The cough reflex is upregulated by lisinopril microinjected into the caudal nucleus tractus solitarii of the rabbit

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ContentslistsavailableatScienceDirect

Respiratory

Physiology

&

Neurobiology

j o ur na l ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / r e s p h y s i o l

The

cough

reﬂex

is

upregulated

by

lisinopril

microinjected

into

the

caudal

nucleus

tractus

solitarii

of

the

rabbit

Elenia

Cinelli,

Fulvia

Bongianni,

Tito

Pantaleo,

Donatella

Mutolo

∗

DipartimentodiMedicinaSperimentaleeClinica,SezioneScienzeFisiologiche,UniversitàdegliStudidiFirenze,VialeG.B.Morgagni63,50134Firenze,Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received9June2015

Receivedinrevisedform27July2015 Accepted27July2015

Availableonline30July2015 Keywords:

Lisinopril Cough Bradykinin SubstanceP

Nucleustractussolitarii

a

b

s

t

r

a

c

t

WehavepreviouslyshownthatcoughpotentiationinducedbyintravenousadministrationoftheAT1

receptorantagonistlosartanislowerthanthatinducedbytheACEinhibitorlisinoprilinanesthetized andawakerabbits.Sincelosartanandlisinoprilcrosstheblood–brainbarrier,theircentralactionon thecoughreﬂexcanbehypothesized.Mechanicalstimulationofthetracheobronchialtreeandcitric acidinhalationwereusedtoinducecoughreﬂexresponsesinpentobarbitalsodium-anesthetized, spon-taneouslybreathingrabbits.Bilateralmicroinjections(30–50nl)oflosartan(5mM),lisinopril(1mM), bradykinin(0.05mM),HOE-140(0.2mM,abradykininB2receptorantagonist)andCP-99,994(1mM,an

NK1receptorantagonist)wereperformedintothecaudalnucleustractussolitarii,thepredominantsite

ofterminationofcough-relatedafferents.Lisinopril,butnotlosartanincreasedthecoughnumber.This effectwasrevertedbyHOE-140orCP-99,994.Coughpotentiationwasalsoinducedbybradykinin.The resultssupportfortheﬁrsttimeacentralprotussiveactionoflisinoprilmediatedbyanaccumulationof bradykininandsubstanceP.

1. Introduction

Coughisaveryimportantairwayprotectivereﬂex,butalso char-acterizesadebilitatingdiseaseunderchronicconditions.Drycough islargelyconsideredoneofthemajorsideeffectsof angiotensin-convertingenzyme(ACE)inhibitors(BerkinandBall,1988),aclass of drugs widely used for hypertension, heart failure and post-infarctiontreatment(Pauletal.,2006).AngiotensinIIisthemain effectoroftherenin-angiotensinsystem(RAS)thatcontrols cardio-vascularhemodynamicandbloodﬂuidhomeostasis.Inmammals therearetwoprimaryangiotensinIIreceptorsubtypes,AT1andAT2 (Pauletal.,2006;WrightandHarding,2011).Losartanandother sartansblockAT1receptors(McIntyreetal.,1997).Theyhavebeen associatedwithlowcoughincidenceinhumans,sothattheyare usuallyemployedtosubstituteACEinhibitorsinpatients display-ingassideeffectpersistentcough(e.g.,Pasteretal.,1998;Mutolo etal.,2013alsoforfurtherRefs.).ThemechanismofACE inhibitor-induced cough remains unclear, but likely involves mainlythe protussivemediatorsbradykininandsubstanceP,agentsthatare degradedbyACEandthereforeaccumulatedintherespiratory sys-temwhentheenzymeisinhibited(e.g.,Balietal.,2014;Foxetal.,

∗ Correspondingauthor.

E-mailaddress:donatella.mutolo@uniﬁ.it(D.Mutolo).

1996;Moreauxetal.,2001;Tomakietal.,1996;Mutoloetal.,2010, 2013alsoforfurtherRefs.).

In a previous study (Mutolo et al., 2013), we have shown thatcoughupregulationinducedbyintravenousadministrationof losartan islower thanthat inducedbylisinopril both inawake and anesthetizedrabbits.Since losartan and lisinoprilcross the blood–brainbarrier(Pediconietal.,2005;Ranadiveetal.,1992; Tanetal.,2005),acentralactionofthesedrugsonthecoughreﬂex canbehypothesizedespeciallyatthelevelofthecaudalnucleus tractussolitarii(NTS),themaincentralterminusofcough-related afferents(KubinandDavies,1995;Kubinetal.,2006;Mutoloetal., 2007).

Apartfromtheirrolein cardiovascularregulation,RAS com-ponentswithinthecentralnervoussystemhaveotherfunctions mediatedbyAT1 and/orAT2receptors(Pauletal.,2006;Premer etal.,2013;WrightandHarding,2011).Inparticular,previous stud-ies havedemonstrated that severalneuromodulators, including angiotensinIIactingonAT1receptors,areinvolvedin cardiovascu-larregulationwithintheNTS(Arnoldetal.,2010;Chengetal.,2010, 2012;Kasparovetal.,1998;Mosqueda-Garciaetal.,1990). Accord-ingly,highlevelsofACE(Rogersonetal.,1995)andhighdensitiesof AT1receptorshavebeenfoundattheleveloftheNTSintherabbit (Aldredetal.,1993)aswellasinmanyothermammalsincluding humans(Pauletal.,2006;Premeretal.,2013;WrightandHarding, 2011).

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10 E.Cinellietal./RespiratoryPhysiology&Neurobiology219(2015)9–17 Anattemptwasmadetoascertainacentralactionoflosartan

andlisinoprilatthelevelofthecaudalNTSandprovideevidence supportingthehypothesisthatupregulationofthecoughreflexin therabbittreatedwithlisinoprilcouldbedue,atleastinpart,to thesamemechanismsalreadysuggestedtobeactiveatthe periph-erallevel.Thus,wecarriedoutthepresentstudyonpentobarbital sodium-anesthetized,spontaneouslybreathingrabbitsbyevoking thecoughreflexinresponsetobothmechanicalandchemical stim-ulationofthetracheobronchialtree.Changesinthecoughreflex inducedbymicroinjectionsoflisinopril,losartan,bradykinin, HOE-140(abradykininB2receptorantagonist)andCP-99,994(anNK1 receptorantagonist)intothecaudalNTSwereinvestigated. 2. Materialsandmethods

2.1. Ethicalapproval

Allanimalcareandexperimentalprocedureswereconductedin accordancewiththeItalianlegislationandtheofﬁcialregulationsof theEuropeanCommunityCouncilontheuseoflaboratoryanimals (Directive86/609/EECand2010/63/UE).Thestudywasapproved bytheAnimalCareandUseCommitteeoftheUniversityofFlorence. Alleffortsweremadetominimizeboththenumberofanimalsused andtheirsuffering.Experimentalproceduresanddetailsaboutthe methodsemployedhavepreviouslybeendescribed(Cinellietal., 2013;Mutoloetal.,2007,2008,2009,2010,2012,2013,2014). 2.2. Animalpreparation

Experiments were performed on 43 male New Zealand white rabbits (2.8–3.5kg) anesthetized with pentobarbital sodium(40mg/kgi.v.,supplementedby2–4mg/kgevery30min; Sigma–Aldrich, St. Louis, MO). Atropine (0.15mg/kg i.m.) was administered to reduce mucosal secretion in the airways. The adequacy of anesthesia was continuously assessed during the experimentasinourpreviousstudies.Thetracheawascannulated andpolyethylene catheterswere insertedintoa femoralartery andveinformonitoringarterialbloodpressureanddrugdelivery, respectively.TheC3orC5phrenicrootononesidewasprepared for recordings.The animal wasplaced in a proneposition and ﬁxed by a stereotaxic head holder and vertebral clamps. The headwasventroﬂexedforoptimalexposureofthedorsalsurface of themedulla byoccipital craniotomy.Body temperaturewas maintainedat38.5–39◦C.

2.3. Recordingprocedures

Bipolar platinum electrodes were used to record efferent phrenic nerve activity from the central stump of one cut and desheathed phrenic root. Wire electrodes were used to record abdominalmuscleelectromyographic(EMG)activity.Phrenicand abdominalactivitieswereamplified,full-waverectified,and “inte-grated”(low-passRCfilter,timeconstant100ms).Arterialblood pressureandend-tidalCO2 partialpressurewererecorded. Car-diorespiratory variables were acquired and analyzed using a personalcomputer,equippedwithananalog-to-digitalinterface (Digidata1440,MolecularDevices,Sunnyvale,CA,USA)and appro-priatesoftware(Axoscope,MolecularDevices).

2.4. Microinjectionprocedures

Bilateralmicroinjectionswereperformedattwodifferentsites alongtherostrocaudalextentofthecaudalNTS,andparticularly intothelateralcommissuralNTS.Theﬁrstwasatthelevelofthe caudal-mostendoftheareapostrema,0.6–0.8mmlateraltothe midlineand0.7–0.8mmbelowthedorsalmedullarysurface.The

secondwas0.5mmmorecaudal,0.4–0.5mmlateraltothemidline and0.7–0.8mmbelowthedorsalmedullarysurface.The stereo-taxiccoordinateswereselectedaccordingtotheatlasofMeessen andOlszewski(1949).

Microinjections (30–50nl) of the following drugs were per-formed:losartan(5mM;Fluka–Sigma–Aldrich),lisinopril(1mM; Sigma–Aldrich), bradykinin (0.05mM;TocrisBioscience, Bristol, UK), HOE-140 (0.2mM; Tocris Bioscience), a potent and selec-tivebradykininB2receptorantagonist,CP-99,994(1mM;giftfrom Pfizer, Groton, CT, USA), an NK1 receptor antagonist and d,l-homocysteicacid(DLH,20mM;Sigma–Aldrich),abroad-spectrum excitatoryaminoacidagonist.Onlyoneofthesedrugswastested ineachpreparationunlessotherwisestated.Drugconcentrations wereinthesamerangeasthosepreviouslyusedininvivo prepa-rations(Arnoldetal.,2010;Caligiorneetal.,1996;Kasparovetal., 1998; Mutolo et al., 2008). In particular, the concentrations of lisinoprilandlosartanwerebasedontheknowledgethatthe anti-hypertensivepotencyoflisinoprilis∼5timeshigherthanthatof losartan(see e.g.,Mutoloetal.,2013).Alldrugs weredissolved in0.9%NaClsolution.Controlinjectionsofequalvolumesofthe vehiclesolutionattheresponsivesiteswerealsoperformed.The localizationof injectionsites isillustratedin Fig.1.Green fluo-rescentlatexmicrospheres(LumaFluor,NewCity,NY,USA)were injectedforposthocconfirmationofinjectionsiteswithinthe cau-dalNTSofsomepreparations(3forlosartanand4forlisinopril). 2.5. Stimulationprocedures

Both mechanical and chemical stimulation of the tracheo-bronchial tree were employed to induce cough. Mechanical stimulation was delivered by a custom-built device recently describedandvalidated(Mutoloetal.,2014)usinga0.5mm diam-eternylonﬁberwithasmoothed tipinsertedthrough a lateral portofthetrachealcannula.Thedeviceallowedtosetthenumber offorthandbackmovementsorcycles(1–3cycles),shaft veloc-ity(10–20mm/s),andshaftdisplacement(10–20mm).Mechanical stimulation wasadjusted tothe following parameters:1 cycle, 15mm/s velocity, and 15mm displacement. These parameters provedtoproduceaboutof2–4coughs.Thestimulation proto-colcomprisedthreestimulationtrialsperformedinsuccession(at ∼1mininterval)beforedrugadministration,repeated∼5minafter thecompletionofallthemicroinjectionsandatappropriate inter-vals(atleast5min)untilcompleterecoverywasobserved.

Chemical stimulation of thetracheobronchial tree was per-formedbymeansofcitricacidinhalation(fordetailsseeMutolo etal.,2009).Citricacid(1M,Sigma–Aldrich)wasfreshlydissolved in0.9%NaClsolutionandnebulized.Theopeningofthetracheal cannula,throughwhichtherabbitswerespontaneouslybreathing, wasexposedtoasteadystreamofthenebulizedcitricacid solu-tionfor∼3s.Thisshortperiodaswellastimeintervalsbetween chemicalchallenges>10minprovedtobeadequatetoavoid tachy-phylaxis.Chemicalstimulationwasalwaysapplied2–3minafter mechanically-inducedcoughandcausedaboutofseveralcoughs usuallyimmediatelyfollowedbyatachypneicresponse.Asarule, chemical stimulation was performed both before and ∼15min afterthecompletionoftheinjectionsandrepeatedat appropri-ateintervalstofollowthetimecourseoftherecoveryprocess.All stimulationprocedureswereperformedatleast5–6minaftereach supplementaldoseofpentobarbitaltoavoiditspossibleimmediate inﬂuenceontherecordedvariables.

2.6. Histology

The histologicalcontrol of pipette tracks and injectionsites wasperformedas previously described (fordetails,see Mutolo et al., 2007, 2012). Medullary sectionsin which injectionsites

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Fig.1.Localizationofinjectionsitesandhistologicalcontrol.A:adiagrammaticrepresentationofadorsalviewofthemedullaoblongataoftherabbitshowingthesiteswhere multiplebilateralmicroinjections()ofdifferentdrugshavebeenperformedintothecaudalaspectofthenucleustractussolitarii(NTS).AP:areapostrema;DRG:dorsal respiratorygroup.B:diagramofacoronalsectionofthemedullaoblongataatthelevelindicatedinpanelA(dashedline)showingthelocationofrepresentativesiteswhere themicroinjectionshavebeenperformed.Tosimplifythepresentationofresults,onlythedistributionofsiteswhere1mMlisinopril(䊉)wasinjectedhasbeenreported. Thediagramalsoshowsthelocationofinjectionsites(䊏)intosomecontrolregionswherelisinoprildidnotcauseappreciablechangesinthepatternofbreathingandcough responses.Representativesitesofcontroldrugmicroinjectionsatalocation>0.8mmcaudaltotheresponsivesitesofthecaudalNTShavenotbeenillustrated.NCM:nucleus cuneatusmedialis;NDV:nucleusdorsalisnervivagi;NG:nucleusgracilis;NOI;nucleusolivarisinferior;NV:nucleustractusspinalisnervitrigemini;NXII:nucleusnervi hypoglossi;P:tractuspiramidalis.TheatlasofMeessenandOlszewski(1949)andtheatlasofSheketal.(1986)wereusedforcomparison.C:photomicrographofacoronal sectionofthemedullaoblongataatapproximatelythesamelevelasinBshowingthelocationofthefluorescentbeads(green,indicatedbythewhitearrow)microinjected intothecaudalNTS.ThesectionsarecounterstainedwithCresylViolet.Light-fieldandfluorescencephotomicrographshavebeensuperimposed.Somerelevantstructures havebeenindicated.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)

weremarked by fluorescent microsphereswereexamined in a light and/or epifluorescence microscopy (Eclipse E400, Nikon, Japan)equippedwiththeNikonIntensilightC-HGFImercury-fiber illuminator.Photomicrographs weretaken witha NikonDS-Fi1 digital camera.Illustrationswere preparedin AdobePhotoshop CS3(AdobeSystemsIncorporated,SanJose,CA,USA).Theatlasof MeessenandOlszewski(1949)aswellasthemorerecentatlasof Sheketal.(1986)wereusedforcomparison.

2.7. Datacollectionandanalysis

Respiratoryvariablesweremeasuredduringeupneicbreathing andreﬂexresponses.Theinspiratory(TI)andexpiratory(TE)times, aswellasthetotaldurationoftherespiratorycycle(TT)were mea-suredonrecordingsofrawphrenicnerveactivity(Mutoloetal., 2014;Poliaceketal.,2011).Therespiratoryfrequencywas subse-quentlycalculated(breaths/min).Peakamplitude(arbitraryunits) ofthephrenicnerve activityandabdominal EMGactivitywere measuredonintegratedtracesandnormalizedbyexpressingthem

asafraction(orpercentage)ofthehighestachievableamplitude observedineachanimal,i.e.,inrelativeunits(RU).Breathing pat-ternvariablesweremeasuredforanaverageofﬁveconsecutive breathspriortoandfollowingdrugmicroinjections.Furthermore, systolicanddiastolicbloodpressuresweremeasuredat2s inter-vals and mean arterialpressure wascalculated as thediastolic pressureplusone-thirdofthepulsepressure.Themeasurement periodsofcardiorespiratoryvariableswerethesameselectedfor cough-relatedvariables(seebelow).Owingtothesmallvariations inrespiratoryandcardiovascularvariableswithineach measure-mentperiod,averagevaluesweretakenassinglemeasurements forthepurposeofanalysis.Similarly,cardiorespiratoryvariables werealsomeasuredbeforeandafterDLHmicroinjectionsatthe timewhenthemaximumresponseoccurred.

Thecoughmotorpatterninresponsetomechanicalor chem-icalstimulationofthetracheobronchialtreeis characterizedby repeatedcoughs.Respiratoryvariablesofcoughs (cough-related variables)includedthecough-relatedTT,TIandTE,peakphrenic amplitude (RU), peak abdominal activity (RU) and the cough

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12 E.Cinellietal./RespiratoryPhysiology&Neurobiology219(2015)9–17

Fig.2.Influencesof5mMlosartanand1mMlisinoprilmicroinjectedintotheselectedsitesofthecaudalNTSoncoughreflexresponseselicitedbymechanicalstimulation ofthetracheobronchialtreeinanesthetizedspontaneouslybreathingrabbits.Originalrecordingsillustratetheabsenceofappreciableeffects∼30minafterlosartan microin-jectionsandthepotentiationofthecoughreflex∼30minafterlisinoprilmicroinjections(maximumeffect).Theonsetofmechanicalstimulationisindicatedbyarrows. PhrIN:phrenicintegratedneurogram;PhrN:phrenicneurogram;AbdIEMG:abdominalintegratedelectromyographicactivity;AbdEMG:abdominalelectromyographic activity.

number,i.e., thenumber of coughs following each stimulation. Cough-relatedvariablesweremeasuredandaveragedbeforeand afterdrugadministrationatthetimewhenthemaximumresponse occurredas wellas atthetime whena completerecoverywas observed(threetrialsformechanicalstimulationandasingletrial forcitricacidinhalation).Theaveragevaluesofcough-related vari-ablesweretakenassinglemeasurementsforsubsequentstatistical analysis(GraphPadPrism5,LaJolla,CA,USA).Onsomeoccasions, anexpirationreflexcouldoccurasthefirstmotoreventinacough epoch(KorpáˇsandTomori,1979;WiddicombeandFontana,2006 forfurtherdetailsseeTataretal.,2008;Mutoloetal.,2007,2008, 2009,2012;Cinellietal.,2013).Expirationreflexeswerenot con-sideredfordataanalysis.

Comparisons were performed by means of the one-way repeated-measuresANOVAfollowedbyStudent–Newman–Keuls tests.Thesamestatisticalanalysiswasemployedtoevaluatethe reversionof thelisinopril-induced effects by thebradykinin B2 receptorantagonistHOE-140andtheNK1receptorantagonist CP-99,994.DLH-inducedchangesincardiorespiratoryvariablesaswell asthebradykinineffectsonthecoughreﬂexinthepresenceof CP-99,994wereevaluatedbytheStudent’spairedt-test.Allreported valuesaremeans±SE;p<0.05wastakenassigniﬁcant.

3. Results

Bilateral microinjections (n=7) of 5mM losartan (30–50nl; 150–250pmol) into thecaudal NTS didnot produceany effect

onthe coughresponse toboth mechanical and chemical stim-ulation of the tracheobronchial tree (Figs. 2 and 3; Table 1). In contrast, bilateral microinjections (n=7) of 1mM lisinopril (30–50nl;30–50pmol)increasedthenumberofcoughsinduced bymechanicalandchemicalstimulationsofthetracheobronchial tree(Figs.2and3;Table1).Theseeffectswerealreadyapparenton mechanically-inducedcoughwithin∼5minandwerefully devel-opedforbothtypesofstimulationat∼30minafterthecompletion ofthemicroinjections.Cough-relatedvariables resumedcontrol valueswithin60min.

Sincelisinopril-inducedcoughpotentiationcouldbelinkedto anaccumulationofbradykinin(Balietal.,2014;Foxetal.,1996; Moreauxetal.,2001),bilateralmicroinjections(n=7)of0.05mM bradykinin(30–0nl;1.5–2.5pmol)wereperformedintothecaudal NTS.About10minafterthemicroinjections(maximumeffect),the coughnumberincreasedfrom3.13_±0.40to4.33_±0.35(p<0.001) duringmechanicalstimulationandfrom3.71±0.42to5.86±0.45 (p<0.001)duringchemicalstimulation(seealsoFig.4).Nochanges intheothercough-relatedvariableswereobserved(notshown). Therecoveryoccurredwithin∼60min.Accordingly, microinjec-tionsof 0.2mM HOE-140 (30–50nl;6–10pmol) into the same caudalNTSsitesperformed∼30minafterlisinopril(n=5)reverted within∼10minlisinopril-inducedpotentiationofthecoughreﬂex (Fig.5A).

ToverifywhethersubstanceP accumulationisalsoinvolved in the lisinopril-induced potentiation of the cough reﬂex (Bali

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Fig.3. Influencesof5mMlosartanand1mMlisinoprilmicroinjectedintotheselectedsitesofthecaudalNTSoncoughreflexresponseselicitedbycitricacidinhalationin anesthetizedspontaneouslybreathingrabbits.Originalrecordingsshowtheabsenceofappreciableeffects∼30minafterlosartanmicroinjectionsandthepotentiationof thecoughreflex∼30minafterlisinoprilmicroinjections(maximumeffect).Stimulationperiodsmarkedbyfilledbars.PhrIN:phrenicintegratedneurogram;PhrN:phrenic neurogram;AbdIEMG:abdominalintegratedelectromyographicactivity;AbdEMG:abdominalelectromyographicactivity.

etal.,2014;Moreauxetal.,2001;Tomakietal.,1996),wetriedto revertlisinopril-inducedeffectsbymicroinjectionsofCP-99,994, anNK1 receptorantagonist.Ithasalreadybeenshownthat sub-stancePmicroinjectedintothecaudalNTSmarkedlypotentiates coughreﬂexresponses(Mazzoneetal.,2005;Mutoloetal.,2007) while10mMCP-99,994microinjectedintothesamesiteabolishes them(Mutoloetal.,2008).Thus,wesearchedforaconcentration oftheNK1receptorantagonistthatinjectedintothecaudalNTS didnotcauseanyantitussiveeffectundercontrolconditions,but couldabolishlisinopril-induced potentiation.Aconcentrationof 1mMwasselectedinpreliminarytrials(n=4).Microinjectionsof

1mMCP-99,994(30–50nl;30–50pmol;n=5)intothesamecaudal NTSsitesexecuted_∼30minafterlisinoprilblockedwithin_∼10min lisinopril-inducedpotentiationofthecoughreﬂex(Fig.5B).

TheresultsshowthatboththeB2receptorantagonistandthe NK1 receptor antagonist areable to completelyabolish cough-potentiatingeffects (thefacilitatory effects onthecough reﬂex responses)inducedbylisinopril.Sincebradykininisknownto facil-itatethereleaseofsubstancePfromratsensoryneuronsinculture aswellasfromperipheralterminalsofcapsaicine-sensitive pri-maryafferents(forareview,seeCoutureetal.,2001),anattempt wasmadetoascertainwhetherbradykinincouldberesponsible

Table1

Cough-relatedvariablesduringmechanicalstimulationofthetracheobronchialtreeandcitricacidinhalationbeforeand∼30minafterbilateralmicroinjectionsof5mM losartan(n=7)and1mMlisinopril(n=7)intothecaudalNTS.

CN TT(s) TI(s) TE(s) PPA(RU) PAA(RU)

Mechanicalstimulation

Control 3.16±0.22 0.53±0.04 0.36±0.03 0.17±0.01 0.63±0.03 0.58±0.03

5mMLosartan 3.07±0.27 0.55±0.03 0.37±0.02 0.18±0.01 0.61±0.03 0.58±0.02

Control 2.81±0.19 0.55±0.03 0.37±0.02 0.18±0.01 0.62±0.02 0.57±0.04

1mMLisinopril 4.38±0.35* _0.56_±_0.02 _0.38_±_0.02 _0.17_±_0.01 _0.66_±_0.03 _0.57_±_0.03

Citricacidinhalation

Control 3.86±0.51 0.51±0.01 0.35±0.02 0.17±0.01 0.62±0.03 0.49±0.03 5mMLosartan 3.79±0.51 0.54±0.02 0.35±0.01 0.19±0.01 0.60±0.05 0.48±0.03 Control 3.43±0.48 0.55±0.02 0.37±0.02 0.18±0.01 0.61±0.03 0.50±0.03 1mMLisinopril 5.71±0.64* 0.56±0.03 0.38±0.03 0.17±0.02 0.62±0.05 0.49±0.02 Valuesaremeans±SE;n:numberofanimals;CN:coughnumber;TT:cycleduration;TI:inspiratorytime;TE:expiratorytime;PPA:peakphrenicactivityinrelativeunits

(RU);PAA:peakabdominalactivityinrelativeunits(RU).

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14 E.Cinellietal./RespiratoryPhysiology&Neurobiology219(2015)9–17

Fig.4. Changesinducedby0.05mMbradykininmicroinjectedintotheselectedsitesofthecaudalNTSincoughreﬂexresponsesofoneanesthetizedspontaneouslybreathing rabbit.Potentiationofcoughresponsesinducedbymechanicalstimulationofthetracheobronchialtreeandbyinhalationof1Mcitricacid∼10minafterbilateral microin-jectionsofbradykinin.Onsetofmechanicalstimulation(arrows)andperiodsofcitricacidinhalation(ﬁlledbars)areindicated.PhrIN:phrenicintegratedneurogram;Phr N:phrenicneurogram;AbdIEMG:abdominalintegratedelectromyographicactivity;AbdEMG:abdominalelectromyographicactivity.

ofthe release of substanceP atthe levelof NTS.Thus, in four experiments bilateral microinjections of 0.05mM bradykinin (30–50nl; 1.5–2.5pmol) were performed∼5min afterbilateral microinjectionsof1mMCP-99,994(30–50nl;30–50pmol)intothe samecaudalNTSsites.About10minafterbradykinin microinjec-tions,thecough reﬂexwasnotpotentiated:the coughnumber remained fairly constant during mechanical stimulation (from 3.05±0.27to2.85±0.17;p>0.05)andduringchemicalstimulation (from4.10±0.31to3.87±0.37;p>0.05).

Althoughthemainfocusofourstudywasoncoughresponses, wealsoobservedthatbilateralmicroinjectionsoflosartan, lisino-prilorbradykinin intothecaudalNTSdidnotaffectrespiratory variablesduringeupneiccontrolbreathing(seee.g.,control recor-dings before reﬂex responses in Figs. 2–4; statistical data not shown).Similarly,noconsistentchanges inarterialblood pres-surewereobserved(meanarterialpressurewasalwaysbetween 96and105mmHg).Ageneralevaluationofcardiorespiratory vari-ables under control conditions has been provided in previous reports (Mutolo et al., 2007, 2008, 2009). The correspondence of lisinopril injection sites tothe NTSregions involved in car-diovascular regulation, where glutamate microinjections cause decreases in arterial blood pressure (e.g., Cheng et al., 2012; Mosqueda-Garcia et al., 1990), was also investigated. Bilateral microinjections of 20mM DLH (30–50nl;600–1000pmol),

per-formed in four preparations before lisinopril administration, consistentlydecreasedwithin3–4minmeanarterialblood pres-sure(−22.25±1.49mmHg;p<0.05).Theseinjectionsalsocaused increases in the respiratory rate (from 51.2±3.1 to 72.4±4.1 breaths/min; p<0.005) mainly due to decreases in TE (from 0.83±0.05 to 0.53±0.05 s; p<0.005) associated withthe sup-pression of the abdominal muscle activity. No attempts were made to elicit cough reﬂex responses during DLH-induced effects.

Infouradditionalpreparations,bilateralcontrolmicroinjections of1mMlisinoprilwereperformedatdifferentmedullarylocations sufﬁcientlyfarfromtheresponsivesites(Cinellietal.,2013;Lipski etal.,1988;Mutoloetal.,2007,2008,2009,2012,2014;Nicholson, 1985;SykovaandNicholson,2008).Fourmicroinjectionsof1mM lisinoprilwereexecutedintoeachofthefollowingneural struc-tures (forcomparison, see Meessenand Olszewski, 1949; Shek etal.,1986):thenucleuscuneatusmedialis,thenucleustractus spinalisnervitrigeminiandtheregionlocated>0.8mmcaudalto theresponsiveNTSsites.Inallinstances,noappreciablechanges inthepatternofbreathingandcoughresponseswereobserved. InaccordancewiththeresultsofourpreviousstudiesontheNTS region(Mutoloetal.,2007,2008,2012,2014),controlinjectionsof equalvolumesofthevehiclesolutionattheresponsivesites(5trials

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Fig.5. Blockadeofthelisinopril-inducedeffectsbythebradykininB2receptor antagonistHOE-140andtheNK1receptorantagonistCP-99,994microinjectedinto thecaudalNTS.A:histogramsshowingthenumberofcoughsinducedbymechanical andchemicalstimulationsundercontrolconditions,∼25minafter1mMlisinopril, and∼10minafter0.2mMHOE-140(n=5).B:histogramsshowingthenumberof coughsinducedbymechanicalandchemicalstimulationsundercontrolconditions, ∼25minafter1mMlisinoprilmicroinjections,and∼10minafter1mMCP-99,994 (n=5).Eachantagonistwasmicroinjected∼30minafterlisinopril,i.e.,duringthe maximumcough-potentiatingeffect.Valuesaremeans±SE.*p<0.001compared withcontrol;#p<0.001comparedwithlisinopril.ThecoughnumberafterHOE-140 orCP-99,994wasnotsigniﬁcantlydifferentfromcontrol.

performedin5differentpreparationsbeforedrugadministration) wereineffective.

Thelocalizationoftheinjectionsiteswasconfirmedbythe his-tologicalcontrol(fordetailsseealsoMutoloetal.,2007,2012).An exampleofthelocationoffluorescentbeadsmicroinjectedintothe caudalNTSisshowninFig.1.Thesamefigureillustratesthe local-izationofinjectionsitesonadorsalviewofthemedullaoblongata aswellasthedistributionofinjectionsiteswithinthecaudalNTS andsomecontrolregions.Onlythedistributionofsiteswhere1mM lisinoprilwasinjectedhasbeenreported.

4. Discussion

Thisstudyisthefirsttoprovideevidencethat,incontrastto losartan,theACEinhibitorlisinoprilmicroinjected intothe cau-dalNTSinducesapotentiationofthecoughreflexevokedbyboth mechanicalandchemicalstimulationofthetracheobronchialtree. Inouropinion,themainnoveltyofthepresentresultsisthatthey supportthenotionofacentralactionoflisinoprilthatinterferes withthecatabolismofbradykininandsubstancePatthelevelofthe caudalNTS,i.e.,atthelevelofaneuralstructurewithavery impor-tant,orevencrucial,roleinthemedullaryregulationofthisreflex (e.g.,Bolseretal.,2006;Bonhametal.,2004;CanningandMori, 2010,2011;Mazzoneetal.,2005;Mutoloetal.,2007,2008,2009, 2012).Inotherwords,theRAScanbesuggestedtobeinvolvedinthe centralcontrolofthecoughreflex.WhilesubstancePiswellknown

toupregulatethecoughreflexattheleveloftheNTS(Mazzoneetal., 2005;Mutoloetal.,2007),itseemsworthmentioningthatforthe firsttimeithasbeenshownthatalsobradykininwithinthecaudal NTSenhancescoughreflexresponses.

4.1. Methodologicalconsiderationandgeneralremarks

Wehavealreadydiscussedindetailsthereliabilityof microin-jections techniques and the characteristic of the spread of the injectate(Cinellietal.,2013;Mutoloetal.,2007,2008,2009,2012, 2014).Relativelyhighdrugconcentrations(mM)havebeenshown tobeasarulenecessarywhenusingmicroinjectiontechniques,as fullydiscussedinapreviousreport(Bongiannietal.,2002).The specificityofdrug-inducedeffectsissupportedbytheabsenceof changesincough-reflexresponsesinducedbybilateral microin-jectionsof thevehicle solutionintothecaudal NTS orby drug microinjections intoregions sufficientlyaway fromthe respon-sivesites.Foramoreexhaustiveappraisalanddiscussiononthe spread ofinjected drugs seealsoLipskietal., 1988;Nicholson, 1985;SykovaandNicholson,2008.Interestingly,changesinmean arterialbloodpressurethatmayaffectthecoughreflex(Poliacek etal.,2011)werenotobserved.Theabsenceofconsistentchanges inmeanarterialbloodpressurefollowingmicroinjectionsof losar-tanand lisinoprilis in agreementwithpreviousstudiesonthe effectsofdifferenttypesofACEinhibitorsorsartansmicroinjected intotheNTSofnormotensivecontrolanimals(Arnoldetal.,2010; ChitravanshiandSapru,2011;Katsunumaetal.,2003;Wangetal., 2007).Thelocalizationofresponsiveinjectionsiteswasconfirmed bythehistologicalcontrol.Itappearstocorrespondfairlywellto thatofNTSsitesofactionofagentsaffectingthecardiovascular system(e.g.,Arnoldet al.,2010;Chengetal.,2012; Mosqueda-Garciaetal.,1990;Tsengetal.,1996).Thiscorrespondencewas alsoconfirmedbyDLH-induceddepressorresponses.

4.2. Drug-inducedchangesinthecoughreﬂex

Theresultssupportourhypothesisthatlisinoprilcould potenti-atethecoughreﬂexatthecentrallevelthroughanaccumulationof protussivemediatorssuchasbradykininandsubstanceP.All com-ponentsnecessaryfortheformationandmetabolismofbradykinin and related kinins have been described in the brain, including the NTS(Bhoola et al., 1992; Diz,1985). In more detail, high-afﬁnitybindingsitesforbradykininwerefoundintheNTS(Fior and Fuxe, 1995;Privitera et al.,1992).Thebiological effects of bradykinin are mediated by its interactions with two distinct bradykinin receptor subtypes, the bradykinin B1 and B2 recep-tors.Thebradykinin B1 receptoris expressedatverylow levels andhasalimiteddistributionundernormalconditions,butcan be induced and upregulated under certain pathological condi-tions (see e.g., Albert-Weissenberger et al., 2013).Of note, the bradykininB2receptorshavebeenfoundtobeexpressedinthe NTS(Muroneetal.,1997;Priviteraetal.,1992).Thiswasthereason whyweusedtheB2receptorantagonistHOE-140tocorroborate bradykinininvolvementinlisinopril-inducedeffects.Theabsence of bradykinin-inducedpressor effects isconsistent withsimilar resultsobtainedwithlisinoprilmicroinjectionsintothecaudalNTS thatactuallymayinducebradykininaccumulation.Onthecontrary, decreasesinmeanarterialbloodpressurehavebeenpreviously obtainedbymicroinjectionsofsimilardosesofbradykininintothe medialNTSoftherat.However,thesiteofthesemicroinjections appearstobefairlydifferent,locatedmorerostrallyatthelevelof theareapostrema(Caligiorneetal.,1996).

ThepeptidesubstancePdisplaysawidespreaddistributionin boththecentralandperipheralnervoussystem.Afterbindingto theNK1 receptors,substancePregulatesmany functionsin the central nervoussystem (fora review, see Munozand Covenas,

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16 E.Cinellietal./RespiratoryPhysiology&Neurobiology219(2015)9–17 2014). In particular, at thelevel of the NTSan important role

ofsubstancePinthecentralplasticityinducedbyvagalafferent fibersaswellasincoughpotentiationhasbeensuggested(Bonham etal.,2004).Accordingly,substancePmicroinjectionsintothe cau-dalNTSpotentiatethecoughreflexinducedbybothmechanical andchemicalstimulationofthetracheobronchialtree(Mazzone etal.,2005;Mutoloetal.,2007),aneffectthatwascounteracted byintracerebroventricularadministrationofNK1receptor antago-nists(Mazzoneetal.,2005).TheantitussiveroleofNK1 receptor antagonismwas confirmedby theobservationthat microinjec-tionsof10mMCP-99,994intothecaudalNTSabolishedthecough reflexinducedbymechanicalstimulationofthetracheobronchial tree(Mutoloetal.,2008).Inthepresentstudy,relativelylow con-centrationsofthesameNK1receptorantagonistdidnotaffectthe coughreflexundercontrolconditions,butcounteracted lisinopril-inducedeffects,thussupportinganinvolvementofsubstancePin theirmediation.Inaddition,theresultsstronglysuggestthatthe completeblockadeoflisinopril-inducedcoughpotentiationeither bythebradykininB2 receptorantagonistorbytheNK1 receptor antagonistcouldbeduetoabradykinin-inducedreleaseof sub-stancePprobablyfromNTSsensoryneuronsorfromtheterminals ofprimaryafferentfibers.

Thepotentialsources of bradykinin totheNTS mayinvolve several sites in the central nervous system. Immunoreactive bradykinin-likeactivitywasdetectedinthehypothalamus, pitu-itary gland, cerebellum, cortex and, interestingly, also in the medullaoblongata(forareview,seeBhoolaetal.,1992alsofor fur-therRefs.).SubstancePmayoriginatefromdifferentsources,such asbronchopulmonaryC-fiberafferents,esophagealvagalafferents, trigeminalafferents,NTSinterneurons,andhigherbraincenters suchasmedullaryraphénuclei(forRefs.anddiscussiononthe pos-sibleroleofsubstancePseeMutoloetal.,2008).Thepresynaptic orpostsynapticsiteofactionofbradykininorsubstancePin deter-miningcoughpotentiationisamatterofspeculation.However,a probablepresynapticactionofbradykininonsensoryC-fibers lead-ingtosubstancePrelease(Bhoolaetal.,1992;Coutureetal.,2001) aswellasapostsynapticactionofsubstancePonNTSneuronsof thecoughafferentpathway(Bonhametal.,2004alsoforfurther Refs.)canbesuggested.Inagreementwiththisview,our exper-imentsshowthatthebradykinin-inducedpotentiationofcough reflexresponseswascompletelyabolishedbythepresenceofthe NK1receptorantagonistwithintheNTS.

Losartanmicroinjectionsdidnotaffectcoughreflexresponses, consistentlywithpreviousresultsobtainedbyi.v.administration (Mutoloetal.,2013).Thisoutcomestrengthensourinterpretation sinceitsinvolvementintheblockadeofAT1receptorscannotlead persetoanaccumulationofbradykininandsubstanceP.In addi-tion,theactivationofAT2receptorsbyangiotensinIImayinhibit ERK1/2activity(Fischeretal.,1998),aneffectthathasbeenproved todownregulatethecoughreflexatthelevelofthecaudalNTS (Mutoloetal.,2012).Further,theactivationofAT2receptorshas beensuggestedtohavearoleinthereleaseofendogenousopioids (Balietal.,2014)thatarewell-knowntoexertnotonlyanalgesic, butalsoantitussiveeffects(seee.g.Mutoloetal.,2008).In agree-mentwith ourinterpretation,evidencehasbeen provided that AT2receptorsarepresentintheNTS,includingitscaudalportion (Lenkeietal.,1997;LuohandChan,1998;Wangetal.,2012). Nev-ertheless,presentresultsdonotallowustoruleoutcompletely thatlosartanmicroinjectedintodifferentmedullaryareasmayhave someeffectsoncoughreflexresponses.

Consistently with our previous findings on ACE inhibitors (Mutoloetal.,2010,2013),thecoughnumberwastheonly vari-ableaffectedbylisinoprilandbradykinin.Theinterpretationofthis findingisobscure,butitcanberelatedtotheexistenceofa cen-tralgatingmechanismthatdoesnotparticipateinthecontrolof thebreathingpattern,butspecificallyregulatesthecough

num-ber(Bolser etal.,2006).By contrast,inpreviousstudiesbyour groupnotonlythecoughnumber,butalsotheintensityofother cough-relatedvariablesweremodiﬁedbyantitussivedrugs(Cinelli etal.,2013;Mutoloetal.,2007,2008,2009,2012,2014).However, thereasonsofthisdiscrepancyareatpresentonlyspeculative.

Inconclusion,theresultssupportourcontentionthatlisinopril, butnotlosartancausesapotentiationofthecoughreﬂexjustatthe levelofthecaudalNTS,themaincentralterminusofcough-related afferents,thusimplyingaroleoftheRASinthecentralregulation ofcough.TheprotussiveeffectsofACEinhibitorsmayberelated toanactiononNTSsensoryneuronsduetoabradykinin-induced releaseofsubstanceP.Sincebothpro-andantinociceptiveroles oftheRAShavealsobeendescribed(Balietal.,2014),wesuggest thatstudiesontheinvolvementoftheRASinpainandcough mod-ulationcouldleadtonoveltherapeuticapproachesespeciallyfor chronicpathologicalconditions.

Acknowledgements

This study was supported by grants from the Ministry of Education, University and Research. E. Cinelli is supported by a postdoctoral fellowship from the Fondazione Internazionale Menarini.

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