Brit.J.Pharmacol.(1965),24,89-97.THEMODEOFACTIONOFDRUGSBLOCKINGGANGLIO - PDF document

Brit.J.Pharmacol.(1965),24,89-97.THEMODEOFACTIONOFDRUGSBLOCKINGGANGLIONICTRANSMISSIONINTHERATBYD.G.SHANDFromtheDepartmentofPharmacology,MedicalCollegeofSt.Bartholomew'sHospital,London,E.C.1(ReceivedMay18,1964)Quilliam&Shand(1964)foundthattheganglion-blockingactionofdrugsintheratcanbeeitherhighlyselective,moderatelyselectiveornonselective.Asitappearedthatthemodeofblockproducedbydrugswithinthesameselectivitygroupwasnotalwayspreciselythesame,thepresentworkwascarriedouttoinvestigatethesedifferences.METHODSTheisolatedsuperiorcervicalganglionpreparationoftheratwasusedinthemannerdescribedbyQuilliam&Shand(1964).Theganglion,togetherwiththepre-andpostganglionicnerves,wasexcised,theconnectivetissuesheathremovedandthepreparationmountedhorizontallyinabathofKrebssolutionat300Cequilibratedwithamixtureof95%oxygenand5%carbondioxide.Thepreganglionicnervecouldbestimulatedthroughapairofplatinumelectrodesat6shocks/minandtheganglionicactionpotentialrecordedfromapairofnonpolarizablesilver-silverchloride-agarsalineelectrodes,oneontheganglionandtheotheronthecutendofthepostganglionicnerve.TherecordingelectrodeswereconnectedtoaDCamplifier.Thepotentialsweredisplayedonacathode-rayoscilloscope.Torecord,thepreparationwasraisedabovethefluidlevel,givingmoistchamberconditions,andtheactionpotentialinresponsetothethirdstimuluswasphotographed.Insomeexperimentsthedepolarizingactionofacetylcholinewasmeasuredusingthemovingfluidelec-trodetechniqueofPascoe(1956),bywhichthepotentialatanypointalongtheganglioncanbemeasuredrelativetothecutendofthepostganglionicnerve,usingaspacebaseintheoscilloscope.Theeffectsofthefollowingdrugswereobserved:acetylcholinechloride(Roche),hexamethoniumchloride(Geigy),tetraethylammoniumbromide(B.D.H.),tubocurarinechlorideandbretyliumtosylate(BurroughsWellcome),atropinesulphate(Samoore),amylobarbitonesodium(Boots),methylpentynolandmethylpentynolcarbamate(BritishSchering),mephenesin(B.D.H.),paraldehyde(B.D.H.),benactyzinehydrochloride(Levanol,I.C.I.),procainehydrochloride(Samoore),tetramethylammoniumbromide(B.D.H.)andnicotinehydrogentartrate(Samoore).Unlessotherwisestatedthedosesaregivenasthesalts.RESULTSEffectsontheformoftheganglionicactionpotentialThroughoutdrugswereaddedtothebathinacumulativefashion,eachdosebeingincontactwiththepreparationfor5min.Thisallowedrapidcomparisonandtheexposuretimewasconsideredsufficientforequilibration(Quilliam&Shand,1964).Whileallthedrugsusedblockedthespikecomponentoftheganglionicactionpotential,increasingcon-centrationsproduceddifferenteffectsontheganglionicafterpotentials.Thedepolarizing D.G.SHANDagents,nicotine,tetramethylammonium,carbacholandacetylcholine,hadqualitativelythesameactionasthatdescribedbyEccles(1956)ontheisolatedsuperiorcervicalganglionoftherabbitandbyPaton&Perry(1953)foracetylcholine,tetramethylammoniumandnico-tineontheintactganglionofthecat.TheeffectofnicotinewastypicalandisshowninFig.1.AstheamplifierusedwasDC,allupwardshiftsofthebaselineindicatedadepolar-izationoftheganglionrelativetothecutendofthepostganglionicnerve.WithalowCIxIO-62x1o-64x10-6NicotineFig.1.Effectsofnicotineontheganglionicactionpotential.Thepotentialsrecordedwereinresponsetosinglemaximalstimulitothepreganglionicnerve.Cindicatescontrolresponse.Cumulativedosesofnicotinewereaddedtothebathandrecordsweretakenafter5-minexposure.Drugconcentrationsareshownaboveeachresponse.Anupwardshiftofthebaselinerelativetothelowertrace(referencebeam)indicatesadepolarizationoftheganglion.Calibration,25cycles/secwithanamplitudeequivalentto2.5mV.Thechangesintheshapeoftheganglionicactionpotentialaretypicalnotonlyoftheactionofnicotinebutalsooftheotherdepolarizingdrugs,tetramethylammonium,carbacholandacetylcholine.Initially(1x106)theNwavewasdecreasedandthePwaveaugmented,withlittledepressionofthespike.Then(2x10-6)thePwavewasshortenedandboththeNwaveandthespikewerefurtherdepressed.Finally(4x10-6)allthepotentialsbecamemarkedlyreducedasfullblockoftransmissionwasapproached.concentration(1x10-6),thenegativeafterpotential(Nwave)wasreducedinsizeandthepositiveafterpotential(Pwave)wasaugmentedwithlittleornoreductionintheheightofthespikepotential.Withgreaterconcentrations,thespikepotentialwasdecreasedandtheNwavewasabolished.ThePwavewasfirstshortenedinduration,returningmorerapidlytoisopotentialthanwithlowdoses,beforebeinggraduallyabolishedbyhighdrugconcentrations(4x10-6).Tubocurarine(Fig.2a)reducedallthecomponentsoftheganglionicactionpotentialequally.Atfullblock,asynapticpotentialwasobtainedwhichwasabout10to20%oftheinitialspikeheightor30to50%oftheinitialsizeoftheNwave.Thisisnotillustrated,butthesynapticpotentialwithtubocurarinewasthesameinformasthatproducedbytetraethylammoniuminFig.2d.Depolarizationwasneverobserved.Theactionoftetraethylammoniumandbretyliumappearedtobemidwaybetweenthoseoftubocurarineandnicotineasregardstheireffectsontheshapeoftheganglionicpotential(Fig.2candd).Initially,aswithsmalldosesofthedepolarizingagents,theNwavewasdecreasedandthePwaveincreasedwithonlyasmalldecreaseinthespikepotential.IncreaseindosagedecreasedthePwaveandfinallyproducedasynapticpotentialverylikethatseenaftertubocurarine.Inkeepingwiththeireffectsontheganglionicpotential,bothtetraethylammoniumandbretyliumcausedamodestdepolarizationoftheganglion.Hexamethonium(Fig.2b)hadmuchthesameactionastubocurarine,butwithlowdosestendedtodepresstheNwaveandcauseslightdepolarization,thoughnottothesameextentasdidtetraethylammonium.Therewasagraduatedincreaseinthestimulatoryeffects,as90 MODESOFGANGLIONICBLOCKC4x10-58x10-51.6x10-4(a)TubocurarineC2x10-54x10-58x10-5(b)HexamethoniumCIx10-42x10-44x10-'(c):BretyliumC4x10-58x10-53.2x10-4(d)TetraethylammoniumFig.2.Effectsof(a)tubocurarine,(b)hexamethonium,(c)bretyliumand(d)tetraethylammoniumontheganglionicactionpotential.RecordsasinFig.1.Cumulativedosesofthedrugs(concentrationsgivenabovetraces)addedafterobtainingcontrolrecords(C).Calibrations,25cycles/secand3mV.Hexamethonium,bretyliumandtetraethylammoniumproducedsomedepolarization.Lowdoses(secondcolumnoftraces)depressedtheNwaveandaugmentedthePwavewithlittlereductionofspikeheightasdolowdosesofthedepolarizingdrugs(Fig.1).Thesestimulatorypropertiesbecomemoremarkedasonepassesfromtubocurarine,throughhexamethoniumtobretyliumandtetraethyl-ammoniumbutwerealwayslessmarkedthanthoseobservedwiththedepolarizingdrugs,Puresynapticpotentialsareillustratedfortetraethylammoniumandbretyliumatcompleteblockoftransmission,butnotfortubocurarineorforhexamethonium,forwhichblockwasincomplete.judgedbythedepolarizationandeffectontheshapeoftheactionpotentialcomplex,asonepassedfromtubocurarine,throughhexamethoniumtobretyliumandtetraethyl-ammonium.Amylobarbitone(Fig.3c),methylpentynolandmethylpentynolcarbamatehadthesameeffectontheganglionicpotentialastubocurarine,butthetime-courseofthepotentialwasincreasedslightly,probablyduetoaslowingofconductioninthepre-andpostganglionicnervefibres,sincethesedrugsproducesomeneuronaldepressioninblockingconcentrations(Quilliam&Shand,1964).Increasingdosesofprocaine(Fig.3a)abolishedthespikepotentialandtheafter-positivity,leavingtheNwaveunalteredinamplitude,butincreasedinduration.Thelongertime-courseoftheNwavewasprobablyduetothedecreaseinthePwave,forthesetwoafter-potentialsappeartosumalgebraically(Eccles,1935;Paton&Perry,1953).FurtherincreaseindosegraduallydiminishedandfinallyabolishedtheNwave.Paraldehydeandmephenesinproducedthesamechangeinshapeoftheganglionicactionpotentialasdidprocaine.91 D.G.SHANDC2x10-54x10-58XI0-5(a)ProcaineC2X10-54x10-58x10-5(b)AtropineC8x10-1.6x10-43.2x10-4(c)AmylobarbitoneCIX10-52x10-54x10-5(d)BenactyzineFig.3.Effectsofprocaineandbenactyzineontheganglionicactionpotential.RecordsasforFig.1.ControlrecordsareindicatedbyCandcumulativedrugconcentrationsaregivenabovetracings.Calibrations,25cycles/secand3mV.IncreasingconcentrationsofprocainegraduallyreducedandabolishedthespikeandPwave,leavingtheNwave.BenactyzineinlowconcentrationreducedtheNwavewithlittlechangeinthespikeorPwavebutfurtherincreaseinconcentrationreducedallthepotentials.Atropinereducedthespike,NwaveandPwaveleavingasynapticpotential,whichhadalongertime-coursethanthatseenwithcurarization.Theeffectsofamylobarbitonewereindistinguishablefromthoseoftubocurarine(compareFig.1,firstrow).Withatropine(Fig.3b),thespikewasreducedandthenabolished,leavingasynapticpotentialsimilartothataftertubocurarine,butwithamoreprolongedtime-course.ThisincreaseddurationmayberelatedtothereductionofthePwavewhichoccurredwithlowconcentrationsofatropine,resultinginanincreaseddurationoftheNwave.Benactyzine(Fig.3d)wasuniqueinitseffects,forinlowdosageitdepressedtheNwavewithlittleornoeffectonthePwave.Increaseindosegraduallyblockedallcomponentsoftheganglionactionpotential.RelationshipbetweendepolarizationandblockWithDCamplification,depolarizationandblockoftransmissionproducedbygradedconcentrationsofdrugscanbemeasuredatthesametime.Nicotine,tetramethyl-ammonium,acetylcholine,carbachol,tetraethylammonium,bretyliumandhexamethoniumproduceddepolarizationoftheganglionbutthemagnitudeoftheganglionicblockwasnotalwaysdirectlyrelatedtotheamountofdepolarizationproduced.ThisisillustratedinFig.4inwhichdepolarization,expressedasapercentageofthevoltageofthecontrolspike92 MODESOFGANGLIONICBLOCK100C._08008C0060°-40C0C*.2000~~~~40O-0~~~~~~~0~~~~~~~~~020406080100Ganglion-block(%)Fig.4.Relationbetweendepolarizationandganglion-blockfornicotine,tetramethylammonium,acetyl-choline,tetraethylammonium,bretyliumandhexamethonium.ActionpotentialswererecordedasforFig.1.Oneexperimentwasperformedwitheachdrugandthecollectiveresultshavebeenplotted.Ordinates:depolarization(measuredfromshiftofbaseline)andexpressedasapercentageoftheheightofthecontrolactionpotentialsothatdifferentexperimentsmaybecompared.Abscissae:percentageblockofganglionictransmission.Filledcircles,nicotine;filledtriangles,tetramethylammonium;filledsquares,acetylcholine;emptycircles,tetraethylammonium;emptytriangles,hexamethonium;emptysquares,bretylium;crosses,tubocurarine(ascontrol).Thesolidlinewasdrawnbyeyeasthebestfitforthesolidsymbolsandthedottedlineasthebestfitfortheopensymbols.Note:Twogroupsofdrugscanbedistinguished:(1)Nicotine,tetramethylammoniumandacetylcholinegavevaluesaroundthesteepersolidregressionlineanddepolarizationwasdose-dependent.(2)Tetraethylammonium,hexamethoniumandbretyliumgavevaluesaroundthehorizontaldottedregressionlineanddepolarizationwasnotdose-dependent.potential,hasbeenplottedagainstthepercentageblockofganglionictransmission.Pointsfortubocurarine(crosses),whichdidnotdepolarize,havebeenincludedasacontrol.Thedrugscouldbedividedintotwogroups;ontheonehandthoseshownasfilledsymbolsinFig.4,namelynicotine,tetramethylammonium,acetylcholineandcarbachol(thelast-namednotbeingincludedinFig.4)andontheotherthoserepresentedbyemptysymbols,tetraethylammonium,hexamethoniumandbretylium.Whiletetraethylammonium,brety-liumandhexamethoniumallcausedsomedepolarizationoftheganglion,itwasinvariablysmallerthanthatproducedbytheotherdrugsand,furthermore,increasingthedoseofthedruggaveverylittle,ifany,increaseinthedepolarization.Theabsolutemagnitudesoftheslightdepolarizationsproducedbythesethreedrugstendedtovarysomewhatfromexperi-menttoexperiment.Althoughthedifferencesweresmall,usuallythedepolarizationproducedbyhexamethoniumwasleast,thatbytetraethylammoniumwasgreatestandthatbybretyliumintermediate.Itwasconcluded,therefore,thatthedepolarizingactivityofthesedrugsprovidednoimportantcontributiontotheirblockingaction.93 D.G.SHANDWithnicotine,tetramethylammoniumandacetylcholine,alinearrelationshipexistedbetweendepolarizationandganglionicblock;withincreasingconcentrationsofthesedrugsboththedepolarizationandtheblockincreasedproportionately.Itwaspossible,therefore,that,intheseshort-termexperiments,persistentdepolarizationoftheganglioncellbodiescouldaccountforallor,atleast,asubstantialproportionoftheganglion-blockingactivityofthesedrugs.AntiacetylcholineactionApartfromnicotine,tetramethylammoniumandcarbacholwhichwerenotinvestigated,allthedrugsintheaboveexperimentsdepressedthedepolarizingactivityofacetylcholinewhentestedwiththemovingfluidelectrodetechniqueofPascoe(1956).Anantiacetyl-cholineactionhasnotbeenshownpreviouslyforparaldehydeandbretylium.Quantitativemeasurementoftheantiacetylcholineactionofthedrugscouldnotbeattemptedwiththepresentmethod,sincethelargedosesofacetylcholinenecessarytodepolarizetheganglioninthepresenceofblockingdosesofthedrugsdesensitizedthepreparationeventhoughaconcentrationof1x10-6ofneostigminewasaddedtothebathfluid.Furthermore,theresponseoftheisolatedgangliontosuccessive,identicaldosesofacetylcholinealonedeclinedmarkedlyaftersixtoeightdoses.DISCUSSIONPaton&Perry(1953)recognizedtwotypesofhighlyselectiveganglion-blockingagents,thoseactingbydepolarizationandthoseactingbycompetitionwiththetransmitter,acetylcholine,forreceptorsitesontheganglioncells.Thepresentfindingssuggestthatdivisionbetweenthetwotypesislesssharplydefinedthantheyinferred.Forinstance,wefoundthatbothtetraethylammoniumandhexamethoniumcandepolarizetheratgangliontoamoderateextent.Indeed,closeinspectionofFig.7cofthepaperofPaton&Perry(1953)ontheeffectsoftetraethylammoniumappearstorevealaslightdepolarizingaction.Inourstudy,tubocurarinedidnotdepolarizetheganglioncells,butAlonso-deForida,Cato,Ramirez&Pardo(1960)havereportedthatthisdrug,aswellashexamethoniumandtetramethylammonium,causedaslightpotentiationofsubmaximalstimulationthroughtheciliaryandsuperiorcervicalgangliaoftheintactcat.Thepotentiationoftransmissionwithtubocurarinewhichtheyreportedinthesuperiorcervicalganglionwasnotsomarkedasthatintheciliaryganglion,whichmightaccountfortheabsenceofanydepolarizationwithtubocurarineinourexperiments,ifindeedtubocurarinehasanysucheffectonthenormalratganglion.However,iftherelationbetweendepolarizationandblockisdeterminedoverthewholerangeofblockingconcentrationsthereislittledifficultyinrecognizingthetwotypesofblockingdrug.Depolarizingdrugs,suchasnicotine,tetramethylammoniumandacetyl-choline,showedasteepregressionline,whilehexamethoniumandtetraethylammoniumdidnotexhibitadose-dependentdepolarization.Itwasconcludedthatthedepolarizationduetohexamethoniumandtetraethylammoniumdidnotcontributetotheirblockingaction,whichwasprobablyrelatedtotheirantiacetylcholineproperties,becauseneitherdrugblocksnervousconduction(Quilliam&Shand,1964)norpreventsacetylcholinelibera-tionfromtheperfusedganglion(Paton&Zaimis,1951;Matthews&Quilliam,1964).Tubocurarinealsoappearstoactbyacetylcholineantagonismforitalsodoesnotaffect94 MODESOFGANGLIONICBLOCKnervousconduction(Quilliam&Shand,1964)noraffectacetylcholineliberation(Brown&Feldberg,1936).Whethersuchantiacetylcholineactionistrulycompetitiveinthebio-chemicalsensehasyettobedetermined,butitwouldseemthatthepreventionofthetransmitteractionbyoccupationofreceptorsbyantagonistmoleculesprovidesthemostplausibleexplanationoftheactionofthecompetitiveganglion-blockingagents.Theweakdepolarizingactionofthecompetitivedrugs,hexamethoniumandtetraethyl-ammonium,mayalsobeexplainedintermsofthereceptortheoryofdrugaction,forifadrugpossessesthemolecularconfigurationrequiredforagoodreceptorfitsoastodenyaccesstoacetylcholinemolecules,thenitmightalsobeexpectedtoshowsomeweakstimu-lant(depolarizing)properties.Thisexplanationholdsforboththeclassical"occupation"conceptofdrugactionandforthe"rate"hypothesisofPaton(1961)inwhichthedrugactionisrelatedtotherateatwhichadrugmoleculecombineswiththereceptor,anaturalcorollarybeingthatalldrugswhichcombinewiththereceptormaystimulate(thatis,depolarize)atleasttosomeextent.Tetraethylammoniumhasanadditionalstimulantproperty,namelythatofincreasingtheamplitudeofthepreganglionicactionpotentialinblockingdoses(Quilliam&Shand,1964).ThisisprobablyrelatedtotheincreasedtransmitteroutputfollowingpreganglionicnervestimulationwithtetraethylammoniumobservedbyDouglas&Lywood(1961)andMatthews&Quilliam(1964).Collier&Exley(1963)alsofoundthattetraethylammoniumincreasedtheacetylcholineoutputfromtheratphrenicnerve-diaphragmpreparation.Whileanincreaseintheoutputofacetylcholinefollowingpreganglionicnervestimulationinthepresenceoftetraethylammoniumcouldconceivablyaccountforapotentiationoftransmission,thedepolarizationoftheganglionintherestingstateiscompatiblewithadirectactionontheganglioncells.Inthisregarditisinterestingtonotethattetraethyl-ammoniumincreasedtheantidromicspikepotential(Quilliam&Shand,1964),butthiseffectwasonlymarkedwithdoseslargerthanthosecausingthedepolarizationdescribedhere.Theslightdepolarizingactionofbretyliuminlowdosesalsodoesnotaccountforitsblockingactionwhichhasbeenattributedtoadepressionoftheconductioninthepost-ganglionicnerve(Quilliam&Shand,1964),inkeepingwithitsactionasanadrenergicneuroneblockingagent(Green,1961).Thispostganglionicdepressioncouldalsoaccountforthereductionofdepolarizationinthehigherdosage(4x10-4)depictedinFig.2c.Itsslightstimulant(depolarizing)actionisprobablyrelatedtoitsquaternaryammoniumstructure.Thefindingthattheganglionicblockingactionofnicotine,tetramethylammoniumandacetylcholinewasdirectlyrelatedtotheirdepolarizingeffectaccordswiththeclassificationoftheseagentsbyPaton&Perry(1953)asdepolarizingganglion-blockingdrugs.Carbacholwouldappeartoactinthesamemanner.ThesedrugsalsoproducedsimilarchangesintheshapeoftheganglionicactionpotentialintheratasdidelectricaldepolarizationofthefrogsympatheticganglioncellsintheelegantexperimentsofNishi&Koketsu(1960)whichprovidesfurthersupportforthecontentionthatthesedrugsactbydepolarization.However,theworkofPaton&Perry(1953),Lundberg&Thesleff(1953)andTrendelenberg(1957)inthecatsuggeststhatthesedrugsmayhaveadualmodeofactionandthisproblemawaitsstudyintherat.D95 D.G.SHANDQuilliam&Shand(1964)foundthattheblockingactionofprocaine,mephenesin,benactyzine,methylpentynolandmethylpentynolcarbamatewasnonselectiveandattributedthistoageneraldepressionofnervousconductioninthepre-andpostganglioniccom-ponentsoftheisolatedratsuperiorcervicalganglionpreparation.Thesedrugsdidnotalwaysaffecttheshapeoftheactionpotentialinthesameway,whichimpliesthattheirprecisemodesofactiondiffer.ProcaineandmephenesininlowdosesabolishedthespikepotentialwithonlyasmallreductionoftheNwave.Itmaybethatthesedrugspreventthesynapticpotentialfromgeneratingaspike,forintheconcentrationsgivinganisolatedNwave,nervousconductionwouldbeonlyslightlyimpaired(Quilliam&Shand,1964).TheseeffectsandthegradualabolitionoftheNwaveandnervousconductionwithhigherdosesseemmostlikelyduetomembranestabilizationwhichhasbeensuggestedasthemodeofactionofthelocalanaestheticagentsbyShanes(1958).Methylpentynolanditscarbamateesterreducedallcomponentsoftheactionpotentialequallyandtheirmodeofactionisoneofgeneralneuronaldepression.ThestimulantactiononthecatganglionofmethylpentynolcarbamatereportedbyMarley&Paton(1959)andconfirmedbyBrown&Quilliam(personalcommunication)onthecatganglionwasnotobservedinthepresentstudyontherat.BenactyzinereducedtheNwaveconsiderablybeforeblockingtransmissionofthespikepotential.Althoughthereasonforthisisobscure,itmayindicatethatthesynapticpotentialiscapableofconsiderablereductionwithoutimpairingthegenerationofthespike,thatis,thereisasubstantialmarginofsafetyfortransmission.Theanti-acetylcholinepropertiesofbenactyzinehavebeensuggestedasabasisforitscentralaction(Jacobsen,1958).Thisdoesnotappeartrueoftheganglionicsynapse.Themoderatelyselectivedrugsamylobarbitoneandatropineproducedmuchthesamechangesinshapeoftheactionpotentialasdidtubocurarineandappeartoactbyacetyl-cholineantagonism,althoughwithlargedosessomeneuronaldepressionisadded.TheobservationthatparaldehydeproducedthesamechangesintheactionpotentialasthenonselectivedrugsprocaineandmephenesinmayberelatedtothefindingofQuilliam&Shand(1964)thatithadthelowestselectivityratiooftheirmoderatelyselectivedrugs.SUMMARY1.Themodeofganglionicblockproducedbyseveraldrugswasinvestigatedbymeasuringtheireffectsonpotentialsrecordedexternallyfromtheisolatedsuperiorcervicalganglionoftherat.2.Nicotine,tetramethylammonium,carbacholandacetylcholinedepolarizedtheganglion.With10-minexposures,thisdepolarizationwasdirectlyproportionaltothedegreeofganglionicblock.3.Hexamethonium,tetraethylammoniumandbretyliumcausedaslightdepolarizationwhichremainedconstantdespiteincreasingconcentrationswhichincreasedthedegreeofblock.4.Theblockingactionofhexamethoniumandtetraethylammoniumwasattributedtoantiacetylcholineactivityandthatofbretyliumtodepressionofthepostganglionicneurones.5.Tubocurarinedidnotdepolarizetheganglion.6.Themoderatelyselectivedrugs,atropine,amylobarbitoneandparaldehyde,andthenonselectivedrugs,procaine,mephenesin,benactyzine,methylpentynolandmethylpentynol96 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