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Endothelial nitric oxide synthase activation contributes to post-exercise hypotension in spontaneously hypertensive rats
Biochemical and Biophysical Research Communications 382 (2009) 711–714
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Endothelial nitric oxide synthase activation contributes to post-exercise hypotension in spontaneously hypertensive rats Sang Ki Lee 1, Cuk Seong Kim 1, Hyo Shin Kim, Eun Jung Cho, Hee Kyoung Joo, Ji Young Lee, Eun Ji Lee, Jin Bong Park, Byeong Hwa Jeon * Infection Signaling Network Research Center, Research Institutes of Biomedical Science, Department of Physiology, College of Medicine, Chungnam National University, 6 Munhwa-dong, Jung-gu, Daejeon 301-131, Republic of Korea
a r t i c l e
i n f o
Article history: Received 16 March 2009 Available online 21 March 2009
Keywords: Post-exercise hypotension Nitric oxide Endothelial nitric oxide synthase Spontaneously hypertensive rats
a b s t r a c t We investigated the role that endothelial nitric oxide synthase plays in post-exercise hypotension in spontaneously hypertensive rats. To accomplish this, rats were subjected to a single bout of dynamic exercise on a treadmill at 15 m/min for 20 min. L-nitroarginine methyl ester (L-NAME, 40 mg/kg, i.p.) significantly inhibited post-exercise hypotension (25 ± 11 and 5 ± 3 mm Hg, respectively; P < 0.05). In addition, the superoxide anion generation was decreased, while the plasma nitrite production and serine phosphorylation of endothelial nitric oxide synthase were significantly elevated in spontaneously hypertensive rats at 30 min after the termination of exercise. Taken together, these data demonstrate that the increased phosphorylation of endothelial nitric oxide synthase plays a crucial role in the reduction of arterial pressure following a single bout of dynamic exercise in spontaneously hypertensive rats. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Regular physical activity is an important factor in the prevention and treatment of cardiovascular diseases. Post-exercise hypotension (PEH) is a phenomenon in which a prolonged decrease in resting blood pressure occurs in the minutes and hours following acute exercise [1,2]. PEH is observed in both normotensive and hypertensive humans, as well as in spontaneously hypertensive rats (SHR); however, it is generally greater in magnitude in hypertensive subjects [3]. This phenomenon has been widely investigated due to the importance of the treatment and prevention of hypertension [1]. Vascular reactive oxygen species, which are produced in endothelial, adventitial, and vascular smooth muscle cells, are derived primarily from NADPH oxidase [4]. In general, moderate physical exercise leads to reduced oxidative stress, whereas exhaustive exercise causes oxidative stress that overwhelms the cellular antioxidant defenses [5]. However, the level of change that occurs in the reactive oxygen species during the PEH period has not yet been defined. Exercise increases nitric oxide production by increasing the level of endothelial shear stress [6]. However, endothelial nitric oxide synthase (eNOS) activity is regulated at multiple levels. In addition to occurring in response to an influx of calcium, eNOS activity is also regulated by post-translational modifications. Activation of eNOS * Corresponding author. Fax: +82 42 585 8440. E-mail address: [email protected] (B.H. Jeon). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.03.090
results in calcium sensitization via its phosphorylation at serine 1177. The production of endothelial nitric oxide increases in the vascular bed of the exercising skeletal muscle during and after exercise due to the increased shear stress [7]. Recently, it was reported that acute resistant exercise has hypotensive effect on SHR and that NO plays a crucial role in PEH [8]. However, the role of the phosphorylation of eNOS on the PEH in hypertensive rats is not known. Therefore, this study is conducted to determine whether the phosphorylation of eNOS contributes to PEH in SHRs. Material and methods Surgical procedures. All experiments were performed on 12– 16 week old male spontaneously hypertensive rats (SHR, Samtaco, Korea). The Animal Care Committee of Chungnam National University approved the animal care and all experimental procedures conducted in this study. All instrumentation was conducted using aseptic procedures. Rats were anesthetized with an intraperitoneal injection of Ketamine (80 mg/kg)/Xylazine (12 mg/kg), after which a polyurethane catheter was inserted into the descending aorta via the left common carotid artery to measure the arterial pressure and heart rate. The catheter was tunneled beneath the skin and exteriorized at the back of the neck. The arterial catheter was flushed daily with heparinized saline, filled with heparin (1000 U/ml), and then plugged with a stainless steel pin. The animals were allowed at least 3–4 days to recover from the surgery. During the recovery period, the animals were monitored for signs of infection and weight loss.
S.K. Lee et al. / Biochemical and Biophysical Research Communications 382 (2009) 711–714
Experimental setup. The arterial pressure was determined by connecting the arterial catheter to a Gould P23XL pressure transducer that was coupled to a preamplifier (Grass Model PI-1, ACDC, USA). On the day of the experiment, the rats were placed unrestrained in a large Plexiglas box. The animals were allowed to acclimate to their new environment for 60 min prior to collection of the pre-exercise data. After the 60 min adaptation period, the blood pressure and heart rate were measured at 5 min intervals for 20 min. The rats then ran on the treadmill at 15 m/min for 20 min, during which time the blood pressure and heart rate were analyzed using the Chartpro software (Adinstruments, USA). Detection of superoxide production by lucigenin chemiluminescence. Vascular superoxide production was measured by lucigenin-enhanced chemiluminescence as described previously [9]. Briefly, aortas were cleared of adherent fat and cut into 3–4 mm rings. Aortic segments were transferred into tubes containing in aerated Krebs– HEPES buffer solution (containing 99 mM NaCl, 4.7 mM KCl, 1.0 mM KH2PO4, 1.2 mM MgSO4, 1.2 mM CaCl2, 25 mM NaHCO3, 20 mM Na-HEPES, and 11 mM glucose, pH 7.4). The aortic rings were maintained at 37 °C for at least 30 min before dark-adapted lucigenin (bis-N-methylacridinium nitrate, Sigma, USA) solution (5 lmol/L) was added. Aortic rings were immersed in lucigenin solution and chemiluminescence was detected with a Monolight luminometer (Lumat LB9507, Berthold Technologies, Germany). The chemiluminescence signal was integrated over 2 min. Freshly prepared NADPH (100 lmol/L) was added for 2 min where indicated, prior to measuring luminescence. Values are expressed as relative light units per milligram dry weight of samples (RLU/mg dry aortas). Western blotting. Thoracic aortas were isolated and homogenized on ice with a tissue homogenizer (Biospec Products Inc., USA) in the lysis buffer containing of 20 mM Tris HCl, 0.5% NP40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM phenylmethylsulfonylfluoride, 2 mM b-glycerophosphate, 1 mM sodium vanadate, 1 lg/ml leupeptin and pH 7.5. The homogenized tissues were centrifuged at 10,000g for 30 min at 4 °C and the supernatants were then used for determination of total protein concentration with the Bradford protein assay. Forty micrograms of protein were separated by 7.5 10% SDS–PAGE and then transferred onto nitrocellulose membranes. Next, the membranes were blocked for 1 h in 5% skim milk solution, after which they were incubated with specific antibodies to eNOS (SC-654, Santa Cruz, USA), phospho-ser1177-eNOS (#9571, Cell Signaling, USA), and b-actin (SC-1616, Santa Cruz, USA). The protein expression was then detected using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, USA). Finally, the Western blots were quantified densitometrically using the ImageQuant software (Molecular Dynamics, USA). Plasma nitrite measurement. For the plasma measurement of NO metabolites, 0.8–1.0 ml of whole blood was obtained by direct puncture of the right ventricle of the anesthetized animals. The plasma was then deproteinized using a 10-kDa cutoff filter. Next, the plasma was processed for measurement of the NO intermediary metabolites, nitrite and nitrate, which are the stable breakdown products of NO, based on the specific light absorbance following the manufacturer’s recommendations (Calbiochem, USA). Statistical analysis. All data are expressed as the mean ± SEM. Statistical evaluation was conducted using two-way repeated measured ANOVA for the MBP analysis and one-way ANOVA, followed by a Turkey post hoc test. A P < 0.05 was considered to be statistically significant.
mechanisms involved in mediating PEH are proposed [1,3,10]. Especially, PEH is closely associated with the reduction of peripheral vascular resistance [11–13]. However, the role of the endothelial nitric oxide synthase activation on dynamic exercise-induced PEH is not clear in SHR. In this study, we investigated the effect of L-nitroarginine methyl ester (L-NAME, 40 mg/kg, i.p.) on the mean blood pressure during dynamic exercise on treadmills in SHRs. As shown in Fig. 1, the mean blood pressure after the cessation of exercise was decreased when compared to the pre-exercise blood pressure levels. In addition, PEH peaked at 30 min and then recovered at 60 min after the cessation of exercise. Furthermore, treatment with LNAME (40 mg/kg, i.p.) led to an increase in basal blood pressure; however, the post-exercise hypotension was significantly reduced (25 ± 11 and 5 ± 3 mm Hg, respectively, P < 0.05) in the L-NAMEtreated SHRs. These findings suggest that the post-exercise hypotension was mediated by nitric oxide production that occurred after the cessation of exercise and that it was a transient phenomenon in SHR. These results are similar to the results of a recently conducted study that found that acute resistance exercise exerts a hypotensive effect on SHR and that these effects were related to nitric oxide production [8]. In addition, it is known that nitric oxide may be partially responsible for the decreased adrenergic receptor sensitivity that occurs in PEH [14]. Taken together, these findings indicate that nitric oxide plays a crucial role in PEH. In both animal models and humans, increased blood pressure has been found to be associated with oxidative stress in the vasculature. Excessive endothelial production of superoxide radicals may be one of cause of hypertension. Indeed, it is known that NADPH oxidase is abundant in the vasculature [4,15]. Therefore, we investigated to determine the NADPH-derived superoxide anion generation in aortic rings of non-exercise control group (SHRC), exercise group of SHR (SHR-E) and exercise group in L-NAMEtreated SHRs (SHR-E, L-NAME) at 30 min after the cessation of exercise. As shown in Fig. 2, NADPH-derived superoxide production of the aortic rings of SHR-E was decreased at 30 min after cessation of exercise; however, it was not in the L-NAME-treated SHRE. These data suggest that decreased superoxide anion production, which may reflect decreased NADPH oxidase activity or increased peroxynitrite formation as discuss below, is an important factor in the decrease in blood pressure that occurs after the cessation of exercise in hypertensive rats. Furthermore, the results of the present study indicate that subjecting SHRs to an exercise intensity
280 Basal L-NAME
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Time (min) Results and discussion Post-exercise hypotension (PEH) plays an important role in the non-pharmacological treatment of hypertension. The possible
Fig. 1. Change in the mean arterial pressure (MAP) before, during, and after a single bout of exercise with and without L-nitroargnine methyl ester (L-NAME, 40 mg/kg) in spontaneously hypertensive rats (SHR). Exercise was conducted on a treadmill at 15 m/min for 20 min. Each data point shows the mean ± SEM. (n = 5). *P < 0.05 vs. L-NAME.
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of 15 m/min may be a useful method to reduce the superoxide anion generation in SHRs. Nitric oxide suppresses NADPH oxidase-dependent oxidative stress via S-nitrosylation of the crucial subunit, p47 phox, in the vasculature [16,17]. Therefore, we measured the plasma nitrite levels at 30 min after the cessation of exercise in the SHRs. As shown in Fig. 3, the plasma nitrite levels at 30 min after the cessation of exercise in SHR-E were significantly higher than that of pre-exercise (SHR-C); however, its production was decreased in response to pretreatment with L-NAME (SHR-E, L-NAME). These findings suggest that increased plasma nitric oxide production acts as an important mediator of PEH in SHRs. Finally, we evaluated the phosphorylation of eNOS at serine residue 1177 in the aortic homogenates of exercise group of SHR. As
Superoxide production n (RLU U/mg ao orta)
# 8000
*
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0 SHR-C SHR-E SHR-E L-NAME
NADPH
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-
-
shown in Fig. 4, the phosphorylation of eNOS (S1177) was markedly higher in the aortic homogenate at 30 min after the cessation of exercise in SHR-E and L-NAME-treated SHR-E, but the eNOS expression was not changed. However, pretreatment with L-NAME did not inhibit the phosphorylation of eNOS when compared to the exercise group of SHRs (SHR-E). Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. NO production from endothelial cells is stimulated by a variety of mechanical forces such as shear stress [18,19]. These findings suggest that high shear stress induced by a single bout of exercise activates the phosphorylation of eNOS. L-NAME is an inhibitor of NO synthesis from L-arginine by competing with L-arginine [20], and does not inhibit the phosphorylation of eNOS which is regulated by several kinds of kinase such as phosphatidyl-inositol-3-kinase or Akt kinase [21]. Therefore, the treatment of L-NAME did not reduce the enhanced eNOS phosphorylation produced by exercise. Taken together, increased endothelial nitric oxide production through the activation of eNOS at serine 1177 may be responsible, at least in part, for the PEH that occurs in SHRs. It is known that a reduction of NO bioavailability in the endothelium-impaired function disorders is associated with an increase in endothelial production of superoxide anion. Moreover, our data indicate that the increased eNOS activity through the activation of eNOS at serine 1177 and the reduced superoxide production can lead to an increase of NO bioavailability in SHRs. In the present study, the decreased NADPH-derived superoxide anion production in PEH may reflect the decreased NADPH oxidase activity, however, it may not exclude the possibility of increased scavenging of superoxide anion through peroxynitrite formation [22] due to increased NO production in PEH.
SHR-C SHR-E SHR-E L-NAME
+
+
+
Fig. 2. Effect of a single bout of exercise on basal and NADPH-derived superoxide production by the aortic rings of spontaneously hypertensive rats (SHR). Rats were subjected to a single bout of exercise (E) on a treadmill at 15 m/min for 20 min. The rats were pretreated with L-nitroargnine methyl ester (L-NAME, 40 mg/kg) 1 h prior to being subjected to the exercise. The aortas of SHRs were isolated at 30 min postexercise, after which the superoxide production was determined based on the lucigenin chemiluminescence. Each data point represents the mean ± SEM. (n = 4). * P < 0.05 vs. SHR-C. #P < 0.05 vs. SHR-E.
SHR-C
eNOS actin β-actin β
P Plasma nitrite (uM)
25
#
20 15 10
Phospho-e P eNOS/eNO OS (arbitray)
*
30
SHR-E + L-NAME
Phospho PhosphoeNOS (S1177)
*
1.5 35
SHR-E
*
1.0
0.5
5 0.0 SHR-C
0 SHR-C
SHR-E
SHR-E L-NAME
Fig. 3. Effect of a single bout of exercise on plasma nitrite levels by spontaneously hypertensive rats (SHR). Rats were subjected to a single bout of exercise (E) on a treadmill at 15 m/min for 20 min. The rats were pretreated with L-nitroargnine methyl ester (L-NAME, 40 mg/kg) 1 h prior to being subjected to the exercise. Each data point represents the mean ± SEM. (n = 4). *P < 0.05 vs. SHR-C; #P < 0.05 vs. SHR-E.
SHR-E
SHR-E L-NAME
Fig. 4. Effect of a single bout exercise on the phosphorylation of endothelial nitric oxide synthase in the aortas of spontaneously hypertensive rats (SHR). Rats were subjected to a single bout of exercise (E) on a treadmill at 15 m/min for 20 min. The rats were pretreated with L-nitroargnine methyl ester (L-NAME, 40 mg/kg) 1 h prior to being subjected to the exercise. The aortas of the SHRs were harvested after 30 min of the cessation of exercise for Western blotting. Each data point represents the mean ± SEM. (n = 4). *P < 0.05 vs. SHR-C.
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S.K. Lee et al. / Biochemical and Biophysical Research Communications 382 (2009) 711–714
Taken together, the increased activation of eNOS and/or decreased production of superoxide anion that occur after the cessation of exercise appear to underlie the drop in blood pressure in the hypertensive animals.
[10]
Acknowledgments
[12]
This work was supported by Korea Science & Engineering Foundation through the Infection Signaling Network Research Center (R13-2007-020-01000-0), Korea Science & Engineering Foundation (R01-2008-000-20623-0, R01-2007-000-10974-0) and Chungnam National University.
[11]
[13]
[14]
[15]
References [1] M.J. Kenney, D.R. Seals, Postexercise hypotension. Key features, mechanisms, and clinical significance, Hypertension 22 (1993) 653–664. [2] J.M. Overton, M.J. Joyner, C.M. Tipton, Reductions in blood pressure after acute exercise by hypertensive rats, J. Appl. Physiol. 64 (1988) 748–752. [3] J.R. MacDonald, Potential causes, mechanisms, and implications of post exercise hypotension, J. Hum. Hypertens. 16 (2002) 225–236. [4] R.M. Touyz, Reactive oxygen species, vascular oxidative stress, and redox signaling in hypertension: what is the clinical significance? Hypertension 44 (2004) 248–252. [5] S. Sachdev, K.J. Davies, Production, detection, and adaptive responses to free radicals in exercise, Free Radic. Biol. Med. 44 (2008) 215–223. [6] M.H. Yen, J.H. Yang, J.R. Sheu, Y.M. Lee, Y.A. Ding, Chronic exercise enhances endothelium-mediated dilation in spontaneously hypertensive rats, Life Sci. 57 (1995) 2205–2213. [7] G.M. Rubanyi, J.C. Romero, P.M. Vanhoutte, Flow-induced release of endothelium-derived relaxing factor, Am. J. Physiol. 250 (1986) H1145–H1149. [8] J.H. Lizardo, E.A. Silveira, D.V. Vassallo, E.M. Oliveira, Post-resistance exercise hypotension in spontaneously hypertensive rats is mediated by nitric oxide, Clin. Exp. Pharmacol. Physiol. 35 (2008) 782–787. [9] B.H. Jeon, G. Gupta, Y.C. Park, B. Qi, A. Haile, F.A. Khanday, Y.X. Liu, J.M. Kim, M. Ozaki, A.R. White, D.E. Berkowitz, K. Irani, Apurinic/apyrimidinic endonuclease
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1 regulates endothelial NO production and vascular tone, Circ. Res. 95 (2004) 902–910. C. Mach, C. Foster, G. Brice, R.P. Mikat, J.P. Porcari, Effect of exercise duration on postexercise hypotension, J. Cardiopulm. Rehabil. 25 (2005) 366–369. M.G. Howard, S.E. DiCarlo, Reduced vascular responsiveness after a single bout of dynamic exercise in the conscious rabbit, J. Appl. Physiol. 73 (1992) 2662– 2667. M.G. Howard, S.E. DiCarlo, J.N. Stallone, Acute exercise attenuates phenylephrine-induced contraction of rabbit isolated aortic rings, Med. Sci. Sports Exerc. 24 (1992) 1102–1107. R.D. Patil, S.E. DiCarlo, H.L. Collins, Acute exercise enhances nitric oxide modulation of vascular response to phenylephrine, Am. J. Physiol. 265 (1993) H1184–H1188. S.P. Rao, H.L. Collins, S.E. DiCarlo, Postexercise alpha-adrenergic receptor hyporesponsiveness in hypertensive rats is due to nitric oxide, Am. J. Physiol. Regul. Integr. Comp. Physiol. 282 (2002) R960–R968. K. Irani, Oxidant signaling in vascular cell growth, death, and survival: a review of the roles of reactive oxygen species in smooth muscle and endothelial cell mitogenic and apoptotic signaling, Circ. Res. 87 (2000) 179–183. S. Selemidis, Suppressing NADPH oxidase-dependent oxidative stress in the vasculature with nitric oxide donors, Clin. Exp. Pharmacol. Physiol. 35 (2008) 1395–1401. S. Selemidis, G.J. Dusting, H. Peshavariya, B.K. Kemp-Harper, G.R. Drummond, Nitric oxide suppresses NADPH oxidase-dependent superoxide production by S-nitrosylation in human endothelial cells, Cardiovasc. Res. 75 (2007) 349– 358. Y.C. Boo, H. Jo, Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases, Am. J. Physiol. Cell Physiol. 285 (2003) C499–C508. Y.C. Boo, G. Sorescu, N. Boyd, I. Shiojima, K. Walsh, J. Du, H. Jo, Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A, J. Biol. Chem. 277 (2002) 3388–3396. D.D. Rees, R.M. Palmer, R. Schulz, H.F. Hodson, S. Moncada, Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo, Br. J. Pharmacol. 101 (1990) 746–752. S. Dimmeler, I. Fleming, B. Fisslthaler, C. Hermann, R. Busse, A.M. Zeiher, Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation, Nature 399 (1999) 601–605. H. Cai, D.G. Harrison, Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress, Circ. Res. 87 (2000) 840–844.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Endothelial nitric oxide synthase activation contributes to post-exercise hypotension in spontaneously hypertensive rats Sang Ki Lee 1, Cuk Seong Kim 1, Hyo Shin Kim, Eun Jung Cho, Hee Kyoung Joo, Ji Young Lee, Eun Ji Lee, Jin Bong Park, Byeong Hwa Jeon * Infection Signaling Network Research Center, Research Institutes of Biomedical Science, Department of Physiology, College of Medicine, Chungnam National University, 6 Munhwa-dong, Jung-gu, Daejeon 301-131, Republic of Korea
a r t i c l e
i n f o
Article history: Received 16 March 2009 Available online 21 March 2009
Keywords: Post-exercise hypotension Nitric oxide Endothelial nitric oxide synthase Spontaneously hypertensive rats
a b s t r a c t We investigated the role that endothelial nitric oxide synthase plays in post-exercise hypotension in spontaneously hypertensive rats. To accomplish this, rats were subjected to a single bout of dynamic exercise on a treadmill at 15 m/min for 20 min. L-nitroarginine methyl ester (L-NAME, 40 mg/kg, i.p.) significantly inhibited post-exercise hypotension (25 ± 11 and 5 ± 3 mm Hg, respectively; P < 0.05). In addition, the superoxide anion generation was decreased, while the plasma nitrite production and serine phosphorylation of endothelial nitric oxide synthase were significantly elevated in spontaneously hypertensive rats at 30 min after the termination of exercise. Taken together, these data demonstrate that the increased phosphorylation of endothelial nitric oxide synthase plays a crucial role in the reduction of arterial pressure following a single bout of dynamic exercise in spontaneously hypertensive rats. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Regular physical activity is an important factor in the prevention and treatment of cardiovascular diseases. Post-exercise hypotension (PEH) is a phenomenon in which a prolonged decrease in resting blood pressure occurs in the minutes and hours following acute exercise [1,2]. PEH is observed in both normotensive and hypertensive humans, as well as in spontaneously hypertensive rats (SHR); however, it is generally greater in magnitude in hypertensive subjects [3]. This phenomenon has been widely investigated due to the importance of the treatment and prevention of hypertension [1]. Vascular reactive oxygen species, which are produced in endothelial, adventitial, and vascular smooth muscle cells, are derived primarily from NADPH oxidase [4]. In general, moderate physical exercise leads to reduced oxidative stress, whereas exhaustive exercise causes oxidative stress that overwhelms the cellular antioxidant defenses [5]. However, the level of change that occurs in the reactive oxygen species during the PEH period has not yet been defined. Exercise increases nitric oxide production by increasing the level of endothelial shear stress [6]. However, endothelial nitric oxide synthase (eNOS) activity is regulated at multiple levels. In addition to occurring in response to an influx of calcium, eNOS activity is also regulated by post-translational modifications. Activation of eNOS * Corresponding author. Fax: +82 42 585 8440. E-mail address: [email protected] (B.H. Jeon). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.03.090
results in calcium sensitization via its phosphorylation at serine 1177. The production of endothelial nitric oxide increases in the vascular bed of the exercising skeletal muscle during and after exercise due to the increased shear stress [7]. Recently, it was reported that acute resistant exercise has hypotensive effect on SHR and that NO plays a crucial role in PEH [8]. However, the role of the phosphorylation of eNOS on the PEH in hypertensive rats is not known. Therefore, this study is conducted to determine whether the phosphorylation of eNOS contributes to PEH in SHRs. Material and methods Surgical procedures. All experiments were performed on 12– 16 week old male spontaneously hypertensive rats (SHR, Samtaco, Korea). The Animal Care Committee of Chungnam National University approved the animal care and all experimental procedures conducted in this study. All instrumentation was conducted using aseptic procedures. Rats were anesthetized with an intraperitoneal injection of Ketamine (80 mg/kg)/Xylazine (12 mg/kg), after which a polyurethane catheter was inserted into the descending aorta via the left common carotid artery to measure the arterial pressure and heart rate. The catheter was tunneled beneath the skin and exteriorized at the back of the neck. The arterial catheter was flushed daily with heparinized saline, filled with heparin (1000 U/ml), and then plugged with a stainless steel pin. The animals were allowed at least 3–4 days to recover from the surgery. During the recovery period, the animals were monitored for signs of infection and weight loss.
S.K. Lee et al. / Biochemical and Biophysical Research Communications 382 (2009) 711–714
Experimental setup. The arterial pressure was determined by connecting the arterial catheter to a Gould P23XL pressure transducer that was coupled to a preamplifier (Grass Model PI-1, ACDC, USA). On the day of the experiment, the rats were placed unrestrained in a large Plexiglas box. The animals were allowed to acclimate to their new environment for 60 min prior to collection of the pre-exercise data. After the 60 min adaptation period, the blood pressure and heart rate were measured at 5 min intervals for 20 min. The rats then ran on the treadmill at 15 m/min for 20 min, during which time the blood pressure and heart rate were analyzed using the Chartpro software (Adinstruments, USA). Detection of superoxide production by lucigenin chemiluminescence. Vascular superoxide production was measured by lucigenin-enhanced chemiluminescence as described previously [9]. Briefly, aortas were cleared of adherent fat and cut into 3–4 mm rings. Aortic segments were transferred into tubes containing in aerated Krebs– HEPES buffer solution (containing 99 mM NaCl, 4.7 mM KCl, 1.0 mM KH2PO4, 1.2 mM MgSO4, 1.2 mM CaCl2, 25 mM NaHCO3, 20 mM Na-HEPES, and 11 mM glucose, pH 7.4). The aortic rings were maintained at 37 °C for at least 30 min before dark-adapted lucigenin (bis-N-methylacridinium nitrate, Sigma, USA) solution (5 lmol/L) was added. Aortic rings were immersed in lucigenin solution and chemiluminescence was detected with a Monolight luminometer (Lumat LB9507, Berthold Technologies, Germany). The chemiluminescence signal was integrated over 2 min. Freshly prepared NADPH (100 lmol/L) was added for 2 min where indicated, prior to measuring luminescence. Values are expressed as relative light units per milligram dry weight of samples (RLU/mg dry aortas). Western blotting. Thoracic aortas were isolated and homogenized on ice with a tissue homogenizer (Biospec Products Inc., USA) in the lysis buffer containing of 20 mM Tris HCl, 0.5% NP40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM phenylmethylsulfonylfluoride, 2 mM b-glycerophosphate, 1 mM sodium vanadate, 1 lg/ml leupeptin and pH 7.5. The homogenized tissues were centrifuged at 10,000g for 30 min at 4 °C and the supernatants were then used for determination of total protein concentration with the Bradford protein assay. Forty micrograms of protein were separated by 7.5 10% SDS–PAGE and then transferred onto nitrocellulose membranes. Next, the membranes were blocked for 1 h in 5% skim milk solution, after which they were incubated with specific antibodies to eNOS (SC-654, Santa Cruz, USA), phospho-ser1177-eNOS (#9571, Cell Signaling, USA), and b-actin (SC-1616, Santa Cruz, USA). The protein expression was then detected using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, USA). Finally, the Western blots were quantified densitometrically using the ImageQuant software (Molecular Dynamics, USA). Plasma nitrite measurement. For the plasma measurement of NO metabolites, 0.8–1.0 ml of whole blood was obtained by direct puncture of the right ventricle of the anesthetized animals. The plasma was then deproteinized using a 10-kDa cutoff filter. Next, the plasma was processed for measurement of the NO intermediary metabolites, nitrite and nitrate, which are the stable breakdown products of NO, based on the specific light absorbance following the manufacturer’s recommendations (Calbiochem, USA). Statistical analysis. All data are expressed as the mean ± SEM. Statistical evaluation was conducted using two-way repeated measured ANOVA for the MBP analysis and one-way ANOVA, followed by a Turkey post hoc test. A P < 0.05 was considered to be statistically significant.
mechanisms involved in mediating PEH are proposed [1,3,10]. Especially, PEH is closely associated with the reduction of peripheral vascular resistance [11–13]. However, the role of the endothelial nitric oxide synthase activation on dynamic exercise-induced PEH is not clear in SHR. In this study, we investigated the effect of L-nitroarginine methyl ester (L-NAME, 40 mg/kg, i.p.) on the mean blood pressure during dynamic exercise on treadmills in SHRs. As shown in Fig. 1, the mean blood pressure after the cessation of exercise was decreased when compared to the pre-exercise blood pressure levels. In addition, PEH peaked at 30 min and then recovered at 60 min after the cessation of exercise. Furthermore, treatment with LNAME (40 mg/kg, i.p.) led to an increase in basal blood pressure; however, the post-exercise hypotension was significantly reduced (25 ± 11 and 5 ± 3 mm Hg, respectively, P < 0.05) in the L-NAMEtreated SHRs. These findings suggest that the post-exercise hypotension was mediated by nitric oxide production that occurred after the cessation of exercise and that it was a transient phenomenon in SHR. These results are similar to the results of a recently conducted study that found that acute resistance exercise exerts a hypotensive effect on SHR and that these effects were related to nitric oxide production [8]. In addition, it is known that nitric oxide may be partially responsible for the decreased adrenergic receptor sensitivity that occurs in PEH [14]. Taken together, these findings indicate that nitric oxide plays a crucial role in PEH. In both animal models and humans, increased blood pressure has been found to be associated with oxidative stress in the vasculature. Excessive endothelial production of superoxide radicals may be one of cause of hypertension. Indeed, it is known that NADPH oxidase is abundant in the vasculature [4,15]. Therefore, we investigated to determine the NADPH-derived superoxide anion generation in aortic rings of non-exercise control group (SHRC), exercise group of SHR (SHR-E) and exercise group in L-NAMEtreated SHRs (SHR-E, L-NAME) at 30 min after the cessation of exercise. As shown in Fig. 2, NADPH-derived superoxide production of the aortic rings of SHR-E was decreased at 30 min after cessation of exercise; however, it was not in the L-NAME-treated SHRE. These data suggest that decreased superoxide anion production, which may reflect decreased NADPH oxidase activity or increased peroxynitrite formation as discuss below, is an important factor in the decrease in blood pressure that occurs after the cessation of exercise in hypertensive rats. Furthermore, the results of the present study indicate that subjecting SHRs to an exercise intensity
280 Basal L-NAME
260
MAP (mmHg)
712
240 220 200
* * *
180
*
* *
160
-20 20
0
20
40
60
80
Time (min) Results and discussion Post-exercise hypotension (PEH) plays an important role in the non-pharmacological treatment of hypertension. The possible
Fig. 1. Change in the mean arterial pressure (MAP) before, during, and after a single bout of exercise with and without L-nitroargnine methyl ester (L-NAME, 40 mg/kg) in spontaneously hypertensive rats (SHR). Exercise was conducted on a treadmill at 15 m/min for 20 min. Each data point shows the mean ± SEM. (n = 5). *P < 0.05 vs. L-NAME.
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of 15 m/min may be a useful method to reduce the superoxide anion generation in SHRs. Nitric oxide suppresses NADPH oxidase-dependent oxidative stress via S-nitrosylation of the crucial subunit, p47 phox, in the vasculature [16,17]. Therefore, we measured the plasma nitrite levels at 30 min after the cessation of exercise in the SHRs. As shown in Fig. 3, the plasma nitrite levels at 30 min after the cessation of exercise in SHR-E were significantly higher than that of pre-exercise (SHR-C); however, its production was decreased in response to pretreatment with L-NAME (SHR-E, L-NAME). These findings suggest that increased plasma nitric oxide production acts as an important mediator of PEH in SHRs. Finally, we evaluated the phosphorylation of eNOS at serine residue 1177 in the aortic homogenates of exercise group of SHR. As
Superoxide production n (RLU U/mg ao orta)
# 8000
*
6000
4000
2000
0 SHR-C SHR-E SHR-E L-NAME
NADPH
-
-
-
shown in Fig. 4, the phosphorylation of eNOS (S1177) was markedly higher in the aortic homogenate at 30 min after the cessation of exercise in SHR-E and L-NAME-treated SHR-E, but the eNOS expression was not changed. However, pretreatment with L-NAME did not inhibit the phosphorylation of eNOS when compared to the exercise group of SHRs (SHR-E). Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. NO production from endothelial cells is stimulated by a variety of mechanical forces such as shear stress [18,19]. These findings suggest that high shear stress induced by a single bout of exercise activates the phosphorylation of eNOS. L-NAME is an inhibitor of NO synthesis from L-arginine by competing with L-arginine [20], and does not inhibit the phosphorylation of eNOS which is regulated by several kinds of kinase such as phosphatidyl-inositol-3-kinase or Akt kinase [21]. Therefore, the treatment of L-NAME did not reduce the enhanced eNOS phosphorylation produced by exercise. Taken together, increased endothelial nitric oxide production through the activation of eNOS at serine 1177 may be responsible, at least in part, for the PEH that occurs in SHRs. It is known that a reduction of NO bioavailability in the endothelium-impaired function disorders is associated with an increase in endothelial production of superoxide anion. Moreover, our data indicate that the increased eNOS activity through the activation of eNOS at serine 1177 and the reduced superoxide production can lead to an increase of NO bioavailability in SHRs. In the present study, the decreased NADPH-derived superoxide anion production in PEH may reflect the decreased NADPH oxidase activity, however, it may not exclude the possibility of increased scavenging of superoxide anion through peroxynitrite formation [22] due to increased NO production in PEH.
SHR-C SHR-E SHR-E L-NAME
+
+
+
Fig. 2. Effect of a single bout of exercise on basal and NADPH-derived superoxide production by the aortic rings of spontaneously hypertensive rats (SHR). Rats were subjected to a single bout of exercise (E) on a treadmill at 15 m/min for 20 min. The rats were pretreated with L-nitroargnine methyl ester (L-NAME, 40 mg/kg) 1 h prior to being subjected to the exercise. The aortas of SHRs were isolated at 30 min postexercise, after which the superoxide production was determined based on the lucigenin chemiluminescence. Each data point represents the mean ± SEM. (n = 4). * P < 0.05 vs. SHR-C. #P < 0.05 vs. SHR-E.
SHR-C
eNOS actin β-actin β
P Plasma nitrite (uM)
25
#
20 15 10
Phospho-e P eNOS/eNO OS (arbitray)
*
30
SHR-E + L-NAME
Phospho PhosphoeNOS (S1177)
*
1.5 35
SHR-E
*
1.0
0.5
5 0.0 SHR-C
0 SHR-C
SHR-E
SHR-E L-NAME
Fig. 3. Effect of a single bout of exercise on plasma nitrite levels by spontaneously hypertensive rats (SHR). Rats were subjected to a single bout of exercise (E) on a treadmill at 15 m/min for 20 min. The rats were pretreated with L-nitroargnine methyl ester (L-NAME, 40 mg/kg) 1 h prior to being subjected to the exercise. Each data point represents the mean ± SEM. (n = 4). *P < 0.05 vs. SHR-C; #P < 0.05 vs. SHR-E.
SHR-E
SHR-E L-NAME
Fig. 4. Effect of a single bout exercise on the phosphorylation of endothelial nitric oxide synthase in the aortas of spontaneously hypertensive rats (SHR). Rats were subjected to a single bout of exercise (E) on a treadmill at 15 m/min for 20 min. The rats were pretreated with L-nitroargnine methyl ester (L-NAME, 40 mg/kg) 1 h prior to being subjected to the exercise. The aortas of the SHRs were harvested after 30 min of the cessation of exercise for Western blotting. Each data point represents the mean ± SEM. (n = 4). *P < 0.05 vs. SHR-C.
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Taken together, the increased activation of eNOS and/or decreased production of superoxide anion that occur after the cessation of exercise appear to underlie the drop in blood pressure in the hypertensive animals.
[10]
Acknowledgments
[12]
This work was supported by Korea Science & Engineering Foundation through the Infection Signaling Network Research Center (R13-2007-020-01000-0), Korea Science & Engineering Foundation (R01-2008-000-20623-0, R01-2007-000-10974-0) and Chungnam National University.
[11]
[13]
[14]
[15]
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