Chronic exercise does not prevent hypoxia-induced increased aortic sensitivity to endothelin in rats

Vascular Pharmacology 44 (2006) 333 – 337 www.elsevier.com/locate/vph

Chronic exercise does not prevent hypoxia-induced increased aortic sensitivity to endothelin in rats C. Reboul a

a,⁎

, S. Tanguy b , M. Dauzat a , P. Obert

b

EA2992, Dynamique des Incohérences Cardio-Vasculaires, Faculté de Médecine de Nîmes, Montpellier, France JE 2426, Physiologie des Adaptations Cardiovasculaires à l’Exercice, Faculté des Sciences, Avignon, France

b

Received 24 November 2005; received in revised form 9 January 2006; accepted 11 January 2006

Abstract Objectives: We report in the present study the effect of regular exercise on vascular reactivity alterations to endothelin (ET-1) following prolonged exposure to hypoxic stress. Methods: Male Dark Agouti rats were randomly assigned to N (sedentary rats), NCE (normoxic exercised rats), CH (chronic hypoxic sedentary rats) and CHCE (chronic hypoxic exercised rats) groups. The effects of ET-1 in the presence or not of the endothelium and/or of the specific inhibitor, bosentan, have been investigated in an isolated model of rat thoracic aorta. Results: Prolonged exposure to hypoxia induced a significant increase in aortic sensitivity to ET-1 (− log ED50 in CH = 8.15 ± 0.01 vs in N = 7.98 ± 0.02, p b 0.05). Despite exercise training reduced the sensitivity to ET-1 in normoxic rats, it has no effects in hypoxic rats (−log ED50 in CH = 8.15 ± 0.01 vs in CHCE = 8.19 ± 0.01, NS). Moreover, although the removal of endothelium has no effect in N rats, it leads, in NCE rats, to a significant increase in sensitivity to ET-1 (− log ED50 in endothelium intact rings = 7.89 ± 0.04 vs in denuded rings = 8.04 ± 0.02, p b 0.05). The implication of ET1 receptors on both endothelial and smooth muscle cells is confirmed by the significant reduced sensitivity to ET-1 in the four groups when bosentan is present in organ bath. Conclusion: Our study clearly suggests that part of the beneficial effect of chronic exercise could be mediated by enhancing endothelial function associated with endothelin reactivity in peripheric vessels. However, chronic exercise training does not seem to be able to limit the increased vasoconstriction to ET-1 stimulation induced by chronic hypoxia exposure. © 2006 Elsevier Inc. All rights reserved. Keywords: Vascular; Hypoxia; Endothelin; Bosentan; Rodent; Hypertension

1. Introduction Prolonged hypobaric hypoxia is classically described as a new model leading to the development of a sustained systemic hypertension (Ni et al., 1998; Barton et al., 2003). Today, several studies clearly show that the hypertension induced by chronic hypoxia is not due to the associated erythocythosis (Vaziri and Wang, 1996) but rather to endogenous arterial properties including modification in both endothelial and smooth muscle cells (Thomas and Wanstall, 2003). This phenomenon appeared to be mediated either by a reduction in endothelial vasodilator ⁎ Corresponding author. JE 2426, Physiologie des Adaptations Cardiovasculaires à l'Exercice, Faculté des Sciences-Dpt STAPS, 33 rue Louis Pasteur, 84000 AVIGNON, France. Tel.: +33 4 32 74 32 00; fax: +33 4 90 14 44 09. E-mail address: [email protected] (C. Reboul). 1537-1891/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.vph.2006.01.006

function (Reboul et al., 2005b), and/or by an increased sensitivity to vasoconstricting factors, including catecholamins (Caudill et al., 1998) and endothelins (ET-1) (Goerre et al., 1995). ET-1, the most potent vasoconstrictor known, seems to play a major role in the explanation of hypoxia-induced systemic hypertension. Indeed, Bialecki et al. (1998) reported in rat, that hypoxic exposure progressively increased ET-1 plasma concentration, and, Zacour et al. (1998) showed that following prolonged hypoxia, aortic contractility was enhanced by ET-1 release. Moreover, as recently reviewed by Schiffrin (2005), the effect of ET-1 release has been described to be different accordingly with the stimulated subtype receptors. While the endothelial ET-1 receptors (ET-B) are associated with nitric oxide (NO) and/or prostacyclin-stimulated vasorelaxation (De Nucci et al., 1988; Haynes and Webb, 1993), the smooth muscle cells ET-1 receptors (ET-A and ET-B) induced vasoconstriction.

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Furthermore, Dschietzig et al. (2003) have recently shown that the endothelial ET-B receptor could be up-regulated, independently from the smooth muscle cells ET-B, by ET-1 antagonist. Then the endothelial cells ET-1 receptors, elicited endotheliumdependent vasorelaxation, could be of interest in order to improve the regulation of ET-1 induced vasoconstriction. To the best of our knowledge, no study has been previously performed to assess whether chronic exercise could prevent cardiovascular pathologies by beneficial effects on endothelial ET-1 receptors-induced vasodilatation. Indeed, exercise training is classically described as a useful way to limit and prevent cardiovascular pathophysiological situations. One of the most described properties of chronic exercise is the significant increase in endothelium mediated vasorelaxation (Maiorana et al., 2003), related to an improvement of the NO pathway (Delp and Laughlin, 1997). Although, recently Donato et al. (2005) reported that exercise training does not affect ET-1 vasoconstriction in resistance vessels, most of the few studies dealing with the effect of exercise training on ET-1 metabolism, reported a significant reduction in ET-1 induced vasoconstriction and/or a reduced ET-1 production (Jones et al., 1999; Latorre et al., 2002). It is also of note to highlight that routine aerobic exercise was reported to counteract abnormal release of plasma ET-1 in normotensive offspring of hypertensive parents (Tanzilli et al., 2003). In this context, because chronic exercise can improve endothelial-dependent vasorelaxation but also reduced the sensitivity to ET-1 action, the present study was specifically designed to assess the implication of endothelium and ET-1 receptors in exercise- and/or hypoxia-induced vasoreactivity alterations of isolated aortic rings. Therefore, we hypothesized that following chronic exercise, endothelial ET-1 receptor alterations could counteract the abnormal hypoxia induced ET-1 vasoconstriction. 2. Methods 2.1. Animals Sixteen week old male Dark Agouti rats, obtained from Harlan Laboratories (Gannat, Puy de Dôme, France) were randomly assigned to live continuously in hypobaric hypoxia or normoxia including or not aerobic exercise sessions. Four experimental groups were each constituted with 10 rats: normoxic rats (N), normoxic exercised rats (NCE), hypoxic rats (CH) and chronic hypoxic exercised rats (CHCE). 2.2. Experimental hypoxia and training Environments were obtained by using steel chambers fitted with a clear plastic glass door to illuminate and observe the animals (Reboul et al., 2005c). Hypobaric hypoxia was obtained by using a specific vacuum pump (Becker Mot63, Rambouillet, France). In each chamber, barometric pressure, humidity and temperature conditions were continuously recorded by using electronic sensors. All rats were maintained for 5 weeks in their own environment, at a barometric pressure of 760mm Hg (PIO2 = 159 mm

Hg) for N and NCE, or of 550 mm Hg (PIO2 = 105 mm Hg) for CH and CHCE. Animals were housed under conditions of constant temperature, humidity and standard light–dark cycle (12 h / 12 h). They had free access to tap water and standard food. All animals were treated according to the guidelines of the recommendations from the declaration of Helsinki and the guiding principles in the care and use of animals (L358-86/609/EEC). Exercise sessions were conducted 5 times/week in NCE and CHCE rats during the 5week of environmental exposure. The exercise sessions were performed in a driven wheel. They lasted about 20 min the first week and reached 40min in the last 2weeks. The exercise intensity was set at 80% of the maximal aerobic velocity (MAV) measured in the exposure environment (i.e. 80% of normoxic MAV for NCE and 80% of hypoxic MAV for CHCE) as previously described in our laboratory (Reboul et al., 2005a,b,c). This exercise training protocol was classically used in our laboratory and was previously reported by our team to improve citrate synthase activity of the soleus muscle (Reboul et al., 2005b; Goret et al., 2005). 2.3. Isolated rings of aorta Under anesthesia the thoracic aorta was quickly removed and placed in Krebs–Henseleit bicarbonate buffer (Composition in mM: NaCl 118, NaHCO3 25, KCl 4.8, KH2PO4 1.2, CaCl2 2.5, Glucose 11). After removal of adherent tissue the vessels were cut in 2–3mm rings. In some aortic rings, the endothelium was mechanically removed (Reboul et al., 2005b). Aortic rings were mounted onto stainless steel supports, suspended in the tissue bath containing Krebs–Henseleit buffer at 37°C, continuously bubbled with O2–CO2 (95%–5%) gas mixture. The rings were connected to an isometric force transducer (EMKA technologies, EMKA Paris, France), linked to an amplifier (EMKA technologies, EMKA Paris, France) and a computerized acquisition system, to record changes in isometric force. The resting tension was adjusted to 2 g and corresponded to the optimal length for tension development in aorta of 4 month old rats. The rings were then equilibrated for 60min. After a 60 min equilibration period, test doses of KCl (80 mM), norepinephrine (NE, 10 − 6 M) and acetylcholine (ACh, 10− 6 M) were added to the organ baths, to ensure reproducibility of contraction and endothelial integrity. When necessary the endothelium was mechanically removed. The vasoconstrictor effect of endothelin-1 (ET-1) was assessed by cumulative dose–response curves (10− 10 to 10− 7 M) on aortic rings with or without endothelium in the presence or not of bosentan (10− 7 M, pre-incubated during 5min), a specific ET-A and ET-B receptors inhibitor. 2.4. Drugs and chemicals All concentrations of the drugs used in aortic ring experiments are expressed as final molar concentration in Krebs– Henseleit solution. All biochemicals were obtained in the highest purity available from Sigma (St. Quentin–Fallavier, France).

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Table 1 Endothelin with endothelium (ET-1endo) or without endothelium (ET-1endo(-))-induced constrictions in aortic rings after 5weeks of environmental exposure with (NCE and CHCE) or without training (N and CH) N

ET-1endo

ED50 (−log) Cmax (%K+) ED50 (−log) Cmax (%K+) ED50 (−log) Cmax (%K+) ED50 (−log) Cmax (%K+)

ET-1endo(−) ET-1endo bos ET-1endo(−) bos

NCE

CH

CHCE

N=9

N=9

N=8

N = 10

7.97 ± 0.02 147 ± 5 8.01 ± 0.03 156 ± 10 7.72 ± 0.05£ 125 ± 8£ 7.85 ± 0.05§ 125 ± 11§

7.86 ± 0.05⁎ 142 ± 6 8.04 ± 0.02£ 152 ± 6 7.69 ± 0.07£ 124 ± 12£ 7.81 ± 0.05§ 130 ± 8§

8.17 ± 0.02$ 144 ± 8 8.15 ± 0.01$ 160 ± 12 7.77 ± 0.06£ 118 ± 7£ 7.79 ± 0.04§ 125 ± 7§

8.19 ± 0.03$ 146 ± 6 8.16 ± 0.03$ 155 ± 6 7.66 ± 0.07£ 116 ± 7£ 7.76 ± 0.09§ 128 ± 9§

Endothelin, in the presence of bosentan, with endothelium (ET-1endo bos) or without endothelium (ET-1endo(−) bos)-induced constrictions in aortic rings after 5weeks of environmental exposure with (NCE and CHCE) or without training (N and CH). Values are means ± SEM. ED50, dose inducing 50% of maximal constriction; Cmax , maximal constriction; N, rats exposed to normoxia; NCE, rats exposed to and trained in normoxia; CH, rats exposed to hypoxia; CHCE, rats exposed to and trained in hypoxia;*, p b 0.05 vs the three other group; $, p b 0.05 vs N and NCE rats; £, p b 0.05 vs ET-1endo in the same group; §, p b 0.05 vs ET-1endo(−) in the same group.

Constriction responses were expressed as percentage of the response to KCl. Values are expressed as mean ± S.E.M. of n experiments with segments from different arteries. Data were analysed using a one-way ANOVA followed when appropriate by post hoc tests of Scheffe. P b 0.05 was considered as statistically significant.

CH = 8.17 ± 0.02 versus in N = 7.97 ± 0.02, p b 0.05). When performed in normoxia exercise training resulted in a significant reduction of aortic ring sensitivity to ET-1, as assessed by the significant reduction in − log ED50 (7.86 ± 0.04, p b 0.05 versus N, CH and CHCE). However, when performed by hypoxic rats (CHCE), chronic exercise does not prevent the increase in ET-1 sensitivity (− log ED50 in CHCE = 8.19 ± 0.03 versus in CH = 8.17 ± 0.01, ns).

3. Results

3.3. Effect of ET-1 in endothelium denuded aortic rings

3.1. Effect of KCl

When endothelium was removed there is a similar significant increase in sensitivity to ET-1 in both sedentary and trained hypoxic rats when compared with normoxic groups (p b 0.05) (Table 1, Fig. 1). Again, this phenomenon was not abolished by chronic exercise in hypoxic rats (− log ED50 in CHCE = 8.16 ± 0.03 versus in CH = 8.15 ± 0.01, ns). Interestingly, in NCE group, the removal of endothelium led to a significant increased sensitivity to ET-1 (− log ED50 in endothelium intact rings = 7.86 ± 0.04 versus in endothelium denuded rings = 8.04 ± 0.02, p b 0.05). Then, the potential beneficial reduction induced by chronic exercise performed in normoxia disappeared in

2.5. Data and statistical analysis

In all experimental groups the maximal contraction induced by 80mM KCl was similar (N: 1.38 ± 0.13; NCE: 1.33 ± 0.10; CH:1.51 ± 0.16; CHCE: 1.44 ± 0.15mg). 3.2. Effect of ET-1 in endothelium intact aortic rings Although the maximal contraction induced by ET-1 reached a similar value (Table 1, Fig. 1), there is a significant increase of sensitivity to ET-1 induced by chronic hypoxia (− log ED50 in

A

B 150

100

50 N rats NCE rats CH rats CHCE rats

0 -10

-9

-8

ET-1 (log mol/L)

-7

Contraction (%K+)

Contraction (%K+)

150

100

50 N rats NCE rats CH rats CHCE rats

0 -10

-9

-8

-7

ET-1 (log mol/L)

Fig. 1. Endothelin (ET-1) with (A) or without (B) endothelium-induced vasoconstriction in aortic rings from normoxic sedentary (N) or exercised (NCE) rats and from chronic hypoxic sedentary (CH) or exercised (CHCE) rats. Results are expressed as mean ± S.E.M. percentage of the reference response to KCl (80mM).

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endothelium denuded aortic rings (− log ED50 in NCE = 8.04 ± 0.02 versus in N = 8.01 ± 0.03, ns). 3.4. Effect of bosentan The addition of the dual ET-A and ET-B receptor inhibitor, bosentan, did significantly reduce the maximal response to ET-1 (Table 1) in the four experimental groups. Moreover it significantly decreases the sensitivity of aortic rings to ET-1 (shown in Table 1 by the significant reduction in − log ED50). The same results were observed in endothelium-denuded aortic rings (Table 1). 4. Discussion To the best of our knowledge this is the first study that investigated the preventive role of chronic exercise on hypoxiainduced ET-1 vasoconstriction. The main result of the present work is that the beneficial effects of chronic exercise on aortic sensitivity to ET-1 are blunted, when exercise was performed in chronic hypoxic rats, by an increase of aortic ET-1 sensitivity. The specific adaptation, resulted from regular exercise, seems to be mainly mediated by the endothelium, as suggested by the lack of specific effects of exercise in endothelium denuded aortic rings. Finally, the specific response of aortic rings in the presence of bosentan confirms an ET-1 receptor mediated mechanism. 4.1. Normoxic exercised rats As we (Reboul et al., 2005a) and others (Latorre et al., 2002) have previously reported, in the present study, regular exercise is able to reduce aortic sensitivity to ET-1 action. Similar results have been also reported in porcine coronary arteries, by Jones et al. (1999) who showed that the sensitivity to ET-1 action is reduced by exercise training. It is of note to highlight that in presence of bosentan, an ET-A and ET-B specific antagonist, the maximal response to ET-1 action in the four experimental groups was significantly reduced and became similar among the various groups. This result strongly suggests an alteration of ET-1 aortic receptors in the specific vascular adaptations reported here. Bowles et al. (1995) proposed that exercise attenuated the Ca2+ contractile response to ET-1 action, this could contribute to the explanation of the reduced sensitivity to ET-1 consecutive to chronic exercise. However, the major result of the present study is that, in endothelium removed aortic rings, sensitivity to ET-1 action was similar between N and NCE rats. This result clearly suggests that, following regular exercise, the reduced sensitivity to ET-1 action was mainly related to endothelial mechanisms. We could therefore postulate that this adaptation is related to an improvement of the specific endothelial ET-1 receptor induced NO vasorelaxation. Indeed, exercise training was classically reported to improve the NO pathway (Delp and Laughlin, 1997; Maiorana et al., 2003) via an increase in endothelial NO Synthase (NOS) gene expression (Delp et al., 1993; Delp and Laughlin, 1997) and NOS protein levels in aortic and arterial endothelial cells of various animal

models (Delp and Laughlin, 1997; Laughlin et al., 2003). To our knowledge, no study has been previously performed to assess whether chronic exercise could also enhance the receptor induced NOS activation. As endothelial ET-1 receptors (ET-B) have been described as a trigger of NO vasorelaxation, the pathophysiological situation associated with increased ET-1 vasoconstriction could be regulated by an improvement of endothelial ET-B receptors mediated NO vasorelaxation. Therefore, the improvement of the endothelial ET-B vasodilator function could be involved in the reduced sensitivity to ET-1 action associated with chronic exercise in normoxic rats. Similarly, several authors (Fukuroda et al., 1994; Reinhart et al., 2002) proposed that endothelial ET-B receptors also plays an important role in clearing circulating ET-1, thereby reducing ET-A mediated vasoconstrictions. 4.2. Hypoxic exercised rats The second major result of the present study is that chronic exercise, when conducted in hypoxic rats, does not prevent the hypoxia-induced increased aortic sensitivity to ET-1 action. Most of the differences between normoxic and hypoxic chronically exercised rats could be explained by the effects of hypoxia per se. This seems to be related to the ET-1 receptors of vascular smooth muscle cells, since in endothelium removed aortic rings of hypoxic groups, the sensitivity to ET-1 action was not different from those observed in intact endothelium aortic rings and remained higher than those observed in the two normoxic groups. Moreover, in endothelium denuded aortic rings and/or in presence of bosentan, the sensitivity to ET-1 action was similar among various groups, suggesting therefore a major role of the vascular smooth muscle cells ET-1 receptors in those specific adaptations. Those results were in part in accordance with the literature. Indeed, although Bialecki et al. (1998) reported no alteration of the aortic sensitivity to ET-1 action following 14 days at 10% O2, Li et al. (1994) reported that following 4 weeks at 10% O2, ET-A and ET-B receptor mRNA levels were increased in the thoracic aorta. Moreover, we have previously shown that hypoxic exposure, with or without exercise, resulted in a significant increase in aortic sensitivity to ET-1 (Reboul et al., 2005a). It is reasonable to postulate that following chronic exercise conducted in hypoxic rats, the lack of regulation of the ET-1 induced vasoconstriction by the endothelial ET-1 receptors, was related to the well described hypoxia-induced alteration of the NO pathway (Barton et al., 2003; Reboul et al., 2005b). Indeed, considering that endothelial ET-B receptors are associated with NO stimulated vasorelaxation (Haynes and Webb, 1993), this hypothesis is consistent with previous studies reporting that chronic hypoxia exposure was associated, in absence or presence of chronic exercise, with reduced endothelial vasodilator capacity (Tahawi et al., 2001; Trang et al., 2001), through NO pathway alterations (Ni et al., 1998; Barton et al., 2003; Reboul et al., 2005b). Obviously, it is also possible that other endothelium-dependent vasorelaxation mechanisms, those including prostacyclin-mediated vasorelaxation, could contribute to this phenomenon. However further studies will be needed to

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clarify the mechanisms involved in such specific endothelium adaptations. 4.3. Conclusion To conclude, our study clearly suggests that part of the beneficial effect of regular exercise could specifically be mediated by enhancing endothelial function associated with ET-1 reactivity in aortic vessels. However, chronic exercise does not limit the increased ET-1 induced vasoconstriction in chronic hypoxic rats. This result could be mainly related, either to a major increase in specific smooth muscle ET-1 action, and/ or to hypoxia-induced impairment of endothelial NO vasodilator functions. However, further biochemical experiments will be needed to clarify the mechanisms involved in such specific adaptations. Acknowledgements The Bosentan has been gracefully provided by Actelion Pharmaceuticals Ltd (Switzerland). References Barton, C.H., Ni, Z., Vaziri, N.D., 2003. Blood pressure response to hypoxia: role of nitric oxide synthase. Am. J. Hypertens. 16, 1043–1048. Bialecki, R.A., Fisher, C.S., Murdoch, W.W., Barthlow, H.G., Stow, R.B., Mallamaci, M., Rumsey, W., 1998. Hypoxic exposure time dependently modulates endothelin-induced contraction of pulmonary artery smooth muscle. Am. J. Physiol. 274, L552–L559. Bowles, D.K., Laughlin, M.H., Sturek, M., 1995. Exercise training alters the Ca2+ and contractile responses of coronary arteries to endothelin. J. Appl. Physiol. 78, 1079–1087. Caudill, T.K., Resta, T.C., Kanagy, N.L., Walker, B.R., 1998. Role of endothelial carbon monoxide in attenuated vasoreactivity following chronic hypoxia. Am. J. Physiol. 275, R1025–R1030. de Nucci, G., Thomas, R., D'Orleans-Juste, P., Antunes, E., Walder, C., Warner, T.D., Vane, J.R., 1988. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc. Natl. Acad. Sci. U. S. A. 85, 9797–9800. Delp, M.D., Laughlin, M.H., 1997. Time course of enhanced endothelium-mediated dilation in aorta of trained rats. Med. Sci. Sports Exerc. 29, 1454–1461. Delp, M.D., McAllister, R.M., Laughlin, M.H., 1993. Exercise training alters endothelium-dependent vasoreactivity of rat abdominal aorta. J. Appl. Physiol. 75, 1354–1363. Donato, A.J., Lesniewski, L.A., Delp, M.D., 2005. The effects of aging and exercise training on endothelin-1 vasoconstrictor responses in rat skeletal muscle arterioles. Cardiovasc. Res. 66, 393–401. Dschietzig, T., Bartsch, C., Richter, C., Laule, M., Baumann, G., Stangl, K., 2003. Relaxin, a pregnancy hormone, is a functional endothelin-1 antagonist: attenuation of endothelin-1-mediated vasoconstriction by stimulation of endothelin type-B receptor expression via ERK-1/2 and nuclear factor-kappaB. Circ. Res. 92, 32–40. Fukuroda, T., Fujikawa, T., Ozaki, S., Ishikawa, K., Yano, M., Nishikibe, M., 1994. Clearance of circulating endothelin-1 by ETB receptors in rats. Biochem. Biophys. Res. Commun. 199, 1461–1465.

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