resistance trained aged rats in the right atrial cardiomyocytesof Glutamine supplementation inﬂuences the secretoryapparatus CIÊNCIAS DO ESPORTE

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www.rbceonline.org.br

Revista Brasileira de

CIÊNCIAS DO ESPORTE

ORIGINAL ARTICLE

Glutamine supplementation inﬂuences the secretory apparatus in the right atrial cardiomyocytes

of resistance trained aged rats

Romeu Rodrigues de Souza

^a^,^b^,^c^,∗

, Cristiano Ferreira Pacheco

^c

, Erico Chagas Caperuto

^d^,^c

, Laura B.M. Maifrino

^d^,^c

, Eliane F. Gama

^d^,^c

aUniversidadeSãoJudasTadeu,CursodePós-Graduac¸ãoemCiênciasdoEnvelhecimento,SãoPaulo,SP,Brazil

bUniversidadedeSãoPaulo,DepartamentodeAnatomia,SãoPaulo,SP,Brazil

cUniversidadeSãoJudasTadeu,DepartamentodeAnatomia,SãoPaulo,SP,Brazil

dUniversidadeSãoJudasTadeu,CursodePós-Graduac¸ãoemEducac¸ãoFísica,SãoPaulo,SP,Brazil

Received25November2017;accepted11August2018 Availableonline16October2018

KEYWORDS Atrialmusclecells;

Aminoacid supplementation;

Anaerobictraining;

Natriureticpeptide

Abstract Weinvestigatedtheeffectsofglutaminesupplementationonthesecretoryappara- tusofnatriureticpeptidesinatrialcardiomyocytesofagedratsundergoingresistancetraining.

Twogroupsofresistance-trainedratswerestudied:resistancetrained,andresistancetrained andsupplementedwithglutaminegroup.Bothgroupsofratsweretrainedtoclimba1.1m vertical ladder withweights tied totheir tail. The cardiomyocytesfrom resistancetrained andsupplemented ratsshowed increaseddensityandsectional areaofnatriureticpeptides granules,higherrelativevolumesofthemitochondria,endoplasmicreticulum,Golgicomplex andnucleareuchromatin,andnuclearporedensitycomparedwithresistancetrainedrats.In conclusion,glutaminesupplementationcausedhypertrophyofthesecretoryapparatusinthe cardiomyocytesofagedratsundergoingresistancetraining.

PALAVRAS-CHAVE Célulasmuscularesdo átrio;

Suplementac¸ãode aminoácidos;

Treinamento anaeróbio;

Peptídeonatriurético

Asuplementac¸ão deglutaminainﬂuencia oaparelho secretordos cardiomiócitosdo átriodireitoderatosidosossubmetidosatreinamentoderesistência

Resumo Foi investigado oefeito dasuplementac¸ãode glutaminanoaparelho secretorde peptídeosnatriuréticosdoscardiomiócitosdoátrioderatosidosossubmetidosatreinamento de resistencia. Foramestudados dois grupos:grupo de treinamento deresistência e grupo de treinamento de resistência suplementado comglutamina. Osratos de ambos os grupos escalaramumaescadaverticalde1,1mcompesosprogressivamentemaioresatreladosàcauda.

∗Correspondingauthor.

E-mail:souzarrd@uol.com.br(R.R.Souza).

https://doi.org/10.1016/j.rbce.2018.08.005

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Osresultados mostraram queoscardiomiócitos de ratosdogrupotreinado esuplementado apresentaram maiordensidade emaior áreade seção degrânulos depeptideos natriuréti- cos,maioresvolumesrelativosdemitocôndrias,retículoendoplasmático,complexodeGolgi eeucromatina nuclear emaiordensidadede porosnuclearesem comparaçãocomratosdo grupodetreinamentoderesistência.Emconclusão, asuplementaçãocomglutaminacausou hipertrofiadoaparelhosecretordoscardiomiócitosderatosidosossubmetidosaotreinamento deresistência.

by-nc-nd/4.0/).

PALABRASCLAVE Célulasdelmúsculo delatrio;

Suplementaciónde aminoácidos;

Entrenamiento anaeróbico;

Péptidos natriuréticos

Lasuplementacióndeglutaminainfluyeenelaparatosecretordeloscardiomiocitos delaaurículaderechaderatonesviejossometidosaentrenamientoderesistencia Resumen Seinvestigólainfluênciadelasuplementacióndeglutaminaenelaparatosecretor depéptidosnatriuréticosdecardiomiocitosauricularesderatonesviejossometidosaentre- namiento de resistencia.Se formarondos grupos:grupo de entrenamientode resistencia y grupodeentrenamientoderesistenciasuplementadoconglutamina.Losratonesescalaronuna escaleraverticalde1,1mconpesosatadosalacola.Losresultados mostraronqueloscar- diomiocitosderatonesdelgrupodeentrenamientoderesistênciasuplementadopresentaron mayordensidadyáreadeseccióndegránulosdepéptidosnatriuréticos,mayoresvolúmenes relativosdemitocondriasydeeucromatinanuclearymayordensidaddeporosnuclearesen comparaciónconlosratonesdelgrupodeentrenamientoderesistencia.Enconclusión,lasuple- mentacióndeglutaminacausóhipertrofiadelaparatosecretorenloscardiomiocitosderatones viejossometidosalentrenamientoderesistencia.

by-nc-nd/4.0/).

Introduction

It is well known that feeding athletes before and/or immediately after training with nutritional supplements such as creatine, l-carnitine and l-glutamine positively affectsstrengthdevelopmentandresistancetrainingrecov- ery (Raastad et al., 2000; Williams et al., 2002; Volek andRawson, 2002; Rawsonand Volek, 2003; Volek, 2004;

Kraemeretal.,2007;Bentetal.,2011).Amongnutritional supplements,glutamineisoneofthemostusedbyathletes (Novellietal.,2007).Glutaminesupplementationincreases musclecellvolumeandstimulatesproteinandglycogensyn- thesis(Varnieretal.,1995;Lowetal.,1996;Antonioand Street,1999;Fontanaetal.,2003;Gleeson,2008;Coqueiro etal.,2017).

Atrialmusclecells(cardiomyocytes)produce,store,and secrete natriuretic peptide hormones (NP). NP plays an important role in the normal regulation of arterial blood pressure(Debold,1999;DanielsandMaisel,2007;Casserly, 2010; De Vito, 2014). Previous studies showed that spe- ciﬁcatrium granules contain NP pro-hormones (O’Donnell etal.,2003).TheproductionandsecretionofNPsintothe bloodstreamdependsonthestructuralcomponentsofthe cardiomyocyte,namedthesecretory apparatusofthecar- diomyocyte(Maksimovetal.,2004;DeSouzaetal.,2014).

Previousstudieshaveshownthatthefunctionofthesecre- tory apparatusof cardiomyocytes is inﬂuencedby several factorssuchasfattyacids, hypertension,proteindepriva- tion,training,hormones,hydrogenperoxideandglutamine (Jingetal.,2000;Maksimovetal.,2004;Gamaetal.,2007;

Pan,2008;Firmesetal.,2012;DeVitoetal.,2013;Ferro etal.,2013;DeSouzaetal.,2014;DeSouzaetal.,2015).

Thepurposeofthepresentstudywastoextendprevious findingsintwoimportantways.First,itisknownthatdur- ingaerobictraining,bloodpressurerises,whichcausesan increaseinthesecretionofNPsinthebloodstream(Marie et al., 2004). A previous study of young animals demon- strated thatglutamine significantlyenhancescardiacANP, thus implicating the beneficial effects of glutamine supplementation tothe ANP system (De Souza et al., 2015).

However,therearenostudiesofNPsystem,whereagedani- malsundergoingresistancetrainingandsupplementedwith glutamine are compared with non-supplemented trained controls. The ﬁrst objective of the present study was to observetheinﬂuenceofglutaminesupplementationonthe effectsofresistancetrainingonthenumberandsizeofNP granulesofagedrats.

Thesecond objective ofthis workwastoevaluate the effectsofglutaminesupplementationonthesecretoryappa- ratus of the atrial cardiomyocytes of resistance trained rats, includingthe number ofpores per 10␮m of nuclear

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membrane,therelativevolumes(%)ofeuchromatin,mitochondria, endoplasmic reticulum and Golgi complex. We hypothesizethatsupplementationcouldenhancetheeffects ofresistancetrainingbypromotinghypertrophyofthesecre- toryapparatusincardiomyocytesandinﬂuencingpositively thesynthesisofNPs.

Materials and methods

SixteenagedmaleWistarrats(15monthsold)wereobtained fromtheInstituteofBiomedicalSciences,UniversityofSão Paulo, São Paulo, Brazil. Rats were maintained at 23^◦C underacycleof12hlight/12hdarkness.Attheendofthe 15thmonth,ratswererandomlyassignedtotheresistance trainedgroup(RT,n=8)orresistancetrainedsupplemented withglutamine (RTG,n=8)group. An aqueoussolutionof l-glutaminewasgiventoratsfromtheRTGgroupbygavage (1gkg⁻¹bodyweightin3mLsaline)1hbeforethetraining sessionaccording toShewchuk etal.(1997) andLagranha et al. (2004). Glutamine solution was freshly prepared before administrationto avoid glutamine hydrolysis. Rats fromRT group received 3mL of saline (0.1mol/L citrate, pH 4.5) as placebo. The University Ethics Committee approved handling of animals (Ethical Protocol number 015/2006), in accordance with the International Guiding PrinciplesforBiomedicalResearchinvolvingAnimals.

Trainingprotocol

Allanimalsunderwentapre-adaptationtothetrainingpro- tocolandequipmentfor ﬁvedays.Theequipmentusedto carryoutthestrength-trainingprogramwithratswasaver- ticalladdermadeofwoodwithironsteps.Theequipment (ladder) heightis 110cm(43.3 inches)withan inclination angleof80^◦.Onthetopoftheequipment,thereisaplastic boxlinedwithnewspaperfortherats’accommodationinthe intervalbetween sets (Campbell etal., 1998; Hornberger andFarrar,2004).Thetrainingprotocolwasprogressive,and theloadwasadjustedeveryweek.Theloadwascomposed ofleadweightsattachedtotherats’tailswithaVelcrotape.

Animalsweresupposedtoclimbtheladdertoreachtherest- ingareaatthe top,whichwasconsideredonerepetition.

Theadaptationprocesslastedoneweekwithsixrepetitions everyday. Thetrainingof animalsincludedsix continuous repetitionsperday,witharestintervalof45sbetweenrepe- titions,ﬁvedaysaweekfor12weeks.Theloadincreasewas establishedfromHeyward’sproposal(Heyward,1998).After measurement of the maximum weightlifted (1 maximum carriedloadtest),thetrainingloadwassetat60%of1max- imumcarriedloadtest.Astheloadwasrelatedtotheweight ofanimals,everyweekallanimalswereweighed,andtheir loadswereadjustedaccordingtotheirbodyweight.

BodyweightandSystolicBloodPressure measurement

Atthemomentofthesacriﬁce,(i.e.,18monthsofage)rats were anesthetized (ketamine-xylazine 80:40mg/kg i.p.), weighed,andbloodpressurewasevaluatedbyindirectmea- surementusingthetail-cuffmethod(Brittoetal.,1997).

DeterminationofthenumberandsizesofNP granules,andtherelativevolumes

oftheendoplasmicreticulum,mitochondria, andGolgicomplex

Underanesthesia, trainedanimals were heparinizedprior tofixationtooptimizeperfusion-fixation.Therightandleft atriawere perfused through the left and right ventricles at a constant pressure of 80mmHg, using 0.1M cacody- latebuffer(3min)followedby2.5%glutaraldehydesolution dilutedinthesamebuffer.Next,theright atriumwasiso- latedanddividedintoslicesapproximately3mmwideand 5mm long. These tissue sliceswere post-fixed in osmium tetroxide in sodium cacodylate buffer for 1h. The tissue wasdehydratedingraded alcoholsandembedded inEpon resin, and sectioned so that myocytes were cut in lon- gitudinal section. Thin sections for transmission electron microscopywerestainedwithuranylacetate,andleadcit- rate(Mifuneetal.,2004).Tworandomlychosenblocksfrom eachatrium,inwhichthe myocytesweresectionedlongi- tudinallywereusedforquantitativeanalysis.Theultrathin sectionswereplacedonacoppergridandrandomlychosen fieldswereselectedformicrographstakenwithaJeoltrans- missionelectronmicroscope.

Tenelectron micrographs per animal weretaken, then chosenbysystematicrandomsamplingofsquaresatafinal magnificationof15,000×,andwereusedtoobtainthefol- lowing: the number of granules per 96␮m², the area of granules (␮m²) and the relative volume of mitochondria, endoplasmic reticulum, and Golgi complex. The number of granules present in each field wasdetermined and its areameasuredusinganimageanalysisprogram(AxioVision, Zeiss).

Determinationofthenumberofpores inthenuclearmembrane

Tenelectronmicrographsperrat,examinedwithaﬁnalmag- niﬁcationof15,000×wereusedtodeterminethenumber ofporesper10␮mofnuclearmembrane.

Calculationoftherelativevolumes

Therelativevolumeofastructurecorrespondstothearea occupiedbythestructurepresentedasa%.Itwasobtained usingatestsystemequippedwith110points(consideredas 100%),whichwasallocatedoneachelectronicimagewhere thepointsforeachcomponentwerecounted(Mandarim-De- Lacerda,2003)(Fig.1).

The following formula wasused toobtain therelative volumes:Vv[struct]=P[struct]100/PT;whereVv[struct]=relative volume,P[struct]=numberof pointsthathitthe structure in question, and PT=total number of points (110) of the testsystem(Gundersen,1986;MayhewandLucocq,2008).

Themeasurementswereperformedusingtheimageanalysis program,AxioVision(Zeiss,2009).

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Figure1 Showsthetestsystem(A)equippedwith110points(consideredas100%),whichwasallocatedontheelectronicimage wherethepointsforeachcomponentwerecounted(B).N,nucleus;M,mitochondria;ER,endoplasmicreticulum;G,Golgicomplex.

Table1 Bodyweightandsystolicbloodpressureinthetwo studiedgroupofratsafter12-wkoftraining.

Parameters RT RTG p-Value

BW(g) 408±42 412±38 >0.05

SBP(mmHg) 112±10 115±14 >0.05 Dataareexpressedasmeans±SE.

Student’sttestwasusedforthestatisticalanalysis.

BW,bodyweight;SBP,systolicbloodpressure.

Statisticalanalysis

Allresultsaremeans±SE.Datafromthetwogroupswere comparedusingunpairedStudent’st-testwithp<0.05asthe levelofsigniﬁcance.TheGraphPadPrism5program(Graph PadSoftware,SanDiego,CA,USA)forWindowswasusedfor dataanalysis.

Results

Thebodyweightandthemeansystolicbloodpressuremea- sured at the end of the experiment at rest showed no signiﬁcantdifferencesbetweengroups(p>0.05)(Table1).

Onerepetitionmaximum

Fig.2shows an increasein the absolute valuesof weight liftedbythe trainedgroupthatwere obtainedduringthe repetitionmaximumtest.RTandRTGratshadsimilarvalues forrepetitionmaximumatthebeginningoftheexperiment (day0).After6weeks,theloadliftedbyratsintheRTgroup was19%higherthantheloadliftedinthefirsttest,andthat liftedbyratsintheRTGgroupwas22%higherthanthatin thefirst test (p<0.05).At the end of 12weeks, the load liftedbyRTratswas24%higherthanthatinthefirsttest, andthatliftedbyratsintheRTGgroupwas26%higherthan thatinthefirsttest(p<0.05).Insummary,atallstagesof training,theloadliftingcapacitywasalwayshigherinthe RTGgroupthanintheRTgroup.

*

**

1500

1000

500

0

0 6 12

RTG RT

RM (g)

Weeks

Figure2 Valuesfor1repetitionmaximumtest.Resultsare mean±SD. *p<0.05, compared with the RT group at 6 mo.

**p<0.05,comparedwiththeRTgroupat12mo.Groupswere comparedusingone-wayANOVAandStudent’sttest.

NumberandsizesofNPgranules

Cytoplasmic secretory granules were predominantly arranged in groups among the mitochondria (Fig. 3) and neartheendoplasmicreticulumandGolgicomplex(Fig.4).

ThenumberofNPgranules/cardiomyocytewassigniﬁcantly higher in RTG rats (64±3) compared to RT rats (53±4) (p<0.05). The number and size (areas) of granules was signiﬁcantlyhigherinRTGrats(0.09±0.01)␮m²)compared toRTrats(0.13±0.02)(p<0.05)(Fig.5).

RelativevolumeofGolgicomplex,mitochondria, andendoplasmicreticulum

RTG rats showed higher relative volumes of the Golgi complex,mitochondria,andendoplasmicreticulum(%)com- paredwiththeRTgroup(inallcases,p<0.05)(Table2).

Numberofporesinthenuclearmembrane

In the two groups, the euchromatin appeared disperse, whereastheheterochromatinappearedasaperipheralband nearthenuclearmembrane(Fig.4).Theultrastructuralsec- tionsshowedthenuclearmembraneastwoultrathinparallel membranesshowing pores asspaces where the inner and outernuclearmembranesjoin(Fig.4).Thedensityofpores

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Figure3 ElectronmicrographsofleftatriumcardiomyocytesinRT(A)andRTG(B)rats.IntheRTGrat(B)moregranulesare observedinthecytoplasmthanintheRT.N,nucleus;M,mitochondria.

Figure4 ElectronmicrographsofleftatriacardiomyocytesintheRT(A)andRTG(B)ratsobservedwithhighermagniﬁcation.

NPgranuleswithvariedsizes(whitearrows)aredispersedamongmitochondria(M),endoplasmicreticulum(ER)andGolgicomplex (G).Thenucleus(N)showstheeuchromatindisperse(E)andtheheterochromatin(HE)asabandnearthenuclearmembrane.The nuclearmembraneappearsastwothinmembranesshowingthenuclearpores(blackarrows).

* *

80

60

40

20

0 0.00

0.05 0.10 0.15

RT RTG RT RTG

Number of grannules cardiomiocyte Section area for grannule (µm2)

Figure5 Meandatashowingtheeffectsofglutaminesupple- mentationonthenumber(A)andsize(B)ofNPgranulesinRA cardiomyocytesofrats.RT,resistancetrainedgroup;RTG,resis- tancetrainedandsupplementedwithglutaminegroup.*p<0.05 vs. RT. Values aremeans±SD. Student’s t-test was used for statisticalanalysis.

inthenuclearmembranewassigniﬁcantlyhigherintheRTG groupcomparedtotheRTgroup(p<0.05)(Table2).

Discussion

There are two major ﬁndings in the present study. First, theNPlevelsintheatrialcardiomyocytesweresigniﬁcantly increasedinresistancetrainedagedanimalssupplemented withglutaminecomparedtothetrainednon-supplemented

Table 2 Morphometric indexes of the cardiomyocyte nucleusandcytoplasmintherightatrialcardiomyocytesof RTandRTGrats.

Morphometricindexes RT RTG p-Value RVGC(%) 2.8±0.2 3.9±0.2^a 0.05

RVM(%) 25±4 32±2^a 0.05

RVER(%) 69±5 78±3^a 0.05

Npo/10␮mNM 4.6±0.2 6±0.2^a 0.05 RT,resistancetrainedgroup;RTG,resistancetrainedandsup- plementedwithglutaminegroup;RVGC,relativevolumeofGolgi complex;RVM,relativevolumeofmitochondria;RVER,relative volume ofendoplasmicreticulum;NPo/10␮mNM, numberof pores/10␮mofnuclearmembrane.

a p<0.05vs.RTrats.

group,whichindicatesthatglutamineenhancestheeffects of resistance training on the atrial biosynthetic process.

Second,resistancetrainedagedratssupplementedwithglu- tamineexhibitedasigniﬁcantincreasein relativevolumes forcomponentsofthesecretoryapparatusinRAcardiomy- ocytescomparedtoresistance-trainedcontrols.Presented datasuggestthatglutaminesupplementationinducedadis- tinctpatternofthesecretoryapparatus.

In the present study, resistance trained aged animals supplementedwithglutamineshowedasigniﬁcantimprove- mentinthenumberandsizeofcardiacNP granuleswhen compared to the RT group. The mechanism by which glutamine associatedwith resistance trainingsigniﬁcantly

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increased NP levels in cardiomyocytes is unknown. It is knownthatglutamineis themostabundant aminoacidin plasma,aswellasinmuscle,andaccountsformorethan60%

ofthetotalintramuscularfreeaminoacidpool(Antonioand Street,1999;Kreider,1999;Gleeson,2008).Itisalsoapre- cursorforthesynthesisofproteins,nucleotides,andmany other biological molecules (Smith, 1990; Watford, 2000;

CurIet al.,2005).Studies in skeletal muscleshowedthat glutamine supplementation contributesas a substrate for gluconeogenesisbyresultingin increasedcellcomponents (AntonioandStreet,1999; Fontanaetal.,2003; Coqueiro etal.,2017).Itispossiblethatthesamemechanismcould beresponsiblefortheresultsofthepresentstudy.

Inaddition,anothermechanismforenhancedcardiomy- ocyteproductionofNPsobservedinsupplementedtrained rats might relate to oxytocin. During resistance training, thereis an increase in blood pressure that promotes the atriumwalldistentionandcausescardiomyocytestosecrete NPintotheplasma,decreasingNPlevelsincardiomyocytes (Schiebingerand Greening, 1992; Seul etal., 1992; Ohba et al., 2001; Chien et al., 2008). After training, as car- diacoxytocinreceptorofcardiomyocytesiscoupledwithNP release(Gutkowskaetal.,1997),thehigherproductionof NPsmaybeduetoactivationofcardiacoxytocinreceptor and subsequent improvement of NP synthesis (Gutkowska et al., 2007). Possibly, this mechanism was enhanced by glutamine,which mayhave inﬂuencedthe increaseof NP productioninsupplementedrats.

In the present study, resistance trained rats receiv- ingglutamineexhibitedthemoresigniﬁcant enhancement of components of cardiomyocyte secretory apparatus (mitochondria,endoplasmicreticulum, Golgicomplexand numberofporesinthenuclearmembrane)comparedwith theRTgroup,whichindicatesthatglutamineincreasesthe effects of resistance training on the secretory apparatus of NPsin cardiomyocytes. The effect of supplementation with glutamine on the secretory apparatus of cardiomyocytes in trained animals can be explained in two ways.

First,resistancetrainingisassociatedwithsigniﬁcanteleva- tionsinanabolichormones.Therearefourmajoranabolic hormones that indirectly or directly affect the secretory apparatusandproteinsynthesisofcardiomyocyte.Theyare thegrowthhormone(GH),insulin-likegrowthfactor-1(IGF- 1)andtestosterone(KraemerandRatamess,2005;Kraemer etal.,2007),whichincreaseproteinsynthesis(Herbstand Bhasin,2004;Olsen etal., 2006).Second, severalstudies indicatethatglutaminesupplementationstimulatesprotein and glycogen synthesis (Varnier et al., 1995; Low et al., 1996;AntonioandStreet,1999).Probably,theeffectsofglu- taminesupplementationonthesecretoryapparatusofNPs incardiomyocytesofresistancetrainedanimalsobservedin thepresentstudycouldberelatedtothesetwomechanisms.

Conclusion

Inconclusion,theresultsofthepresentworkindicatethat inagedratswithnormalarterialpressureundergoingresis- tancetraining,glutamineingestion promoted hypertrophy ofatrialcardiomyocytesandintensivesynthesisofNPs.

Funding

UniversidadeSãoJudasTadeu.

Conﬂicts of interest

Theauthorsdeclarenoconﬂictsofinterest.

References

AntonioJ,StreetC.Glutamine:apotentiallyusefulsupplementfor athletes.CanJApplPhysiol1999;24:1---14.

BentRR,NygaardH,RaastadT.Physiologicalelevationofendoge- noushormonesresultsinsuperiorstrengthtrainingadaptation.

EurJApplPhysioly2011;111:2249---59.

BrittoRR,SantosRAS,Fagundes-MouraCR,KhoslaMC,Campagnole- Santos MJ. Role of angiotensin-(1-7) in the modulation of thebaroreﬂexinrenovascularhypertensiverats.Hypertension 1997;30:549---56.

Campbell DJ, Anastasopoulos F, Duncan A-M, James GM, Kladis A,BriscoeTA.Briscoe-effectsofneutralendopeptidaseinhibi- tionandcombinedangiotensinconvertingenzymeandneutral endopeptidaseinhibitiononangiotensinandbradykininpeptides inrats.JPharmacolExpTher1998;287:567---77.

CasserlyB.CardiacatriaaretheprimarysourceofANPreleasein hypoxiaadaptedrats.LifeSci2010;87:382---9.

ChienJY,LinMS,HuangYCT,ChienYF,YuCJ,YangPC.Changesin B-typenatriureticpeptideimproveweaningoutcomepredicted byspontaneousbreathingtrial.CritCareMed2008;36:1421---6.

Coqueiro AY,Raizel R, Hypólito TM, TirapeguiJ. Effects ofsup- plementation with L-glutamine and L-alanine in the body compositionofratssubmittedtoresistanceexercise.RevBras CienEsport2017;39(4):417---23.

CurIR,LagranhaCJ,DoiSQ.Molecularmechanismsofglutamine action.JCellPhysiol2005;204:392---401.

Daniels LB, Maisel AS. Natriuretic peptides. J Am Coll Cardiol 2007;50:2357---68.

Debold MLK. Estrogen, natriuretic peptides and the renin---angiotensinsystem.CardiovascRes1999;1:524---31.

DeSouzaRR,DeOliveiraVC,Pithon-CuriTC,MaldonadoD.Effects ofovariectomy onthesecretory apparatusintheright atrial cardiomyocytesofmiddle-agedmice.Clinics2014;69:554---8.

DeSouza RR,Caldeira CAV, CarbonePO, PiancaEV.Inﬂuence of glutamineontheeffectofresistanceexerciseoncardiacANPin rats.RevBrasCiencEsp2015;37:74---9.

DeVito P,Incerpi S,AffabrisE,Percario Z,BorgattiM, Gambari RZ,PedersenJ,LulyP.Effect ofatrialnatriuretic peptideon reactiveoxygenspecies-inducedbyhydrogenperoxideinTHP- 1 monocytes: role in cell growth. Migrat Cytokine Rel Pept 2013;50:100---8.

De Vito P. Atrial natriuretic peptide: an oldhormone or a new cytokine?JPept2014;58:108---16.

FerroM,WitterC,DeSouzaRR,BuritiMA.Meta-analysisaboutAtrial NatriureticPeptide(ANP).JMorpholSci2013;30:121---5.

Firmes LB, Belo NO, Reis AM. Conjugated equine estrogens and estradiolbenzoatedifferentiallymodulatethenatriureticpep- tide system in spontaneously hypertensive rats. Menopause 2012;20:554---60.

Fontana KE,ValdesH,Valdissera V.Glutamina comosuplemento ergogênico.RevBrasCiênMov2003;11:91---6.

GamaEF,LibertiEA,DeSouzaRR.Effectsofpre-andpostnatalpro- teindeprivationonatrialnatriureticpeptide(ANP)granulesof therightauricularcardiocytes.Anultrastructuralmorphometric study.EurJNutr2007;46:245---50.

Gleeson M.Dosingand efﬁcacyof glutamine supplementationin humanexerciseandsporttraining.JNutr2008;138:2045S---9S.

(7)

GundersenHJ.Stereologyofarbitraryparticles.Areviewofunbi- asednumberandsizeestimatorsandthepresentationofsome new ones, in memory of William R. Thompson. J Microsc 1986;143:3---45.

GutkowskaJ,JankowskiM,LambertC,Mukaddam-DaherS,Zingh HH, Mccann SM. Oxytocin releases atrial natriuretic peptide by combining with oxytocin receptors in the heart. PNAS 1997;94:11704---9.

GutkowskaJ,PaquetteA,WangD,LavoieJM,JankowskiM.Effect ofexercisetrainingoncardiacoxytocinandnatriureticpeptide systemsinovariectomizedrats.AmJPhysiolRegulIntegrComp Physiol2007;293:R267---75.

HerbstKL,BhasinS.Testosteroneactiononskeletalmuscle.Curr OpinClinNutrMetabCare2004;7:271---7.

HeywardVH.Practicalbodycompositionassessmentforchildren, adults,andolderadults.IntJSportNutr1998;8(3):285---307.

Hornberger TAJ,Farrar RP.Physiological hypertrophy oftheFHL musclefollowing8weeksofprogressiveresistanceexercisein therat.CanJApplPhysiol2004;29:16---31.

JingX,Kang,Leaf A. Preventionoffatal cardiacarrhythmiasby polyunsaturatedfattyacids.AmJClinNutr2000;71:202S---7S.

KraemerWJ,RatamessNA.Hormonalresponsesandadaptationsto resistanceexerciseandtraining.SportsMed2005;35:339---61.

KraemerWJ,HatﬁeldDL,SpieringBA,VingrenJL,FragalaMS,Ho JY,VolekJS,AndersonJM,MareshCM.Effectsofamulti-nutrient supplementonexerciseperformanceandhormonalresponsesto resistanceexercise.EurJApplPhysiol2007;101:637---46.

KreiderR.Dietarysupplementsandthepromotionofmusclegrowth withresistanceexercise.SportsMed1999;27:97---110.

Lagranha CJ, Senna SM, De Lima TM, Silva E, Doi SQ, Curi R, Pithon-Curi TC. Beneﬁcial effect of glutamine on exercise- induced apoptosis of rat neutrophils. Med Sci Sports Exerc 2004;36:210---7.

LowSY,TaylorPM,RennieMJ.Responsesofglutaminetransportin culturedratskeletalmuscletoosmoticallyinducedchangesin cellvolume.JPhysiol(London)1996;492:877---85.

Mandarim-De-Lacerda CA. Stereological tools in biomedical research.AnaisAcadBrasCienc2003;75:1---25.

MifuneH,HondaJ,TakamoriS,SugiymaF,YagamiK,SuzukiS.A- typenatriureticpeptidelevelinhypertensivemice.ExpAnim 2004;53:11---9.

MaksimovVF,KorostyshevskayaIM,MarkelAL,ShmerlingMD,Yakob- sonGS.Structuralcharacteristicsofcardiomyocytesintheright atriumofNISAGrats.BullExpBiolMed2004;138:1---4.

Marie PY, Martes PM,Hassan-SebbagN, de TalenceN, Djaballah W, Friberg J, et al. Exercise Release of cardiac peptides is markedly enhanced when patients with coronary artery dis- easearetreatedmedicallybybeta-blockers.JAmCollCardiol 2004;43:353---9.

MayhewTM,LucocqJM.Quantifyingimmunogoldlabellingpatterns of cellular compartments when they comprise mixtures of

membranes (surface-occupying) and organelles (volume- occupying).HistochCellBiol2008;129:367---78.

NovelliM,StrufaldiMB,Rogero MM,Rossi L.Supplementationof glutamine applicated for physical activity. R Bras Cien Mov 2007;15:109---17.

O’Donnell PJ, Driscoll WJ, Bäck N, Muth E, Mueller GP.

Peptidylglycine-alpha-amidatingmonooxygenaseandpro-atrial natriureticpeptideconstitutethemajormembrane-associated proteins of rat atrial secretory granules. J Mol Cell Cardiol 2003;35:915---22.

OlsenS,AagaardP,KadiF,TufekovicG,VerneyJ,OlesenJL,Suetta C,KjaerM.Creatinesupplementationaugmentstheincreasein satellitecellandmyonucleinumberinhumanskeletalmuscle inducedbystrengthtraining.JPhysiol2006;573:525---34.

OhbaH,TakadaH,MushaH,NagashimaJ,MoriN,AwayaT,OmiyaK, MurayamaM.Effectsofprolongedstrenuousexerciseonplasma levelsofatrialnatriureticpeptideandbrainnatriureticpeptide inhealthymen.AmHeartJ2001;141:751---8.

PanSS.Alterationsofatrialnatriureticpeptideincardiomyocytes and plasmaofratsafterdifferentintensityexercise.ScandJ MedSciSports2008;18(3):346---53.

Raastad T, Bjoro T, Hallen J. Hormonal responses to high and moderate-intensitystrength exercise.Eur J ApplPhysiol 2000;82:121---8.

Rawson ES, Volek JS. Effects of creatine supplementation and resistancetrainingonmusclestrengthandweightliftingperfor- mance.JStrengthCondRes2003;17:822---31.

SchiebingerRJ,GreeningKM.Interactionbetweenstretchandhor- monally stimulatedatrial natriureticpeptide secretion.AmJ Physiol1992;262:H78---83.

SeulKH,ChoKW,KimSH.Rightatrialpredominanceofatrialnatri- ureticpeptidesecretioninisolatedperfusedratatria.RegulPept 1992;39:67---81.

Shewchuk LD, Baracos VE, Field CJ. Dietary l-glutamine supplementation reduces the growth of theMorris hepatoma in exercise-trainedandsedentaryrats.JNutr1997;127:158---66.

SmithRJ.Glutaminemetabolismanditsphysiologicimportance.J ParenterEnterNutr1990;14:40S---4S.

VarnierM,LeeseGP,ThompsonJ,RennieMJ.Stimulatoryeffectof glutamineonglycogenaccumulationinhumanskeletalmuscle.

AmJPhysiol1995;269:E309---15.

VolekJS.Inﬂuenceofnutritiononresponsestoresistancetraining.

MedSciSportsExerc2004;36:689---96.

Volek JS, Rawson ES. Scientiﬁc basis and practical aspects of creatine supplementation for athletes. Eur J Clin Nutr 2002;56:585---92.

WatfordM.Glutamineandglutamatemetabolismacrosstheliver sinusoid.JNutr2000;130:983S---7S.

WilliamsAG,IsmailAN,SharmaA,JonesDA.Effectsofresistance exercise volume and nutritionalsupplementation onanabolic andcatabolichormones.EurJApplPhysiol2002;86:315---21.