Nicotinic Activity of Arecoline, the Psychoactive Element of "Betel Nuts"

18 Pages·2015·2.27 MB·English

RESEARCHARTICLE
Nicotinic Activity of Arecoline, the
Psychoactive Element of "Betel Nuts",
Suggests a Basis for Habitual Use and Anti-
Inflammatory Activity
RogerL.Papke1*,NicoleA.Horenstein2,ClareStokes1
1 DepartmentofPharmacologyandTherapeutics,UniversityofFlorida,POBox100267Gainesville,Florida,
32610–0267,UnitedStatesofAmerica,2 DepartmentofChemistry,UniversityofFlorida,POBox117200,
Gainesville,Florida,32611–7200,UnitedStatesofAmerica
*[email protected]
Abstract
Habitualchewingof"betelnut"preparationsconstitutesthefourthmostcommonhuman
self-administrationofapsychoactivesubstanceafteralcohol,caffeine,andnicotine.The
primaryactiveingredientinthesepreparationsisarecoline,whichcomesfromtheareca
nut,thekeycomponentofallsuchpreparations.Arecolineisknowntobearelativelynon-
OPENACCESS selectivemuscarinicpartialagonist,accountingformanyoftheovertperipheralandcentral
Citation:PapkeRL,HorensteinNA,StokesC(2015) nervoussystemeffects,butnotlikelytoaccountfortheaddictivepropertiesofthedrug.We
NicotinicActivityofArecoline,thePsychoactive reportthatarecolinehasactivityonselectnicotinicacetylcholinereceptor(nAChR)sub-
Elementof"BetelNuts",SuggestsaBasisfor
types,includingthetwoclassesofnAChRmostrelatedtotheaddictivepropertiesofnico-
HabitualUseandAnti-InflammatoryActivity.PLoS
tine:receptorscontainingα4andβ2subunitsandthosewhichalsocontainα6andβ3
ONE10(10):e0140907.doi:10.1371/journal.
pone.0140907 subunits.Arecolineisapartialagonistwithabout6–10%efficacyfortheα4*andα6*recep-
torsexpressedinXenopusoocytes.Additionally,arecolineisasilentagonistofα7nAChR;
Editor:SidneyArthurSimon,DukeUniversity
MedicalCenter,UNITEDSTATES whileitdoesnotactivateα7receptorswhenappliedalone,itproducessubstantialactivation
whenco-appliedwiththepositiveallostericmodulatorPNU-120696.Someα7silentago-
Received:August21,2015
nistsareeffectiveinhibitorsofinflammation,whichmightaccountforanti-inflammatory
Accepted:October1,2015
effectsofarecoline.Arecoline'sactivityonnAChRassociatedwithaddictionmayaccount
Published:October21,2015
forthehabitualuseofarecanutpreparationsinspiteofthewell-documentedrisktoper-
Copyright:©2015Papkeetal.Thisisanopen sonalhealthassociatedwithoraldiseasesandcancer.Thecommonlinkbetweenbeteland
accessarticledistributedunderthetermsofthe
tobaccosuggeststhatpartialagonisttherapieswithcytisineortherelatedcompoundvare-
CreativeCommonsAttributionLicense,whichpermits
niclinemayalsobeusedtoaidbetelcessationattempts.
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare
credited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaper.
Introduction
Funding:ThisworkwassupportedbyNIHgrant
GM57481. "BloodyMary'schewingbetelnuts..."FormanyWesterners,theselinesfromRogersand
Hammerstein'smusicalistheextentoftheirknowledgeofthefourthmostcommondrughabit
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. intheworld,afteralcohol,nicotineandcaffeine[1].Thepsychoactiveagentsassociatedwith
PLOSONE|DOI:10.1371/journal.pone.0140907 October21,2015 1/18
NicotinicBasisforBetelNutAddiction
Fig1.Theworldofbetelnuts.(A)ThecarvedhiltofaDayakheadhuntersword.Thechewingofarecanut
isanimportantanessentialelementofthecultureofthesetribesontheIslandofBorneo,andhasbeen
throughouttheirknownhistory.Itwasreflectiononthisabstract,almostpsychedelic,carvingthatinspiredthe
seniorauthor(RLP)toinvestigatetheactivityofarecanutsandarecoline.(B)Anarecanutandtraditionalnut
cutter,anessentialpieceoftheparaphernaliaassociatedwiththisdrughabit.(C)AwallinBarabanki,Uttar
Pradesh,Indiastainedbytheexpectorateofabetelchewer.PhotobypermissionAshokKumar.
doi:10.1371/journal.pone.0140907.g001
thishabitcomefromtheseedoftheArecacatechupalm,whichissliced,oftencombinedwith
spices,andwrappedinleavesofthevinePiperbetlethathavebeenspreadwithslakedlime,
makingpacketssuitableforchewingknownas"betelquids".Muchofthehistoryandcultureof
SouthAsiainvolvestheuseofarecanuts,fromhallucinogeniccarvingsofDyakheadhunter
swords(Fig1A),totheelegantaccoutrementsforuseofbetelquids(Fig1B).Itisstillcommon
toseesidewalksandwallsbesmirchedwithcrimsonbetelspittleinpoorneighborhoodsof
IndiaandpartsofAsia(Fig1C).Thedetailsofpreferredarecapreparationsvarysignificantly
acrossAsia,andinrecentdecadeshaveincludedsomeformoftobaccoforabout50%ofthe
users[1],althoughthekeyingredientisalwaysarecanut.
Therearenumerousalkaloidsinarecanuts,thepredominantpsychoactiveagentbeingarec-
oline[2],amuscarinicacetylcholinereceptoragonist[3–5].Whilesomeeffectsofthearecanut
areobvious,mostnotablythecopiousproductionofbrightredsaliva,othersaremoresubtle
and,aswithnicotine,relyonsubjectiveaccounts.Euphoricoranxiolyticeffectsarereported,
aswellasbothsedationandarousal,andtherearefrequenthistoricalreferencestobetelasan
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NicotinicBasisforBetelNutAddiction
aphrodisiac[6,7].Theseeffectsmostlikelyaccountfortheshort-termreinforcingaspectsof
thedruguse,anddependenceandmildwithdrawalhavefrequentlybeenreported.However,
whatmightunderlietheaddictivepropertiesofbetelusehasbeenunclear.Historically,habit-
ualarecausewassociallyacceptable,evenexpected,inAsiancultures,butnowbeteluseistran-
sitioningtothesituationwhereitistoleratedbutnolongerwidelyencouraged.Inlarge
measurethischangeinpublicattitudeshascomefromappreciationofthehealthliabilityasso-
ciatedbeteluse,especiallyinregardtooraldiseasesandcancers[8–10].
Whiletobaccouseisrightlymalignedforalloftheassociatedhealthrisks,nicotinereceptors
arebeingassignednewpotentialrolesastherapeutictargets,especiallyinregardtocognitive
disorders[11–13]and,morerecently,inflammatorydiseasesandpain[14,15].Likewise,there
maybetherapeuticdirectionssuggestedbythepropertiesofarecolinethatarenotduetomus-
carinicagonistactivity.Therehavebeennumerousreportsofanti-inflammatoryorimmuno-
suppressantactivityassociatedwitharecoline[4,16,17].Interestingly,ithasalsobeensuggested
thatbetelnutusemaybeoftherapeuticvalueforschizophrenia[18,19].Thesearebothareas
inwhichα7nAChRhaverecentlybeenidentifiedasanewtherapeutictarget,andα7-targeting
drugsthathavelowefficacyforionchannelactivation,suchastheweakpartialagonistGTS-
21,appeartobeamongthemostpromisingcandidates[12,20–23].Indeed,inregardtoinflam-
mationandpain,agentsrecentlyidentifiedasα7"silentagonists"[24,25],whichproduceno
significantchannelactivationbutmayregulateintracellularsignaltransduction,showbetter
therapeuticpotentialthantypicalagonistssuchasnicotine[21,26,27].Althoughsilentagonists
producelittleornochannelactivation,theyinducedesensitizedconformationssimilartothose
producedbyefficaciousagonists,whichcanbeconfirmedbyco-applicationwithapositive
allostericmodulator(PAM)suchasPNU-120596,whichconvertsthosedesensitizedstates
intoveryactivelyconductingstates.Wethereforeinvestigatedtheeffectsofarecaandarecoline
onα7nAChRandextendedthosestudiestoidentifyalikelymechanismforbeteladdiction.
MaterialsandMethods
Commercialreagents
Acetylcholinechloride(ACh),arecoline,muscarine,methacholine,oxotremorine,carbachol,
mecamylamine,andatropinewerepurchasedfromSigma-AldrichChemicalCompany
(St.Louis,MO).FreshAChstocksolutionsweremadeinRinger'ssolutioneachdayofexperi-
mentation.StocksolutionsofthetestdrugsweremadeinRinger'ssolutionandkeptat4°Cand
usedwithintwodays.Workingsolutionswerepreparedfreshlyatthedesiredconcentration
fromthestoredstock.
Arecanutinfusion
WholeArecacatechunutswerepurchasedthrougheBayfromRiderInternationalHealthFoods,
Nuts&More,ChicagoIL.Thedryarecanuts,weighingapproximately10geach,werebroken
intosmallbits(roughly0.5cmcubes)withahammerandpruningshears,thenpulverizedinan
electriccoffeegrinder.TheroughpowderwasaddedtoRinger'ssolution(pH7.2)at200mgper
ml.Themixturewasstirred10minutesatroomtemperature,thendrip-filteredwithP8coarse
filterpaper.ThepHwasmeasuredatthispointtobe5.4andwasthenbroughtupto7.2.
HeterologousexpressionofnAChRsinXenopuslaevisoocytes
HumannAChRclonesandconcatamerswereobtainedfromDr.J.Lindstrom(Universityof
Pennsylvania,Philadelphia,PA).Thehumanresistance-to-cholinesterase3(RIC-3)clone,
obtainedfromDr.M.Treinin(HebrewUniversity,Jerusalem,Israel),wasco-injectedwithα7
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NicotinicBasisforBetelNutAddiction
toimprovethelevelandspeedofα7receptorexpressionwithoutaffectingthepharmacological
propertiesofthereceptors[28].Subsequenttolinearizationandpurificationoftheplasmid
cDNAs,cRNAswerepreparedusingthemMessagemMachineinvitroRNAtransfectionkit
(Ambion,Austin,TX).
OocytesweresurgicallyremovedfrommatureXenopuslaevisfrogs(Nasco,Ft.Atkinson,
WI)andinjectedwithappropriatenAChRsubunitcRNAsasdescribedpreviously[29].Frogs
weremaintainedintheAnimalCareServicefacilityoftheUniversityofFlorida,andallproce-
dureswereapprovedbytheUniversityofFloridaInstitutionalAnimalCareandUseCommit-
tee.Inbrief,thefrogwasfirstanesthetizedfor15–20minin1.5Lfrogtankwatercontaining1
gof3-aminobenzoatemethanesulfonatebufferedwithsodiumbicarbonate.Theharvested
oocytesweretreatedwith1.25mg/mlcollagenase(WorthingtonBiochemicals,Freehold,NJ)
for2hatroomtemperatureincalcium-freeBarth’ssolution(88mMNaCl,1mMKCl,2.38
mMNaHCO ,0.82mMMgSO ,15mMHEPES,and12mg/ltetracycline,pH7.6)toremove
3 4
thefollicularlayer.StageVoocytesweresubsequentlyisolatedandinjectedwith50nlof5–20
ngnAChRsubunitcRNA.Recordingswerecarriedout1–7daysafterinjection.
Two-electrodevoltageclampelectrophysiology
ExperimentswereconductedusingOpusXpress6000A(MolecularDevices,UnionCity,CA)
[29].Boththevoltageandcurrentelectrodeswerefilledwith3MKCl.Oocyteswerevoltage-
clampedat-60mV.Theoocyteswerebath-perfusedwithRinger’ssolution(115mMNaCl,2.5
mMKCl,1.8mMCaCl ,10mMHEPES,and1μMatropine,pH7.2)at2ml/minforα7recep-
2
torsandat4ml/minforothersubtypes.Toevaluatetheeffectsofexperimentalcompounds
comparedtoACh-evokedresponsesofvariousnAChRsubtypesexpressedinoocytes,baseline
conditionsweredefinedbytwoinitialapplicationsofAChmadebeforetestapplications.The
solutionswereappliedfroma96-wellplateviadisposabletips,andthetestcompoundswere
appliedalone,co-appliedwithACh,orco-appliedwithPNU-120596.Fortheconcentration-
responsestudy,drugapplicationsalternatedbetweenAChcontrolsandexperimentalcom-
pounds.Unlessotherwiseindicated,drugapplicationswere12sindurationfollowedbya181
swashoutperiodforα7receptorsand6switha241swashoutforothersubtypes.Atypical
recordingforeachoocyteconstitutedtwoinitialcontrolapplicationsofACh,anexperimental
compoundapplication,andthenafollow-upcontrolapplicationofAChtodeterminethe
desensitizationorrundownofthereceptors.ThecontrolAChconcentrationswere60μMfor
α7,100μMforα3β4,and30μMforα4β2.Theresponsesofα4β2andα3β4-expressingcells
weremeasuredaspeakcurrentamplitudes,andtheα7datawerecalculatedasnetcharge,as
previouslydescribed[30].
Datawerecollectedat50Hz,filteredat20Hz,analyzedbyClampfit9.2(Molecular
Devices)andExcel2003(Microsoft,Redmond,WA),andnormalizedtotheaveragedpeak
currentornet-chargeresponseofthetwoinitialAChcontrolsforeachoocyte[30].Datawere
expressedasmeans±SEMfromatleastfouroocytesforeachexperimentandplottedbyKalei-
dagraph3.0.2(AbelbeckSoftware,Reading,PA).Multi-cellaverageswerecalculatedforcom-
parisonsofcomplexresponses.Topermitbettercomparisonsbetweenexperimentseachsingle
cellresponsewasnormalizedtotheaverageofthetwoinitialcontrolsobtainedfromthatcell.
Averagesofthenormalizeddatawerecalculatedforeachofthe10,500pointsineachofthe
210straces(acquiredat50Hz),aswellasthestandarderrorsforthoseaverages.
Results
Anaqueousextractofarecanut(arecanutinfusion,ANI)waspreparedasdescribedabove,
andafterobtainingcontrolAChresponsesANIwasappliedtooocytesexpressinghumanα7
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NicotinicBasisforBetelNutAddiction
Fig2.Theeffectsofarecanutinfusion(seeMethods)onoocytesexpressingα7nAChR.Cellswereinitiallytestedfortheirresponsestocontrol
applicationsof60μMAChpriortotheapplicationofthefilterednutinfusedsolution.Aftera4-minutewash,theinfusionsolution±10μMPNU-120596was
applied(0.4mlover12seconds)followedbyanotherapplicationof60μMACh.Thecellswerevoltageclampedat-60mV,andthetracesshownrepresent
theaverageresponse(blackline)±theS.E.M.(shadedband)calculatedforeachofthe10,500pointsinthe210straces(acquiredat50Hz).Forapplication
oftheinfusionsolutionalonen=8,andforthedataobtainedinthepresenceofPNU-120596(n=5cells).
doi:10.1371/journal.pone.0140907.g002
nAChR.ANIaloneevokedminimalresponsescomparedtoAChbutsuppressedsubsequent
responsestoACh(Fig2).ANIproducednodetectableresponsesinoocytesthatwerenot
injectedwithRNAfornAChRsubunits(notshown).When10μMoftheα7-selectivePAM
PNU-120596[31]wasaddedtotheANI,theα7-expressingcellsshowedresponsesthatwere
muchlargerthanthoseevokedbyAChalone.PNU-120596isknowntodestabilizedesensi-
tizedstatesofα7nAChRandsotypicallyevokesresponsesthataremuchmoreprolongedthan
thosestimulatedbyAChorotherα7agonists[32].WhenusedincombinationwithAChin
oursystem,PNU-120596-potentiatedresponsesnormallydecaywellbacktobaselineduring
thenormalwashoutprocedure[32].However,potentiatedANIresponseswerebiphasicand
onlypartiallydecayedthroughthewashoutperiod.When60μMAChwasapplied4minutes
aftertheinitialANIapplication,therewasatransientincreaseincurrentaddedtothestill
decayingresponsestothepreviousapplicationofANIplus10μMPNU-120596.
Therearemanypotentiallyactivemoleculesinthearecanut,includingnumerousalkaloids,
withthemostabundantandactiveforproducingresponsesincentralandperipheralnervous
systemtissuesbeingarecoline,amuscarinicagonistwithactivityatM1,M2,andM3-type
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NicotinicBasisforBetelNutAddiction
Fig3.Effects100μMarecoline(structureillustrated)onoocytesexpressingα7nAChR.Cellsweretestedfortheirresponsestocontrolapplicationsof
60μMAChpriortotheapplicationofthetestsolution.Thesecondoftwosuchcontrolapplicationsisshown.Aftera4minutewashperiod,100μM
arecoline±10μMPNU-120596wasapplied(0.4mlover12seconds)followedbyanotherapplicationof60μMACh,asshown.Priortothecalculationofthe
multi-cellaverages,eachsinglecellresponsewasnormalizedtotheaverageofthetwoinitialcontrolsobtainedfromthatcell.Thecellswerevoltageclamped
at-60mV,andthetracesshownrepresenttheaverageofthenormalizedresponses(blackline)±theS.E.M.(shadedband)calculatedforeachofthe10,500
pointsinthe210straces(acquiredat50Hz).Forarecolinealone(n=8),andforarecolineplusPNU-120596(n=7).Inordertoallowforcomparison
betweenexperiments,thedatafortheresponsestothenutinfusionplusPNU-120596showninFig2werealsonormalizedtotheirrespectivecontrolsand
aredisplayedalongwiththearecolineplusPNU-120596dataintheinsert.
doi:10.1371/journal.pone.0140907.g003
receptors.Arecolineisatertiaryamine(Fig3)withgoodbrainpenetration,asevidencedbyits
numerouscentralnervoussystemeffects[7].Thereforewetestedarecolinespecificallyforits
effectsonα7nAChR.AsshowninFig3,100μMarecolineappliedalonedidnotproducesig-
nificantactivationofα7nAChR.SubsequentACh-evokedresponseswerealsolargelyunaf-
fected.However,whenarecolinewasco-appliedwith10μMPNU-120596,responseshada
peakamplitude9.6±3.4timeslargerthantheAChcontrols(n=7),withnetcharge37±10
timeslarger.Inordertofacilitatecomparisons,theresponsesevokedbyANIplusPNU-
120596showninFig2werenormalizedtotheirAChcontrolsandareshownintheinsertof
Fig3comparedtothePNU-120596-potentiated100μMarecolineresponses.Thearecoline
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NicotinicBasisforBetelNutAddiction
responseshadalargerpeakamplitude,but,astypicalforthepotentiatedresponsesofACh,
theydecayedmorerapidlyandcompletelybacktotheoriginalbaseline.TheACh-evoked
responsesfollowingthe100μMarecolineapplications(±PNU-120596)werenotsignificantly
differentfromtheoriginalACh-evokedcontrols.Thesedataindicatethatarecolineisanα7
silentagonist[24,25],essentiallyineffectiveatactivatingtheionchannelthroughthenormal
orthostericagonistbindingsite,butabletoinducethenon-conductingconformationalstates
thataredestabilizedbyPNU-120596.
Aconcentration-responsestudyofarecolineresponsespotentiatedby30μMPNU-120596
(Fig4A)indicatedEC valuesof60±7and93±2μMforpeakcurrentsandnetcharge,
50
respectively.Aswehavepreviouslyreported[24,25,27],intheabsentofaPAM,silentagonists
canfunctionasantagonistsofACh-evokedresponses.However,asshowninFig4B,witha
simpleco-applicationprotocolarecolinehadverylowpotencyforinhibiting60μMACh-
evokedresponses(IC >1000μM).Thisisconsistentwiththehypothesisthatsomestructural
50
requirementfeaturesofsilentagonistsmaybedistinctfromthoseforeffectivebindingand
inhibitionattheACh(i.e.orthosteric)bindingsite[25].
Inordertofurtherinvestigatethehypothesisthattheremaybecommonelementssharedby
thepharmacophoresofmuscarinicagonistsandα7silentagonists,wetestedotherknownacti-
vatorsofmuscarinicAChR,includingthenon-selectiveagonistcarbachol,withandwithout
PNU-120596.AsshowninFig5,carbacholstimulatedα7receptorsunderbothconditions.
Interestingly,oxotremorine,whileabletoproducesmallbutsignificantactivationinthe
absenceofthePAM,producedrelativelylittleadditionalactivationinthepresenceofPNU-
120596.Althoughnotasactiveasarecoline,methacholinewasalsoasilentagonistforα7,
whilemuscarinefailedtoactivatethereceptorsundereithercondition.
Thissmallpanelofmuscariniccompoundsisstructurallydiverse.Arecolinemostresembles
carbacholandmethacholineintermsoftherelativedistancebetweenthepositivechargeand
thehydrogenbondacceptor.Itdiffersfromthesetwointermsofnothavinga“hard”quater-
naryammoniumchargedcenter,butthisappearsnottobeafactorgiventhatmethacholine
maybeclassifiedasasilentagonist,whileoxotremorinecannot.Oxotremorineisinfact
unusualforbeingabletoproducesignificantorthostericactivationwithrelativelylittlepotenti-
ationbyPNU-120596.Thisisincontrasttotheusualcase,aswithcarbachol,whereeffective
orthostericactivationpredictseffectiveallostericallypotentiatedactivation.Theorthosteric
agonismofcarbacholisnotexhibitedbytherelativelyhomologouscompoundmethacholine,
andmaybeareflectionofconformationalbiasingofthemethylgroupwhenboundtothe
nAChRintheabsenceofthePAM.
WealsotestedwhetherarecolinewasabletoactivateothernAChRsubtypes.Asshownin
Fig6,althougharecolineshowedverylittleactivityattheganglionandmuscle-typeanalogs
(α3β4andα1β1εδ,respectively),itshowedsmallbutsignificantactivitywithoocytesinjected
withα4β2andtheα6-containingconcatamer(α6β2β3α4β2)[33].
NotethatintheexperimentshowninFig6,cellswereinjectedwithmonomericformsofα4
andβ2whichisknowntoproduceaheterogeneouspopulationofreceptorswithtwodifferent
ratiosofα4andβ2subunits.Byco-expressingaβ2-α4concatamerwithmonomericα4orβ2
subunits,itispossibletoobtainhomogeneouspopulationsofreceptorswithdefinedsubunit
composition,eitherα4(3)β2(2)orα4(2)β2(3),respectively[34].BasedonthepotencyofACh
andnicotineforactivatingthesereceptors,theyhavebeencharacterizedaslowsensitivity(LS)
orhighsensitivity(HS)subtypes.Chronicnicotineexposurehasbeenshowntospecifically
upregulatetheHSformofα4β2.Thiseffectisbelievedtobeimportantforthedevelopmentof
nicotineaddictionanddependence.Theexpressionofα6andβ3subunitsishigh,andlargely
restrictedto,dopaminergicneuronsbelievedtomediatethechemicalrewardpromoting
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Fig4.Arecolineconcentration-responsestudies.(A)Oocytesexpressingα7weretestedwithco-applicationsof30μMPNU-120596plusvarying
concentrationsofarecoline.Bothpeakcurrentsandnetchargeresponseswerecalculatedandnormalizedtotheaverageoftwoinitial60μMAChcontrol
responsesinthesamecells.TheEC valueswere60±7and93±2μMforpeakcurrentsandnetcharge,respectively.RelativetoAChcontrols,theI
50 max
valueswere15±1and49±1forpeakcurrentsandnetcharge,respectively.(B)SinceintheabsenceofaPAM,silentagonistscanfunctionasantagonists
oftypicalagonists,thepotencyofarecolineforantagonizing60μMACh-evokedresponseswastested.Arecolinewassurprisinglyineffectiveatinhibiting
AChresponses,withanIC >1000μM.
50
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Fig5.Agonistandsilentagonistactivityofmuscariniccholinergicagonists.Thepharmacophoreforsilentagonismofα7isdistinctfromthatfor
activationintheabsenceofaPAM[25].Sincearecolineisknowntobeamuscarinicagonist,wetestedadditionalcompoundswithmuscarinicactivityfor
theirabilitytoactivateα7intheabsenceandpresenceof10μMPNU-120596.Thestructuresofthetestcompoundsareshown,aswellas,thatofnicotinefor
comparison.
doi:10.1371/journal.pone.0140907.g005
nicotineself-administration(i.e.smoking).Thereforeα4-andα6-containingreceptorsarecon-
sideredimportanttargetsforsmokingcessationtherapies.
AsshowninFig7,arecolineisarelativelypotent,albeitlowefficacy,partialagonistforHS
α4β2andα6-containingreceptors,suggestingthatactivityatthesereceptorsmaysubtlymedi-
atesomeoftherewardassociatedwithhabitualbeteluse,andmoreovermayaccountforsome
oftheaddictivepropertiesofareca.Thetypicalpatternofbeteluseinvolvesprolongedchewing
thepreparedquidandsowouldbeexpectedtoproduceprolongeddeliveryofarecolineand
otherfactorsatlowlevels.Thissortofpresentationofapartialagonistwillhavetwoeffects:it
willdown-regulatethephasicactivityofotherstimuli,suchasoscillationsinendogenousACh,
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Fig6.ArecolineactivationofothernAChRsubtypes.100μMarecolinewasappliedtocellsexpressingthenAChRsubunitsindicated.Responsesof
humanα3β4,α7,andmousemuscle(α1β1εδ)subunitswerebarelyatthethresholdofdetection,lessthan1%theAChmaximum,extrapolatedfrom
comparisonstoAChcontrolsandAChconcentration-responsestudiesconductedpreviously.Theresponsesofcellsexpressingα4β2oraconcatamer
containingα6andβ3inadditiontoα4andβ2weresubstantiallylargerandwellabovethethresholdofdetection.
doi:10.1371/journal.pone.0140907.g006
anditmayalsopromotelowlevelsofsteady-stateactivationasreceptorspassinandoutof
desensitizedstates[35,36].AsshowninFig8,theprolongedbathapplicationof3μMarecoline
tocellsexpressingHSα4β2receptorsproducedsubstantialinhibitionofAChresponseanda
steady-statecurrentthatwassensitivetothenAChRantagonistmecamylamine.Thissmolder-
ingcurrentwassmallbutnon-trivial,asitwasapproximatelyequalto1%ofthemaximaltran-
sientcurrentassociatedwithAChactivation.Thetime-integratedeffectofthisactivationcould
besubstantial.
Theeffectsofarecanutinfusiononα7receptorswereonlypartiallymimickedbyarecoline,
sowealsotestedANIoncellsexpressingα4β2nAChR(mixedpopulationsformedfrom
monomers)ortheα6-containingconcatamer.Asexpected,theapplicationofANIproduced
smalltransientactivationofboththesereceptorsubtypes(Fig9),and,similartotheeffectson
α7receptors,therewasalargeinhibitionofsubsequentACh-evokedresponses.ANIco-appli-
cationalsoantagonizedthetransientactivationofthesereceptorsbyACh.
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