Vampire Venom: Vasodilatory Mechanisms of Vampire Bat (Desmodus rotundus) Blood Feeding


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Vampire Venom: Vasodilatory Mechanisms of Vampire Bat (Desmodus rotundus) Blood Feeding

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Title:
Vampire Venom: Vasodilatory Mechanisms of Vampire Bat (Desmodus rotundus) Blood Feeding
Series Title:
Toxins
Creator:
Kakumanu, Rahini
Hodgson, Wayne C.
Ravina, Ravi
Alagon, Alejandro
Harris, Richard J.
Brust, Andreas
Alewood, Paul F.
Kemp-Harper, Barbara K.
Fry, Bryan G.
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MDPI AG
Publication Date:
Language:
English

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Subjects / Keywords:
Vasodilatation ( local )
Potassium Channels ( local )
Desmodus Rotundus ( local )
Vampire Bat ( local )
Venom ( local )
Calcitonin Gene-Related Peptide ( local )
Genre:
serial ( sobekcm )

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Abstract:
Animals that specialise in blood feeding have particular challenges in obtaining their meal, whereby they impair blood hemostasis by promoting anticoagulation and vasodilation in order to facilitate feeding. These convergent selection pressures have been studied in a number of lineages, ranging from fleas to leeches. However, the vampire bat (Desmondus rotundus) is unstudied in regards to potential vasodilatory mechanisms of their feeding secretions (which are a type of venom). This is despite the intense investigations of their anticoagulant properties which have demonstrated that D. rotundus venom contains strong anticoagulant and proteolytic activities which delay the formation of blood clots and interfere with the blood coagulation cascade. In this study, we identified and tested a compound from D. rotundus venom that is similar in size and amino acid sequence to human calcitonin gene-related peptide (CGRP) which has potent vasodilatory properties. We found that the vampire bat-derived form of CGRP (i.e., vCGRP) selectively caused endothelium-independent relaxation of pre-contracted rat small mesenteric arteries. The vasorelaxant efficacy and potency of vCGRP were similar to that of CGRP, in activating CGRP receptors and Kv channels to relax arteriole smooth muscle, which would facilitate blood meal feeding by promoting continual blood flow. Our results provide, for the first time, a detailed investigation into the identification and function of a vasodilatory peptide found in D. rotundus venom, which provides a basis in understanding the convergent pathways and selectivity of hematophagous venoms. These unique peptides also show excellent drug design and development potential, thus highlighting the social and economic value of venomous animals.
Original Version:
Toxins, Vol. 11, no. 1 (2019-01-08).

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University of South Florida Library
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University of South Florida
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Unknown
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K26-05229 ( USFLDC: LOCAL DOI )
k26.5229 ( USFLDC: LOCAL Handle )

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Article VampireVenom:VasodilatoryMechanismsof VampireBat Desmodusrotundus BloodFeeding RahiniKakumanu 1 ,WayneC.Hodgson 1 ,RavinaRavi 1 ,AlejandroAlagon 2 , RichardJ.Harris 3 ,AndreasBrust 4 ,PaulF.Alewood 4 ,BarbaraK.Kemp-Harper 1, and BryanG.Fry 3, * , 1 DepartmentofPharmacology,BiomedicineDiscoveryInstitute,FacultyofMedicine, Nursing&HealthSciences,MonashUniversity,Clayton,Victoria3800,Australia; rahini.r.ragavan@monash.eduR.K.;wayne.hodgson@monash.eduW.C.H.; ravina.ravi@monash.eduR.R.;barbara.kemp@monash.eduB.K.K.-H. 2 DepartamentodeMedicinaMolecularyBioprocesos,InstitutodeBiotecnolog a,UniversidadNacional Aut nomadeM xico,Av.Universidad2001,Cuernavaca,Morelos62210,Mexico;alagon@ibt.unam.mx 3VenomEvolutionLab,SchoolofBiologicalSciences,UniversityofQueensland,St.Lucia,Queensland4067,Australia;rharris2727@googlemail.com 4 InstituteforMolecularBiosciences,UniversityofQueensland,StLucia,QLD4072,Australia; Andreas.brust@iinet.net.auA.B.;p.alewood@imb.uq.edu.auP.F.A. * Correspondence:bgfry@uq.edu.au Jointseniorauthors. Received:20November2018;Accepted:2January2019;Published:8January2019 Abstract:Animalsthatspecialiseinbloodfeedinghaveparticularchallengesinobtainingtheirmeal,wherebytheyimpairbloodhemostasisbypromotinganticoagulationandvasodilationinordertofacilitatefeeding.Theseconvergentselectionpressureshavebeenstudiedinanumberoflineages,rangingfromeastoleeches.However,thevampirebatDesmondusrotundusisunstudiedinregardstopotentialvasodilatorymechanismsoftheirfeedingsecretionswhichareatypeofvenom.ThisisdespitetheintenseinvestigationsoftheiranticoagulantpropertieswhichhavedemonstratedthatD.rotundusvenomcontainsstronganticoagulantandproteolyticactivitieswhichdelaytheformationofbloodclotsandinterferewiththebloodcoagulationcascade.Inthisstudy,weidentiedandtestedacompoundfromD.rotundusvenomthatissimilarinsizeandaminoacidsequencetohumancalcitoningene-relatedpeptideCGRPwhichhaspotentvasodilatoryproperties.Wefoundthatthevampirebat-derivedformofCGRPi.e.,vCGRPselectivelycausedendothelium-independentrelaxationofpre-contractedratsmallmesentericarteries.ThevasorelaxantefcacyandpotencyofvCGRPweresimilartothatofCGRP,inactivatingCGRPreceptorsandKvchannelstorelaxarteriolesmoothmuscle,whichwouldfacilitatebloodmealfeedingbypromotingcontinualbloodow.Ourresultsprovide,forthersttime,adetailedinvestigationintotheidenticationandfunctionofavasodilatorypeptidefoundinD.rotundusvenom,whichprovidesabasisinunderstandingtheconvergentpathwaysandselectivityofhematophagousvenoms.Theseuniquepeptidesalsoshowexcellentdrugdesignanddevelopmentpotential,thushighlightingthesocialandeconomicvalueofvenomousanimals. Keywords:vasodilatation;potassiumchannels;Desmodusrotundus;vampirebat;venom;calcitoningene-relatedpeptide KeyContribution:Inthisstudy,weidentiedacompoundfromD.rotundusvenomvCGRPthatinducesvasodilationofresistancevesselssuchasmesentericarteriespartlyviavoltage-gatedpotassiumchannelsandendotheliumindependentmechanisms.ThehumanformofCGRPisapotentvasodilatorthatactspartiallyviaendotheliumdependentandindependentmechanisms.Toxins 2019 , 11 ,26;doi:10.3390/toxins11010026www.mdpi.com/journal/toxins

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Toxins 2019 , 11 ,26 2of10Hence,theselectivityofvCGRPcouldbeusedfortherapeuticinterventionsindiseasessuchashypertensionanddiabetes. 1.IntroductionCommonvampirebatsDesmondusrotundusarefoundinCentralandSouthAmerica,andfeedexclusivelyonmammalianblood[1,2].Theypreferentiallyfeedonlivestockanimalssuchascattle[3]andproducevenomcomponentsthatdisruptthebloodcoagulationcascade,enablingaconstantbloodowforfeeding[4–7].However,therearereportsofrareincidentsofhumaninteractionswhichhaveledvampirebatstobecomemoremedicallyrelevanttohumans[8,9].Outbreaksofrabiesinhumanpopulationsduetothevampirebatsbeingvectorsofthedisease[10],haveledtoanti-vampirebatcampaignsandcullingofbatpopulations[11,12].PreviousstudieshavedemonstratedthatD.rotundusvenomcontainstwoimportantanticoagulanttoxins:Draculin[6,7,13];andDSPADesmodusrotundassalivaryplasminogenactivator[14,15].DraculinisaglycoproteinthatirreversiblybindstofactorsIXaandX,andinhibitstheconversionofprothrombintothrombin[6,7,13].Thispreventsbrinogenbeingconvertedintobrinandthusinhibitscoagulationofbloodduringfeeding[5].DSPAcomponentsalsoaidinensuringcontinuousbloodowbybreakingupthebrinmeshofanybloodclotsthatareformed[16].WhiletherearerelativelyextensivestudiesonDraculinandDSPA,littleisknownabouttheothercomponentsofD.rotundus venom,withvasodilationapredictedbutuntestedactivity[15,16].Otherhematophagousanimalsinduceanticoagulantandvasodilatoryeffectsthroughthedeliveryofbioactivecompounds,thusensuringefcientbloodowforfeeding.Forexample,mosquitospossesstachykinin-likepeptidessialokinins[17,18],whilstbedbugspossessnitrosyl-hemoproteinsnitrophorins[19,20].Inaddition,sandiescontainapotentvasodilatormaxadilanthatactsviathePAC1receptor[21,22],andhorseydisintegrinsinhibitplateletaggregationlikethosefromsnakevenoms[23].Interestingly,tickprostaglandinsconstrictbloodvessels[24].Themaintenanceofbloodowduringfeedingisamajorratelimitingstepandchallengeforbloodfeederstoovercome.Therefore,thelongertheytaketofeed,thehigherthechancesthehostorpreywillnotice,makingthemmorevulnerable[25].Thus,duetothesimilaritiesinfeedingmechanismsbetweenhematophagousanimals,ithasbeenpostulatedthatvasodilatorsmayplayakeyroleinthevenomofD.rotundus,targetingskincapillaries,tocomplementcoagulationinhibition[15,16].However,suchactionshaveremainedspeculativeuntilthecurrentstudywhichdemonstratedselectiveandpotentactionforresistance-likearteries.PreviouslyweshowedthatthetranscriptomeandproteinaceousproductsoftheD.rotundushematophagoussecretionglandsarerichincalcitoningenerelatedpeptidevariants[26],whicharesimilarinsizeandaminoacidsequencestoCGRPbutwithmodicationsinkeyresiduesFigure1.CGRPisapotentvasodilatorthatactsviaactivationofCGRP1receptorsoneitherendothelialorsmoothmusclecells[27–30].Thesignicanceofthispeptidetypeinrelationtotheobtainingofblood-meals,andtheimpactofresidues,wastestedinordertoascertaintheroleinsecuringblood-mealsbyD.rotundus.Inthisstudy,wehavedemonstratedthatvCGRPalsocausesvasodilationofresistance-likearteriesviasimilarpathwaystoCGRPbutwithgreaterselectivity. Figure1.AlignmentsofvCGRPVampirebat,rCGRPRat,andhCGRPhumanwithcysteinesshadedinblackandvampirebatspecicmodiedresiduesingreen.

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Toxins 2019 , 11 ,26 3of10 2.Results 2.1.VasorelaxantResponsestoD.rotundusvCGRPandrCGRPInratsmallmesentericarteries,D.rotundusvCGRPwasapotentvasorelaxantpEC50=9.470.32)]TJ/F180 9.9626 Tf 1.007 0 0 1 84.854 687.832 Tm [(logM,Rmax=94.62.4%withapotencyandefcacysimilartothatofratcalcitoningene-relatedpeptiderCGRP;pEC50=9.160.17-logMRmax=93.82.6;Figure2A.InthepresenceoftheratCGRP1receptorantagonistCGRP8-37,thepotencyofD.rotundusvCGRPFigure2BandrCGRPFigure1Cwasdecreasedby6-foldp<0.05and5-foldp<0.05respectively,withnochangeinR max Figure2B. Figure2.D.rotundusvCGRPcausesvasodilationsimilartorCGRPviaCGRP1receptors.Cumulativeconcentration-responsecurvestoAD.rotundusvCGRPn=23andratCGRPn=23aloneandBD.rotundusvCGRPn=10andCratCGRPn=7intheabsenceandpresenceofCGRP8-37nM,n=7inratsmallmesentericarteries.Valuesareexpressedas%reversalofpre-contractionandgivenasmeanSEM,wheren=numberofanimals.*p<0.05pEC50versuscontrol,student'sunpaired t -test.2.2.ContributionofNO-SGCandAdenylateCyclasetoD.rotundusvCGRPandrCGRPMediatedRelaxationVasorelaxationtoD.rotundusvCGRPwasunchangedfollowingendothelialdenudationortreatmentwithL-NAMEMFigure3A.Incontrast,potencytorCGRPwasdecreased5-foldinthepresenceofL-NAMEMfrom9.160.17to8.620.09pEC50=0.01withnodifferenceinmaximumrelaxationFigure3D.ThepresenceofthesolubleguanylylcyclaseinhibitorODQMortheadenylylcyclaseinhibitorSQ22536MFigure3B,C,E,Fhadnosignicanteffecton D.rotundus vCGRPorrCGRPrelaxationcurves. 2.3.ContributionofPotassiumChannelstoD.rotundusvCGRPandrCGRPMediatedRelaxationRaisingtheextracellularconcentrationofK+to30mMmarkedlyattenuatedtherelaxantresponsetoD.rotundusvCGRPFigure4A.Blockingvoltage-dependentK+channelswith4-aminopyridinemMmarkedlyattenuatedD.rotundusvCGRP-inducedrelaxation,reducingthepotencybyapproximately30-foldp<0.05andreducingtheresponseat10nMto53.717.3%p<0.01.However,vasorelaxationtoD.rotundusvCGRPwasunchangedinthepresenceoftheATP-sensitiveK+channelinhibitor,glibenclamideM,ortheCa2+activatedK+channelinhibitor,TEAmM.Similarly,vasorelaxationtorCGRPwasattenuatedinthepresenceof30mMK+or4-aminopyridinemMyetunchangedinthepresenceofTEAmMorglibenclamide MFigure4B.

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Toxins 2019 , 11 ,26 4of10 Figure3.ThesolubleguanylylcyclaseoradenylylcyclasepathwaysdonotplayaroleinvasorelaxationinducedbyD.rotundusvCGRPorrCGRP.Cumulativeconcentration-responsecurvestoD.rotundusvCGRPA–CorratCGRPD–FinratsmallmesentericarteriesintheabsenceD.rotundusvCGRP,n=6;ratCGRP,n=5)]TJ/F180 8.9664 Tf 0.991 0 0 1 202.311 127.012 Tm [(9orpresenceofeitherL-NAMEM,n=9,ODQM,n=5,SQ22536M,n=6orfollowingendothelialdenudationn=7.Valuesareexpressedas%reversalofpre-contractionandgivenasmeanSEM,wheren=numberofanimals.*p<0.05pEC50versuscontrol,student'sunpaired t -test.

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Toxins 2019 , 11 ,26 5of10 Figure4.Voltage-gatedpotassiumchannelssignicantlyattenuatethevasodilatoryeffectsofD.rotundusvCGRPandrCGRP.Cumulativeconcentration-responsecurvestoAD.rotundusvCGRPn=17orBratCGRPn=12inratsmallmesentericarteriesfromratsintheabsenceorpresenceofeither30mMK+n=7,TEAmM,n=6)]TJ/F180 8.9664 Tf 1.02 0 0 1 287.582 533.286 Tm [(8,glibenclamideM,n=6or4-aminopyridinemM,n=6.Valuesareexpressedas%reversalofpre-contractionandgivenasmeanSEM,wheren=numberofanimals.*p<0.05,concentration-responsecurvesignicantlydifferentascomparedtocontrol-WayANOVA.#p<0.05,responseat30nMor10nMsignicantlydifferentascomparedtocontrol-WayANOVA,Bonferroni'sposthoc. 3.DiscussionD.rotundusvenomiswellknowntocontainanticoagulatingpropertiesinordertofacilitatebloodfeeding[26].Indeed,aglycoprotein,Draculin,whichinhibitsactivatedcoagulationfactorsIXIXaandXXahasbeenisolatedfromD.rotundusvenom[5].Inthecurrentstudy,weisolatedandcharacterisedapeptidevCGRPfromthevenom,whichissimilarinsizeandaminoacidsequencetoCGRPfoundinhumansandrats.CGRPisapotentvasodilatorthatactsviaactivationofCGRP1receptorsoneitherendothelialorsmoothmusclecells[27,31].Therefore,theaimofthisstudywastodeterminewhethervCGRPalsocausesvasodilationviasimilarpathways.WeidentiedvCGRPasadilatorofratsmallmesentericarterieswithapotencyandefcacysimilartorCGRP.Importantly,likerCGRP,thevasorelaxationwasattenuatedbytheCGRP1receptorantagonist,CGRP8-37,indicativeofanabilityofthepeptidetotargetthisreceptortomediateitsresponsethoughdirectactivationofCGRP1receptorscanbefurthersupportedbyradioactiveligandbindingassaysinthefuture.NextweexaminedtheroleofendothelialcellsinvasorelaxationviavCGRP.GiventhevasorelaxationtovCGRPwasunchangedfollowingendothelialdenudationorinhibitionofnitricoxidesynthasebyL-NAME,itislikelythatvCGRPtargetsCGRP1onvascularsmoothmusclecellsVSMCtocauseendothelium-independentrelaxation.Incontrast,relaxationtorCGRPappearedtobe,inpart,dependentonendothelial-derivednitricoxideNOasthepotencywasattenuatedfollowingNOSinhibition.ThesendingshighlightapotentialpointofdifferencewithregardtoCGRPderivedfromdistinctspecies.Thuswhilstanendothelium-dependentcomponentofvasorelaxationtorCGRPhasbeenobservedinmesenteric[32]andretinal[33]arteries,wehavedemonstratedthatvCGRP,likehumanCGRP[34],mediatesrelaxationviaendothelium-independentmechanisms.ThissimilarityinmechanismofactionbetweenhumanCGRPandvCGRPsupportsthenotionofvCGRPbecomingapotentialcandidatefortherapeuticdrugdiscovery.PreviousstudieshavealsodemonstratedthatactivationofCGRPreceptorscanleadtotheactivationoftheguanylylcyclasepathwayendothelium-dependentoradenylylcyclasepathwayendothelium-independent[33–38].However,thepresenceofODQguanylylcyclaseinhibitororSQ22536adenylylcyclaseinhibitor,hadnosignicanteffectonrCGRPorD.rotundusvCGRPrelaxationcurves.DifferencesbetweenCGRPendothelium-independentand-dependentmechanismsarerelatedtotheregion,sizeofthevesseltestedandspeciesofCGRP.Forinstance,human

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Toxins 2019 , 11 ,26 6of10orratCGRPtestedinpigcoronaryleadstoincreasedcAMPandcausesvasorelaxationviaendothelium-independentpathways[34].However,humanCGRPtestedinhumanvesselsareendothelium-dependent[28].Therefore,wenextsoughttocharacterisethemechanismsviawhichvCGRPmediatesendothelium-independentrelaxation.OurndingthatraisingtheextracellularK+concentrationto30mMmarkedlyattenuatedtherelaxationtovCGRPsuggeststhatthepeptidemodulatesrelaxationofratsmallmesentericarteriesinpartviaactivationofK+channels.Indeed,weidentiedanabilityofvCGRPtoactivatevoltage-dependentK+channelsasrelaxationresponsesweredecreasedby4-AP.ThiswasinagreementtondingswithrespecttorCGRP.NeitherKATPnorKCachannelsappearedtobeinvolvedinrelaxationtovCGRPorrCGRPasglibenclamideandTEAwerewithouteffect.Indeed,thereisevidencethatactivationofCGRPreceptorscouldleadtodirectopeningofK+channels,inparticularKvchannels[33].ThereareconictingreportsontheinvolvementofKATPandKCachannelsinvasorelaxation,whichcouldberelatedtothetypeofvesselstudied.Forinstance,studiesusingbovineretinalarteriesandrabbitmesentericarteriesreportthatactivationofKATPchannels,butnotKCachannels,leadstovasorelaxation[37,39,40].However,studiesinsmoothmusclecellsfromratmesentericarterieshaveshownCGRPdirectlyactivatesBKCachannels[41].ThesedatafurtherhighlightthatCGRPcausesvasorelaxationthroughavarietyofmechanismswhichisdependentuponthespeciesandvesselinvolved.ConsideringthemedicalrelevancetohumansofD.rotundusandothervampirebatspeciesasdiseasevectorsforrabies[1],itissurprisingthatmoreindepthstudieshavenotbeenconductedontheintricatemechanismsemployedintheirfeedingbehaviour,despitestudiesonotherbloodfeedinganimalssuchaseasandleeches[13,26,42].Suchsecretionstwithinthedenitionofvenomas`Asecretionproducedinspecializedcellsinoneanimal,deliveredtoatargetanimalthroughtheinictionofawoundandthatdisruptsendophysiologicalorbiochemicalprocessesinthereceivinganimaltofacilitatefeeding,defenseorcompetitionby/oftheproducinganimal'[42].Aspeptidesusedbyvenoms/hematophagous-secretionsaremodiedversionsofthoseroutinelyexpressedinothertissues[43]futureworkincludingtheothertwospeciesofvampirebatandnon-hematophagousbatswouldbeenlighteninginregardstothetimingoftherecruitmentforuseinblood-feedingandthemoleculardiversicationevents.Thisstudyhasopenedthewayforfurtherresearchtoinvestigatethepathwaysandintricatemechanismsofhematophagousvenoms,inparticularvampirebats.Therefore,wehavemadecleartheabilityofvCGRPtoselectivelymediateendothelium-independentvasorelaxationinpartviaactivationofKvchannels.ThisselectivityofvCGRPtotargetonlyvascularsmoothcellssimilartothatofhumanCGRPhighlightstheinterestingpossibilitythatvCGRPmayconferbenetinthecontextofcardiovasculardiseasessuchashypertension,heartfailureandkidneydiseases[44].FurtherfunctionalstudiesarerequiredforvCGRPtobecomeatherapeuticinterventionwithpotentialpharmacologicalapplications.Thisresearchalsopavesthewayforfurtherevolutionarystudiesintohematophagousvenoms. 4.MaterialsandMethodsSynthesisofvCGRPwasaccomplishedusingprotocolspreviouslydescribedbyusforotherpeptides[45]. 4.1.IsolationofRatSmallMesentericArteriesMaleSprague-DawleyratsgwereeuthanizedviaCO2inhalation%CO2,5%O2followedbyexsanguination.Smallmesentericarteriessecond-orderbranchofthesuperiormesentericarterywereisolated,cutinto2mmlengths,andmountedon40mwiresinsmallvesselmyographs[46].Vesselsweremaintainedinphysiologicalsaltsolution[composedofinmM119NaCl,4.7KCl,1.17MgSO4,25NaHCO3,1.8KH2PO4,2.5CaCl2,11glucose,and0.026EDTA]at37Candwerebubbledwithcarbogen%O2,5%CO2.Inasubsetofarteries,theendotheliumwasgentlydenudedviainsertionofa40mwireinsidethelumenandrubbingthevesselwalls.Themesenteric

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Toxins 2019 , 11 ,26 7of10arterieswereallowedtoequilibratefor30minunderzeroforceandthena5mNrestingtensionwasapplied.ChangesinisometrictensionwererecordedusingMyographInterfaceModel610Mversion2.2DMT,Aarhus,DenmarkandPowerLab/835ADInstrumentsInc,BellaVista,NSW,Australia.DatawasrecordedwiththedataacquisitionprogramChartV5,ADInstruments.Followinga30minequilibrationperiodat5mN,themesentericarterieswerecontractedmaximallyFmaxusingaK+depolarizingsolution[K+ )]TJ/F180 9.9626 Tf 0.98 0 0 1 194.538 676.495 Tm [(containingphysiologicalsaltsolutionKPSS;composedofinmM123KCl,1.17MgSO4,1.18KH2PO4,2.5CaCl2,25NaHCO3,and11glucose].TheintegrityoftheendotheliumwasconrmedbyrelaxationtoacetylcholineACh,10M[46]intissuespre-contractedwiththethromboxaneA2mimetic,U46619M[46].Arterieswerewashedwithphysiologicalsaltsolutionandthetensionallowedtoreturntobaseline. 4.2.VasorelaxationExperimentsCumulativeconcentration-responsecurvestoD.rotundusvCGRP)]TJ/F180 7.5716 Tf 6.227 0 Td [(1210)]TJ/F180 7.5716 Tf 6.227 0 Td [(8MorrCGRP10)]TJ/F180 7.5716 Tf 6.228 0 Td [(12)]TJ/F180 7.5716 Tf 6.228 0 Td [(8M[32,34,37]wereconstructedinvesselspre-contractedsubmaximally~50%FmaxwithtitratedconcentrationofU46619.01M.2M.ResponsestoD.rotundusvCGRPandrCGRPwereobtainedinendothelium-intactmesentericarteriesintheabsenceorpresenceofeitherODQM[47],SQ22536M[48],L-NAME.1M[46],CGRP8-37.1M[49,50],30mMK+[33],TEAM,4-aminopyridineM[46]orglibenclamideM[32,51].Alltreatmentswereaddedfor30minpriortoprecontractionwithU46619.Inasubsetofendothelium-denudedarteries,vasorelaxationtoD.rotundusvCGRPwasalsoexamined.SodiumnitroprussideSNP;10M[47]wasaddedattheendofeachconcentration-responsecurvetoensuremaximumrelaxation.Onlyoneconcentration-responsecurvetoD.rotundusvCGRPorrCGRPwasobtainedineachvesselsegment[47,52]. 4.3.DataAnalysisandStatisticalProceduresRelaxationresponseswereexpressedasapercentagereversaloftheU46619pre-contraction.IndividualrelaxationcurveswerettedtoasigmoidallogisticequationandpEC50valuesconcentrationofagonistresultingina50%relaxationcalculatedandexpressedas–Logmol.L)]TJ/F180 7.5716 Tf 6.227 0 Td [(1.Statisticalcomparisonsbetweentheexperimentalgroups'meanpEC50andmaximumrelaxationRmaxvaluesweremadeusingaStudent'sunpairedt-testorone-wayANOVAwithBonferroni'sposthoccomparison.WherepEC50valuescouldnotbeobtained,concentration-responsecurveswerecomparedbymeansofatwo-wayANOVA.n=numberofarterysegmentsfromseparateanimals.DatarepresentthemeanSEMerrorbarsongraph.Statisticalsignicancewasdenedas*p<0.05.AlldataanalysiswasperformedusingGraphPadPrismversion5.02GraphPadSoftware,SanDiego,CA,USA,2009[46]. 4.4.ReagentsReagentsandtheirsourceswereU46619CaymanChemicalcompany,AnnArbor,Michigan,USA,SQ22356Tocrisbioscience,Bristol,UK,ODQ,Glibenclamide,TEA,4-aminopyridine,L-NAME,SNP,ACh,CGRP8-37Sigma-Aldrich,StLouis,MO,USA,andCGRPratPeptideInstitute,Osaka,Japan.StocksolutionsofODQmmol/LandU46619mMweredissolvedinabsoluteethanol.Allsubsequentdilutionsofstocksolutionswereindistilledwater.Allotherdrugsweremadeupindistilledwaterandalldilutionswerepreparedfreshdaily. AuthorContributions:Conceptualization,B.G.F.andB.K.K.-H.;Methodology,B.G.F.,B.K.K.-H.andR.K.;validation,B.K.K.-H.,R.K.andR.R.;formalanalysis,B.K.K.-H.andR.K.;investigation,B.G.F.,B.K.K.-H.andR.K.;resources,B.G.F.,A.A.,A.B.,P.F.A.,andB.H.;datacuration,B.K.K.-H.andR.K.;writing—originaldraftpreparationR.K.;writing—reviewandediting,W.C.H.,R.J.H.,B.K.K.-H.,B.G.F.,andR.K.;visualization,B.G.F.,B.K.K.-H.andR.K.;supervision,W.C.H.,andB.K.K.-H.;projectadministration,B.G.F. Funding: Thisresearchreceivednoexternalfunding. ConictsofInterest: Theauthorsdeclarenoconictofinterest.

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