Arq Bras CardiolabcArquivos Brasileiros de CardiologiaArq. Bras. Cardiol.0066-782X1678-4170Sociedade Brasileira de Cardiologia - SBCzHfxrZbvND9sBLrNySvGQnSS0066-782X201700030022810.5935/abc.20170023Original ArticlesExercisingA Single Resistance Exercise Session Improves Aortic Endothelial Function in Hypertensive RatsFariaThaís de OliveiraAngeliJhuli KeliMelloLuiz Guilherme MarchesiPintoGustavo CostaStefanonIvanitaVassalloDalton ValentimLizardoJuliana Hott de FúcioUniversidade Federal do Espírito SantoVitóriaESBrazilUniversidade Federal do Espírito Santo (UFES), Vitória, ES - BrazilUniversidade Federal do Espírito SantoMailing Address: Juliana Hott de Fúcio Lizardo • Rua Luíza Grinalda. Postal Code 29100-240, Centro, Vila Velha, ES - Brazil. E-mail: julianahfl@gmail.com, julianahott@yahoo.com
Author contributions
Conception and design of the research: Stefanon I, Vassallo DV, Lizardo JHF; Acquisition of data: Faria TO, Angeli JK, Mello LGM, Pinto GC, Lizardo JHF; Analysis and interpretation of the data: Faria TO, Angeli JK, Mello LGM, Stefanon I, Vassallo DV, Lizardo JHF; Statistical analysis: Faria TO, Angeli JK, Lizardo JHF; Obtaining financing: Stefanon I, Vassallo DV; Writing of the manuscript: Faria TO, Lizardo JHF; Critical revision of the manuscript for intellectual content: Faria TO, Angeli JK, Mello LGM, Pinto GC, Stefanon I, Vassallo DV, Lizardo JHF.
Potential Conflict of Interest
No potential conflict of interest relevant to this article was reported.
0320171083228236130620162610201627092016This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.AbstractBackground:
Physical exercise is an important tool for the improvement of endothelial function.
Objective:
To assess the effects of acute dynamic resistance exercise on the endothelial function of spontaneously hypertensive rats (SHR).
Methods:
Ten minutes after exercise, the aorta was removed to evaluate the expression of endothelial nitric oxide synthase (eNOS), phosphorylated endothelial nitric oxide synthase (p-eNOS1177) and inducible nitric oxide synthase (iNOS) and to generate concentration-response curves to acetylcholine (ACh) and to phenylephrine (PHE). The PHE protocol was also performed with damaged endothelium and before and after NG-nitro-L-arginine methyl ester (L-NAME) and indomethacin administration. The maximal response (Emax) and the sensitivity (EC50) to these drugs were evaluated.
Results:
ACh-induced relaxation increased in the aortic rings of exercised (Ex) rats (Emax= -80 ± 4.6%, p < 0.05) when compared to those of controls (Ct) (Emax = -50 ± 6.8%). The Emax to PHE was decreased following exercise conditions (95 ± 7.9%, p < 0.05) when compared to control conditions (120 ± 4.2%). This response was abolished after L-NAME administration or endothelial damage. In the presence of indomethacin, the aortic rings' reactivity to PHE was decreased in both groups (EC50= Ex -5.9 ± 0.14 vs. Ct -6.6 ± 0.33 log µM, p < 0.05 / Emax = Ex 9.5 ± 2.9 vs. Ct 17 ± 6.2%, p < 0.05). Exercise did not alter the expression of eNOS and iNOS, but increased the level of p-eNOS.
Conclusion:
A single resistance exercise session improves endothelial function in hypertensive rats. This response seems to be mediated by increased NO production through eNOS activation.
The vascular endothelium has been considered to be a major target organ of arterial hypertension.1 Several reports have shown that endothelial dysfunction is involved in the genesis or the development of arterial hypertension and may be either the cause or the consequence of the problem.2,3 In the presence of arterial hypertension there is an imbalance in the production of endothelial factors, thus vasoconstrictors are produced in greater quantity than vasodilators. It explains the impaired endothelium-dependent relaxation in hypertensive animals and human subjects.3-5
The main cause of this endothelial dysfunction in arterial hypertension seems to be the decreased bioavailability of nitric oxide (NO).2,3,6 It is well known that by interacting with NO, superoxide anions (O2.-) form peroxynitrite, which decreases NO availability for smooth muscle relaxation.7 Endogenous inhibitors of NO synthase (NOS) are also found in the blood of hypertensive individuals, and their increased expression has been associated with greater cardiovascular risk.3
Physical exercise is an important tool for the improvement of endothelial function, because improves the balance between the release of vasodilators and vasoconstrictors. It has already been shown that chronic or acute exercise protocols have important effects on the release of vasoactive substances resulting in better endothelium-dependent control of vascular tone.8-11 Nevertheless, these data concerns endurance exercise. Thus, the effects of a single resistance exercise session on endothelial function are still poorly understood. We have previously demonstrated that a single resistance exercise session decreased the reactivity to phenylephrine (PHE) and increased the endothelium-dependent relaxation to acetylcholine (ACh) in the tail arteries of spontaneously hypertensive rats (SHR).12 Cheng et al.13 have also demonstrated similar response, however after endurance exercise.
The vascular function improvement after acute endurance exercise seems to be mediated by increased NO release.8-11,13 Our results suggested that acute resistance exercise also potentiates the production of that vasoactive agent, and the response was associated with the release of vasodilator prostanoids. More studies are necessary to clarify the underlying mechanisms of endothelial function after acute resistance exercise.
Thus, the present study aimed to investigate endothelial function after a single resistance exercise session in SHR.
MethodsAnimals
The experiments were conducted using 22 male SHR that weighed 250-300 g. The rats were housed in an environment that was controlled for room temperature, humidity, light cycles (12 h light/dark). They had free access to tap water and were fed a standard rat chow ad libitum. The care and use of laboratory animals and all of the experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the Ethics Committee Escola Superior de Ciências da Santa Casa de Misericórdia de Vitória, Brazil (CEUA- EMESCAM).
Experimental designExperimental groups
The animals were submitted to surgery for direct measurement of blood pressure. All of the surgical procedures were performed using aseptic techniques. Anesthesia was induced with chloral hydrate (400 mg/kg, i.p.) and supplementary doses were administered if the rat regained a blink reflex. The left carotid artery was carefully isolated to avoid damage to any nearby nerves. A tapered polyethylene cannula (PE 50) filled with heparinized saline (100 units/ml) was inserted into the left common carotid artery for blood pressure measurement. The free end of a catheter was plugged in a stainless steel obturator and inserted subcutaneously to exit from the back of the neck. The animals were placed in separate cages and were allowed to recover for 24 hours before the initiation of the experimental procedures. The rats were monitored for any signs of infection.
Blood pressure and heart rate were continuously recorded in conscious rats before the resistance exercise session to confirm the presence of arterial hypertension. The blood pressure was determined by connecting the arterial catheter to a TSD104A pressure transducer that was coupled to a DA100C amplifier. An acquisition system (MP 100 Biopac Systems, Inc., CA, USA) was used for real-time blood pressure and heart rate recording and for subsequent analysis.
On the day of the experiment, the rats were allowed to adapt to the laboratory environment for 1 hour before their resting hemodynamic measurements were recorded. After the adaptation period, baseline blood pressure levels were measured in conscious animals for 10 minutes before exercising. Subsequently, the animals were randomly divided into two experimental groups: the exercise group (n = 11), in which rats were submitted to a single resistance exercise session; and the control group (n = 11), where the animals were submitted to a single simulation of a resistance exercise session. Ten minutes after exercise training, the animals from both groups were anesthetized with sodium thiopental (50 mg/kg, i.p.) and were euthanized by exsanguination. The thoracic aorta was carefully dissected for the analysis of vascular reactivity and protein expression.
Exercise protocol
Initially, all animals were adapted to the exercise apparatus for 4 - 5 days. For adaptation, the rats were placed on the exercise apparatus without weight in the rest position, and, therefore, the animals did not move, although received tail electrical stimulus. Afterwards, one repetition of the maximal test was performed. The maximum repetition (RM) was determined to be the maximum weight that was lifted by each rat using the exercise apparatus. After 2 days of rest, the animals were submitted to an exercise protocol. The rats performed the resistance exercise according to a model adapted from previous studies.12-15 Rats that were wearing a canvas jacket were able to regulate the twisting and flexion of their torsos and were fixed by a holder in a standing position on their hindlimbs. An electrical stimulation (20 V for 0.3-second duration and at 3-second intervals) was applied to the rat's tail through a surface electrode. As a result, the animals extended their legs repeatedly, which lifted the weight on the arm of the exercise apparatus. This apparatus was chosen because mimics traditional squat exercises that are performed by humans, and the results obtained in rat skeletal muscles are similar to those observed in humans.15 The rats were exercised for 20 sets with 15 repetitions per set in the exercise apparatus. The repetitions were performed at 3-second intervals with a 1-minute rest between the sets. The exercise intensity was 50% of one RM. The control group received the same stimulus for the same frequency and duration and at the same intensity and intervals as the exercise group. However, the exercise apparatus was unweighted and in the rest position, and therefore, these animals did not lift a load.
Vascular reactivity measurements
The thoracic aorta was carefully dissected out and cleaned of fat and connective tissue. For the reactivity experiments, the aorta was divided into 3-4 mm cylindrical segments. The functional testing of the aortic rings was performed as previously described.16 Briefly, 4 mm-long segments of thoracic aorta were mounted in an isolated tissue chamber containing Krebs-Henseleit solution (in mM: 118 NaCl; 4.7 KCl; 23 NaHCO3; 2.5 CaCl2; 1.2 KH2PO4; 1.2 MgSO4; 11 glucose and 0.01 EDTA), gassed with 95% O2 and 5% CO2, and maintained at a resting tension of 1 g at 37ºC. The isometric tension was recorded using an isometric force transducer (TSD125C, CA, USA) that was connected to an acquisition system (MP100 Biopac Systems, Inc., Santa Barbara, CA, USA).
After a 45-min equilibration period, all of the aortic rings were initially exposed twice to 75 mM KCl, the first time to check their functional integrity and the second time to assess the maximal tension that developed. Afterwards, 10 µM ACh was used to test the endothelial integrity of the segments that had been previously contracted with 1 µM PHE. A relaxation response that was equal to or greater than 90% was considered to be demonstrative of functional endothelial integrity. After a 45-min washout, concentration-response curves to PHE were determined. Single curves were generated for each segment. The role of select, local vasoactivators on the PHE-elicited contractile response was investigated. The effects of the following drugs were evaluated: (1) the nonspecific NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) (100 µM) and (2) the nonspecific cyclooxygenase (COX) inhibitor indomethacin (10 µM). These drugs were added to the bath 30 min before generating PHE concentration-response curves.
The influence of the endothelium on the response to PHE in the absence or presence of exercise was investigated after its mechanical removal was achieved by rubbing the vessel lumen with a needle. The absence of endothelium was confirmed by the inability of 10 µM ACh to induce relaxation.
In another set of experiments, after a 45-min equilibration period, the aortic rings of control and exercise rats were pre-contracted with 1 µM PHE and concentration-response curves to ACh (0.1 nM - 30 mM) were determined.
Western blot analyses
After performing euthanasia as previously described, the thoracic aorta was obtained. To analyze the endothelial nitric oxide synthase (eNOS) expression, phosphorylated endothelial nitric oxide synthase (p-eNOS) expression and inducible nitric oxide synthase (iNOS) expression, the arteries were rapidly frozen and kept at -80ºC. From each homogenized artery, 80 µg of protein were separated by 10% SDS-PAGE. The protein was transferred to nitrocellulose membranes that were incubated with blocking buffer, and then incubated with antibodies for eNOS, eNOS that was phosphorylated on the amino acid serine at position 1177 (p-eNOS1177) (1:250; BD Transduction Laboratories™, Lexington, UK), and iNOS (1:250; BD Transduction Laboratories™, Lexington, UK). After washing, the membranes were incubated with anti-mouse immunoglobulin antibody (1:5,000; StressGen, Victoria, Canada) that was conjugated to horseradish peroxidase. After a thorough washing, the immunocomplexes were detected using an enhanced horseradish peroxidase/luminol chemiluminescence system (ECL Plus, Amersham International, Little Chalfont, UK) and film (Hyperfilm ECL International). The signals on the immunoblot were quantified using the ImageJ computer program, and the same membrane was used to determine α-actin expression with a mouse monoclonal antibody (1:5,000; Sigma, USA).
Data analysis and statistics
The contractile responses were expressed as a percentage of the maximal response that was induced by 75 mM KCl. The relaxation responses to ACh were expressed as the percentage of relaxation of the maximal contractile response. For each concentration-response curve, the maximal effect (Emax) and the concentration of agonist that produced 50% of the maximal response (-log EC50) were calculated using a non-linear regression analysis. Thus, the sensitivity (50% of the maximal response) of the agonists was expressed as EC50 (-log EC50) and the maximal contractile response to drug was expressed as Emax. To compare these variables (EC50 and Emax) between groups, unpaired Student's t-test was used.
To compare the effects of endothelial denudation or L-NAME on the contractile responses to PHE, the results were expressed as the differences in the area under the concentration-response curve (dAUC) for the control and experimental groups.
For protein expression, the data were expressed as the ratio between the signals on the immunoblot that correspond to the protein of interest and to α-actin. The differences were analyzed using unpaired Student's t-test. All the results were expressed as mean ± SE (standard error). P < 0.05 was considered to be significant. For all analyses, GraphPad Prism Software (Inc., San Diego, CA, USA) was used.
ResultsThe effect of exercise on aortic reactivity
To investigate the attenuation of aortic reactivity after exercise, the endothelium-dependent relaxation was elicited by the addition of ACh (Figure 1). A single resistance exercise increased the endothelium-dependent relaxation as observed in the concentration-response curve to ACh. Moreover, after exercise, there was an increase in the Emax to ACh (p < 0.05), however, the EC50 was not altered (p > 0.05) (Table 1).
EC<sub>50</sub> and E<sub>max</sub> values for each protocol
EC50
Emax
Ct
Ex
Ct
Ex
ACh
8.9 ± 0.2
9.0 ± 0.1
50 ± 6.8
80 ± 4.6*
E+
6.7 ± 0.09
6.7 ± 0.1
120 ± 4.2
95 ± 7.9*
E-
7.6 ± 0.1
7.7 ± 0.2
159 ± 7.2†
162 ± 7.1†
LN
7.0 ± 0.2
7.0 ± 0.2
149 ± 7.9†
148 ± 5.1†
Indo
6.6 ± 0.3
5.9 ± 0.1
17 ± 6.2†
9.5 ± 2.9*†
EC50: 50% of the maximum effect of the drug; Emax: maximum effect of the drug; Ach: acetylcholine; E+: phenylephrine without damaged endothelium; E-: phenylephrine with damaged endothelium; LN: L-NAME; Indo: indomethacin on the isolated aortic rings in control (Ct, n = 11) and exercise (Ex, n = 11) conditions in spontaneously hypertensive rats;
p < 0.05 vs. Emax Ct.
p < 0.05 vs. Emax E+ Ct and Ex.
Effects of exercise training on concentration-response curve in aortic rings. Concentration-response curve to acetylcholine (ACh) obtained in aortic rings pre-contracted with phenylephrine (PHE) in control (Ct, n = 17) and exercise rats (Ex, n = 15). *p < 0.05 vs. Ct.
Aortic reactivity to PHE was attenuated after exercise (Table 1). In the presence of L-NAME, the decrease in vascular reactivity to PHE after exercise was abolished, and there was a significant increase in vascular response in both groups (Table 1). Figure 2 shows the concentration-response curves to PHE, as well as the percentage of the dAUC after L-NAME or indomethacin administration, and following endothelial damage. Vascular reactivity was increased significantly in both groups after endothelial damage (p < 0.05) (Figure 2; Table 1). Under this condition, the percentage change of the dAUC was also greater in the exercised rats, suggesting that there was an important endothelial modulation on the vascular reactivity to PHE (Figure 2). Aortic reactivity to PHE was significantly decreased in both groups in the presence of indomethacin, suggesting that there is an increased COX-mediated vasoconstrictor prostanoid production in hypertensive rats. This result is reinforced by the percentage change of the dAUC, which demonstrated a greater effect in the exercise group (Figure 2, Table 1).
Effects of exercise training on concentration-response curve in aortic rings. Concentration-response curve to phenylephrine (PHE) obtained in aortic rings in control (Ct, n = 22) and exercise rats (Ex, n = 22) (A) before and after L-NAME administration (Ct+LN, n = 11; Ex+LN, n = 12); (B) after endothelial damage (Ct E–, n = 7; Ex E–, n = 6); and (C) after indomethacin administration (Ct+Indo, n = 7; Ex+Indo, n = 7). dAUC, difference in the area under the curve. *p < 0.05 Ex vs. other conditions. +p < 0.05 Ct+LN and Ex+LN vs. other conditions. §p < 0.05 Ct E– and Ex E– vs. other conditions. #p < 0.05 Ct+Indo and Ex+Indo vs. other conditions. The values are expressed as percentage of maximal response to KCl.
Expression of iNOS, eNOS and p-eNOS
As shown in Figures 3 and 4, the protein expression level of iNOS and eNOS was not altered after acute exercise. However, the level of p-eNOS protein was 38% higher (p < 0.05) in the exercised rats as compared to the controls (Figure 5), suggesting that there is increased NO production after a single resistance exercise session.
iNOS as determined by Western blot analysis in the aorta of control (Ct) and exercise training rats (Ex). Mean ± SEM (n = 7).
Effects of exercise on protein level. eNOS as determined by Western blot analysis in the aorta of control (Ct) and exercise training rats (Ex). Mean ± SEM (n = 7).
Effects of exercise on protein level. eNOS phosphorylation at Ser1177 as determined by Western blot analysis in the aorta of control (Ct) and exercise training rats (Ex). Mean ± SEM (n = 7). *p < 0.05 vs. CT.
Discussion
The present study demonstrated that a single resistance exercise session that was conducted at 50% of one RM increases the endothelium-mediated vasodilatation and decreases the vascular responsiveness to PHE. This response was associated with an increase in the level of p-eNOS117, indicating that NO has an important role in the improvement of endothelial function following acute exercise.
Using a similar exercise protocol, we previously demonstrated that a single resistance exercise session decreases blood pressure in conscious SHRs,17 reduces responsiveness to PHE and increases endothelium-dependent relaxation12 in the tail arteries of SHR. These responses appear to be primarily mediated by NO. It has been demonstrated that chronic exercise, as well as acute aerobic exercise, decreases α-adrenergic vascular responsiveness18-23 and increases endothelium-dependent relaxation in humans and in normotensive and hypertensive animals.18,24,25 This response is thought to be mediated by NO production and by other vasodilators, such as prostacyclin.18,24,26 Definitive data on the effects of acute resistance exercise on vascular function are limited. Two previous studies investigated the effects of isometric exercise using a handgrip27,28 and aimed to evaluate brachial artery function, primarily in patients with endothelial dysfunction. There is no other study about vascular function and acute resistance exercise in conductance vessels.
We initially evaluated the endothelium-dependent vasodilatation that was elicited by ACh on the isolated aortic rings and showed that a single resistance exercise session evoked an increase in this response (Figure 2). These results corroborate the previous findings in normotensive and hypertensive rats after acute dynamic exercise.18-21 Additionally, Maiorana et al.29 investigated the response of the brachial artery to ACh in patients with heart failure after 8 weeks of endurance and resistance training and also demonstrated a significant increase in the vasodilatation response to ACh.
We also demonstrated that acute resistance exercise decreases the vasoconstriction response to PHE mediated by increased endothelial NO production (Figure 2). Using similar methods, Howard et al.20 have demonstrated that a single aerobic exercise session reduced the response to PHE in normotensive rabbits. Additionally, Patil et al.30 have shown in vivo a significant attenuation of the maximal vasoconstriction response to PHE in the iliac arteries of Sprague-Dawley rats after a single running session. This response was abolished with the inhibition of NO synthesis. Similarly, in the present study, the responsiveness to PHE after exercise returned to control levels following L-NAME administration, suggesting that NO production is increased during post-exercise recovery. Rao et al.22 have also demonstrated a significant reduction in the responsiveness to PHE in the femoral arteries of SHR after acute exercise on a treadmill, which was abolished after the inhibition of NO synthesis with L-NAME. In humans, NO synthesis inhibition also abolishes the vasodilatation response after acute exercise.31 Our results are in agreement with the data obtained from chronic exercise experiments as well. Chen et al.19 have shown that the reduction of the vascular response to norepinephrine after treadmill training was mediated by NO. Similarly, Chen et al.18 has demonstrated a reduction in sensitivity to norepinephrine and PHE in the aorta of SHR and Wistar Kyoto rats after treadmill training, due to increased NO production.
Thus, it appears that both acute and chronic exercise elicit a reduction in vascular responsiveness that is primarily mediated by the increase in NO synthesis. Nevertheless, the underlying mechanisms that generate the increased NO production after chronic and acute exercise are different. It has been previously reported that exercise training increases eNOS expression.26,32 We investigated whether eNOS and iNOS protein expression were increased after a single resistance exercise session. As expected, the expression of these isoforms was not altered after acute exercise because it is unlikely that a single exercise session represents a sufficient stimulus to induce protein expression. Because eNOS activation is dependent on the phosphorylation pattern of well-characterized sites,33 we hypothesized that eNOS phosphorylation could be the mechanism involved in NO production after acute exercise; therefore, we measured p-eNOS1177 protein levels. The amino acid serine at position 1177 is the primary activating eNOS phosphorylation site, and when it is modulated by the Akt kinase (also known as kinase protein B) and eNOS, it demonstrates an increased sensitivity to baseline Ca2+/calmodulin concentrations.34 The level of p-eNOS1177 protein was significantly increased after acute resistance exercise when compared to control rats, indicating that eNOS is activated after acute resistance exercise. This finding confirms our hypothesis that the decrease of vasoconstriction and the increase of ACh-stimulated post-exercise vasodilatation were mediated by NO. Some of the factors that are involved in eNOS activation and, consequently, NO synthesis, such as shear stress, hypoxia and catecholamine release, are present during exercise and during the post-exercise recovery. Therefore, after exercise, the eNOS activity could remain increased for an extended time, resulting in a reduction in the vascular reactivity that is mediated by NO.
To investigate the possible role of vasodilator prostanoids in the reduction of vascular responsiveness after exercise, we evaluated the response to PHE in the presence of indomethacin, a COX inhibitor. In contrast to the results obtained in the tail artery in our previous study,12 the aortic responsiveness was significantly decreased after COX inhibition (Figure 2C) in control and exercised rats. This response may be explained by the increase of the COX-induced vasoconstrictor prostanoid production in hypertensive rats.35 Moreover, the increased prostanoid synthesis was shown to be due to an increase of COX-2 activity. Our data indicate that there is an important effect provoked by a single resistance exercise session. As observed in Figure 2, the percentage of the area under the curve was greater after the exercise, suggesting that acute resistance exercise decreased the vasoconstrictor prostanoid release in conductance vessels. The endothelial dysfunction present in arterial hypertension evokes an increase in vasoconstrictor prostanoid production,35-38 and a single resistance exercise session has an important impact on vascular function improvement, because it decreases the vasoconstrictor prostanoid release. Moreover, it is well established that NO can regulate the activity of COX enzymes39 and the activity of NOS is increased when the COX pathway is inhibited by indomethacin.40
Conclusions
This study demonstrated that a single resistance exercise session decreased the vascular response to PHE and increased the endothelium-dependent relaxation mediated by ACh in SHR. This adaptation appears to be mediated by NO, due to the increase in the p-eNOS1117 protein levels. Moreover, the present investigation also showed that acute resistance exercise may decrease the production of vasoconstrictor prostanoids in the aortic rings of SHR. Thus, our findings suggest that the practice of resistance exercise, even in a single session, might have great clinical relevance for hypertension control.
Sources of Funding
This study was funded by CNPq.
Study Association
This article is part of the thesis of Doctoral submitted by Juliana Hott de Fúcio Lizardo, from Universidade Federal do Espírito Santo.
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Postexercise α-adrenergic receptor hyporesponsiveness in hypertensive rats is due to nitric oxide. Am J Physiol Integrative Comp Physiol. 2002;282(4):R960-8.RaoSPCollinsHLDiCarloSEPostexercise α-adrenergic receptor hyporesponsiveness in hypertensive rats is due to nitric oxideAm J Physiol Integrative Comp Physiol20022824R960R96823 Spier SA, Laughlin MH, Delp MD. Effects of acute and chronic exercise on vasoconstrictor responsiveness of rat abdominal aorta. J Appl Physiol (1985). 1999;87(5):1752-7.SpierSALaughlinMHDelpMDEffects of acute and chronic exercise on vasoconstrictor responsiveness of rat abdominal aortaJ Appl Physiol (1985)19998751752175724 Goto C, Higashi Y, Kimura M, Noma K, Hara K, Nakagawa K, et al. Effect of different intensities of exercise on endothelium-dependent vasodilation in humans: role of endothelium-dependent nitric oxide and oxidative stress. Circulation. 2003;108(5):530-5.GotoCHigashiYKimuraMNomaKHaraKNakagawaKEffect of different intensities of exercise on endothelium-dependent vasodilation in humans: role of endothelium-dependent nitric oxide and oxidative stressCirculation2003108553053525 Goto C, Nishioka K, Umemura T, Jitsuiki D, Sakagutchi A, Kawamura M, et al. Acute moderate-intensity exercise induces vasodilation through an increase in nitric oxide bioavailiability in humans. Am J Hypertens. 2007;20(8):825-30.GotoCNishiokaKUmemuraTJitsuikiDSakagutchiAKawamuraMAcute moderate-intensity exercise induces vasodilation through an increase in nitric oxide bioavailiability in humansAm J Hypertens200720882583026 Sessa WC, Pritchard K, Seyedi N, Wang J, Hintze TH. Chronic exercise in dogs increases coronary vascular nitric oxide synthase production and endothelial cell nitric oxide synthase gene expression. Circ Res. 1994;74(2):349-53.SessaWCPritchardKSeyediNWangJHintzeTHChronic exercise in dogs increases coronary vascular nitric oxide synthase production and endothelial cell nitric oxide synthase gene expressionCirc Res199474234935327 McGowan CL, Visocchi A, Faulkner M, Rakobowchuk M, McCartney N, et al. Isometric handgrip training improves endothelial function in persons medicated for hypertension. [Abstract]. Exp Clin Cardiol. 2004;9:68.McGowanCLVisocchiAFaulknerMRakobowchukMMcCartneyNIsometric handgrip training improves endothelial function in persons medicated for hypertension. [Abstract]Exp Clin Cardiol200496828 McGowan CL, Levy AS, Millar PJ, Guzman JC, Morillo CA, McCartney N, et al. Acute vascular responses to isometric handgrip exercise and effects of training in persons medicated for hypertension. Am J Physiol Heart Circ Physiol. 2006;291(4):H1797-802.McGowanCLLevyASMillarPJGuzmanJCMorilloCAMcCartneyNAcute vascular responses to isometric handgrip exercise and effects of training in persons medicated for hypertensionAm J Physiol Heart Circ Physiol20062914H1797H180229 Maiorana A, O'Driscoll G, Dembo L, Cheetham C, Goodman C, Taylor R, et al. Effect of aerobic and resistance exercise training on vascular function in heart failure. Am J Physiol Heart Circ Physiol. 2000;279(4):H1999-2005.MaioranaAO'DriscollGDemboLCheethamCGoodmanCTaylorREffect of aerobic and resistance exercise training on vascular function in heart failureAm J Physiol Heart Circ Physiol20002794H1999H200530 Patil RD, DiCarlo SE, Collins HL. Acute exercise enhances nitric oxide modulation of vascular response to phenylephrine. 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J Appl Physiol (1985). 1994;76(5):2241-7.SunDHuangAKollerAKaleyGShort-term daily exercise activity enhances endothelial NO synthesis in skeletal muscle arterioles of ratsJ Appl Physiol (1985)19947652241224733 Mount PF, Kemp BE, Power DA. Regulation of endothelial and myocardial NO synthesis by multi-site eNOS phosphorylation. J Mol Cell Cardiol. 2007;42(2):271-9.MountPFKempBEPowerDARegulation of endothelial and myocardial NO synthesis by multi-site eNOS phosphorylationJ Mol Cell Cardiol200742227127934 Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, et al. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature. 1999;399(6736):597-601. Erratum in: Nature 1999;400(6746):792.FultonDGrattonJPMcCabeTJFontanaJFujioYWalshKRegulation of endothelium-derived nitric oxide production by the protein kinase AktNature19993996736597601Erratum in Nature. 1999;400(6746):79235 Tang EH, Vanhoutte PM. Gene expression changes of prostanoid synthases in endothelial cells and prostanoid receptors in vascular smooth muscle cells caused by aging and hypertension. Physiol Genomics. 2008;32(3):409-18.TangEHVanhouttePMGene expression changes of prostanoid synthases in endothelial cells and prostanoid receptors in vascular smooth muscle cells caused by aging and hypertensionPhysiol Genomics200832340941836 Dohi Y, Kojima M, Sato K. Benidipine improves endothelial function in renal resistance arteries of hypertensive rats. Hypertension. 1996;28(1):58-63.DohiYKojimaMSatoKBenidipine improves endothelial function in renal resistance arteries of hypertensive ratsHypertension1996281586337 Taddei S, Vanhoutte PM. Endothelium-dependent contractions to endothelin in the rat aorta are mediated by thromboxane A2. J Cardiovasc Pharmacol. 1993;22 Suppl 8:S328-31.TaddeiSVanhouttePMEndothelium-dependent contractions to endothelin in the rat aorta are mediated by thromboxane A2J Cardiovasc Pharmacol199322Suppl 8S328S33138 Zhou MS, Nishida Y, Chen QH, Kosaka H. Endothelium-derived contracting factor in carotid artery of hypertensive Dahl rats. Hypertension. 1999;34(1):39-43.ZhouMSNishidaYChenQHKosakaHEndothelium-derived contracting factor in carotid artery of hypertensive Dahl ratsHypertension1999341394339 Salvemini D, Misko T, Masferrer J, Seibert K, Currie M, Needleman P. Nitric oxide activates cyclooxygenase enzymes. Proc Natl Aca. Sci USA. 1993;90(15):7240-4.SalveminiDMiskoTMasferrerJSeibertKCurrieMNeedlemanPNitric oxide activates cyclooxygenase enzymesProc Natl Aca. Sci USA199390157240724440 Illiano S, Marsault R, Descombes JJ, Verbeuren T, Vanhoutte PM. Regulation of nitric oxide-like activity by prostanoids in smooth muscle of the canine saphenous vein. Br J Pharmacol. 1996;117(2):360-4.IllianoSMarsaultRDescombesJJVerbeurenTVanhouttePMRegulation of nitric oxide-like activity by prostanoids in smooth muscle of the canine saphenous veinBr J Pharmacol19961172360364Artigos OriginaisExercícioUma Única Sessão de Exercício Resistido Melhora a Função Endotelial Aórtica em Ratos HipertensosFariaThaís de OliveiraAngeliJhuli KeliMelloLuiz Guilherme MarchesiPintoGustavo CostaStefanonIvanitaVassalloDalton ValentimLizardoJuliana Hott de FúcioUniversidade Federal do Espírito Santo (UFES), Vitória, ES - BrasilCorrespondência: Juliana Hott de Fúcio Lizardo • Rua Luíza Grinalda. CEP 29100-240, Centro, Vila Velha, ES - Brasil. E-mail: julianahfl@gmail.com, julianahott@yahoo.com
Contribuição dos autores
Concepção e desenho da pesquisa: Stefanon I, Vassallo DV, Lizardo JHF; Obtenção de dados: Faria TO, Angeli JK, Mello LGM, Pinto GC, Lizardo JHF; Análise e interpretação dos dados: Faria TO, Angeli JK, Mello LGM, Stefanon I, Vassallo DV, Lizardo JHF; Análise estatística: Faria TO, Angeli JK, Lizardo JHF; Obtenção de financiamento: Stefanon I, Vassallo DV; Redação do manuscrito: Faria TO, Lizardo JHF; Revisão crítica do manuscrito quanto ao conteúdo intelectual importante: Faria TO, Angeli JK, Mello LGM, Pinto GC, Stefanon I, Vassallo DV, Lizardo JHF.
Potencial conflito de interesse
Declaro não haver conflito de interesses pertinentes.
ResumoFundamento:
O exercício físico é uma importante ferramenta para o aprimoramento da função endotelial.
Objetivo:
Avaliar os efeitos do exercício dinâmico resistido agudo na função endotelial de ratos espontaneamente hipertensos (SHR).
Métodos:
Após 10 minutos de exercício, a aorta foi removida para avaliação da expressão de óxido nítrico sintase endotelial (eNOS), óxido nítrico sintase endotelial fosforilada (p-eNOS1177) e óxido nítrico sintase endotelial induzível (iNOS), e para a construção de curvas concentração-resposta de acetilcolina (ACT) e fenilefrina (FEN). O protocolo FEN foi também realizado com lesão endotelial e antes e depois da administração de N-nitro-L-arginina metil éster (L-NAME) e indometacina. A resposta máxima (Emax) e a sensibilidade (EC50) a esses fármacos foram avaliadas.
Resultados:
Houve aumento do relaxamento induzido por ACT nos anéis aórticos dos ratos exercitados (Ex) (Emax = -80 ± 4,6%; p < 0,05) quando comparado àquele dos controles (Ct) (Emax = -50 ± 6,8%). A Emax à FEN diminuiu após exercício (95 ± 7,9%; p < 0,05) quando comparada àquela dos controles (120 ± 4,2%). Tal resposta foi abolida após administração de L-NAME ou lesão endotelial. Na presença de indometacina, a reatividade dos anéis aórticos à FEN diminuiu nos dois grupos (EC50= Ex -5,9 ± 0,14 vs. Ct -6,6 ± 0,33 log µM; p < 0,05/ Emax = Ex 9,5 ± 2,9 vs. Ct 17 ± 6,2%; p < 0,05). O exercício não alterou a expressão de eNOS e de iNOS, mas aumentou o nível de p-eNOS.
Conclusão:
Uma única sessão de exercício resistido melhora a função endotelial em ratos hipertensos. Essa resposta parece ser mediada por elevação da produção de NO através de ativação de eNOS.
O endotélio vascular é considerado um importante órgão-alvo na hipertensão arterial.1 Há vários relatos sobre o envolvimento da disfunção endotelial na gênese ou no desenvolvimento de hipertensão arterial, podendo ser tanto a causa quanto a consequência do problema.2,3 Na hipertensão arterial, há um desequilíbrio na produção de fatores endoteliais, com maior produção de vasoconstritores do que de vasodilatadores. Isso explica o comprometimento do relaxamento dependente de endotélio em hipertensos, tanto animais quanto seres humanos.3-5
A principal causa dessa disfunção endotelial na hipertensão arterial parece ser a diminuição da biodisponibilidade de óxido nítrico (NO).2,3,6 Sabe-se que, ao interagirem com o NO, os ânions superóxido (O2.- formam peroxinitrito, que reduz a disponibilidade de NO para o relaxamento de músculo liso.7 Os inibidores endógenos da NO sintase (NOS) são ainda encontrados no sangue de hipertensos, sendo o aumento de sua expressão associado com maior risco cardiovascular.3
O exercício físico é uma ferramenta importante para o aprimoramento da função endotelial, pois melhora o equilíbrio entre a liberação de vasodilatadores e de vasoconstritores. Já foi demonstrado que os protocolos de exercício crônico ou agudo têm importantes efeitos na liberação de substâncias vasoativas, resultando em melhor controle do tônus vascular endotélio-dependente.8-11 Esses dados, entretanto, referem-se ao exercício aeróbio. Portanto, os efeitos de uma única sessão de exercício resistido na função endotelial são pouco conhecidos. Demonstramos anteriormente que uma única sessão de exercício resistido diminuía a reatividade à fenilefrina (FEN) e aumentava o relaxamento endotélio-dependente em resposta à acetilcolina (ACT) na artéria da cauda de ratos espontaneamente hipertensos (SHR).12 Cheng et al.13 demonstraram resposta similar, entretanto, após exercício aeróbio.
O aprimoramento da função vascular após exercício aeróbio agudo parece ser mediado pela maior liberação de NO.8-11,13 Nossos resultados sugerem que o exercício resistido agudo também potencializa a produção daquele agente vasoativo, sendo a resposta associada com a liberação de prostanoides vasodilatadores. Estudos adicionais são necessários para esclarecer os mecanismos subjacentes da função endotelial após exercício resistido agudo.
O presente estudo teve por objetivo investigar a função endotelial após uma única sessão de exercício resistido em SHR.
MétodosAnimais
Os experimentos foram conduzidos em 22 SHR machos, pesando 250-300 g e acomodados em ambiente com controle de temperatura e umidade e ciclos claro/escuro de 12 horas. Os animais tiveram livre acesso a água potável e receberam ração padrão ad libitum. O cuidado e o uso dos animais e todos os experimentos foram conduzidos conforme o Guia de Cuidado e Uso de Animais de Laboratório, tendo os protocolos sido aprovados pelo Comitê de Ética da instituição Escola Superior de Ciências da Santa Casa de Misericórdia de Vitória, Vitória, ES, Brasil (CEUA- EMESCAM).
Desenho do experimentoGrupos experimentais
Os animais foram submetidos a cirurgia para medida direta da pressão arterial. Todos os procedimentos cirúrgicos foram realizados com técnicas assépticas. A anestesia foi induzida com hidrato de cloral (400 mg/kg, i.p.), doses suplementares tendo sido administradas se o rato recuperasse o reflexo de piscar. A artéria carótida esquerda foi cuidadosamente isolada para evitar dano aos nervos adjacentes. Uma cânula afunilada de polietileno (PE 50) contendo solução salina heparinizada (100 unidades/ml) foi inserida na artéria carótida comum esquerda para medida da pressão arterial. A extremidade livre do cateter foi conectada a obturador de aço inoxidável e inserida por via subcutânea para sair da parte posterior do pescoço. Os animais foram colocados em gaiolas separadas e deixados para se recuperar por 24 horas antes de iniciar o procedimento experimental. Os ratos foram monitorados para sinais de infecção.
A pressão arterial e a frequência cardíaca foram continuamente registradas nos ratos conscientes antes da sessão de exercício resistido para confirmar a presença de hipertensão arterial. Determinou-se a pressão arterial conectando-se o cateter arterial ao transdutor de pressão, modelo TSD104A, que foi acoplado ao amplificador DA100C. Utilizou-se um sistema de aquisição (MP 100 Biopac Sistemas, Inc., CA, EUA) para registro em tempo real da pressão arterial e da frequência cardíaca e para análise subsequente.
No dia do experimento, os ratos foram deixados para se adaptar ao ambiente do laboratório por 1 hora antes da tomada de suas medidas hemodinâmicas. Após o período de adaptação, mediram-se os níveis basais de pressão arterial nos animais conscientes por 10 minutos antes do exercício. Subsequentemente, os animais foram divididos aleatoriamente em dois grupos: grupo de exercício (n = 11), cujos ratos foram submetidos a uma única sessão de exercício resistido; e grupo controle (n = 11), cujos animais foram submetidos a uma única simulação de sessão de exercício resistido. Após 10 minutos de exercício, os animais dos dois grupos foram anestesiados com tiopental sódico (50 mg/kg, i.p.) e submetidos a eutanásia por exsanguinação. A aorta torácica foi cuidadosamente dissecada para análise da reatividade vascular e expressão proteica.
Protocolo do exercício
Inicialmente, todos os animais foram adaptados ao aparelho de exercício por 4 - 5 dias. Para adaptação, os ratos foram colocados no aparelho de exercício sem peso na posição de repouso, e, portanto, os animais não se moveram, embora tivessem recebido o estímulo elétrico na cauda. Depois, realizou-se uma repetição do teste máximo. Determinou-se que a repetição máxima (RM) seria o peso máximo levantado por cada rato usando o aparelho de exercício. Após 2 dias de repouso, os animais foram submetidos ao protocolo de exercício. Os ratos realizaram o exercício resistido conforme modelo adaptado de estudos prévios.12-15 Os ratos vestindo uma jaqueta de lona puderam regular a girada e a flexão de seus torsos, tendo sido fixados por um suporte na posição ereta em seus membros posteriores. Aplicou-se um estímulo elétrico (20 V por 0,3 segundos e em intervalos de 3 segundos) na cauda do rato através de um eletrodo de superfície. Como resultado, os animais estenderam suas pernas repetidamente, levantando a carga no aparelho, que foi escolhido por imitar os exercícios tradicionais de agachamento realizados pelo homem, sendo os resultados obtidos no músculo esquelético do rato similares àqueles observados no homem.15 Os ratos se exercitaram no aparelho por 20 séries de 15 repetições cada. As repetições foram realizadas em intervalos de 3 segundos com 1 minuto de descanso entre as séries. A intensidade do exercício foi de 50% de 1 RM. O grupo controle recebeu o mesmo estímulo com a mesma frequência e duração, na mesma intensidade e mesmos intervalos do grupo de exercício. Entretanto, o aparelho de exercício não teve peso e foi mantido na posição de repouso. Esses animais, portanto, não levantaram carga.
Medidas de reatividade vascular
A aorta torácica foi cuidadosamente dissecada, com eliminação da gordura e do tecido conjuntivo. Para os experimentos de reatividade, a aorta foi dividida em segmentos cilíndricos de 3-4 mm. O teste funcional dos anéis aórticos foi realizado como previamente descrito.16 Resumidamente, segmentos de aorta torácica de 4 mm de largura foram montados em câmara de tecido isolada contendo solução de Krebs-Henseleit (em mM: 118 NaCl; 4,7 KCl; 23 NaHCO3; 2,5 CaCl2; 1,2 KH2PO4; 1,2 MgSO4; 11 glicose e 0,01 EDTA), gaseificados com mistura contendo 95% O2 e 5% CO2, e mantidos sob uma tensão de repouso de 1 g a 37°C. Registrou-se a tensão isométrica com um transdutor isométrico de força (TSD125C, CA, EUA) conectado a um sistema de aquisição (MP100 Biopac Sistemas, Inc., Santa Barbara, CA, EUA).
Após 45 minutos de estabilização, todos os anéis aórticos foram inicialmente expostos por duas vezes a 75 mM KCl, a primeira vez para checar sua integridade funcional, e a segunda para avaliar a tensão máxima desenvolvida. Depois, 10 µM de ACT foram usados para testar a integridade endotelial dos segmentos que haviam sido previamente contraídos com 1 µM de FEN. A resposta de relaxamento igual a 90% ou maior foi considerada como demonstrativa de integridade funcional endotelial. Após uma lavagem de 45 minutos, determinaram-se as curvas concentração-resposta para FEN. Curvas únicas foram geradas para cada segmento. Investigou-se o papel de certos vasoativadores locais sobre a resposta contrátil determinada pela FEN. Os efeitos dos seguintes fármacos foram avaliados: (1) inibidor não específico da NOS, N-nitro-L-arginina metil éster (L-NAME) (100 µM); e (2) inibidor não específico da ciclooxigenase (COX), indometacina (10 µM). Esses fármacos foram adicionados ao banho 30 minutos antes da produção das curvas concentração-resposta de FEN.
A influência do endotélio na resposta à FEN na ausência ou presença de exercício foi investigada após sua remoção mecânica obtida ao se friccionar a luz do vaso com uma agulha. Confirmou-se a ausência de endotélio pela inabilidade de 10 µM de ACT induzir relaxamento.
Em outro conjunto de experimentos, após estabilização de 45 minutos, os anéis aórticos dos grupos controle e de exercício foram pré-contraídos com 1 µM de FEN, determinando-se as curvas concentração-resposta para ACT (0,1 nM - 30 mM).
Análise Western blot
Após eutanásia conforme descrição anterior, obteve-se a aorta torácica, cujas amostras foram rapidamente congeladas e mantidas a -80°C para análise das seguintes expressões: da NOS endotelial (eNOS); da NOS endotelial fosforilada (p-eNOS); e da NOS induzível (iNOS). De cada artéria homogeneizada, 80 µg de proteína foram separados por 10% SDS-PAGE. A proteína foi transferida para membranas de nitrocelulose, que foram incubadas com tampão bloqueador, e então incubadas com anticorpos para eNOS, eNOS fosforilada na posição 1177 do aminoácido serina (p-eNOS1177) (1:250; BD Transduction Laboratories™, Lexington, UK), e iNOS (1:250; BD Transduction Laboratories™, Lexington, RU). Após lavagem, as membranas foram incubadas com anticorpo anti-imunoglobulina de camundongo (1:5.000; StressGen, Victoria, Canadá) conjugado com peroxidase de rábano. Após lavagem exaustiva, os imunocomplexos foram detectados com um sistema peroxidase de rábano/quimiluminescência luminol realçada (ECL Plus, Amersham International, Little Chalfont, RU) e filme (Hyperfilm ECL International). Os sinais no 'immunoblot' foram quantificados com o programa ImageJ, sendo a mesma membrana usada para determinar a expressão da α-actina com anticorpo monoclonal de camundongo (1:5.000; Sigma, EUA).
Análise de dados e estatística
A resposta contrátil foi expressa como porcentagem da resposta máxima induzida por 75 mM KCl. A resposta de relaxamento à ACT foi expressa como porcentagem do relaxamento da resposta contrátil máxima. Para cada curva concentração-resposta, calcularam-se, usando análise de regressão não linear, o efeito máximo (Emax) e a concentração do agonista que produziu 50% da resposta máxima (-log EC50). Assim, a sensibilidade (50% da resposta máxima) do agonista foi expressa como EC50 (-log EC50), e a máxima resposta contrátil ao fármaco, como Emax. O teste t de Student não pareado foi usado para comparar essas variáveis (EC50 e Emax) entre os grupos.
Para comparar os efeitos da desnudação endotelial ou do L-NAME na resposta contrátil à FEN, os resultados foram expressos como a diferença na área sob a curva concentração-resposta (dAUC) para os grupos controle e experimental.
Para a expressão proteica, os dados foram expressos como a razão entre os sinais no immunoblot correspondendo à proteína de interesse e à α-actina. As diferenças foram analisadas usando-se o teste t de Student não pareado. Todos os resultados foram expressos como média ± EP (erro padrão). Adotou-se o nível de significância de p < 0,05. Para todas as análises, usou-se o programa GraphPad Prism Software (Inc., San Diego, CA, EUA).
ResultadosEfeito do exercício na reatividade aórtica
Para investigar a atenuação da reatividade aórtica após exercício, desencadeou-se relaxamento endotélio-dependente pela adição de ACT (Figura 1). Uma única sessão de exercício resistido aumentou o relaxamento endotélio-dependente como observado na curva concentração-resposta da ACT. Além disso, após o exercício, houve aumento da Emax à ACT (p < 0,05); entretanto, a EC50 não se alterou (p > 0,05) (Tabela 1).
Valores de EC50 e Emax para cada protocolo
EC50
Emax
Ct
Ex
Ct
Ex
8,9 ± 0,2
9,0 ± 0,1
50 ± 6,8
80 ± 4,6*
E+
6,7 ± 0,09
6,7 ± 0,1
120 ± 4,2
95 ± 7,9*
E-
7,6 ± 0,1
7,7 ± 0,2
159 ± 7,2†
162 ± 7,1†
LN
7,0 ± 0,2
7,0 ± 0,2
149 ± 7,9†
148 ± 5,1†
Indo
6,6 ± 0,3
5,9 ± 0,1
17 ± 6,2†
9,5 ± 2,9*†
EC50: 50% do efeito máximo do fármaco; Emax: efeito máximo do fármaco; ACT: acetilcolina; E+: fenilefrina sem lesão endotelial; E-: fenilefrina com lesão endotelial; LN: L-NAME; Indo: indometacina. Em anéis aórticos isolados de ratos espontaneamente hipertensos controles (Ct, n = 11) e exercitados (Ex, n = 11).
p < 0,05 vs. Emax Ct.
p < 0,05 vs. Emax E+ Ct e Ex.
Efeitos do exercício na curva concentração-resposta em anéis aórticos. Curva concentração-resposta da acetilcolina (ACT) obtida em anéis aórticos pré‑contraídos com fenilefrina (FEN) em ratos controles (Ct, n = 17) e exercitados (Ex, n = 15). *p < 0,05 vs. Ct.
A reatividade aórtica à FEN foi atenuada após exercício (Tabela 1). Na presença de L-NAME, a redução da reatividade vascular à FEN após exercício foi abolida, tendo havido um significativo aumento da resposta vascular nos dois grupos (Tabela 1). A Figura 2 mostra as curvas concentração-resposta de FEN, e a porcentagem da dAUC após administração de L-NAME ou indometacina e após lesão endotelial. A reatividade vascular elevou-se significativamente nos dois grupos após lesão endotelial (p < 0,05) (Figura 2; Tabela 1). Nessa condição, a mudança percentual da dAUC foi também maior nos ratos exercitados, sugerindo haver uma importante modulação endotelial na reatividade vascular à FEN (Figura 2). A reatividade aórtica à FEN mostrou-se significativamente diminuída nos dois grupos na presença de indometacina, sugerindo haver uma maior produção de prostanoide vasoconstritor mediado pela COX em ratos hipertensos. Tal resultado é reforçado pela mudança percentual da dAUC, que mostrou maior efeito no grupo de exercício (Figura 2, Tabela 1).
Efeitos do exercício na curva concentração-resposta em anéis aórticos. Curva concentração-resposta da fenilefrina (FEN) obtida em anéis aórticos em ratos controles (Ct, n = 22) e exercitados (Ex, n = 22) (A) antes e depois da administração de L-NAME (Ct+LN, n = 11; Ex+LN, n = 12); (B) após lesão endotelial (Ct E–, n = 7; Ex E-, n = 6); e (C) após administração de indometacina (Ct+Indo, n = 7; Ex+Indo, n = 7). dAUC, diferença na área sob a curva. *p < 0,05 Ex vs. outras condições. +p < 0,05 Ct+LN e Ex+LN vs. outras condições. §p < 0,05 Ct E- e Ex E– vs. outras condições. #p < 0,05 Ct+Indo e Ex+Indo vs. outras condições. Valores expressos como porcentagem de resposta máxima ao KCl.
Expressão de iNOS, eNOS e p-eNOS
Como mostram as Figuras 3 e 4, o nível de expressão da proteína de iNOS e eNOS não se alterou após exercício agudo. Entretanto, o nível da proteína de p-eNOS foi 38% maior (p < 0,05) nos ratos exercitados se comparado ao dos controles (Figura 5), sugerindo haver aumento da produção de NO após uma única sessão de exercício resistido.
iNOS determinado por análise Western blot na aorta de ratos controles (Ct) e exercitados (Ex). Média ± EPM (n = 7).
Efeitos do exercício no nível de proteína. eNOS determinado por análise Western blot na aorta de ratos controles (Ct) e exercitados (Ex). Média ± EPM (n = 7).
Efeitos do exercício no nível de proteína. Fosforilação da eNOS na posição Ser1177 como determinado por análise Western blot na aorta de ratos controles (Ct) e exercitados (Ex). Média ± EPM (n = 7). *p < 0,05 vs. CT.
Discussão
O presente estudo demonstrou que uma única sessão de exercício resistido conduzida a 50% de uma RM aumenta a vasodilatação endotélio-mediada e diminui a reatividade vascular à FEN. Tal resposta associou-se com aumento do nível de p-eNOS117, indicando que NO tem importante papel no aprimoramento da função endotelial após exercício agudo.
Usando um protocolo de exercício similar, demonstramos anteriormente que uma única sessão de exercício resistido reduz a pressão arterial em SHRs conscientes,17 reduz a resposta à FEN e aumenta o relaxamento endotélio-dependente12 na artéria da cauda de SHR. Tais respostas parecem ser primariamente mediadas por NO. Foi demonstrado que exercício crônico, assim como exercício aeróbio agudo, reduz a resposta vascular α-adrenérgica18-23 e aumenta o relaxamento endotélio-dependente em seres humanos e animais normotensos e hipertensos.18,24,25 Acredita-se que tal resposta seja mediada pela produção de NO e outros vasodilatadores, como prostaciclina.18,24,26 Dados definitivos sobre os efeitos do exercício resistido agudo na função vascular são limitados. Dois estudos prévios investigaram os efeitos do exercício isométrico de 'pegada de mão'27,28 com o objetivo de avaliar a função da artéria braquial em pacientes com disfunção endotelial. Não há outro estudo sobre função vascular e exercício resistido agudo.
Inicialmente avaliamos a vasodilatação endotélio-dependente desencadeada pela ACT em anéis aórticos isolados e mostramos que uma única sessão de exercício resistido aumentou essa resposta (Figura 2). Tais resultados corroboram os achados prévios em ratos normotensos e hipertensos após exercício dinâmico agudo.18-21 Além disso, Maiorana et al.29 investigaram a resposta da artéria braquial à ACT em pacientes com insuficiência cardíaca após 8 semanas de treinamento aeróbio e de resistência, tendo ainda demonstrado significativo aumento da resposta vasodilatadora à ACT.
Além disso, demonstramos que o exercício resistido agudo diminui a resposta vasoconstritora à FEN mediada pelo aumento da produção de NO endotelial (Figura 2). Usando métodos similares, Howard et al.20 mostraram que uma única sessão de exercício resistido reduziu a resposta à FEN em coelhos normotensos. Ainda, Patil et al.30 relataram in vivo uma significativa atenuação da resposta vasoconstritora máxima à FEN nas artérias ilíacas de ratos Sprague-Dawley após uma única sessão de corrida. Tal resposta foi abolida com a inibição da síntese de NO. Da mesma forma, no presente estudo, a resposta à FEN após exercício retornou aos níveis de controle com a administração de L-NAME, sugerindo haver aumento da produção de NO durante a recuperação após exercício. Rao et al.22 também demonstraram uma significativa redução na resposta à FEN nas artérias femorais de SHR após exercício agudo em esteira, que foi abolida após inibição da síntese de NO com L-NAME. Em seres humanos, a inibição da síntese de NO também abole a resposta vasodilatadora após exercício agudo.31 Nossos resultados estão de acordo com os dados obtidos de experimentos com exercício crônico. Chen et al.19 mostraram que a redução da resposta vascular a norepinefrina após treino em esteira foi mediada por NO. Da mesma maneira, Chen et al.18 demonstraram uma redução na sensibilidade à norepinefrina e à FEN na aorta de SHR e ratos Wistar Kyoto após treino em esteira, devido ao aumento da produção de NO.
Portanto, parece que tanto o exercício agudo quanto o crônico desencadeiam uma redução na resposta vascular primariamente mediada por aumento da síntese de NO. Entretanto, os mecanismos subjacentes que aumentam a produção de NO após exercício agudo e crônico são diferentes. Foi relatado que o exercício aumenta a expressão de eNOS.26,32 Investigamos se a expressão de proteína de eNOS e iNOS aumentava após uma única sessão de exercício resistido. Como esperado, a expressão dessas isoformas não se alterou após exercício agudo, porque é improvável que uma única sessão de exercício represente suficiente estímulo para induzir expressão de proteína. Como a ativação de eNOS depende de um padrão de fosforilação de sítios bem caracterizados,33 levantamos a hipótese de que a fosforilação de eNOS pudesse ser o mecanismo envolvido na produção de NO após exercício agudo; consequentemente, medimos os níveis de proteína p-eNOS1177. O aminoácido serina na posição 1177 é o sítio primário de ativação da fosforilação de eNOS, e, quando modulado por Akt quinase (também conhecida como proteína B quinase) e eNOS, demonstra aumento da sensibilidade às concentrações basais de Ca2+/calmodulina.34 O nível da proteína p-eNOS1177 aumentou significativamente após exercício resistido agudo quando comparado ao de ratos controles, indicando que eNOS é ativado após exercício resistido agudo. Tal achado confirma nossa hipótese de que a diminuição da vasoconstricção e o aumento da vasodilatação após exercício estimulada por ACT foram mediados por NO. Alguns fatores envolvidos na ativação de eNOS e, consequentemente, na síntese de NO, como estresse de cisalhamento, hipóxia e liberação de catecolamina, estão presentes durante exercício e na recuperação depois do exercício. Logo, após exercício, a atividade de eNOS poderia permanecer elevada por maior tempo, reduzindo a reatividade vascular mediada por NO.
Para investigar o possível papel dos prostanoides vasodilatadores na redução da resposta vascular após exercício, avaliamos a resposta à FEN na presença de indometacina, um inibidor da COX. Diferentemente dos resultados obtidos na artéria da cauda em estudos prévios,12 a resposta aórtica foi significativamente diminuída após inibição da COX (Figura 2C) em ratos controles e exercitados. Tal resposta pode ser explicada pela elevação da produção de prostanoide vasoconstritor induzida por COX em ratos hipertensos.35 Além disso, o aumento da síntese de prostanoide mostrou resultar do aumento da atividade de COX-2. Nossos dados indicam um importante efeito de uma única sessão de exercício resistido. Como observado na Figura 2, a porcentagem da área sob a curva foi maior após o exercício, sugerindo que o exercício resistido agudo diminua a liberação de prostanoide vasoconstritor nos vasos. A disfunção endotelial presente na hipertensão arterial aumenta a produção de prostanoide vasoconstritor,35-38 e uma única sessão de exercício resistido tem importante impacto no aprimoramento da função vascular, pois reduz a liberação de prostanoide vasoconstritor. Além disso, está bem estabelecido que o NO pode regular a atividade de enzimas COX,39 e a atividade de NOS é aumentada quando a via COX é inibida por indometacina.40
Conclusões
Este estudo demonstrou que uma única sessão de exercício resistido diminuiu a resposta vascular à FEN e aumentou o relaxamento endotélio-dependente mediado por ACT em SHR. Tal adaptação parece ser mediada por NO, devido à elevação dos níveis da proteína p-eNOS1117. Além disso, a presente investigação mostra que o exercício resistido agudo pode reduzir a produção de prostanoides vasodilatadores nos anéis aórticos de SHR. Assim, nossos achados sugerem que a prática de exercício resistido, mesmo de uma única sessão, possa ter grande relevância clínica para o controle da hipertensão.
Fontes de financiamento
O presente estudo foi financiado pelo CNPq.
Vinculação acadêmica
Este artigo é parte de tese de Doutorado de Juliana Hott de Fúcio Lizardo pela Universidade Federal do Espírito Santo.