Role of Atrial Natriuretic Peptide in Oxytocin Induced Cardioprotection

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Role

of

Atrial

Natriuretic

Peptide

in

Oxytocin

Induced

Cardioprotection

Fariba

Houshmand,

PhD

a

,

Mahdieh

Faghihi,

PhD

b*

,

Saleh

Zahediasl,

PhD

c

aDepartmentofPhysiology,SchoolofMedicine,ShahrekordUniversityofMedicalSciences,Shahrekord,IslamicRepublicofIran bDepartmentofPhysiology,SchoolofMedicine,TehranUniversityofMedicalSciences,Tehran,IslamicRepublicofIran

cEndocrineResearchCenter,ResearchInstituteforEndocrineSciences,ShahidBeheshtiUniversityofMedicalSciences,Tehran,IslamicRepublicof

Iran

Received4January2014;receivedinrevisedform30April2014;accepted19May2014;onlinepublished-ahead-of-print30June2014

Introduction

Oxytocin, neurohypophyseal nonapeptide hormone, plays important roles in regulation of blood pressure, vascular toneandcardiovascularfunction[1]. Oxytocinisalso syn-thesisedincardiac myocytesand exerts important cardio-vasculareffectsdirectlyviaactiononitsreceptors[2],which arecoupledtoGproteins[1].

Previousstudyhasshownthathormone preconditioning withOTprotectstheheartagainstischaemiaandOTreceptors contributetotheprocessofcardioprotection[3].The cardio-protectiveeffectofOTmayincludedirecteffectsonthe ischae-micmyocardium.Itmayalsoindirectlyimpactthereleaseof

othermediatorssuchasatrialnatriureticpeptide(ANP).This wasbasedontheobservationsthatOTvianeuroendocrine/ endocrine/paracrinepathwaysreleaseANPfromtheheart, whichthenexertsanegativechrono-andinotropiceffectvia activationofguanylylcyclaseandreleaseofcGMP[4–6].

Natriureticpeptidesareimportantfactorsinvolvedinthe regulation of hydroelectrolyte balance and vascular tone, inducinghypotension,natriuresisanddiuresisaswellas car-dioprotectioninmyocardialischaemiabothascirculating hor-monesandaslocalautocrineand/orparacrinefactors[7].ANP exertsitsbiologicalactionsthrough,andtheinteractionwith, two specific membrane-bound guanylyl cyclase receptors: natriuretic peptidereceptor A(NPR-A) andB (NPR-B) [8].

Background Thepurposeofthisstudywastodeterminewhetherendogenousatrialnatriuretic peptide(ANP) con-tributestotheprotectiveeffectofneurohypophysialhormoneoxytocin(OT)inheartpreconditioning.

Methods Sprague–Dawleymaleratsweresubjectedto25minregionalischaemiaand120minreperfusionandwere dividedintoeightgroups.Oxytocinoranequivalentvolumeofsalinewasadministratedintraperitoneally, 30minbeforeischaemia.TheOTreceptorantagonist(atosiban),ANPreceptorantagonist(anantin)and nitricoxidesynthaseinhibitor(L-NAME)wereinjected10minbeforeOT.Inothergroups,atosiban,anantin andL-NAMEwereonlyadministered40minpriortoischaemia.

Results Comparedwiththeischaemia/reperfusiongroup(I/R),alterationsininfarctsize,biochemicalparameters [LDH(lactatedehydrogenase),CK-MB(creatinekinase-MB),MDA(malondialdehyde)plasmalevels]and severityofventriculararrhythmiaduetoI/RinjurywereattenuatedandVFwasabolishedbyOTtreatment. TheseOTeffectswereeliminatedbyOTandANPreceptorblockersandnitricoxidesynthaseinhibitor,but anantindidnotreversetheeffectofOTinlipidperoxidation.

Conclusions These findings demonstrate an important contributory role of ANP in the OT induced protection in myocardialischaemiareperfusion.OTalsoreducedlipidperoxidationwithaNO-dependentmechanism. Keywords Oxytocin  Atrialnatriureticpeptide  Ischaemiareperfusion  Oxidativestress  Preconditioning.

©2014AustralianandNewZealandSocietyofCardiacandThoracicSurgeons(ANZSCTS)andtheCardiacSocietyofAustraliaandNewZealand(CSANZ).PublishedbyElsevier Inc.Allrightsreserved.

*Correspondingauthorat:DepartmentofPhysiology,SchoolofMedicine,TehranUniversityofMedicalSciences,Tehran,IslamicRepublicofIran. Tel.:+982166419484;facsimile:+982166419484.,Email:faghihim@tums.ac.ir

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Ontheotherhand,recentevidencedemonstratedthattheANP exertsitsvasodilatoryactionthroughnitricoxide(NO) gener-ation[9].

Asoxytocin and ANPact inconcert tocontrolvascular homeostasisandthebody’sinternalenvironment,itis pos-sible that theprotective effect of OT ismediated through endogenousANP;therefore,thepurposeofthisstudywasto determinewhetherendogenousANPcontributestothe pro-tectiveeffectofOTinthepreconditioningoftheheart.Totest thishypothesis,weusedthecompetitiveNPR-Aantagonist anantin toblockany ANP-mediatedeffect.Thereasonfor thiswasduetothefactthatANPexertsitscardioprotective effectsbymeansofNPR-A/cGMPpathway[7].

Haemodynamic parameters, infarct size, biochemical indexes as well as theventricular arrhythmia assessments wereusedtoevaluatetheeffects.

Materials

and

Methods

Animals

MaleSprague–Dawleyratsweighing280–310g,were main-tained under controlledconditions (temperature 20–228C, humidity 40–50% and 12hrs light- dark cycle), fed on a standard diet with free access to water. All experimental procedureswerecarriedinaccordancewiththeinstitutional guidelinesofTehranUniversityofMedicalSciences(Tehran, Iran).

Surgical

Preparation

Theanimalswereanaesthetisedwithsodiumpentobarbital (60mg/kg,i.p.).Aftertrachealintubation,artificial respira-tionwithroomair-and-oxygenmixturewasinstitutedbyuse ofarodentventilator(model683,HarvardApparatus)witha strokevolumeofapproximated1.2ml100g1bodyweight atarateof60–70strokemin1.Therightcarotidarterywas cannulated for blood sampling and cardiac monitoring throughout the experiment, using a computerised data acquisitionsystem(ML750PowerLab/4sp,ADInstruments). Aleftthoracotomywasperformedatthefourthintercostal spacefollowedbyapericardiotomy.A6-0silksuturewas passed below theleft anterior descending coronary artery (LAD)forlaterinductionofcoronaryarteryocclusion.After 25min, thesuturewasremoved,andanimals were reper-fusedfor120min.Heartrateandbloodpressurewere sta-bilised for 10min before the following protocols were initiated. At the end of 120min reperfusion, infarct size andmyocardialenzymeleakageweremeasured.

Experimental

Protocol

Theanimalsweredividedintoeightgroups.(i)Thecontrol group(cardiacI/R):followinga10minstabilisationperiod, rats(n=10)weresubmittedto25minofischaemia,120min reperfusion. Saline was administered i.p. 30min before heartsweresubjectedtoLADocclusion.(ii)Oxytocintreated group:sixanimalsreceived oxytocin(Sigma ChemicalCo. St.Louis,MO,USA)0.01mg/rat[3],30minbeforeischaemia.

(iii)Atosiban treated group (ATO+OT, n=6);this group was pretreated with OT-receptor antagonist, atosiban (0.5mg/rat,i.p., Sigma Chemical Co. St.Louis,MO, USA) 10minbeforeOT.(iv)Anantinetreatedgroup(ANA+OT, n=7): anantin (1.2mg/kg, i.p., Sigma Chemical Co. St.Louis,MO, USA) wasadministrated10minbefore OT toexploretheroleofANPreceptor-mediatedin OT-cardio-protection. (v) L-NAME treated group (L-NAME+OT, n=7):thisgroupreceived0.2mg/kgnitricoxidesynthase inhibitor(Sigma Chemical Co.St.Louis,MO,USA), intra-peritoneally10minpriortotheOTtreatment.

In(vi)ATO(n=7),(vii)L-NAME(n=8)and(viii)ANA (n=8) groups alone,inhibitorswere used 40min priorto LADocclusion.

Haemodynamic

Variables

Arterial bloodpressure andheartrate(HR) were continu-ouslymonitoredandrecordedthroughouttheexperiment; 15minbaseline, preocclusion, 25min LAD occlusion, and 120minreperfusion.Atthesametime-points,therate pres-sureproduct(RPP),systolicbloodpressuremultipliedbythe heart rate, also were calculated by the data acquisition system.

Area

at

Risk

and

Myocardial

Infarct

Size

Determination

Theareaatrisk(ischaemic-reperfusedregion)andtheinfarct sizeweremeasuredusingEvansblueand2,3,5- triphenylte-trazoliumchloridedouble stainingmethod(TTC). Inbrief, LADwasreoccludedand2mlofa2%Evansbluesolution wasinjectedviathetailveinattheendof120min reperfu-sion.Then,theheartwasremoved,andimmediatelyfrozen at -208C. To define infarct size, hearts were sliced trans-verselythenstainedintriphenyltetrazoliumchloride(TTC, in0.1Mphosphatebuffer,pH7.4)at378Cfor15min, fol-lowed by incubation in 10% formalin for 24h to increase contrast.Totalareaatriskwasexpressedasapercentageof the left ventricles (AAR/LV). The infarct size was deter-minedas theratiobetweenthepixel ofnecroticarea(NA) and ischaemic area at risk (AN/AAR), using Adobe Photoshop7.0.

Biochemical

Analysis

Attheendofreperfusionperiod,bloodsampleswere col-lectedtomeasurethemyocardialenzymeleakage,including lactate dehydrogenase (LDH), creatinine kinase (CK-MB) andmalonyldialdehyde(MDA).Theheparinisedblood sam-pleswerecentrifugedandobtainedplasmawasanalysedby usinganautoanalyser(RocheHitachiModularDPSystems, Mannheim,Germany)fordeterminationofCK-MBandLDH levels,MDAwasdeterminedbyspectrophotometrically.

Arrhythmia

Analysis

Electrocardiograms (ECGs) were continuously monitored with standard limb lead-II throughout the experiment. Bothischaemiaandreperfusioninducedventricular arrhyth-mias were determined in accordance with the Lambeth

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Conventions[10].Prematureventricularcontractions(PVC) weredefinedasidentifiableprematureQRScomplexes. Ven-triculartachycardia(VT)wasdefinedasfourormore conse-cutiveprematureventricularcontractions(PVC).Ventricular fibrillation(VF)wasdefinedasasignalthat changedfrom beattobeatinrateand configurationorwhere individual QRS-deflectionscouldnoteasilybedistinguishedfromone another.Theseverityofarrhythmiaswasquantifiedbythe followingscoringsystem[11]0:<10PVC,1:10PVC,2:VT (duration<30s),3:VT(duration30s),4:VFstarting15min aftertheonsetofischaemia,5:VFstarting5–15minafterthe onsetofischaemia,6:VFstartingwithin5minaftertheonset ofischaemia.

Statistical

Analysis

AlldatawerepresentedasmeanSEMorthepercentageof incidence. The repeated measures ANOVA were used to compare the haemodynamic parameters within groups. One-wayANOVAfollowedbytheDuncan’stestwasused tocomparedifferencesinhaemodynamicparameters,infarct size, CK-MB, LDH and MDA plasma content between groups.P<0.05wasconsideredstatisticallysignificant.

Results

Haemodynamic

Variables

Therewere nosignificantdifferencesamonggroupsatthe baseline before treatment. In controlanimals (I/R group), coronary occlusion decreased RPP significantly (P<0.05). During 120min of reperfusion, systolic arterial pressure (SAP) and RPP reduced (P<0.05) without a significant changesintheheartrate.

Before coronary artery occlusion, neither atosiban nor anantinenorL-NAMEpretreatmentinfluencedsignificantly

thehaemodynamicparametersincomparisonwiththe base-line(datanotshown).Anantin(ANA+OTgroup)induceda significantreductioninHRascomparedtobaselinewhenOT wasadministrated(P<0.05,Table1).

In theOT group,ischaemia had nearlyno influenceon haemodynamicvariablesexceptforSAPwhichwas signifi-cantly increased compared to the control group, after a 120min reperfusion period (P<0.05). Pretreatment with anantin (ANA+OTgroup) significantly decreasedHR and RPPduringischaemia/reperfusioncomparedtotheir base-line. ItalsohadsignificantlyreducedSAPduring reperfu-sion.AscomparedtoOTgroup,anantininducedsignificant reductioninHRduringischaemia.AsshowninTable1, L-NAME(L-NAME+OT)causedsignificantincrease inRPP andSAPduringreperfusion;anantin(ANA+OT)and ato-siban(ATO+OT)alsosignificantlyenhancedSAPcompared to control this time period. No significant differences of haemodynamicparameterswereobservedinothergroups.

Area

at

Risk

and

Infarct

Size

Measurements

Fig.1representstheareaatrisk(AAR/LV)andthenecrotic area(NA/AAR)asapercentageofareaatrisk.Therewereno significant differences in the area at risk among groups. Oxytocin significantly reduced myocardial infarction com-pared with control group (P<0.05). This protectiveeffect was totally abolished by atosiban, anantin and L-NAME. Treatmentwithatosiban,anantineandL-NAMEalonehad nosignificanteffectoninfarctsizecomparedtoI/Rgroup.

Biochemical

Analysis

Comparedtothecontrolgroup,administrationofOTcould preventelevationofLDHandCK-MBactivityinplasmaafter ischaemia reperfusion injury. This reduction was signifi-cantly abolished by atosiban, anantin and L-NAME

Table1 Haemodynamicvalues.

Group Basal OT Ischaemia Reperfusion

SAP HR RPP SAP HR RPP SAP HR RPP SAP HR RPP

Control 1167 36612 433 10410 34916 374y 897y 32415 313y OT 1268 3638 462 1325 36010 473 1288 35813 435 1166* 33816 383 ATO+OT 1152 34511 401 1244 3207 401 1177 32010 373 1155* 3228 372 ANA+OT 1245 36010 453 1287 3402y 444 11010 31614y, 353y 1185* 32614y 392y L-NAME+OT 1235 34114 423 1305 3348 432 1124 3257 371 1177* 33913 403* ATO 1175 3508 412 1115 32514 362 1107 31917 353 ANA 1214 34511 423 1027 32512 322 1076 32318 342 L-NAME 1245 35512 442 1085 32816 352 1116 32611 363

SAP(systolicarterialpressure,mmHg),HR(heartrate,beats/min),RPP(ratepressureproduct,beatmin1mmHg103).OT(oxytocin),ATO(atosiban),ANA

(anantin).ValuesarepresentedasthemeanSEM.

yP<0.05,vs.baselinewithinthesamegroup. *P<0.05vs.control,

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administration. The level ofLDH wassignificantly higher thancontrolgroupinL-NAME+OTgroup(Table2).

Malonyldialdehyde (MDA) plasma level in OT treated group was significantly reduced compared to control group. When atosiban and L-NAME was administrated before OT, the levelof MDAwas significantlyincreased, but theanantin-receivinganimalshad MDAlevelsimilar tothoseoftheOTgroup.Inhibitoralonehadnosignificant effecton MDA,LDH andCK-MBplasmalevelcompared toI/R group.

Ventricular

Arrhythmias

During

Ischaemia

In the control group, all rats experienced VT, while VF occurred in80% of the hearts, and thearrhythmias score (AS)averaged4.70.2.

In OTtreatedgroup,thetotalPVCandASwere signifi-cantlydecreasedduringocclusion periodcompared tothe control(P<0.05).Inaddition,noventriculararrhythmia(VT andVF)wasobservedduringischaemiaintheOTgroup.

Oxytocin and ANP receptor blockers in ATO+OT and ANA+OT groups and nitric oxide inhibitor in L-NAME +OTgroupincreasedarrhythmiasscoreandabolishedthe cardioprotectiveeffectofOTonarrhythmias(Figs.2–4).The numberofepisodesofVTinL-NAME+OTanddurationof VTinANA+OTgroupwasmarkedlyenhancedcompared toATO+OTgroup(Fig.3).

TheincidenceofVTwas0%intheOTgroupand100%inthe control,ATO+OT,ANA+OTandL-NAME+OTgroups.

Comparedwiththecontrolrats,OTabolishedVF. Admin-istration of atosiban, anantin and L-NAME prior to OT increased the incidence of VF to 33.3% (P>0.05), 57.2% and57.1%(P<0.05)respectively,(vs. to0%inOT). More-over,durationofventricularfibrillation(VF)inANA+OT groupwasmarkedlyenhancedcomparedtocontrolgroup (Fig.4).Atosiban,anantinandL-NAMEalonehadno signif-icanteffectonventricular arrhythmias(Figs.2–4).

Ventricular

Arrhythmias

During

Reperfusion

Thedurationofventriculartachycardia(VT)intheOTgroup wasshorterthantheothergroupsandonlytwooutofsixrats

Figure1 Myocardial area atrisk (AAR/LV %)and necroticarea (NA/AAR %). OT (oxytocin), ATO (atosiban),ANA (anantin)andL-NAME.ThevaluesareexpressedasthemeanSEM.

*

P<0.05vs.control,P<0.05vs.OT.

Table2 PlasmalevelsofLDH,CK-MBandMDAatthe endofreperfusion.

Group LDH(U/L) CK-MB(U/L) MDA(nmol/ml)

Control 1836215 2273.8238 3.30.2 OT 85075* 1462138* 2.70.15* ATO+OT 923154 2604365 3.30.2 ANA+OT 1979145 2741220 2.30.15* L-NAME+OT2239160*, 2523288 3.40.25 ATO 1064109 2522256 3.10.2 ANA 1996155 2487277 2.70.2 L-NAME 1916155 2486276 3.50.3

LDH(lactatedehydrogenase),CK-MB(creatinekinase-MB),MDA (mal-ondialdehyde).OT(oxytocin),ATO(atosiban),ANA(anantin).Thevalues areexpressedasthemeanSEM.

*P<0.05,vs.control. P<0.05,vs.OTgroup.

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hadVTwithameanof0.40.2episodes.Ratspretreatedwith OTexperiencednotonlyasignificantlyreducedPVC,butalso experiencedasignificantreductionintheAS(0.80.5) com-paredwiththecontrolgroup.

TheincidenceofVTwassignificantlylessinOTandATO +OT groups than in L-NAME+OT group. The atosiban (ATO+OT)grouphadfewerPVCthantheL-NAME+OT and ANA+OT groups. Both anantin and L-NAME

administration beforeOTresultedinamarkedincrease in thedurationofVT(Table3).Atosiban,L-NAMEandanantin bythemselveshadnosignificanteffectonarrhythmias.

Discussion

In order to explore the mechanisms involved in the OT-induced early preconditioning against ischaemia and

Figure2 Distributionofthescoreofarrhythmiaandlog10PVCduringischaemia.OT(oxytocin), ATO(atosiban),ANA

(anantin).ThevaluesareexpressedasthemeanSEM. *P<0.05vs.control,P<0.05vs.OT,+P<0.05vs.ATO+OT.

Figure3 Duration(s)andepisodenumberofVT(ventriculartachycardia)duringischaemia.OT(oxytocin),ATO(atosiban), ANA(anantin).ThevaluesareexpressedasthemeanSEM.

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ventriculararrhythmiasintheratheartinvivo,we investi-gatedthepotentialroleofnatriureticpeptidepathways.In thepresentstudy,wefoundthatblockadeoftheANP recep-torsbyanantin,acompetitive NPR-Aantagonist[12], pre-ventedthecardioprotectivepropertiesofOT,indicatingthat the ANP pathway plays a prominent role in OT-induced cardiac protection. The involvement of NO in oxytocin reducedlipidperoxidationwasalsodetermined.

Althoughthereisevidencesuggestingtheprotectiverole forANPagainstI/Rinjuryintheheart,thisisthefirststudy toourknowledgethatshowsalinkbetweenOTandANPin

theregulationofprotectiononmyocardialI/Rinjury.Thisis alsosupportedbyarecentstudywhichsuggeststheroleof the oxytocin-ANP system in cardiomyogenesis [13]. In a recent study on diuresis and natriuresis effects of OT, ANP pathway involvement was proposed [6]. Similarly, othershavedemonstratedthatOTplaysanimportantrole incardiovascularhomeostasisbyregulating bloodvolume viaANPreleasefromcardiacatria[5,6].

Inthepresentstudy,OTsignificantlyreducedI/R-induced heartdamagewithlowerchangesofhaemodynamicvalues thanthoseincontrolgroup.Inspiteofvasodilatoryeffectsof

Figure4Theincidenceanddurationofventricularfibrillation(VF)duringischaemia.OT(oxytocin),ATO(atosiban),ANA (anantin).ThevaluesareexpressedasthemeanSEM.

*P<0.05vs.control,P<0.05vs.OT,+P<0.05vs.ATO+OT.

Table3 Theseverityofarrhythmiasduringreperfusion.

Group Log10PVC Durationof(s)Arrhythmia(s) EpisodeofArrhythmias Incidenceof(%) ArrhythmiaScore

VT VF VT VF VT VF Control 1.30.2 63 00 20 00 20 OT 0.60.1* 1.61.4 00 0.40.2 00 33.3 0 0.80.5* ATO+OT 0.80.3 8.14 00 0.70.3 00 50 0 1.50.2 ANA+OT 1.70.3 169 6.96.9 3.42 0.40.4 71.4 14.3 30.3 L-NAME+OT 1.20.1 15.15 00 20.6 00 100,+ 0 2.10.1 ATO 0.80.2 9.43 00 10.4 00 71 0 1.70.2 ANA 1.50.2 115 5.73.7 30.8 0.70.5 57 25 2.30.5 L-NAME 1.40.2 16.35 0.90.9 2.40.7 0.10.1 86 14.3 2.40.3

PVC(prematureventricularcontraction),VT(ventriculartachycardia),VF(ventricularfibrillation).OT(oxytocin),ATO(atosiban),ANA(anantin).Dataare presentedasmeanSEM.

*P<0.05vs.control,

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ANP, blockade of ANP receptor (NPR-A), in ANA+OT group,hadnoeffect on haemodynamicparameters before inducedocclusion, butsignificantlyreducedthese parame-tersduringI/R.In agreementwithourresults, Chowdrey etal.[14]andTakataetal.[15]havedemonstratedthatANP has no significant influence on haemodynamic variables whenitwasaddedbeforeischaemia.

TheplasmalevelofLDHandCK-MB,associatedwithheart ischaemiareperfusion injury, were also significantly lower afterreperfusionintheOTgroupthaninthecontrolgroup. Furthermore,OTpreventslipidperoxidationintheischaemic reperfused myocardium. Malondialdehude (MDA), a bio-markerofcardiacoxidativeinjury,isaproductoflipid per-oxidation that can be produced by a variety of oxidative damages[16].Biyiklietal.hasbeenreportedthatOTreduces oxidantrenalinjuryinpyelonephriticratsbyitsantioxidant actions[17].

In addition, OT exerts antioxidant effects on vascular smoothmusclecells,aorticECs,andmacrophagesthrough attenuationofNADPH-oxidase-dependentsuperoxide pro-duction[18].TheantioxidantabilityofOTwasalso respon-siblefortherenoprotectivepropertiesofOT[19].

Inourstudy,unlikeincreasedLDHandCK-MBlevels,the plasmalevelofMDAwasnotsignificantlychangedby anan-tin,ascomparedtoOT.Therefore,possiblyothersignalling pathway(s)thanANPareinvolvedinantioxidativeproperties ofOT.On the otherhand, OTreceptoractivationandNO productionhave asignificantroleinthiseffectofoxytocin. Nitricoxidecanfunctionasaninvivoantioxidant[20];hence, OT,throughNO,mayfunctionasaneffectiveantioxidant.

OT-inducedinfarctsizereductionwasreversedbyanantin and L-NAME. These results may imply that infarct size reductioninresponseto oxytocinmediated byOT/ANP/ NO pathway,as it was previously demonstrated that OT leadstothereleaseofANP,whichthenstimulatestheNO production[9].Inagreement,Okawaetal.demonstratedthat NOis involvedinthe ANPinduced infarctsize reducing effectthroughNO-mitochondrialATP-sensitiveK+(mK

ATP)

channel-dependentpathway[21].

However, in the control group, both regional coronary occlusionanditsreperfusioninducedventriculararrhythmia, reperfusion of the ischaemic myocardium led to less severity of arrhythmias in all groups under study. The results of the present study showed that OT completely preventedepisodes of VF inducedboth in occlusion and reperfusionperiod.Oxytocin significantlydecreasedPVC and lowered the arrhythmias severity index compared withcontrolandpreventedVTinocclusionperiod. Treat-ment with atosiban abolished OT-reduced arrhythmia, indicating a receptor-mediated effect. Our data also revealedthatblockadeof ANPand NOpathway(s) abol-ishedthisantiarrhythmiceffect.Thisfindingdemonstrates the role of ANP and NO system on the anti-arrhythmic effects of OT during both ischaemia and reperfusion phases.Althoughdrug(atosiban,anantinandL-NAME) pre-treatment prevented OT effectsand increased the severity of lethal arrhythmias during ischaemia and reperfusion

periods, the major effectwasobserved inischaemiaphase assignificantincreaseinVTandVF.Ontheotherhand,since during the reperfusion, VF was increased by anantin, we assumethatANPpathwayplaysanimportantrolein anti-VFeffectsofOT.

Itseemsthatanti-lipidperoxidationpropertyofOT, func-tioningasanantioxidantisnotthecauseofantifibrillation effect,becausenatriureticpeptideisnotinvolvedinthisOT action.IncontrasttoANP,nitricoxidenotonlyeliminated the anti-lipid peroxidation effect of oxytocin but also decreasedtheproductionoffibrillation.Sinceanti-VFeffect ofoxytocinappearstobeindependentoflipidperoxidation, thus, possibly,anti-lipidperoxidation,anti-arrhythmicand anti-ischaemiceffectsofOTinducedbydifferentpathways (ormechanisms).

Inaddition,thecardioprotectiveactionofANPinI/Rinjury was shown to be mediated through the nitric oxide [21], elevationofcGMPlevels[22]andbyPKC,PKGdependent transductionmechanisms[21,23]followedbymKATPchannel

activation.Okawaetal.alsodemonstratedthatpreischaemic infusionofsyntheticANPhasmyoprotectiveeffectsagainst I/R throughNO-mKATPchannel-dependentpathway[21].

Then,itispossiblethatOTeffectonmKATPchannels[24]was

indirect and comes from the ANPaction on this channel. Furtherstudiesarerequiredtodiscriminatethespecificrole ofOTandANPinmediatingKATPchannelactivity.

Based on our observation, anantin abolished OT effects through blockade of NPR-A receptor activation, which is coupled toanincreaseintheintracellular concentrationof cGMP. Thus, decreasedcGMP generationby inhibition of NPR-AreceptormaybeinvolvedinabolishingOT precon-ditioning.However,theliteraturehasproposedan involve-mentofcGMPasamessengerinmultiplephysiologicactions ofOT[1],butthedataobtainedinthisstudydonotallowus to conclude the involvement of cGMP-mechanism in the OT-preconditioneffect.

Additionally,intracardiacOTisessentialforbasalrelease ofANPintheheart[6]andOTreceptorsareassociatedwith theANP-cGMPandNO-cGMPpathways,whichreducethe forceandrateofcontractionandincreasevasodilatation[1]. Sincenosignificantdifferenceswereobservedbetweenthe twogroupsofANA+OTandL-NAME+OTininfarctsizeand ventriculararrhythmia,itispossiblethatOTexerts cardio-protectiveeffectsthroughOT/ANP/NOpathways.In addi-tion, unlikeL-NAME, anantin cannot change theeffect of oxytocininreducinglipidperoxidation.Therefore,the pos-siblemechanismofOT/ANP/NOpathwayisnotinvolved inboththeanti-ischaemicandanti-lipidperoxidationeffects ofoxytocin.Hence,basedonourfindings,itseemsthatOT exerts cardioprotective effects through OT/ANP/NO and OT/NOpathway independently;however, further investi-gationsareneeded.

Conclusion

Althoughfurtherstudiesareneededtoclarifythe contribu-tion of other transduction pathways or mechanisms, our

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findingsindicatethatOTexertsitsprotectiveeffectthrough ANPpathwayandalsoviaattenuatinglipidperoxidation.

Acknowledgments

Thisstudywasfinanciallysupportedbythegrantno.82from TehranUniversityofMedicalSciences.

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