluorescencechangeafterterpenoidadditiontoAtT20-CB1cellsisshowninFigure3(closedcircles).
Nodifferencebetweenvehicleandterpenoidswasobserved.Further,noneoftheterpenoidschangedthe
membranepotentialofcellsexpressingCB2(SupplementaryFig.S1).Thechangeinfluorescenceproduced
bythesubsequentadditionofthenon-selectivecannabinoidagonistCP55,940(1μM)wasalsounaffected
inbothAtT20-CB1and-CB2(Fig.3andSupplementaryFig.S1–opencircles).
CP55,940isahighefficacyagonistofbothCB1andCB2receptors.20However,inCannabis,∆9-THCisthe
principlecannabinoidagonistandithasalowerefficacythanCP55,940,whichisapparentinthe
hyperpolarizationassayasalowermaximalresponse.20Wenexttestedtheeffectofalowandhigh
concentrationofterpenoids(100nMand10μM)onthehyperpolarizationproducedbythree
concentrationsof∆9-THC(100nM,1μMand10μM).Afterfiveminutesofindividualterpenoidapplication,
applicationof∆9-THCproducedfluorescencechangesthatwerenotsignificantlydifferentfromthose
producedby∆9-THCaloneinbothAtT20-CB1and-CB2cells(10μM∆9-THCFigs.5and6,100nM∆9-THC
SupplementaryFigs.S2andS3).Toexplorethepossibilityofanemergententourageeffect,wecombined
allsixterpenoids(10μMeach)andtestedtheeffectofthemixtureonthe∆9-THC-induced
hyperpolarization.Similartoindividuallytestedterpenoids,theeffectsof∆9-THCwerenotchangedbythe
mixture(Fig.7).
TerpenoidsanddesensitizationinAtT20-CB1
Wehavepreviouslyreporteddesensitizationcannabinoid-mediatedcellularhyperpolarizationinAtT20
cellsexpressingratorhumanCB1receptors21,22,andwefoundthisreversalofCP55,940-induced
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hyperpolarizationwasacceleratedbynegativeallostericmodulatorssuchasORG27569andPSNCBAM-1.
Therefore,wetestedwhetherterpenoidsmayactinasimilarwaytoORG27569andothernegative
allostericmodulators,alteringdesensitizationtime-course.Weused∆9-THCinsteadofCP55,940,as∆9-
THCisthemainphytocannabinoidagonist.Prolongedapplicationof∆9-THC(10μM)produceda
hyperpolarizationthatreversedsubstantiallyover30minutes.Representativetracesforthisexperiment
areillustratedinFigure8A.Wemeasuredthepeakresponseto∆9-THCandthesignalremaining30
minutesafteragonistexposureandquantifieddesensitizationasapercentdeclineinthepeakresponse.
The∆9-THC(10μM)signaldesensitizedby63.3±6.3%,inthepresenceoftheterpenoidmixdesensitization
was60.8±4.9%(Fig.8B).Thus,terpenoidsdidnotinterferewithdesensitizationofCB1signallingproduced
by∆9-THC.Wealsoassessedthecapacityof∆9-THCalone,terpenoidsalone(10μMeach)orterpenoids
combinedwith∆9-THCtoaffectsomatostatinreceptorsignallinginAtT20-CB1cells(heterologous
desensitization).Somatostatin(100nM)wasapplied30minutesafterfirstdrugapplication(Figs.8Aand
9Aandthehyperpolarizationproducedbysomatostatinafter∆9-THC,terpenoidsaloneor∆9-THCwith
terpenoidswerenotsignificantlydifferenttosomatostatinalone(p>0.05,Fig.8B).
Discussion
TheprincipalfindingofthisstudyisthatagonistactivationofCB1andCB2receptorsisnotobviously
alteredbyanyorallofthe6majorterpenoidsfromCannabissativa.Theterpenoidstesteddidnot
activateCB1orCB2bythemselves,nordidtheymodifythesignallingofthehighefficacyagonistCP55,940
orthelowerefficacyagonistΔ9-THC.Inparticular,Δ9-THCeffectswouldbeexpectedtobeverysensitive
tothepresenceofdrugswhichinhibited(orenhanced)signallingatthereceptor.Therearenospare
receptorsforΔ9-THCinthisassay,andchangesinligandbindingwouldbedirectlyreflectedasachange
inthemaximumresponse.Thelackofeffectofterpenoidsontheresponsetothesyntheticcannabinoid
CP55,940indicatesthatterpenoidsdonotinterferewithmaximalcannabinoidreceptor-mediated
hyperpolarization,suggestingnodirectmodulationofthepotassiumchannelresponse.Thiswas
confirmedbythelackofeffectofterpenoidsontheresponsetosomatostatin.
Apreviousstudyprovidedevidencethatβ-caryophylleneisaCB2agonist23.However,wewereunableto
detectanyeffectofβ-caryophylleneonCB2signallinginthepresentstudy.Thereasonsforthisare
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unclear,buttheefficacyofβ-caryophyllenehasnotbeendefinedincellularassaysandmaybelower
thanΔ9-THC.TheCB2responsetoevenhighconcentrationsofΔ9-THCinourassayissmall,suggesting
thatproductivecouplingofCB2toendogenouspotassiumchannelsinAtT-20cellsrequireshighefficacy
agonists.Theaffinityofβ-caryophylleneforCB2(155nM)hasbeendeterminedinmembranesfrom
HEK293cellsheterologouslyexpressingCB223,butisnotknowninintactcells.ItsEC50forinhibitionof
forskolin-inducedadenylylcyclaseinCHO-K1expressingCB2wasaround2µM,23suggestingalow
functionalaffinity,whichmaynotbesufficienttosignificantlyaffecttherapidresponsetothehigher
affinityagonistΔ9-THC.
PositiveandnegativeallostericmodulatorshavebeenreportedforCB124,25,andtheeffectsofseveral
negativeallostericmodulatorshavebeendefinedinthehyperpolarizationassayusedhere.21Both
PSNCBAM-1andORG27569enhancedCP55,940signaldesensitization,whilePSNCBAM-1alsoinhibited
theinitialCP55,940hyperpolarization.Co-applicationoftheterpenoidswithΔ9-THCfailedtoaffectthe
peakresponse,orthedegreeoftachyphylaxisobservedovera30-minuteexposuretodrug,suggesting
thattheyarenotactingasallostericmodulatorsofthisCB1signallingpathway.
AlimitationofthepresentstudyisthatweonlyexaminedCB1andCB2signallingthroughonepathway,
involvingGi/o.ThehyperpolarizationoftheAtT20cellslikelyrepresentsG-proteinmediatedactivationof
inwardlyrectifyingpotassiumchannels(GIRK),aspreviouslydescribedforCB1andotherGPCRinthese
cellsaswellasinseveraldifferentneurons.26,27,28CannabinoidreceptorscoupletomultipleGproteinsas
wellassignallingthroughotherpathwayssuchasthosedependentonarrestinsandItispossiblethat
entourageeffectsofterpenoidsaremediatedthroughmodulationofasubsetofthecannabinoid
receptorsignallingrepertoire26.CB1andCB2receptorscanbeactivatedinaligandbiasedmanner–the
phenomenonwhereadrugpreferentiallyactivatesasubsetofthesignallingpathwaysthatthereceptor
canaccess.29Ingeneral,thisbiashasbeenbestdefinedforGproteincouplingversusactivationof
arrestin-mediatedsignalling,buttoourknowledgetherearenoexamplesofcannabinoidligandsonly
affectingarrestin-mediatedsignalling.20,30Itremainspossiblethatterpenoidshavesuchanabsolutebias,
butthiswouldbeunprecedented,andinanycaserecruitmentofarrestinwouldbeexpectedtoproduce
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enhanceddesensitizationoftheCB1responsestoprolongedagonistexposure21,29.Anysubtlechangeto
receptorsignallingshouldbeclearwithuseofthelowefficacyagonistΔ9-THC.
Overall,ourdatasuggestthatitisunlikelythattheterpenoidsstudiedhereaffect∆9-THCinteractions
withcannabinoidreceptors.However,thisisnotadefinitiverebuttaloftheentourageeffect.Thereare
manyotherwaysthatthesemoleculescouldinteractwithcannabinoidstoinfluencetheoverall
therapeuticandsubjectiveoutcomesofcannabisadministrationanditshouldbeacknowledgedthat∆9-
THCinfluencessignallingatawidevarietyofothernon-cannabinoidreceptortargets(seeBanisteretal31
forreview).Theseincludeinteractionwithmetabolicpathways,otherG-proteincoupledreceptors,
ligand-gatedionchannels,signallingcascadespresentonthesamecellsthatexpresscannabinoid
receptors,oronothercellsupordownstreamofthecannabinoid-receptorexpressingcells.Terpenoids
mayevenhaveprimaryeffectsondistinctfunctionalmodulesthattogetherwithcannabinoidreceptor-
modulatedpathwaysareultimatelyintegratedintoabehaviouralorphysiologicaloutput.Sothequest
forentouragedoesnotendhere;inmanywaysithasonlyjustbegun.
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Fig.1.TerpenoidandsomatostatinmediatedfluorescencechangeinAtT20-WT.Representativetraces
showingchangeinfluorescencesignalafterterpenoidandsomatostatin(SST,100nM)application.Adecrease
insignalcorrespondstomembranehyperpolarization.Additionofterpenoids(A.β-pinene,β-caryophyllene
andβ-myrcene;B.α-pinene,linaloolandlimonene)didnotchangebaselinefluorescence,whilesomatostatin
mediatedaclearhyperpolarization.C.Percentagechangeoffluorescencefrombaselineaftereachterpenoid
(opencircle)andsomatostatin(closedcircles)application.Terpenoidswereaddedattwominutes;five
minutesbeforesomatostatin.Whencomparedtopositive(SST)ornegative(vehicle)controls,noneofthe
terpenoidstestedaffectedbaselinemembranepotentialorpeaksomatostatinresponse.β-Car=β-
caryophyllene.n=5,SEM,one-wayANOVAp>0.05.Drugswereaddedforthedurationofthebar.
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Fig.2.Representativetracesofβ-caryophylleneandCP55,940inAtT20-CB1and-CB2.Fluorescencewas
recordedfor10minuteswhereβ-caryophyllene(100nMand100µM)wasaddedat2minutesfollowedby
incubationfor5minutes,before1µMCP55,940application.β-caryophyllenedidnothyperpolarizeA.AtT20-
CB1andB.AtT20-CB2cells,oraffecttheresponsetoCP55,940(1µM).Drugswereaddedforthedurationof
thebar.
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Fig.3.EffectofterpenoidsatvaryingconcentrationsonAtT20-CB1membranepotentialandon1μM
CP55,940inducedhyperpolarization.Terpenoids(A.α-pinene,B.β-pinene,C.β-caryophyllene,D.β-myrcene,
E.linaloolandF.limonene)wereaddedtoAtT20-CB1cellsandincubatedfor5minutes.Maximum
fluorescencechangeswerenotdifferentfromnegativecontrol(closedcircles,n=5,SEM,one-wayANOVAp>
0.05).CP55,940(1µM)additiontoAtT20-CB1cellsinducedfluorescencechangesfrom33.1±1.7%to34.6
±0.7%.PeakCP55,940responseswerenotaffectedbythepresenceofterpenoids(opencircles,n=5,SEM,
one-wayANOVAp>0.05).V=Vehicle.
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted March 6, 2019. . https://doi.org/10.1101/569079doi: bioRxiv preprint
Fig.4.Representativetracesofβ-myrceneandΔ9-THCinAtT20-CB1andAtT20-CB2.Fluorescencechange
mediatedbytwosub-maximalconcentrationsofΔ9-THC(100nMand1µM)inthepresenceofβ-myrcene
(10µM).Terpenoidwasaddedat1minutesandincubatedfor5minutesbeforeΔ9-THCapplication.CP55,940
addedaspositivecontrol.Drugswereaddedforthedurationofthebar.
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Fig.5.Effectof10μMterpenoidsonΔ9-THCinducedhyperpolarizationinAtT20-CB1.ResponsetoΔ9-THCat
twosub-maximalandonemaximalconcentration(n=6-7,SEM,unpairedt-testp>0.13).Datapresentedas%
ofmaximumCP55,940(1µM)response.
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.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted March 6, 2019. . https://doi.org/10.1101/569079doi: bioRxiv preprint
Fig.6.Effectof10μMterpenoidsonΔ9-THCinducedhyperpolarizationinAtT20-CB2.ResponsetoΔ9-THCat
twosub-maximalandonemaximalconcentration(n=6-7,SEM,unpairedt-testp>0.26).Datapresentedas%
ofmaximumCP55,940(1µM)response.
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted March 6, 2019. . https://doi.org/10.1101/569079doi: bioRxiv preprint
Fig.7.Testingthe“Entourageeffect”.Effectofcombinationofsixterpenoidsat10μMeachonΔ9-THC
inducedhyperpolarizationinA.AtT20-CB1andB.AtT20-CB2.ResponsetoΔ9-THCattwosub-maximalandone
maximalconcentration(n=5,SEM,unpairedt-testp>0.13).Datapresentedas%ofmaximumCP55,940(1µM)
response.
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Fig.8.TerpenoidsonΔ9-THCmediateddesensitizationinAtT20-CB1.A.Representativetracesof
hyperpolarizationandsignaldesensitizationmediatedbyΔ9-THCalone(10µM,black)orwithterpenoids
(10µMeach,red).Cellswerethenchallengedwithsomatostatin(100nM)after30minutestoexamine
heterologousdesensitization.B.Percentagedesensitizationafter30minutesexposuretoΔ9-THCalone(10µM)
orinthepresenceofterpenoids(10µMeach),comparedtopeakfluorescenceresponse.Terpenoidsdidnot
affectΔ9-THCmediateddesensitization(n=5,SEM,unpairedt-testp=0.76).SST=somatostatin.Drugswere
addedforthedurationofthebar.
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted March 6, 2019. . https://doi.org/10.1101/569079doi: bioRxiv preprint
Fig.9.SomatostatinchallengeofAtT20-CB1cellstoinvestigateheterologousdesensitization.A.
Representativetracesofcellspreincubatedwith(black)orwithout(red)terpenoidsfor30minutesbefore
somatostatin(100nM)challenge.B.Comparisonofpeakhyperpolarization(%fluorescencechange)obtained
aftersomatostatin(100nM)challenge(n=5,one-wayANOVAp>0.05).SST=somatostatin.Drugswereadded
forthedurationofthebar.
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SupplementaryFig.S1.EffectofterpenoidsatvaryingconcentrationsonAtT20-CB2membranepotential
andon1µMCP55,940inducedhyperpolarization.Terpenoids(A.α-pinene,B.β-pinene,C.β-caryophyllene,
D.β-myrcene,E.linaloolandF.Limonene)wereaddedtoAtT20-CB2cellsandincubatedfor5minutes.
Maximumfluorescencechangesweredeterminedandcomparedtonegativecontrol(HBSS,closedcircles).No
significantfluorescencedifferencewasobservedwhencomparingmeansofterpenoidsandHBSS(n=5,SEM,
unpairedt-testp=0.72).CP55,940(1µM)additiontoAtT20-CB2cellsinducedfluorescencechangesfrom30.4
±2.4%to32.2±2.5%.PeakCP55,940responseswerenotaffectedbythepresenceofterpenoids(opencircles,
n=5,SEM,unpairedt-testp>0.09).V=Vehicle
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted March 6, 2019. . https://doi.org/10.1101/569079doi: bioRxiv preprint
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SupplementaryFig.S2.Effectof100nMTerpenoidsonpeakhyperpolarizationinducedbyΔ9-THCinAtT20-
CB1cells.ResponsetoΔ9-THCattwosub-maximalandonemaximalconcentration(n=5,SEM,unpairedt-test
p>0.24).Datapresentedas%ofmaximumCP55,940(1µM)response.
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted March 6, 2019. . https://doi.org/10.1101/569079doi: bioRxiv preprint
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SupplementaryFig.S3.Effectof100nMTerpenoidsonpeakhyperpolarizationinducedbyΔ9-THCinAtT20-
CB2cells.ResponsetoΔ9-THCattwosub-maximalandonemaximalconcentration(n=5,SEM,unpairedt-test
p>0.50).Datapresentedas%ofmaximumCP55,940(1µM)response.
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted March 6, 2019. . https://doi.org/10.1101/569079doi: bioRxiv preprint