Brit.J.Pharmacol.(1961),16,352-359.EFFECTOFCOCAINEANDRELATEDDRUGSONTHE - PDF document

Brit.J.Pharmacol.(1961),16,352-359.EFFECTOFCOCAINEANDRELATEDDRUGSONTHEUPTAKEOFNORADRENALINEBYHEARTANDSPLEENBYE.MUSCHOLLFromtheDepartmentofPharmacology,UniversityofMainz,Germany(ReceivedApril10,1961)Noradrenalineuptakebyheartandspleenafterintravenousinfusionofnoradren-alinewasmeasuredinthepithedrat.Cocaine,givenbeforetheinfusion,inhibitedthenoradrenalineuptakeinrelation(a)tothedoseadministeredand(b)totheamountofnoradrenalineinfused.Therewasanassociationbetweenincreaseinthepressorresponsetoatestdoseofnoradrenalineandinhibitionoftheuptakebytheheart.Drugsrelatedchemicallytococaine,suchasa-cocaine,amethocaine,andatropine,didnotalterthenoradrenalineuptakeorpotentiatethebloodpressureresponsetonoradrenaline.Thenoradrenalineuptakebytheheartwasunchangedafterdibenamine,butblockedbythedichloro-analogueofisoprenaline.Itwasconcludedthatcocainespecificallypreventedtheuptakeofnoradrenalinebytissues,thusincreasingtheamountofnoradrenalineavailableforcombinationwithadrenergicreceptors.Thedichloro-analogueofisoprenalineappearedtoblockbothuptakebytheheartandthecombinationwithreceptors.SinceFrohlich&Loewi(1910)firstdescribedsensitizationbycocaineofvariousorganstoadrenaline,differentexplanationsforthepotentiatingeffectofcocaineonresponsestocatecholamineshavebeenoffered.RecentlyTrendelenburg(1959)reinvestigatedthequestionwhethercocainecausessupersensitivitybypreventingthedestructionofinjectednoradrenaline.Hefoundthatcocainedelayedtheinactivationofnoradrenaline,andthattheelevatedplasmaconcentrationsofnor-adrenalinefullyaccountedfortheincreaseinbloodpressureresponses.Trendelen-burg(1959)suggestedthatinactivationmaybecaused,amongstothermeans,byabindingprocess,andsuchbindingbymanytissueswasdemonstratedbyAxelrod,Weil-Malherbe&Tomchick(1959)toinactivatecirculatingadrenaline.Fromindirectevidence,Macmillan(1959)concludedthatcocainepreventedtheuptakeofcatecholaminesbytissuestores,therebyincreasingtheamountofnoradrenalineactingonsympatheticreceptors.Inadirecttestofthishypothesis,Muscholl(1960a)showedthatanoradrenalineinfusiongiventoaratapproximatelydoubledtheconcentrationofnoradrenalineinheartandspleen,andthatcocaineblockedthisuptake.Blockingbycocaineoftheaccumulationoftritium-labellednoradrenalineinvariousorganshasalsobeenobservedbyWhitby,Hertting&Axelrod(1960).Thepurposeofthepresentinvestigationwastoinvestigatethespecificityoftheeffectofcocaineontheuptakeofnoradrenalineandthequantitativerelationbetweenthisuptakeandthepotentiationoftheeffectsofnoradrenaline. COCAINEANDNORADRENALINEUPTAKEMETHODSMaleorfemalerats,weighing170to250g,wereanaesthetizedwithether,pithedandmaintainedbyartificialrespiration(Muscholl&Vogt,1957).ThebloodpressurewasrecordedfromthecarotidarterywithaCondon-typemercurymanometer.Afemoralveinwascannu-latedforinjectionofdrugs.Infusionsofnoradrenalinedissolvedinsalineweregivenintothiscannulaataconstantrateforaperiodof20minusingamotor-drivensyringe.Afterinfusionsorinjectionsthecannulawasrinsedthroughwithsaline.Thetotalvolumeofsalineadministeredinanyoneexperimentneverexceeded3ml.Fiveortwentyminaftertheendoftheinfusionofnoradrenalinetheheartwasexcised.Atnaandlargevesselswereremoved,themyocardiumdriedonfilterpaperandweighed.Thetissuewashomogenized,extractedinacidethanol,theextractpurifiedandchromato-graphedonpaper.Theeluatesoftheregionscontainingthenoradrenalineandadrenalinewereassayedindividuallyonthebloodpressureofthepithedratandontheisolatedatropinizedratuterusstimulatedwithoxytocin,asdescribedbyMuscholl(1959).Insomeexperimentstheconcentrationofnoradrenalineinthespleenwasestimatedbypreparingextractsasdescribedforcatheart(Muscholl,1959).Thefollowingsubstanceswereused:(-)-Noradrenalinebasedissolvedinhydrochloricacid;(-)-adrenaline-(+)-bitartrate(dosesandconcentrationsrefertothebases).Cocainehydrochloride;amethocaine(Tetracaine)hydrochloride;atropinesulphate;a-cocainehydro-chloride;dibenamine(N,N-dibenzyl-,8-chloroethylaminehydrochloride);thedichloro-analogueofisoprenaline,1-(3',4'-dichlorophenyl)-2-isopropylaminoethanolhydrochloride(dosesrefertothesalts).RESULTSActionofcocaineontheuptakeofnoradrenalinebyheartandspleen.Thecon-centrationofnoradrenalineintheheartof9normalratswasfoundtobe0.59+0.02tg/g(mean+s.e.ofthemean).Thisagreeswellwiththevalueof0.61fkg/greportedpreviouslybyMuscholl(1959).Likewise,theconcentrationofadrenalineinthenormalhearts(46+5ng/g)fellwithintherangefoundbyMuscholl(1959).TABLE1CONTENTOFNORADRENALINEANDADRENALINEINTHEHEARTANDOFNORADRENALINEINTHESPLEENOFTHEPITHEDRATAFTERINFUSIONConcentrationsinuigorng/gfreshtissue,meanzs.e.ofthemean.Numberofestimationsinbrackets.*Individualfigures.Ratskilled5minaftercessationofintravenousinfusionofsalineor30minafterintravenousadministrationofthedrug.DichloroisoprenalinewasinjectedintotailveinHeartSpleenDoseNoradrenalineAdrenalineNoradrenalineTreatmentDrugmg/kgug/gng!gUg/g-Untreatedcontrols059±0-02(9)46±5(5)019±0-04(6)PithedSalineinfusion,3ml./20min0-58±0-04(4)38±10(4)0-19±0-07(3)PithedCocaine100-61*;0-60(2)40;21(2)0-09;0-17(2)PithedDibenamine25-50055±0-02(3)Dichloroisoprenaline300-60±0*01(3)Theconcentrationofnoradrenalineinthespleenandtheconcentrationsofthecatecholaminesintheheartwerenotalteredbythepithingprocedureused(Table1).ControlvaluesgiveninTable2representthemeansofuntreatedratsandofpithedcontrolanimalsinfusedwithsaline.353 E.MUSCHOLLInheartsexcised5minaftertheendofaninfusionof20jigofnoradrenalineadministeredduringaperiodof20min,theconcentrationofnoradrenalinewas1.05+0.04ug/g.Thiscorrespondedtoanetuptakeof0.46ttg/gor78%ofthecontrolvalue.Theadrenalineconcentrationoftheheartwasnotaffectedbythenoradrenalineinfusion(Table2),butthenoradrenalineconcentrationofthespleenhadrisenby0.15ttg/g(79%increase).TABLE2EFFECTOFCOCAINEONTHENORADRENALINEUPTAKEBYTHEHEARTANDBYTHESPLEENAFTERANINFUSIONOFNORADRENALINEConcentrationsinjpgorng/gfreshtissue,mean±s.e.ofthemean.Numberofestimationsinbrackets.Organswereremoved5minafterendofinfusionpgofHeartDoseofnoradrenalineSpleencocaineinfusedNoradrenalinaAdrenalineNoradrenalinemg/kgin20minUg/gng/gfg/g--0-59±0-02(13)42±5(9)019±0-03(9)-20l-05±004(21)36±9(10)0-34±0-02(10)10200-85±0-03(4)32:5(4)0-26±004(4)20200-74±0-08(8)35±8(6)0-22±0-02(8)5085±003(10)-2-550.62±0-03(5)Inexperimentsinwhichtheheartwasexcised20minaftertheinfusiontermi-nated,theconcentrationofnoradrenalinewasstillhigh,being1.06+0.08ptg/g(n=5).Adoseof10or20mg/kgofcocainegiven5to15minbeforethenoradrenalineinfusionreducedsignificantlythenetuptakeofnoradrenalinebyheartandspleenmeasured5minaftertheendoftheinfusion(Table2).Again,theadrenaline0c220O0U0.60.81.01.2Noradrenaline(Lg/g)Fig.1.Potentiationofthepressorresponsetonoradrenalinebycocaineplottedagainstthenoradrenalineconcentrationintheratheartafteranoradrenalineinfusion.Eachpointrepresentsanestimationonapithedratinjectedwithcocaine(10to20mg/kg)andgivenanoradrenalineinfusionof20ugin20min.Ordinate:Ratioofthepressorresponsetoatestdoseofnoradrenaline(2to5ng)aftercocaine/controlresponse.Abscissa:Noradrenalineconcentrationoftheheartmeasured5to20minafterterminatinginfusion.Thecalculatedregressionissignificantatthe1%level.354 COCAINEANDNORADRENALINEUPTAKEconcentrationoftheheartwasunchanged.Concentrationsofnoradrenalinemeasured20minafterterminatingtheinfusion(0.74+±0.05jtg/g,n=6)werealsoloweredbythepreviousinjectionof10mg/kgofcocaine.In14outof18experimentsthepressorresponsetoasmalltestdoseofnoradren-alinewasrecordedbeforeandaftercocaine(10to20mg/kg)hadbeeninjected.Subsequently,20jugofnoradrenalinewasinfused.Thepressorresponsetonor-adrenalineaftercocainevariedinverselytoitsconcentrationmeasuredintheheartaftertheinfusion,andthisrelationcouldbeexpressedasaregression(Fig.1).Sinceincreasesofconcentrationsofnoradrenalineintheheartsabovecontrolvalues(0.59jfg/g)gaveanestimateoftheuptakebytheheart,theresultsinFig.1showthat,withincreasingdepressionoftheuptakeofnoradrenalinebycocaine,therewasincreasingsensitizationofthepressorresponsetonoradrenaline.Controlexperimentsweremadetoeliminatethepossibilitythatcocaineactedbyreducingtheconcentrationofnoradrenalineintheheartperseratherthanbypreventingtheuptakeofexogenousnoradrenaline.Table1showsthatcocainedidnotaltertheconcentrationsofcatecholaminesinheartandspleensignificantly.Intwootherexperimentstheamountofnoradrenalineaccumulatedbytheheartduringanoradrenalineinfusionof20jugdidnotdecreaseafterthesubsequentinjectionof10or20mg/kgofcocaine;theconcentrationsofnoradrenalineintheheartswere1.04and1.054g/g,respectively,andthusdidnotdifferfromthemeanof1.05tg/gforratsreceivinganoradrenalineinfusionwithoutpretreatmentwithcocaine.Injectionofasmalldose(2.5mg/kg)ofcocainehadnoeffectontheuptakeofnoradrenalinebytheheartif20,agofnoradrenalinewasinfused,butcompletelyblockedtheuptakeofnoradrenalinewhenonly5/-gwasinfused(Table2).Theresultsofexperimentsusingdosesof0.15to2.5mg/kgofcocaineandinfusions1o_(4)\~~~~~~~~~~~~~~(2)\(3)0.-g35(4)\r__.0.c(U-\(8)0*5)00.150.30.61.252.55.01020Doseofcocaine(mg/kg)Fig.2.Doseofcocaineplottedagainst%inhibitionofnoradrenalineuptakebytheheart.Heartsremoved5minafterterminatingnoradrenalineinfusionof5,ug(0)or20Pg(A)perratgivenin20min.Numberofestimationsinbrackets.NoradrenalineuptakecalculatedasY.ofcontrolvaluesfromTable2(for5uginfusions0%=0.59and100%=0.85ug/g;for20jpginfusions0%=0.59and100%=1.05pg/g).355 E.MUSCHOLLof5/AgofnoradrenalinearesummarizedinFig.2.Itcanbecalculatedfromtheregressionlinesthat0.8mg/kgofcocainecaused50%inhibitionofuptakewhen5jugnoradrenalinewasinfused,whereas11.6mg/kgofcocainewasrequiredtoproducethesamepercentageinhibitionwithinfusionof20ugofnoradrenaline.Sincetherewaslittlevariationintheheartweightsbetweentheexperimentalgroups,changesinconcentrationsalsorepresentedchangesintotalcontentofcatecholamines.Drugschemicallyrelatedtococaine.Theseeffectsofcocainemightberelatedtoitslocalanaestheticaction.Amethocaine,whichhasabout4timesthelocalanaestheticactivityofcocaine,didnotincreasethebloodpressureresponsetonoradrenalineinthepithedrat,nordiditinhibittheuptakeofnoradrenalinebytheheart(Table3).Atropine,whichisstructurallyrelatedtococaine,hadnosensitizingeffectcnthenoradrenalinepressorresponseandneitherdiditaltertheuptakeofnoradrenaline(Table3).TABLE3EFFECTOFDRUGSONTHENORADRENALINEUPTAKEINTOTHEHEARTAFTERANINFUSIONOFNORADRENALINEMeanofnoradrenalineconcentrationinug/gs.e.ofthemean.Numberofestimationsinbrackets.Heartswereremoved5minaftercessationofinfusionof20pgofnoradrenalinein20minDruggivenbeforeHeartnoradrenalinenoradrenalineinfusionmg/kgpg/gNodrug_105±004(21)Amethocaine5-101-24±0-10(3)Atropine201-02±0-04(3)a-Cocaine201-04±0-05(5)Dibenamine25-50091±010(5)Dichloroisoprenaline30064±005(5)Foster,Ing&Varagic'(1955)haveshownthata-cocaine,astructuralisomerofcocaine,hassomelocalanaestheticproperties,inhibitsmonoamineoxidasetothesameextentascocainebutlacksthepotentiatingactiononthecatecholaminesinthespinalcat.In5rats,a-cocainehadnoactiononthebloodpressureresponsetonoradrenalineorontheuptakeofnoradrenalinebytheheart(Table3).Adrenaline-blockingagents.AccordingtoBrown&Gillespie(1957),blockingoftheadrenalinereceptorsincreasedthenoradrenalineoutputofthespleen,presumablybypreventingthecombinationofnoradrenalinewiththereceptors.Inthepresentstudytwoblockingagentsweretested.Dibenamine(25to50mg/kg),whichblocksonlya-receptors(Ahlquist,1958),andthedichloro-analogueofiso-prenaline(30mg/kg),whichblocksonly,B-receptors(Powell&Slater,1958;Moran&Perkins,1958).Bythemselvesneithermodifiedtheconcentrationofnoradren-alineintheheart(Table1).Inthepithedratthemaximumpressoreffectofnoradrenalineinfusedatarateof20jig/20minwas88+4mmHg,anditoccurredwithinthefirstfewminutesofstartingtheinfusion.Dibenamine,injectedbefore356 COCAINEANDNORADRENALINEUPTAKEthenoradrenalineinfusion,abolishedcompletelytheinitialpressoreffectofnor-adrenaline.However,duringthecourseoftheinfusionthebloodpressurerosesteadily,reachingamaximumattheendoftheinfusion.Nevertheless,thisriseofbloodpressure(24+4mmHg)wassignificantlylowerthanthemaximumriseincontrolanimalsnotgivendibenamine.Theuptakeofnoradrenalinebytheheartwasnotsignificantlyaffectedbydosesofdibenaminewhicheffectivelyblockedthea-receptors(Table3).Thedichloro-analogueofisoprenaline,injectedbeforethenoradrenalineinfusion,significantlyincreasedthemaximumbloodpressurerisecausedbynoradrenaline(114+4mmHg).Theuptakeofnoradrenalinebytheheartwascompletelyblockedbytheanalogue(Table3).DISCUSSIONTheresultsreportedhereshowthatcocaineblockedthenoradrenalineuptakefromthecirculatingbloodbyheartandspleenwithoutaffectingtheendogenousconcentrationsofnoradrenalineandadrenalineintheseorgans.Muscholl(1960b)foundreserpineblockedtheuptakeofnoradrenalinebytheheart,but,incontrasttococaine,itdecreasedtheendogenousconcentrationofnoradrenaline.Theremaybe,therefore,adifferenceinthemechanismsbywhichcocaineandreserpinepreventtheuptakebytissuesofnoradrenalinefromthecirculatingblood.ThepresentresultsareinagreementwiththeworkofWhitbyetal.(1960),whofoundthataninjectionof5pjg/kgofcocainedepressedtheuptakeoftritium-labellednoradrenalinebytheadrenalglands,heartandspleenofthecat.Therewasacorrespondingelevationofbloodconcentrationsofnoradrenalinewithinthefirst3minfollowingtheinjection,asnotedbyTrendelenburg(1959).Theinhibitionoftheuptakeofnoradrenalinewasrelatedtothepotentiationoftheresponsestonoradrenalineobservedaftercocaine(Fig.1).Moreover,withincreasingdosesofnoradrenaline,muchlargerdosesofcocainewererequiredtoinhibituptake(Fig.2).Finally,thespecificityoftheactionofcocainewasdemon-strated.Ofthefourchemicallyrelateddrugs,cocaine,atropine,amethocaine,anda-cocaine,onlycocaineinhibitedthenoradrenalineuptakebytheheartandcausedsupersensitivitytowardsnoradrenaline.Dosesassmallas0.2mg/kgofcocaineareknowntopotentiatetheactionsofnoradrenalineonthenictitatingmembraneandspleenofthespinalcat(Trendelen-burg,1959),but,inthepresentexperiments,dosesof0.15and0.3mg/kgdidnotsignificantlyreducetheuptakeofnoradrenalinebytheheart(Fig.2).Thisapparantlackofcorrelationbetweentheeffectsofcocaineontheuptakeandthepotentiationofnoradrenalinemaybeexplainedbythefollowingconsiderations.Withinfusionsof20and5jugofnoradrenaline,thedosesofcocaineneededtoproducea50%inhibitionoftheuptakeofnoradrenalinewere11.6and0.8mg/kg,respectivelyThereforerelativelymuchsmallerdosesofcocainearerequiredtoblocktheuptakeofsomewhatsmalleramountsofinfusednoradrenaline.Thedosesofnoradren-alineinjectedbyTrendelenburg(1959)weresmaller(3to16jug/kg)thanthelowestdoseinfusedinthepresentexperiments(5ugperratbeingabout25pug/kg).357 E.MUSCHOLLAlthoughtheactionofcocaineonthenoradrenalineuptakewasspecificinthesensethatsomecloselyrelateddrugsdidnotpossesssuchaneffect,blockingofthenoradrenalineuptakeisalsoknowntooccurwithreserpineandwasseenwiththedichloro-analogueofisoprenaline.Thelatter,likecocaine,didnotaffecttheendogenousconcentrationsofnoradrenalineintheheart,butthetypeofblockexertedbythesetwodrugswasquitedissimilar.Itmightbeassumedthatcocaine,apartfromblockingthesiteswhichtakeupnoradrenalinefromtheblood,doesnotblocktheadrenalinereceptorsoftheheart,sinceitdoesnotblockbutpotentiatesnoradrenalineeffectsonatria(Macmillan,1959).Theisoprenalineanalogue,ontheotherhand,exertsaspecificblockoftheadrenalinereceptorsoftheheart(Moran&Perkins,1958).Bothagentsreducetheuptakeofnoradrenalinebythehearttothesameextentifemployedinasufficientdose.Thisindicatesthatblockofadrenalinereceptorsbythedichloro-analogueofisoprenalinewasnotreflectedinagreaterinhibitionofthenoradrenalineuptake.Possiblythenoradrenalinewhichcombinedwithadrenergicreceptorswasmetabolizedandthereforecouldnotbedetectedbythemethodused,whereasthenoradrenalinetakenupbythetissuestoreswasprotectedandcouldbeestimatedafterextractingthewholeheart.Itisinterestingtonotethatdibenamine,whichdoesnotblockthereceptorsresponsibleforincreaseinrateandforceofcontractionoftheheart(Nickerson,1949),didnotpreventnoradrenalinefrombeingheldbythetissues.Fromthepresentexperimentsthefollowingconclusionsmaybedrawn:Injectednoradrenalinenotonlycombineswithadrenergicreceptorsbutisstoredincon-siderableconcentrationsatothersites.Cocainepreventstheuptakeintothesesites,therebyincreasingtheamountofnoradrenalineavailableforcombinationwiththeadrenalinereceptors.Thedichloro-analogueofisoprenalineblocksboththeuptakeofnoradrenalineintothestoresandthecombinationwiththeadrenalinereceptors.ThisworkwassupportedbyagrantfromtheDeutscheForschungsgemeinschaft.MythanksareduetoDr.H.R.Ingforasampleofa-cocaineandtoMissGiselaIbelingfortechnicalassistance.REFERENCESAHLQUIST,R.P.(1958).PharmacologyinMedicine,ed.V.A.DRILL,2nded.,pp.378-407.NewYork:McGraw-HillBookCompany,Inc.AXELROD,J.,WEIL-MALHERBE,H.&TOMCHICK,R.(1959).ThephysiologicaldispositionofH3-epinephrineanditsmetabolitemetanephrine.J.Pharmacol.exp.Ther.,127,251-256.BROWN,G.L.&GILLESPIE,J.S.(1957).Theoutputofsympathetictransmitterfromthespleenofthecat.J.Physiol.(Lond.),138,81-102.FOSTER,R.,ING,H.R.&VARAGI6,V.(1955).Alpha-cocaine.Brit.J.Pharmacol.,10,436-441.FROHLICH,A.&LoEwi,0.(1910).ObereineSteigerungderAdrenalinempfindlichkeitdurchCocain.Arch.exp.Path.Pharmak.,62,159-169.MACMILLAN,W.H.(1959).Ahypothesisconcerningtheeffectofcocaineontheactionofsym-pathomimeticamines.Brit.J.Pharmacol.,14,385-391.MORAN,N.C.&PERKINS,M.E.(1958).Adrenergicblockadeofthemammalianheartbyadichloroanalogueofisoproterenol.J.Pharmacol.exp.Ther.,124,223-237.MUSCHOLL,E.(1959).DieKonzentrationvonNoradrenalinundAdrenalinindeneinzelnenAbschnittendesHerzens.Arch.exp.Path.Pharmak.,237,350-364.358 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