| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Association pour la Recherche en Physiologie de l'Environnement, UFR Médecine, 93017, Bobigny, France, 2 UFR Médecine Necker, 75014, Paris, France
CORRESPONDENCE: J-P. Richalet, ARPE UFR Médecine, 74, rue M. Cachin, 93017, Bobigny cedex, France. Fax: 33 148387777
Keywords: cyclic guanosine 3',5'-monophosphate, hypoxia, l-citrulline, nitric oxide
Received: August 18, 2000
Accepted March 26, 2001
 |
Abstract |
|---|
 TOP Abstract MethodsResultsDiscussionConclusionAcknowledgementsReferences |
|---|
To evaluate the effect of l-arginine on the NO release in high altitude, 11 subjects were infused with l-arginine (0.5 g·kg–1) during 30 min in normoxia and after 36 h at 4,350 m (hypoxia). The l-citrulline and cyclic guanosine monophosphate (cGMP) concentrations were measured to investigate NO synthesis and guanylyl cyclase activity respectively. l-citrulline concentration, arterial oxygen saturation (Sa,O2), systemic blood pressure, heart rate and acute mountain sickness (AMS) score were measured at rest and 15, 30 and 45 min after starting infusion.
The results showed that baseline l-citrulline was lower in hypoxia (p<0.05). l-arginine infusion increased l-citrulline concentration in both conditions. However, in hypoxia l-citrulline concentration remained lower than in normoxia (p<0.05). The concentration of cGMP was lower in hypoxia (p<0.05). In hypoxia, Sa,O2 increased from 15 min after the start of the infusion to 45 min (p<0.05). Blood pressure and heart rate were not affected by l-arginine infusion.
Subjects who experienced symptoms of AMS showed a slight decrease in AMS score with l-arginine.
The decreased l-citrulline suggests a hypoxia-induced impairment of nitric oxide synthase III or a decrease in l-arginine availability. The improvement of arterial oxygen saturation by pretreatment with l-arginine could be ascribed to an enhancement of the ventilation/perfusion ratio. Collectively, these results are consistent with a decrease in nitric oxide production in hypoxia that could be antagonized by supplying nitric oxide synthase cosubstrate.
Exposure to high altitude causes hypoxaemia, which is often accompanied by the clinical manifestations of acute mountain sickness (AMS) and may lead to the development of pulmonary hypertension and high altitude pulmonary oedema (HAPE). Several factors are incriminated: hypoxic pulmonary vasoconstriction (HPV), water retention, and permeability alterations 1. Increased release of vasoconstrictors, such as endothelin or catecholamines, at high altitude may be partially responsible for HPV 2. However, impairment of nitric oxide (NO) synthesis by pulmonary endothelial cells occurs in the pulmonary hypertensive process suggesting that the increase in pulmonary vascular tone could have its origin in a decline of endothelium-dependent relaxation 3. NO is synthesized in pulmonary artery endothelial cells by the action of the endothelial constitutive NO synthase isoform (NOS III) on the cationic amino acid l-arginine (Arg). NO is produced from the terminal guanidino-nitrogen of Arg upon conversion to l-citrulline by NOS III in a reaction that requires molecular oxygen and cofactors. Direct measurement of NO release from vascular endothelial cells needs an NO-selective electrode. However, l-citrulline that is formed stochiometrically with NO could be more easily measured than the short-lived NO. Lagerwerf et al. 4 have previously shown that determination of l-citrulline enrichments in plasma was a useful tool for assessment of in vivo NO production. Therefore, the release of NO can be measured directly by the l-citrulline concentration as an index of NO synthesis. Synthesis of NO by NOS depends on the availability of its substrate 5, 6. In normal vessels, the amount of substrate is sufficient for the conversion to NO at a maximal rate. However, addition of Arg causes an endothelium-dependent relaxation in pulmonary arteries depleted from Arg 7. Chronic hypoxia may impair the uptake of Arg into endothelium or alter its availability or metabolism. However, Arg deficiency does not appear to account for the decrease in NO production reported in endothelial cells exposed to short-term hypoxia 8, 9. By contrast, Arg restores endothelium-dependent relaxation in pulmonary circulation of chronically hypoxic rats 10, suggesting that the availability of intracellular Arg is a critical factor in the regulation of the rate of NO synthesis in endothelial cells. Furthermore, in pulmonary hypertension, basal plasma level of Arg was below average 11, supporting the hypothesis that a relative deficiency of the Arg pool might contribute to the pathogenesis of pulmonary hypertension. Thus, understanding the metabolism of intracellular Arg might clarify the mechanism by which NO formation decreases in hypoxia. Alternatively, low oxygen tension inhibits NOS activity in biochemical assays of the isolated NOS enzyme 12 and chronic hypoxia could interrupt the NO signalling pathway at the level of guanylyl cyclase (GC) 13. However, chronic hypoxia induced an upregulation of NOS protein expression and activity 14. Thus, in long-term hypoxia, the part of the Arg availability seems to be lower. To explore the hypothesis that exposure to acute hypoxia is associated with a decrease in Arg availability and to confirm whether decreased NO activity in high altitude could be restored by supplying the precursor of NO, plasma l-citrulline (l-cit) and cyclic guanosine monophosphate (cGMP) concentrations were measured in humans during infusion of Arg at sea-level (normoxia) and after 36 h of exposure to altitude of 4,350 m (hypoxia). Accordingly, the effect of exogenous Arg on arterial oxygen saturation (Sa,O2) and AMS score were examined.
|
Methods |
|---|
|
TOPAbstractMethodsResultsDiscussionConclusionAcknowledgementsReferences |
|---|
4 weeks prior to the study, were not acclimatized to altitude before the experiments and did not consume any long-term medication or caffeine before the Arg load. Their age and body weight were (mean±se) 27.8±1.5 yrs and 73.0±2.8 kg, respectively.
Study design
All experiences were performed in two conditions: at sea-level during the first week (Bobigny hospital France, altitude: 60 m, barometric pressure 761±5 mmHg) and 3 weeks later at a field laboratory on Mont Blanc (Observatoire Vallot, altitude: 4,350 m, barometric pressure 457±1 mmHg). Subjects were transported by helicopter from Chamonix (altitude: 1,035 m) to 4,350 m in <15 min. The temperature in the laboratory was kept constant at 20–23°C during the whole study. Response to Arg infusion was determined at sea-level and 36 h after arriving at 4,350 m. After 1-h rest in supine position, each subject had a short plastic catheter introduced in a cubital vein 15 min before beginning the infusion. A single dose of Arg (l-arginine-chloride; Laboratoire Veyron et Froment, Marseille, France) was infused (0.5 g·kg–1). Arg was diluted in 250 mL of a 5% glucose solution and the duration of the infusion was 30 min. Each dose was administered early in the morning before breakfast. Administration of Arg was designed as time 0 (t0). Arterial oxygen saturation (Sa,O2), heart rate (HR) and supine systolic and diastolic blood pressure (SBP and DBP, respectively) were measured by an ear pulse oximeter (Biox II, Ohmeda, Montreuil, France) and an automatic sphygmomanometer (Dinamap 1846 SX P, Critikon, Blanquefort, France). One venous blood sample was collected for the measurement of l-cit concentration and cGMP concentration prior to medication. Then 15, 30 and 45 min after the beginning of the infusion, in resting supine position (t15, t30 and t45 respectively), Sa,O2, HR and BP were measured and blood was withdrawn. Clinical assessment of AMS was performed by the same observer using the Lake Louise score at the same time as blood sampling. Four symptoms were monitored: headache, gastrointestinal upset, dizziness and asthenia. The response to each of the four items was rated with a three-point scale in which a score of 0 indicated no symptoms, 1: slight symptoms, 2: moderate symptoms and 3: severe symptoms. The AMS score is the sum of the scores for the four items 15.
Chromatographic measurements of l-citrulline
Ten millilitres of blood were sampled without venostasis for l-cit and cGMP concentration measurements in plastic tubes containing ethylenediamine tetraacetic acid (EDTA). Blood was immediately centrifuged for 20 min at 900xg at 4°C. Plasma was carefully removed, frozen and stored in liquid nitrogen within 30 min for subsequent analysis. l-cit concentration was measured by ions-exchange chromatography with ninhydrin detection adapted from the technique of Slocum and Cummings 16. Plasma was deproteinized by precipitation with sulphosalicylic acid and centrifuged at 10,000xg. The supernatant was added Beckman Li-S buffer and injected into the amino acid analyser Beckman 6300 (Beckman Instruments, Roisy, CDG, France) calibrated by analysing calibration standard of known concentration. Retention time was used for amino acid identification and integration was used for quantification.
Radioimmunoassay measurements of cyclic guanosine monophosphate
NO stimulates guanylyl cyclase to produce cGMP, an intermediate in the endothelium-dependent relaxation of blood vessel. To study the NO-cGMP pathway, cGMP concentration was measured by immunoassay. The nuclear radioimmunoassay kit for the determination of cGMP is adapted from the procedures of Steiner et al. 17. Increase in sensitivity was achieved by succinylating samples with succinic anhydride, then the tracer was added and incubated for 22 h at 4°C (kit Beckman-Coulter, Immunotech SA, Marseille, France). Radioimmunoassays of cyclic nucleotides (e.g. cGMP) in biological fluids currently involve the use of specific antibodies and 125I-labelled tracers. Femtomole levels could be measured when 125I-labelled tracers and 2'-O-derivatization procedures are used.
Statistical analysis
All data are expressed as means±se. A two-way analysis of variance with repeated measures was performed to make comparisons between altitude conditions and across time for clinical data, Sa,O2, cardiovascular data and blood sample. If the F-value indicated significant differences among group means, the overall significance of the variation with time including conditions normoxia and hypoxia were assessed using a paired t-test. Linear regressions were performed by using the least squares method. A p-value <0.05 was considered as significant.
|
Results |
|---|
|
TOPAbstractMethodsResultsDiscussionConclusionAcknowledgementsReferences |
|---|
. Subjects who experienced symptoms of AMS showed a slightly decreased AMS score with Arg infusion, mean minimal score was observed at the end of Arg infusion but no significant correlation was found. Fifteen minutes after the end of the infusion, the AMS score retrieved its baseline level, whereas Sa,O2 stayed higher. In the present study, the experiments were performed in the morning, which corresponds to the period of maximal AMS symptoms during the daytime 18. The most frequent symptom was headache. No subject experienced clinical signs of pulmonary or cerebral oedema.
|
). In normoxia, Sa,O2 was not modified by Arg infusion. In hypoxia, Sa,O2 tended to decrease before and in the first 15 min of Arg infusion, and then increased from 15–45 min. Sa,O2 was higher by 3.2±0.4% from t15 to t30 and by 5.3±0.3% from t15 to t45 (p<0.05). This increase was parallel to l-cit concentration but no significant correlation was found. SBP and DBP remained unchanged at 4,350 m (table 1). In both conditions, SBP and DBP did not vary during Arg infusion. HR increased by 25±1% (p<0.01) from sea-level to 4,350 m (table 1). HR did not vary during Arg infusion in both conditions.
|
respectively. Baseline l-cit concentration decreased from 33.1±1.7 µM·L–1 at sea-level to 28.5±2.1 µM·L–1 at 4,350 m (p<0.05). In both conditions, l-cit concentration increases during Arg infusion from respective baseline to 46.2±2.5 µM·L–1 at sea-level and 36.3±3.2 µM·L–1 at 4,350 m (p<0.05). Moreover, concentration remained higher after 15 min recovery (t45). Therefore, l-cit concentration increased by 49±1% at sea-level and 35±2% at 4,350 m from t0 to t45 (p<0.01). However, l-cit concentration in hypoxia remained below the normoxia value by 22±1% throughout Arg infusion and recovery (p<0.05). cGMP concentration was lower at 4,350 m than at sea level (p<0.05). In normoxia, cGMP concentration increased from 2.8±0.5 nM.L–1 at t0 to 4.2±0.6 nM·L–1 at t45 (53±6%, p<0.05). Moreover, cGMP concentration increase was positively correlated to l-cit concentration increase (r=0.96, p<0.05) (fig. 4). In hypoxia the concentration of cGMP remained unchanged during Arg infusion and the correlation was not significant.
|
|
|
|
Discussion |
|---|
|
TOPAbstractMethodsResultsDiscussionConclusionAcknowledgementsReferences |
|---|
l-arginine availability
The availability of intracellular Arg is a critical factor in the regulation of the rate of NO synthesis in endothelial cells. The Arg content of endothelial cells is derived primarily from plasma membrane-dependent transport of extracellular Arg, but can also be synthesized from l-cit 19. Block et al. 20 showed that exposure to hypoxia causes significant reduction in the transport of Arg, only accompanied by a decrease in intracellular Arg content after long-term exposure to hypoxia. Su and Block 21 demonstrated that hypoxia time dependently inhibits the synthesis of Arg from l-cit in pulmonary artery endothelial cells. These results suggest that hypoxia-induced l-cit concentration decrease might not be due to the capacity of endothelial cell to recycle l-cit and since the Arg-NO pathway represents the major metabolic pathway, it can be assumed that decreased l-cit was caused by an effect of hypoxia on Arg metabolism. However, NO production is linked to the rate of transformation of Arg to l-cit, which depends on cardiac blood flow and arteriovenous difference. In acute hypoxia, cardiac blood flow increases and l-cit concentration decreases suggesting that NO production might not be correlated to l-cit concentration. Although, hypoxia resulted in a decrease in the conversion of l-cit, intracellular Arg content was increased in the hypoxic pulmonary artery endothelial cells 22, 23. Thus, lack of availability of intracellular Arg does not appear to account for the decrease in NO production unless the intracellular pools of Arg are inaccessible to NOS III in hypoxia. This is of particular relevance to endothelium because NOS III is membrane-associated and thus, a membrane-related pool of Arg may be critical to the activity of the enzyme. In normal vessels, the amount of the substrate is sufficient for conversion to NO at a maximal rate. However, addition of Arg causes an endothelium-dependent relaxation in pulmonary artery depleted of Arg 7. Administration of substrate for NOS restores endothelium-dependent vasodilatory response in hypoxia-induced pulmonary hypertension 10, 24. In patients with pulmonary hypertension, Arg reduced the pulmonary vascular resistance and the magnitude of the pulmonary vasodilatory response was correlated to the level of l-cit 11. Furthermore, although NOS expression was upregulated in the early course of development of HPV 25, NOS activity was not increased if the substrate for the NOS enzyme was limiting 26, 27. Thus, the increase in l-cit and the short-term oxygenation effect of exogenous Arg at high altitude may be due to enhanced NO production in the pulmonary vasculature, and increase in Arg availability can promote l-cit/NO synthesis in high altitude. The vascular effect of Arg may be mediated through NO-independent mechanisms such as the effect of pH. Although alkaline solutions of Arg can induce vasodilatation, Arg hydrochloride solution was slightly acidic, weakening an effect of the pH. As Arg was infused with glucose, the direct effect of carbohydrates on ventilation/perfusion ratio should not be ruled out. However, this effect could be NO-independent since hyperglycaemia did not affect endothelium-dependent vasoreactivity in humans 28. Alternatively, a nutritional deficiency might not have contributed to the concentration decrease of l-cit during 36 h of hypoxia and Arg availability is low owing to extensive protein binding.
Ventilation/perfusion ratio
Pison 29 demonstrated that NO may improve ventilation and perfusion matching, as well as excretion and retention dispersion indexes, suggesting an improvement in gas exchange because of a redistribution of both ventilation and blood flow. In HAPE, inhalation of NO redistributed blood flow in the lungs away from oedematous regions, which improved the matching of ventilation and perfusion and reducing the alveolar-arterial oxygen difference 30. Thus, the improvement of the Sa,O2 with Arg suggests that the NO substrate could be a useful adjuvant in the treatment of high altitude pulmonary hypertension, which is implicated in the pathogenesis of HAPE. Alternatively, NO induces vasodilatation of bronchial artery in both normoxia and hypoxia 31 and modulates agonist-induced bronchoconstriction 32. The improvement of Sa,O2 may be due to the vasodilatory effect of NO on the small vascular pulmonary arteries thereby increasing the perfusion/ventilation ratio. However, Arg-induced hyperventilation might not be ruled out. Furthermore, as subjects prone to HAPE have a less compliant pulmonary circulation due to multifactorial mechanism, including reduced NO synthesis 33, Arg infusion may prevent, at least in part, the complication of HPV. The initial decrease in the Sa,O2 may be due to reclined position during the procedure, since lying flat in bed raises intrathoracic blood volume and consequently, pulmonary arterial pressure 18.
Guanylyl cyclase
In bovine arterial rings, a depletion of tissue Arg is associated with a decrease in basal cGMP levels and impairment of endothelium-dependent cGMP formation 7. However, basal guanylyl cyclase activity did not differ significantly between normoxic and hypoxic lungs 25. The decreased cGMP at 4,350 m assumes that Arg content was lower at 4,350 m. However, in hypoxia no correlation was found between l-cit and cGMP concentration variation, suggesting that the cGMP metabolism differs from sea-level to 36 h of exposure to altitude of 4,350 m. Conversely, the nonspecificity of the plasma cGMP level may explain the discrepancy of these results in hypoxia.
Systemic vessel
Administration of Arg results in an increase in the concentration of NO in the exhaled air matched by an increase in the concentration of NO3– in the plasma 34. Bode-Böger et al. 35 showed that Arg induced vasodilatation in healthy humans in normoxia and Blitzer et al. 36 demonstrated that NOS inhibition increased systemic vascular resistance and BP and decreased cardiac output in short-term hypoxia. However, the presented results show that Arg infusion was not associated with any measurable change in systemic haemodynamics, suggesting the absence of a direct effect of NOS substrate on heart and vascular smooth muscle of the systemic vessels. The discrepancy of the findings may be explained by the baseline level of SBP and DBP (150/90 for Bode-Böger et al. 35 and 120/70 for the present study) and by inactivation of free NO by high-affinity binding to haemoglobin in hypoxia. Furthermore, in the protocol of Blitzer et al. 36, decrease in the fraction of inspired oxygen was induced by breathing hypoxic mixture and the duration of hypoxia was less than the present study.
|
Conclusion |
|---|
|
TOPAbstractMethodsResultsDiscussionConclusionAcknowledgementsReferences |
|---|
|
Acknowledgements |
|---|
|
TOPAbstractMethodsResultsDiscussionConclusionAcknowledgementsReferences |
|---|
|
References |
|---|
|
TOPAbstractMethodsResultsDiscussionConclusionAcknowledgementsReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  G. F. Gonzales, F. A. Chung, S. Miranda, L. B. Valdez, T. Zaobornyj, J. Bustamante, and A. Boveris Heart mitochondrial nitric oxide synthase is upregulated in male rats exposed to high altitude (4,340 m) Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H2568 - H2573. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-P. Richalet, P. Gratadour, P. Robach, I. Pham, M. Dechaux, A. Joncquiert-Latarjet, P. Mollard, J. Brugniaux, and J. Cornolo Sildenafil Inhibits Altitude-induced Hypoxemia and Pulmonary Hypertension Am. J. Respir. Crit. Care Med., February 1, 2005; 171(3): 275 - 281. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. R. S. Steiner, N. C. Gonzalez, and J. G. Wood Interaction between reactive oxygen species and nitric oxide in the microvascular response to systemic hypoxia J Appl Physiol, October 1, 2002; 93(4): 1411 - 1418. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) during l-arginine infusion at sea-level (•) and after 36 h of exposure to altitude of 4,350 m (
). Values are mean±se; n=11. l-arginine infusion (0.5 g·kg–1 in 30 min) occurred after 15 min of rest in supine position and was followed by 15 min of recovery. *: p<0.05, significantly different from 15 min after the beginning of the infusion (t15).
