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1Dynamique des Incohérences Cardio-Vasculaires, Faculté de Médecine de Nîmes, Montpellier; and 2Physiologie des Adaptations Cardiovasculaires à l'Exercice, Faculté des Sciences, Avignon, France
Submitted 6 October 2004 ; accepted in final form 8 April 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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nitric oxide; endothelial function
In their constant search to improve performance, many athletes invest in considerable resources to train at altitude because hypoxia exposure enhances erythropoiesis. However, there is clear scientific evidence in both humans and animals that training at altitude does not provide any advantage over training at sea level on maximal O2 uptake and aerobic performances (14, 33). Although cardiac central factors play a major role (25), the peripheral vasomotricity could also be involved. Impairment of endothelium-dependent vasodilatation has been shown after chronic hypoxia exposure (26, 29, 31), probably as a result of alteration in NO pathway. Basal production of NO is essential for maintenance of endothelial vasodilatory properties. Ni et al. (21) and Barton et al. (2) reported that acclimatization to moderate altitude was accompanied by a marked reduction in urinary excretion of NO metabolites. NO is produced from L-arginine by NOS in the vascular endothelium (23). Therefore, L-arginine as well as NOS could play a major role in hypoxia-induced endothelial dysfunction. Several studies have demonstrated a significant downregulation of endothelial NOS expression with hypoxia in cultured endothelial cells (19, 24). Moreover, Eddahibi et al. (10) demonstrated that treatment with L-arginine restored the vasodilatory response to acetylcholine in the pulmonary circulation of chronically hypoxic rats. In addition, Ni et al. (21) have shown in rats chronically exposed to moderate hypoxia that L-arginine supplementation mitigated the fall in urinary NO metabolites (nitrate, nitrite). Finally, it must be noted that Calbet et al. (6) and Bender et al. (3) reported a decrease in leg blood flow during exercise after hypoxia acclimatization in humans. To the best of our knowledge, no report is available on the consequences of training at altitude on the NO vasodilator system. Whether endothelium-dependent vasorelaxation is enhanced after altitude training has never been challenged. We may, however, postulate that the beneficial effects of sea-level training on the vascular function would be blunted when training is performed under hypoxic conditions because of a predominant effect of hypoxia.
The present study was specifically designed to assess the effect of living and training at moderate altitude on endothelium-dependent vasorelaxation and determinants of NO bioavailability in isolated aortic rings. We hypothesized that hypoxic training would not improve NO-mediated vasorelaxation to the same extent as sea-level training because of deleterious effect of chronic hypoxia on L-arginine and/or NOS. For practical and ethical reasons, experiments were carried out in rats, an animal model frequently used in altitude and training studies that shares with humans several features of acclimatization to hypoxia and training (11).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Ten-week-old, sea-level-native Dark Agouti male rats, obtained from Harlan laboratories (Gannat, Puy de Dôme, France), were randomly assigned to live continuously in hypobaric hypoxia with hypoxic aerobic training sessions (CHT rats, n = 7), hypobaric hypoxia (CH rats, n = 7), normoxia with normoxic aerobic training sessions (NT rats, n = 7), or normoxia (N rats, n = 7). Environments were obtained by using steel chambers fitted with a clear plastic glass door to illuminate and observe the animals, as previously described in our laboratory (25). Hypobaric hypoxia was obtained by using a specific vacuum pump (model Mot63, Becker, Rambouillet, France). In each chamber, barometric pressure, hygrometry, and temperature conditions were continuously recorded by electronic sensors.
All rats were maintained for 5 wk in their own environment, at a barometric pressure of 760 Torr [inspired PO2 (PI
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159 Torr, altitude 80 m] for N and NT rats or of 550 Torr (PI 105 Torr, altitude 2,800 m) for CH and CHT rats. CH and CHT animals were fed ad libitum with free access to tap water. Because of altitude impact on food intake and consequently animal growth (7), a pair-fed model was applied to the two other groups, with free access to tap water. Room temperatures were maintained at 21°C using air conditioning. Four animals were kept in each cage on a 12:12-h light-dark cycle at the same time. All procedures were performed in agreement with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85-23, revised 1996) and with the approval of the French Ministry of Agriculture.
Training Program
Training sessions were conducted in NT and CHT rats during the 5-wk environmental exposure. To precisely monitor training intensity, the maximal aerobic velocity (MAV) of each rat was evaluated for each rat before the study period in normoxia (PI 159 Torr) and hypoxia (PI 105 Torr). MAV was also evaluated during the third week of exposure in the animal's living environment to adapt training intensity. Both normoxic and hypoxic MAV were evaluated using a driven wheel during a continuous and progressive maximal exercise test. Under normoxia, the driven wheel was set at a speed of 10 m/min for 2–3 min, after which the speed was increased by 4 m/min every 90 s until 85–90% of the expected MAV was reached (11, 14). The speed was then increased by 0.5–1 m/min every 60 s until MAV. Hypoxic MAV was evaluated using the same protocol but with a starting speed of 7 m/min. Training lasted 5 wk and was conducted at the same relative intensity for both groups (i.e., 80% of normoxic MAV for NT and 80% of hypoxic MAV for CHT). Training sessions lasted 
20 min the first week and reached 60 min in the last week. Rats trained at the same PI they were subjected to during normal living conditions. To get an insight into the effect of the training program, MAV was evaluated on the fifth week in normoxia. Also, muscle samples were taken from the soleus, frozen in liquid nitrogen, and stored at –80°C until processed for citrate synthase activity as described by Srere (28).
Analytical Methods
Hematological analyses. Blood samples (200 µl) were collected before and after exposure in normoxic conditions at the tail of the rats lightly anesthetized with intraperitoneal ketamine HCl (50–75 mg/kg) and xylazine (10–15 mg/kg). Samples were quickly analyzed for hemoglobin concentration using a CO-oximeter (Avoximeter 4000, AVOX, San Antonio, TX).
Aortic ring preparation. Under anesthesia, the thoracic aorta was quickly removed and placed in Krebs-Henseleit bicarbonate buffer (composition in mM: 118 NaCl, 25 NaHCO3, 4.8 KCl, 1.2 KH2PO4, 1.2 MgCl2, 2.5 CaCl2, and 11 glucose). After removal of adherent tissues, the vessels were cut into 2- to 3-mm rings. Aortic rings were mounted onto stainless steel supports, suspended in the tissue bath containing Krebs-Henseleit bicarbonate buffer at 37°C, and bubbled continuously with 95% O2 to maintain a PO2 of 670–680 Torr and 5% CO2 to maintain a pH of 7.4 in the incubation bath. The rings were connected to an isometric force transducer (EMKA Technologies, EMKA, Paris, France), linked to an amplifier (EMKA Technologies, EMKA) 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 the aorta of 4-mo-old rats. The rings were then equilibrated for 60 min.
After equilibration, test doses of KCl, norepinephrine (NE), and acetylcholine (ACh) were added to the rings, to ensure reproducibility of constriction, relaxation, and endothelial integrity. Each vessel ring was contracted with KCl (80 mM) until the constriction reached a plateau. After being rinsed, each vessel ring was precontracted with NE (10–6 M). After the preconstriction reached a plateau, endothelium-dependant relaxation was produced with ACh (10–6 M). After being rinsed, the aortic rings were exposed to cumulative ACh concentrations in the absence or presence of three different doses of L-arginine (10–5, 10–4, and 10–3 M). After 15-min incubation with L-arginine, NE (10–6 M) was applied. After a plateau of contraction was reached, cumulative concentrations of ACh (10–9 to 10–4 M) were introduced. After the rings were rinsed, the same protocol was performed in the presence of nitro-L-arginine methyl ester (L-NAME; 10–5 M). After 20-min incubation of L-NAME and 15-min incubation of L-arginine (10–5, 10–4, 10–3 M), NE (10–6 M) was reapplied until a plateau of contraction was reached. Cumulative ACh concentrations were then introduced (10–9 to 10–4 M). The relaxation response to cumulative doses of sodium nitroprusside (10–9 to 10–4 M) was assessed on other rings preconstricted with NE (10–6 M). Vessel rings were rinsed to stable resting tension level between each drug intervention.
Drugs. All biochemicals were obtained in the highest purity available from Sigma (St. Quentin-Fallavier, France). All drugs were dissolved in distilled water, and concentrations were expressed as final molar concentration in Krebs-Henseleit bathing solution.
Statistics
Dilator responses were given as percentage dilatation relative to the preconstriction level. ED50 values were calculated by computer interpolation from individual cumulative concentration-response curves. The effects of hypoxia and training on body weight, MAV, as well as aortic ring preparation data were assessed by a two-way ANOVA, with repeated measures. In cases of interaction between the main factors, a two-way ANOVA examined (independently of time or dose) the effect of training and hypoxia exposure, followed by post hoc Tukey-Kramer tests when appropriate. EC50 data were analyzed using a two-way ANOVA followed when appropriate by post hoc Tukey-Kramer tests. Values are expressed as means ± SE of n experiments with segments from different arteries. P < 0.05 was considered as statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Before exposure, no significant difference was found between groups for body weight (N: 218 ± 7; NT: 220 ± 6; CH: 222 ± 7; CHT: 216 ± 11 g). Body weight increased significantly (P < 0.05) at the end of exposure with no difference between groups (N: 246 ± 11; NT: 238 ± 11; CH: 248 ± 16; CHT: 239 ± 15 g). Before exposure, no significant difference was found between groups for total hemoglobin (N: 14.5 ± 0.5; NT: 14.3 ± 0.5; CH: 14.5 ± 0.7; CHT: 14.5 ± 0.3 g/dl). Total hemoglobin markedly increased (P < 0.05) in CH and CHT rats at the end of exposure, whereas no modification in this parameter was observed in N and NT rats (N: 14.8 ± 0.6; NT: 14.4 ± 0.7; CH: 15.5 ± 0.7; CHT: 15.6 ± 0.7 g/dl).
MAVs
Before exposure, there was no significant difference between the two trained groups regarding normoxic MAV (NT: 39.7 ± 0.9; CHT: 40.8 ± 0.7 m/min). Whatever exercise conditions, training induced a significant (P < 0.05) improvement in MAV (NT: 43.7 ± 1.2; CHT: 45.7 ± 1.1 m/min). CHT rats improved their MAV to the same extent as NT rats. Training resulted also in a higher citrate synthase activity of the soleus muscle in exercise-trained rats when compared with their sedentary counterparts (n = 35.1 ± 4.5; NT = 62.2 ± 7.0; CH = 36.8 ± 3.9; CHT = 63.5 ± 7.1 IU/g wet weight; P < 0.01 trained vs. sedentary rats). It was of note to highlight that a similar increase in citrate synthase was obtained in the two trained groups.
Vasoconstrictor Responses of Aortic Rings
The maximal tension developed by KCl was similar among the various groups (N: 1.37 ± 0.16; NT: 1.39 ± 0.11; CH: 1.36 ± 0.09; CHT: 1.40 ± 0.12 g). Moreover, the tension developed by NE (10–6 M) was also similar among the four groups (N: 1.18 ± 0.27; NT: 1.14 ± 0.22; CH: 1.20 ± 0.27; CHT: 1.18 ± 0.15 g).
Vasodilator Responses in Aortic Rings
In NE-preconstricted aortic rings, the concentration-response curve of the endothelium-independent vasodilator sodium nitroprusside was the same in the four groups (maximal relaxation: N: 81.2 ± 3.0; NT: 84.6 ± 1.3; CH: 81.2 ± 2.8; CHT: 82.8 ± 2.9 g). In NE-preconstricted aortic rings, ACh-induced vasorelaxation was improved in NT rats, whereas it was depressed in CH rats (Fig. 1, Table 1). No difference in sensitivity to ACh was observed between the four groups (Table 1). L-Arginine incubation did not affect the degree of aortic relaxation in N and NT rats (Fig. 2, Table 1). On the other hand, L-arginine incubation (10–5 to 10–3 M) significantly improved the vasorelaxation response to ACh in both CH and CHT rats (Fig. 2, Table 1). Whatever ACh concentration, aortic ring vasorelaxation did not differ between the two trained groups (i.e., NT and CHT) or between the two sedentary ones (i.e., N and CH) in the presence of L-arginine (10–3 M). Moreover, the presence of L-arginine restored ACh-induced maximal relaxation in CH and CHT rats to the level obtained in N and NT rats, respectively (Fig. 3, Table 1).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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In our study, regular endurance training conducted at sea level resulted in an improvement in NO-mediated vasodilator function through increased endothelial NO synthesis. Similar results have been previously reported in the literature. In peripheral vessels as well as the aorta, exercise training improved vasodilator response to ACh but not to sodium nitroprusside, highlighting enhanced endothelium-dependent function but unchanged smooth muscle cell sensitivity to NO (9, 18). The specific components of NO bioavailability that are sensitive to exercise training are not completely understood. Because NO is produced from L-arginine by NOS in the vascular endothelium, both could be involved in the process of increased NO production. Several studies reported that endurance training increased endothelial NOS gene expression in aortic endothelial cells (8, 9) and NOS protein levels in whole peripheral arteries (9, 16) of various animal models. In the present work, results obtained using NOS inhibitor L-NAME served as validation that the in vitro ACh-effects were mainly related to NO synthesis. Moreover, considering that in the presence of L-arginine the bioavailability of NO substrate does not constitute a limiting factor for NO production, and keeping in mind that NO is produced from L-arginine by NOS, it is therefore very likely that the improvement in the NO vasodilator response to ACh in NT rats was related to improvement in NOS activity. This would be consistent with increase in the genetic expression of NOS in aortic tissues after endurance training, previously shown by Delp and Laughlin (9) and related to mechanical factors involving shear stress via training-induced high blood flow (12). Supporting this theory, Noris et al. (22) in cultured endothelial cells and Nadaud et al. (20) in an in vivo model of chronic high blood flow have clearly demonstrated that blood flow can modulate NOS expression via alteration in shear stress. The improvement in NO vasodilator function in NT rats could also have been related to increased L-arginine bioavailability. However, acute supplementations of L-arginine in this group did not result in an improvement in the NO vasodilator system. It seems therefore very likely that intracellular L-arginine bioavailability did not constitute a limiting factor for NO production in NT rats and did not contribute to their enhanced endothelium-dependent vasorelaxation. This assumption is supported by results from previous studies showing that intracellular amounts of L-arginine in normal endothelial cells are at least 100 µM (1, 13). Because these amounts exceed the Michaelis-Menten constant of the NOS enzyme [5 µM (34)], excess L-arginine would not alter NO production in normal aortic endothelial cells. It can be reasonably postulated that intracellular L-arginine pool in N as well as in NT rats was much higher than the level of L-arginine required by NOS.
The main finding of the present study was that chronic hypoxia exposure severely depressed maximal ACh-vasorelaxation in both hypoxic sedentary and trained rats. However, chronic hypoxia did not interfere with training-induced increases in the maximal ACh responses of aortic rings. Indeed, maximal ACh vasorelaxation was improved to the same extent in normoxic and hypoxic trained rats (NT and CHT, respectively) compared with their sedentary counterparts (N and CH, respectively). This specific adaptation of the endothelial cell function to altitude training may be explained by deleterious effects of acclimatization to hypoxia on NO pathway. In the present study, ACh-induced vasorelaxation was depressed in CH rats compared with N rats. This is consistent with previous studies reporting that chronic hypoxia exposure was associated with reduced endothelium-dependent vasodilator capacity (26, 29, 31) through NO pathway alterations (2, 21). This phenomenon would seem to be due to hypoxia depressed L-arginine availability (27, 35) and/or NOS downregulation (19, 24). In the present study, acute L-arginine supplementation resulted in improvement in the NO vasodilator system in both CH and CHT rats. In these specific experimental conditions, endurance training improved endothelium-dependent vasorelaxation in the two trained groups to the same extent, and no difference was observed between the two sedentary groups. Moreover, the presence of L-arginine (10–3 M) restored the maximal vasodilatory responses to ACh in CH and CHT rats to levels of their normoxic counterparts. These findings strongly suggest that, ex vivo, impairment in L-arginine availability is essentially responsible for the depressed NO-mediated vasorelaxation function in CH and CHT rats compared with their normoxic counterparts. Eddahibi et al. (10) similarly demonstrated in the pulmonary vascular bed that treatment with L-arginine restored vasodilatory response to ACh in chronically hypoxic rats to the level of normoxic ones. Furthermore, it can also be noted that, in an in vivo study, Ni et al. (21) reported that the decrease in NO production after prolonged hypobaric hypoxia can be mitigated by L-arginine supplementation. The mechanisms responsible for impairment in L-arginine availability after chronic hypoxia exposure are not fully understood but may involve the L-arginine transport system. Reduced L-arginine content and impaired L-arginine uptake in endothelial cells from pig pulmonary arteries cultured under hypoxic conditions for 3–5 wk have been reported (4). Moreover, it has recently been found that long-term exposure to hypoxia resulted in calpain-mediated fodrin proteolysis that, in turn, disrupted the functional association between cationic amino acid transporter-1 and actin microfilaments, leading to inhibition of L-arginine transport in the pulmonary artery (35). In addition to reduced substrate availability, the limitation of improvement in NO vasodilator function after altitude training could also be explained by deleterious effects of hypoxia per se on NOS. Indeed, several studies reported that hypoxia resulted in significant downregulation of endothelial NOS expression and/or NOS protein level (19, 24, 30) of various tissues, including aorta. However, in the presence of L-arginine (10–3 M) and/or L-NAME (10–5 M), CH and N rats presented a similar endothelium-dependent vasorelaxation. These results strongly support the notion that the in vitro ACh effects reported here were probably not related to impairment in NOS activity. However, because the NOS activity and/or density were not evaluated in the present work, a potential effect of hypoxia on these two components cannot be dismissed. Discrepancies between aforementioned studies and ours may be explained by the duration of hypoxia exposure. Results of previous studies were obtained under acute (24–48 h) and/or short-term (7 days) exposure to hypoxia. However, Barton et al. (2) showed a specific NOS kinetic in aorta from rats exposed to hypobaric hypoxia (5,000 m), NOS values going back to normal at the end of the study period (21 days).
As a result of lower absolute training intensity, the total distance (i.e., training volume) run by CHT rats (34,500 m over 5 wk) was slightly lower (14%) than that of NT rats (40,000 m over 5 wk). However, it is very unlikely that this could be responsible for the lack of training adaptations in CHT rats. Indeed, Delp and Laughlin (9) reported enhancement in endothelium-dependent ACh-mediated dilation of aorta in rats trained for 4 (training volume 35,000 m) and 10 (training volume 90,000 m) wk. Moreover, vascular adaptations to training were similar between the two groups despite a total workload 60% lower in the 4-wk-trained group compared with the 10-wk-trained group. Furthermore, it should be noted in the present work that when L-arginine (which is not affected by aerobic training) was added into the bath, a similar endothelium-dependent vasorelaxation was obtained between NT and CHT rats. An effect of training intensity could have been suspected if this had not been the case.
In agreement with several studies (9, 17), and despite the marked increase in the O2-carrying capacity generated by chronic hypoxia exposure, CHT rats improved their aerobic performance to the same extent as NT rats. Although cardiac central adaptations play a major role (25), peripheral adaptations may also be involved. Indeed, if extrapolated to the peripheral vascular bed, adaptations of aortic tissues after altitude training, as shown by the present study, could play a major role in the limitation of blood delivery to exercising muscles and subsequently maximal O2 uptake improvement. In this context, it should be noted that Calbet et al. (6) reported that, after acclimatization to hypoxia, leg blood flow was lower during exercise but higher in noncontracting tissues. Moreover, Bender et al. (3) showed that regulating mechanisms of increased peripheral resistance could play a major role in the control of O2 transport during exercise after altitude acclimatization. However, because several vasoactive compounds are involved in vasodilatory mechanisms in contracting skeletal muscle, extrapolating results obtained ex vivo to in vivo conditions should be done with caution.
To conclude, the results of the present study indicate that acclimatization to hypoxia had deleterious effects on the NO vasodilator system in both sedentary and trained rats. However, chronic hypoxia exposure did not interfere with training-induced improvement of the NO vasodilator system. This specific adaptation of the endothelial function to altitude training was mediated by a hypoxia-induced decrease in L-arginine bioavailability. These results could be of importance for aerobic performance as the specific vascular adaptations after training at altitude may participate to limit peripheral vasodilatation and subsequently blood flow during exercise.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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O2 max after altitude acclimatization still reduced despite normalization of arterial O2 content? Am J Physiol Regul Integr Comp Physiol 284: R304–R316, 2003.O2 max more than living and training in normoxia. J Appl Physiol 90: 2057–2062, 2001.This article has been cited by other articles:
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  B. Roels, C. Reggiani, C. Reboul, C. Lionne, B. Iorga, P. Obert, S. Tanguy, A. Gibault, A. Jougla, F. Travers, et al. Paradoxical effects of endurance training and chronic hypoxia on myofibrillar ATPase activity Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2008; 294(6): R1911 - R1918. [Abstract] [Full Text] [PDF]
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 S. Keslacy, R. S. Mazzeo, D. A. Giussani, A. S. Thakor, G. Insalaco, M. R. Bonsignore, F. A. Rodriguez, K. S. Mark, C. Reboul, S. Tanguy, et al. Commentary on Point-Counterpoint J Appl Physiol, January 1, 2006; 100(1): 363 - 363. [Full Text] [PDF]
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P < 0.05 vs. N and CH rats. 
P < 0.05 vs. N and CHT rats.
