cell iochemistry nd function Cell Biochem Funct 2013; 31: 136 151. Pulished online 7 Septemer 2012 in Wiley Online Lirry (wileyonlinelirry.com) DOI:.02/cf.2868 Physicl trining prevents oxidtive stress in -induced hypertension rts Andréi Mchdo Crdoso 1, Croline Curry Mrtins 1, Fernndo d Silv Fiorin 1, Roert Schmtz 1, Fátim Husein Adll 1, Jessié Gutierres 1, Dniel Znini 1, Amnd Mino Fiorenz 1, Nir Stefnello 1, Jons Dci d Silv Serres 1, Fino Crvlho 1, Verônic Piv Cstro 1, Cinthi Melzzo Mzznti 1, Luiz Fernndo Freire Royes 1, Adrine Belló-Klein 3, Jeferson Ferrz Goulrte 3, Ver Mri Morsch 1, Mrgrete Dulce Bgtini 2 * nd Mri Ros Chitolin Schetinger 1 * 1 Post-Grdution Progrm in Toxicologicl Biochemistry, Deprtment of Chemistry of the Center of Nturl nd Exct Sciences, Federl University of Snt Mri, Snt Mri, RS, Brzil 2 Collegite of Nursing, University of Southern Frontier, Chpecó Cmpus, SC, Brzil 3 Helth Bsic Sciences Institut, Deprtment of Physiology, Federl University of Rio Grnde do Sul, Porto Alegre, RS, Brzil The present study investigted the effects of 6-week swimming trining on lood pressure, nitric oxide (NO) levels nd oxidtive stress prmeters such s protein nd lipid oxidtion, ntioxidnt enzyme ctivity nd endogenous non-enzymtic ntioxidnt content in kidney nd circulting fluids, s well s on serum iochemicl prmeters (cholesterol, triglycerides, ure nd cretinine) from No-nitro-L-rginine methyl ester hydrochloride ()-induced hypertension treted rts. Animls were divided into four groups (n = ):,, nd. Results showed tht exercise prevented decrese in NO levels in hypertensive rts (P < 005). An increse in protein nd lipid oxidtion oserved in the -treted group ws reverted y physicl trining in serum from the group (P < 005). A decrese in the ctlse (CAT) nd superoxide dismutse (SOD) ctivities in the group ws oserved when compred with normotensive groups (P < 005). In kidney, exercise significntly ugmented the CAT nd SOD ctivities in the group when compred with the group (P < 005). There ws decrese in the non-protein thiols (NPSH) levels in the -treted group when compred with the normotensive groups (P < 005). In the group, there ws n increse in NPSH levels when compred with the group (P < 005). The elevtion in serum cholesterol, triglycerides, ure nd cretinine levels oserved in the group were reverted to levels close to norml y exercise in the group (P < 005). trining hd hypotensive effect, reducing lood pressure in the group (P < 005). These findings suggest tht physicl trining could hve protector effect ginst oxidtive dmge nd renl injury cused y hypertension. Copyright 2012 John Wiley & Sons, Ltd. key words hypertension; physicl trining; oxidtive stress; kidney INTRODUCTION Currently, hypertension hs ffected more thn 1 illion dults worldwide nd 90 to 95% of these ptients hve essentil hypertension. 1 This disese is considered s n independent risk fctor for stroke, myocrdil infrction, hert filure, rteril neurysm nd it hs een the leding cuse of chronic renl filure. 2,1 It is well estlished tht nitric oxide (NO) produced in the vsculr endothelil cells shows potent vsodiltor effect 3,4 nd plys n importnt role in the locl regultion of pltelet-vessel wll interctions nd in vsculr resistnce *Correspondence to: PhD. Mrgrete Dulce Bgtini, Curso de Enfermgem, Universidde Federl d Fronteir Sul, Chpecó, SC, Brzil - 89812-000. E-mil: mrgretegtini@yhoo.com.r; PhD. Mri Ros Chitolin Schetinger, Deprtmento de Químic, Centro de Ciêncis Nturis e Exts, Universidde Federl de Snt Mri, Av. Rorim, 975-900, Snt Mri, RS, Brzil. E-mil: mrichitolin@gmil.com nd growth. 5 On the sis of these effects, NO hs een proposed to hve ntihypertensive, ntithromotic nd ntitherosclerotic properties. 3,4 Chronic inhiition of NO produces volume-dependent elevtion of lood pressure (BP) nd its physiologicl nd pthologicl chrcteristics resemle essentil hypertension. 6 8 Severl studies hve dministered in vivo n inhiitor of NO iosynthesis, the No-nitro-L-rginine methyl ester hydrochloride (), n L-rginine nlogue, to induce hypertension in rts. 9 15 In hypertension, the production of rective oxygen species (ROS) is incresed y vrious mens nd it cn lso cuse other vsculr diseses nd disorders. 16 However, it still remins uncler whether incresed ROS levels re cuse or consequence of high BP. There is proly feed-forwrd system wherey oxidtive stress induces BP elevtion, which in turn promotes incresed ROS genertion nd oxidtive dmge. 17,16 At first glnce, compelling Copyright 2012 John Wiley & Sons, Ltd. Received 15 Mrch 2012 Revised 26 June 2012 Accepted 30 July 2012
exercise in hypertensive rts 137 explntion to this link is tht the incresed ROS production, specilly superoxide rdicl (O 2 ), oxidizes NO to peroxynitrite nd susequently nitrite nd nitrte. This, plus n inhiition of NO synthesis (y dministrtion), results in very low concentrtion of NO-medited vsodilttion, n increse in vsoconstriction, nd susequently n increse in systemic vsculr resistnce, which contriutes to BP elevtion. 16 The incresed genertion or decresed scvenging or metolism of ROS is defined s oxidtive stress. 17 ROS nd free rdicls re meditors of severl forms of tissue dmge, such s ischemic injuries to different orgns, inflmmtory response nd injury resulting from intrcellulr metolism of drugs nd chemicls. 18,17 One of the iologicl molecules to suffer oxidtive dmge in cells re proteins; their side chins cn e cronylted y rective compounds. 19 In ddition, memrnes re composed mostly of phospholipids nd proteins. Incresing ROS led to the peroxidtion of lipid memrnes nd loss of memrne integrity, resulting in necrosis nd cell deth. 19,20 The effect of ROS is lnced y the ntioxidnt ction of non-enzymtic ntioxidnts s well s y ntioxidnt enzymes. Superoxide dismutse (SOD) nd ctlse (CAT) re prt of the ntioxidnt enzyme system. SOD ctlyses the dismuttion of superoxide nion (O 2 )toh 2 O 2. Susequently, H 2 O 2 is reduced to H 2 O nd O 2 y peroxidses such s glutthione peroxidse or CAT. 19,21 Ascoric cid nd non-protein thiols (NPSH) re importnt non-enzymtic ntioxidnt defences. They present vriety of functions in ioreduction nd detoxifiction processes, eing extremely importnt to redox lnce. 22 25 Moderte eroic exercise hs een recognized s codjuvnt on hypertension tretment or prevention. 26,13,27 This wy, mny people re engged in orgnized group exercise rehilittion progrmmes or pursue individul exercise with or without mediction to mintin good crdiovsculr helth. 26,28 Acute exercise increses the utiliztion of oxygen in the ody nd seems to enhnce the free rdicl formtion in vrious tissues of nimls 23,24 nd in circulting fluids, such s serum, plsm nd totl lood in humn. 29 s tht generte free rdicls seem to increse the ctivity of ntioxidnt enzymes in the ody. 30,13,23 Also, it hs lredy een demonstrted tht physicl trining improves the ntioxidnt cpcity in the ort of hypertensive rts. 13 trining lso genertes NO y the induction of nitric oxide synthse (NOS). 13,31 The enefit of regulr physicl ctivity hs een reported to improve crdiovsculr functions in ptients with chronic hert filure nd other crdiovsculr diseses, 26,28 esides hving n importnt nd well documented effect on reducing BP in hypertensive sujects nd nimls. 32,30,33 37 It is hypothesized tht the chronic NO-deficient hypertension is ssocited with the depletion of ntioxidnts nd oxidtive injury to the vsculr tissue nd kidney, nd exercise conditioning normlizes the hypertensive response y scvenging free rdicl/ros through the up-regultion of the ntioxidnt system in circulting fluids nd kidney of rt. As mentioned, moderte eroic exercise hs een relted to reduce BP nd improve ntioxidnt defence, nd oxidtive stress hs een ssocited with the pthogenesis of hypertension. Therefore, this study ws imed to investigte the effect of exercise trining on BP, kidney NO levels nd oxidtive stress prmeters, such s protein nd lipid oxidtion, ntioxidnt enzyme ctivity nd endogenous non-enzymtic ntioxidnt content in serum, plsm, totl lood nd kidney from -induced hypertension treted rts. MATERIALS AND METHODS Chemicls The, 5,5 0 -dithio-is(2-nitroenzoic cid) (DTNB), reduced glutthione (GSH), tris (hydroxymethyl)-minomethne GR, thiorituric cid (TBA) nd Coomssie Brillint Blue G were otined from Sigm Chemicl Co (St. Louis, MO, USA). The ovine serum lumin ws otined from Regen. All the other chemicls used in this experiment were of the highest purity. Animls Adult mle Wistr rts (70 90 dys; 220 300 g) from the Centrl Animl House of the Federl University of Snt Mri were used in this experiment. The nimls were mintined t constnt temperture (23 1 C) on 12 h light/ drk cycle with free ccess to food nd wter. All niml procedures were pproved y the Animl Ethics Committee from the Federl University of Snt Mri (protocol under numer: 029/2011). All protocols re in ccordnce with the guidelines of the Colégio Brsileiro de Experimentção Animl, sed on the Guide for the Cre nd Use of Lortory Animls (Ntionl Reserch Council), nd ll efforts were mde to minimize the numer of nimls used in this study nd their suffering. Experimentl protocol Rts were rndomly divided into four groups, normotensive (), normotensive plus exercise (), hypertensive () nd hypertensive plus exercise ( L- NAME). In the hypertensive groups, hypertension ws induced y the orl dministrtion of the NOS inhiitor (). L- NAME dministrtion includes vi drinking wter, gvge nd sucutneous injection. Although most of the works induce hypertension through drinking wter, 38,6,9 12,8,15 Rieiro et l. 8 oserved tht verge diry wter intke per ox ws 26% lower in -treted rts thn in controls. This wy, we chose gvge dministrtion to e sure on the dose ingested y rts nd to e sure ll rts were receiving the sme dose. In ddition, despite of hypertension induction, high doses 38,8 or long-term dministrtion 6 of my induce severe proteinuri nd renl injury. This wy, we decided to use medium dose s used y Furstenu et l. (30 mg kg 1 dy 1 ) nd t reltive medium time, understnding tht our rts would ecome hypertensive ut the renl injury would hppens nd not e too deleterious. In the normotensive groups, the nimls received wter y gvge throughout the entire experiment to e sumitted to the sme stress (control groups). These rts
138. m. crdoso ET AL. were euthnized 24 h fter the lst exercise session. 33,14 Blood ws collected y crdic puncture nd kidneys were crefully removed. protocol Swimming ws the exercise chosen for this study. The use of swimming rts s model of exercise presents dvntges over tredmill running, ecuse swimming is nturl ility of the rts nd it voids the selection of nimls, which is necessry in experimentl protocols using tredmill running. The protocol used ws ccording to Souz et l. 39 s follows: Swimming protocol All rts were dpted to wter efore the eginning of the trining. The dpttion ws to keep the nimls in shllow wter t 31 1 C 39 for 5 dys, with durtion of 1 h. This procedure ws performed lwys t the sme time, etween :00 h nd 00:00 h. The djustment reduces stress, without, however, promoting dpttions to the trining. Animls were trined five times per week in n dpted swimming system with wter heted to 31 1 ºC for 6 weeks with durtion of 60 min, performed lwys etween :00 h nd 00:00 h. The trining tnk used for this study ws 80 cm in length, 50 cm in width nd 90 cm in depth. 39 The worklod (weight on the ck) ws grdully incresed up to 5% of the niml s ody weight (Tle 1). Sedentry nimls were plced in shllow wter (5 cm in depth), heted to 31 1 ºC, for 60 min, 5 dys week without the work lod (5% of ody weight) to e sujected to the sme stress, however, without eing sumitted to the effects of physicl trining. Hemodynmic prmeter determintion In ll rts, systolic lood pressure (SBP) nd hert rte (HR) were mesured in wke nimls, y til-cuff plethysmogrphy (Kent Scientific; RTBP01 Rt Til Blood Pressure System for rts nd mice, Litchfield, USA). Rts were conditioned with the pprtus efore mesurements were tken. During the mesurements, the nimls were kept for pproximtely 20 min in n crylic retiner within n enclosure tht mintins the temperture etween 30 31 C. SBP ws recorded t the end of experiment (lst tretment week). Hert rte vlues were derived from the pulstions detected y SBP. Tle 1. Swimming protocol, with trining time from 1st week to 6th week, held from Mondy to Fridy Week Mondy Tuesdy Wednesdy Thursdy Fridy 1st 20 min 30 min 40 min 50 min 60 min wo wo wo wo wo 2nd 40 min 50 min 60 min 60 min 60 min 2% w 2% w 2% w 2% w 2% w 3rd 40 min 50 min 60 min 60 min 60 min 5% w 5% w 5% w 5% w 5% w 4th, 5th, 6th 60 min 60 min 60 min 60 min 60 min 5% w 5% w 5% w 5% w 5% w wo, without overlod; w, ody weight (n = to ech group). Blood nd tissue preprtion Twenty-four hours fter the lst tretment, nimls were previously nesthetized with hlothne nd sumitted to euthnsi. Hlothne ws dministered y the closed technique in dose of 05%, ccording to Hlothne ull (Tnohlo 1:1 ml; CRISTÁLIA Produtos Químicos Frmcêuticos LTDA) dpted to rts. Animls were kept in closed chmer, hving n environment sturted with nesthetic, for pproximtely 2 min. The exct time spent in the chmer depended on the clinicl signs of ech niml. Immeditely fter collection, rts were killed y decpittion. Blood ws collected y crdic puncture in tues with nd without nticogulnt system to determine oxidtive stress prmeters nd to perform iochemicl nlysis. In tues without n nticogulnt system, lood ws centrifuged t 1800 g for min, the precipitte ws discrded, nd the serum ws used to determine sustnces rective to thiorituric cid (TBARS), protein cronyl, levels of cholesterol, triglycerides, ure nd cretinine. CAT nd SOD ctivities were determined using whole lood collected in citrted tues nd diluted to rtio of 1: in sline solution Plsm ws seprted from tues with ethylenediminetetrcetic cid (EDTA) nticogulnt to determine NPSH content. The smples of kidneys were quickly removed, plced on ice nd homogenized within min in cold 50 mm Tris-HCl ph 74 (1/, w/v). The homogente ws centrifuged t 2000 g, t 4 ºC, for min to yield the low-speed superntnt (S1) tht ws used immeditely for TBARS, vitmin C nd NPSH groups. Furthermore, during ll procedures, S1 ws mintined on ice. It is importnt to note tht the immedite use of smple cn prevent possile ltertion in the smple cused y storge time nd y freezing process. All the procedures for the test smples nd control smple were crried out together. In order to perform SOD nd CAT ssy, kidneys were diluted nd homogenized s descried further. Biochemicl nlysis The levels of ure nd cretinine were determined using stndrd methods on Cos MIRA W (Roche Dignostics, Bsel, Switzerlnd) utomted nlyzer. In ddition, serum totl cholesterol nd triglyceride concentrtions were mesured using stndrd enzymtic methods using Ortho-Clinicl Dignostics W regents on the fully utomted nlyzer (Vitros 950 W dry chemistry system; Johnson & Johnson, Rochester, NY, USA). NO determintion NO content in kidney superntnt ws estimted in medium contining 400 ml of2%vcl 3 (in 5% HCl), 200 ml of01% N-(l-nphthyl) ethylene-dimine dihydrochloride, 200 ml of 2% sulfnilmide (in 5% HCl). After incuting t 37 ºC for 60 min, nitrite levels, which corresponds to n estimtive of levels of NO, were determined spectrophotometriclly t 540 nm, sed on the reduction of nitrto to nitrite
exercise in hypertensive rts 139 y VCl 3. 40 Kidney nitrite nd nitrte levels were expressed s nnomole of NO/milligrm of protein. Cronyltion of serum protein The cronyltion of serum proteins ws determined y modified Levine method. 41 Firstly, from 1 ml of serum, proteins were precipitted using 05 ml of % trichlorocetic cid (TCA) nd centrifuged t 1800 g for 5 min, discrding the superntnt. One hlf millilitre of mmol l 1 2,4-dinitrophenylhydrzine (DNPH) in 2 mol l 1 HCl ws dded to this protein precipitte nd incuted t room temperture for 30 min. During incution, the smples were mixed vigorously every 15 min. After incution, 05 ml of % TCA ws dded to the protein precipitte nd centrifuged t 1800 g for 5 min. After discrding the superntnt, the precipitte ws wshed twice with 1 ml of ethnol/ethylcette (1:1), centrifuging out the superntnt in order to remove the free DNPH. The precipitte ws dissolved in 15 ml of protein dissolving solution (2 g sodium dodecyl sulfte nd 50 mg EDTA in 0 ml 80 mmol l 1 phosphte uffer, ph 80) nd incuted t 37 ºC for min. The colour intensity of the superntnt ws mesured using spectrophotometer t 370 nm ginst 2 mol l 1 HCl. Cronyl content ws clculted y using the molr extinction coefficient (21 3 1mol 1 cm 1 ), nd results were expressed s nnomoles per milligrm protein. Determintion of lipid peroxidtion Lipid peroxidtion ws estimted y mesuring TBARS in serum smples ccording to modified method of Jentzsch et l. 42 Briefly, 02 ml of serum ws dded to the rection mixture contining 1 ml of 1% ortho-phosphoric cid nd 025 ml lkline solution of thiorituric cid (finl volume 20 ml), followed y 45 min heting t 95 ºC. After cooling, smples nd stndrds of mlondildehyde (MDA) were red t 532 nm ginst the lnk of the stndrd curve. Results were expressed s nnomoles MDA per millilitre of serum. Lipid peroxidtion in kidney ws estimted colorimetriclly y mesuring thiorituric cid rective sustnces (TBARS) using the method descried previously y Ohkw et l. 43 In short, the rection mixture contined 200 ml of smples of S1 from liver nd kidney or stndrd (MDA 003 mm), 200 ml of 81% sodium dodecylsulfte, 750 ml of cetic cid solution (25 M HCl, ph 35) nd 750 ml of08% TBA. Mixtures were heted t 95 ºC for 90 min. TBARS tissue levels were expressed s nmol MDA/mg protein. Ctlse nd superoxide dismutse ctivities The determintion of CAT ctivity in totl lood ws crried out in ccordnce with modified method of Nelson nd Kiesow. 44 This ssy involves the chnge in sornce t 240 nm due to CAT-dependent decomposition of hydrogen peroxide. An liquot (002 ml) of lood ws homogenized in potssium phosphte uffer, ph 70. The spectrophotometric determintion ws initited y the ddition of 007 ml in n queous solution of 03moll 1 hydrogen peroxide. The chnge in sornce t 240 nm ws mesured for 2 min. Ctlse ctivity ws clculted using the molr extinction coefficient (00436 cm 2 mmol), nd results were expressed s nnomoles per milligrm protein. For CAT ssy in kidney, the tissue ws homogenized in 50 mm potssium phosphte uffer, ph 75, t proportion of 1:9 (w/v) nd 1:5 (w/v), respectively. The homogente ws centrifuged t 2000 g for min to yield superntnt tht ws used for the enzyme ssy. The rection mixture contined 50 mm potssium phosphte uffer (ph 7), mm H 2 O 2 nd 20 ml of the superntnt. The rte of H 2 O 2 rection ws monitored t 240 nm for 2 min t room temperture, the sme s in totl lood ssy. The enzymtic ctivity ws expressed in units mg 1 protein (one unit of the enzyme is considered s the mount of CAT tht decomposes 1 mmol of H 2 O 2 per min t ph 7 t 25 C.) Superoxide dismutse ctivity mesurement in totl lood is sed on the inhiition of the rdicl superoxide rection with drenlin s descried y Misr nd Fridovich. 45 In this method, SOD present in the smple competes with the detection system for superoxide nion. A unit of SOD is defined s the mount of enzyme tht inhiits the rte of drenlin oxidtion y 50%. Adrenlin oxidtion leds to the formtion of the coloured product, drenochrome, which is detected y spectrophotometer. Superoxide dismutse ctivity is determined y mesuring the rte of drenochrome formtion, oserved t 480 nm, in rection medium contining glycine-noh (50 mm, ph ) nd drenlin (1 mm). With the purpose of performing the SOD ssy in kidney, 45 the tissue ws dequtely diluted with Tris-HCl ph 74 t proportion of 1:40 (w/v) nd 1:60(w/v), respectively. Briefly, epinephrine undergoes uto-oxidtion t ph 2 to produce drenochrome, coloured product tht ws detected t 480 nm. The ddition of smples (, 20, 30 ml) contining SOD inhiits the uto-oxidtion of epinephrine. The rte of inhiition ws monitored for180 s. The mount of enzyme required to produce 50% inhiition ws defined s one unit of enzyme ctivity, the sme s in totl lood ssy. NPSH determintion NPSH ws mesured spectrophotometriclly with Ellmn s regent. 46 An liquot of 200 ml for kidney in finl volume of 900 ml of solution ws used for the rection. The rection product ws mesured t 412 nm fter the ddition of mm DTNB (005 ml). A stndrd curve using cysteine ws dded to clculte the content of thiol groups in smples nd ws expressed s mmol SH/g tissue. Aliquots (01 ml) of plsm were dded to phosphte uffer 03 mol L 1 (085 ml), ph 74 nd the rection ws red t 412 nm fter the ddition of mm DTNB (005 ml). Results were expressed s mmol ml 1 of plsm.
140. m. crdoso ET AL. Ascoric cid quntifiction Renl vitmin C nlysis ws determined y the modified method descried y Jcques-Silv et l. 3 Proteins of kidney were precipitted in cold % TCA solution t proportion of 1:1 (v/v) nd sumitted to centrifugtion gin. This superntnt ws then used for nlysis. A 300 ml liquot of smple in finl volume of 575 ml of solution ws incuted for 3 h t 37 C, then 500 ml H 2 SO 4 65% (v/v) ws dded to the medium. The rection product ws determined using colour regent contining 45mgml 1 dinitrophenyl hydrzine (DNPH) nd CuSO 4 (0075 mg ml 1 ). Vitmin C levels re expressed s mg scoric cid/g tissue. Protein determintion Protein ws mesured y the method of Brdford 47 using ovine serum lumin s stndrd. Sttisticl nlysis Dt were nlysed sttisticlly using nlysis of vrince followed y the Duncn multiple test using SPSS 80 for Windows (SPSS, Chicgo, IL, USA). Some dt were nlysed y Person s correltion. Differences were considered significnt when P < 005. Vriles re presented s men SD. RESULTS Systolic lood pressure In the present study, the orl dministrtion of (n inhiitor of NOS) y gvge ws ssocited with significnt increse in SBP when compred with control groups, vlidting the hypertensive model. On the other hnd, we oserved tht exercise clerly presented hypotensive effect, reducing SBP in group (P < 005) (Figure 1). NO levels Levels of NO content in kidney re shown in Figure 2(A), which displys significnt decrese in NO levels in the group when compred with other groups (P < 005). As cn e oserved, NO levels in the group remined close to the vlues found in normotensive groups. Figure 2(B) shows negtive Person s correltion etween kidney NO levels nd SBP (r = 0819, P < 005), indicting tht the NO levels decrese s the ugment of BP. Biochemicl nlysis Tle 2 shows lipid profile nd renl mrkers of,, nd groups. Cholesterol nd triglyceride levels were significntly incresed in the group when compred with the other groups (P < 005). As cn e oserved, exercise trining prevented the increse of cholesterol nd triglyceride levels in mmhg 200 180 160 140 120 0 Finl Systolic Blood Pressure Figure 1. Finl systolic lood pressure (SBP) mesurements of group, group, group nd group. SBP ws followed s descried in mterils. Dt re presented s men SD. Groups with different letters re sttisticlly different (P < 005, n = to ech group). ANOVA Duncn s Test hypertensive rts (P < 005). per se did not lter significntly the cholesterol or triglyceride levels when compred with the control group, lthough the vlues were lower thn other groups. Levels of renl function mrkers, ure nd cretinine lso presented significnt increse in the -treted group when compred with other groups (P < 005) (Tle 2). However, exercise trining prevented this increse of ure nd cretinine levels in hypertensive rts (P < 005). per se showed no effects in ure or cretinine levels in the group when compred with the control group. Body weight nd hert rte No difference ws oserved in the food nd wter consumption fter the dministrtion of in the experimentl groups (dt not shown). The sme results were oserved to ody weight (dt not shown). Moreover, the hert rte, expressed s cycles minute 1 (cpm), remined unchnged in the -treted group nd in the exercise groups compred with the respective control groups. However, lthough not sttisticlly significnt, we could oserve tht in the exercise groups, hert rte vlues were lower thn control nd -treted groups, which ws n exercise expected result (Figure 3). Protein oxidtion Regrding the oxidtive stress prmeters, Figure 4(A) shows the protein oxidtion, determined y protein cronyl content in serum. As cn e oserved, there ws sttisticlly significnt increse in protein oxidtion in the -treted group (P < 005) when compred with the control nd exercise groups. In the group, it is cler tht exercise hs the potentil to reduce c
exercise in hypertensive rts 141 A Nitric Oxide Levels 0.6 nmol/mg protein 0.4 0.2 0.0 B 0.5 NOx (nol/mg protein) 0.4 0.3 0.2 0.1 r=-0.819 0.0 120 140 160 180 200 Systolic Blood Pressure (mmhg) Figure 2. (A) Nitric oxide levels in the kidney of group, group, group nd group. Dt re presented s men SD. Groups with different letters re sttisticlly different (P < 005, n = to ech group). ANOVA Duncn s Test. (B) Person s correltion etween kidney levels of nitric oxide nd systolic lood pressure (r =-0819, P < 005) protein oxidtion in hypertensive rts (P < 005). Figure 4(B) shows positive Person s correltion etween protein oxidtion nd SBP (r =0841, P < 005). As cn e oserved, the protein oxidtion increses s BP ugments. Lipid peroxidtion Figure 5 shows TBARS vlues from serum (Figure 5A) nd kidney (Figure 5B). In oth cses, rise in the lipid oxidtion in hypertensive nd sedentry rts (P < 005) ws oserved when compred with normotensive groups. Moreover, the exercise trining in the hypertensive group reduced the TBARS vlues in serum when compred with the group (P < 005); however, these results were not oserved in TBARS from kidney. Figure 5(C) shows positive Person s correltion etween kidney TBARS vlues nd SBP (r =0799, P < 005). As cn e oserved, the lipid peroxidtion increses s the BP ugments. CAT nd SOD ctivities CAT determined in totl lood nd kidney is shown in Figure 6 (A,B). We oserved sttisticlly significnt decrese in the CAT ctivity in -treted group when compred with the control nd exercise groups (P < 005) in smples of totl lood nd kidney. In the group, lthough with no sttisticl significnce, it ws clerly tht exercise hd the potentil to increse this enzyme ctivity in totl lood of hypertensive rts when compred with the hypertensive sedentry group. In kidney, exercise hd the ility to ugment significntly the CAT ctivity in the exercise group when compred with the group (P < 005). The sme results were found concerning the ctivity of SOD enzyme, s shown in Figure 6. In totl lood nd in kidney (Figure 7A,B), we oserved sttisticlly significnt decrese in the SOD ctivity in hypertensive sedentry group when compred with the control nd exercise groups (P < 005). In the group, lthough with no sttisticl
142. m. crdoso ET AL. Tle 2. Lipid profile nd renl mrkers of,, nd groups Cholesterol (mmol l 1 ) 151 017ª 139 021ª 221 013 165 020ª Triglycerides (mmol l 1 ) 083 005ª 078 007ª 117 006 094 004 c Ure (mg dl 1 ) 512 97 471 45 872 75 599 82 Cretinine (mg dl 1 ) 043 007 038 009 059 003 048 005 Dt re expressed s mens SD. Different letters in the sme line indicte differences mong the groups nd sme letter in the sme line indictes groups re not sttisticlly different. (P < 005; n = nimls per group). ANOVA Duncn s test. cpm 600 500 400 Hert Rte Ascoric cid content Figure 9 shows the scoric cid content in kidney. As oserved in the figure, the groups studied did not present sttisticl differences. However, we cn see n indictive of decrese in the scoric cid levels in hypertensive sedentry rts. 300 200 Figure 3. Hert rte vlues of group, group, group nd group. Dt re presented s men SD. Groups with different letters re sttisticlly different (P < 005, n = to ech group). ANOVA Duncn s Test significnce, it ws cler tht exercise hd the potentil to increse this enzyme ctivity in totl lood of hypertensive rts when compred with the hypertensive sedentry group. In kidney, exercise ws le to significntly improve the CAT ctivity in the exercise group when compred with the group (P < 005). Figures 6(C) nd 7(C) show negtive Person s correltion etween kidney CAT nd SOD ctivities nd SBP (r = 0723 nd r = 0791, respectively; P < 005). As cn e oserved, oth enzyme ctivities decrese s the BP increses. NPSH levels NPSH levels mesured in plsm nd kidney re shown in Figure 8(A,B). There ws decrese in the NPSH levels in the -treted group in oth plsm nd kidney when compred with normotensive groups (P < 005). In the group, there ws sttisticlly significnt increse in the NPSH levels when compred with the group (P < 005). Plsm nd kidney hd the sme ehviour in this mesurement. DISCUSSION Severl studies hve shown the eneficil effects of regulr physicl ctivity in reducing the elevted BP in oth humn 26,2 nd niml hypertension models. Regrding to the experimentl models of hypertension, physicl trining ws efficient to reduce BP in spontneously hypertensive rts, 37 Dhl sltsensitive nd slt-resistnt rts, 35 deoxycorticosterone cette (DOCA)-induced hypertension, 32 hypertension due to the mnipultion of kidney rteries 34 nd hypertension induced y dministrtion. 13,33,14 Regulr physicl exercises hve een recommended y helth professionls to mintin good crdiovsculr fitness nd prevent or tret hypertension. For the evlution of trining-relted effects in hypertension models, severl kinds of exercise protocols hve een pplied. This wy, it hs ecome evident tht regulrly performed eroic exercise significntly reduces the high BP in rts with spontneous hypertension 32,36 nd in rts with hypertension induced y dministrtion 13,33,14 s in the present study, where rts performed 6 weeks of swimming protocol, corresponding to moderte eroic exercise. Chronic dministrtion of, esides the induction of dose-dependent nd time-dependent sustined elevtion in BP, 6 is ssocited with renl dmge such s incresed renl vsculr resistnce nd proteinuri, coincident with the ppernce of renl histopthology s glomerulr sclerotic injury nd rteriolr nrrowing. 6,48 These dmges in kidneys re ssocited with the incresed production of ROS nd decresed levels of NO, 48 50 s found in this work. In the present study, the dministrtion of significntly decresed the levels of NO in kidney nd displyed negtive correltion with the BP corroorting the literture 48,50 nd indicting prole vsoconstriction tht induced hypertension y the sence of ioctive NO. However, our dt showed tht 6 weeks of swimming trining significntly prevented the decrese of kidney NO
exercise in hypertensive rts 143 A Protein Cronyl Vlues nmol/mg protein 2.0 1.5 1.0 0.5 c 0.0 B Cronyl (nmol/mg protein) 2.5 2.0 1.5 1.0 0.5 r=0.841 0.0 120 140 160 180 200 Systolic Blood Pressure (mmhg) Figure 4. (A) Protein cronyl vlues content in serum of group, group, group nd group. Dt re presented s men SD. Groups with different letters re sttisticlly different (P < 005; n = to ech group). ANOVA Duncn s Test. (B) Person s correltion etween protein cronyl vlues content in serum nd systolic lood pressure (r = 0841, P < 005) levels in hypertensive rts, indicting eneficil role of exercise conditioning in NO levels. conditioning is well relted to the improvement of NO levels in -treted rts. 9 NO hs crdioprotector nd vsodiltdor effects, eing extremely importnt to the regultion of BP. Incresed levels of NO, s evidenced lso y our study, hve een considered the min mechnism for lowering BP due to physicl exercise. 51,13,31,52 Hypertension is disese chrcterized y multiple ltertions in the structure nd function of the cell memrne, nd it is often ssocited with importnt metolic normlities including those concerning lipid metolism. 53 Dyslipidemi ccompnying essentil hypertension consists of elevted plsm triglycerides, low high-density lipoprotein (HDL) cholesterol nd incresed levels of therogenic low-density lipoprotein (LDL) cholesterol prticles. 54,13,55 The ltered memrne microviscosity seen in hypertensive sujects reflects the chnges of memrne lipid composition resulting from the intensive exchnge etween circulting nd memrne lipids, s well s from norml cellulr lipid synthesis nd metolism, which includes oxidtive stress nd lipid peroxidtion 54 s verified in our study y TBARS levels nd the other iomrkers of tissue dmge tht discussed further. Our results show significnt rise in serum cholesterol nd triglyceride levels in -treted rts. However, exercise trining ws efficient to prevent lipid ltertions. This prevention my e explined y the incresed demnd of the working muscle for ftty cids s n energy-yielding sustrte s well s the replenishment of ftty cid contining stores for the regenertion of dmged muscle fires. 56,57 Lower cholesterol nd triglyceride levels cn lso e explined through the iochemistry of exercises ecuse there is n incresed ctivity of lipoprotein lipse, which ugments the degrdtion of triglycerides from very low-density lipoproteins nd cuses the lipoprotein prticles to shrink. 56,57 According to Herzerg, 56 eroic exercise hs een shown to reduce the risk of crdiovsculr disese, nd this reduction is t lest prtilly medited y chnges in circulting lipoproteins resulting from dptive chnges in enzymes involved in
144. m. crdoso ET AL. A Serum TBARS Vlues nmol MDA/ml serum 40 30 20 c B Kidney TBARS Vlues nmol MDA/g tissue 40 30 20 C TBARS (nmol MDA/mg tissue) 50 40 30 20 r=0.799 0 120 140 160 180 200 Systolic Blood Pressure (mmhg) Figure 5. Levels of thiorituric cid rective sustnces (TBARS) in serum (A) nd kidney (B) of group, group, group nd group. Dt re presented s men SD. Groups with different letters re sttisticlly different (P < 005; n = to ech group). ANOVA Duncn s Test. (C) Person s correltion etween kidney TBARS levels nd systolic lood pressure (r = 0799, P < 005)
exercise in hypertensive rts 145 A nmol/mg protein 8 6 4 2 Totl Blood CAT Activity 0 B Kidney CAT Activity nmol PBG.mg -1 protein.h -1 8 6 4 2 0 c C CAT (nmol PBG.mg -1 protein.h -1 ) 8 6 4 2 r=-0.723 0 120 140 160 180 200 Systolic Blood Pressure (mmhg) Figure 6. Ctlse (CAT) ctivity in totl lood (A) nd kidney (B) of group, group, group nd group. Dt re presented s men SD. Groups with different letters re sttisticlly different (P < 005; n = to ech group). ANOVA Duncn s Test. (C) Person s correltion etween kidney CAT ctivity nd systolic lood pressure (r =-0723, P < 005)
146. m. crdoso ET AL. A Totl Blood SOD Activity U SOD/mg protein 15 5 0 B Kidney SOD Activity 15 U SOD/mg protein 5 c 0 C Kidney SOD (U SOD/mg protein) 15 5 r=-0.791 0 120 140 160 180 200 Systolic Blood Pressure (mmhg) Figure 7. Superoxide dismutse (SOD) ctivity in totl lood (A) nd kidney (B) of group, group, group nd group. Dt re presented s men SD. Groups with different letters re sttisticlly different (P < 005; n = to ech group). ANOVA Duncn s Test. (C) Person s correltion etween kidney SOD ctivity nd systolic lood pressure (r = 0791, P < 005)
exercise in hypertensive rts 147 A Plsm NPSH Levels 40 Ascoric Acid Content mol/ml plsm 2.0 1.5 1.0 0.5 c ug scoric cid/g tissue 35 30 25 20 B 0.0 Kidney NPSH Levels 15 Figure 9. Ascoric cid in kidney of group, group, group nd group. Dt re presented s men SD. Groups with different letters re sttisticlly different (P < 005; n = to ech group). ANOVA Duncn s Test umol SH/g tissue 2.0 1.5 1.0 0.5 0.0 Figure 8. Non-protein thiols (NPSH) levels in plsm (A) nd kidney (B) of group, group, group nd group. Dt re presented s men SD. Groups with different letters re sttisticlly different (P < 005; n = to ech group). ANOVA Duncn s Test their metolism. Moreover, eroic exercise is ssocited with reductions in LDL, totl cholesterol nd tricylglycerol nd increses in HDL. Exposure to oxygen cn oxidtively dmge LDL. 56,57 Oxidized LDL is risk fctor for therosclerosis. Although eroic exercise cn cuse oxidtive dmge when performed cutely, there re dptive chnges resulting from chronic exercise, s in our study, which cn result in the improvement of the lipid profile. 56,57 In ddition to dyslipidemi, in the current study, hypertensive rts lso presented renl dmges tht were evidenced y the elevtion in serum ure nd cretinine levels, which re considered s significnt mrkers of renl dysfunction. 58,59 Of gret importnce, physicl trining prevented the increse in the levels of ure nd cretinine in -treted rts. c These findings suggest tht moderte eroic exercise possesses the potentil to ttenute renl injury cused y hypertension. This cn e ssocited directly with the effect of physicl trining on improving the ody ntioxidnt cpcity, which protects the kidneys ginst oxidtive dmge, s evidenced in this nd other studies. 58 60 On the sis of these results, we could suggest tht physicl trining improves the dyslipidemi while inhiiting the progression of renl dysfunction in -induced hypertension in rts. Chnges in the lipid profile nd the evidence of renl injury verified in this study in hypertensive rts cn e linked to the high production of ROS nd oxidtive stress, which hs een shown to ccompny this disese. 17,16 It is well estlished tht there is link etween hypertension nd oxidtive stress. 16,13 The reltionship etween the development of hypertension nd the incresed iovilility of ROS, decresed ntioxidnt cpcity, or oth, hs een demonstrted in severl experimentl models of hypertension, s well s in humn hypertension. 61,16,13,62 However, it still remins uncler whether incresed ROS levels re cuse or consequence of high BP. 17 Furthermore, growing evidence from niml studies suggests tht oxidtive stress in kidney could e key fctor in the development nd persistence of hypertension. 49,50 For this reson, in this study, we focused the investigtion on kidney ltertions s well s on circulting fluids tht reflect ltertions in the whole orgnism. It is relevnt to highlight the importnce of this study on the investigtion of the enefits of the physicl trining on kidney nd circulting fluids minly, ecuse we fce lck of informtion even nowdys when it is well estlished tht exercise trining improves the hypertensive suject helth. Results of the present study confirm the ssertive tht gret production of ROS is slightly linked to hypertension development. Proteins re one of the mjor trgets for ROS
148. m. crdoso ET AL. ecuse of their high overll undnce in iologicl systems. 19 Oxidtive dmge to proteins cn occur directly y the interction of the protein with ROS or indirectly y the interction of the protein with secondry product (resulting from the interction of the rdicl with lipid or sugr molecule). 19 Cronyltion of proteins is n irreversile oxidtive dmge, often leding to the loss of protein function, which is considered widespred indictor of severe oxidtive dmge nd disese-derived protein dysfunction. 19 This wy, the formtion nd ccumultion of protein cronyls hs een one of the most commonly used methods for ssessing overll protein oxidtion. 19 In our study, hypertensive rts showed n increse in protein oxidtion, which suggests severe dmge in severl tissues. 19 This dmge in overll protein cn contriute to mjor development nd ggrvtion of this disese, 19,16 s shown y the positive correltion etween protein oxidtion nd BP. However, it ws clerly showed tht physicl trining hd the ility to prevent protein oxidtion, nd the mechnisms of this prevention re discussed hereinfter. Another common method to ccess oxidtive dmge in tissues is to verify the lipid peroxidtion through the contents of TBARS, where MDA levels re the min sustnces. MDA is physiologic ketoldehyde produced y peroxidtive decomposition of unsturted lipids. 19 MDA cn e produced s result of norml metolism, ut high production of MDA hs een reported s n indictive of oxidtive stress. 19 In this study, -treted showed significnt increse on lipid peroxidtion in oth serum nd kidney, which indictes severe dmge in tissues. To confirm the link etween lipid peroxidtion nd hypertension, positive correltion in these vriles ws oserved, indicting tht the oxidtive injury ecomes worse with incresing BP, s lredy documented in hypertension. 17,16,13 On the other hnd, our dt showed tht exercise trining depleted serum MDA levels indicting the dptive response of the exercise conditioning ginst peroxidtive injury to vsculr tissue. Previous reports hve lso demonstrted the suppression of lipid peroxidtion in other tissues s result of exercise trining. 63,64,13 In ddition, it is well estlished tht there is n elevtion in NO production fter exercise trining 31,52, nd the elevted NO levels fter trining my lso inhiit lipid peroxidtion. 51,13 On the other hnd, exercise trining cnnot prevent lipid peroxidtion in kidney, ut n indictive of this prevention ws oserved. Thus, in this line of resoning, we could infer tht proly if the period of trining hd een extended, the lipid peroxidtion in kidney could e reverted y trining. Free rdicl-scvenging enzymes such s SOD nd CAT re prt of the first line of cellulr defence ginst oxidtive injury, decomposing O 2 nd H 2 O 2 efore intercting to form the more rective hydroxyl rdicl ( OH). 23 In our study, chronic dministrtion significntly decresed oth totl lood nd kidney ntioxidnt enzyme ctivities, indicting the inility of these tissues to scvenge ROS in hypertensive rts. Furthermore, our results displyed negtive correltion etween kidney CAT nd SOD ctivities nd BP, indicting tht with the ggrvtion of the levels of BP, the enzymtic ntioxidnt cpcity of the kidneys ecme worse. This enzyme suppression in -treted rts could lso e result of protein oxidtion, tking into ccount tht the protein cronyls were cutely elevted in hypertensive rts. Moreover, Alvrez et l. 65 hve demonstrted tht some ROS, such s peroxinitrite (ONOO _ ) generted during oxidtive stress, cn nitroste nd inctivte SOD. In summry, there re severl pthwys to explin the suppression of ntioxidnt enzyme ctivities in hypertension. Other reports hve found the sme suppression in SOD nd CAT ctivities relted to niml models of hypertension. 66,13,67 AccordingtoPnicoet l. 67, down-regultion of SOD nd CAT proly contriutes to the pro-oxidnt stte of kidneys in hypertension, which is importnt ecuse redox lnce influences proximl tuule fluid resorption in the hypertensive kidney. This decrese in the CAT nd SOD ctivities in kidney ws prevented y exercise trining in our study. Our findings indicte tht the kidney ntioxidnt enzyme ctivities significntly enhnced fter 6 weeks trining to rts, indicting the oxidtive conditioning of orgnism. Incresed ntioxidnt enzyme ctivities hve lso een previously reported in nimls fter exercise trining. 13,68,24,69 Furthermore, study crried out y Cludino et l. 9 showed tht exercise reversed theimpirmentonthesodctivityin-tretedrts, corroorting our results. It ws suggested tht this improvement in the enzymtic ntioxidnt system my enhnce vsculr diltion y llowing for more rpid elimintion of superoxide nion tht would prolong the hlf-life of NO in the cells. 13,50 In diphrgm muscle, short term (5-dy) trining of rts incresed oth the enzymtic nd non-enzymtic ntioxidnt defence mechnisms of the tissue, with increses eing recorded in CAT, glutthione peroxidse nd SOD ctivities. 69 Regrding exercise trining effects in lood ntioxidnt enzymes, we oserved tht exercise improve prtilly the decrese of SOD nd CAT ctivities in group. According to Nikolidis nd Jmurts, 70 the exct origin of rective species nd oxidtive dmge detected in lood ecuse of exercises is lrgely unknown. Blood intercts with ll orgns nd tissues nd, consequently, with severl possile sources of rective species. It is known tht the fctors regulting the ctivity of lood ntioxidnt enzymes in vivo relted to exercise re poorly understood. 70 This wy, we encourge more studies iming to elucidte these fctors, enling us to interpret more clerly the effects of exercise on lood ntioxidnt enzymes. However, our study mkes cler tht exercise trining hs the potentil do void lood dmge, lthough there is lck of mechnisms s descried erlier. In our study, it is interesting to note tht in kidney, exercise hd the potentil to improve enzyme ctivities, ut TBARS levels remined high when compred with control groups. It indictes tht kidney is gretly ffected y ROS in hypertension nd suggests tht this up-regultion in the SOD nd CAT ctivities could e ody compenstory mechnism trying to scvenge the high production of
exercise in hypertensive rts 149 ROS in kidney, esides eing n exercise response. In opposite of these, our results from lood did not present the sme reltion, wht could indicte tht the dmge in other ody tissues were more esily reversed y exercise trining when compred with kidney dmge. Literture shows tht during exercise there is gret production of ROS y minly the mitochondri ecuse of moderte exercise, such s swimming, s in this study, seems to increse the oxygen consumption rte y 20-fold. 24 Furthermore, Hnsford et l. 71 found n increse on H2O2 production y rt hert mitochondri with lower vlues of O2 in the respirtory chin nd using inhiitors, which cn e similr to wht hppens in n exercise session. Thus, the ROS generted during exercise ct s signls to increse the production of enzymes relevnt to the dpttion of cells to exercise 72,13,23 nd improve the ody ntioxidnt cpcity. Noting this effect of exercise trining, we cn discuss the redox chnges in GSH (the mjor non-enzymtic endogenous ntioxidnt), mesured y NPSH levels, which hve een reported to e good mrker of hypertension oth in nimls nd humns. 13,73,62 The chronic dministrtion of significntly decresed plsm nd kidney NPSH levels proly y depleting the GSH nd incresing glutthione disulfide (GSSG), which clerly demonstrte the oxidtive stress response during hypertension. Interestingly, depletion of tissue GSH y uthionine sulfoximine in rts hs een demonstrted to increse BP. 62 Moreover, centrl GSH hs lso een shown to regulte the BP in rts. 74 Husin 13 lso found the sme reduction on ort GSH levels in -treted rts. The results of the present study show tht exercise trining significntly incresed the plsm nd kidney NPSH levels in group when compred with group, indicting the eneficil effects of the trining in the ody ntioxidnt cpcity. There re evidences tht the skeletl muscle dpts exercise trining y incresing the GSH content nd reducing GSSG. 23,75,24 The enhnced NPSH levels y physicl conditioning seem to e essentil in mintining the redox stte nd to cope with the oxidtive stress during trined exercise. The present dt nd the forementioned reports scertin the role of GSH in the regultion of BP nd therpeutic importnce using moderte exercise trining s n ntioxidnt in hypertensive ptients in the clinic. Vitmin C, referred to s ntioxidnt, is considered the primry ntioxidnt in plsm nd cells to e depleted under conditions of oxidtive stress nd hs een suggested to limit oxidtive dmge in humns, therey lowering the risk of certin chronic diseses, such s hypertension nd crdiovsculr diseses in generl. 22,25 Under certin conditions, L-scoric cid cn exhiit ntioxidnt properties nd thus my reduce the formtion of oxidized smll molecules, proteins nd lipids, which re possile cuse of cellulr de-regultion. 25 In this line of resoning, it is common to find the content of scoric cid reduced in severl pthologies s consequence of oxidtive stress. 76 78 Although not reching sttisticl relevnce, our findings lso showed slight decrese in the vitmin C content in hypertensive rts, which could e due to the incresing utiliztion of vitmin C s n ntioxidnt defence ginst ROS. Six weeks of swimming trining, lthough not significnt, prevented this reduction in the vitmin C content in hypertensive rts. Previous reports tht proposed to ccess the content of scoric cid relted to exercise re generlly performed with the supplementtion of these ntioxidnts in order to prevent oxidtive stress cused y cute exercise. 79,80 There re no studies reporting chnges in content of scoric cid due to trining dpttion, nd this is the first one tht hd this im. The findings of the present work re of growing importnce ecuse ll of the estlished or non-trditionl crdiovsculr risk fctors hve een ssocited in clinicl or experimentl studies with evidence of incresed ROS, which my e linked not only to vsoconstriction, slt retention, nd hypertension ut lso to severl other dverse long-term consequences. With this, we cn highlight the gret potentil of moderte exercise trining in preventing or treting hypertension, ecuse we found tht exercise hd the ility to improve kidney NO levels nd prevent oxidtive dmge in kidney, serum nd plsm of rts treted with nd exposed to swimming. Furthermore, it lso improved the ntioxidnt defences in kidney, serum, plsm nd whole lood of these rts. These oservtions suggest tht trining up-regulted the ntioxidnt defence system, therey scvenging free rdicls/ros, preserving good levels of lipid profile nd preventing kidney dmge. We cn conclude tht eroic exercise conditioning provided significnt protection ginst oxidtive injury in treted hypertensive rts y up-regulting the NO nd ntioxidnt systems, scvenging free rdicl/ros nd preserving the BP. CONFLICT OF INTEREST The uthors hve declred tht there is no conflict of interest. ACKNOWLEDGEMENTS The uthors wish to thnk Conselho Ncionl de Desenvolvimento Científico e Tecnológico (CNPq), Fundção de Ampro à Pesquis do Rio Grnde do Sul (FAPERGS), Fundção Coordenção de Aperfeiçomento de Pessol de Nível Superior (CAPES), Instituto Ncionl de Ciênci e Tecnologi (INCT) nd Rede Instituto Brsileiro de Neurociênci (IBN-Net). 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