-AdrenergicReceptorsAugmentP2XNociceptiveResponsesintheUninjuredStateJ - PDF document

-AdrenergicReceptorsAugmentP2XNociceptiveResponsesintheUninjuredStateJasonG.Meisner,JamesB.Waldron,andJanaSawynokDepartmentofPharmacology,DalhousieUniversity,Halifax,NovaScotia,Canada.Inthepresentstudy,theadrenergicreceptor(AR)subtypemediatingadrenergicaugmen-tationofP2Xreceptor–mediatednociceptiveresponsesonsensorynerveendingswasexaminedbyusingselectiveARreceptoragonistsandantagonistsinSpragueDawleyratsintheuninjuredstate.Localadministrationof ReceivedSeptember7,2006;RevisedJanuary19,2007;AcceptedFebru-ary25,2007.SupportedbytheCanadianInstitutesofHealthResearch.AddressreprintrequeststoDr.JanaSawynok,DepartmentofPharma-cology,DalhousieUniversity,Halifax,NovaScotia,CanadaB3H1X5.E-mail:jana.sawynok@dal.ca©2007bytheAmericanPainSociety TheJournalofPain,Vol8,No7(July),2007:pp556-562Availableonlineatwww.sciencedirect.com reectsdirectinteractionsonsensorynerveendings,whereasthelatterinvolvesanindirecteffect.2-adrenergicreceptorshavebeenimplicatedinsympatheticinuencesonnociception,andstraindiffer-encesareprominentintheseinuences(seeDiscussion).Bothadrenergicaugmentationofinchingandintrinsicinchinginducedby-methyleneATPexhibitastraindifference,beingmorepronouncedinWistarthaninSpragueDawleyrats.Inthepresentstudy,wecharacterizedadrenergicre-ceptor(AR)subtypesinvolvedintheadrenergic-puriner-gicfacilitationofnociceptionatsensorynerveendingsinSpragueDawleyrats.Drugswerecoadministeredintothehindpaw,andobservationsareofparticularrele-vancetoadrenergic-sensorycouplingthatcanoccuratperipheralsensorynerveendings.AlthoughSpragueDawleyratsdonotexhibitthemostprominentexpres-sionoftheadrenergic-purinergicinteraction,thisisthemostcommonlyusedstrainforneuropathicpainstudies,andwewishedthedatageneratedtoberelevanttoasignicantbodyofpriorobservations.WeexaminedtheabilityofselectiveARagonists(2)tomimictheabilityofNAtoaugmentinchingbehaviorsandthermalhyperalgesiaproducedbyeneATPandtheneffectsofselectiveadrenergicreceptorantagonists()oneffectsproducedbyNAorphenylephrineincombinationwithMaterialsandMethodsExperimentswereperformedwithmaleSpragueDaw-leyrats(150to250g)obtainedfromCharlesRiver,Mon-treal.ExperimentsreportedinthisarticlewereapprovedbytheUniversityCommitteeonLaboratoryAnimalsandperformedinaccordancewithCanadianCouncilonAn-imalCareguidelines.Ratswerehousedinpairsandmaintainedona12/12-hourlight/darkcycleat22withfoodandwaterfreelyavailable.Eachhindpawwastestedonce,withanintervalof3to5daysbetweensuccessivetrials;apreviousstudyindicatedahighdegreeofreproducibilitybetweensuchtrials.Alllocallyadministereddrugsandcombinationswereinjectedinatotalvolumeof50L;injectionsweremadesubcutaneouslyintotheplantarhindpawwhileratswerelooselyrestrained.ThefollowingselectiveagentswereusedtocharacterizeARs:phenylephrine(1-ARag-onist),clonidineandUK14,304(2-ARagonists),prazo-sinandterazosin(1-ARantagonists),yohimbine(antagonist),andtimololandpropranolol(-ARantago-nists).AlldrugsweredissolvedinsalineexceptforUK14,304,yohimbine,andprazosin,whichweredissolvedin10%DMSO/10%HO/80%saline;appropriatevehiclecontrolswereusedthroughout.Alldrugswerepur-chasedfromSigma(St.Louis,MO).FlinchingBehaviorsIndividualratswereacclimatizedforaminimumof15minutesinaPlexiglasobservationchamber(28).Tworatswereobservedatatimeinadjacentchambersinalternating2-minutebinsfor32minutes,andspontaneousinching(pawelevationorshakingofthehindpaw)behaviorswererecordedasacumulativenum-berofepisodes.Thetimecourseforbehaviorsproducedbyadrenergic/purinergiccombinationsiscontinuous,andthisalternatingmethodrecordsone-halfofthetotalincidenceofbehaviors.Inthatearlierstudy,behaviorsweredeterminedoveraperiodof60minutes,dosesof25nmolNA,whereeffectsarelocal,behaviorsoccurprimarilyoveraperiodof30minutes,andtheshorterintervalwasusedinthecurrentstudy.ThermalHyperalgesiaAthermalhyperalgesiatestapparatus(DepartmentofAnesthesiology,UniversityofCaliforniaSanDiego)wasused.RatswereplacedinPlexiglasobservationchamberspositionedonaglasssurfacemaintainedat30Candacclimatizedfor30minutes,bywhichtimespontaneousexplorationhadceased.Radiantheat,intheformofafocusedlightbeam,wasdirectedthroughtheglassattheplantarsurfaceofthehindpaw,and3baselinehindpawwithdrawallatenciesweredeterminedat10-minuteintervals.Afterbaselinedeterminations,ratswerere-movedforinjectionofdrugs;theywerethenreturnedtothechambersandlatenciesdetermined5,15,30,45,and60minutesafterinjections.Quiescentintervalsbetweenspontaneousepisodeswereselectedfordeterminingthermallatencies.Themeanof2latencymeasurementstakenateachtimeintervalwasusedtocalculatecumu-lativedifferencesfrombaseline,andthisapproximatesanareaunderthecurve.Acutoffvalueof20secondswasimposedonthethermalstimulustopreventtissuedam-DataandStatisticsDataarepresentedasmeanandSEMforthetimecourse.Cumulativebehaviorsaredepictedasthetotalnumberofinchesorcumulativedifferencefrombase-lineduringtheindicatedtimeinterval.Datawereana-lyzedbyuseofanalysisofvariancefollowedbytheStu-dentNewmanKeulstest.ReceptorCharacterizationofFlinchingBehaviorsUsingARAgonistsLocaladministrationof500nmolintotheplantarsurfaceofthehindpawproducesfewinchingresponsesinSpragueDawleyrats(10to15inches,asdoessaline);incombinationwithNA(whichalsoproducesminimalresponseswhengivenalone),methyleneATPleadstoamarkedexpressionofinchingoveraperiodof30to60minutes.Theseeffectsarelocallymediated,asinjectionofNAintothecontralat-eralhindpawdoesnotaugmentinchesproducedbyORIGINALREPORT/Meisner,Waldron,andSawynok -methyleneATP(25/250nmolrespectivedrugdoses;22inchesoveraperiodof32minutesforcoin-jectionvs73forcontralateralinjection;.01,nWhenadrenalineiscombinedwiththereisalsoamarkedexpressionofinchingbehaviorsFig1).Althoughthedose-responsecurveliestotheleftoftheNAcurve,adrenalinealsoproducessomeintrinsicinchingbehaviors(hollowsymbols)thatcontributetothetotaleffectofthedrugcombination.Whenselective1-ARand2-ARagonistsareused,phenylephrine1-ARagonist)butnotclonidineorUK14,304(agonists)mimicstheeffectofadrenalineandNAinaug--methyleneATPresponses(Fig1).(Higherdosesof2-ARagonistswerenotexamined,astheseclearlyleadtosystemicsedativeeffects.)Apartfromadrenaline,allotherARagonistslackedintrinsicactivitycomparedwithsalinewhentestedatthemaximaldoseusedincombination(datanotshown).ReceptorCharacterizationofFlinchingBehaviorsUsingARAntagonistsPrazosin,widelyusedasaselective1-ARantagonist,leadstoinhibitionofinchesproducedbyNA/yleneATPoverawiderangeofdoses(0.03to3nmol;A).Incontrast,yohimbine,aselective2-ARantagonist,hasnosignicanteffectoverthisdoserange,andthereisa1to2order-of-magnitudedifferenceintheactionoftheseagentsinreducinginching(Fig2A).Prazosinalsoinhibitsinchesproducedbyphenylephrine/eneATP,andyohimbineappearstoexhibitsomeactivityatthehighestdose(30nmol;Fig2B).Itisofinteresttonotethatprazosinwasmuchmoreabletoinhibitthe-methyleneATPcombinationthanthephenyleph-rine/-methyleneATPcombination.The-ARantago-niststimolololandpropranolol,at100nmol,didnotsignicantlyalterinchesproducedbyNA/eneATP(datanotshown).ReceptorCharacterizationofThermalHyperalgesiaResponsesPeripheraladministrationof-methyleneATPintothehindpawalsoleadstoatransientthermalhyperal-gesiainSpragueDawleyrats;thisisincreasedbycoad-ministrationofNA,whichaugmentsboththemagnitudeanddurationofhyperalgesia.NAalsoleadstoaprominenthyperalgesiaincombinationwithaninactivedose-methyleneATP(150nmol;Fig3A).Thermalhyper-algesiaisalsoelicitedbyphenylephrinegivenincombi-nationwith-methyleneATP(Fig3B),butnotbyclonidineorUK14,304incombinationwitheneATP(Fig3C).Thetime-responsecurvesindicatethattheeffectofphenylephrineislesssustained(resolvesby30minutes)thanthatobservedwithNA(stillprominentat45min)(cfFigs3BandA).WithclonidineandUK14,304,latenciesattheendofthetimecourseareele-vatedcomparedwithcorrespondingtimepointsinthe-methyleneATPgroup(Fig3C),suggestiveofintrinsicanalgesicactions;however,thesearenotprominentin Figure1.Dose-responsecurvesforinchesproducedbymethyleneATP(500nmol)coinjectedincombinationwithARagonistsintotheplantarsurfaceofthehindpawofSpragueDawleyrats.Solidsymbolsindicatecumulativenumberofinchesover32minutesproducedbycombinationwithindi-cateddrugs;hollowsymbolindicatestheintrinsiceffectofadrenaline.[DashedlineindicatesdataforNAredrawnfortheshortertimecourseusedinthepresentstudyandisprovidedforthepurposeofdirectcomparison.]DataindicatemeanSEMvalues;n5to7pergroup.Inallcasesinwhichmeans60,valuesareincreased(.05)comparedwithsaline.ADR,Adrenaline;NA,noradrenaline;PE,phenylephrine;UK,UK14,304;CLON,clonidine. Figure2.InhibitionofinchesproducedbyNAwithmethyleneATP(25/300nmol,respectively),and,phenyleph-rine(PE)with-methyleneATP(300/300nmol,respectively)overaperiodof32minutesbyprazosin(PRZ)andyohimbine(YOH).*.05,***.001comparedwithcontrolgroup,indicatedbyhollowsymbolineachpanel(n6pergroup).1-ARsandPain controlexperiments(Fig3D).Whenhyperalgesiaresultsareexpressedascumulativereductionsinbaseline,adrenalineaugmentsthermalhyperalgesiabyyleneATPwithanactivitysimilartoNA,withphenyleph-rineproducingalessercumulativeeffectduetotheshortertimecourseofaction(Fig4Infurtherthermalhyperalgesiaexperiments,terazo-sin,anotherselective1-ARantagonist,producesadose-relatedreductioninthesustainedthermalhyperalgesiaproducedbyNAincombinationwithalowdoseofmethyleneATP(150nmol;Fig5A),andthisisdose-re-lated(Fig5B);however,ithasnoeffectonthemoretransienthyperalgesiaproducedbyahigherdoseofmethyleneATP(500nmol)givenalone(Fig5ThepresentstudyusedselectiveARagonistsandan-tagoniststocharacterizeadrenergicreceptorsinvolvedinaugmentingperipheralpainsignalinginducedbyreceptoractivation(vialocalhindpawadministrationof-methyleneATP)inSpragueDawley1-ARs,butnot2-ARs,areimplicatedin2differentfunctionalendpointsthatreectsensorynerveendingactivation(inchingandthermalhyperalgesia)intheun-injuredstate.Bothpainbehaviors(inching)andther-malhyperalgesiaproducedbyplantarinjectionsofmethyleneATPreectactivationofreceptorsonC-bers,basedonsensitivitytocapsaicin-pretreatment;bothdirectandindirectinuencesofNAonsuchactiva-tioncanoccur.Recently,MaruoetalpublishedastudyinwhichelectrophysiologicinteractionsbetweenadrenergicreceptorsandP2XreceptorswereexaminedinWistarratsaftersciaticnervetransection(axotomy);theysuggestedthat1B-ARsareinvolvedinpotentia-tionofP2X-mediatedresponsesinDRGs,basedonmRNAanalysisofadrenergicreceptors.Althoughthe1-ARsubtypeinvolvedinSpragueDawleyratscannotbedeterminedonthebasisoftheagonistsandantago-nistsusedinthepresentstudy,thereisaclearconsistencyintheessentialconclusionofimplicating1-ARsinaug-mentingP2X-mediatedresponsesonsensoryneuronsinthetwostudiesusingquitedifferentexperimentalap-proachesand2differentstrainsofrats.TherehasbeenlittledirectcharacterizationofinDRGneurons.AnearlystudynotedaninabilitytoobservemRNAfor1-ARsinsensorygangliainSpragueDawleyrats.Inmorerecentstudies,mRNAformultiple1-ARshasbeenidentiedinsensorygangliainLewis(thispaperdiscussedpossiblereasonsforthelackofdetectioninthatearlierstudy)andWistarrats,1-ARsareimplicatedinfunctionalstudiesofperipheraladrenergicinuencesonnociceptionusingLewisandWistarrats.IntrinsiceffectsofNAonnociceptionafterinammationaremorepronouncedinLewisratscomparedwithSpragueDawleyratsandintheuninjuredstateinWistarratscomparedwithSpragueDawleyrats.Collectively,theseobservationssuggestaprominentroleof1-ARsinmediatingperiph-eraladrenergicnociceptiveresponsesinbothLewisandWistarstrainsofrats;therearefewerdataimplicating1-ARsinsuchactionsinSpragueDawleyrats.Thereis1datasetinwhich1-ARsdofeaturepromi-nentlyinfacilitatingperipheralnociceptiveresponsesinSpragueDawleyrats,andthatiswithinteractionswithTRPV1receptors.Thisresponseismaintainedaftersympathectomy,andthepresenceof1-ARsonnocicep- Figure3.Timecourseofthermalhyperalgesiaproducedby-methyleneATPinjectedintotheplantarsurfaceofthehindpawofSpragueDawleyratscombinedwithNAandclonidineorUK14,304IndicateddosesareinEffectsofadrenergicagonistsareshownincompari-sontosaline(SAL).*.05,**.01comparedwithgroupdepictedbyhollowsymbols(n6pergroup).NA,noradrena-line;PE,phenylephrine;CLON,clonidine;UK,UK14,304. Figure4.Cumulativechangesfrombaseline,representingtheareaunderthecurve,forthermalhyperalgesiaproducedby-methyleneATP(150nmol)incombinationwithanumberofARagonists.Negativevaluesindicatecumulativereductionsinlatency.n6pergroup.ADR,Adrenaline;NA,noradrenaline;PE,phenylephrine;CLON,clonidine;UK,UK14,304.ORIGINALREPORT/Meisner,Waldron,andSawynok tiveafferentneuronshasbeenemphasizedininterpret-ingresults.GiventhatbothP2XandTRPV1receptorscolocalizeinasignicantpopulationofsensoryandthatactivationofbothreceptorsallowsforcationentryintosensoryafferentsandleadstoen-hancednociception,aprominentinvolvementofinfacilitatingcationchannelfunctiononsensoryaffer-entsisplausible.BothTRPV1andP2XreceptorsarepositivelyregulatedbyproteinkinaseCandotherCadependentprocesses,andtheseintracellulareventscanbeactivatedasaresultof1-ARactivation.Inpreviousstudies,2-ARs(particularly2Aand2C)havebeenexamineddirectlyinDRGneuronsinSpragueDawleyrats.Thisemphasiswasbasedonthemanypreviousfunctionalstudiesthathadimpli-2-ARsinpronociceptiveresponsesaftervariousformsofnerveortissueinjury.Thepresentstudydoesnotprovideclearevidencefor2-ARin-volvementinaugmentinginchingorthermalhyper-algesiaresponsesproducedbyP2Xreceptoractivation,as1-AR(butnot2-AR)agonistsclearlymimictheeffectofNA,and1-ARantagonistsshowaclearselectivityinblockingtheeffectsofNAcombinedwith-methyleneATP.Theabilityofahighdoseofyohim-binetoblockinchingproducedbyNA/eneATPmayreectlossofreceptorselectivityathigherdoses,asresponsestophenylephrine,aselec-1-ARagonist,incombinationwitheneATP,alsowasreducedatahighdose.Itisinterest-ingtonotethatprazosinismuchmorepotentinblockingtheeffectofNAthanphenylephrineincom-binationwith-methyleneATP,anditispossiblethattherecouldbean2-ARinteractionexpressedwiththelessselectiveagonist(NA).Withthermalhyperal-gesiaresponses,thereistheadditionalpossibilitythatintrinsicantinociceptiveeffectsof2-ARagonistscould,insomeinstances,confoundincreasednocicep-tionproducedby2-ARsincombinationwithmethyleneATP(cfFig3C).Whereas-ARsareimpli-catedinfacilitationofsomenociceptiveresponsesinSpragueDawleyrats,-ARantagonists(propranololandtimolol)didnotinhibitresponsesproducedbyNA/-methyleneATP,anditisunlikelythatthesereceptorscontributetoeffectsobservedinthepresentstudy.TheinvolvementofsympatheticnervesinadrenergicaugmentationofP2Xreceptor–mediatedthermalhyperalgesiaprovidesamodelofneuronal-neuro-nal(sympathetic-sensory)interactionsthatoccuratthelevelofthesensorynerveterminal.Otherpoten-tialindirectmechanisms,includingnon-neurogenicones,needtobeconsideredasperhapscontributingtothepresentobservations.(1)NAcouldleadtovascularconstrictionvia1-ARs,whichleadstoretentionof-methyleneATPinthetissueforlonger,allowingforapharmacokinetic,ratherthanapharmacodynamic,mechanismtocontributetoobservedinteractions.InSpragueDawleyrats,thisisunlikelytobetheonlymechanisminvolved,asNAclearlypotentiatesmethyleneATPresponsesevenwhenthereisnointrin-siceffectproducedby-methyleneATP(eg,Fig3InWistarandLongEvansrats,inwhichintrinsiceffectsofboth-methyleneATPandNAareobservedwithinching,thereisnopotentiationoftheimmediatepeakeffectbutaprogressiveaugmentationwithtimeandamarkedprolongationofaction;althoughthispatterncouldreectapharmacokineticinteraction,theprogressivenatureofdevelopmentofthepeakeffect(doublingoftheinitialeffect)clearlysuggestsapharma-codynamicinuenceinwhichtimeisrequiredforfullexpressionofpeakaction.(2)NAcouldactonothercelltypestoreleasechemicalmediatorsthatthenactonsen-soryneuronstointeractwiththeP2Xatedresponses;thisrepresentsanon-neurogenicindi-rectmechanism.Forexample,phenylephrine(agonist)hasbeenshowntoreleasenervegrowthfactorfromculturedvascularsmoothmusclecells,andthismediatorcouldcontributetoacutefacilitatoryeffectsonnociceptionbyactionsonsensoryafferents,itsmoreprominentroleisinregulationoflonger-termFurtherclaricationofthenatureofthemarkedpositiveinteractionobservedbetweenandP2Xreceptorsonperipheralnociceptionwillrequiretheuseofadditionalexperimentalapproaches. Figure5.Effectofterazosin(TER)onthermalhyperalgesiaproducedbyNAcombinedwith-methyleneATP(25/150nmol,respectively).Theleftpaneldepictsthetimecourseandthemiddlepaneldepictsthedose-responserelationforterazosininreducingsustainedhyperalgesiaproducedbyNAcombinedwith-methyleneATP.Therightpaneldepictsthelackofeffectofterazosininreducingthetransienthyperalgesiaproducedbyahighdoseof-methyleneATPalone.Indicateddosesareinnmol.*.05,**.01comparedwithNA/-methyleneATP(n6pergroup).1-ARsandPain Insummary,thepresentstudyrevealsaprominentpos-itiveinteractionbetween1-ARsandP2Xtorsonsensorynerveendings.Directeffectsonsensorynervesdoappeartooccur,butindirecteffectsonsympa-theticneuronsand/orvascularsmoothmuscle,involvingreleaseofintermediaryagentswhichthenactonsensoryafferents,alsomaycontributetosucheffects.Giventhat1-ARmechanismsareimplicatedinhumanneuropathicpainstatesandthatantagonistsforP2Xreceptorsarereceivingconsiderableattentionasapo-tentialnovelanalgesic,thisinteractionmaybeofimportancetoaddressusingfurtherinvivoandinvitrotech-niques.Suchstudieswillneedtoattendtotheprominentstraindifferencethatoccursinratswiththisinteraction.WethankAllisonReidfortechnicalassistance.1.AleyKO,LevineJD:Multiplereceptorsinvolvedinperiph-,andAantinociception,tolerance,andwithdrawal.JNeurosci17:735-744,19972.BanikRK,SatoJ,YajimaH,MizumuraK:Differencesbe-tweentheLewisandSprague-Dawleyratsinchronicinam-mationinducednorepinephrinesensitivityofcutaneousC-bernociceptors.NeurosciLett299:21-24,20013.Bland-WardPA,HumphreyPPA:Acutenociceptionmedi-atedbyhindpawP2Xreceptoractivationintherat.BrJPharmacol122:365-371,19974.BradburyEHJ,BurnstockG,McMahonSB:TheexpressionofP2Xpurinoreceptorsinsensoryneurons:Effectsofaxotomyandglial-derivedneurotrophicfactor.MolCellNeuro-sci12:256-268,19985.BurnstockG:NoradrenalineandATP:Cotransmittersandneuromodulators.JPhysiolPharmacol46:365-384,19956.ChenY,LiGW,WangC,GuY,HuangLYM:MechanismsunderlyingenhancedP2Xreceptor-mediatedresponsesintheneuropathicpainstate.Pain119:38-48,20057.ChoHJ,KimDS,LeeNH,KimJK,LeeKM,HanKS,KangYN,KimKJ:Changesinthe-adrenergicreceptorsubtypesgeneexpressioninratdorsalrootganglioninanexperimen-talmodelofneuropathicpain.NeuroReport8:3119-3122,8.GoldMS,DastmalchiS,LevineJD:-Adrenergicreceptorsubtypesinratdorsalrootandsuperiorcervicalganglionneurons.Pain69:179-190,19979.GuoA,VulchanovaL,WangJ,LiX,EldeR:Immunocyto-chemicallocalizationofthevanilloidreceptor1(VR1):Rela-tionshiptoneuropeptides,theP2X3purinoceptorandIB4bindingsites.EurJNeurosci11:946-958,199910.HamiltonSG,McMahonSB,LewinCR:Selectiveactiva-tionofnociceptorsbyP2Xreceptoragonistsinnormalandinamedratskin.JPhysiol543.2:437-445,200111.HamiltonSG,WadeA,McMahonSB:Theeffectsofin-ammationandinammatorymediatorsonnociceptivebe-haviourinducedbyATPanaloguesintherat.BrJPharmacol126:326-332,199912.JänigW,HäblerHJ:Sympatheticnervoussystem:contri-butiontochronicpain.BrainRes129:451-468,200013.JarvisMF:ContributionsofP2X3homomericandhet-eromericchannelstoacuteandchronicpain.ExpertOpinTherTargets7:513-522,200314.KageK,NiforatosW,ZhuCZ,LynchKL,HonoreP,JarvisM:AlterationofdorsalrootganglionP2X3receptorexpres-sionandfunctionfollowingspinalnerveligationintherat.ExpBrainRes147:511-519,200215.KennedyC,AssisTS,CurrieAJ,RowanEG:Crossingthepainbarrier:P2receptorsastargetsfornovelanalgesics.JPhysiol553.3:683-694,200316.KennedyC:P2Xreceptors:targetsfornovelanalgesics?Neuroscientist11:345-356,200517.KhasarSG,GreenPG,ChouB,LevineJD:Peripheralno-ciceptiveeffectsof-adrenergicreceptoragonistsintherat.Neuroscience66:427-432,199518.KhasarSG,McCarterG,LevineJD:Epinephrineproduces-adrenergicreceptor-mediatedmechanicalhyperalgesiaandinvitrosensitizationofratnociceptors.JNeurophysiol81:1104-1112,199919.KimC,ChungJM,ChungK:Changesinthegeneexpres-sionofsixsubtypesofP2Xreceptorsinratdorsalrootgan-glionafterspinalnerveligation.NeurosciLett337:81-84,20.KinnmanE,LevineJD:Involvementofthesympatheticpostganglionicneuronincapsaicin-inducedsecondaryhy-peralgesiaintherat.Neuroscience65:283-291,199521.KoshimizuT,TanoueA,HirasawaA,YamauchiJ,Tsuji-motoG:Recentadvancesin-adrenoceptorpharmacology.PharmacolTherapeutic98:235-244,200322.LeeDH,LiuX,KimHT,ChungK,ChungJM:Receptorsubtypemediatingtheadrenergicsensitivityofpainbehav-iorandectopicdischargesinneuropathicLewisrats.JNeu-rophysiol81:2226-2233,199923.LevineJD,TaiwoYO,CollinsSD,TamJK:Noradrenalinehyperalgesiaismediatedthroughinteractionwithsympa-theticpostganglionicneuroneterminalsratherthanactiva-tionofprimaryafferentnociceptors.Nature323:158-160,24.LewinGR,RitterAM,MendellLM:Nervegrowthfactor-inducedhyperalgesiaintheneonatalandadultrat.JNeu-rosci13:2136-2148,199325.LinQ,ZouX,FangL,WillisWD:Sympatheticmodulationofacutecutaneousareinducedbyintradermalinjectionofcapsaicininanesthetizedrats.JNeurophysiol89:853-861,26.MaruoK,YamamotoH,YamamotoS,NagataT,Fu-jokawaH,KannoT,YaguchiT,MarioS,YoshiyaS,NishizakiT:ModulationofP2Xreceptorsviaadrenergicpathwaysinratdorsalrootganglionneuronsaftersciaticnerveinjury.Pain120:106-112,200627.McGaraughtyS,JarvisMF:Antinociceptivepropertiesofaselectivenon-nucleotideP2X3/P2X2/3receptorantago-nist.DrugNewsPerspect19:501-507,2005ORIGINALREPORT/Meisner,Waldron,andSawynok 28.MoonDE,LeeDH,HanHC,XieJ,CoggeshallRE,ChungJM:Adrenergicsensitivityoftheratsensoryreceptorsmod-ulatingmechanicalallodyniainaratneuropathicpainmodel.Pain80:589-595,199929.NorthRA:TheP2Xsubunit:Amoleculartargetinpaintherapeutics.CurrOpinInvestigDrugs4:833-840,200330.OusephAK,LevineJD:Alpha1-adrenoceptor-mediatedsympatheticallydependentmechanicalhyperalgesiaintherat.EurJPharmacol273:107-112,199531.ParkSK,ChungK,ChungJM:Effectsofpurinergicandadrenergicantagonistsinaratmodelofpainfulperipheralneuropathy.Pain87:171-179,200032.PaukertM,OsterothR,GeislerHS,BrändleU,GlowatzkiE,RuppersbergJP,GründerS:Inammatorymediatorspo-tentiateATP-gatedchannelsthroughtheP2XJBiolChem276:21077-21082,200133.PieriboneVA,NicholasAP,DagerlindA,HökfeltT:Dis-tributionofalpha-1adrenoceptorsinratbrainrevealedbyinsituhybridizationexperimentsutilizingsubtypespecicprobes.JNeurosci14:4252-4268,199434.PremkumarLS,AhernGP:Inductionofvanilloidrecep-torchannelactivitybyproteinkinaseC.Nature408:985-990,35.RamerMS,BradburyEJ,McMahonSB:NervegrowthfactorinducedP2Xexpressioninsensoryneurons.JNeurochem77:864-875,200136.RenY,ZouX,LinQ:SympatheticmodulationofactivityinA-andC-primarynociceptiveafferentsafterintradermalinjectionofcapsaicininrats.JNeurophysiol93:365-377,37.SatoJ,PerlER:Adrenergicexcitationofcutaneouspainreceptorsinducedbyperipheralnerveinjury.Science251:1608-1610,199138.ShiTJ,Winzer-SerhanU,LeslieF,HökfeltT:Distributionandregulationof-adrenoceptorsinratdorsalrootganglia.Pain84:319-330,200039.TeassellRW,ArnoldJMO:Alpha-1adrenoceptorhyper-responsivenessinthreeneuropathicpainstates:Complexregionalpainsyndrome,diabeticperipheralneuropathicpainandcentralpainstatesafterspinalcordinjury.PainResManage9:89-97,200440.TraceyDJ,CunninghamJE,RommMA:Peripheralhyper-algesiainexperimentalneuropathy:Mediationbynoreceptorsonpost-ganglionicsympatheticterminals.Pain60:317-327,199541.TsudaM,KoizumiS,KitaA,ShigemotoY,UenoS,InoueK:MechanicalallodyniacausedbyintraplantarinjectionofP2Xreceptoragonistinrats:Involvementofheteromericreceptorsignalingincapsaicin-insensitiveprimaryafferentneurons.JNeurosci20RC90:1-5,200042.TsuzukiK,KondoE,FukuokaT,YiD,TsujinoH,Sak-agamiM,NoguchiK:DifferentialregulationofP2Xexpressionbyperipheralnerveinjuryinintactandinjuredneuronsintheratsensoryganglia.Pain91:351-360,200143.TuttleJB,EtheridgeR,CreedonDJ:Receptor-mediatedstimulationandinhibitionofnervegrowthfactorsecretionbyvascularsmoothmuscle.ExpCellRes208:350-361,199344.VellaniV,MapplebeckS,MoriondoA,DavisJB,Mc-NaughtonPA:ProteinkinaseCactivationpotentiatesgatingofthevanilloidreceptorVR1bycapsaicin,protons,heatandanandamide.JPhysiol534:813-825,200145.WaldronJB,SawynokJ:PeripheralP2Xreceptorsandnociception:Interactionswithbiogenicaminesystems.Pain110:79-89,200446.XieJ,LeeYH,WangC,ChungJM,ChungK:Differentialexpressionofalpha1-adrenergicsubtypemRNAsinthedor-salrootganglionafterspinalnerveligation.MolBrainRes93:164-172,200147.XuGY,HuangLYM:PeripheralinammationsensitizesP2Xreceptor-mediatedresponsesinratdorsalrootganglionneurons.JNeurosci22:93-102,200248.XuGY,HuangLYM:Ca/calmodulin-dependentproteinkinaseIIpotentiatesATPresponsesbypromotingtraf-ckingofP2Xreceptors.ProcNatlAcadSc11873,200449.ZhouJ,ChungK,ChungJM:Developmentofpurinergicsensitivityinsensoryneuronsafterperipheralnerveinjuryintherat.BrainRes915:161-169,20011-ARsandPain