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CA²⁺-activated potassium channels in smooth muscle cell from cerebral artery of adult rat Wang, Yihong 1992-12-17

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CA2+ACTIVATEDPOTASSIUMCHANNELSINSMOOTHMUSCLECELLFROMCEREBRALARTERYOFADULTRATByYIHONGWANGM.D.,BeijingSecondMedicalCollege,1983M.Sc.,PekingUnionMedicalCollege,1985ATHESISSUBMITthDINPARTIALFULFILLMENTOFTHEREQUIREMENTSFORTHEDEGREEOFDOCTOROFPHILOSOPHYinTHEFACULTYOFGRADUATESTUDIESFACULTYOFMEDICINEDEPARTMENTOFPHYSIOLOGYWeacceptthisthesisasconformingtherequiredstandardTHEUNIVERSITYOFBRITISHCOLUMBIAApril1992©YihongWang,1992InpresentingthisthesisinpartialfulfilmentoftherequirementsforanadvanceddegreeattheUniversityofBritishColumbia,IagreethattheLibraryshallmakeitfreelyavailableforreferenceandstudy.Ifurtheragreethatpermissionforextensivecopyingofthisthesisforscholarlypurposesmaybegrantedbytheheadofmydepartmentorbyhisorherrepresentatives.Itisunderstoodthatcopyingorpublicationofthisthesisforfinancialgainshallnotbeallowedwithoutmywrittenpermission.Departmentofphf((7TheUniversityofBritishColumbiaVancouver,CanadaDate__________________DE-6(2/88)AbstractPatchclampmethodswereusedtostudythebiophysicalpropertiesofCa 2+-activatedpotassium(KCa)channelsincerebrovascularsmoothmusclecells(CVSMCs)derivedfromthecerebralarteriesofadultrats.ProceduresweredevelopedfortheenzymaticdissociationofCVSMCsfromcerebralarteries.Dissociatedcellsweremaintainedat4°Corculturedat37°Cfor1-3dayspriortouse.CVSMCswereidentifiedusingamonoclonalantibodyspecificforsmoothmusclea-actin.Thecalcium-sensitivefluorescentprobefura-2wasemployedtomeasurethtrestingandserotonin(5-HT)stimulatedlevelsoffreeintracellularcalcium,[Ca 2+]inthesecells.Aresting[Ca 2+]ilevelof41j5.6nMwasobtainedfromatotalof110CVSMCsinculture.Serotonin(5-HT,10nMto100M)inducedatransientincreaseof[Ca 2 +Jiaboverestinglevels.Theeffectsofthreeknownblockersofvoltage-dependentCa 2+channels,nifedipine,La3+andCo 2+weretestedbycoapplicationwith5-HT.Itwasfoundtheneither10mMLaC13nor10mMC0C12reducedtherisein[Ca 2+ftevokedbyapplicationof1p..M5-HT.Nifedipine(10M)alsofailedtosignificantlyreducetherisein[Ca 2+]iactivatedby1M5-HT.Theeffectsofthepartiallyselective5-HT2receptorantagonist,ketanserin(5nM)reversiblyattenuatedthe5-HTresponseinallcellstested.Theeffectof5-HTon[Ca 2+Jiwasclearlydose-dependentandtheconcentrationof5-HTproducingahalf-maximalincreasein[Ca 2+]iwasfoundtobe10nM.Duringcell-attachedpatchclamprecordingsfromCVSMCskeptat4°Cuntiluse,5-HT(1-10p.M)inducedtheappearanceofinwardlydirected,8-10pAsinglechannelcurrentsstudiedatapipettepotentialof0mV.5-HT(10-100p.M)alsoinducedtheappearanceofbriefbiphasiccurrentspikeswithpeak-to-peakamplitudeof10pA.TheseobservationsindicatethatisolatedCVSMCsretainedUelectro-responsivenessto5-HTafter2-3daysinvitro.Duringwhole-cell,currentclamprecordingsfromthesecells,therestingmembranepotentialofCVSMCswasfoundtobe-41±11.7mV.Applicationofstrongdepolarizingstimulievokedonlysmall(10-15mV)regenerativeresponsesandactionpotentialswerenotobserved.Whenmeasuredatzeroappliedcurrent,theslopemembraneresistance,RmofCVSMCswas3.2±0.48Go.Theaveragemembranetimeconstant,rmwas78±26msandcellcapacitance,Cmwas24±2.3pF.Isolated,inside-outmembranepatchesexcisedfromCVSMCsdisplayedtwoclassesofKCachannel.Thefirstclassofchannels,designatedasK(Ca)Lchannels,hadameanconductanceof207±10pSinsymmetrical140mMKC1solutions.ThepermeabilityoftheopenchanneltopotassiumwascalculatedusingtheGoldman-Hodgkin-KatzconstantfieldequationatPK=3.9x10-13cm 3 /s.Thesechannelsshowedahighdegreeofselectivityforpotassiumoversodiumions(PNa/PK<0.05)andforpotassiumovercesiumions(PCs/PK<0.05).Cs+causedavoltage-dependentflickeringblockofopenK(Ca)Lchannels.Theeffectofvarying[Ca 2+]iontheopenprobabilityandopentimedistributionsofK(Ca)Lchannelswasstudied.Thethresholdlevelof[Ca 2+]jatwhichchannelopeningswerejustdetectablewasabout0.01jIM.Themeanvalueof[Ca 2+]jatwhichsingleK(Ca)Lchannelswereopenhalfofthetime,Po(o.5)was23Mwhenthemembranepotential(V)was+40mV.Overtherangeofmembranepotentials(-80to+80mV)and[Ca 2+Jilevels(0.01Mto1mM)studied,opentimedistributionsforK(Ca)Lchannelswerewellfittedbythesumoftwoexponentials,indicatingthepresenceofatleasttwokineticallydistinguish4bleopenstatesforthischannel.Raising[Ca 2+]iincreasedthetimeconstantoftheslowcomponent,whilehavingnoeffectonthatofthefastcomponent.Theeffectofmembranepotentialontheopenprobabilityandopentimedistributionwerealsostudied.Atlow[Ca 2+Ii(<50M),a14mV111depolarizationinducedane-foldincreaseinPo.At[Ca 2+]j>50M,Poapproachedunityandshowedlittlefurtherincreaseonmembranedepolarization.At[Ca 2 +]j=10PM,thevalueoftheslowtimeconstantderivedfromopentimedistributionswassignificantlyincreasedatpositivemembranepotentials,whilethatofthefasttimeconstantwasunaffected.Theeffectoftetraethylammoniumions(TEA)oncurrentflowinK(Ca)Lchannelswasstudied.TEAcausedadose-dependent,reversiblereductionintheamplitudeofcurrentintheK(Ca)Lchannels,whenappliedtothecytoplasmicfaceofthemembrane.Thiseffectwascharacterizedbyadissociationconstant,Kdof0.83±0.09mMatamembranepotentialof+40mV,andwith[Ca 2+=100,LM.TEAhadnosignificanteffectontheprobabilityofK(Ca)Lchannelsadoptingtheopenstate.ThesecondclassofKCachannels,designatedasK(Ca)Ichannels,wasdetectedonaminorityofpatchesstudied.K(Ca)Ichannelsshowedaconductanceof92±2.6pSinsymmetrical140mMKC1solutions.Thepotassiumpermeabilityoftheopenchannelwas1.75.±0.12x10-13cm/s.K(Ca)Ichannelsarehighlyselectiveforpotassiumoversodiumions(PNa/PK<0.05)andforpotassiumovercesium(PCs/PK<0.05).Theopenprobability,PoofK(Ca)Jchannelsincreasedas[Ca 2+]jwaselevatedovertheconcentrationrange0.1Mto100M.Poalsoincreasedondepolarizationofthemembranepatch.TEAcausedareversible,dose-dependentreductionintheamplitudeofcurrentintheK(Ca)Ichannel,whenappliedtothecytoplasmicmembraneface(Kd=0.31mM±0.03).Thepresentpreparationrepresentsausefulmodelwithwhichtostudytheionicconductancespresentincerebrovascularsmoothmusclecells.Thispreparationcanalsobeemployedtoinvestigatetheeffectofvasoconstrictorsandvasodilatorsoncalciummobilizationinsmoothmusclecellsofthecerebralvasculature.lvTableofContentsAbstractiiListofFiguresixListofTablesxiiiListofAbbreviationsxivAcknowledgementsxviCHAPTER1:INTRODUCIION11.1.Theroleofvascularsmoothmusclecells(VSMCS)inthecontrolofbloodflow11.1.1.ContractilemechanismsinVSMCs21.1.2.Factorsinfluencing[Ca2+]iinVSMCs31.1.3.HormonalandneuralregulationofcontractioninVSMCs51.1.4.VasoconstrictoractionofserotoninonVSMCs71.2.ElectrophysiologicalpropertiesofVSMCs81.3.PotassiumcurrentsinthemembraneofVSMCs101.3.1.Thedelayed(outward)rectifierK-current111.3.2.Thetransientoutwardcurrent(A-current)111.3.3.InwardlyrectifyingK-currents121.3.4.ATP-sensitiveK-current141.3.5.ça-activatedpotassiumchannels141.4.Calcium-activatedK-channels151.4.1.Largeconductancecalcium-activatedpotassiumchannels151.4.2.Intermediateconductancecalcium-activatedpotassiumchannels181.4.3.Smallconductancecalcium-activatedpotassiumchannels191.4.4.ModulationofioniccurrentsthroughKCachannels201.5.Rationale21VCHAPThR2:EXPERIMENTALPROCEDURES.242.1.Dispersalofcerebrovascularsmoothmusclecells242.1.1.Coverslippreparation272.2.IdentificationofisolatedCVSMCs282.2.1.Massontrichrometest282.2.2.ImmunoflourescentandinimunoperoxidasestainingofCVSMCs282.3.DeterminationoffreeintracellularcalciumconcentrationinisolatedCVSMCs302.3.1.Loadingofthefura-2-AMindicatorintoCVSMCs302.3.2.Cellchamberandperfusionsystemforfura-2measurements312.3.3.Measurementof[Ca2+]jinCVSMCs322.3.4.CalculationoffreeintracellularcalciuminCVSMCs382.3.5.Calibrationofthefura-2systemfordeterminationof[Ca2+]jinCVSMCs392.4.ElectrophysiologicalstudiesonisolatedCVSMCs392.4.1.Preparationofpatchelectrodes402.4.2.Gravityperfusionsystemforapplicationofexperimentalsolutions432.4.3.Experimentalsolutions442.4.4.Analysisofpatchclampdata45CAHPTER3:RESULTS503.1.MorphologicalcharacteristicsofCVSMCsinvitro503.2.IntracellularfreecalciumofCVSMCsinculture533.2.1.Resting[Ca2+]iofCVSMCsinculture53vi3.2.2.Effectofserotoninon[Ca2+]jofCVSMCsinculture.533.3.ElectricalpropertiesofisolatedCVSMCs783.3.1.BasicelectrophysiologicalpropertiesofisolatedCVSMCsstudiedwithcell-attachedandwhole-cell,currentclamprecordings783.4.KCachannelsininside-outmembranepatchesexcisedfromCVSMCs943.4.1.ConductanceandionicselectivityoftheK(Ca)Lchannel973.4.2.EffectofK+replacementbyCs+oncurrentflowthroughK(Ca)Lchannels1093.4.3.Effectofvaiying[Ca2+]iontheopenprobabilityofK(Ca)Lchannels1153.4.4.Effectofvarying[Ca2+]jontheopentimedistributionofK(Ca)Lchannels1183.4.5.EffectofmembranepotentialontheopenprobabilityofK(Ca)Lchannels1283.4.6.EffectofmembranepotentialontheopentimedistributionofK(Ca)Lchannels1313.4.7.1ffectofinternallyappliedTEAonK(Ca)Lchannels1343.5.K(Ca)Ichannelsininside-outmembranepatchesexcisedfromisolatedCVSMCs141CHAPTER4:DISCUSSION1624.1.MorphologicalcharacteristicsofCVSMCsinvitro1624.2.IntracellularfreecalciuminculturedCVSMCs1644.2.1.Modulationof[Ca2+]ibyserotonininCVSMCs1654.3.ElectrophysiologicalpropertiesofisolatedCVSMCs1674.4.Ca2+-activatedKchannelsininside-outmembranepatches1704.4.1.PropertiesofK(Ca)Lchannels171vu4.4.1.1.IonicselectivityoftheK(Ca)Lchannel.1714.4.1.2.Ca 2 +dependenceofK(Ca)Lchannelopening1744.4.1.3.VoltagedependenceofopeningofK(Ca)Lchannels1784.4.1.4.BlockadeofK(Ca)LchannelsbyinternallyappliedTEA1794.4.2.PrepertiesofK(Ca)Ichannels1814.4.3.ThepossibleroleofK(Ca)LandK(Ca)Ichannels1824.5.Conclusionsandsignificance184REFERENCES187yinListofFiguresFigure1.Ventralaspectoftheadultratbrainshowingpositionsofthebasilar,posteriorcommunicating,posteriorandmiddlecerebralarteriesusedinthisstudy25Figure2.Schematicillustrationofthesystemformeasuring[Ca 2+Iiusingthefluorescentprobefura-234Figure3.Excitationspectraof1mMfura-236Figure4.Schematicillustrationoftheproceduresusedtomakethewhole-cellandinside-outpatchrecordingsreportedinthisstudy41Figure5...Blockdiagramofthepatchclamprecordingsystem46Figure6.Photomicrographsofdispersedratcerebralarterysmoothmusclecellsinvitro,treatedwiththeMassontrichromestain51Figure7.PhotomicrographsofdispersedCVSMCsafterincubationwithamonoclonalantibodydirectedagainstsmoothmusclea-actinandvisualizedusingaflourescentmarker54Figure8.PhotomicrographsofdispersedCVSMCsafterincubationwithamonoclonalantibodydirectedagainstsmoothmusclea-actinandvisualizedusingaperoxidasemarker56Figure9.Theincreasein[Ca 2 +]itriggeredbyapplicationof5-HTtoaCVSMC59Figure10.Averagetimecourseoftheincreasein[Ca 2 +]itriggeredbyapplicationof5-HTtoanisolatedCVSMCs61Figure11.Increasein[Ca 2+]itriggeredbyapplicationofK+toaCVSMCFigure12.EffectofLa3+andCo2+onserotonin-inducedchangesin[Ca2+]jofaCVSMC65Figure13.Effectofnifedipineontherisein[Ca 2 +]itriggeredby5-HTinaCVSMC67lxFigure14.Effectofketanserinontherisein[Ca 2+Jievokedby5-HTinaCVSMCafter2daysinvitro71Figure15.DecreasingresponsivenessofaCVSMCtorepeatedapplicationsof5-HT73Figure16.Logdose-responserelationshowingthepercentageincreasein[Ca 2+]itriggeredbyvariousconcentrationsof5-HT75Figure17.Spontaneous,smallamplitudesinglechannelcurrentsrecordedincell-attachedpatches78Figure18.Largeamplitude,inwardlydirectedsinglechannelcurrentsevokedinacell-attachedpatchduringapplicationof5-HT80Figure19.BiphasiccurrentsrecordedfromaCVSMCusingthecell-attachedconfiguration82Figure20.RestingmembranepotentialsrecordedfromCVSMCs85Figure21.VoltagechangesinducedbyapplicationofconstantcurrentpulsesinaCVSMCstudiedusingthewhole-cell,currentclamptechnique87Figure22.Current-voltagerelationshipsobtainedinthewhole-cell,currentclamprecordingmode89Figure23.Semi-logarithmicplotofthedecayphaseofahyperpolarizingelectrotonicpotentialevokedinaCVSMCunderwholecell,current-clampconditions91Figure24.Plotofcurrent-voltage(I-V)relationshipsofsingleK(Ca)LandK(Ca)Ichannelcurrentsfrompatchesbathedinsymmetricalpotassiumsolutions94Figure25.Single-channelcurrentsflowingthroughasingleK(Ca)Lchannelinaninside-outmembranepatchvoltage-clampedatvariouspotentials97xFigure26.AmplitudedistributionsforK(Ca)Lchannelcurrentsobtainedfromaninside-outpatch99figure27.Current-voltage(I-V)relationshipofsingleK(Ca)Lchannelcurrentsinsymmetrical140mMpotassiumsolutions101Figure28.EffectsofNa+orCs+replacementofK+onsinglechannelcurrentsinK(Ca)Lchannels104Figure29.Current-voltage(I-V)relationshipofsingleK(Ca)Lchannelcurrentswhen80mMKC1atcytoplasmicorexternalmembranefacewasreplacedbyNaCl106Figure30.Current-voltage(I-V)relationshipofsingleK(Ca)Lchannelcurrentswhen80mMKC1atcytoplasmicorexternalmembranefacewasreplacedbyCsC1110Figure31.Single-channelK(Ca)Lcurrentsrecordedfromaninside-outpatchatdifferentfilterbandwidths112Figure32.Effectofvarying[Ca 2+]iontheopeningofK(Ca)Lchannels116Figure33.Effectofvarying[Ca 2+]iontheopenprobabilityoftheK(Ca)Lchannel118Figure34.DistributionofopentimesfortheK(Ca)Lchannelstudiedinanisolatedpatchcontainingoneactivechannel121Figure35.Effectofvarying[Ca 2 +]ionthetimeconstantsgoverningopentimedistributionsofK(Ca)Lchannels124Figure36.Effectofvarying[Ca 2+]ionrelativenemberofopeningsofK(Ca)Lchannelsgovernedbythetimeconstantsrfandrs126Figure37.DependenceoftheopenprobabilityofK(Ca)Lchannelsonmembranepotentialand[Ca 2+Ii129Figure38.EffectofmembranepotentialonthetimeconstantsgoverningtheopentimedistributionsofK(Ca)Lchannels132xlFigure39.BlockingeffectofinternalTEAontheK(Ca)Lchannel134Figure40.Dose-responsecurvefortheblockofcurrentflowintheK(Ca)LchannelbyTEA137Figure41.EffectofinternallyappliedTEAontheopenprobabilityoftheK(Ca)Lchannel139Figure42.Single-channelcurrentsflowinginK(Ca)Ichannelsatvariousmembranepotentials141Figure43.AmplitudedistributionsobtainedforK(Ca)Ichannelcurrents144Figure44.ConductanceandionicselectivityoftheK(Ca)Ichannel146Figure45.Effectofvarying[Ca 2 +]iontheactivityofK(Ca)Ichannels...149Figure46.Effectof[Ca 2+]jontheopenprobabilityoftheK(Ca)Ichannel151Figure47.DependenceoftheopenprobabilityofK(Ca)Ichannelsonmembranepotentialand[Ca 2+]i153Figure48.BlockingeffectofTEAoncurrentflowintheK(Ca)Ichannel155Figure49.Dose-responsecurveforblockoftheK(Ca)IchannelbyinternallyappliedTEA157Figure50.EffectofTEAontheopenprobabilityoftheK(Ca)Ichannel159xliListofTablesTableI:TheinfluenceofCo2+,La3+,andnifedipineonthepercentageincreasein[Ca2+]iinducedby5-HTinculturedratCVSMCs69TableII:CharacteristicsofBKchannelsinvariouspreparations174XII’ListofAbbreviations[Ca 2+]j:Concentrationofintracellularfreecalcium4-AP:4-aminopyridine5-HT:5-hydroxytryptamine,serotoninACH:AcetyicholineADP:AdenosinediphosphateANOVA:AnalysisofvarianceANP:AtrialnatriureticpeptideAP:ActionpotentialATP:AdenosinetriphosphateATPases:EnzymesthatcatalyzethehydrolysisofATPcAMP:Cyclicadenosine-3’,5’-monophosphatecGMP:Cyclicguanosine-3’,5’-monophosphateCVSMC:CerebrovascularsmoothmusclecellCVSMCsCerebrovascularsmoothmusclecellsDAB:3,3’-diaminobenzidineDAG:DiacyiglycerolDMSO:DimethylsulfoxideEBSS:Earl’sbalancedsaltsolutionEDRF:Endothelium-derivedrelaxingfactorEGTA:Ethyleneglycol-bisQ3-aminoethylether)N,N,N’,N’-tetraaceticacidFITC:FluoresceinisothiocyanateG-protein:Guaninenucleotidebindingprotein.GIP:GastricinhibitorypeptideHBSS:Hank’sbalancedsaltsolutionHEPES:N-2-Hydroxyethylpiperazine-N’-2-ethanesulfonicacidxiv1P3:Inositol-1,4,5-triphosphateKCachannel:Calcium-activatedpotassiumchannelKCacurrent:Calcium-activatedpotassiumcurrentK(Ca)Ichannel:Intermediateconductancecalcium-activatedpotassiumchannelK(Ca)Lchannel:Largeconductancecalcium-activatedpotassiumchannelKGsolution:PotassiumglutamatesolutionMLCK:Myosinlight-chainkinaseMW:MolecularweightPBS:PhosphatebuffersalinePIP2:L--phosphatidylinositoldiphosphatePKC:ProteinkinaseCPLC:PhospholipaseCPo:OpenprobabilitySEM:StandarderrorofthemeanSMC:SmoothmusclecellSMCs:SmoothmusclecellsSR:SarcoplasmicreticulumTEA:TetraethylammoniumTTX:.TetrodotoxinVSMC:VascularsmoothmusclecellVSMCs:VascularsmoothmusclecellsxvAcknowledgementsIwishtoexpressmydeepestgratitudetomysupervisor,Dr.DavidA.Mathers,forhisencouragement,knowledgeabledirection,patience,andsupportthroughoutthecourseofresearchinhislaboratoryandduringthepreparationofthisthesis.TheconfidencethatDr.Mathersshowedinmyabilitiesallowedmetocontinuethisstudyatatimewhenmyownconfidencewaswaning,andenabledmetocompletethework.Iwouldliketothankothermembersofmythesiscommittee,Dr.RalphKeeler,Dr.SteveKehi,andDr.DavidQuastelfortheirvaluablesuggestionsandconstructivecommentswhichmadethisprojectinterestingandrelevant.IwouldliketoexpressmysincerethankstoDr.KennethG.Baimbridgeforprovidingmewithlaboratoryequipmentandchemicalsforintracellularfreecalciummeasurements.IthankDr.MorleySutterforhisvaluableadviceandencouragement.AlsothanksareduetothoseintheDepartmentofPhysiologyfortheirhelpandfriendship.TheyareDr.AlisonBuchan,Dr.TonyPearson,Dr.PeterVaughan,Mr.JohnSariker,Mr.JoeTay,MissStellaAtmadja,andMissNarinderDhatt.IespeciallythankVeronicaCorvalan-Grosslingforhermanyhoursofgraphicworkonthecomputer.Peopleworkinginthedepartmentadministration,workshopandanimalroomwerealwaysverykindandhelpfulandIthankthemverymuch.xviThisthesisisdedicatedtomyparentsZenggaoWangandZhengzhuangYao,myhusbandPingandmydaughterDianafortheiremotionalsupportandencouragement.Chapter1IntroductionAlteredcontractilityofcerebrovascularsmoothmusclecells(CVSMCs)isbelievedtoplayaroleinseveralcerebrovasculardiseasesofclinicalimportance(Michel.etal.1990).ThecontractilestateofCVSMCsisinturnregulatedbytheleveloffreeintracellularcalcium([Ca2+]i)andbytheflowofioniccurrentinseveraltypesofmembranechannels(Johnsetal.1987;Tomita1988;Hathawayetal.1991).Amongthesearetheso-calledcalcium-activatedpotassiumchannels(KCachannels),whichareprobablyimportantinthemodulationofcellrestingmembranepotentialandcellmembranerepolarizationafterexcitation(Tomita1988;Latorreetal.1989;EdwardsandWeston1990;Koib1990).Todate,however,mostdetailedstudiesonfreeintracellularcalciumandcalcium-activatedpotassiumchannelsinsmoothmusclecellshavebeenperformedoncellsisolatedfromperipheralvesselsorfromnonvasculartissues.Thegoalofthisstudy,thereforewastoinvestigateusingpatchclamptechniques(Hamilletal.1981)thepropertiesofKCachannelsinCVSMCsderivedfromthecerebralvasculatureofadultrats:Thecalciumsensitivedyefura-2wasalsousedtostudytheleveloffreeintracellularcalciuminthesecells.1.1.Theroleofvascularsmoothmusclecells(VSMCs)inthecontrolofbloodflow.Vascularsmoothmusclecells(VSMCs)playapredominantroleinregulatingthebloodflowofthebrainandotherorgans.Thecontractionandrelaxationofsmoothmusclecellsinthebloodvesselwallcontributetothe1maintenanceofnormalvascularfunction.ThefollowingparagraphssummarizecurrentknowledgeofthebasiccontractilemechanismsfoundinVSMCs.1.1.1.ContractilemechanismsinVSMCsThecontractilityofvascularsmoothmuscleisdependentontheinteractionbetweentwomajortypesofcontractileproteins,actinandmyosin.Mostinvestigatorsenvisagesmoothmusclecontractionintermsofthesliding-filamentmodeloriginallyproposedforstriatedmuscle(Huxley1990).Inthismodel,theheadsofmyosinmolecules,whicharebundledtogetherintothick(myosin)filaments,undergoacycleofhigh-andlow-affinitybindingtothinfilaments(actin,tropomyosinandotherproteins)inareactiondrivenbyadenosinetriphosphate(ATP)hydrolysis.Tensiongenerationoccursasaresultofrotationoftheheadsofmyosin,whichareboundincross-bridgestructurestoactinmolecules,fromanangleof900to450withrespecttothelongaxisofactin-andmyosin-containingfilaments.Thedirectionofforcegenerationisparalleledtotheorientationofthesefilaments.InVSMCs,contractionmediatedbymyosinandactinfilamentsisregulatedbyCa 2 +throughanumberofdifferentmechanisms(Bolton1979;Johnseta!.1987;Hathawayetal.1991).Themostimportantoftheseinvolvesthephosphorylationofmyosinmolecules(AdeisteinandSellers1987).Inthismechanism,Ca 2+bindstocalmodulinandtheresultingcomplexactivatestheenzymemyosinlight-chainkinase(MLCK),whichinturncatalyzesmyosinphosphorylationandinitiatesrapidshortening.Afterdephosphorylation,theactomyosinpassesthrougha“latch”statebacktorest(HaiandMurphy1989).Vascularmyosiniscomposedoftwoheavychains(MW,200,000and204,000)2andtwosetsoflight-chainsubunits[regulatory,or20,000Da,lightchains(i.e.,LC2O)andalkali,or17,000Da,lightchains].Theserine-19residueofLC2OisthesiteofphosphorylationbyMLCK.InvestigationswiththefluorescentCa2+indicatorfura-2orthephotoproteinaequorinhavedemonstratedthattheleveloffreeintracellularCa2+closelyregulatestheextentofLC2Ophosphorylation(Remboldetal.1988;Tayloretal.1989).PhosphorylationofLC2OactivatestheATPaseactivityactomyosin(Sellers1985)initiatingthecyclingofcross-bridgeformationand,hence,ofcontraction.ThestateofphosphorylationinsmoothmusclemyosinisalsoregulatedbytheopposingactionofmyosinphosphataseandbythemodulationofMLCKactivity.Invitro,MLCKcanbephosphorylatedbyseveralproteinkinases,includingcyclicadenosine3’,5’-monophosphate(cAMP)-dependentproteinkinase(Payneeta!.1986),cyclicguanosine3’,5’-monophosphate(cGMP)-dependentproteinkinase(Nishikawaeta!.1984;Hathawayeta!.1985),Ca2/calmodulin-dependentproteinkinaseII(IkebeandReardon1990),andproteinkinaseC(Nishikawaetal.1985).Invascularsmoothmuscle,tensioncanbemaintainedwhileLC2Odephosphorylationand[Ca2+]levelsdecrease.Thismechanicalstateoftensionmaintenancewithslowlycyclingcross-bridgeshasbeencalledthe“latchstate”byHaiandMurphy(1989).ThelatchstateisenergyefficientbecausetensionismaintainedatreducedATPconsumption.Themolecularmechanismsthatproducethelatchstateareunknown.HaiandMurphy(1989)hypothesizedthatdephosphorylationofLC2Owhileinahigh-affinitybindingconformationmightalterthekineticsofcross-bridgedetachment.Slowingofcross-bridgedetachmentwouldprolonghigh-affinitybindingandtension.1.1.2.Factorsinfluencing[Ca2+]iinVSMCs3ThegenerationandmaintenanceoftensioninvascularsmoothmusclearedependentonprocessesthatmodulateintracellularlevelsoffreeCa 2+.CalciummayenterthecytoplasmiccompartmentofVSMCsinanumberofways,includingtrans-sarcolemmalentrythoughvoltage-dependentCa 2+channels,Na+-Ca 2 +exchange,andreceptor-operatedCa 2 +channels.Thedepolarizationofsmoothmusclecellsbyelectricalstimulation,increasedexternalpotassium,orvariousneurotransmittersevokesaninwardcurrentcarriedbyCa 2+ionsthroughvoltage-dependentCa 2+channelswhichsharecertainfunctionalcharacteristicswithL-typeCa 2 +channelsofstriatedmuscle(Nelsonetal.1988).Calciummayalsobereleasedfrominternalstoresviaryanodinereceptor-likeCa 2 +channelsofthesarcoplasmicreticulum(SR)(Ashida1988)andreleasedfromotherinternalsitesviaaninositol-1,4,5-triphosphate(1P3)-activatedchannel(Nelsonetal.1990).SeveraltypesofK+channelshavebeendetectedinvascularmusclecells,includingCa 2+-activatedK+channels,delayedrectifierK+channels,andATP-sensitiveK+channels.IncreasesinK+conductanceasaresultofactivationofanyofthesechannelsleadstomembranehyperpolarizationwithsubsequentclosureofvoltage-dependentCa 2+channels(Standenetal.1989)andenhancedCa 2 +extrusionviaNa+-Ca 2 +exchange(Lauger1987).Inallcases,thephysiologicalresponseisareductioninvascularsmoothmuscletone.Varioushormonesactingthroughmembrane-boundguaninenucleotidebindingproteins(G-proteins)(Freissmutheta!.1989)activatephospholipaseC(PLC),whichconvertsL-a-phosphatidylinositoldiphosphate(PIP2)todiacyiglycerol(DAG)andinositol-1,4,5-triphosphate(1P3).1P3inturn4stimulatesreleaseofCa 2 +fromthesarcoplasmicreticulumviaand1P3-receptorprotein,whichisatypeofCa 2 +channel(Hashimotoetal.1986;Berridge1989).DAGactivatestheenzymeproteinkinaseCinthesarcolenimawhichinturnphosphorylatesmanyothermembraneproteins,suchasreceptorsandtheNa+-K+exchanger(Nishizuka1988).Othersecondmessengers,suchascAMPandcGMPregulateintracellularCa 2+and/orchangetheCa 2+-sensitivityofcontractileproteinsinsmoothmusclecells(KammandStull1989).cAMPisthoughtmainlytostimulateCa 2 +uptakeintotheSR,whereascGMPisthoughttoactprincipallybystimulatingsarcolemmalATPases(Lindemanneta.1983;Vrolixetal.1988).1.1.3.HormonalandneuralregulationofcontractioninVSMCsThecontractileactivityofvascularsmoothmusclecellsisregulatedbyavarietyoflocalandsystemicmechanisms.Sympatheticandparasympatheticnervesplayamajorroleinthesystemiccontrolofvascularsmoothmuscletone,whileanumberofotheragentsmodulatetheresponsivenessofVSMCsatalocallevel.Knowledgeconcerningtheselocalregulatingmechanismshasprogressedrapidlyinrecentyears.Localregulationisbroughtaboutbytheintrinsiccontractilresponseofsmoothmuscletostretch,andbytheactionsofvasodilatorymetabolitesandlocalvasoconstrictors.SinceFurchgottandZawadski(1980)firstreportedthatthevasodilatoryresponseofvascularsmoothmuscletoacetyicholinerequiresthepresenceofanintactendothelium,theroleoftheendotheliumintheregulationofvasculartonehasattracted5considerableinterest.Activationofendothelialreceptorsbyanumberofvasoactivesubstancesstoredandreleasedbyplatelets,endothelium,orsurroundingtissuesstimulatestheproductionofendothelium-derivedrelaxingorconstrictingfactors(Vanhoutte1981;DeClerckandDavid1981;Burnstock1990).Thesesubsequentlymodifyvasculartonebycontractingorrelaxingthevascularsmoothmuscle(Burnstock1990).Theendothelium-derivedrelaxingfactor(EDRF)hasnowbeenidentifiedasnitricoxide(Ignarroetal.1986,Palmeretal.1987).Inadditiontoacetyicholine,endothelium-dependentvasodilationhasalsobeenshowntooccurinresponsetoothervasoactivesubstances,includingATP,adenosinediphosphate(ADP),arachidonicacid,substanceP,neurokininA,5-hydroxytryptamine(5-HT),bradykinin,histamine,neurotensin,vasopressin,angiotensinIIandthrombin(Mioneetal.1990).Someofthevasoactivesubstanceswhichrequirethepresenceofendotheliumtoproducevasodilationalsoactasvasoconstrictorswhenreleasedfromperivascularnerves.Thereappears,therefore,tobearestingdynamicbalancebetweenendotheliumderivedvasodilatorytoneandsympatheticvasoconstrictortone,whichisalteredunderdifferentphysiologicalandpathophysiologicalcircumstances.Althoughthenatureoftheendothelium-dependentcontractingfactorisstillunclear,atleastthreedifferentclassesofendothelialvasoconstrictorsubstanceshavebeenrecognized(Luscher1988).Theseconsistofmetabolitesofarachidonicacid,polypeptide-likefactors,suchasendothelin,a21-residuepeptideisolatedfromporcineaorticendothelialcells(Yanagisawa,1988)andanunidentifieddiffusiblefactorreleasedfromanoxic/hypoxicendotheliumcells.6Systemicregulationofvascularsmoothmuscletoneisbroughtaboutbycirculatingsubstancesandbytheactionofvasomotornerves.Circulatingvasodilatoryagentsincludekininsandatrialnatriureticpeptide(ANP).Circulatingvasoconstrictorsincludevasopressin,norepinephrine,epinephrine,andangiotensinII.IntheneuralregulationofVSMCcontraction,sympatheticnervesplayadominantrole.Withveryfewexceptions,norepinephrinereleasedfromadrenergicnerveterminalsactivatesaipha-adrenoceptorsatthemembraneofVSMCs(postjunctionalalpha-adrenoceptors)(Burnstock1986;1990).1.1.4.VasoconstrictoractionofserotoninonVSMCsSerotoninisoneofthemostpotentvasoconstrictoragentsinthecerebralcirculationofmammals(Edvinssonetal.1984;Youngetal.1986a,1986b;ChangandOwman1987).ItwasfirstisolatedandcrystallizedbyRapport,GreenandPage(1948)andwassynthesizedbyHamlonandFisher(1951).Serotoninmayplayaroleintheetiologyofseveralimportantdisordersofthevascularsystem,includingmigraine,hypertension,vasospasmassociatedwithsubarachnoidhaemorrhageandischemia(BohrandWebb1988;Micheletal.1990;Lance1982;Vanhoutte1985;Wilkins1980).Nervefiberscontaining5-HThavebeendemonstratedimmunohistochemicallyaroundthepialarteriesofmanyspecies(Chang,OwmanandSteinbusch1988).Serotoninreceptorsubtypescanbecategorizedintothreemajorfamilies,5-HT1,5-HT2and5-HT3receptors,andeachfamilyconsistsofmultiplereceptorsubtypesthatsharesimilaritiesintheirmolecularbiological,pharmacological,biochemical,and/orphysiologicalproperties(SchmidtandPeroutka1989).7Intheperipheralvasculature,itiswellestablishedthat5-HTinducedcontractionofteninvolvesthebindingofserotoninto5-HT2receptorsonthemembraneofVSMCs.Inthesevessels,5-HT2receptoroccupationtriggersphosphoinositidehydrolysis,yieldingthesecondmessenger1P3.1P3theninitiatescontractionbyreleasingCa 2 +fromintracellularstores(PeroutkaandSnyder1979;ConnandSaunders-Bush1987;Peroutka1987).Inthecaseofcerebrovascularvessels,however,themechanismsbywhichserotoninproducescontractioninsmoothmusclearelesswellunderstood.Inconductingcerebralarteriesoftherat,serotonin-inducedcontractionsareblockedbynanomolarconcentrationsofthe5-HT2receptorantagonistketanserin,suggestingtheinvolvementof5-HT2receptorsinthistissue(ChangandOwman1987).However,insimilarstudiesconductedoncaninebasilararteries,serotonineffectshavebeenascribedtoactivationof5-HT1receptors(PeroutkaandKuhar1984),5-HT2receptors(MullerSchweinitzerandEngel1983),ortoanasyetunclassifiedreceptortype(CohenandColbert1986).Contractileresponsesto5-HTwhichcannotbereadilyascribedtotheactivationof5-HT2receptorshavealsobeenreportedincerebralarteriesfromthecat(Youngetal.1986b)andtherabbit(Bradleyetal.1986).Factorswhichinfluence5-HT2receptormediatedchangesinintracellularfreecalciumhavebeenstudiedinseveraltypesofVSMCfromperipheralvesselsusingcalcium-sensitivefluorescentdyes(Nabikaetal.1985;Capponietal.1987;Takataetal.1988).TherehaveasyetbeennodirectmeasurementsontheinfluenceofserotoninonfreeintracellularCa 2 +in8cerebrovascularVSMCstopermitcomparisonwiththedataobtainedfromperipheraltissues.1.2.ElectrophysiologicalpropertiesofVSMCsVascularsmoothmusclecellsfromdifferentpreparationsexhibitwidevariationsintheirpassiveandactiveproperties.IntracellularrecordingsfromCVSMCsshowrestingmembranepotentialswithintherangeof-40mVand-70mVinintactguinea-pigcerebralartery(KarashimaandKuriyama1981;YamamotoandHotta1986),dogcerebralartery(SuzukiandFujiwara1982;Fujiwaraetal.1982),andcatmiddlecerebralartery(Harder1980;Harderetal.1981).Inintactguinea-pigcerebralartery,themembraneofCVSMCswasfoundtobeelectricallyquiescent,andneitherspontaneousactionpotentialsnorminiatureexcitatoryjunctionpotentialswereobserved.Asingle,briefstimulusinducedaspikepotentialfollowedbyadepolarizingslow-potential,andtheseeventswereassociatedwithmusclecontraction.Externalapplicationoftetraethylammonium(TEA),aK+channelblocker,enhancedtheamplitudeofthespikepotential(KarashimaandKuriyama1981;YamamotoandHotta1986).Intheintactdogcerebralartery,CVSMCsonlygeneratedspikesinresponsetooutwardcurrentpulsesunderconditionsofpretreatmentwith10mMTEA.InCVSMCsofintactrabbitbasilarartery,themeanrestingpotentialwas-61mV,membraneinputresistanceandtimeconstantwere5-40Mf2and5-30ms,respectively.Actionpotentialswerereadilyevokedbydepolarizingcurrents,evenintheabsenceofTEA(Surprenantetal.1987).9Whole-cell,patchclamprecordingshavebeenmadeinVSMCsisolatedfrommesentericartery(Boltonetal.1985),aorta(Toroeta!.1986),andratcerebralarterySteeleetal1991;Zhangetal.1991).SingleaortaVSMCswerefoundtoexhibitinputresistancesinthe3G(]range,muchhigherthanfoundusingconventionalintracellularmicroelectrodes.Thesecellshadameancapacitanceof17pFaiidinmanycasescouldgenerateCa 2+-dependentactionpotentialSondepolarization(Toroeta!.1986).CVSMCsisolatedfromratcerebralarteriesexhibitedinputresistancesintherange4-9Go,againmuchhigherthantypicalofcerebralarteriesstudiedwithintracellularmicroelectrodes(Steeleetal.1991).Invascularsmoothmusclecells,thedepolarizingphaseoftheactionpotentialhasbeenregardedmainlyastheresultofinwardflowofCa 2 +throughvoltage-dependentCachannels(Bolton1979;Tomita1982).InwardmembranecurrentsgeneratingtheactionpotentialarelittleeffectedbycompletereplacementofNawithTEAorTris+.Tetrodotoxin,aselectiveblockerofvoltage-sensdveNachannelsisalsoineffective.Incontrast,theseinwardcurrentsarestronglydependentontheexternalconcentrationofCa 2+orBa 2+(Tomita1988).Twodistinctclassesofvoltage-dependentcalciumchannelsexistinratvascularsmoothmusclecells.Oneofthesechannelshasalowthresholdandafastinactivationrate(“fastchannel”),whiletheotherdisplaysahighthresholdandaslowinactivationrate(“slowchannel”)(Loirandetal.1986).These“fast”and“slow”Cachannelsseemtocorrespondtothe“T(transient)-type”and“L(long-lasting)-type”Cachannelsseeninothertissues(McCleskeyetal.1986).Astudyonguinea-pigthoracicaortasmoothmusclecells,however,onlyshowedtheexistenceoftheslowchanneltype(Caffreyetal.1986).TheinabilityofsomeVSMCstogenerateactionpotentialson10membranedepolarizationbyinjectedcurrentprobablyreflectstheirmarkedoutwardrectificationduetoenhancedKconductance,ratherthanalackofCachannels(Mekata1976).1.3.PotassiumcurrentsinthemembraneofVSMCsThegeneralroleofpotassium(K)currentsinthemodulationofvascularexcitabilityisnowwellrecognized.Insmoothmusclecells,potassiumcurrentsplayacrucialroleinthevascularresponsetoendogenousandpharmacologicalvasodilatorsbydeterminingtherestingmembranepotential,andthetimecourse,amplitudeandpolarityofelectricalchanges(Braydenetal.1991).Ingeneral,anincreaseinKconductanceraisesthemembranepotential(hyperpolarization)anddepresseselectricalexcitability(BulbringandTomita1987).Thephysiologicalresponseisrelaxationorreductionofvasculartone.K-currentsmaybeclassifiedintovariouscategoriesusingthecriteriaofchannelgatingproperties,conductanceandpharmacologicalcharacteristics.Basedonthesecriteria,K-currentscanbedividedintothefollowingclasses.1.3.1.Thedelayed(outward)rectifierK-currentThiscurrentcontributestothemacroscopicoutwardK-currentwhichismainlyresponsiblefortherepolarizingphaseoftheactionpotentialinmanycelltypes.Itwasfirstdescribedinthesquidgiantaxon(HodgkinandHuxley1952).Singlechannelrecordingsconductedinneurones(ContiandNeher1980)andinskeletalmuscle(Standenetal.1985),haveshownanelementary11conductanceof15-20pS.Whole-cellcurrentflowinginthesechannelsincreasessigmoidallyonmembranedepolarization.Underaconstantdepolarizingstimulus,aslow,exponentialinactivationoccurs,requiringuptoseveralsecondsforcompletion.Thereisevidencethatthevoltagedependenceofactivationandinactivationcanbemodulatedbyproteinphosphorylation(Bezanillaetal.1985).Noselectivepharmacologicalagonistsareknownforthiscurrent.LikemostKcurrents,thedelayedrectifiercanbeblockedbyCs+,Ba2+(WagonerandOxford1987),intracellularTEA(Armstrong1971),andexternalTEA(Hille1992).Insmoothmusclecells,delayedrectifierchannelshavebeenidentifiedinpulmonaryartery(Okabeetal.1987),rabbitjejunum(BenhamandBolton1983;Benhametal.1%7),bladder(KiocknerandIsenberg1985)andinintestinalsmoothmuscle(Ohyaetal.1986).Inthesetissues,thedelayedrectifierchannelsmayopenduringtherepolarizationphaseofslowelectricalwaves.1.3.2.Thetransientoutwardcurrent(A-current)Thetransientoutwardcurrent,alsoknownas“theA-current”(IA)wasfirstdescribedinmolluscanneurons(ConnorandStevens1971).Patchclampstudieshaveshownthatthiscurrentismediatedbysinglechannelsofconductance15-20p5whichcanbeblockedbyexternalapplicationof4-aminopyridine(4-AP,Taylor1987).A-currentchannelsactivateandinactivateveryrapidlyduringmembranedepolarizations.A-currentchannelsareopeninthesubthresholdregionofthemembranepotential,andcanthereforeplayaroleindeterminingcellfiringfrequency.Inneurones,thiscurrentparticipates12inthecontrolofneuronalfiringrate,spikelatencyandactionpotentialrepolarization(Hille1984).Insmoothmusclecells,acurrentwithpropertiessimilartoneuronalA-currentshasbeendescribedinpulmonaryartery(Ohyaeta!.1986).ThefunctionalsignificanceofthiscurrentinVSMCsremains,however,unclear.1.3.3.InwardrectifyingK-currentsTheexistenceofpotassiumcurrentswhichareactivatedbymembranehyperpolarizationhasbeenreportedinvarioustissues.Ingeneral,thesecurrentsfallintotwobroadcategories.Thefirsttypeofcurrentischaracterizedbyahighselectivityforpotassiumoversodiumions.Thevoltagerangeoverwhichthiscurrentisactivatedshiftsmarkedlywhentheexternalconcentrationofpotassiumisaltered.Thiscurrentisblockedbylow(1mM)concentrationsofBa 2+orCs+.Currentsofthistypehavebeenclassicallydescribedinthemembraneofstarfisheggs(Hagiwaraetal.1976,1978,1979),skeletalmuscle(Adrian1969)andcardiacmuscle(Noble1984).Singlechannelstudiesusingthepatchclamptechniqueshowthatmembranechannelscarryingthistypeofinwardrectifiercurrenthaveamodestconductance(20-30pS)whenmeasuredinphysiologicallyappropriatepotassiumsolutions(Brismaretal.1989;BurtonandHutter1990;Clarkeetal.1990).Asecondclassofinwardrectifiercurrenthasbeenfoundinmanytypesofneurone(HalliwellandAdams1982;MayerandWestbrook1983;Crepeletal.1986)aswellasingutsmoothmusclefibres(Benhametal.1987).ThisclassofionicconductanceshowsappreciablepermeabilitytosodiumionsaswellastoK+.Thevoltagerangeforactivationofthisconductanceisinsensitivetochangesintheconcentrationof13extracellularpotassium.Bariumionsarerelativelyineffectiveinblockingthiscurrent.Todate,therehavebeenrelativelyfewdetailedstudiesconductedontheinwardrectifierchannelsfoundinsmoothmusclecells.However,inmanytypesofSMC,electrotonicpotentialsinducedbylargehyperpolarizingcurrentsreachapeakandthendecaywithtime.ThisinwardrectificationhasbeenobservedinSMCsisolatedfromguinea-pigtaeniacaeci(Tomita1966),toadstomach(Simseta!.1985)andrabbitjejunum(Benhametal.1987).Theinwardr&ctifiercurrentpresentinrabbitjejunumSMCshasbeenanalyzedusingthewhole-cellpatchclamptechnique(Hamilletal.1981).Thiscurrentbelongstothesecondclassdescribedabove.AninwardlyrectifyingchannelpermeabletoK+,Na+andCa 2 +hasalsobeendescribedinSMCsisolatedfromthestomachofthetoad(Hisadaetal.1991).Thischannelhasaconductanceof64pSinphysiologicalsolutionsandisactivatedbymembranehyperpolarization.Incontrasttothesefindingsingutsmoothmuscle,evidenceindicatesthatinwardrectifiersofthefirstclassarepresentinCVSMCstheproximalsegmentofratcerebralarterioles(Edwardsetal.1988)andinCVSMCsinsubmucosalarteriolesoftheguinea-pigileum(EdwardsandHirst1988).ThesecurrentsdreunaffectedbyremovalofexternalNa+butareblockedhylowconcentrationsofBa2+.1.3.4.ATP-sensitiveK-current.AnATP-sensitiveK-current(KATp)wasfirstidentifiedincardiacmuscle(Noma1983).ThiscurrentwasfirstdemonstratedatthesinglechannellevelinVSMCsbyStandenetal.(1989).Aprimarycharacteristicofthis14currentisitsactivationbythehyperpolarizingvasodilatorcromakalimanditsirihibition.bycytoplasmicATP,aswellasbysulfonylureacompounds,suchasglibenclamideandbylowconcentrationsofBa2+.MostKATPchannelsdescribedshownoapparentintracellularcalciumdependencyandonlyslightvoltagesensitivity.Somerecentreports,however,havedemonstratedalargeconductancevoltageandCa 2+-sensitivechannelinVSMCsfromporcinecoronaryartery(SilberbergandBreemen1990)andrabbitaorta,rabbittracheaandpigcoronaryartery(Gelbandetal.1990)whichisalsosensitivetocytoplasmicATPandtocromakalim.ThischannelhasbeenisdesignatedastheBKATpchannel.KATPchannelsinvarioustypesofSMCsmayplayaroleinprotectionagainstischemicandanoxicinsults(Gelbandetal.1990;SilberbergandBreemen1990).1.3.5.Ca-activatedpotassiumchannelsAtpresentthreegroupsofcalcium-dependentK-channels,KCahavebeendescribedinmanytissues,basedontheirsinglechannelconductance,calciumsensitivity,voltagedependence,andpharmacologicalproperties.ThesethreegroupsofKCachannelsaredesignatedaslargeconductancecalcium-activatedchannels(BK),intermediateconductancecalcium-activatedchannels(1K),andsmallconductancecalcium-activatedchannels(SK).Anumberofreviewsaboutthesechannelshavebeenpublished(Tomita1988;Latorreetal.1989;EdwardsandWeston1990;KoIb1990).Propertiesofthesechanneltypesarediscussedinthefollowingsection.1.4.Calcium-activatedpotassiumchannels151.4.1.Largeconductancecalcium-activatedpotassiumchannels,BKBKormaxi-Kchannelshavebeenextensivelystudiedinmanycelltypes.ElementarycurrentsflowinginBKchannelswerefirstobservedinbovineadrenalcciromaffincells(Marty,1981)andlaterincellsfromskeletalmuscle(MethfesselandBoheim1982;Latorreetal.1982;VergaraandLatorre1983;Vergaraetal.1984;BlatzandMagleby1984,1986;Moczydlowski1983,1985),cardiacmuscle(Callewaertetal.1986),endocrinecells(Wongetal.1982;Marty1983;Yellen1984;WongandAdler1986),exocrinecells(Maruyamaetal.1983;Cooketal.1984;Findlayetal.1985;Gitteretal.1987;Grayetal.1990),epithelialcells(ChristensenandZeuthen1987)andimmunecells(Gallin1984).BKchannelshavebeenidentifiedinSMCsderivedfromtheintestine(Benhametal.1985,1986;Cecchietal.1986;Mayereta!.1990),stomach(Bergeretal.1984;Carletal.1990),peripheralarteries(Benhameta!.1985,1986;Boltonetal.1985;Inoueetal.1985),airway(McCannandWelsh,1986),ureter(Shuba1981)andtaeniacaeci(InomataandKao1979).BKchannelsexhibitthefollowingproperties:(1)largesinglechannelconductanceintherangeof150-300pSatasymmetrical,highKconcentrations;(2)highselectivityforK+overothermonovalentcations;(3)activationbyintracellularcalcium(1-10M)andmembranevoltage(e-foldincreaseincurrentfor9-15mVdepolarization);(4)blockadebyexternallyappliedTEA(Kd<1mM),quinine,Ba2+and,inmostcasesbythescorpiontoxincharybdotoxin.BKchannelsarehighlyselectiveforK±overNa+(PNa/K<0.05).Ingeneral,thepermeabifltysequenceforthemonovalentcationsinthesechannels16appearstobesubstantiallyindependentofthecelltype(Tonilinsetal.1984;Benhametal.1986;Akbarakietal.1989;Emeranetal.1990).ThissequenceisTI+>K+>Rb+>NH4+>>Cs+>Na+>Lj+.ThesensitivityofBKchannelsto[Ca 2+]ivariessignificantlyamongdifferentcelltypes.Forexample,inrabbitjejunumandguinea-pigmesentericarterySMC,10-9M[Ca 2 +]jissufficienttoactivateBKchannels(Benhameta!.1986).However,incanineairwaySMC,BKchannelsrequire[Ca 2 +Jj>10-7Mforactivation(McCannandWelsh1986).BKchannelopenprobabilityisalsosensitivetomembranepotential.Again,however,thevoltagedependencyofchannelopeningvariesamongdifferentcelltypes.ThisvoltagedependenceseemstoresultfromthepotentialdependentbindingofCa 2 +tothechannel,ratherthanfromanintrinsicchanneldipole(Kolb1990).ThegatingkineticsofBKchannelscanbeverycomplex.BKchannelsinculturedratmuscleshowfourdistinctmodesofgating,definedasnormal,intermediateopen,briefopenandbuzz(McManusandMagleby1988).Thenormalgatingmode(96%ofalltransitions)itselfdisplaysatleastthreetofouropenstatesandsixtoeightshutstatesofthechannel,asindicatedbythemulti-exponentialnatureofdwelltimedistributions.Incontrast,BKchannelsinVSMCsisolatedfrommesentericarterydisplayapparentlysimplerkinetics(twoopenstatesandthreeclosedstates,Benhameta!.1986).However,itisnotclearwhetherthissimplicityreflectstheuseofshorterlengthsofdataforanalysis,whichwouldeliminateinfrequentlyoccurringopenstatesandlongdurationclosedstates(McManusandMagleby1988).17ExperimentswithBKchannelsreconstitutedintolipidbilayersstronglyindicatethatCa2+actsasasimpleligandtopromotechannelopening,ratherthanactivatingacalcium-dependentreactionpathway,suchasCa 2+-calmodulinorproteinkinaseC-dependentphosphorylation(Latorreetal.1985).ComplexstatediagramshavebeendevelopedfortheBKchannelinwhich1-3calciumionsbindsequentiallytothechannel,stabilizingoneofanumberofopenorclosedstates.Uniligandedchannelsarepresumedtoberesponsibleforthemajorityofshortdurationchannelopenings,whilethebindingofthesecondandthirdcalciumionsstabilizesthelonger-livedopenstates(Barrettetal.1982;MoczydlowskiandLatorre1983:McManusandMagleby1988).InBKchannelsofskeletalmusclefibers,intracellularmagnesiumionscanpromotechannelopeningatphysiologicalconcentrationsofthiscation(0.4-3mM)(SquireandPetersen1987).Thefunctionalsignificanceofthisphenomenonisasyetunclear.BKchannelsinmost,butnotallpreparationsareblockedbynanomolarconcentrationsofthescorpiontoxincharybdotoxinappliedtotheexternalmembraneface(Milleretal.1985).Thistoxinappearstoactasanopenchannelblocker.Inmostpreparations,lowconcentrations(0.1-1mM)ofTEAappliedtotheexternalmembranefacereversiblyblockcurrentflowinBKchannelsinamanneroniyweaklydependentonmembranevoltage.Thisinteractionmaybedescribedasamonomolecularreactionwithanapparentdissociationconstant,Kd=0.3mM(Yellen1984a,1984b;VergaraandLatorre1983;BlatzandMagleby1984).TEAalsoblockscurrentflowinBKchannelswhenappliedtotheinternalmembraneface.Inculturedmyotubes(BlatzandMagleby1984),18chromaffincells(Yellen1984a,1984b),andinmesentericarteryVSMCs(BenhametaL.1985),theKdforblockbyinternallyappliedTEAis12mMorhigher.Inmarkedcontrast,BKchannelsinclonalpituitarycells(WongandAdler1986)andinbr&nsynaptosomalmembranes(FarleyandRudy1988)displayamuchhighersensitivitytointernalTEA,withaKdinthe0.1mMrange.BKchannelsarealsoblockedbyCs+appliedtoeithermembraneface.ThisblockisintensifiedatmembranevoltageswhichdriveCsionsintotheopenchannel.Typically,theblockistoofasttobekineticallyresolved,andresultsinanapparentreductioninmeansinglechannelcurrent(Yellen1984a,1984b).Inneurones,currentflowinBKchannelsservestorepolarizethemembranetowardsEK,terminatingvoltage-dependentcalciumentryandrestoringfiringfrequencytorestinglevels.BKchannelsapparentlymediatethemacroscopicmembranecurrentdesignatedasIcinmanytypesofexcitablecell.Thiscurrçntgeneratesthefastafter-hyperpolarizationseenduringactionpotentialfiringinthesecells(KaczmarekandLevitan1987).IthasalsobeenproposedthatBKchannelsmayplayaroleintheafter-hyperpolarizationseeninCVSMCsofcerebralarteries(Hirsteta!.1986;Steeleetal.1991).1.4.2.Intermediateconductancecalcium-activatedpotassiumchannels,1K1Kchannelshavebeenidentifiedinagreatvarietyoftissuesincludingneurones(Ewaldetal.1985),ratkidneytubules(FrindtandPalmer1987)andratbrainsynaptosomes(FarleyandRudy1988).ThesechannelssometimescoexistwithBKchannels(Akbaralieta!.1990).Channelconductancevaries19from30to120pS.Quinine,quinidine,TEAandcharybdotoxinactasblockersof1Kchannels(Koib1990;EdwardsandWeston1990).1Kchannelsofconductancearound100pShavebeendetectedinVSMCsisolatedfromrabbitportalvein(Inoueetal.1985,1986)andhumancysticartery(Akaralietal.1990).Thesechannelsshowanincreasedopenprobabilityoncelldepolarizationorincreasing[Ca 2 +Ii,buttheopenprobabilityisquitelowatnormalrestingpotentialsof-50mVto-55mV.These1Kchannelsmaythereforebemoreimportantinrepolarizationaftertheactionpotentialthaninmaintainingtherestingmembranepotential(Inoueetal.1985).1.4.3.Smallconductancecalcium-activatedchannels,SK.TheSKchannelsexhibitaconductanceintherangeof10-14pSandwerefirstdescribedinculturedratskeletalmusclecells(BlatzandMagleby1986)andinguinea-pighepatocytes(CookandHaylett1985).SKchannelsexhibitnovoltagesensitivityandingeneralthesechannelsaremoresensitivethanBKchannelsto[Ca 2+]j.SKchannelsmediatetheslowafterhypepoIarization(AHP)whichfollowstheactionpotentialinmanycelltypes(Pennefatheretal.1985).Theslowafter-hyperpolarizationplaysakeyroleinspikefrequencyadaptationinneurones(BarrettandBarrett1976;Barrettetal.1981;KawaiandWatanabe1986).ExternallyappliedTEAatconcentrationsupto25mMhasnosignificantblockingeffectonSKchannelsinskeletalmusclecells(RomeyandLazdunski1984;BlatzandMagleby1986)orinthesympatheticneurones20(Pennefatheretal.1986).SKchannelsarehoweverblockedbynanomolarconcentrationsofthebeetoxinapamin(Kolb1990).Amongsmoothmusclecells,SKchannelshaveyettobestudiedatthesinglechannellevelofresolution.However,apaminisknowntoblockneurotensininducedrelaxationinratileumsmoothmuscle,suggestingthatSKchannelsareindeedpresentinsomeSMCs(Kullacketa!.1987)1.4.4.ModulationoftheioniccurrentthroughKCachannelsBothsinglechannelandwhole-cellstudieshavesuggestedthatthecurrentthroughKCacl’annelscanbemodulatedbyneurotransmittersorintracellularsecondmessengers,andbyputativeK+channelopeners,suchascromakalimandpenacidil.Therelaxationofguinea-pigtaeniacolimediatedbya-adrenergicagonistsinvolvesactivationofKCachannels,withsubsequentmembranehyperpolarization(BuibringandTomita1987).Asnotedabove,neurotensininducedrelaxationofratileumsmoothmusclecanbeblockedbyapamin,suggestingtheinvolvementofSKchannelsinthisprocess(Kullacketal.1987).cAMPhasbeenshowntoactivateKCachannelsinrataortaVSMCs,suggestingthatphosphorylationmayplayaroleintheactivationofthesechannels(Sadoshimaeta!.1988).Intrachealmyocytes,proteinkinaseAactivatesKCachannels(Kumeetal.1989)whilethemuscarinicactivationofcaninecolonicsmoothmuscleappearstoinvolvesuppressionofKCachannelactivityviaapertussistoxinsensitiveGprotein(ColeandSanders1989).Inporcinecoronaryartery,thenewlydiscoveredvasoactivepeptideendothelinhasbeenfoundtoenhancetheprobabilityofopeningofBKchannels,whilehigherdoses21ofthepeptide(>10nM)irreversiblyinhibitedthischannel.Theseactionscouldplayrolesinthevasodilatorandvasoconstrictoreffectsofthispeptide(Huetal.1991).ThepotentvasodilatornitroprussideappearstorelaxaorticVSMCsbyactivatingKCachannels(Williamseta!.1988).ThepotassiumchannelopenercromakalimactivatesBKchannelsinhumanmesentericarterybydecreasingthemeanshuttimeofthesechannels(Kiockneretal.1989).1.5.RationaleItisevidentfromthisreviewthatKCachannelshavebeenwellstudiedinVSMCsfromisolatedperipheralvessels,aswellasinsmoothmusclecellsfromnon-vasculartissues(Tomita1988;SperelakisandOhya1989;Alcbarakietal.1990;EdwardsandWeston1990).Incontrast,however,KCachannelshavebeenlittlestudiedinSMCsderivedfromthecerebralcirculationofmammals.ThefewpaperswhichhaveappearedonthistopichaveclearlydemonstratedthepresenceofKCacurrentsinCVSMCsisolatedfromratcerebralarteries,butasyetfewdetailsofthebiophysicalpropertiesofthesechannelsareavailable(Zhangeta!.1991;Steeleeta!.1991).Thisabsenceofknowledgeisregrettable,inviewoftheapparentimportanceofthesechannelsinregulatingthecontractilestateofVSMCs.KCachannelsmayalsoplayaimportantroleintheetiologyofthevasospasmthatoccursinmanypatientsfollowingsubarachnoidhemorrhage(Micheletal.1990).Ithasbeenproposedthatfreeradicalsmaymediatethevasoconstrictoractionofoxyhemoglohinoncerebrovascularsmoothmuscle.OxyhemoglobininducedcbntractionsareaccompaniedbyanincreaseinKCacurrentsin22CVSMCs,presumablyasaresultofanelevatedleveloffreeintracellularcalcium(Steeleetal.1991).Withtheseconsiderationsinmind,itwasdecidedtoperformastudydesignedtoelucidatethebiophysicalpropertiesofKCachannelsinCVSMCsderivedfromadultrats.Conventionalintracellularrecordingtechniqueswerenotsuitableforthispreect,sincethesemethodscannotresolvecurrentflowinindividua’membranechannels.Forthispurposeitwasnecessarytoemploythepatchclampmethod,devisedbyNeherandSakmann(Hamilletal.1981;SakmannandNeher1983).ThisdecisioninturnrequiredtheuseofenzymaticallydissociatedCVSMCs,sincethemembraneofsmoothmusclecellsisinvestedbyatoughconnectivetissuematrixinintactvessels(RossandReith1985).Thegoalsofthisprojectmaythenbestatedasfollows:1.TodevisemethodsfortheenzymaticdispersionofCVSMCsfromthecerebralarteriesofadultrats.IsolatedcellswerefoundtoremainviableinVitroforseveraldaysafterisolation.HistologicalandimmunocytochemicalmethodswereemployedtoidentifydispersedCVSMCSinthesepreparations.2.Toperformpatchclamprecordingsinthewhole-cell,currentclampmodetodeterminethebasicelectrophysiologicalpropertiesofdispersedCVSMCs.233.Toperformpatchclamprecordingsintheisolated,inside-outpatchmodeinordertoinvestigatethebiophysicalpropertiesofsingleKCachannelsintheCVSMCmembrane.Thesepropertiesincludedsinglechannelconductance,channelkinetics,modulationofchannelgatingbymembranevoltageandintracellularfreecalcium,andthesensitivityofthesechannelstothepotassiumchannelblockersTEAandCs+.4.SincethegatingofKCachannelsiscriticallydependentonthelevelofintracellularfreecalcium,itwasalsodecidedtoestimatethevalueofthisparameterindispersedCVSMCs,usingthecalcium-sensitiveprobefura-2.5.Asnotedpreviously,thereissomeuncertaintyastothemodeofactionofthevasoconstrictor5-HToncerebrovascularsmoothmuscle.Itwasthereforedecidedtoinvestigatethismatterusingthefura-2assayofintracellularcalcium.24Chapter2ExperimentalProcedures2.1.DisprsalofcerebrovascularsmoothmusclecellsExperimentswereperformedoncerebrovascularsmoothmusclecells(CVSMCs)isolatedfromthemiddle,basilar,posteriorcommunicatingandposteriorcerebralarteriesofadultWistarrats(200-250g,CharlesRiver,Montreal).Ratsweresacrificedbydecapitationundersodiumpentobarbitalanaesthesia(30mg/kg).Thecerebralhemispheresandcerebellumwereexposedbyremovingtheparietalbone.Thebrainwascarefullyremovedunderasepticconditionsandplacedina60mmculturedishwithbraindissectingbuffersolutioncontainingCa2+andMg2+freeHank’sBalancedSaltsolution(HBSS,GibcoLaboratories,GrandIsland,NY)ofthefollowingcomposition,inmM:138NaC1,5KC1,0.3KH2PO4,0.3Na2HPO4.7H20,18Dextrose,4NaHCO3,15.7HEPESwithpenicillin100U/miandstreptomycin100g/ml(SigmaChemicalCompany,St.Louis,MO),pH7.4.Underthelowpowerofadissectinglightmicroscope,thebasilar,middle,posteriorcommunicatingandposteriorcerebralarteriesandtheirfirstorderandsecondordersidebranches(Fig1)wereremoved(Fig1)usingiridectomyscissorsandfineforcepsandplacedina65mmculturedishfilledwithpotassiumglutamate(KG)buffersolutioncontaining,inmM:140glutamicacidmonopotassium,16NaHCO3,0.5NaH2PO4,16.5Dextroseand25HEPES,pH7.4.AfterincubationinKGsolutionfor10minutesat370C,thevesselsweremincedwithiridectomyscissorsinto0.5mmfragments.Thefragmentswerethentransferredintoa15mlcentrifugetubecontaining3mlof0.1%trypsin(TypeC,Sigma,dissolvedinKGsolution)andincubatedat37oCfor10minutes.Thetissuesuspensionwasthen25Figure1.Vet4ralaspectoftheadultratbrainshowingpositionsofthebasilar,posteriorcommunicating,posteriorandmiddlecerebralarteriesusedinthisstudy.26-MiddleCerebralArteryPosteriorCommunicatingArteryPosteriorCerebralArteryBasilarArtery27centrifuged,andresuspendedinatubecontaining3mlof0.3%collagenase(Type1A,Sigma,dissolvedinKGsolution)and0.2mlof0.5%trypsininhibitor(Sigma,dissolvedinKGsolution).Followingincubationat37°Cfor15minutes,thecellsuspensionwasallowedtosedimentfor3minutesandthesupernatantwasremoved.Cellswereresuspendedin3mlofhorseserum(heat-inactivated,Gibco)at4°Cinordertoinhibittheactivityofenzymes.Thissuspensionwascentrifugedandthesupernatantwasdiscarded.A6mlvolumeofKGsolutionwasplacedinthetubeandcellswereresuspended,washedandcentrifuged.Afinalresuspensionin1mlofKGsolutionwasmade.A0.2mlofthiscellsuspensionwaspipettedontoapre-coatedglasscoverslip(seeSection2.1.1.)andallowedtosettleforatleast30minutesatroomtemperature(21-23°C).Ifintendedforuseinfura-2determinationofintracellularcalcium,thecoverslipwastransferredintoa35mmculturedishwith2mlofMinimumEssentialMedium(MEM,Gibco)containing,inmM:4L-glutamine,16NaHCO3,20HEPES,and15%horseserum(Gibco)(SturekandHermsmeyer1986).Thesecultureswereincubatedina10%C02incubatorat37°Cfor2-4dayspriortouse.Inthecaseofelectrophysiologicalstudies,coverslipsweretransferredinto35mmculturedisheswith2mlofmaintenancesolutioncontaining,inmM:133NaC1,5KC1,0.8CaC12,1.3MgC12,5Glucose,10HEPESwithpenicillin(100U/mi,Sigma)andstreptomycin(100j.g/m1,Sigma)(Zhangetal.1991).Cellsweremaintainedat4oCinarefrigeratorandusedwithin72hoursofplating.2.1.1.Coverslippreparation18mmcircleglasscoverslips(FisherScientificCompany,cat.no.12-545-100)werecleanedinconcentratednitricacidat60°Cfor30minutesandthen28sterilizedinanautoclavefor40minutes.Aftersoakingovernightin10g/mlpoly-D-lysine(Sigma)dissolvedin0.15Mboratebuffersolution(pH8.4),coverslipswererinsedinsteriledistilledwater.0.2mlHBSS(Gibco)containing16.7glaminin(Sigma)wasaddedtoeachcoverslip2-3hoursbeforeuse.2.2.IdentificationofisolatedCVSMCs2.2.1.MassontrichrometestSmoothmusclecellscanbeidentifiedbytheuseoftheMassontrichromestain(Masson1929;Spatzetal.1983).Inthismethod,thecellsonthecoverslipwerefirstfixedwith2.5%formalininphosphatebuffersolution(PBS)containing,inmM:149NaCl,2KH2PO4,4.2Na2HPO4,pH7.4for10minutes.Nucleiwerestainedwith50%ironhematoxylin(1:1inH20)in5minutesfollowedbydifferentiationwith1%acidalcoholfor1second.Afterwashingwithdistilledwater,thecellsweretreatedwith2%Ponceauacidsolution(dissolvedin1%aceticacid)for2.5minutes.Thisdyestainsthecytoplasmofsmoothmusclecells.Thecellswerewashedwithdistilledwaterandfinallydifferentiatedwith1%phosphotungsticacidfor5minutes.Stainedcellsweredehydratedbysequentialapplicationof70%,95%,and100%ethanolsolutions,cleanediuxyleneandmountedonprecleanedglassmicroscopeslides(Fisher)withmountingmedia(Coverbond,ScientificProducts,McGrawPark,Illinois).ThenucleiofCVSMCsandbackgroundcells(endothelialcells,fibroblasts)werestainedblack.ThecytoplasmofCVSMCswasstainedred,whilethecytoplasmofcellsofconnectivetissueoriginwasstainedblue.292.2.2.ImmunofluorescentandimmunoperoxidasestainingofCVSMCsSmoothmusclecellscanalsobeidentifiedbyapplicationofamonoclonalantibodyspecificforsmoothmusclea-actin(Frankeetal.1980).Inthismethod,coverslipswererinsedthreetimeswithPBS,fixedin2.5%formalin(dissolvedinPBS)for5minutes,airdried,andwashedthreetimeswithPBS.Afterblockingnonspecificbindingwith50%goatserum(Gibco)for20minutesatroomtemperature,coverslipswereincubatedwiththeprimaryantibody,amonoclonalantibodyspecificforsmoothmusclea-actin(CGA7,Sigma,diluted1:400withPBS)overnightat4°C.CoverslipswererinsedthreetimeswithPBSandincubatedwitha1:5000dilutioninPBSofthesecondaryantibody,biotin-APaffinipuregoatanti-mouseIgG(JacksonImmunoresearchLaboratoriesInc.,WestGrove,PA).Forimmunofluorescentstudies,thebiotinylatedIgGwaslocalizedbyincubationwitha1:5000dilutionoffluorescentconjugatedegg-whiteavidin(JacksonImmunoresearchLab.Inc.)fortwohoursatroomtemperature.StainedcoverslipsweregivenafinalrinseinPBSandmounted.onglassmicroscopeslideswithmountingmedia(Coverbond).Thesepreparationswerevisualisedandphotographedusingafluorescentmicroscope(Zeiss,WestGermany).Forimmunoperoxidasestaining,thebiotinylatedIgGwaslocalizedbyincubatingwithaperoxidaseconjugatedegg-whiteavidin(VectorLab)fortwohoursatroomtemperature.CoverslipswererinsedthreetimesinPBS.Theperoxidasereactionwasdevelopedbyincubatingthecoverslipswiththeperoxidasesubstrate3,3’-diaminobenzidine(DAB,10mg/miinPBS)for1030minutesand0.03%hydrogenperoxide(H202)for30minutes.ThesecoverslipsweregivenafinalrinseinPBS.Cellsweredehydratedingradedethanol-watersolutions,clearedinxyleneandmountedontomicroscopeslideswithmountingmedia.Immunoperoxidasepreparationswerevisualizedandphotographedunderlightmicroscopy(OlympusCCK-1,x300magnificaton).Thefollowingcontrolsforspecificityofstainingweredoneforboththeimmunoperoxidaseandimmunofluorescentproceduresdescribedabove:(a)replacementoftheprimaryantibody,CGA7,byanirrelevantantibodyspecificforgastricinhibitorypeptide(GIP,MRCpeptidegroup,Dept.Physiology,UBC),(b)omissionoftheprimaryantibody,(c)incubationwith50%goatserum(Gibco).2.3.DeterminationoffreeintracellularcalciumconcentrationinisolatedCVSMCsThefluorescentCa 2+indicatorfura-2AM(Grynkiewiczetal,1985)wasusedtomeasuretheleveloffreeintracellularcalcium[Ca 2+IipresentinisolatedCVSMCs.Thismethodwasalsoappliedtodeterminethesensitivityofthecellstothevasocontrictorserotonin(5-HT)(Cappometa!.1987).Theseexperimentswereperformedon2-3dayoldculturesincubatedat370C,asdescribedpreviously.Thesecultureswereused,sincethesphericalCVSMCsbecamemorphologicallydistinctfromtheflatbackgroundcellsafter2-3daysincubationat370C,asrevealedbyMassonstainingandimmunocytochemicalstainingforsmoothmusclea-actin(seeResultssection).312.3.1.Loadingofthefura-2-AMindicatorintoCVSMCs.Fura-2AM(MolecularProbesInc.,Eugene,OR)1mgwasdissolvedin1mlofchloroform,and501aliquotsofthissolutionwerepipettedinto20smallplasticampules.Theseampuleswereplacedinadissicator,andvacuum-driedfor3hours.Thedriedaliquotswerestoredat-700C.Foreachcoverslipused,5goffura-2AMperplasticampulewasdissolvedin501ofdimethylsulfoxide(DMSO,Sigma),producingan1mMstocksolution.2Ojdofthisstocksolutionwereaddedto2mlofEarl’sbalancedsaltsolution(EBSS)containing(mM):5KC1,0.8MgC12,1.8CaC12,117NaCl,26NaHCO3,1NaH2PO4,5.6Dextrose,and10HEPES,pH7.4,temperature370C.1mlofthissolutionwasimmediatelyaddedtoa35mmculturedishcontaining1mlEBSS.Thecoverslipwiththesmoothmusclecellswasplacedface-upinthedish.Fura2AMishydrophobicandthereforepenetratestheplasmamembranewithoutdifficulty.Cellswereincubatedwithfura-2AMfor1.5hoursat37°Ctoallowtheuptaketoreachequilibrium.Onceinsidethecell,cytosolicesterasescleavetheacetoxymethylgroupsfromtheindicatortoreleasefreefura-2whichismembraneimpermeantandisthereforetrappedinsidethecells.AfterarinsewithEBSS,afurtherincubationof30minutesat37°Cwascarriedoutin2mloffreshEBSStowashoutexcessfura-2AM.Theseloadingconditionswerechosentominimizetheriskofoverloadingthecellswithdye,andalsoservedtosuppresstheformationofpartiallyhydrolyzedfura-2derivatives(Tsien1988).2.3.2.Cellchamberandperfusionsystemforfura-2measurements.Afterloading,coverslipsweremountedface-downintoaspeciallydesignedlaminarflow-throughchamber(volume350l)filledwithHank’s32balancedsaltsolution(HBSS).consistingof(mM):150NaC1,4KC1,1CaC12,1MgC12and10HEPES,pH7.4.Siliconegreasewasusedtocompleteawatertightsealbetweenthecoverslipandchamber.Thechamberwasinsertedintoastainlesssteelholder,andthentheentireassemblywasmountedontothestageofaJenalumarZeissfluorescentmicroscope.CellswereviewedunderoilimmersionatlOOxmagnificationonthemicroscope.Duringdeterminationofintracellularfreecalcium,cellswerecontinuouslyperfusedwithHBSSat21-23°Cusingaflowrateof4mI/mm.TheHBSScontained1mMCa 2+inallexperimentsutlilizingfura-2.ThisconcentrationofCa 2 +wasselectedtoallowcomparisonofresultswithapreviousstudyofthistype,conductedonaorticVSMCs(Nabikaetal.,1985).Ketanserin(JanssenBiotech,Beerse,Belgium),lanthanumchloride(Sigma),cobaltchloride(Sigma)andnifedipine(Sigma)weredissolvedinHBSSandperfusedoverthecellsinplaceofdrug-freeHBSS.Serotonin(5-hydroxytryptamine,5-HT,Sigma)wasdissolvedin100lHBSSandinjectedintotheinlettubeoftheperfusionchamber.Whentheeffectsofantagonistswerebeingstudied,theseagentswerealsopresentinthe100p.lvolumecontaining5-HT,atthesamedoseasappliedtothecellchamber.Applicationofdrug-freeHBSSinthiswayproducedatransientincreasein[Ca 2+]iinmostcellstested.Thepeakincreasein[Ca 2+]iseenduringtheseartifactualresponsesaveraged23±5%overbaselinelevelsoffreeintracellularcalcium(n=39cells,mean±SEM).Forquantitativeanalysis,thisartifactualincreasewassubtractedfromthesignalinducedbyserotoninapplicationtoyieldamoreaccurateestimateoftheeffectof5-HTon[Ca 2+]i.Toreducetheinfluenceoferrorsintroducedbyartifactsubtraction,onlycorrectedincreasesin[Ca 2+]ithatexceededbaseline33levelsbyatotalof50%ormorewereconsideredduringquantitativeanalysesof5-HTresponses..2.3.3.Measurementof[Ca 2+ftinCVSMCsFreeintracellularcalciumwasdeterminedinindividualCVSMCsbyusingaZeissJenalumarmicroscopeequippedwithanepifluorescencedetector.Thelightsourcewasa200WmercuryarclamppoweredbyaDCpowersupply.Thelightwasfirstpassedthroughoneofthreedifferentialinterferencefilterswhichselectivelypassedthefollowingwavelengths:350±10,380±10or365±10urn(Fig.2).Thesefilterswereheldinaturretwhichcouldberotatedbyacomputer-controlledsteppingmotor.Thelightwasthenpassedthrougha410nmdichroicmirrorandalOOxapochromaticoilimmersionlenswithanumericalapertureof1.4andanadjustablediaphragmtoreducethelightintensity.AfielddiaphragminthelightpathpriortothedichroicmirrorwasusedtoreducetheareaofilluminationtothesizeofasingleCVSMC.Allfluorescentlightpassedbackthroughthedichroicmirroranda450urnbandpassfiltertoreducebackgroundfluorescence.The365nmfilterwasemployedduringcellselection,sinceboththeCa 2+-boundandunboundfura-2moleculesemitasimilarintensityoffluorescenceatthisexcitationwavelength(Fig.3)(Gryiikiewiczetal.1985).Theemittedfluorescencetakenat350urn(indicatorfluorescenceenhancedbyCa2+binding)and380rim(decreasedbyCa2+binding)waseitherobservedbydeflectingthelighttothemicroscopeeyepiece,orquantifiedbydeflectingthelighttoaphotomultipliertubeplacedinthecamerapositionofthemicroscope.ThephotomultiplierconvertedthelightsignalintoaDCvoltage(Fig.2).ThisvoltagewasthenconvertedtodigitalformbyananaloguetodigitalconverterboardinanIBMcompatiblecomputer,andstoredonthe34Figure2.Schematicillustrationofthesystemformeasuring[Ca 2+Iiusingthefluorescentprobefura-2.351.MercuryArcLampweightbalances(nofilters)9.PhotomultiplicrTube8.ifwavelengthoflights>410nm3.Dich.roicMirror5.Excitationat350or380nm10.DATAANALYSISDigitaldataconvertedto(Ca 2 iI.IIIIComputerconvertsfluorescenceintensitytodigitalform2.Filterturretj7.G251filter365nm3SOnsn—4.ifwavelengthoflightis<410nm3SOnm6.Emissionat500n.mII36Figure3.Excitationspectraof1mMfura-2.BoththeCa 2+boundandtheunboundfura-2emitasimilarintensityoffluorescenceatanexcitationwavelengthof365mn.377.peakat335nm6—Ca2-BOUNDFURA-2—Ca2-UNBOUNDFURA-24.pcaka3ó2nm00LE1O(/JjV1equalintensityat356nmII300320340360380400EXCITATIONWAVELENGTH(nm)EXCITATIONSPECTRAOF1mMFURA—2.AdaptedfromGrynkicwiczet.al.1985.38computerharddisk.Measurementsoffluorescenceratioswereobtainedona5secondtimebase.Transientchangesin[Ca2+Iioccurringinlessthan5secondscouldhavegoneundetectedbythissystem.Fu.ra-2measurementsweremadefromatotalof110cellsin40culturesderivedfrom20dissections.Nosignificanttrendswerenotedinthebaselineor5-HTstimulatedcalciumlevelsasafunctionofthenumberofdayspassedinvitro.Thisobservationmakesitunlikelythat5-HTsensitivitywasbeinggraduallydown-regulatedbythepresenceofserotoninintheculturemedium.However,culturesderivedfromdifferentdissectionsdidshowappreciablevariabilityintheproportionofcellsresponsiveto5-HT,andinthedegreeofresponsivenessthecellsshowedtoserotonin.Thisvariabilitycouldhaveresultedinpartfromtheuseofproteolyticenzymes,sinceitwasverydifficulttoensurethateachdissectionexposedthecellstothesamedegreeofenzymeactivity.However,thepossibilitythatsomeofthevariabilitynotedisalsopresentinvivocannotbediscounted.2.3.4.CalculationoffreeintracellularcalciuminCVSMCs.Theconcentrationoffreeintracellularcalcium[Ca2+Jiinthecellsstudiedwascalculatedusingthefollowingformula(Grynkiewiczeta!.,1985):[Ca2+=KdxxR-Rmin/Rmax-RwhereKd=theequilibriumdissociationconstantfortheassociationoffura-2withcytosolicfreecalcium:224M.=ratioofvalues:thefluorescenceintensityat380nmwithzero[Ca2+]/380nmwithinfinite[Ca2139R=experimentallydeterminedratioofthefluorescenceintensityat350nm/380nm.Rmin=ratioofvalues:thefluorescenceofintensityat350mn/380rimwithzero[Ca 21.Rmax=ratioofvalues:thefluorescenceofintensityat350nm/380rimwithinfinite[Ca 2+1.Thevaluesofi3,RminandRmaxweredeterminedbycalibrationofthesystem,asdescribedinthefollowingsection.Forthisstudy,f3=7.464,Rmjn=0.584,andRmax=5.549.2.3.5.Calibrationofthefura-2systemfordeterminationof[Ca 2+]jinCVSMCs.Calibrationprocedureswerecarriedoutusing2-4dayoldCVSMCculturesloadedwithfura-2AMinthemannerpreviouslydescribed.ApairofcultureswasfirstplacedinthemeasuringchamberfilledwithHBSS,containing1.0mMCa 2 +and0.8mMMg 2 +.OneculturewasthenexposedtoCa 2 +freeHBSScontaining5mMEGTAand10PMBr-A23187,anon-fluorescentcalciumionophore(HSCReseachDevelopmentCorporation,Toronto,Canada).After15minutes,theratioofflourescenceintensityat350rimand380rimwasmeasuredin30cells,underconditionsinwhich[Ca 2+]iwaseffectively0mM.ThisratioyieldedthevalueofRmin.ThesecondculturewasnowexposedtostandardHBSScontaining1mMCa 2+and10JLMBr-A23187.Theratiooffluorescenceintensitiesat350nmand380nmwasmeasuredin30cells,asextracellularCa 2+quicklyenteredthecelland[Ca 2+Jibecameeffectively1mM.ThisratioyieldedthevalueofRmax.Thevalueof13wascalculatedby40dividingthefluorescenceintensityat380nmobtainedfromthefirstculturebythevalueobtainedfromofthesecondculture.Thisentireprocedurewasrepeatedintwofurtherculturespreparedfromadifferentdissection.ThevaluesofRnijn,Rmaxandf3obtainedfromtheseculturesdidnotdifferfromtheoriginalestimatesbymorethan10%.Theaveragevalueswereusedinsubsequentcalculationsof[Ca 2+]j.2.4.ElectrophysiologicalstudiesonisolatedCVSMCs.Patchclamprecordingswerecarriedoutatroomtemperature(21-23oC).Atthetimeofrecording,oneculturedishcontainingcellswastakenoutoftherefrigeratpr.Themaintenancesolutionwasdrawnoffandreplacedwith2mlofabathingsolutionappropriatetotheexperimentaldesign.Theculturedishwasthenmountedonthestageofaninverted,phase-contrastmicroscope(OlympusCK,Tokyo,x300magnification).Themicroscope,patchclampamplifierhead-stageandtheperfusionsystemweremountedonavibrationdampingtable(KineticSystemsInc.,Boston,Massachusetts)andshieldedfromelectromagneticradiationbyaFaradaycage.Recordingsweremadeusingthecell-attached,whole-cell(current-clamp),inside-outpatchconfigurations(Fig.4)ofthepatchclamptechnique.Inthistechnique,alowresistancesealisproduceduponmechanicalcontactbetweentheelectrodeandthecellmembrane.Withgentlesuction,thesealincreasesinresistanceby2-3ordersofmagnitude,resultinginacell-attachedgigaohmseal.Backgroundnoiseisgreatlyreducedwiththisseal,leadingtoimprovedresolutionofrecordings.Additionalsuctionappliedtothepatchcreatedthewhole-cellrecordingconfigurationforstudyofbasic41Figure4.Scbematicillustrationoftheproceduresusedtomakethewhole-cellandinside-outpatchrecordingsreportedinthisstudy.42LOWRESISTANCESEAL(50Mfl)SUCTIONJGIGAOHMSEALCELLATTACHEDPULSEOFSUCTION,,//\PULL//v17AIREXPOSURELWHOLECELLpINSIDE-OUTRECORDINGPATCH43electrophysiologicalpropertiesofthecells.Toformaninside-outpatch,theelectrodewasdrawnawayfromthecellsurface,creatingamembranevesicleattheelectrodetip.Theoutermembraneofthisvesiclewasrupturedbybriefpassagethroughthesolution/airinterface(Hamilletal.1981).2.4.1.PreparationofpatchelectrodesPatchelectrodeswerepulledfromcapillarytubesofborosilicateglass(1.5mmODx0.75mmID,FrederickHaerCorp,Brunswick,ME)intwosuccessivestepsonamodifiedDavidKopf700verticalmicroelectrodepuller.Electrodetipdiametersrangedfrom1-2iLm.Electrodeswerecoatedtowithin50moftheirtipswith3140RTVsealant(DowCorningCorporation,Midland,Michigan)whichreducedpipette-bathcapacitancebyformingahydrophobicsurfaceandincreasingthewallthickness(Hamilletal.1981).PatchclamprecordingsofKCachannelactivityweremadefromCVSMCsculturedfor2-3daysat370Coralternativelyfromcellsmaintainedat40C,asdescribedpreviously.Itwasevidentthatthelatterprocedureproducedahigherprobabilityofobtaininginside-outpatchescontainingfunctionalKCachannels.Itwasthereforedecidedtoemploycellsmaintainedat4°Cforallelectrophysiologicalstudiesreportedinthisthesis.AdisadvantageofusingthecooledcellpreparationwasthatbothCVSMCsandothercelltypesretainedasphericalmorphologyovertheincubationperiod(seeResults).Forthisreason,itwasnecessarytoconfirmtheidentityofeachcellbymeansoftheMassontrichrometestperformedattheendoftherecordingsession.Recordingsweremadefromseveralcellsinthesamefield.Aftercompletionofrecording,theentireplateofcellswasstainedusing44theMassontechnique,withoutchangingthefieldofcellsvisibleinthemicroscope.Datafromcellswhichfailedtoreactpositivelytothistest,orwhichdissociatedfromthecuturedishduringstainingwerenotusedinthisstudy.Atthetimeoftheexperiment,thetipofeachelectrodewasfire-polishedtoproduceacleanandsmoothtiprim.Thisfacilitatedtheformationofalargeresistancesealbetweentheelectrodeandthecellmembrane(Hamilletal.1981).Tocarryoutthisstep,theelectrodewasplacedonthestageofacompoundmicroscope(x480).Thetipofelectrodewasbroughttowithin160mofaplatinumfilamentheatedbythepassageofDCcurrentforabout1second.Afterfire-polishingandfillingwithphysiologicalsaline,electrodeshadresistancesof5-10M2.2.4.2.Gravityperfusionsystemforapplicationofexperimentalsolutions.Aperfusionsystemwasdesignedtoapplyexperimentalsolutionstothecytoplasmicfaceofisolatedmembranepatches.Thesystemcomprisedofeight60mlsyringebarrelsmountedabovethestageofthemicroscope.Plastictapswereconnectedtightlytotheendofeachcylindertoallowforselectionoftheappropriatetestsolution.AshortlengthofplastictubingranfromthisassemblytoanL-shapedglasstubeheldinamicromanipulator.Followingpatchisolation,theorificeofthistubewasmovedtowithin200jmofthepatchelectrodetip.Testsperformedusingisolatedinside-outpatchescontainingKCachannelsandsolutionscontainingvariousconcentrationsofCa2+showedthatmembranepatchesequilibratedwithanewtestsolutionwithin25seconds.2.4.3.Experimentalsolutions45Priortorecording,themediumbathingthecellswaschangedtoastandardextracellularsolutioncontaining(inmM):140NaC1;4KC1;1CaC12;1MgC12;10HEPES,pH7.4.Incell-attachedandinwhole-cellrecordings,patchpipetteswerefilledwithasolutionofcomposition(mM):140KC1;3NaC1;0.65CaC12;3EGTA;10HEPES,pH7.4.ThefreeCa2+concentrationinthissolutionwascalculatedat10-8MusingproceduresdevisedbyStockbridge(1987).TheleveloffreeCa2+presentasacontaminantinothersaltswasfoundtobebelow10-5M,asmeasuredbyacalcium-sensitiveelectrode(Model93-20,Orion,Massachusetts).ThisvaluewassmallcomparedtothetotalcalciumconcentrationneededinalltheEGTAbufferedsalines.Acontaminationlevelof8x10-6MwasassumedwhencalculatingthetotalconcentrationofCaCl2requiredinexperimentalsolutions.Duringrecordingsfrominside-outmembranepatches,pipettesusuallycontainedasolutionofcomposition(inmM):140KC1;10HEPES;3EGTA;0.65CaC12,pH7.4.Thefreecalciumconcentrationinthissolutionwascalculatedat10nM(Stockbridge1987).Thecytoplasmicfaceoftheisolatedmembranepatcheswasnormallyexposedtoasolutioncontaining(inmM):140KC1,10HEPES,3EGTA,pH7.4,andthefollowingconcentrationsoftotalandfreeCa2+:0.36mM,5nM;0.65mM,10nM;1.72mM,50nM;2.19nM,0.1MM;2.54mM,0.2MM;2.79mM,0.51M;2.7mM,1MM;2.98mM,51LM;3mM,1OJLMand3.1mM,100MM(Stockbridge1987).TodeterminethepotassiumselectivityofKCachannels,80mMKC1wasreplacedbyanequimolaramountofNaC1orCsC1,ineitherthepipettesolutionorinthesolutionbathingthecytoplasmicmembraneface.ReplacementofK+byionsofdifferentmobilityinsolutionleadtotheappearanceofliquidjunction46potentials.{Hubbardetal.1969).Thesevoltagesweremeasuredbyflowingexperimentalsalinespastthetipofapatchelectrodefilledwithstandardextracellularsolution.Liquidjunctionpotentialswereindicatedasthechangeinzero-currentpipettevoltagemeasuredbythepatchclampamplifier.Appropriatecorrectionswereappliedtothepatchpotentialsmeasuredduringexperimentalsolutionchanges.Correctionfactorswerelessthan±5mV.Theeffectofthepotassiumchannelblockertetraethylammonium(TEA)onKCachannelswasdeterminedbydissolvingthechloridesaltofthedrug(EastmanKodak,Rochester,NewYork)inthesolutionbathingthecytoplasmicfaceofisolatedmembranepaLhes.2.4.4.AnalysisofpatchclampdataThegeneralarrangmentoftheanalysissystemisshowninFigure5.PatchelectrodecurrentsandvoltageswererecordedusingaListEPC-5amplifier(MedicalSystemCorp,NewJersey).SignalswerecapturedonFMwidebandtapeatabandwidthofDC-3kHz(-3dB,Bessel)usinganinstrumentationrecorder(Store4DS,Racal-Decca,England)anddisplayedonarectilinearpenrecorder(DC-100Hz,Gould,Ohio).Dataanalysisproceededoff-lineusinganAtariMega4computer(Atari,Sunnyvale,California)andsoftwaredevisedbyInstrutechCorporation,NewYork.Afteranalogue-todigital(A-D)conversionat8kHz,currentrecords8kBytesinlengthweredisplayedonahigh-resolutionmonitor,andobviousartifactswereeditedout.Gaussiandigitalfilteringwasappliedtoyieldafinalcornerfrequency,f=2kHz(-3dB)usingtherelation1/fc 2=1/f12+1/f22wherefiistheGaussianfiltercutoffandf2isthatofthetaperecorderoutputfilter(CoiquhounandSigworth,1983).Athresholdforeventdetectionwassetat50%ofthemean47Figure5.Blockdiagramofthepatchclamprecordingsystem.48[1PatChCurrentCellCultureDishDC-3KHzPipetteVoltageC, 0,00cJo’-oS049singlechannelcurrentforKCachannelsencounteredinthepatchunderstudy.Under-estimationofthedurationsofbriefopeningsandclosingsbarelycrossingthisthresholdwasaddressedbyapplyingafrequencycorrectionfactor.Opentimedistributionswerevaliddowntothedead-timeoftherecordingsystem,givenasTd=0.179/fci.e.90J.Ls(ColquhounandSigworth1983).Frequencyhistogramsofchannelopentimesandamplitudeswereconstructedusingbinwidths0.4%ofthetotalrangeoftheparameterbeingplotted.Opentimedistributionswereplottedonalogarithmictimeaxis.Thistransformedtheexponentialfunctiony=A.e-t/rintoacurvewithpeakamplitudeatthetimeconstantrandanareaproportionaltothenumberofeventsinthatcomponent(SigworthandSine1987).Simplexmaximizationoflikelihoodwasusedtofitone,twoorthreeexponentialcomponentstotheobservedopentimedistributions(CoiquhounandSigworth1983).Curvefittingdisregardedchannelopeningsbrieferthantwicethesystemdead-time.Frequencydistributionsofcurrentamplitudeswerefittedbyeyewithcomputer-generatedGaussianfunctions.Currentsandvoltagesweredenotedwithrespecttothecytoplasmicfaceofthemembraneinallrecordingconfigurationsused.Reversalpotentialsforsinglechannelcurrentsweredeterminedasthezerocurrentinterceptsoftheoreticalcurvesfittedtothedatapointsbylinearornonlinearregression,asappropriate.Theprobability,PooffindingasingleKCachannelintheopenstateduringarecordingoftotalduration,TttwascalculatedfromtherelationP0=(Ti+T2+...+TN)/NTtotHere,NisthetotalnumberoffunctionalKCachannelsinthepatch.Ti,T2,TNarethetimeswhenatleast1,2...Nchannelswereopen(Mayeretal.1990).50Ntookvaluesintherange0to4,asestimatedbydepolarizingeachpatchto+50mVwith1mMCa2+presentatthecytoplasmicmembraneface.Activepatchesgenerallyhad1-2KCachannelspresent.AsnotedearlierCVSMCswhichhadbeenculturedat37°Ctypicallyshowedmoreinside-outpatchesdevoidofKCaactivitythanwasthecaseincellsmaintainedat4oCuntiluse.Forthisreason,thelatterprocedurewasadaptedinallsinglechannelstudiesonKCacurrentspresentedinthisthesis.Resultswereexpressedasmean±S.E.M.TheStudent’st-testorANOVAwasusedtoevaluatedifferencesbetweenexperimentalgroups.51Chapter3Results3.1.MorphologicalcharacteristicsofCVSMCsinvitro.Collagenase-trypsintreatmentofisolatedvesselfragmentsyieldedaheterogeneouspreparationconsistingofvariouscelltypesanddebris.ThesedispersedCVSMCsdidnotreadilyattachtotheculturesubstrate,makingitdifficulttoconductfura-2orelectrophysiologicalstudiesonfreshlyisolatedcells,However,after2-3daysinvitro,manyCVSMCsdidbecomefirmlyattachedtothesubstrate.Theappearanceofthesepreparationswasdependentontheincubationprotocolfollowed.After2-3daysmaintenanceat4°C,CVSMCsusuallyappearedasMassonpositivecellsofsphericalshapewithanaveragediameterof12±O.6m(mean±SEM,n=31).Occasionally,CVSMCswereslightlyfusiforminshapeontheseplates.SmallclustersofincompletelydissociatedCVSMCswerealsopresentinthesepreparations.TheseplatescontainedanumberofMasson-negativecells,whichwerealsoofsphericalshape(Fig.6A).Inpreparationsculturedfor2-3daysat37°C,MassonpositiveCVSMCsretainedthesphericalmorphologyseenafterlowtemperatureincubation(Fig.6B).However,theMassonnegativecellsintheseculturesadoptedflattened,extendedmorphologiescharacteristicoffibroblasts(Rose1970)andpolygonalendothelialcellsmaintainedinvitro(Warren1990).Thecytoplasmofthesebackgroundcellsstainedblue-greyundertheMassontrichrometest(Fig.6B).52Figure6.Photoniicrographsofdispersedratcerebralarterysmoothmusclecellsinvitro,treatedwiththeMassontrichromestain.A,Preparationincubatedat40Cfor3days.MassonpositiveCVSMCsarestainedredandappearassinglecells(blackarrows)orassmallclustersofcells(openarrows).Massonnegativecells(whitearrows)anddebrisarealsopresent.B,Asecondplateincubatedat37°Cfor3days.TheMassonpositiveCVSMCs(red)haveretainedasphericalshape(blackarrows).TheMassonnegativebackgroundcells(blue-grey)havedifferentiatedintoflattened,extendedshapes(whitearrows).53E DC 0I•0‘“p.,.,.S.I•“SE D0 0IP‘.90,;:,mCVSMCsincubatedeitherat4°C(Fig.7A)orat37°C(Fig.7B)for2-3daysalsoreactedpositivelyonincubationwithanti-a-actinantibody,andwerereadilyvisualizedusingthefluorescentFITCmarker.Omissionoftheprimaryantibodyfromthisprocedureleadtothelossofspecificlabellinginallpreparationstested(Fig.7C).CVSMCsexposedtotheanti-a-actinantibodywerealsoreadilyvisualizedusingtheperoxidasemarker(Fig.8).Themorphologyandsizeofantibody-positivecellsagreedwellwithresultsobtainedusingtheMassontest.3.2.IntracellularfreecalciumlevelofCVSMCsinculture3.2.1.Resting[Ca2+]iofCVSMCsinculture[Ca2+]iwasdeterminedusing2-3dayoldculturesofCVSMCsincubatedat37°C,sincethesmoothmusclecellsintheseplatesweremorphologicallydistinctfrombackgroundcells.Measurementsoffluorescenceratiosfromatotalof31CVSMCsinthesamecultureplatesusedforcalibrationyieldedanaveragevalueofresting[Ca2+]iof39.±3.6nM.Fluorescenceratioswerealsomeasuredin110additionalcellsin28othercultures.[Ca2+]jwascalculatedinthesecellsusingthecalibrationfactorsdeterminedfromthefirstgroupofplates.Thesecellsyieldedaresting[Ca2+]ilevelof41±5.6nM.Thisvaluewasnotsignificantlydifferentfromthatdeterminedinthecalibrationplates(P>0.05,Studeit’st-test),indicatingthatfluorescenceratiomeasurementswerestableoverthecourseofthisstudy.3.2.2.Effectofserotoninon[Ca2+]jofCVSMCSinculture55Figure7.PhotomicrographsofdispersedCVSMCsafterincubationwithamonoclonalantibodydirectedagainstsmoothmusclecr-actinandvisualizedusingthefluorescentmarkerFITC.A,Acultureincubatedat4°Cfor3days.AntibodypositiveCVSMCsappearbrightgreen(whitearrows).B.Acultureincubatedfor3daysat37°C.AntibodypositiveCVSMCsagainappearbrightgreen(whitearrows).Faintlystainedbackgroundcellsexhibitedadegreeofauto-fluorescence.C.Auto-fluorescenceisalsopresentinanegativecontrolculture,incubatedat37oCfor3daysandprocessedintheabsenceoftheprimaryantibody.Calibrationbaris100jmforallmicrographs.56Lc3SVwn001Figure8.PhotomicrographsofdispersedCVSMCsafterincubationwithamonoclonalantibodyagainstsmoothmusclea-actinandvisualizedusingtheperoxidasemarker.A,Acultureincubatedat37°Cfor3days.AntibodypositiveCVSMCsappeardarkbrownandappearedassinglecells(blackarrows)orsmallclusters(openarrows).Antibodynegativebackgroundcellsarealsopresent.B,Anegativecontrolcultureincubatedat370Cfor3daysintheabsenceoftheprimaryantibody.Calibrationbaris100cmforbothmicrographs.584.wrj1C0C3S400C35-HT(10nMto1001 AM)wasappliedbyinjecting1001volumesoftheagonistintotheinletportofthecellchamber.Undertheseconditions,5-HTinducedarapid,transientincreaseof[Ca 2+]iaboverestinglevelsfrom84outof110cellstested.Atypicalresponseofthistype,inducedbyapplicationof1M5-HT,isshowninFig.9.Theaveragetimecourseoftheresponseof13cellsto1M5-HTisshowninFig.10.Noevidencewasseenforoscillatoryorlong-lastingchangesoffreeCa 2+levelsfollowingthetransientapplicationofserotonintoCVSMCs.Itseemedpossiblethatthe5-HTinducedrisein[Ca 2+IisimplyreflectedaninfluxofextracellularCa 2+throughvoltage-sensitivecalciumchannelsinthesmoothmusclecellmembrane.ThatsuchchannelswereindeedpresentonCVSMCmembraneswasindicatedbythefactthatdepolarizationbyhighpotassiumsolutionsalsoevokedaincreaseinintracellularfreecalcium(Fig.11).Toaddressthispossibility,theeffectofthreeknownblockersofvoltage-dependentCa 2+fluxes,nifedipine,La 3+andCo2+(Suprenanteta!.1987)weretestedbycoapplicationwith5-HT.Itwasfoundthatneither10mMLaC13(4outof4cells)nor10mMC0C12(4outof4cells)reducedtherisein[Ca 2 +Iievokedbyapplicationof1M5-HT(Fig.12,TableI).Nifedipine(1OJLM)alsofailedtosignificantlyreducetherisein[Ca 2+]iactivatedby1M5-HTin4outof4cellstested(Fig.13,TableI).Increasesin[Ca 2+]iwerealsoseenwhen5-HTwasappliedtoCVSMCsbathedinacalcium-freemedium(0mMCa 2 ±+0.1mMEGTA).However,undertheseconditionscellresponsivenesswastooerraticforquantitativecomparisonswithnormalmedium.TheTrypanBluedyeexclusiontestcomfirmedthattheviabilityofCVSMCscellswasgreatlyreducedonexposuretoCa 2+-freemedium.60Figure9.Theincreaseinintracellularfreecalcium,[Ca2+Ii,triggeredbyapplicationof5-HTtoasinglesmoothmusclecellculturedfor3dayspriortouse.Atthetimemarkedbythearrow,5-HTwasappliedbyrapidinjectionofa100jL1volumeofHBSSsolutioncontaining1Magonistintotheinlettubeofthecellchamber.Notetheexpandedtimescalecomparedwith5-HTresponsesshowninmostsubsequentfigures.Thelonglatencyoftheresponseislargelytheresultofperfusiondelaysinthedrugdeliverysystem.Temperature21°C.612001,uM5—HT.150C.‘iooL—JNA0•00.40.81.21.62.0Time(mm)62Figure10.Averagetimecourseoftheincreasein[Ca 2+]itriggeredbyapplicationofljM5-HTto13CVSMCsin12differentcultureplates.Theseresponseshavebeennormalizedbypeakamplitudeandalignedtemporallybydenotingthetimeatonsetofeachresponseast=0.Datapointsrepresentthemeanresponseofthe13cellsmeasuredataparticulartimeafterresponseonset.63/\ 0(1)Io0.5NIIII.I0Iz0...i..—,--00.40.81.21.62.0Time(mm)64Figure11.Increasein[Ca2+Jitriggeredbyapplicationof100mMK+salinetoaculturedsmoothmusclecellafter3dayinvitro.Thissolutionwaspreparedbyreplacingsodiumchlorideisotonicallywithpotassiumchloride.A100.tlvolumeofthissolutionwasinjectedintotheinlettubeofthecellchamberatthetimeindicatedbythearrow.65200100mMK+150C100+c\JQ0L_JIs000.40.81.21.62.0Time(mm)66Figure12.EffectofLa 3+andCo2+onserotonin-inducedchangesin[Ca 2+]jofaCVSMCculturedfor3dayspriortouse.5-HT(1M)wasappliedbyinjectionof100lvolumesoftheagentdissolvedinHBSSintotheinlettubeofthecellchamber.Thetimesoftheseinjectionsareindicatedbythearrows.Applicationofdrug-freeHBSSsolutioninthismanner(control)resultedinasmallrisein[Ca 2+]iwhichwasbelowthethresholdlevelrequiredforaresponse.Duringthetimesindicatedbythehorizontalbar,La 3+orCo 2+wasperfusedthroughthecellchamberas10mMsolutionsoftheirrespectivechloridesalts.67200LaCI3CoCI2(10mM)(10mM)5—HT5—HT5—HTControl5—HT150(1pM)100c’10(3‘‘50AAAA0010203040Time(mm)68Figure13.Effectofnifedipine(10M)ontherisein[Ca2+]itriggeredby1M5-HTinaCVSMCculturedfor3dayspriortouse.Serotoninapplicationsweremadeasshownbythearrows,usingtheproceduredescribedforFig.12.NifedipinewasdissolvedinHBSSandperfusedoverthecells,asindicatedbythehorizontalbar.69150lOuMNifedipine120luM5—HTluM5—HT.1C1’./\__60(_)LJ30AA0;1.Time(mm)70TableITheinfluenceofCo 2 +,La 3 +,andnifedipineonthepercentageincreasein[Ca 2 jinducedby5-HTinculturedratCVSMCsTreatment%increasein[Ca 2 jStudent’st-testoverbaselinelevel5-HT(1M)115±18P<O.O15-HT+ketanserin(5nM)63±9(n=5)V5-HT(1 1 LM)187±38P>O.055-HT+Co 2 (lOmM)190±33(n=4)5-HT(1 1 M)162±48P>0.055-HT+La 3 (10mM)165±41(n=4)5-HT(1M)141±16P>0.055-HT+nifedipine(10M)138±27(n=4)71Theeffectsofthepartiallyselective5-HT2receptorantagonist,ketanserin,wereexaminedin10cells.Ketanserin(5nM)reversiblyattenuatedthe5-Hi’responseinall5cellstested(Fig.14,TableI).Whenappliedataconcentrationof10M,ketanserinproducedacompleteblockoftherisein[Ca 2h]jtriggeredby1jM5-HTinSoutof5cellstested.Inmanycases,repeatedexposureof5-HTreactivecellstoaconstantdoseofserotoninleadtoagraduallossofresponsivenesstotheagonist.Thelossofresponseseenduringrepeatedapplicationof5-HTcouldoccurintheabsenceofanyincreaseinthebaselineleveloffreecalciuminthecell(Fig.15).Thisdeclinecouldnotbecompletelyreversed,evenbywashingthecellunderstudyinserotonin-freesalineforaperiodof30minutes.However,robustresponsesto5-HTcouldstillbedetectedinothercellsinthesameculture,whichhadnotpreviouslybeenexposedtophotoexcitation.Theeffectof5-HTon[Ca 2+]jwasclearlydose-dependent,asisshowninFig.16.Thislogdose-responserelationwasobtainedfrom84CVSMCsexposedtoasingle,transientapplicationof5-HT,tominimizethedeleteriouseffectsofphotoexcitantwavelengths.Quantitativeaspectsofthislogdose-responserelationshould,however,beinterpretedwithcaution.The1001 ilvolumesofagonistusedmayhavebeensubjecttodilutionduringpassagethroughthecellchamber.Inaddition,theartificialincreasein[Ca 2+]icausedbyfluidinjectionprecludedaccuratemeasurementsforserotonindosesbelow10nM.Undertheconditionsused,however,itwasobservedthattheresponseofmostCVSMCswasalreadymaximalataserotonindoseof1jLM.Theconcentrationof5-HTproducingahalf-maximalincreasein[Ca 2+]iwas10nM(Fig.16).The72Figure14.Effectof5nMketanserinontherisein[Ca2+]ievokedby1M5-HTinaCVSMCstudiedafter2daysinvitro.5-HTwasappliedbyinjectionofa100JL1volumeofdrug,asdescribedinpreviousfigures.Duringthetimeindicatedbythehorizontalbar,theculturewasperfusedwithHBSSsolutioncontaining5nMketanserin.Theantagonistreduced,butdidnotabolishtheresponseofthiscellto5-HT.Injectionofdrug-freeHBSSproducedonlyasmall,artifactualincreasein[Ca2+Jj(control).73200ketanserin(5nM)control5—HT(1M)5—HT(1,iM)5—HT(1iiM)160.S120.ri+0180ooL_J40-AAAA0II04812162024Time(mm)74Figure15.DecreasingresponsivenessofaCVSMCculturedfor3daystorepeatedapplicationsof0.1jM5-HT.Serotoninwasappliedbyinjectionof1001volumesoftheagentatthetimesindicatedbythearrows.752000.1uM5—HT150C+100\(“100IJ50____•N_Y.AAAA0510115W2025Time(mm)76Figure16.Log-doseresponserelationshowingthepercentageincreasein[Ca 2 jtriggeredbyvariousconcentrationsof5-HT,testedinatotalof84CVSMCsafter2-4daysinvitro.Symbolsshowthemean±SEMvaluesforeach5-HTconcentrationused.Thesmoothcurvewasdrawntotheequation:Response=100%I(1+K/[5-HTI)withK=1OnM.77100-a)ci:o80/60.—ciC,,40C)—a)C)200ioio 8ioio 61o1oIoSerotonin(M)78maximallevelof[Ca 2+]iattainedduringstimulationby5-HTwasapproximately170nM.3.3.ElectricalpropertiesofisolatedCVSMCsTheelectrophysiologicalstudiesdescribedinthisthesiswereconductedonCVSMCsmaintainedfor2-3daysat4°Cpriortouse.ItwasfoundthatcellstreatedinthismannerconsistentlyyieldedagreaterpercentageofmembranepatcheshavingfunctionalKCachannelsthanwasthecaseforCVSMCsincubatedat37°C.3.3.1.BasicelectrophysiologicalpropertiesofisolatedCVSMCsstudiedwithcell-attachedandwhole-cell,currentclamprecordings.Duringpatchclamprecording,patchelectrodesformedhighresistance(10-50GO)sealsonthemembraneofCVSMCsinabout70%ofattempts.Patchelectrodeswerefilledwithasolutionofcomposition(mM):140KC1,3NaC1,0.65CaC12,3EGTAand10HEPES,pH7.4.Thepipettevoltagewas0mV.Undertheseconditions,about25%ofthecell-attachedpatchesshowednodiscernablesinglechannelactivity(Fig.17A).Intheremaining75%ofthepatches,inwardlydirectedsmallamplitude(1-2pA)singlechannelcurrentswereobserved(Fig.17B).Singlechannelactivitycouldbemodifiedincell-attachedpatchesbybathapplicationof5-HTinthestandardexternalsalinebathingthecells.5-HT(1-10M)inducedtheappearanceofinwardlydirected,8-10pAsinglechannelcurrentsin6/10cell-attachedpatchesstudiedatapipettepotentialof0mV(Fig.18).Theidentityofthesechannelswasnotinvestigated79Figure17.Recordingsobtainedfromtwocell-attachedpatchesstudiedintwoCVSMCsincubatedat4°Cfor3dayspriortouse.Patchelectrodeswerefilledwithasolutioncontaining140mMKC1.BandwidthDC-2kHz,pipettepotential0mV.A.Nodiscernablesinglechannelcurrentswerepresentinthispatch.B.Smallamplitudesinglechannelcurrentswerepresentinthisrecording.Inthisandinallsubsequenttraces,inwardmembranecurrentisdenotedbydownwarddeflectionfrombaseline.8018SW0gElVFigure18.Upperpanel.Largeamplitude,inwardlydirectedsinglechannelcurrentsevokedinacell-attachedpatchduringbathapplication(bar)of100M5-HTtotheexposedmembranesurfaceofaCVSMC.BandwidthDC-200Hz.Lowerpanel.Partoftherecordingshowninupperpanelatincreasedtimeresolution(DC-2kHz).Pipettepotential0mV.Thepatchelectrodecontained140mMKCI.82SWVdOlS01ydoi0JA.Jrl001 LH5furtherinthisstudy.Whenappliedatahigherconcentrationrange(10-100MM),5-HTalsoinducedtheappearanceofbrief,biphasiccurrentspikesin6/10cell-attachedpatchesstudied(Fig.19).Theseeventswereabout10pAinpeak-to-peakamplitude,whenmeasuredatapipettepotentialof0mV.Thesecurrentsweresimilarinformandamplitudetothoseassociatedwithactionpotentialdischargeinhighinputimpedancecellsstudiedusingthecell-attachedconfiguration(Fenwicketal.1982).TheseobservationsindicatethatisolatedCVSMCsretainedelectro-responsivenessto5-Hi’after2-3daysinvitro.TherestingmembranepotentialofalargesampleofCVSMCsweremeasuredusingthewhole-cell,current-clamprecordingtechnique.Inthesestudiesthepatchpipetteswereagainfilledwithasalineofcomposition(mM):140KC1,3NaC1,0.65CaC12,3EGTA,10HEPES,pH7.4.TherestingpotentialsofCVSMCswereunimodallydistributedwithanaverageof-4111.7mV(n=69),andrangedfrom-18mVto-69mV(Fig.20).Theresistance,capacitanceandtimeconstantoftheCVSMCmembranewereestimatedbyapplyingsmalldepolarizingandhyperpolarizingcurrentstothepatchelectrode(Fig.21).Fluctuationsinmembranepotentialwerenotedatallmembranevoltages,andthesewereparticularlyapparentwhenthemembranewasstronglyhyperpolarizedordepolarized.Applicationofstrongdepolarizingstimulievokedonlysmall(10-15mV)regenerativeresponsesandactionpotentialswerenotobserved.ThisresultisinagreementwithpreviousstudiesconductedonratCVSMCsinintactvessels(Hirstetal.1986).Thecurrent-voltagerelationsoftwotypicalCVSMCsareshowninFig.22.Therestingmembranepotentialsofthesecellswere-31mVand-49mV.Itcanbeseenthatthemeansloperesistanceofthesecellsdecreasedmarkedlyat84Figure19.BiphasiccurrentsrecordedfomaCVSMCafter2daysinvitrousingthecell-attachedconfiguration.Thesolutionbathingthecellcontained1OOM5-HT.Pipettepotential0mV.BandwidthDC-2kHz.Thepipettesolutioncontained140mMKC1.8598‘Vd01SJA.T11001 in-cFigure20.Restingmembranepotentialsrecordedfrom69CVSMCsafter2-3daysinvitroat4°C.Whole-cellrecordingmethodswereusedtoobtainthesedata.Patchpipettescontained140mMKC1and3mMNaC1.871412(I,co10-4-,Ca)U)-o°6-d4.z2-—,—0—10—20—30—40—50—60—70—80—100RestingMembranePotential(mV)88Figure21.Voltagechanges(lowertraces)inducedbyapplicationofconstantcurrentpulses(uppertraces)inaCVSMCstudiedusingthewhole-cell,currentclamptechnique.Restingpotential-31mV,celldiameter12.5JLm.Pipettesolutioncontained140mMKC1and3mMNaCl.BandwidthDC-200Hz.8906SIAWifdozFigure22.Current-voltagerelationshipsfortwoCVSMCsstudiedinthewhole-cell,currentclamprecordingmode.Therestingmembranepotentialsofthesecellswere-31mV(closedcircles)and-49mV(opencircles).Thegraphshowsthechangein.restingmembranepotential,zV,evokedbyapplicationofconstantcurrentpulsesofamplitude,I.Lineswerefittedtodatapointsbyeye.Recordingpitettescontained140mMKC1and3mMNaCl.Temperature21°C.91V(mV)6025-10-y3I(pA)17-60-80II—i20—14092potentialsnearto,anddepolarizedfromtherestingmembranevoltage.TheslopeoftheI-Vrelationshipattherestingpotentialsindecatedamembraneresistance,Rof3.2±0.48G)(n=20).Cellcapacitanceandtimeconstantwereassessedfromthedecayofhyperpolarizingelectrotonicpotentialsofabout20mVamplitudein7cells.Thesedecayswerewellfittedbyasingleexponentialterm,yieldinganaveragemembranetimeconstantofm=78±26ms(n=7)(Fig.23).CellcapacitancewascalculatedfromtherelationCm=Tm/Rm.Ameanvalueof24±2.3pFwasobtainedfrom7cells.Thisvalueislargerthanthatcalculatedforl2jLmdiametercells,assumingsphericalshapeandaspecificmembranecompacitanceof1jLF/cm 2 .3.4.KCachannelsininside-outmembranepatchesexcisedfromCVSMCsIsolatedinside-outmembranepatchesexcisedfromCVSMCsdisplayedavarietyofsinglechannelcurrentswhenexposedto140mMKC1solutionsatbothmembranefaces.Insomecases,smallcurrentscorrespondingtosinglechannelconductancesintherange10-25pSwereobserved.TheseeventscouldreadilybedistinguishedfromtheactivityofKCachannelsbytheirsmallerconductanceandinsensitivitytochangesincalciumionconcentrationatthecytoplasmicmembraneface.EvidencewasobtainedforthepresenceoftwoclassesofKCachannelinthesepatches.Theseweredistinguishedonthebasicofalargedifferenceinsinglechannelconductance,asmeasuredinsymmetrical140mMK+solutions(Fig.24).Thefirstclassofchannels,designatedasK(Ca)L(largeconductance)channels,wascharacterizedbyameansinglechannelconductanceof207±10pS(n=16).Thesecondcategory,designatedasK(Ca)I(intermediate93Figure23.Semi-logarithmicplotofthedecayphaseofahyperpolarizingelectrotonicpotentialevokedinaCVSMCmembraneunderwholecell,current-clampconditions.Thiscellhadarestingmembranepotentialof-39mV.Thegraphshowsthedeviation,Vofthemembranepotentialfromthisrestinglevelasafunctionoftimesincetheterminationofthestimulatingcurrentpulse.Datapointswerefittedbyastraightlineusinglinearregression.Thearrowindicatesthemembranetimeconstanti-=98ms.94302010•e.....1•050100150200250300350Time(ms)95Figure24.Current-voltage(I-V)relationshipsofsingleK(Ca)Lchannels(circles,n=16patches)andK(Ca)Ichannels(triangles,n=10patches)studiedininside-outmembranepatchesbathedinsymmetrical140mMpotassiumsolutions.Thestraightlineswerefittedtothedatabylinearregression,andrepresentthemeansinglechannelconductanceforK(Ca)Lchannels(207±10pS)andforK(Ca)Ichannels(92±2.6pS).96L691—..(Aw)A09 090179—..170— 017—09— 09—9.91(yd)Iconductancechannels)displayedasignificantlysmallerconductance,92±2.6pS(n=10),P<0.05inStudent’st-testwithK(Ca)Lclass.AsshowninFig.24,therewaslittleoverlapintheconductancerangescharacterizedbythesemeanvalues,suggestingthatindeedtwodistinctclassesofeventswerepresent.ItwasalsonotedthatthemeanK(Ca)LchannelconductancewasnotsimplyanintegermultipleoftheK(Ca)Ichannelconductance,indicatingthattheformereventsdidnotresultfromthesimultaneousopeningoftwoormoreK(Ca)Ichannels.Forthesereasons,itwasdecidedtotreatK(Ca)LandK(Ca)Icurrentsastwodistinctclassesofevent,andtoassesstheirpropertieswithregardtoionicselectivity,Ca 2+andvoltagedependenceandsensitivitytotheblockingactionofTEA.Theprope-tiesoftheK(Ca)Lchannelwillbedescribedfirst,sincethistypewasencounteredmorefrequentlythanthesmallerK(Ca)Ichannel.3.4.1.ConductanceandionicselectivityoftheK(Ca)LchannelSinglechannelcurrentsascribedtotheopeningofK(Ca)Lchannelswereobservedin16/20inside-outpatchesstudied.Fig.25showscurrentsflowingthroughasingleK(Ca)Lchannelinaninside-outpatchvoltage-clampedatvariousmembranepotentials.Thispatchwasbathedinsymmetrical140mMK±solutionsand[Ca 2 +]iwas10AM.Fig.26showstheamplitudedistributionsobtainedforK(Ca)Lchannelcurrentsrecordedfromapatchatmembranepotentials-30mVand+30mV.Atmembranepotentialsintherange-80mVto+80V,thesedistributionswerewellfittedbyasingleGaussianterminall16patchesstudied.ThisresultshowsthattheK(Ca)Lchanneldidnotexhibitsignificantsubstateconductancebehaviorwhenstudiedinexcisedmembranepatches.98Figure25.Single-channelcurrentsflowingthroughaK(Ca)Lchannelinaninside-outpatchvoltage-clampedatvariousmembranepotentials,V.Thispatchwasbathedinsymmetrical140mMK+solutions,[Ca2+]jand[Ca2+Jowere10JLMand0.01Mrespectively.0indicateschannelclosedand1indicateschannelopen.BandwidthDC-200Hz.9965U’ 145rrTwrflr1111251T t1P’I’frrTTlI‘‘rrgwn1r’i1rLV‘jrv1Tr’1T.7IqTrI1j’irrnp’If’T‘TI’11nq’0—25iiIlhllLHHLLhL.LI1iiI.dLiIILIliiiLILLliL.iJ 1 LLiiiLilItIlLJdIiiiiII.lI.1111111IA.I-45-65.-——--————-.-——--—--..-*1IaIIS5slOpAV(mV)0100100Figure26.AmplitudedistributionsofK(Ca)Lchannelcurrentsobtainedfromainside-outpatchvoltage-clampedtomembranepotentialsof(A)+30mV,(B)-30mV.BothdistributionswerewellfittedbysingleGaussianterms(smoothcurves)withmodalvaluesaccurringat6.27pAin(A)andat-6.48pAin(B).Patchwasbathedinsymmetrical140mMK+solutionsand[Ca2+]iwas1OM.[Ca2+Jo=0.O1,LM.101A120-,,100-C0>L..a)C’)-040-2:]1’21’60Amplitude(pA)B1Oo0800600L.ci)040z 200—-j-----OpA510Amplitude(pA)102Inpatchesbathedinsymmetrical140mMKC1solutions,theI-VrelationshipoftheK(Ca)Lchannelwaslinearoverthevoltagerange-80mVto+80mV,showedareversalpotentialof0mVandameanslopeconductanceof207±10pS(n=16,Fig.27).Therangeofconductancevaluesencounteredindifferentpatcheswasfrom170pSto267pS.Thepermeabilityoftheopenchanneltopotassium,PKwascalculatedfromtheI-Vrelationinsymmetrical140mMK+,usingtheGoldman-Hodgkin-Katzconstant-fieldequation(Goldman,1943;Hodgkin&Katz,1949):‘K=PK.VF2/RT{[K+]o-[K+Jiexp(VF/RT)}/{1-exp(VF/RT)}(1)When[K+]o=[K+Jj,Equation1simplifiesto1K=PK.VF2[K+]/(RT)(2)Here,1Kisthecurrentthroughanopenchannel,Vismembranepotential,[K+]o=[K+Ji=140mM,FisFaraday’sconstant,Risthegasconstant,Tistheabsolutetemperatureandoandiindicateoutsideandinside(cytoplasmic)concentrationsofionsrespectively(Benhametal.1986).UsingEquation2,aPKof3.9x10-13cm/swascalculatedfromtheI-Vplotsaveragedfromsixteenpatches(Fig.27).When80mMKC1inthesolutionbathingtheexternalmembranefacewasreplacedbyanequimolaramountofNaCI,currentsflowinginsingleK(Ca)Lchannelsreversedatapotentialaround-21mV(Fig.28A).AnaverageVrevof21.20.50mVwasobtainedfrom5patches(seeFig.29).Thisvalue103Figure27.Current-voltage(I-V)relationshipofsingleK(Ca)Lchannelcurrentsaveragedfrom16patchesbathedinsymmetrical140mMpotassiumsolutions.ItwasfoundthattheI-VrelationshipoftheK(Ca)Lchannelwaslinearoverthevoltagerange-80mVto+80mV,showedareversalpotentialat0mV,andameanslopeconductanceof207±10pS.Errorbarsare±SEM.Inthisandinallsubsequentfiguresshowingstandarderrors,thisparameterisomittedifitsvalueislessthanthesizeofthesymbol.10416i(pA)12Outward.4—80—60—40—20LIIIIIIIIIII1III20406080V(mV).412Inward16105Figure28.EffectsofNa+orCs+replacementofK+onthesinglechannelcurrentsinK(Ca)Lchanneis.Singlechannelcurrentsatvariousmembranepotentials,V;wereobtainedfromapatchwithoneactivechannelwhen80mMKC1inthepipettewasreplacedbyanequimolaramountofNaC1inA,andofCsC1inB.0indicateschannelclosedand1indicateschannelopen.BandwidthDC-2kFIz.[Ca 2+Jj,100Mand[Ca 21o,0.01M.AandBwereobtainedfromdifferentpatches.106SW001yds:‘W001yd11::z0$,(wALOI—I—I——-I_ — -. —.---—- ‘ - -.:.09-I ZLILLLVFigure29.Current-voltage(I-V)relationshipofsingleK(Ca)Lchannelcurrentswhen80mMKCIatthecytoplasmic(opensquares)orexternalmembraneface(filleddiamonds)wasreplacedbyNaC1.Thesymbolsshowmeancurrentscalculatedfrom5patches.Standarderrorbarswereomittediflessthanthesymbolsize.Thethicksolidlinesshowcurrentspredictedfromconstant-fieldtheory,assumingPK=3.5x10-13cm/s(externalK+replacement)orPlC4.0x10-13cm/s(cytoplasmicK+replacement).Allthecurrentsobservedfallwithintherangeofvaluespredictedfromtheconstant-fieldequation,usingtheextremevaluesofPKseeninsymmetrical140mMsolutions(thinlines).[Ca2+]j=100PM.[Ca2+]o0.0lM.10816i(pA)12Outward10-o-o8V(mV)406080•8Inward1216109isveryclosetothereversalpotentialexpectedforapotassiumselectivechannelundertheseconditions(Vrev=-21.4mV).When80mMKC1inthesolutionbathingthecytoplasmicfacewasreplacedbyanequimolaramountofNaC1,currentsinK(Ca)Lchannelreversedatamembranepotentialof+21.2±0.37mV(n=5,seeFig.29),againveryclosetotheNernstianreversalpotentialvalueforK+(+21.4mV).ThesereversalpotentialswereusedtocalculatethepermeabilitytatioPNa/PK,employingtherelationVrev=(RT/zF)ln[(PNa[Na]o+PK[K]o)/(PNa[Na]j+PK[K]j)](3)(Benhametal.1986).HereVrevistheobservedreversalpotential,andzisthevalence.AvalueofPNa/PK<0.05wasobtained,indicatingahighdegreeofselectivityforpotassiumoversodiumionsinthischannel.Itwasalsonotedthat,sinceEQwas0mVthroughouttheseexperiments,thepermeabilityoftheK(Ca)LchanneltoCl-mustbeverylow.ThepotassiumselectivenatureofthischannelwaslaterconfirmedinexperimentswiththepotassiumchannelblockersCs+andTEA,aswillbedescribedinalatersection.UndertheassumptionofnegligiblepermeabilitytoNa+,theconstant-fieldrelation(Equation1)wasusedtocalculatetheexpectedcurrentthroughK(Ca)Lchannelswhen80mMK+wasreplacedbyanequimolaramountofNa+atthecytoplasmicorattheexterialmembraneface.Incalculatingthesecurrents,theparameterPKwasallowedtovarybetween3.3x10-13cm/sand4.9x10-13cm/s,theextremevaluesnotedforK(Ca)Lchannelsstudiedinsymmetrical140mMK+solutions.Itcanbeseenthatthemeancurrentsobservedin5patcheswerewellpredictedbyconstant-fieldtheorywhenPK=3.5x10-13cm/s(replacementof80mMexternalK+byNa+)andPK=4.0x10-13cm/s(replacementof80mMinternalK±byNa+)(Fig.29).Fig.29alsoshows(asthinsolidlines)the110currentthroughtheK(Ca)LchannelcalculatedusingtheextremevalueofPKnotedearlier,namely3.3x10-13cm/sand4.9xlO-l 3cm/s.ItisevidentthatallthecurrentsobservedduringNa+replacementofK+fallwithintherangeofvaluespredictedfromtheconstant-fieldequation.ItwasthereforeconcludedthatNa+ionsdonotinterferewiththepassageofK+ionsthroughtheK(Ca)Lchannel,undertheconditionsusedinthisstudy.3.4.2.EffectofK+replacementbyCs+oncurrentflowthroughK(Ca)Lchannels.Theeffectsofreplacing80mMKC1byanequimolaramountofCsC1ineitherthecytoplasmicortheexternalbathingsolutionwereeachstudiedin5patches.WhenCs+replacedK+attheexternalmembraneface,thesinglechannelcurrentshadareversalpotentialcloseto-21mV(Fig.28B).AnaveragevalueofVrev=-21.3±0.46mVwasobtainedfrom5patches(seeFig.30).Replacementof80mMK±inthecytoplasmicbathingsolutionbyequimolarCs+resultedinareversalpotentialof+21.2±0.51mV(5patches,seeFig.30).ThesereversalpotentialswereusedtocalculatethepermeabilityratioPCs/PK,employingEquation3.AvalueofPCs/PK<0.05wasobtained,indicatingthattheK(Ca)LchannelisverypoorlypermeabletoCs+.ItwasalsonotedthatcurrentsinsingleK(Ca)LchannelsrecordedinthepresenceofCs+exhibitedagreaternoisevariance,whencomparedwithcurrentsrecordedinsymmetricalK+gradients,orduringreplacementofK+byNa+(seeFigs.25and28).ThisobservationisconsistentwithaflickeringblockofopenK(Ca)LchannelsbyCs+,ashaspreviouslybeenobservedinothertypes111Figure30.Current-voltage(I-V)relationshipofsingleK(Ca)Lchannelcurrentswhen80mMKC1atthecytoplasmic(filledtriangles)orexternalmembraneface(opentriangles)wasreplacedbyCsCl.Symbolsshowmeancurrentsobtainedfrom5patches.Standarderrorbarswereomittedifsmallerthansymbol.Solidlinesshowpredictedcurrentsassumingconstant-fieldbehavior,calculatedfortheextremevaluesofPKseeninsymmetrical140mMK+solutions.Meancurrentsweresmallerthanpredictedbytheconstant-fieldequation,especiallyunderconditionspromotingtheentryofCs+intotheK(Ca)Lchannel(i.e.atnegativepotentialswhenC+waspresentintheexternalsolutionandatthepositivepotentialswhenCs+wasappliedtothecytoplasmicmembraneface.[Ca2+]j=100PM.[Ca2±]o=O.0lM.11216i(pA)12Outward-80-60—40—2/,,,,,,VV406080V(mV)12Inward16113ofKCachannel(Benhametal.1986).Individualblockingandunbiockingevent;wereatleastpartiallyresolvedata2kHzbandwidth.Thisindicatedthattherateconstantsforchannelblockandunblockwerenotveryfast,sincethiswouldresultinasmoothed,time-averagedvalueofcurrentthroughthechannel(Yellen1984a,b).TheoccurrenceofsignificantnoiseduringcurrentflowinthepresenceofCs+madeitdifficulttodetermineanappropriatemeanchannelcurrentforuseinI-Vplots.Forthisreason,thesecurrentsweresubjectedtofurtherdigitalfilteringtoreducetheireffectivebandwidthtoDC-400Hz.AsshowninFig.31,thisprocedureresultedinasmooth,time-averagedcurrentsuitablefortheconstructionofI-Vplots(Benhameta!.1986).Meanvaluesofsuchcurrentsobtainedfrom5patchstestedwithcytoplasmicorexternalsubstitutionof80mMK+byCs+areshowninFig.30.ThisFigurealsoshowstherangeofexpectedcurrentsthroughtheK(Ca)Lchannel,undertheassumptionthatCs+isnon-permeablebutdoesnotblockthepassageofK+throughthechannel.AswasthecaseinFig.29,thesetheoreticalcurrentswerecalculatedfromtheconstant-fieldrelation,usingtheextremevaluesofPKnotedinsymmetrical140mMK+solutions.Itwasnotedthattheobservedcurrentsweresmallerthanpredictedbyconstant-fieldtheory,especiallyunderconditionspromotingtheentryofCs+intotheK(Ca)Lchannel,thatisatnegativemembranepotentialswithCs+intheexternalsolution,andatpositivemembranepotentialswithCs+atthecytoplksmicmembraneface(Fig.30).AssumingthattheflickerblockbyCs+isnotinfluencedbyotherions,thenthetime-averagedcurrentthroughtheblockedchannelisgivenas:114Figure31.SinglechannelK(Ca)Lcurrentrecordingsfromaninside-outpatchbathedin140mMK±solutionwith1001LM[Ca2+ft.Thepatchelectrodecontained60mMK+and80mMCs+0indicateschannelclosedand1indicateschannelopen.ThesamegroupofsinglechannelcurrentsisseenatafilterbandwidthofDC-2kHz(uppertrace),andatafilterbandwidthofDC400Hz(lowertrace).Membranepotential+60mV.1159L1SWOS-0-I0—IiiII I.II Ii liiiIiIII!‘1’1•iIr’ir• T1J.I[.1<i(V)>=io(V)[1+[Cs]/K(o)exp(FÔV/RT)]-1(4)whereio(V)isthecurrentthroughtheunblockedchannel,Vispotential,K(o)isthezerovoltageequilibriumconstantand.3isthefractionofthemembranefieldfeltbyCs+atitsblockingsite,sometimesreferredtoasthe“effectivevalence”(Woodhull1973;Yellen1984a).Plotsofln(io/i-1)versusVshouldthereforeyieldastraightlineofslopeF.3/RT.Thevalueof.3providesameasureofthesteepnessofvoltagedependentblocking.Valuesofi 0wereobtainedfromthe5patchesinwhichNa+replacedK+,sincenoevidenceforNa+blockoftheK(Ca)Lchannelwasseen.UsingthedatashowninFig.29andFig.30,plotsofthistypewerefount’tobeapproximatelylinearoverthevoltagerange-70mVto+70my.WhenCs+waspresentatthecytoplasmicmembraneface,avalueof.3=0.07wasobtained,indicatingthatthecationdetectsverylittleofthemembranefieldatthissite.Incontrast,externallyappliedCs+yieldedavalueof6=-0.66,suggestingthattheionmusttransverseanappreciablefractionofthemembranetoreachitsbindingsite,ifappliedexternally.3.4.3.Effectofvarying[Ca 2+]iontheopenprobabilityofK(Ca)LchannelsTodeterminetheeffectof[Ca 2+JiontheprobabilityofthesingleK(Ca)Lchannelsbeingintheopenstate,recordingsweremadefromisolatedinside-outpatchesexposedtosymmetrical140mMK+solutions.[Ca 2 +Iiwasvariedwhilethemembranepotentialofthepatchwasvoltage-clampedto+40mV.Fig.32showssinglechannelcurrentsobtainedfromapatchcontainingasingleactiveK(Ca)Lchannel.When[Ca 2+]iwas0.5,LM,fewchannelopenings117Figure32.Effectofvarying[Ca 2+ftontheopeningofK(Ca)Lchannels.Singlechannelcurrentrecordingswereobtainedfromanisolatedinside-outpatchcontainingoneactivechannelandbathedinsymmetrical140mMK+solutions.Therecordingpipettecontained0.01MCa 2 +.Membranepotentialwas+40mV.0indicateschannelchisedand1indicateschannelopen.BandwidthDC200Hz.118ydSiS61!rl$.I001‘I”3IPS00—---I ——0—0--—-rimr annrm1,rr1rnrw01‘njifI———wereseen(Po=0.0045)andthemeanchannelopentimewasclearlyverybrief.Onincreasing[Ca 2+]ito1,10and100M,Poincreasedmarkedly(Po=0.60at100MM[Ca 2 +]i).ThemeanshuttimeoftheK(Ca)Lchannelwasgreatlyreducedat[Ca 2+]j=100M,whilethemeanopentimeappearedgreaterthanthatseenatlowcytoplasmiccalcium.Theeffectofvarying[Ca 2 +]ifrom0.01Mto1mMonPovaluesmeasuredin5patchesatV=+40mVisshowninFig.33A.Thethresholdlevelof[Ca 2 +]iatwhichchannelopeningswerejustdetectablewasabout0.01M.Themeanvalueof[Ca 2+]iatwhichsingleK(Ca)Lchannelswereopenhalfofthetime,Po(0.5)was23JLM.Fig.33BshowsthesedatapresentedasaHillplot,i.e.logPo/(1-Po)versuslog[Ca 2+]j.Alinearregressionlinehasbeenfittedtothedatapointsobtainedfor[Ca 2+]jvaluesbetween0.05LMand1jLM,theprobablephysiologicalrangeofthisparameterinsmoothmusclecells(Kiockneretal.1989).Overthisrange,thislinehadameanslopeof2.00Thisresultshowsthat2calciumionsmustbindtofullystabilizeeachK(Ca)Lchannelintheopenstate(Barrettetal.1982).Atverylowandveryhigh[Ca 2 +Ji,theslopefactorwasgreatlyreduced,ashaspreviouslybeennotedinKCachannelsstudiedinratskeletalmusclefibres(Barrettetal.1982).3.4.4.Theeffectofvarying[Ca 2 +]iontheopentimeoftheK(Ca)Lchannel.Overtherangeofmembranepotentials(-80to+80mV)and[Ca 2+levels(0.01Mto1mM)usedinthisstudy,opentimedistributionsforK(Ca)Lchannelswerewelldescribedbythesumoftwoexponentialterms,indicatingthe120Figure33.Effectof[Ca2+IiontheopenprobabilityoftheK(Ca)Lchannel.(A)MeanopenprobabilityPoisplottedasafunctionof[Ca2+]ionsemilogaritbmicco-ordinates.Thesmoothcurvewasfittedtodatapointsusingpolynomialregression.(B)Hillplotofthesedata,i.e.logPo/(1-Po)againstlog[Ca2+Ii.Overrangeof[Ca2+Jifrom0.0511Mto1jIM,datawerewellfittedbylinearregressionline(solidline)ofslopen2.00.Datawereobtainedfromfivepatches.Membranepotentialwas+40mVduringtheserecordings.[Ca2+]o=0.01121AP 00.30.6-0.4I0.2100.0010.010.11101001000[Ca 2 J 1(jj4)P(1-P 0 )10///0.010.0010.00010.0010.010.11101001000[Ca 2 J 1(J.LM)122•qpresenceofatleasttwokineticallydistinguishableopenstatesforthischannel(Fig.34).Thesedistributionswerethereforewellfitbyanequationoftheformy=Nf.e-t/rf+Ns.et/rs(5)HereNfandNsarethezerotimeamplitudesoftwoexponentialfunctionsgoverned,respectively,byafasttimeconstantrfandaslowtimeconstantrs.Fromtheseparameters,thetotalnumberofopeningsrepresentedbyeithercomponentwascalculatedusingtherelationsAf=Nf.rf/Binwidth(6)andAs=Ns.rs/Binwidth(7)Here,AfandAarethenumbersofopeningsintheexponentialtermsgovernedbythefasttimeconstant,rfandtheslowtimeconstant,TSrespectively.Thevalueofthebinwidthparameterwasobtaineddirectlyfromtheopentimehistograms.Theeffectofchanging[Ca 2+]iontheopentimedistributionsofK(Ca)Lchannelswasinvestigatedin5patchesvoltage-clampedatamembranepotentialof+40mV(Fig.35).Itwasfoundthatincreasing[Ca 2+Iihadnosignificanteffectonthemeanvalueofthefasttimeconstantrfinthesepatches(Fig.35A).At[Ca 2 +]i=O.O1M,ameanvalueofrf=0.58±0.08mswasobtainedwhileat[Ca 2+Ii=10M,Tf=0.520.15mswasseen(P>0.05,ANOVA).Incontrast,rswasfoundtoincreaseonelevating[Ca 2 +]i(Fig.35B).At[Ca 2 ±]i=0.01MM,ameanvalueofrs=5.9±0.85mswasobtained,whileat[Ca 2 +]j=10AM,s=14.6±2.24mswasobserved(P<0.05,ANOVA).123Figure34.DistributionofopentimesfortheK(Ca)Lchannelstudiedinoneisolatedpatchcontainingasingleactivechannel.[Ca2+]i=1J.LMinAand1mMinB.Bothdistributionswereplottedonalogarithmictimeaxisandfittedbythesumoftwoexponentialterms(smoothcurves)usingtheSimplexalgorithm.InA,thefastfitcomponenthadatimeconstantrf=0.41ms,thiscomponentmadeup29%ofthetotalopenings.Theslowfitcomponentwasgovernedbyatimeconstantrs=11.4ms,andmadeuptheremaining71%ofopenings.CorrespondingvaluesforthedistributionshowninBwererf=0.38ms(19%ofopenings)and7s=14.3ms(81%ofopenings).Membranepotentialwas+40mVinbothAandB.[Ca2+]o=0.01cM.124AU)1000cti>L80a)C’)Do.600U)D40Ez200-5-4-3-2-10123Logopentime(s)B60(I)C0•:;50>(1)40(I)-o030020Sz100-5-4-3-2-10123Logopentime(s)125Figure35.Effectofvarying[Ca 2+]jonthetimeconstantsgoverningopentimedistributionofK(Ca)LchannelsstudiedinSinside-outpatchesvoltage-clampedat+40mV.A,Thefasttimeconstant(i-f)versus[Ca 2+Ii.B,Theslowtimeconstant(rs)versus[Ca 2+ii.[Ca 2+]o=0.011 AM.126A0.00I0.010.11101001000ICa2+Ii(tM)20rB00010.010.1I101001000iCa2+Ii(J.LM)127Thus,increasing[Ca 2+]jincreasedthetimeconstantoftheslowexponentialcomponent,whilehavingnoeffectonthatofthefastexponentialcomponent.Changing[Ca 2 +Jialsohadaneffectontherelativenumbersofopeningsgovernedbythetimeconstantsi-fandi-s.AsshowninFig.36,increasing[Ca 2 +]jfrom0.01Mto1mMleadtoamonotonicincreaseinthevalueoftheratioA 5 /(Af+As)(Fig.36B),withacorrespondingdecreaseintheratioAf/(Af+As)(Fig.36A).Theseobservationsshowthatincreasing[Ca 2+preferentiallystabilizedopeningsoftheK(Ca)Lchannelgovernedbytheslowtimeconstanti-s.Themeanopentime,rmeanoftheK(Ca)Lchannelwascalculatedfromtheabovedatausingtherelationrmean=Af/(Af+As).rf+A 5 /(Af+As).rs(8)Atamembranepotentialof+40mV,i-meanincreasedfrom2.70.23ms(n=5)at0.01MM[Ca 2 +]ito13.5±0.35msat10AM[Ca 2 +]j(P<0.05,Students’t-test).This5foldriseini-meanwasmuchtoosmalltofullyaccountforthe425foldincreaseseeninopenprobabilityonelevating[Ca 2+]ifrom0.01Mto10PM(seeFig.33).ItwasthereforeconcludedthatthemajoreffectofincreasinginternalfreecalciumconcentrationwastodecreasethemeanshuttimeoftheK(Ca)Lchannel.3.4.5.EffectofmembranepotentialontheopenprobabilityoftheK(Ca)Lchannel128Figure36.Effectofvarying[Ca 2+]jontherelativenumberofopeningsofK(Ca)Lchannelsgovernedbythetimeconstantsi-fandi-s.Dataaremean±SEMfrom3-5inside-outpatchesvoltage-clampedat+40mV.InA,therelationshipbetweentheratioAf/(Af+As)and[Ca 2+]iisillustrated.InB,theeffectof[Ca 2 +]jontheratioA 5 /(Af+As)isshown.[Ca 2 +]oO.01M.129AF‘Hs..H0.0010.010.1I101001000lCa2+Ii(M)BUiO.60.00I0.010.11101001000lCa2+Ii(jiM)130TheprobabilityofK(Ca)LchannelsbeingintheopenstatewasalsoafunctionofthepotentialappliedacrossthemembranepatchasshowninFig.37.When[Ca 2+]iwaslessthan50M,thedependenceofPoonmembranepotentialwaswelldescribedbytheBoltzmannrelation(Koib1990)Po=[1+exp(-K(V-Vo))]-l(9)whereVoisthepotentialatwhichPo=0.5(Fig.37).In5patchesexposedto[Ca 2+]i<50.M,themeanvalueoftheconstantKwas0.045+0.006mV-i.At[Ca 2+Jjlevelsbetween0.01Mand50M,theprincipaleffectofincreasingintracellularfreecalciumwastoshifttheBoltzmannrelationtotheleftonthevoltageaxis.In4patchesstudiedinthisrangeof[Ca 2 +Ji,themeanshiftoftheVowas45mVperdecadechangeinintracellularfreecalcium.At[Ca 2+Ii>50jIM,Poapproachedunityandshowedlittlefurtherincreaseonmembranedepolarization(Fig.37).IndeedatendencywasnotedtowardreducedPoatmembranepotentialsmorepositivethan+40mV(Fig.37).ReducedPoatpositivemembranepotentialshaspreviouslybeennotedinKCachannelsstudiedinvascularsmoothmusclecellsoftheguinea-pig(Benhametal.1986)andinKCachannelsincorporatedintolipidbilayers(VergaraandLatorre1983).3.4.6.EffectofmembranepotentialontheopentimeofKCachannels.131Figure37.Dçpendenceoftheopenprobability,P 0ofK(Ca)Lchannelsonmembranepotential(V)and[Ca 2+Iimeasuredin4inside-outpatches.When[Ca 2+Jjwas1mM(circles)and100JLM(diamonds),fittinglinesweredrawnbythepolynomialregression.When[Ca 2+Jiwas50M(squares)and0.5M(triangles),thefittingcurvesweredrawntotheBoltzmannequationP 0=[1+exp(-K(V-V 0 ))]-1whereVoisthepotentialatwhichPo=0.5.Atboth50Mand0.5JLMfreeintracellularcalcium,theconstantKhadthevalue0.045mV-i.At50JLMfreecalcium,Vo=-5mV.at0.5jMcalcium,Vo=+88mV.132(Aw)A08 09Ot Ot080 0ZV090 t°dTheeffectofvaryingmembranepotentialonthedistributionofopentimesforK(Ca)LchannelswasstudiedinSpatchesexposedto[Ca2+]j=10,iM.Thesedistributionswerewelldescribedbythesumoftwoexponentialterms,asnotedpreviously.Itwasfoundthatchangesinmembranepotentialdidnotsignificantlyalterthevalueofthefasttimeconstant,Tfinthesepatches(Fig.38A).ThemeanvalueofifatV=-60mVwas0.45±0.06ms,notsignificantlydifferentfromthevalueobtainedatV=+60mV(if=0.38±0.04ms,P>0.05,ANOVA).Incontrast,theslowtimeconstant,iSdescribingopentimedistributionswassignificantlyalteredbymembranepotentialinthesepatches(Fig.38B).r8wasweaklyvoltage-dependent,increasingfrom8.4±0.55msatV=-60mVto16.6±1.64msatV=+60mV(P<0.05,ANOVA).Itshouldhoweverbenotedthatthesevalueofrfareclosetothedeadtimeoftherecordingsystem.Smallchangesinifcouldthereforehavegoneundetected.3.4.7.TheeffectofTEAappliedtothecytoplasmicmembranefaceoncurrentflowintheK(Ca)Lchannel.TheeffectofTEAontheK(Ca)Lchannelwasstudiedin6inside-outmembranepatchesvoltage-clampedat+40mVwith[Ca2+]i=100PM.Fig.39showssinglechannelcurrentsrecordingduringanexperimentofthiskindinwhichTEAwasappliedbybathperfusiontothecytoplasmicmembraneface.Undertheseconditions,TEAcausedadose-dependent,reversiblereductionintheamplitudeofcurrentintheK(Ca)Lchannel.ItwasnotedthattheblockingactionofTEAwasnotaccompaniedbyalargeincreaseinthevarianceofcurrentnoiseintheopenchannelasobservedatabandwidthofDC-2-kHz(Fig.39).TEAdidnotalterthereversalpotentialofthecurrentthroughK(Ca)Lchannels,whichwas0mVintheseexperiments.134Figure38.EffectofmembranepotentialonthetimeconstantsgoverningtheopentimedistributionofK(Ca)Lchannels.A.Fasttimeconstant(i-f)versusmembranepotential.B.Slowtimeconstant(rs)versusmembranepotential.Datapointsaremean±SEMfromthreetofivepatches.[Ca2+Ji=10p.Mand[Ca2±lo=001p.MinbothAandB.135A‘tf(ms)IIIIIII—80—60—40—20020406080V(mV)B(ms)f----- 15IIIII—80—60—40—20020406080V(my).136Figure39.BlockingeffectofinternalTEAontheK(Ca)Lchannel.Singlechannelcurrentswererecordedfromaninside-outmembranepatchbathedinsymmetrical140mMpotassiumsolutions.TEAwasappliedtothecytoplasmicfaceofthemembraneattheconcentrationsindicated.0indicateschannelclosedand1indicateschannelopen.BandwidthDC-2kHz,membranepotential+40mV.[Ca 2 +]o=O.01$M.137NOTEA1----‘rli9Ld0.5mMTEA1----1mMTEA::::2pA50ms138Thepercentagereduction,RiinsinglechannelcurrentcausedbyvariousconcentrationsofTEAappliedtothecytoplasniicmembranefaceisshowninFig.40.ItwasfoundthatthevalueofRjwaswelldescribedbytherelationRj=100%/(1+Kd/[TEA])(10)whereKd0.83±0.09mM(n=5patches)atamembranepotentialof+40mVand[Ca2+]j=1001AM.ThepresentdataonTEAblockweretransformedintoaHillplotbyreplottingaslog(Ri/i-Ri)versuslog[TEA],whereRisthereductioninsinglechannelcurrentsseeninthepresenceofthedrug.Thisplotyieldedaslopeof0.82,suggestingthatTEAinteractswiththechannelinaone-to-onefashion,presumablybytransientlyenteringthechannelandblockingmovementofK+.Intheseexperiments,TEAhadnosignificanteffectontheprobabilityofK(Ca)Lchannelsadoptingtheopenstate(Fig.41).ThusPo=0.55±0.15(n=3)intheabsenceofTEA,andP 0=0.53±0.17(n=3)inthepresenceof10mMTEAatthecytoplasmicmembraneface(P>0.05,ANOVA).3.5.K(Ca)Ichannelsininside-outmembranepatchesexcisedfromisolatedCVSMCs.139Figure40.Dose-responsecurvefortheblockofcurrentflowintheK(Ca)LchannelbyinternalTEA.ThepercentagereductioninsinglechannelcurrentisplottedagainsttheconcentrationofTEAatthecytoplasmicmembraneface.ThesolidlineplotstherelationRi=100%/(1+Kd/[TEA])wheretheapparentdissociationconstantKdwas0.83mMand[TEA]wastheconcentrationofTEA.Datapointsaremean±SEMfrom3-5patches.Thecalculatedcurvewasfittedtothedatapointsbynon-linearregression.V=+40mV,[Ca 2 +]j=1O0M.[Ca 2 ±]o=0.01,LM.140HruPercentagereductionofsinglechannelcurrent—CH-iFigure41.EffectofTEAontheopenprobabilityoftheK(Ca)Lchannel.TheopenprobabilityofthechannelisplottedagainsttheconcentrationofinternalTEA.Datapointsaremean±SEMfrom3-5patches.NosignificancebetweenthemeanvaluesofP 0calculatedateachTEAconcentration(P>0.05ANOVA).Membranepotential+40mV,[Ca 2 +]j=10OM.[Ca 2 +]j=0.01jM.1421.0-P 00.8-0.6-0.4-0.2-0Control0.1110100TEA(mM)143K(Ca)Ichannelsweredetectedinabout25%oftheinside-outpatchesexaminedinthisstudy.Whenmeasuredinsymmetrical140mMK+solutions,currentsinK(Ca)Lchannelsshowedareversalpotentialof0mV(Fig.42).Undertheseconditions,thecurrent-voltagerelationofthischannelwaslinearwithameansinglechannelconductanceof92±2.6pS(n=10patches).AmplitudedistributionsofcurrentsflowinginK(Ca)IchannelswerewelldescribedbysingleGaussianterms,indicatingthatthischanneldidnotdisplayasignificantsubstateorsuperstateconductance(Fig.43).Fromthesedata,thepotassiumpermeabilityoftheopenchannelwascalculatedusingtheconstant-fieldrelation(Equation2).AmeanvalueofPK=1.75±0.12x10-13cm/swasobtainedfrom10patches.TheinfrequentoccurrenceofK(Ca)Ichannelsinthesepatchesprecludedadetailedquantitativeexaminationoftheionicselectivityofthischanneloritssensitivitytochangesin[Ca 2+]jandmembranepotential.However,itwaspossibletodemonstratequalitativelythatK(Ca)Ichannelsareindeedselectiveforpotassiumions,andaremodulatedbybothmembranepotentialand[Ca2h]j.When80mMKC1wasreplacedbyanequimolaramountofNaC1,currentsthroughsingleK(Ca)Ichannelsreversedat+21.1mV(cytoplasmicfaceKC1replacement)and-21.1mV(externalfaceKC1replacement,Fig.44).TheratioPNa/PKwascalculatedfromthesedatausingEquation3.AvalueofPNa/PK<0.05wasobtained,indicatingthatK(Ca)Jchannelsarehighlyselectiveforpotassiumoversodiumions.InsimilarexperimentswhereCs+replacedK+,theratioPCs/PKwasalsofoundtobelessthan0.05.InthepresenceofCs+,currentflowintheK(Ca)Ichannelwaslessthanpredicted144Figure42.Single-channelcurrentrecordingsoftheactivityofK(Ca)Ichannelsatvariousmembranepotentials(mV).Alltraceswererecordedfromasingleinside-outpatchcontainingfouractivechannels.Thispatchwasbathedinsymmetrical140mMK+solutions.0indicatesallchannelswereclosed,whilethenumbers1,2,3and4indicatecurrentlevelsassociatedwith1,2,3and4channelsopen,respectively.BandwidthDC-200Hz.Calibrationbarsapplytoalltraces.Thecytoplasmicmembranefacewasexposedto[Ca 2+Ii=1LM.Thepipettecontained[Ca2+]o=0.01riM.14509IScVdOZkAlLfb4L L,4L.LL.zt -- 1 rIj11•11I’IIIITI I’0- OLr4 iL4MJw0(Aw)Figure43.AmplitudedistributionsobtainedforK(Ca)Ichannelcurrentsrecordedfromasingleinside-outpatchvoltage-clampedatamembranepotentialof+30mVinAand-30mVinB.BothdistributionswerewellfittedbysingleGaussianterms(smoothcurves)withmodalvaluesof2.44pAinAand-2.43pAinB.Thepatchwasbathedinsymmetrical140mMK+solutions.[Ca2+]j=10M,[Ca2+]o=0.01M.147A120-C’,C10o(‘I>0)C,,I:Z206121‘62’OAmplitude(pA)B70(I)C60ccl>50CI)-Do4000)61’21’6Amplitude(pA)148Figure44.ConductanceandionicselectivityoftheK(Ca)Ichannel.Current-voltageplotsofsingle-channelcurrentsrecordedfromfiveinside-outpatchesinfivedifferentionicgradients:(filledcircles)symmetrical140mMK+solutions;(opensquares)80mMinternalK+replacedbyanequimolaramountofNa+;(filleddiamonds)80mMexternalK+replacedbyanequimolaramountofNa+;(filledtriangles)80mMinternalK+replacedbyanequimolaramountofCs+;(opentriangles)80mMexternalK+replacedbyanequimolaramountofCs+.[Ca2+]oand[Ca2+]iwereO.01Mand100PMrespectively.Continuouslinesaretheoreticalcurvesfitbylinearregression(dataobtainedinsymmetricalpotassiumsolutions)ornon-linearregressiontotheGoldmanHodgkin-Katzconstant-fieldequation,usingthedatapointsobtainedduringreplacementofK+byNa+.149•10i(pA)8v.Outward7.AAA—80—60—40—2III7-?o406080V(mV).4 A•ADAInward-10150fromtheconstant-fieldrelation.ThiseffectwasespeciallymarkedunderconditionstendingtodriveCs+intotheopenchannel,i.e.atnegativemembranepotentialswithCs+presentintheexternalsolution,andatpositivemembranepotentialswhenCs+waspresentatthecytoplasmicmembraneface(Fig.44).TheopenprobabilityofK(Ca)Ichannelsincreasedas[Ca2±]iwaselevatedoverthecoicentrationrange0.1Mto100MM(Fig.45).Fig.46AshowsthedependenceofP0on[Ca2+]iforonemembranepatchvoltage-clampedatV=+4OmV.ThesedatawerereplottedasaHillplot,i.e.aslogPo/(1-Po)againstlog[Ca2+Ji(Fig.46B).ItisevidentthatP0wassteeplydependenton[Ca2+Jiovertheprobablephysiologicalrangeofthisparameter(0.05-1PM).Inthisrange,theslopeoftheHillplotwas2.17,suggestingthatmorethanonecalciumionmustbindtofullyactivateeachK(Ca)Ichannel.TheopenprobabilityofK(Ca)Ichannelsalsoincreasedondepolarizationofthemembranepatch(Fig.47).ThedependanceofPoonmembranepotentialwaswelldescribedbytheBoltzmannrelation(Equation9).Theeffectofincreasing[Ca2+]iwastoshifttheBoltzmannrelationtotheleftonthevoltageaxis,byabout40mVperdecadeincreaseinfreeintracellularcalcium(Fig.47).TheeffectofTEAontheK(Ca)Ichannelwasexaminedquantitativelyin6inside-outpatchesvoltage-clampedatV=+40mVwith[Ca2+]j100M.TEAwasappliedbybathperfusiontothecytoplasmicmembraneface.Undertheseconditions,TEAcausedareversible,dose-dependentreductionintheamplitudeofcurrentintheK(Ca)Ichannel(Fig.48).TEAhadnoeffectonthereversalpotentialofthecurrentinK(Ca)Ichannels(0mV).151Figure45.Effectofvarying[Ca2+JiontheactivityofK(Ca)Ichannels.Singlechannelcurrentrecordingswereobtainedfromanisolatedinside-outpatchwithtwoactivechannelsbathedinsymmetrical140mMpotassiumsolutions.Membranepotentialwas+40mV.0indicateschannelclosed,whilethenumeralsland2indicatecurrentlevelsassociatedwith1and2channelsopen,respectively.BandwidthDC-200Hz.[Ca2+Jo=0.01M.1520.1MCa2JiLIii.fli1-iLILj1bLf’I10M100lOpA153Figure46.Effectof[Ca2+]iontheopenprobabilityoftheK(Ca)Ichannel.(A)Openprobabilityplottedagainst[Ca2+]ionsemilogarithmicco-ordinates.(B)Fullplotofthesedata,i.e.logPo/(1-Po)againstlog[Ca2+]i.Overrangeof[Ca2+]jfrom0.05Mto1M,datawerewellfittedbylinearregressionline(solidline)ofslopen=2.17.Datawereobtainedfromoneinside-outpatch.V=+40mV,[Ca2+Jo0.01tM.154AP 00.80.6-0.40.2-00.0010.010.11101001000[Ca 2 ](iiM)BP 0 /(1-P 0 )0.01-0.001..0.0001III0.0010.010.11101001000[Ca 2 ](tM)155Figure47.Dependenceoftheopenprobability,PoofK(Ca)IchannelsonmembranepotentialV(mY)atdifferentvaluesof[Ca2+Ii.Thedatawereobtainedfrom4inside-outpatchesexposedtothefollowing[Ca2+]j:1M(opensquares);1OtM(filledsquares);1OOM(filledtriangles);1mM(filleddiamonds).ThefitcurvesweredrawntotheBoltzmannequationPo=[1+exp(-K(V-V0))]-1whereVoisthepotentialatwhichPo0.5.Voat1M,1OM,100PMand1mM[Ca2+]iwere+40mV,+18mV,0mVand-65mV,respectively.TheconstantKtookthefollowingvalues:0.16mV-iat[Ca2+]i=1AM;0.096mV-iat[Ca2+]j=i0M;0.035mV-iat[Ca2+]j=i0OMand0.011mV-iat[Ca2+]j=1mM.[Ca2+]o=0.01MM.156P 01.0-0.8-A•44AA0.4ApaaIIIIII-80-60-40-20020406080V(mV)157Figure48.BlockingeffectofTEAoncurrentflowintheK(Ca)Ichannel.Singlechannelcurrentrecordingsfromaninside-outpatchwithoneactivechannel.TEAwasappliedtothecytoplasmicfaceofthemembrane.0indicateschannelclosedand1indicateschannelopen.BandwidthDC-2kHz,V=+40mV.[Ca2+Jj100,LM,[Ca2+]o=O.OlM.Thepatchwasbathedinsymmetrical140mMK±solutions.158NOTEA0.5mMTEA1mMTEAoj5p4lOOms159Thepercentagereduction,RiinsinglechannelcurrentcausedbyvariousconcentrationsofTEAappliedtothecytoplasmicmembranefaceisshowninFig.49.ThevalueofRiwaswellpredictedbytherelationRj=100%/(1+Kd/[TEA]),wheretheconstantKdhadthevalue0.31mMundertheseconditions.ThesedatawerereplottedasaHillplot,log(Ri/i-Ri)versuslog[TEA]whereRisthereductioninmeansinglechannelcurrentcausedbyagivenconcentrationofTEA.Theslopeofthisplotwas0.70,suggestingthatTEAblocksthischannelinaone-to-onefashion,asseenfortheK(Ca)Lchannel.TEAhadnosignificanteffectontheprobabilityofK(Ca)Ichannelsadoptinganopenstate(Fig.50).AnANOVAtestwasperformedtocomparetheseresultswithcomparabledataobtainedfortheK(Ca)Lchannel.Itwasfoundthatnosignificantdifferencesexistedbetweenthesetwodatasets(P>0.05).ThisresultindicatesthattheK(Ca)LandtheK(Ca)IchannelshavecomparablesensitivitiestointernallyappliedTEA.160Figure49.Dose-responsecurveforblockoftheK(Ca)IchannelbyTEA.ThepercentagereductionofsinglechannelcurrentisplottedagainsttheconcentrationofTEAappliedtothecytoplasmicmembraneface.Datapointsarethemean±SEMfrom3-5patchesvoltage-clampedatV=+40mVandexposedtosymmetrical140mMK+solutions.ThesolidlineplotstherelationRi=100%/(1+Kd/[TEA]).TheapparentdissociationconstantKdwas0.31mM.[Ca 2 +]j=100MM,[Ca 2 ±]o=0.01161mPercentagereductionofsinglechannelcurrent00000%00000pHH0Figure50.EffectofTEAontheopenprobabilityoftheK(Ca)Ichannel.Openingprobabilityofthechannel,PoisplottedagainsttheconcentrationofinternalTEAappliedtothecytoplasmicmembraneface.[Ca2+Ii100M,[Ca2+Jo=0.01M.Patcheswerebathedinsymmetrical140mMK+solutions.Datapointsaremean±SEMfrom3-5patchesvoltage-clampedat+40mV.Nosignificantdifferenceswerefoundbetweenthesemeans(P>0.05,ANOVA).163l.0Controlo.i11000[TEA](mM)164Chapter4DiscussionThepresentstudyhasresultedinthedevelopmentofanovelinvitropreparationofCVSMCsderivedfromthecerebralarteriesofadultrats.Thesecellswere,identifiedusingtheMassontrichrometestandanantibodyspecifictosmoothmusclea-actin.Thecalcium-sensitivefluorescentprobefura-2wasemployedtomeasuretherestingleveloffreeintracellularcalciuminCVSMCsandtostudytheeffectof5-HToncalciummobilizationinthesecells.PatchclampmethodswereusedtomeasurethebasicelectrophysiologicalpropertiesofCVSMCsinvitro.ThebiophysicalpropertiesoftwotypesofKCachannelwerestudiedininside-outmembranepatchesexcisedfromthesecells.Thesignificanceoftheseresultswillnowbediscussedinrelationtopreviousfindingsfromotherlaboratories.4.1.MorphologicalcharacteristicsofCVSMCsinvitroSeveralreportshaveappeareddescribingtheinvitrogrowthofCVSMCsfromcerebralresistancevessels(Spatzetal.1983;Diglioetal.1986).However,thepresentstudyisapparentlythefirsttodescribeshort-termculturesofCVSMCsderivedfromconductingcerebrovasculararteriesoftherat.Whenexaminedafter1-4daysinvitro,thecellsobtainedtypicallydisplayedasphericalshape.Theshapeofthesecellswasthesameregardlessofwhetherplateswereincubatedat37oCinculturemediumorkeptat40Cinamaintenancesolution.Similarmorphologyhasalsobeenobservedinshort-termculturesofCVSMCsderivedfrompenetratingvesselssupplyingtheratbrain,wheremorethanfour165daysinvitrowererequiredforthecellstoadoptafusiformorpolygonalshape(Spatzetal.1983;Diglioetal.1986).Cerebralarteriesmaybeclassifiedasthe“muscular”typeofbloodvessel,inwhichthetunicamediacontainsonlyafewelasticlaminaeandrelativelylittleintercellujarspace,whencomparedto“elastic”arteries,suchastheaorta.CVSMCsarespindleshapedwhenrelaxed,butlackthehighlyfusiformmorphologytypicalofgastrointestinalsmoothmuscle,theratioofmaximumtominimumaxesbeingabout9:1(Rhodin,1980).Steeleetal.(1991)havedescribedaprocedurebywhichCVSMCsapproximatingthisshapecanbeproducedfollowingenzymaticdissociationofratbasilararteries.Thereasonwhythisresultwasnotobtainedinthepresentstudyremainsunclear.Itispossiblethatthedissociationproceduresusedresultedincytoskeletaldamage,asreportedforotherCVSMCsfollowingenzymatictreatmentandplatingontoforeignsubstrates(Spatzetal.1983;Diglioetal.1986).ItisalsoknownthatCVSMCsdissociatedfromratbasilararteriesareunabletorelaxfollowingabriefperiodofactivecontraction,presumablybecauseoftheabsenceofelasticelementsinthesepreparations(Steeleetal.1991).ThepresentstudyindicatesthattheintracellularfreecalciumlevelofCVSMCswasabout40nM,farbelowthemicromolarlevelsrequiredtoactivatemusclecontraction(Johnsetal.1987).Therefore,itisprobablethatthesphericalshapeoftheCVSMCsusedinthisstudywastheresultoffailureofthecellstorelaxpassively,ratherthanofanactivelymaintainedcontraction.ThemajorityofthesphericalcellspresentinourshorttermprimarycultureswerefoundtoreactpositivelytobothMasson’strichromestainandtoamonoclonalantibodydirectedagainstsmoothmusclea-actin.Indifferentiated166SMCs,smoothmuscleoe-actinand‘y-actinpredominateoverother,non-SMCspecfficformsoftheprotein.InvascularSMCsstudiedtodate,mostoftheactinpresentisinthea-isoactinform(Gabbianietal.1981;Owensetal.1986).Inaddition,thea-isoactinofSMCsisimmunologicallydistinctfromthatfoundinskeletalorcardiacmusclecells(Owensetal.1986).SMCa-actinisthereforeconsideredtobeareliablemarkerforsmoothmuscletissuestudiedinvitro(Mooreetal.1984;Diglioetal.1986),althoughcertaintumorsmayalsoexpresstheantigen(Skalleetal.1986).TheseconsiderationsstronglysuggestthatthecellsusedinthepresentstudywereindeedSMCsofvascularorigin.4.2.IntracellularfreecalciuminculturedCVSMCsTherestingleveloffreeintracellularcalciumintheCVSMCsculturedat37°Cwasfoundtobeabout40nM.Thisvalueiscomparablewiththevalueof50nMcytosolicfree[Ca2+]obtainedfromrestingairwaysmoothmusclecellsobtainedpsingfluorescencemeasurements(RodgerandSmall,1991).However,itissignificantlylowerthanthatfoundinVSMCsdispersedfromaperipheralvessel,therataorta,inwhich[Ca2+]jwasfoundtobe126nM,(Takataetal.1988);153nM,(Capponietal.1987);and114nM,(Nabikaetal.1985).Inthepresentstudy,baselinelevelsof[Ca2+]iweredeterminedinthesameculturesusedtocalibratethemeasurementoffreeintracellularcalcium.Inaddition,insitucalibrationusingcellsisgenerallyconsideredtobemorereliablethanmethodsemployingfluorescenceratiosdeterminedincell-freesystems(Ishiietal.1989).Themeanrestinglevelof[Ca2+]jwhichwasobtainedshouldthereforebereliable.167[Ca2+]jisknowntodeclinewhenextracellularcalciumisloweredbelowabout2mM,atleastinthecaseofSMCsderivedfromthegastrointestinaltract(Wiffiamsetal.1985;PritchardandAshley1986).Inboththepresentstudy,andinoneofthestudiesquotedabove,1mMCa2+wasemployedintheexternalbathingsolution(Nabikaetal.1985).Itseemsunlikelythereforethatthesmallervalueof[Ca2+]jobtainedfromculturedcerebrovascularSMCswasdueentirelytotheuseofarelativelylowconcentrationofCa2+intheexternalbathingsolution.Bindingofthefreeacidbycellularproteinscanleadtoachangeinaffinityoffura-2forcalcium,andtoanunderestimateof[Ca2+]j(Konishietal.1988).However,comparisonoffura-2datawithresultsobtainedusingcalciumsensitivemicroelectrodessuggeststhattheunderestimateintroducedbyfura-2isnomorethantwo-fold(SomlyoandHimpens1989).Inaddition,thiserrorshouldapplytoallstudiesinwhichacuvettecalibrationoffura-2calciumaffinitywasemployed.4.2.1.Modulationof[Ca2+]ibyserotonininCVSMCsTheresultspresentedinhereshowthat5-HTincreasesfreeintracellularCa2+levelsinCVSMCsderivedfromratcerebralarteries.ThiseffectshowedanapparentEC5Oof10nM,avalueclosetothatseeninsimilarstudiesconductedonanaortaSMCline,EC50=26nM,(Doyleetal.1986).Inaddition,theeffectof5-lIToncalciummobilizationwasshowntobemaximalataserotonindoseof1uMinallthreeofthesepreparations(Doyleetal.1986;Capponietal.1987).168InstudiesconductedonculturedrataortaVSMCs,itwasfoundthat5-HTincreased[Ca2+]jtoamaximallevelof187nM,similartothatseeninthepresentstudy(Takataetal.1988).However,Capponietal.(1988),alsousingrataortaVSMCsinculture,foundthat5-HTapplicationcouldelevate[Ca2+]ito400nMinthesecells.Thereasonforthisdiscrepancyisnotclear,althoughthesestudiesusedcellsafterdifferingnumbersofpassageinculture(Capponietal.1988;Takataetal.1988).Simultaneousmeasurementsof[Ca2+]andforcedevelopmentinindividualvascularsmoothmusclecellssuggestthatthethresholdlevelof[Ca2+Jifortensionproductionisaround125nMandthatmaximalforcecanbeproducedata[Ca2+]iof500-600nM(Yagietal.1988).Theseresultssuggestthatthemaximallevelsof[Ca2+]ireachedinthepresentstudywouldbesufficientfortensiondevelopmentinisolatedCVSMCs.Itshouldalsobenotedthatthesensitivityofthecontractilesystemto[Ca2+Iihasbeenfoundtovarywiththemannerinwhichvascularsmoothmusclecellsarestimulated.Thus,aortaVSMCsstimulatedwithnorepinephrineexhibitedhalf-maximaltensionata[Ca2+Iiofonly100nM,whilecellsstimulatedbythecalciumionophoreionomycinrequired600nMtoreachacomparabletension(Bruschietal.1988).ItisknownthatmyosinlightchainkinasecanbephosphorylatedbyproteinkinaseC,anenzymeactivatedbythediacylglycerolliberatedonactivationof5-HT2receptors.Ithasbeensuggestedthatthisphosphorylationprocesscouldreducethecalciumrequiredformaintainedcontraction(Bruschietal.1988).Theseconsiderationsmakeitdifficulttoaccuratelyassessthedegreeofcontracturewhichwouldbeproducedbyagivenrisein[Ca2+]i,followingactivationofthepresentcellsby5-HT.169The5-HTinducedincreasein[Ca 2+IiwasnotpreventedbythepresenceoforganicorinorganicCa 2 +channelblockers.Incontrast,itisknownthatactionpotentialgenerationinratbasilararteryCVSMCsispowerfullyblockedbybothCo 2 +andnifedipine(Suprenantetal.1987).Takentogether,theseresultssuggestthattherisein[Ca 2+]ievokedby5-HTdidnotdependontheentryofCa2+throughvoltage-sensitivecalciumchannels,duringactionpotentialfiring.AsimilarconclusionwasreachedinthecaseofserotonininducedcalciumreleaseoccurringinVSMCsdissociatedfromtherataorta(Capponietal.1987).Atdosesof1-5OM,5-HTtriggersasmalldepolarizationandabriefburstofGa 2 +spikesincerebrovascularSMCs(Suprenanteta!.1987).Calciumentrythroughvoltage-gatedCachannelsactivatedinthiswaymighthavebeenexpectedtogiverisetoanenhancedfura-2signalat5-HTdosesof1Morgreater.Thissignalshouldbesensitivetocalciumchannelblockers.Noevidenceforthiseffectwasseeninthepresentresults.ThissuggeststhatCa 2 +entrythroughmembranechannelswasrelativelyminor,incomparisontoCa 2+releasedfrominternalstores.Itremainspossible,however,thatcalciumchannelblockadewouldbemoreeffectiveinreducingtheresponseto5-HTconcentrationsabovethe1Mdoseusedinthepresentexperiments.Inthepresentstudy,theeffectof5-HTon[Ca 2 +Iiwasreducedbyabout50%inthepresenceof5nMketanserin.Thisconcentrationissimilartotheaffinityofketanserinmeasuredatagonistlabelled5-HT2receptorsitesinratbraincortex,whichshowaKd=1.4nM,(Lyonetal.1987).1nMketanserinalsosuppresseswhatappeartobe5-HT2receptormediatedcalciumfluxesintheA7r5smoothmusclecelllinebyabout50%(Doyleetal.1986).Ketanserin170isknowntoblockresponsesmediatedbythe5-HT1Creceptor,buttheaffinityofthissiteisapparentlytwoordersofmagnitudetoolowtoreadilyaccountforthepresentresults(ConnandSanders-Bush1987).Inthepresentstudy,theobservedinsensitivityof5-HTactiontocalciumchannelblockers,andthehighsensitivitytoblockadebyketanserinarecompatiblewithamechanisminwhich5-HT2receptoractivationresultsinthemobilizationofcalciumfrominternalstores.Serotonininducedcontractionsinintactratcerebralarteriesaresuppressedbynanomolarlevelsofketanserin(ChangandOwman1987).However,thesecontractionsarealsostronglyinhibitedbylowconcentrationsofcalciumchannelblockers(ChangandOwman1987).Itseemslikely,therefore,thatfullactivationofthecontractilemechanismincerebrovascularSMCsrequiresCa 2+-dependentprocessesoverandabovethereleaseofcalciumfromintracellularstores.Theeffectsof5-HTonmembranepotentialandoncalciumreleasefrominternalstoreshavebeenstudiedindetailinratmesangialcells,acontractile,smoothmuscle-likecellfromthekidneyglomerulus(Meneetal.1991).Itwasfoundthat5-HTactivateda5-HT2receptor,whichtriggeredimmediateddischargeofcalciumfrominternalstores.Theelevationof[Ca 2+Jiactivatedacalcium-dependentchlorideconductance,producingasmall,maintaineddepolarizationofthecellwhichwasdependentonthecontinuedoccupationof5-HT2receptorsbytheagonist.ThisdepolarizationinturnactivatedvoltagesensitiveCachannels,leadingtotonicentryofcalciumintothecell(Meneetal.1991).IthasbeensuggestedthatatonicinfluxofCa 2 +throughvoltagesensitivechannelsmayalsooccurin5-HTstimulatedVSMCs,andthatthis171influxmaybenecessarytomaintainthecontractilestate,possiblybyfacilitatingtherecyclingofinternalcalcium(Sutter1990).Insomecellsinvestigatedinthisstudy,amarkedlossof5-HTresponsivenesswasseenonrepeatedapplicationoftheagonist.Thisphenomenonhasalsobeenreportedinstudiesof5-HTinducedcalciummobilizationinSMCsfromtheperipheralvasculature(Capponietal.1987;Takataetal.1988).Theprogressivedeclineinresponseto5-HTwasmostmarkedinthecellbeingexposedtothephotoexcitantwavelengths.Itseemslikely,therefore,thatthisphenomenoninvolvedadegreeofphotodynamicdamage,ashasbeennotedinpreviousstudiesusingfura-2(Tsien1988).Inthecellbeingilluminated,photodynamicdamagemayalsohaveenhancedthedeleteriouseffectofelevated[Ca 2+]i,whichisknowntoactivateCa 2+-dependentproteasesinmanytissues(Bond,1988).Thedecreasedresponseto5-HTseenonrepeatedagonistapplicationoccurredintheabsenceofelevatingbaselinelevelsoffreecalcium,suggestingthatmembraneintegritywasnotgreatlycompromisedduringthisphenomenon.Itseemsunlikely,therefore,thatgrossdamagetothecellmembranewasresponsibleforthedecliningresponsivenessto5-HT.Insummary,thepresentresultsshowthatCVSMCsculturedfromratcerebralarteriesprovideanusefulpreparationinwhichtostudyCa 2+mobilizationbyserotoninandotheragentsactiveinthecerebrovascularcirculationofmammals.4.3.ElectrophysiologicalpropertiesofisolatedCVSMCs172Inthepresentstudy,themeanrestingpotentialofisolatedCVSMCsfromratcerebralarterieswasfoundtobeabout-40mV.Thesecellsshowedaninputresistanceofabout3GOattherestingpotentialandatotalmembranecapacitanceof24pF.SimilarvaluesofinputresistancehavebeenreportedinapreviouspatchclampstudyofisolatedCVSMCsderivedfromratbasilarartery(Steeleetal.1991).Assumingaspecificmembranecapacitanceof1pF/cm2,ashasbeensuggestedforVSMCs(Toroetal.1986),a24pFcellshouldpossessasurfaceareaof2400m2.Thisisgreatlyinexcessofthesurfaceareacalculatedfor12jLmdiametercellsofpurelysphericalshape(452m2).Thisresultindicatesthatthemembranesurfaceofthepresentcellsishighlyinvoluted,presumablyasaconsequenceofcellcontractionandofthepresenceofnumerouscaveolaeinvascularsmoothmusclecells(Mulvany1986).Hirstetal.(1986)studiedtheelectricalpropertiesofarteriolarsegmentsofratmiddlecerebralarteryusingintracellularrecordingtechniques.Theyobtainedameanrestingmembranepotentialof-63mV.Similarexperimentsconductedonintactcatbasilararteries(Harderetal.1981)andonguinea-pigbasilarartery(Fujiwaraetal.1982)yieldedmeanrestingpotentialsof-55mVand-50mVrespectively.Theslightlylowervaluesofrestingpotentialobservedinthepresentstudycouldbetheresultofseveralfactors.Followingformationofthewhole-cellrecordingconfiguration,dialysisofthecellularcontentsoccurs,leadingtotheformationofanewequilibriumbetweenthebathingsolutionandthesolutionfillingthepatchpipette(SakmannandNeher1983).Thelattersolutionmustthereforeaccuratelyreflecttheioniccompositionofthesarcoplasmincerebrovascularsmoothmusclecells,ifreliablevaluesofrestingpotentialaretobeobtained.173ThemembraneofvascularsmoothmusclecellsshowssignificantrestingpermeabilitytoNa+andCl-,aswellastoK+(Sutter1990;Fujiwaraetal.1982;JohanssonandSomlyo1980).Electronprobeanalysisstudiesconductedonmesentericportalveinshaveindicatedthefollowingvaluesforintracellularionicconcentrations,inmmole/kgdryweight:Na+167;K+611;Cl-278;Mg 2 +36andCa 2 +1.9(Somlyoeta!.1979).Thesevaluesindicatethat,inintactCVSMCs,[K+Ji/[Na+]jmaybeappreciablysmallerthanthevaluewhichpertainedduringwhole-cellrecordingsreportedinthisstudy(47:1).Wahistrom(1971)estimatedthattheNernstpotentialforNa+invascularsmoothmuscleisprobablynomorepositivethan21mV,whileEKwas-86mV.TheuseoflowinternalNa+inwhole-cellrecordingscouldthereforehaveresultedinanunderestimateofthetruemembranepotentialinisolatedCVSMCs.Inaddition,aouabainsensitiveNa+/K+pumpisknowntoregulateionicconcentrationsinVSMCsandtoelectrogenicallycontributeseveralmillivoltstotherestingpotentialofthesecellsinvivo(Sutter1990;Fujiwaraetal.1982).Thispumpwaspresumablyinhibitedduringmaintenanceofthecellsat4°Canddepressedduringwhole-cellrecordings,sinceexperimentswereconductedat210CandthepipettesolutiondidnotcontainATP.TheinputresistanceoftheisolatedCVSMCsusedinthepresentstudywasfoundtoaverage3GO,avaluecomparabletoresultsobtainedinpatchclampstudiesonSMCsisolatedfromratcaudalvein,1.4GO(Toroetal.1986),rabbitjejunum,2GO(Boltonetal.1985),andratbasilarartery,4GO(Steeleeta!.1991).Themeasuredvalueincludestheleakageresistancebetweenthepipetteandthemembrane,whichwastypicallyabout10GO.Hencethe174measuredinputresistancesslightlyunderestimatedthetruevalueofthisparameter.TheseresistancesinthegigaohmrangegreatlyexceedvaluesdeterminedinisolatedVSMCsusingintracellularrecordingmethods,whicharetypicallyintherange0.45-0.59GO(Toroetal.1986).Thisdifferenceprobablyreflectsthepresenceofappreciableleakagecurrentsaroundthemicroelectrodeinsertion(SakmannandNeher1983).IntracellularrecordingsfromVSMCsinintactbloodvesselsyieldyetlowerestimatesofmembraneresistance,intheorderof10-100MO.Thisprobablyreflectsthesyncytialnatureofsmoothmusclepreparationsinintactvessels(Hirstetal.1986;Hermsmeyer1976;Somlyo1980;Suprenanteta!.1987).CVSMCsisolatedfromratcerebralarteriesshowedamarkedfallininputresistanceonmembranedepolarization,andonlysmallregenerativeresponsescouldbeevokedbydirectelectricalstimulation.ThesepropertieswerealsoseeninCVSMCsstudiedwithintracellularmicroelectrodesinintactcerebralarteriesoftherat(Hirstetal.1986)andguinea-pig(FugiwaraandKuriyama1983).Incontrast,CVSMCsinintactrabbitbasilararterydogeneratelargeactionpotentialsondepolarizationinnormalbathingmedia(Suprenanteta!.1987).InCVSMCsstudiedinintactratbasilararteries,largeactionpotentialswereevokedondirectelectricalstimulation,followingsuppressionofoutwardpotassiumcurrentbyTEA.Inwardcurrentduringtheseactionpotentialwascarriedbycalciumions(Hirsteta!.1986).Theenhancedlevelofintracellularfreecalciumseeninthepresentcellsondepolarizationbyhighpotassiumsolutionssuggeststhatvoltage-sensitivecalciumchannelswerealsopresentinthispreparation.Itseemslikely,however,thattheinwardcurrent175flowinginthesechannelswasinsufficienttoovercomethedampingeffectofrectifiercurrents,duringdirectelectricalstimulation.Duringapplicationofserotonintothepresentcells,biphasiccurrentswereobservedincell-attachedmembranepatches.Thesecurrentsstronglyresembledthecapacitancecurrentsrecordedincell-attachedpatchesduringactionpotentialactivityinothertypesofhighimpedancecell(Fenwicketal.1982).Serotonin(1-5OM)isknowntocauseasmalldepolarizationincerebralarteryVSMCs,andtoinitiateaburstofactionpotentialsinthesecells(Suprenantetal.1987).CalciumentryduringtheseactionpotentialsmightbeexpectedtoactivateKCachannelsinthepresentcells.Largeamplitudesinglechannelcurrentswereindeedobservedincell-attachedpatchesduringexposureofCVSMCstoserotonin.Furtherworkisneeded,however,toidentifythesecurrentsdefinitively.4.4.Ca2+-activatedK+channelsininside-outmembranepatchesInthisstudy,evidencewasobtainedfortwotypesofvoltageandCa-dependentK-channelsinCVSMCsdispersedfromratcerebralarteries.Onthebasisofalargedifferenceinmeansinglechannelconductance,theseweredesignatedasK(cDa)LandK(Ca)Ichannels.4.4.1.PropertiesoftheK(Ca)LchannelTheK(Ca)Lchannelsdescribedinthispapershowedaconductanceof207±10pAinsymmetricalhighpotassiumsolutions,similartothatofBK176channelsstudiedinVSMCsfrommesentericartery,andavarietyofothersmoothmusclepreparations(SeeTableII).MeasuredconductancevaluesofK(Ca)Lchannelsvariedfrom170pSto267pSwhenmeasuredunderconstantionicconditions.SimilarvariationhasbeenseenintheconductanceofBKchannelsinchromaffincells,190-330pS(Marty1981)andinratskeletalmuscle,150-240pS(Barretteta!.1982).Themeaningofthesevariationsintermsofchannelstructureremains,however,unclear.AmoredefinitiveassignmentofthepresentchanneltotheBKcategoryawaitsdeterminationofchannelsensitivitytocharybdotoxin,aselectiveblockerofBKchannelsinmanytissues(LangandRitchie1990;MacKinnonandMiller1988).4.4.1.1.IonicselectivityoftheK(Ca)LchannelK(Ca)LchannelswerehighlyselectiveforK+overNa+(PNa/PK<0.05),ashasbeenobservedforBKchannelsinothersmoothmusclecells(Benhameta!.1986;Inoueetal.1985;Greenetal.1991;Mayeretal.1990).Na+didnotsignificantlyinterferewiththepassageofK+throughopenK(Ca)Lchannels,evenwhenpresentataconcentrationof80mM.SimilarresultshavebeenobtainedfortheBKchannelfoundinguinea-pigmesentericartery(Benhametal.1986).However,currentflowintheBKchannelsofbovinechromaffincellsissubjecttoafastflickerblockby5mMinternalNa+,providing*K+isabsentfromtheexternalmedium(Yellen1984a).Undertheionicconditionsusedinthepresentstudy,aflickerblockoftheK(Ca)LchannelbyinternalNa+mayhavebeenrelievedbythepresenceof140mMK+intheexternalsolution,andcouldthereforehavegoneundetected(Yellen1984b).177TableIICharacteristicsofBKchannelsinvariouspreparationsVoltageCalciumTEATonicConductancedependencedependenceblock(c)Preparationconditionsa(pS)Selectivity(a)(b)Kd(0)iKd(0)0ReferenceBovine160K+265K>Rb>Na,Cs12-15mVlOnM27mM0.2mMMarty1981,1983,chromaffinYellen1984acellRatmyotubes140K+240TI>K>Rb>NH4>>11-16mV4OmV/10-fold60mM0.3mMBarretetaT.1982,Na,Li,CsV0(lsM)=40rnVBlatzandMagleby1984,1986RabbitlOOK+260Ti>K>Rb>NH4>>11-13mV4OmV/10-fold45mM0.29mMLatorreetal.1982,TtubulesbNa>Li>CsV0(1M)=40mVMoczydlowskietal.1983,1985,Vergara1983etal.1984Ratanterior140K+200-9mV6OmV/10-fold0.08mM52.2mMWongandAdler1986,pituitarycellsV0(1iM)=-30mVWongetal.1982(AtT2O/D16-16)Ratanterior150K+250-300K>>Na8mVV0(1M)=50mVKd(60)02mMLangandRitchie1988pituitary(GH3)Mouseparotid145K+250K>>Na12mV1nM-Maruyamaetal1983aciniRatpanceatic140K+244K>>Na15mVllOmV/10-fold-Cooketal.l984,B-cell,V0(1M)=0mVFindlayetaL1985Rabbit140K+180K>>Na17mV100nM-Gitteretal.1987collectingductcellsRatpancreatic150K+200-yes60-7OmV/100-fold-Grayetal.1990ductcellsV0(3jiM)=-4mVRabbit100K+230K>Rb>Na,Li,Cs15mV3OmV/10-fold-Cecchietal.1986intestinalSMCbV0(2jM)60mVToad130K+250K>>Na9mV8OmV/l0-fold-SingerandWalsh1987stomachSMCV0(1JLM)=lOmVFrogandtoad120K+200K>>Nayes10nM20mMforBergeretal.1984stomachSMCtotalblockexternallyRabbit126K+200K>>Nayes10nM12mM-Benhametal.1985intestinalSMCRabbit126K+200TI>K>Rb>>30mV30-60mV/TO-fold-BenhametaT.1986mesenteric‘Na,CsarterySMC178Rabbit126K+210K>>Nayes60mV/l0-fold30mMfortotalMayeretat.1990colonicSMCV0(5tM)=0mVblockexternallyCanine140K+265K>>NayeslOOmV/l0-foldNE0.18mMCarletat.1990gastricSMCRabbit142K273K>>Na-V0(l.4RM)=OmVNE0.1mMInoueetal.1985portalveinSMCRatcerebral140K+207K>>Na,Cs14mV45mV/l0-foldKd(-t-40)1=0.83mMPresentstudyarterySMCV0(23M)=+40mVa=concentrationisinmM.Symmetricalsolutionswereused.b=parametersdeterminedinplanarbitayers.Forallotherchannels,patch-clamptechniquewasused.(a)=expressedasc-foldchangeinP0(fractionofthetimeintheopenstate)perxmV.(b)=expressedasxmVchangeinV0(voltageatwhichP0=0.5)per10-foldincreasein[Ca2+];V0(xmM)1indicatesthevoltageatwhichP0=0.5atgiven[Ca2+]i.Otherwisethelowest[Ca2jisgivenatwhichchannelactivitycanbeseen.(c)Kd(0)1indicatesconcentrationofinternallyappliedTEAcausing50%blockofsinglechannelcurrentat0mV.Kd(0)0iscorrespondingvalueduringexternalapplicationofTEA.Ifdatawereobtainedatothermembranevoltages,thisisindicatedinbrackets.SMC=Smoothmusclecell.NE=NoeffectofinternallyappliedTEA.179TheK(Ca)LchannelalsoshowednegligiblepermeabilitytoCs+(PC/PK<0.05).UnderconditionswhichfavouredtheentryofCs+intothechannel,theamplitudeofpotassiumcurrentswassignificantlydepressed.QualitativelysimilarobservationshavebeenmadeinthecaseofBKchannelsinsmoothmusclecellsderivedfromguinea-pigmesentericartery.Inthispreparation,however,BKchannelsexhibitamuchhighersensitivitytotheblockingeffectofCs+thanwasevidentinthepresentstudy,suggestingthatthesetwochannelpopulations,despitetheirsimilarconductance,areprobablynotidentical(Benhametal.1986).TheactionofCs+onthepresentchannelcanbeexplainedbythetheoryofionicblockade(Woodhull1973;Hille1991).ItisassumedCs+bindstoasiteintheopenchannelandblocksit.Atequilibrium,thechannelwillfluctuatebetweenclosed,openandblockedstates.ThefrequencyofblockingeventsincreasesatpotentialswhichpromotetheentryofCs+intothechannel.Iftheaveragelifetimeoftheblockedstatewerelessthanthetimeresolutionoftherecordingsystem,thentheopenchannelcurrentwouldrepresentatime-averagedcurrent,reflectingtheproportionoftimethechannelspentintheblockedstateduringanopening(Benhametal.1986).TheflickerblockofK(Ca)LchannelsbyCs+showedvoltagedependency.TheeffectivevalenceforinternalCs+blockwasonly0.07,whileexternallyappliedCs+blockedwithaneffectivevalenceôof-0.66.ThistenfolddifferenceindicatesthatthetwositesofCs+bindingarenotlocatedatequaldepthsinthemembranefield.AsimilarresultwasobtainedbyYellen180(1984a)inastudyofCs+blockofBKchannelsinchromaffincells.However.thevaluesof8weredifferent(0.25forinternalblock,-1.0forexternalblock).Benhametal.(1986)reportedavalueof8=-1.4forexternalblockofBKchannelsinmesentericarteryVSMCs.TheseresultssuggestthatthelargeconductanceKCachannelsfoundinthesethreepreparationsexhibitsomefinedifferenceswithrespecttotheirionophorestructures.4.4.1.2.Ca 2 +dependenceofK(Ca)LchannelopeningTheopenprobabilityofK(Ca)Lchannelswassensitivetotheconcentrationofintracellularfreecalciumions.AtamembranepotentialofV=+40mV,theK(Ca)Lchannelwasopenforhalfofthetimeat[Ca 2+]i=23M.AscanbeseeninTableII,thecalciumsensitivityofBKchannelsvariesappreciallyamongdifferentcelltypes.BKchannelsincertainsecretorycells,includingmouseparotidacinarcellsarehighlycalcium-sensitive,beingopen50%ofthetimeat[Ca 2+]ivaluesbetween10-8-10-7M(V=0mV)(Maruyamaetal1983).ForBKchannelsfoundinskeletalmusclefibres,thecorrespondingvalueof[Ca 2+]iisaround5jLM(Barrettetal.1982;MethfesselandBoheim1982),whilefornonvascularandvascularsmoothmusclecells,estimatesintherange0.5-1Mhavebeenreported(Carletal.1990;Beiihametal.1986).ThelowapparentcalciumsensitivityoftheK(Ca)Lchannelhasimplicationsforthefunctionalsignificanceofthischannel,aswillbediscussedlaterinthisthesis.Inihepresentstudy,asecondpowerrelationshipwasfoundbetweentheprobabilityoftheK(Ca)Lchannelopeningandthevalueof[Ca 2+Ji,whenmeasuredovertherangeofintracellularfreecalciumlikelytopertainunder181physiologicalconditions.ThisresultisinagreementwithpreviousstudiesconductedonBKchannelsinothertissues,whichsuggestthat2-4calciumionsarerequiredtofullyactivateeachchannel(Barretteta!.1982;MethfesselandBoheim1982;MoczydlowskiandLatorre1983).OpentimedistributionsforK(Ca)Lchannelswerewellfittedbythesumoftwoexponentialterms,wheninvestigatedfor[Ca 2+]ivaluesovertherange10-8-10-3M.Increasing[Ca 2+]ihadnosignificanteffectonthevalueofthefasttimeconstantfittedtothesedistributions,whiletheslowtimeconstantwaselevatedbythisprocedure.SimilarresultswerereportedinthecaseofBKchannelsinratskeletalmuscle(Barretteta!.1982;MaglebyandPallotta1983a).However,inBKchannelsstudiedinsmoothmusclecellsisolatedfrommesentericartery,opentimedistributionswerewellfitbyasingleexponentialtermatlow[Ca 2+ft(<10-6M).Onraising[Ca 2+]j,asecond,fastfitcomponentappearedinthesedistributions,andthetimeconstantoftheoriginalcomponentincreased(Berthameta!.1986).ThepresentresultsarecompatiblewiththefollowingminimumstatediagramforthegatingoftheK(Ca)Lchannel(closed)K1[Ca](closed)K2[Ca](closed)S<>SCa<>SCa2K111C2a’aSCa*SCa2*(open)(open)Here,mostofthebriefchannelopeningsaretothemonoligandedstateSCa*,whilethemajorityoflongopeningsaretostateSCa2*,withtwocalciumionsboundperchannel.182Thismodelpredictsthattheratioofopeningsinthefastfitcomponentstothenumberintheslowcomponentshouldfallathigher[Ca 2+]i,aswasseenexperimentally.However,thisschemedoesnotaccountfortheobservedincreaseinthetimeconstantoftheslowfitcomponentonraising[Ca 2+]j.Thereareanumberofpossiblemechanismswhichcouldproducethiseffect.Firstly,stateSCa2*couldbindafurthercalciumionandmakeatransitiontoafurtheropenstate,SCa3*,increasingtheobservedopentimeofthelongopendistribution(MaglebyandPallotta1983b).However,thiswouldlikelyresultinopentimedistributionsrequiringthreeexponentialfitcomponents,whichwasnotobservedinthepresentstudy.Neitherwastheslopeoftheopenprobabilityversus[Ca 2+Iiplotsteepenoughtoindicatethreecalciumionswereneededtofullyactivateeachchannel.Alternatively,onemaypostulatetheoccurrenceofdirecttransitionsbetweentheopenstatesSCa*andSCa2*,accompaniedbythelossorgainofcalciumionsasrequired.However,thismodelpredictsthatthetimeconstantofthefastfitcomponentshoulddecreaseas[Ca 2+]iisraised.Atrendofthistypewasseeninthepresentdata,butthisdidnotreachstatisticalsignificance.Theincreaseintheslowtimeconstantwith[Ca 2 +]jcouldalsoresultfromtheoccurrenceofveryshortlivedclosedstatesinterposedbetweenstatesSCa2andSCa2*.Atleastoneofthetransitionsbetweentheseinterposedclosedstateswouldhavetobedependenton[Ca 2+Ji,andthelifetimeofthesestateswouldneedtobetoobrieftoallowdetectionwiththepresentrecordingsystem.Inthisscheme,calciumdrivenreopeningstoSCa2*fromtheseshortlivedclosedstateswouldleadtoanapparentincreaseinthemagnitudeoftheslowtimeconstant.Finally,aslowtimeconstantdependenton[Ca 2+]icanalso183begeneratedbymodelEwhichproposethatintracellularCa 2+bothactivatesandblocksKCachannels(MethfesselandBoheim1982).Thepresentdatadonotallowaclearchoicetobemadebetweenthismodelandthatoftheshort-livedclosedstates.BKchannelsinratskeletalmusclehavebeenobservedoccasionallytoenteralternativemodesofgatinginwhichopenprobabilitiesmaybeverydifferentfromthenormalcondition(McManusandMagleby1988).Whenlargenumbersoftransitionsareanalyzed(>30,000),itisobservedthatabout96%ofthesefallintothenormalgatingmode,theremainderbeingdistributedintheso-calledintermediateopen(3.2%),briefopen(0.5%)andbuzzmodes(0.1%).Thelaterthreemodesreallassociatedwithamuchbriefermeanopentimethanisseeninthenormalgatingmode.Inaddition,analysisoflargenumbersoftransitions(upto106)inthenormalmoderevealthatopentimedistributionsinthismodecontainatleastthreetofourexponentialcomponents,notthetwodetectedinearlierstudies(McManusandMagleby1988).Inthepresentstudy,opentimedistributionswerecalculatedforupto2000transitions,thisbeinglimitedbythelifetimeofexcisedpatches,whichrarelyexceeded20minutes.Forthesereasons,thepossibilitythatraremodesofgatingandadditionalopenstatesalsoexistforK(Ca)Lchannelscannotbeexcluded.4.4.1.3.VoltagedependenceofopeningofK(Ca)LchannelsTheprobabilityofopeningforK(Ca)Lchannelsincreasede-foldfora14mVmembranedepolarization,when[Ca 2+]iwasatlowlevels.Asimilarvoltagedependency(e-foldfor11-16mVchange)hasbeenreportedinBKchannelsstudiedinratskeletalmuscle(Barretteta!.1982;Methfesseland184Boheim1982;BlatzandMagleby1984),renalepithelialcells(Kolbetal.1986)andinchromaffincells(Marty1981),seeTableII.BKchannelsinanteriorpituitarycellsandintoadstomachsmoothmusclecellsshowasteepervoltagedependency(e-foldfor8-9mVchange(WongandAdler1986;LangandRitchie1988;SingerandWalsh1987).BKchannelsinmesentericarterysmoothmusclecellsexhibitedalowervoltagedependencythanseeninK(Ca)Lchannels(e-foldfor30mVchange)(Benhametal.1986).Voltagecouldalterchannelkineticsdirectlybychangingtherateconstantsgoverningstatetransitions,orcouldactindirectlybychangingtheeffectiveconcentrationofCa 2+atbindingsiteslocatedpartwaythroughthemembranefield(Woodhull1973;Barrettetal.1982).UsingBKchannelsfromratskeletalmuscleincorporatedintoplanarlipidbilayers,MoczydloskiandLatorre(1983)foundthatthevoltagedependencyofchannelopeningcouldbeentirelyaccountedforbythepotential-sensitivebindingofCa2+tositesinthemembranefield.Calculationsuggestedthatcalciiimionsbindtositeswhichsense75-95%ofthetotalmembranefield.Suchsitescouldbelocatedinawide,deepatriumleadingtotheionicselectivityfilter,orcouldresideatapartofthechannelnotdirectlyconnectedwithiontranslocation.Thereisevidencewhichfavoursthelatterpossibility,sinceCa 2+activationsitesaremoresensitivetomembranesurfacepotentialthanistheK+conductionprocessinthesechannels.Thus,theadditionofnegativelychargedmembranelipidsmarkedlyincreasestheapparentCa 2+sensitivityofBKchannelgatingwhilelittlechangeisseeninthepotassiumpermeabilityofthechannel(Moczydlowskietal.1985).185Inthepresentstudy.membranedepolarizationatconstant[Ca 2+]ihadnoeffectonthefasttimeconstantofopentimedistributions,whileincreasingthevalueoftheslowtimeconstant.Membranedepolarizationthereforemimickedtheeffectofincreasing[Ca 2+]iataconstantmembranevoltage.ThisresultisconsistentwiththeviewthatthevoltagedependencyofK(Ca)Lchannelgatingsimplyreflectsthepotential-dependentbindingofCa 2+tositesonthechannelcomplex.4.4.1.4.BlockadeofK(Ca)LchannelsbyinternallyappliedTEAInternallyappliedTEAreducedtheapparentsinglechannelcurrentinK(Ca)LchannelswithaKdof0.83mMat+40mVmembranepotential.AsshowninTableII,BKchannelsinchromaffincells(Yellen1984a),ratskeletalmuscle(BlatzandMagleby1984)andrabbitt-tubulecells(VergaraandLatorre1983)showamuchhigherKdforinternallyappliedTEA,valuesbeingintherange27-60mM.BKchannelsinVSMCsfromrabbitportalveinwereunaffectedbyinternallyappliedTEAatconcentrationsupto10mM(Inoueetal.1985).Inmarkedcontrast,BKchannelsinclonalanteriorpituitarycells(WongandAdler1986andBKchannelsfromratbrainsynaptosomes(FarleyandRudy1988)bothexhibitveryhighsensitivitytointernallyappliedTEA,withKdof0.08mMand0.8mMrespectively.ThehighsensitivityofK(Ca)LchannelstotheblockingeffectofinternalTEAisunlikelytobeanartifactcausedbyenzymetreatmentofCVSMCs,sincesimilardissociationprocedureswereemployedinotherstudieswhichdidnotexhibitthisphenomenon(Benhameta!.1985;Inoueeta!.1985).Thepossibilitythatthepresentdataresultedfrominadvertentformationofoutside-out186membranepatchesalsoseemsunlikely,sincetherecordingpipettescontainedonly0.01MCa 2+,insufficienttoyieldtheobservedchannelopenprobability,ifappliedtothecytoplasmicmembraneface.InthecaseoftheA-currentK+channelcodedbytheShakergeneinDrosophila,asingleaminoacidresiduecriticallyaffectstheaffinityofthechannelforinternallyappliedTEA(Yelleneta!.1991).Thisresidueliesatposition441inthesocalledSS1-SS2regionofthechannel,whichishighlyconservedamongvoltage-gatedK+channels,andmaycrossthemembranetwicetoformtheionconductingporeitself(MacKinnonandYellen1990).Wildtypefliesshowedchannelswithathreonineresidueatposition441,andexhibitedaKdforinternallyappliedTEAofabout0.7mM.ThemutantT441Spossessedaserineresidueatposition441andtheKdforinternalTEAincreasedbytenfoldasaresultofthissinglesubstitution(Yellenetal.1991).ThesensitivityofthechanneltoexternallyappliedTEAwasunalteredbythismutation.AlthoughthesestudieswerenotperformedongenecodingforaKCachannel,theydoillustratetheextremesensitivityofTEAaffinitytochangesintheprimarystructureofpotassiumchannels.ItmayberelevanttonotethatallthreeofthecelltypeswhichexhibitBKchannelswithhighinternalsensitivitytoTEAarederivedfrombraintissue,suggestingthataslightlydifferentformoftheBKchannelmaybeexpressedinthisorgan.ThepresentdataonTEAblockweretransformedintoaHillplotbyreplottingaslog(R/1-R)versuslog[TEA],whereRisthereductioninsinglechannelcurrentsseeninthepresenceofthedrug.Thisplotyieldedaslopeof0.82,suggestingthatTEAinteractswiththechannelinaone-to-onefashion,presumablybytransientlyenteringthechannelandblockingmovementofK+.187AnapproximatevalueforthedwelltimeofTEAatitsblockingsitecanbederivedfromtheKdforTEA,whereKd=K-i/Ki(Benhametal.1985).Themaximumvaluefo.theblockingrateconstant,K].islimitedbythedrugdiffusionrateandisthoughttobeabout108s-iM-i(Gutfreund1972).ThusK-1maybeestimatedat8.3x104s-iandhencethemeanblocktimewasabout12s,oneorderofmagnitudetoobrieftobedetectedbythepresentmeasuringsystem.ThisresultisconsistentwiththeobservationthatTEAblockwasnotaccompaniedbyamarkedincreaseincurrentnoiseinthechannel,asstudiedatabandwidthofDC-2kHz.TheblockofKCachannelsbyinternalTEAmaybeexpectedtobevoltage-dependent,sincepositivemembranepotentialsfavourtheentryoftheTEAcationintothechannels.However,thedegreeofvoltage-dependencyisgovernedbythefractio’iofthemembranefieldsensedbytheTEAmoleculeatitsbindingsite(Woodhull1973).ExperimentsperformedonBKchannelsinseveralcelltypeshaveindicatedthatblockbyinternalTEAisonlyweaklyvoltage-dependent,exhibitinganequivalentvalenceof0.06-0.1(Yellen1984a;Langtoneta!.1991;Yellenetal.1991).Thetheoreticalmaximumvalueoftheequivalentvalenceforamonovalentblockingioninteractingina1:1fashionwiththechannelis1.0(Benhametal.1986).Thiscorrespondstoane-foldincreaseinaffinityfora25mVdepolarization(Yellenetal.1984a).LetitbeassumedthepresentKCachannelspossessaTEAaffinityat0mVmembranepotentialtypicalofBKchannelsstudiedinskeletalmuscle,i.e.Kd(0mV)=30mM.Ifmeasuredat+40mM,theKdshouldstillbegreaterthan4mM,evenoftheequivalentvalenceofTEAisassumedtobe1.0.Itseemsunlikely,therefore,188thatthehighsensitivityofK(Ca)LchannelstointernalTEAisduesolelytoaverysteepvoltagedependencyofchannelblock.4.4.2.PropertiesofK(Ca)IchannelsInadditiontotheK(Ca)Lchannel,aKCachannelofintermediateconductance(1Kchannel)wasdetectedintheminorityofinside-outpatchesexcisedfromisolatedCVSMCs.ThisK(Ca)Ichannelshowedasinglechannelconductanceof92pSandwashighlyselectiveforK+overNa+andCs+.Thischannelwasactivatedbyintracellularfreecalciumandbymembranedepolarization.ApplicationofTEAtothecytoplasmicmembranefacereducedthecurrentinthischannelinadose-dependentfashionwithaKdof0.31mM.ThesedatawerereplottedasaHillplot,log(R/1-R)versuslog[TEA]whereRisthereductioninmeansinglechannelcurrentcausedbyagivenconcentrationofTEA.Theslopeofthisplotwas0.70,suggestingthatTEAblocksthischannelinaone-to-onefashion,asseenfortheK(Ca)Lchannel.NosignificantdifferencebetweentheKdvaluesfoundforblockoftheK(Ca)LandK(Ca)IchannelsbyinternalTEA.ThissuggeststhatthesechannelsshareacommonstructureintheregionoftheTEAbindingsite.Itseemsunlikely,however,thattheK(Ca)IchannelsimplyrepresentsaconductancesubstateoftheK(Ca)Lchannel,sinceamplitudedistributionsforcurrentflowinbothspecieswerewellfittedbysingleGaussianterms.KCachannelsofintermediateconductance(30-120pS)havepreviouslybeendetectedinawidevarietyofcelltypes(Latorreetal.1989;EdwardsandWeston1990;Koib1990).1Kchannelsofconductance90-100pShavebeenreportedinVSMCsisolatedfromrabbitportalvein(Inoueetal.1985)andfrom189humancysticartery(Akbaralietal1990).However,the1KchannelreportedinrabbitportalveinisunaffectedbyTEAappliedtothecytoplasmicmembraneface,inmarkedcontrasttothepresentresults(Inoueetal.1985).An1Kchannelhasbeenreportedinaorticsmoothmuscleoftherat,butthischannelshowsaconductanceofonly55pS,appreciablylowerthanthatoftheK(Ca)Ichannel(ShoemakerandWorrell1991).Twotypesof1Kchannelhavebeenobservedfollowingincorporationofratbrainsynaptosomalproteinsintolipidbilayers(FarleyandRudy1988).However,bothofthesechannelsarelittleaffectedbyapplication10mMTEAtothecytoplasmicmembraneface.4.4.3.PhysiologicalrolesforK(Ca)LandK(Ca)IchannelsThelowapparentcalciumsensitivityoftheK(Ca)LandK(Ca)Ichannelswouldseemtoprecludeasignificantcontributionofthesechanneltorestingpotassiumconductance,since[Ca 2+]jinunstimulatedvascularsmoothmusclehasbeenestimatedas40-130nM(Nabikaetal.1985;Takataeta!.1988;Kuriyamaeta!.1982;presentstudy).However,duringthefullycontractedstate,vascularsmoothmusclecellsmayshow[Ca 2+11valuesapproaching10M(Kuriyamaeta!.1982).Micromolarlevelsoffreeintracellularcalciumcanalsobeattainedduringtheactionofcertainvasoconstrictorhormones,includingangiotensinII(Capponietal.1986).[Ca 2+IilevelsinthelowmicromolarrangewouldsufficetoactivateafewpercentoftheavailableK(Ca)Lchannelsatdepolarizedmembranepotentials.ItshouldbenotedthattheopeningofevenasmallfractionoftheavailableK(Ca)Lchannelswouldmarkedlyreducetheinputresistanceofa3GOcell,aswellasprovidingastronghyperpolarizinginfluenceonmembranepotential.Inaddition,itispossiblethatlocallyveryhighconcentrationsof[Ca 2+]iareestablishedneartheinnerfaceofvoltage-sensitive190Ca 2+channels.IfKCachannelswerelocatedclosetothesesites,theircontributiontomembranerepolarizationduringtheactionpotentialcouldbegreatlyenhanced(Benhameta!.1986).ThecalciumsensitivityofK(Ca)LandK(Ca)Ichannelsmayalsobeenhancedinvivo,bythepresenceofintracellularmodulatorssuchasMg 2+(SquireandPetersen1987).Forthesereasons,itseemsprobabletheK(Ca)L,andpossiblyalsoK(Ca)IchannelsplayanimportantroleintherepolarizingphaseoftheactionpotentialinCVSMCs.TheactionpotentialinCVSMCsofintactratbasilararterieshasbeenstudiedfollowingpartialblockadeofpotassiumconductancesbyexternallyappliedTEA(Hirsteta!.1986).Undertheseconditions,thespikeconsistsofarapiddepolarizationofabout56mVamplitudefollowedbyafastrepolarization.Thisisfollowedbyanafter-depolarizationofabout13mV,lasting300-1000ms.Inmostcells,aslowafter-hyperpolarizationofafewmillivoltsisseenattheendoftheactionpotential.Boththeinitialspikedepolarizationandtheafter-depolarizationrequirethepresenceofCa 2 +inthebathingmedium,whiletheslowafter-hyperpolarizationprobablyreflectsanincreaseinK+conductance.ThelatterpotentialisalsoabolishedbyremovalofCa 2 +fromthebathingmedium,suggestingthatitisprobablymediatedbyaKCaconductance.Thisconductanceapparentlyservestoterminatetheafter-depolarization,whichprobablyreflectstheentryofcalciumthroughnon-inactivatingCa 2 +channels.Themajorityofcalciumentryintothefibretakesplaceduringthisafterdepolarizationphaseoftheactionpotential(Hirstetal.1986).TheseobservationsstronglysuggestthatKCachannelsplayanimportantroleintheregulationofactionpotentialdurationandofcalciumentryintocerebrovascularsmoothmusclecells.1914.5ConclusionsandsignificanceAtleast50%ofneurologicalpatientsexhibitsignsofcerebrovasulardisease,oftenincludingthemajorcategoriesofstroke,aneurysm,migraineortransientischemicattack(AdamsandVictor1985).Inthehypertensionoftenassociatedwithstroke,anumberofabnormalitieshavebeendetectedincerebrovascularsmoothmuscle,includingincreasedmembranepermeability,alteredmembranepotentialandelectrogenicpumpactivity,andanincreasedsensitivitytovasoconstrictors,includingserotonin(WebbandBohr1984;BohrandWebb1988;Micheletal.1990).Similarchangeshavebeenfoundtooccurinatheromaandduringthenormalagingprocess(Micheletal.1990;Michel1987).Atheroscleroticdamagetotheendotheliallayerofcerebralvesselsprobablyexacerbateshypersensitivityofsmoothmuscletoserotonin.ThisisbecausetheendotheliumnormallyproducesEDRF,whichmediatesendothelium-dependentvasodilation(Burnstock,1990)andprostacyclin,whichinhibitsplateletaggregation(Moncadaetal.1976).Endothelialcellsalsocontributetothedegreiationofserotoninbymonoamineoxidase(Peachetal.1985).TheenhancedresponsivenessofVSMCstoserotoninmaycontributetoarterialspasminvivo(Beamishetal.1984).Cerebralvasospasmdevelopsinmostpatientsfollowingruptureofanintracranialaneurysm.Thisphenomenonisofgreatclinicalimportance,sincetheresultingischemiacangreatlycompoundtheneurologicaldamagedonebytheinitialhemorrhage,andmayprovefatal(Steeleetal.1991).Inananimalmodelofcerebralvasospasm,evidencehasbeenfoundfordepolarizationandincreasedactionpotentialactivityinVSMCsofthebasilarartery.These192changesappearedtoresultfromdecreasedpotassiumconductanceinthecells.Theobservedvasospasmcouldbepartiallysupressedbyapplicationofthepotassiumchannelopenernicorandil(WatersandHarder1985;Harderetal.1987).However,themechanismsunderlyingvasodilationbypotassiumchannelopenersremainsasubjecttocontroversy.CromakalimhasbeenclaimedtoactivateKCachannelsinVSMCsisolatedfromrabbitaorta(Kreyeeta!.1987)andfromguinea-pigmesentericartery(Nakaoeta!.1988).However,Standeneta!.(1989)attributedthevasodilatoryactionofcromakalimonthelatterpreparationtoactivationofKATpchannels.TherelaxationofintactdogcerebralarteriesbycromakalimhasalsobeenattributedtoactivationofKATp,ratherthantoKCachannels(Masuzawaetal.1990).Inviewofthegreatclinicalimportanceofcerebrovascularsmoothmuscle,andoftheurgentneedforrationalandeffectivemeansofpreventingarterialspasm,itisimperativethatthepotassiumconductancesofCVSMCsbewellunderstood.ThepresentstudyhascontributedtothisgoalbydevelopingapreparationinwhichtostudythebiophysicalpropertiesofKCachannelsincerebrovascularsmoothmusclecells.Themodulationofthesechannelsbyclinicallyimportantdrugsandbyphysiologicalstimulicanalsobestudiedinthepresentinvitropreparation.193REFERENCESAdams,P.R.,Constanti,A.,Brown,D.A.andClark,R.B.(1982).IntracellularCa 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