Role of Pituitary Adenylate-Cyclase Activating Polypeptide and Tac1 gene derived tachykinins in sensory, motor and vascular functions under normal and neuropathic conditions

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Peptides

jo u r n al hom e p ag e :w w w . e l s e v i e r . c o m / l o c a t e / p e p t i d e s

Role of Pituitary Adenylate-Cyclase Activating Polypeptide and Tac1 gene derived tachykinins in sensory, motor and vascular functions under normal and

neuropathic conditions

Bálint Botz

, András Imreh

, Katalin Sándor

, Krisztián Elekes

, János Szolcsányi

, Dóra Regl ˝odi

, John P. Quinn

, James Stewart

, Andreas Zimmer

, Hitoshi Hashimoto

, Zsuzsanna Helyes

aDepartmentofPharmacologyandPharmacotherapy,UniversityofPécs,H-7624Pécs,Szigetiu.12,Hungary

bDepartmentofAnatomy,PTE-MPTE-MTALenduletPACAPResearchTeam,FacultyofMedicine,UniversityofPécs,H-7624Pécs,Szigetiu.12,Hungary

cJánosSzentágothaiResearchCenter,UniversityofPécs,H-7624Pécs,Ifjúságu.20,Hungary

dDepartmentofMolecularandClinicalPharmacology,InstituteofTranslationalMedicine,LiverpoolUniversity,Liverpool,UK

eSchoolofInfectionandHostDefense,UniversityofLiverpool,UK

fLaboratoryofMolecularNeurobiology,DepartmentofPsychiatry,UniversityofBonn,Bonn,Germany

gGraduateSchoolofPharmaceuticalSciences,OsakaUniversity,Japan

a r t i c l e i n f o

Articlehistory:

Received21January2013

Receivedinrevisedform4March2013 Accepted4March2013

Availableonline13March2013

Keywords:

Traumaticmononeuropathy Sciaticnerveligation Mechanicalhyperalgesia Neurogenicvasodilation SubstanceP

TachykininNK1receptor

a b s t r a c t

PituitaryAdenylate-CyclaseActivatingPolypeptide(PACAP)andTac1gene-encodedtachykinins(sub- stance P: SP, neurokinin A: NKA) are expressed in capsaicin-sensitive nerves, but their role in nociception,inflammationandvasoregulationisunclear.Therefore,weinvestigatedthefunctionofthese neuropeptidesandtheNK1tachykininreceptor(fromTacr1gene)inthepartialsciaticnerveligation- inducedtraumaticmononeuropathymodelusinggenedeficient(PACAP^−/−,Tac1^−/−,andTacr1^−/−)mice.

Mechanonociceptivethresholdofthepawwasmeasuredwithdynamicplantaraesthesiometry,motor coordinationwithRota-RodandcutaneousmicrocirculationwithlaserDopplerimaging.Neurogenic vasodilationwasevokedbymustardoilstimulatingsensorynerves.Inwildtypemice30–40%mechani- calhyperalgesiadevelopedoneweekafternerveligation,whichwasnotalteredinTac1^−/−andTacr1^−/− mice,butwasabsentinPACAP^−/−animals.MotorcoordinationofthePACAP^−/−andTac1^−/−groupswas significantlyworsebothbeforeandafternerveligationcomparedtotheirwildtypes,butitdidnotchange inTacr1^−/−mice.BasalpostoperativemicrocirculationontheplantarskinofPACAP^−/−micedidnotdif- ferfromthewildtypes,butwassignificantlylowerinTac1^−/−andTacr1^−/−ones.Incontrast,mustard oil-inducedneurogenicvasodilationwassignificantlysmallerinPACAP^−/−mice,butnotinTacr1^−/−and Tac1^−/−animals.BothPACAPandSP/NKA,butnotNK1receptorsparticipateinnormalmotorcoordina- tion.Tachykininsmaintainbasalcutaneousmicrocirculation.PACAPisacrucialmediatorofneuropathic mechanicalhyperalgesiaandneurogenicvasodilation.Thereforeidentifyingitstargetanddeveloping selective,potentantagonists,mightopenpromisingnewperspectivesforthetreatmentofneuropathic painandvascularcomplications.

©2013ElsevierInc.Allrightsreserved.

1. Introduction

Painfulneuropathyisaveryimportantclinicalproblem,par- ticularlywiththegrowthofanelderlypopulation,thenumberof

Abbreviations:PACAP,PituitaryAdenylateCyclase-ActivatingPolypeptide;DRG, Dorsal,RootGanglion;SP,substanceP;CGRP,calcitoningene-relatedpeptide;Tac1- 4,tachykinin-encodinggenes;VIP,vasoactiveintestinalpeptide;NKA,neurokinin A;TRPA1,TransientReceptorPotentialAnkyrin1receptor.

∗ Correspondingauthorat:DepartmentofPharmacologyandPharmacotherapy, UniversityofPécs,Pécs7624,Szigetiu.12,Hungary.Tel.:+3672536001/5386/5591;

fax:+3672536218.

E-mailaddress:zsuzsanna.helyes@aok.pte.hu(Z.Helyes).

peopleaffectedbytheseconditionsisincreasing.Itstherapyisan emergingissue,becauseevenwiththelatestdevelopmentsonthe fieldofanalgesic-research,itisstillnotsatisfactory.

PituitaryAdenylateCyclase-ActivatingPolypeptide(PACAP)and tachykinins, such as substance P released from the capsaicin- sensitivepeptidergicsensorynerveswereshowntobeinvolved inacuteandchronicpainconditions[2,6,8,42,45,48,52],buttheir roleinchronicneuropathicpainsyndromeshasnotyetbeenclearly elucidated.

PACAP belongs to the vasoactive intestinal polypeptide (VIP)/secretin/glucagon family [31]. It is present in 27 and 38 aminoacid-containing forms, thelatter is more predominantin mammals.Sofarthreereceptorsofthispeptidehavebeencloned, 0196-9781/$–seefrontmatter©2013ElsevierInc.Allrightsreserved.

http://dx.doi.org/10.1016/j.peptides.2013.03.003

all of which belong to thefamily of G-protein coupledrecep- tors mediating their effect through the activation of adenylate cyclase and phospholipase C. PACAP is the main endogenous agonist on the PAC1 receptor, whereas PACAP and VIP have similaraffinities for theVPAC1 and VPAC2 receptors [22]. The PAC1receptoris expressedmainlyonneuraland smoothmus- clecells,whiletheVPAC1/VPAC2arelocalizedprincipallyonthe dorsalrootganglia(DRG),sensorynerveendings,andinflamma- torycells [6,9,50,55,64]. The fact, that PACAP shows increased expressionin thesuperficiallayerofthespinaldorsalhorn and incapsaicin-sensitiveprimarysensoryneurons,suggestedthatit hasarole innociception[33,34,39,61–63].However,theresults obtainedfromtheearlyinvivostudiesprovedtobecontradic- tory[41].IntrathecallyadministeredPACAP-38showedanalgesic effectintheearlyphase,subsequentlyfollowedbyalong-lasting algesia in the formalin test [48]. In several other models cen- trallyappliedPACAPshowedmarked pro-nociceptiveeffects: it decreasedthermalstimulation-evokedpawwithdrawallatencies andpotentiatednociceptivetransmissiontothedorsalhorn[37].

Itfacilitatedspinalnociceptiveflexorreflexes[41,60]andinduced hyperalgesia[35].WeprovidedevidencethatPACAP-38exertsan interestingdivergenteffectonpain-signaling.Peripherallyadmin- istrated PACAP induces anti-nociceptive, anti-hyperalgesic and anti-allodyniceffectsinbothacutesomaticandvisceralpainmod- els.However,it doesnot affectmechanical hyperalgesiain the traumaticmononeuropathy model, but induces sensitization of kneejointprimaryafferents[42].Furthermore,PACAPinhibitsthe releaseofseveralpro-nociceptiveandpro-inflammatorysensory neuropeptides (CGRP, SP and other tachykinins) from periph- eral terminals of capsaicin-sensitive nerves, and it diminishes acute neurogenic and non-neurogenic inflammatory processes [10,17,36].Wealsodescribed,thatPACAPhasadualroleinnocicep- tion.Thermalallodyniamediatedonlybyperipheralmechanisms is increased in PACAP gene-deficientanimals, but somatic and visceralnocifensivebehaviors,andneuropathicmechanicalhyper- algesiaarediminished.Thisdemonstrates thatPACAP canhave bothinhibitoryandexcitatoryrolesdependingonthesiteofaction andthepredominantmechanisminthepainmodel[44].

Tachykinins represent one of the largest families of neu- ropeptides released from the capsaicin-sensitive sensory nerve terminals.Allof thesepeptides arerelatively small,theiraver- agelengthis10–11aminoacids.Sofarthreetachykininencoding geneshave beencloned(Tac1,Tac3, andTac4),butthenumber ofthetachykininsismuchlargerduetopost-translationalmod- ifications[26,38].Thefirstidentified preprotachykinin-A(PPTA;

Tac1)geneencodessubstanceP(SP)andNeurokinin-A(NKA).This geneisexpressedpredominantlyinsensoryneurons,inflamma- toryandimmunecells[40].SPandNKAexertavarietyofeffects underbothnormalandpathophysiologicalconditions.Theyinduce neurogenicinflammation(vasodilation,plasmaproteinextravasa- tion,andstimulationofinflammatorycells),elicitsmoothmuscle contraction,regulatevasculartone,mucussecretionandimmune functions[26]. They inducethe release of histamine and sero- tonin throughmast cellactivation, which in turnincreasesthe neuropeptide-releasefromthesensory nerveterminalsthrough positivefeedbackmechanisms[18,51].

Threetachykininreceptorshavebeencloned,theNeurokinin1, 2,and3(NK₁;NK₂;NK₃,respectively),whichareGs/Gq-protein coupled[26].Whilealltachykininscanactivateallreceptors,SP andNKAarethepreferredligandsfortheNK1andNK2receptors [15,23,27].TheinvolvementoftheTac1gene-encodedtachykinins inseveralmodelsofchronicinflammationandpainhasbeensug- gested[13].SPandNKAaresynthesizedinthedorsalrootganglia, andtheyareinvolved intheregulationofnociceptiveinforma- tion at the first sensory synapse in the spinal cord [7,56]. SP, NKA,andtheNK1receptorhavebeensuggestedtobeinvolvedin

inflammatoryandneuropathicpainsyndromes,suchastraumatic mononeuropathies[2,11,12],streptozotocin-induceddiabetes[5], andpaclitaxel-inducedperipheralneuropathy[52].However,in thesestudiesonlythealterationsofNK₁receptorexpressionwas investigatedortheeffectofnon-peptideNK1receptorantagonists wereanalyzed,asTac1andNK₁genedeficientanimalswereuntil recentlynotavailable.

Since PACAP and tachykinins are co-localized in capsaicin- sensitiveafferents,ouraimwastoelucidatetheirrolesinamodel oftraumaticmononeuropathywiththehelpofgene-deletedmice.

Furthermore,theinvolvementoftheNK1receptor,themaintar- getofSP, wasalsoinvestigated.In ordertohaveanintegrative approach, differentfunctional techniques toassess neuropathic mechanical hyperalgesia, motor coordination, and neurogenic vasodilationwereused.

2. Materialsandmethods 2.1. Animals

Experiments were performed with PACAP, Tac1 and Tacr1 gene-deficient(PACAP^−/−,Tac1^−/−and Tacr1^−/−)miceand their respective,appropriatewildtypecounterparts.Thegenerationof PACAP^−/− miceontheCD1backgroundattheOsakaUniversity hasbeenpreviously describedindetails [14].Theheterozygous mice(PACAP^+/−)werebackcrossed for10 generations withthe CD1strain.Aftergenotypingtheoffspringsofthefirstgeneration ofthePACAP^+/− breedingpairs,micewerebredonaswildtype (PACAP^+/+)andhomozygousgene-deleted(PACAP^−/−)linesinthe LaboratoryAnimalHouseoftheDepartmentofPharmacologyand PharmacotherapyoftheUniversityofPécs.Offspringswithinthe first threegenerations were usedfor the experimentsto mini- mizegeneticvariations.TheSPandNKAdeficient(Tac1^−/−)and NK1receptorgene-deleted(Tacr1^−/−)miceweregeneratedonthe C57Bl/6backgroundat theUniversity ofLiverpoolandtheUni- versity of Bonn,respectively, as previously described [16]. The homozygousgene-deletedanimalswerebackcrossedfor8-10gen- erationstotheC57Bl/6strain,therefore,thesemicewereusedas wildtype(WT)controls.Theiroriginalbreedingpairswerepur- chasedfromCharles-RiverLtd.(Hungary).Micewerebredandkept intheLaboratoryAnimalHouseoftheDepartmentofPharmacology andPharmacotherapyoftheUniversityofPécsat24–25^◦Candpro- videdwithstandardmousechowandwateradlibitumunder12h lightand12hdarkcycles.Allexperimentalprocedureswerecarried outaccordingtothe1998/XXVIIIActoftheHungarianParliament onAnimalProtectionandConsiderationDecreeofScientificPro- ceduresofAnimalExperiments(243/1988)andcompliedwiththe recommendationsoftheInternationalAssociationfortheStudyof Pain[65]andtheHelsinkiDeclaration.Thestudieswereapproved bytheEthicsCommitteeonAnimalResearchoftheUniversityof PécsaccordingtotheEthicalCodex ofAnimalExperiments and licensewasgiven(LicenseNo.:BA02/2000-9-2011).

2.2. Thetraumaticmononeuropathymodel

Anesthesiawasperformedwitha combination of100mg/kg ketaminei.p.(RichterGedeonPlc.,Budapest,Hungary)and5mg/kg xylazinei.m.(LavetLtd.,Budapest,Hungary).Thentherightcom- monsciaticnervewasunilaterallyexposedhighinthethighandits 1/3wastightlyligatedusinganatraumaticsiliconizedsilksuture (Ethicone10-0)[28,47].Thereafterthewoundwasclosed,andthe animalswerenotexaminedforthenext7daysinordernottodis- turbthehealingprocess.Inaccordancewithourpreviousstudies [42,44],thisisareliablemodelofneuropathicmechanicalhyper- algesia,whichisnotinfluencedbyanimpairedweightdistribution.

2.3. Determinationofthemechanonociceptivethresholdby dynamicplantaraesthesiometry

The mechanonociceptive threshold of plantar surface of the hindpawwasmeasuredbydynamicplantaraesthesiometry(Ugo Basile37400,Comerio,Italy).Inthisdevicetheanimalscanfreely moveintheirseparatecompartmentsonametalmesh.Priorto each measurement there was 10–15min of acclimation period untilthecessationoftheanimalsexploratorybehavior.Thenthe stimulatorunitofthedevicewasplacedundertheanimal’spaw, usingan adjustable angled-mirrortoaidthe positioningofthe metalfilamentrightbelowtheplantarsurface.Thenanelectro- dynamicactuatorofproprietary designliftedthestraightmetal filament,whichtouchedtheplantar surfaceandbeganexerting anincreasing upwardforceat a presetrate (5s)of application untileither thenocifensivebehavior (removalof thepaw)was attained,oruntilthemaximal10gwasreached.Afteracondition- ingand3controlpreoperativemeasurements,thepawwithdrawal thresholdsweredeterminedondays7,10,14,19afterthenerve ligation.

2.4. AssessmentofthemotorcoordinationonaRota-Rod

MotorfunctionswerestudiedusinganacceleratingRota-Rod device(UgoBasile7750,Comerio,Italy).Thisinstrumentconsists ofaconstantlyfasterrotatingdrum,whichisdividedintofoursep- aratecompartments,eachforasingleanimal.Beforethebeginning oftheexperiment,weperformed3trainingmeasurementsonthree consecutivedays.Themotor performance wasindicatedbythe durationoftime(s)spentontherotatingdrum[43].Everymea- surementwasrepeated5timesforeachanimal,andtheirmeans wereusedforevaluation.

2.5. MeasurementofcutaneousmicrocirculationbylaserDoppler scanning

Microcirculationintheplantarskinofthehindpawwasmea- sured by laser doppler imaging (Perimed PIM II, Stockholm, Sweden).ThemechanismofthisdeviceisbasedontheDoppler- principle,asitemitsamonochromatic(wavelength:670nm)laser beam,whichisreflectedfromthemovingredbloodcells.Asthe reflected laserbeampasses throughDoppler-shift,detectingits wave-lengthallowsustomeasuretherateofbloodflowinthe superficialcapillaries.Becausemostofthelaserlightisabsorbed inthesuperficialtissues,thebloodflowofthelargerbloodves- selsdoesnotdisturbthemeasurement.Micewereanesthetized withurethane(2.4g/kgi.p.), theirbody temperaturewasmain- tainedat37^◦Cwithacontrolledheatingpad.Atthebeginningofthe experiment3–4controlimagesoftheplantarsurfacesofboththe operatedandtheintactcontralateralhindpawsweretakensimul- taneouslytoobtainstablebaselineflowdata.Then30–30␮lof5%

mustard-oil(allyl-isothiocyanate)wasappliedtopicallyonboth plantarsurfacestoinduceneurogenicvasodilatation.Thischem- icalcompoundisaTransientReceptorPotentialAnkyrin1(TRPA1) receptoragonist,therefore,itinducesthereleaseofvasoactiveneu- ropeptidesfromtheintactsensorynerveendings,whichinturn leadstoanincreasedbloodflowin theinnervatedarea(neuro- genic vasodilation).Theplantar microcirculationwas measured for 60min aftermustard oil application.Altogether an average of 30 imageswererecorded for each animal including theini- tialcontrolmeasurements.5%mustard-oilwaspreparedfreshly beforethebeginning oftheexperiment fromthesameconcen- tratedsolution.Allknockoutandwildtypemiceweremeasured withinatimelimitof48htodecreasethepossibleenvironmental interferences.

2.6. Statisticalanalysis

Alldatawereexpressedasmeanswithstandarderrorsofmeans (s.e.m.).ForstatisticalevaluationrepeatedmeasuresANOVAfol- lowedbyBonferroni’smodifiedt-testwasusedfortheassessment ofmechanicalhyperalgesiaandmotorperformance,whereasthe resultsofthelaser dopplerimagingwereevaluated usingtwo- wayANOVAandBonferroni’smodifiedt-test.Whencomparingthe results*p<0.05wasconsideredstatisticallysignificant.

3. Results

3.1. Mechanicalhyperalgesiainresponsetopartialligationofthe sciaticnerve

TheinitialcontrolmechanonociceptivethresholdsofPACAP^−/− miceweresimilartothewildtypes.Tightligationof1/3ofthesciatic nerveinducedasignificant,about30–40%decreaseofthethreshold oftheaffectedhindpawinwildtypeanimalsbetweenthe7thand 19thpostoperativedays.Incomparison,neuropathicmechanical hyperalgesiawasminimalinthePACAP^−/−group,thenociceptive thresholdremainedsimilartotheinitialcontrolvaluesthroughout thetotaldurationoftheexperiment(Fig.1A).

The preoperative mechanonociceptive thresholdsof C57Bl/6 wildtypemicedidnotdiffersignificantlyfromeitherthePACAP^+/+

wildtypesgeneratedontheCD1backgroundortheTac1^−/−andthe Tacr1^−/−ones.Furthermore,the35–45%mechanicalhyperalgesia detectedinresponsetothenerveligationwasalsoverysimilar inallthethreegroups,SP/NKAorNK₁receptordeficiencydidnot influenceitsdevelopmentduringthewholestudy(Fig.1BandC).

3.2. Changesofthemotorfunctions

ThebasalmotorperformanceontheacceleratingRota-Rodwas significantlyworseinboththePACAP^−/−andTac1^−/−groupscom- pared to the respective wildtypes(Fig. 2A and B). In contrast, deletionoftheNK1tachykininreceptor(Tacr1^−/−)didnotinfluence themotorcoordination(Fig.2C).Thepartialsciaticnerveligation didnotinfluencethemotorcoordinationinanyofthegroups.Dur- ingthecontrolpre-operativeandeventhepost-operativeperiodsa continuouslearningprocesswasobservedinthewildtypeandthe PACAPandTac1gene-deletedgroups,butnotinTacr1^−/−animals.

However,thebasalimpairedmotorcoordinationinPACAP^−/−and Tac1^−/−micewasalsoobservedaftertheoperation(Fig.2AandB).

3.3. Changesofcutaneousbloodflowandneurogenicvasodilation

In both wildtype (PACAP^+/+ and C57Bl/6) and all the three gene-deficient (PACAP^−/−, Tac1^−/−, Tacr1^−/−) groups, the basal microcirculationintheplantarskinwasreducedontheoperated limb,butthedifferencewasnotstatisticallysignificant.Thebasal perfusionwassignificantlyhigherintheblackC57Bl/6,Tac1^−/−,but notTacr1^−/−miceascomparedtothewhitePACAP^+/+andPACAP^−/−

onesgeneratedontheCD1background.Meanwhile,topicalappli- cationof5%mustardoilactivatingthecapsaicin-sensitivesensory nerve terminals in the skinand inducing neuropeptide release evokedsignificantlysmallerneurogenicvasodilatorresponseinthe C57Bl/6-basedgroupscomparedtotheCD1-basedones(15–20%vs.

50–60%).Thisclearlyindicatesaremarkablestraindifferenceinthe regulationofmicrocirculation(Figs.3–5).

Basalperfusionin theplantarskinofboth theoperatedand intactlimbsofPACAP^+/+andPACAP^−/−micedidnotdiffersignif- icantly(3.4±0.32V,vs.3.97±1.04V).Inresponsetoactivationof thecapsaicin-sensitivesensorynerveterminalsbytopicalappli- cationof5%mustardoilmicrocirculationsimilarly increasedby

Fig.1. Changesofmechanonociceptivethresholdofthepawafterpartialsciatic nerveligationin(A)PACAPgene-deficient(PACAP^−/−)micecomparedtotheir wildtypes(PACAP^+/+)generatedontheCD1background,aswellasin(B)Tac1 gene-deletedanimals(Tac1^−/−)lackingsubstancePandneurokininA,and(C)NK1 tachykininreceptordeficient(Tacr1^−/−)miceincomparisonwiththeirC57Bl/6 wildtype(WT)counterparts.Datapointsrepresentmeansofn=12pergroupwith s.e.m.;**p<0.01,***p<0.001vs.respectivewildtypecontrol(repeatedmeasures ANOVA+Bonferroni’smodifiedt-test).

50–60%inbothgroupsonbothsidesafter5–10min.Thisneuro- genicvasodilationresponsepersistedinwildtypemiceduringthe next60minoftheexperiment,whereas inPACAP^−/− animalsit startedtodecreaseafter10–15minSignificantlylowerperfusion

Fig.2. MotorperformanceontheacceleratingRota-Rodwheelbeforeandonthe7th and10thpostoperativedaysaftersciaticnerveligationin(A)PACAPgene-deficient (PACAP^−/−)micecomparedtotheirwildtypes(PACAP^+/+)generatedontheCD1back- ground,aswellasin(B)Tac1gene-deletedanimals(Tac1^−/−)lackingsubstanceP andneurokininA,and(C)NK1tachykininreceptordeficient(Tacr1^−/−)miceincom- parisonwiththeirC57Bl/6wildtype(WT)counterparts.Columnsrepresentmeans ofn=6–12pergroupwiths.e.m.;*p<0.05,**p<0.01,***p<0.001vs.respectivewild- typecontrol;#p<0.05vs.respectiveday1preoperativevalue(repeatedmeasures ANOVA+Bonferroni’smodifiedt-test).

wasdetectedinthePACAP^−/−groupfromthe30thminuteofthe measurement(Figs.3and4AandB).

IntheTac1^−/−andTacr1^−/−groupsthebasalcutaneousmicro- circulationwassignificantlyloweronbothlimbscomparedtothe

Fig.3.RepresentativelaserDopplerimagesoftheplantarskinoftheoperated(right)andtheintact(left)hindlimbsbeforeand20minaftertopicalapplicationof1%mustard oiltostimulatethepeptidergicsensorynerveterminals.PanelsshowdataofPACAPgene-deficient(PACAP^−/−)micecomparedtotheirwildtypes(PACAP^+/+)generatedon theCD1background,aswellasin(B)Tac1gene-deletedanimals(Tac1^−/−)lackingsubstancePandneurokininA,and(C)NK1tachykininreceptordeficient(Tacr1^−/−)mice incomparisonwiththeirC57Bl/6wildtype(WT)counterparts.Thebluecolorrepresentslowperfusionareas,greenandyellowrefertohigherperfusionandredshowsthe highestmicrocirculation.

C57Bl/6wildtypes(Fig.5).Inresponsetomustardoilsmearing theperfusionincreasedsteadilyby15–20%onbothlimbsofthe wildtypemice.Incontrast,incasesofSP/NKAandNK1receptor deficiencytherewasaninitialtransientdecreaseontheintactside, butafterthefirst10min,thebloodperfusiondidnotdiffersignifi- cantlyfromtheresultsoftheC57Bl/6wildtypeanimalsfortherest oftheexperiment(Fig.5).

4. Discussion

Theseresultsprovide clearevidence that:(1) Under normal conditionsPACAPandtachykininsplayimportantrolesinmotor coordination,buttheyarenotinvolvedinmechanonociception.(2) Tachykininsregulatethebasalcutaneousmicrocirculationthrough NK₁ receptor activation, but PACAP is involved in neurogenic vasodilation.(3) Partial ligationof thesciaticnerve, which is a widely usedtraumatic mononeuropathy model, induces purely sensoryneuropathy(mechanicalhyperalgesia)withoutaffecting themotorandthevascularfunctions.(4)PACAPisacrucialmedi- atorofneuropathichyperalgesia.

Ourearlier datashowingthatPACAPhasa hyperalgesicand pro-nociceptiveroleinavarietyofpainmodelsareinagreement withthepresent findings [42].Others alsoshowed thatPACAP mightplayakeyroleinspinalsensitization,andtherefore,inthe developmentof neuropathicpain [8,25] throughPAC1 receptor activationinthedorsalrootganglia[6,20].Furthermore,PACAP islikelytohaveapivotalpro-nociceptivefunctioninanimalmod- elsofmigraineandalsoinhumanmigraneurs[29,46,54].Others suggested that theperipheralnociceptive responseselicited by intradermallyadministeredPACAParemainlymediatedthrough theVPACreceptors[45].Therefore, identificationofthe targets andthemechanismsofthehyperalgesicactionsofPACAPstillneed furtherinvestigations.

While the increased expression of the Tac1 gene-encoded tachykininsinthedorsalrootgangliaunderneuropathicconditions hasbeenestablished[57],ourresultsindicatethatSP/NKAandthe NK₁receptordonotplayanimportantroleinthedevelopmentof neuropathichyperalgesia.Studyingtheanalgesicmechanismsof

theactionoftopicalcapsaicinapplicationinneuropathies,itwas foundthattheincreasedexpressionofSPhasonlymarginaleffect, andcapsaicinexertsitstherapeuticbenefitthroughotherpath- ways[1].OtherresultsalsoindicatethatSP/NKAup-regulationis notacrucialfactorinthedevelopmentofneuropathies:whileitwas foundtobeinvolvedinthedevelopmentofpaclitaxel-inducedneu- ropathy,oxaliplatintreatmentleadstoasimilarconditionwithout influencingtheSP/NKArelease[52].

ThemotorcoordinationofthePACAPandTac1genedeficient animalswassignificantlyworsethantheirwildtypecounterparts underbothnormalandneuropathicconditions,whilethemotor performanceoftheNK₁ receptor deficientgroupwassimilarto thecontrolanimals.Theseresultssuggest,thatPACAPandSP/NKA mighthaveapotentialroleasmediatorsofnormalmotorcoordina- tion.However,thiseffectofthetachykininsisnotmediatedthrough theNK₁ receptor, but theaction ofNKA at theNK₂ tachykinin receptorcanbesuggested.Thesefindingsaresupportedbyear- lierdatashowingthatPACAPmaycontributetotheregulationof motorcoordination,thoughthemechanismandthesiteofaction hasnotbeendescribed[30].Currentlythereislessdataavailable aboutthe potentialrole of theTac1gene encoded tachykinins, butthefew earlierresultsobtainedinmarkedly differentmod- els(lesscomplexspecies)alsoindicatethattachykininsmightalso affectthemotorcoordination[21].SPhasbeensuggestedtohave aprotectiveroleinananimalmodelofamyotrophiclateralscle- rosis,whichisduetomotorneurondegeneration,supportingits roleinmotorcoordination[3].SPandtheNK1receptoractivation mayalsohaveapotentialroleinthedevelopmentofParkinson’s disease, in which the loss of motor coordination is the princi- palsymptom. IncreasedSPexpressioninthismodelaccelerated diseaseprogression,whereasNK₁ receptorantagonisttreatment improvedthemotorperformance [53]. Thesignificantmechan- icalhyperalgesia,butnormalmotorperformanceandcutaneous microcirculationobservedinresponsetothesciaticnerveligation indicates,thatalthoughitcontainssensory,motorandautonomic fibers,thetraumaticmononeuropathyinducedbythisoperative processisexclusivelyofsensorynatureanditdoesnothindereither themotorperformanceorthevascularregulation.

Fig.4.Mustardoil-inducedvasodilationcomparedtotherespectiveinitialreferenceimagesintheplantarskinoftheoperatedandintactpawsofPACAPgene-deficient (PACAP^−/−)micecomparedtotheirwildtypes(PACAP^+/+)generatedontheCD1background(A–meanflux,B–percentage).n=5–6pergroupwiths.e.m.;#p<0.05,##p<0.01 PACAP^+/+vs.PACAP^−/−operatedside,*p<0.05,**p<0.01,***p<0.001PACAP^+/+vs.PACAP^−/−intactside(two-wayANOVA+Bonferroni’smodifiedt-test).

Besides neuropathic hyperalgesia, PACAP is involved in the development of neurogenic vasodilation, particularly in its long-termmaintenance.Asmustardoilactivatingthecapsaicin- sensitiveafferentspredominantlyviaTRPA1ionchannelactivation inducedaremarkableinitialvasodilationinPACAPdeficientmice, which very rapidlydecreased, it is suggested, that PACAPcon- tributes to the later, stable phase of neurogenic vasodilation.

Othersensoryneuropeptidesreleasedfromthestimulatedsensory nerves,suchasCGRParelikelytoberesponsiblefortheearlyphase ofthisresponse.

AsthebasalbloodflowwassignificantlylowerinbothTac1and Tacr1gene-deficientmice,SPthroughNK₁ receptoractivationis likelytoplayanimportantroleinthemediationofthebasalvas- culartone,itdilatesthecutaneousvessels.However,theydonot haveanimportantfunctioninneurogenicvasodilation.Itshouldbe notedthatthewellestablishedeffectofSPinneurogenicinflam- mationisincreasingvenularpermeability,aphenomenwhichwas notmeasuredwiththelaserDopplerscanning[19].Inaddition, CGRPisconsideredasthemostpotentmediatorofvasodilation [24].Itwassuggested,thattheNK₁ receptoractivationservesto

increaseindirectlytheproductionofnitricoxide(NO)andother localvasodilatorysubstances,howeveritisaccepted,thatthereis alotofinteractionsbetweentheneuropeptides,localmediators, andreceptorsinvolvedinthisprocess,andtheresponsiblepath- waysareredundant[59].Itmustbetakenintoaccount,thatSP isnotthesoleendogenousligandoftheNK₁receptor,onwhich theotherknowntachykininsalsoactaspotentagonists,therefore probablybypassingtheroleofthelackingSP/NKA.Inadditionitis established,thatthoughtachykininsareinmostconditionspotent vasodilators,indifferentmodelsorspeciestheycaninducevaso- constriction,andthattheirneteffectisheavilyinfluencedbythe localdifferencesoftheendothelium[58].Thisassumptionalsocor- relateswiththefact,thatsofarnosubstancecouldbelabeledas theexclusivemediatorofcutaneousvasodilation[4,24].

WefoundremarkablestraindifferencesbetweentheC57Bl/6 andCD1mice(PACAP^+/+)intheexaminedsensory,motorandvas- cularparameters.Itisnotsurprising,sincealargenumberofpapers describedsuchfindings,andreviewsfocusontheissueofstraindif- ference[32,49].Describingthesedifferencesisinfactvaluable,and highlightstheimportanceofusingtheappropriatewildtypeswhen

Fig.5.VasodilativeresponseinducedbymustardoilsmearintheplantarskinoftheoperatedandintactpawsoftheTac1gene-deletedanimals(Tac1^−/−)lackingsubstance PandneurokininA,and(AandB)andNK1tachykininreceptordeficient(Tacr1^−/−)mice(CandD)incomparisonwiththeirC57Bl/6wildtype(WT)counterparts.Data pointsrepresentmeansofn=5–6pergroupwiths.e.m.;*p<0.05,**p<0.01,***p<0.001,vs.intactsideofrespectivewildtypecontrol;#p<0.05,##p<0.01,###p<0.001vs.

respectiveoperatedpawofwildtypecontrol(two-wayANOVA+Bonferroni’smodifiedt-test).

interpreting dataof gene-deficientmice.In thepresent experi- mentalserieswedidnotaimtocomparethetachykininandNK₁ deficientmicewiththePACAP knockouts,but onlytocompare themwiththeirrespectiveandappropriatewildtypesinorderto elucidate theirrole intheinvestigated biologicalfunctions.The conclusionsweredrawnfromtheresultsseparately.

It can be concluded that (1) PACAP has an overall pro- nociceptiveroleinperipheraltraumaticneuropathy.(2)PACAPand SP/NKAarebothinvolvedinmotorcoordination,whilethelackof theNK₁receptordoesnotaffectthisfunction.(3)Tac1geneprod- uctsandNK1 receptorhaveapivotalroleinthemaintenanceof basalvasculartone,whereasPACAPisakeymediatorofneurogenic vasodilation,particularlyinvolvedinitslong-termmaintenance.

Acknowledgements

SROP-4.2.2.A-11/1/KONV-2012-0024, SROP-4.2.1.B-10/2/

KONV-2010-0002,SROP-4.2.2.B-10/1/2010-0029,OTKAK104984, ArimuraFoundation,PTE-MTA“Lendület”Program.

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