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Calcitonin gene-related peptide and adrenomedullin release in humans: Effects of exercise and hypoxia
Regulatory Peptides 108 (2002) 89 – 95 www.elsevier.com/locate/regpep
Calcitonin gene-related peptide and adrenomedullin release in humans: Effects of exercise and hypoxia Philip Hasbak a,b,c,*, Carsten Lundby d, Niels Vidiendal Olsen d,e, Søren Schifter b, Inge-Lis Kanstrup a a Department of Clinical Physiology and Nuclear Medicine, University Hospital of Herlev, Herlev, Denmark Department of Clinical Physiology and Nuclear Medicine, University Hospital of Glostrup, Glostrup, Denmark c Department of Clinical Experimental Research, University Hospital of Glostrup, Denmark d Department of Pharmacology, Panum Institute, University of Copenhagen, Copenhagen, Denmark e Department of Neuroanaesthesia, The Neuroscience Centre, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark b
Received 22 April 2002; received in revised form 1 July 2002; accepted 3 July 2002
Abstract Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are potent vasorelaxant peptides. This study examined exerciseinduced changes in CGRP and AM levels in 12 healthy sea level natives at sea level (SL) and subsequently after 24 h (HA1) and 5 days (HA5) in high altitude hypoxia (4559 m). Plasma values of CGRP, AM, calcitonin, noradrenaline, adrenaline, lactate and heart rate were measured at rest and during maximal exercise (Wmax). On each study day, the dopamine D2-receptor antagonist, domperidone (30 mg; n = 6), or no medication (n = 6) was given 1 h before exercise. Wmax at SL, HA1 and HA5 increased CGRP and AM along with heart rate, lactate and catecholamines, whereas, calcitonin remained unchanged. The maximal CGRP levels at Wmax were significantly decreased at HA1 (74.3 F 6.1 pmol/l; p = 0.002) and HA5 (69.6 F 6.0 pmol/l; p < 0.001) compared to maximal CGRP at SL (85.1 F 4.9 pmol/l). A similar pattern was observed for lactate and the relation between CGRP and lactate release showed a close linear correlation (r2 = 0.63, P < 0.0001). Domperidone produced a marked increase in noradrenaline at Wmax, but had no affect on CGRP or AM. In conclusion, CGRP release during hypoxic exercise does not respond to domperidone-induced changes in circulating levels of noradrenaline, rather the release may be directly related to the production of lactate. D 2002 Elsevier Science B.V. All rights reserved. Keywords: CGRP; Calcitonin; Adrenalin; Noradrenaline; Lactic acid; Exercise; Hypoxia; Humans
1. Introduction Calcitonin gene-related peptide (CGRP), calcitonin and adrenomedullin are structurally related peptides belonging to the same peptide family [1]. CGRP is a 37-amino acid vasoactive neuropeptide produced by alternative splicing of the transcript of the calcitonin/aCGRP gene [2]. Whereas, calcitonin is expressed almost exclusively in the C cells of the thyroid gland, aCGRP is widely distributed in the central and peripheral nervous systems in mammals. A
*
Corresponding author. Department of Clinical Physiology and Nuclear Medicine, University Hospital of Copenhagen, Glostrup Hospital, Nordre Ringvej, DK-2600 Glostrup, Denmark. Tel.: +45-4323-2451, +454323-2429; fax: +45-4323-3928. E-mail address: [email protected] (P. Hasbak).
second CGRP isoform, hCGRP, was encoded by a different gene locus and was expressed in specific neuronal sites [3]. The a- and h-CGRP isoforms exhibit overlapping biological activities in most vascular beds [4]. The release of aCGRP from sensory pain fibers has been implicated in the perception of pain [5]. Being a very potent vasodilator [6], aCGRP causes a decrease in blood pressure and an increase in heart rate when administered intravenously to healthy volunteers [7,8]. In most studies, the total concentration of CGRP (a and h CGRP) is given, because no available assay can differentiate between human a and h forms. CGRP levels in plasma were elevated in response to exercise in healthy individuals [9] and has been proposed to counteract the vasoconstrictor response to enhanced release of adrenalin and noradrenaline [10,11]. Moreover, CGRP release can be induced by the release of lactate as described in animal studies [12,13]. Another possible determinant for CGRP
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release is tissue hypoxia. Hypoxia has marked effects on the artery caliber and modifies the release of different vasoactive mediators [14]. Plasma levels of CGRP have been shown to decrease during hypoxia [15]. Adrenomedullin is a 52-amino acid peptide originally extracted from a human pheochromocytoma [16]. Current evidence points to vascular tissue and especially endothelial cells as the major source of circulating adrenomedullin [17]. Adrenomedullin has strong hypotensive properties, and has been demonstrated to induce relaxation in different vascular beds [18]. Exercise has been reported by some studies [19], but not others [20,21], to increase plasma adrenomedullin in man, with a correlation between plasma adrenomedullin and blood pressure [19]. Exposure to high altitude has been reported to be associated with an increase in plasma adrenomedullin in man [22]. The aims of this study were to study the possible exercise- and hypoxia-induced release of CGRP, adrenomedullin and calcitonin in humans and the relationship between these peptides and circulating concentrations of lactate and catecholamines. Preganglionergic dopamine D2 receptor act as autoreceptors that reduce the amount of noradrenaline released from sympathetic nerve endings [23,24] and dopaminergic D2 receptor antagonists have been found to increase the plasma concentration of noradrenaline both at rest and during exercise. Thus, we used the dopaminergic D2 receptor blocker domperidone to further characterize the relation between preganglionic release of noradrenaline and circulating levels of CGRP.
2. Methods The study was carried out as part of an integrated protocol of which some of the results have already been published [25]. Twelve healthy subjects (five females and seven males), aged 26.1 F 1.4 years (mean F S.E.M.), a body mass of 73.5 F 0.8 kg and a height of 179.2 F 4.3 cm, entered the study after having given their written informed consent. The study was approved by the regional Ethical Committee of Copenhagen. The subjects were studied at sea level (SL) in Copenhagen, Denmark, and subsequently 24 h (HA1) and 5 days (HA5) after arrival to high altitude. The subjects were airlifted by helicopter to the Capanna Regina Margherita Hut, Monte Rosa, Italy (altitude = 4559 m). The high altitude experiments were carried out 1 week after the sea level experiments. Room temperature in both laboratories was 19 – 21 jC. At altitude physical activity was kept at a minimum. At sea level, strenuous physical activity was not allowed 72 h before the experiment. The protocols for the three study days were identical and were conducted at the same time of the day. A catheter was inserted in an antecubital vein, and heart rate and arterial blood pressure were recorded after 30 min of supine rest. Thereafter, blood samples were drawn for measurements of plasma concentrations of CGRP, calcito-
nin, adrenomedullin, catecholamines, lactate and hematocrit. Subjects were then divided randomly into two groups, one receiving 30 mg of domperidone orally (four males, two females) and a control group (three males, three females). After a light 5-min warm-up at 120 W and 80 rpm on a Monark 848 cycle ergometer (Monark, Sweden), the subjects started a maximal exercise test. The protocol was designed to exhaust the subjects within 3 –5 min. The start workload was estimated from the warm-up and was then increased by 40 W every 1.5 min until exhaustion. Maximal exercise (Wmax) was the maximal workload corresponding to peak VO2. At sea level, achievement of VO2max was accepted if two of the following criteria were fulfilled: (1) plateau in oxygen uptake ( < 100 ml/min/40 W increase), (2) plasma lactate concentration > 8.0 mmol/l, or (3) respiratory exchange ratio>1.15. Gas exchange was recorded every 15 s by a MedGraphic CPX/d system (St. Paul, MN, USA). Before use, the system was calibrated with calibration gases, which had previously been analysed by the micro-Scholander technique. Heart rate was measured beat by beat with a Polar Vantage XL monitor (Oy, Finland). Oxygen saturation was recorded continuously by a Nellcor pulse oxymeter (Hayward, USA) with the sensor attached to the first or second finger. Blood samples at maximal exercise were drawn immediately (within seconds) after the end of the exercise session. Plasma catecholamines were measured by high-performance liquid chromatography (Waters, Millipore, MA, USA) [26]. Five milliliters of blood were drawn into ice-chilled tubes containing 1.7 mg/ml EGTA and 1.1 mg/ml reduced glutathione. Blood samples for determination of CGRP and calcitonin were collected in tubes containing heparin (15 IU/ ml blood) and aprotinin (‘‘Trasylol’’, Bayer, Leverkusen, Germany, 100 KIU/ml blood). All blood samples were separated at 3000 g for 10 min and stored at 20 jC for subsequent analysis. At high altitude, samples were stored in liquid nitrogen until the return to the sea level laboratory. CGRP and calcitonin were determined by radioimmunoassay (RIA), as previously described [27,28]. As for other CGRP RIA assays, it was not possible to distinguish between a and h forms of CGRP. To determine adrenomedullin in plasma, samples (1.0 ml) were mixed with an equal volume of 1% trifluoroacetic acid (TFA) in H2O and extracted through C-18 Sep-Pak cartridges (Waters, Milford, MA, USA). The proteins were eluted with 60% acetonitrile in 1% TFA and the recovered volume was dried in a centrifugal concentrator. Extracts were reconstituted in 250 Al RIA assay buffer for a two-tube assay, spun at 14 000 rpm for 10 min at 4 jC to remove any solid matter and two 100 Al aliquots from each sample were separated for analysis. The RIA was performed using the Phoenix human adrenomedullin RIA kit (Phoenix Pharmaceuticals, USA) and following the manufacturer’s instructions. The Vitrosk 700 System (Johnson & Johnson Clinical Diagnostics) and Vitros LAC Slides were used for a
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Table 2 Changes in hematocrit and maximal workload at sea level (SL), high altitude day 1 (HA1) and day 5 (HA5)
Hematocrit (%) Workload (W)
rest Wmax
SL
HA1
HA5
41.4 F 0.8 312.2 F 10.2
42.8 F 1.0 246.8 F 8.2 *
45.4 F 1.2 * 253.3 F 6.2 * ,y
Maximal exercise (Wmax) is the maximal workload corresponding to peak VO2. Results are given as mean F S.E.M. * p < 0.05 compared to sea level data. y p < 0.05 compared to Mt. Rosa day 1 data.
Fig. 1. Changes in plasma CGRP concentrations at rest and at maximal exercise (Wmax) (n = 12). Results are given as mean F S.E.M. * p < 0.05 compared to rest.
quantitative measurement of the lactate concentration in plasma. Hematocrit was measured in triplicate by the use of a minicentrifuge at 2000 g for 5 min. 2.1. Statistics Results were given as mean F S.E.M. Statistical difference of variance between groups and study days was analysed by a two-way ANOVA for repeated measures. If allowed for, differences between groups and study days were assessed by unpaired and paired t-tests, respectively. Regression analysis was performed to examine the relationship between plasma concentrations of lactate and CGRP. p < 0.05 was considered significant. Data for noradrenaline were presented separately (n = 6 in each group) depending on whether the subjects received domperidone or not. The remaining data were pooled (n = 12), because no significant difference was found between the groups.
In contrast to adrenomedullin, the absolute CGRP response at Wmax decreased progressively at HA1 and HA5 compared with conditions at SL. No changes in the calcitonin levels were detected during exercise or hypoxia (Table 1). At resting conditions, CGRP, calcitonin and adrenomedullin levels were unaltered by hypoxia. 3.2. Workload, heart rate and lactate The obtained maximal workload was significantly higher at SL compared with HA1 and HA5. Furthermore, a significant workload difference was found between HA1 and HA5 (Table 2). Both the maximal values of heart rate and plasma lactate decreased successively on HA1 and HA5 compared with maximal values at SL (Figs. 2 and 3). Resting values of heart rate increased during acute hypoxia but on HA5 have returned to values not different from those obtained at SL (Fig. 2). 3.3. Catecholamines Exercise in both environments increased the plasma concentrations of noradrenaline and adrenaline (Figs. 4 and 5a,b). Domperidone had no effect on resting plasma
3. Results 3.1. CGRP, calcitonin and adrenomedullin In both environments, exercise increased circulating levels of CGRP and adrenomedullin (Fig. 1 and Table 1).
Table 1 Changes in plasma adrenomedullin and calcitonin concentrations at rest and at maximal exercise (Wmax) Adrenomedullin (pmol/l) Calcitonin (pmol/l)
Rest Wmax Rest Wmax
SL
HA1
HA5
6.9 F 0.7 8.0 F 0.8 * 13.1 F 2.0 14.9 F 1.6
6.8 F 0.6 7.4 F 0.6 * 12.9 F 1.5 15.4 F 1.6
6.9 F 0.7 8.3 F 0.7 * 14.1 F 2.0 15.6 F 2.7
Results are given as mean F S.E.M. * p < 0.05 compared to rest.
Fig. 2. Changes in heart rate at rest and at maximal exercise (Wmax) (n = 12). Results are given as mean F S.E.M. * p < 0.05 compared to rest.
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Fig. 3. Changes in plasma lactate at rest and at maximal exercise (Wmax) (n = 12). Results are given as mean F S.E.M. * p < 0.05 compared to rest.
concentrations of catecholamines. However, at Wmax plasma concentrations of noradrenaline were higher in the domperidone group than in the control group (Fig. 5a and b). 3.4. CGRP and lactate correlation Plasma concentrations of lactate matched the pattern of CGRP release at Wmax at sea level and during hypoxia. Furthermore, a linear correlation plot between the relative increase in CGRP and lactate levels during exercise showed a close linear correlation (r2 = 0.63, p < 0.0001) (Fig. 6). 3.5. Hematocrit levels during hypoxia Hematocrit levels increased progressively at high altitude (Table 2).
Fig. 4. Changes in plasma adrenaline concentrations at rest and at maximal exercise (Wmax) (n = 12). Results are given as mean F S.E.M. * p < 0.05 compared to rest.
Fig. 5. (a) The domperidone group (n = 6). Changes in plasma noradrenaline concentrations at rest and at maximal exercise (Wmax). * p < 0.05 compared to rest, #p < 0.05 compared with non-domperidone group. (b) The non-doperidone group (n = 6). Changes in plasma noradrenaline concentrations at rest and at maximal exercise (Wmax). * p < 0.05 compared to rest, #p < 0.05 compared with non-domperidone group.
Fig. 6. Linear correlation plot between the relative increase in CGRP and lactate levels during exercise (n = 12). The regression line, y = 0.42x + 3.5 ( p < 0.0001); r2 = 0.63. The 95% confidence interval of the regression line is shown with dotted line.
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4. Discussion CGRP is the most potent endogenous vasodilatatory peptide described, thus, far [29] and both CGRP and adrenomedullin play a role not only in the control of vascular tone but also has a direct effect on the heart [30 – 32]. Since the discovery of CGRP and adrenomedullin, the significance of various release factors has been investigated, primarily in animal studies. Hypoxia, exercise, sympathetic activity and lactate are such factors. 4.1. Hypoxia Hypoxia causes vasodilatation in most vascular beds, including the coronary, skeletal muscle and cerebral circulations, whereas, vasoconstriction occurs in the pulmonary circulation [33]. Keith and Ekman [15] have demonstrated a hypoxia-induced decrease in resting CGRP levels in hypoxic rats (FIO2 10%). In the present study, no significant decrease were found in resting CGRP levels during hypoxia, but the subjects were exposed to hypoxia for only 5 days compared to 17 – 21 days in the work of Keith and Ekman [15]. The present study showed that hypoxia in a timedependent manner decreased in the maximal CGRP levels at Wmax. This decrease in exercise-induced release of CGRP was not caused by a reduction in the absolute workload, as this was actually increased from HA1 to HA5. CGRP levels were measured in venous plasma. Hematocrit levels increase during exercise, where the increase may be up to 10%. Hematocrit levels were not determined during exercise in this study, but were probably increased by the same factor in all three Wmax bouts. Thus, changes in hematocrit cannot explain the increase in CGRP values during exercise. It may be hypothesized that the synthesis of CGRP receptors is stimulated by hypoxia causing a decrease in the need for CGRP release. Studies indicate that CGRP receptors are highly expressed especially by vascular smooth muscle cells within the pulmonary vasculature. These receptors have been shown to be up-regulated by hypoxia [34]. Whether CGRP receptors are generally up-regulated in humans during hypoxia is not known. Several studies have shown that hypoxia induce increase of both adrenomedullin mRNA and protein expression in different cell cultures [35 –39]. Plasma levels were raised in experimental pulmonary hypertension [40], a consequence of staying at high altitude. Furthermore, Toepfer et al. [22] reported elevated plasma adrenomedullin concentrations at high altitude compared to sea level conditions. In contrast to Toepfer et al. [22], we do not find any increase in circulating adrenomedullin concentrations during hypoxia, but the study conditions were not the same. In the Toepfer study, the subjects climbed to the summit in 72 h, whereas, our subjects were passively airlifted directly to high altitude within 30 min. The adrenomedullin elevation in the Toepfer study may therefore be explained by the physical strain rather than by the hypoxia per se. It cannot be excluded that
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hypoxia-induced changes on a cellular level in the production of adrenomedullin remained undetectable in plasma from a peripheral vein. Changes in hematocrit can, to some degree, explain the difference in exercise-induced adrenomedullin production, in contrast to CGRP. 4.2. Sympathetic activity Intense exercise in man produces an increased blood flow through contracting skeletal muscle and an increase in heart rate, cardiac output and arterial blood pressure together with an increase in sympathetic nervous activity [41]. Since CGRP is one of the most potent endogenous vasodilators known, several studies have hypothesized that CGRP counteracts the sympathetic vasoconstrictors adrenaline and noradrenaline [10,11,42,43]. Thus, adrenaline infusion in humans increased the plasma concentrations up to 6.4 nM and resulted in CGRP release, whereas, CGRP levels were not affected by noradrenaline infusion [44,45]. In the present study, no significant changes in adrenaline concentration were found when comparing the Wmax values during normoxia and hypoxia, and the release pattern of noradrenaline was not identical to that of CGRP at Wmax. The dopaminergic D2-antagonist domperidone was administered to six subjects. At SL, HA1 and HA5, the domperidone group had significantly higher maximal noradrenaline concentrations compared to the control group [25]. However, this domperidoneinduced increase in noradrenaline did not affect the CGRP release either. Thus, our data do not support that CGRP release was mediated by adrenaline/noradrenaline, and no correlation between changes in circulating levels of catecholamines and CGRP could be found. 4.3. Lactate Lactate is released by exercising skeletal muscle cells as the oxygen requirements of working muscle cells exceed adequate oxygen delivery to the cells. In the present study, the subjects produced high concentrations of lactate. At Wmax, the release of lactate showed a similar pattern as observed for the CGRP release, namely a relative decrease in maximal concentration which was accentuated with time in hypoxia. A linear correlation was found between the increase in CGRP and lactate release, suggesting a relationship between the lactate production and the CGRP release. This is supported by animal studies showing that lactate and low pH induce release of CGRP in rats [12]. In conclusion, the exercise-induced CGRP release is, in contrast to adrenomedullin, modified by hypoxia. The exact signal for the decrease in CGRP release cannot be stated from this study, but lactate and low pH are candidates. Our results do not support the hypothesis that CGRP solely acts as a counter-regulatory mechanism to the sympathetic vasoconstrictor noradrenaline. Lactate formation and low pH in muscle tissue may be mediators of sensory nerve fiber activation and CGRP release.
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Calcitonin gene-related peptide and adrenomedullin release in humans: Effects of exercise and hypoxia Philip Hasbak a,b,c,*, Carsten Lundby d, Niels Vidiendal Olsen d,e, Søren Schifter b, Inge-Lis Kanstrup a a Department of Clinical Physiology and Nuclear Medicine, University Hospital of Herlev, Herlev, Denmark Department of Clinical Physiology and Nuclear Medicine, University Hospital of Glostrup, Glostrup, Denmark c Department of Clinical Experimental Research, University Hospital of Glostrup, Denmark d Department of Pharmacology, Panum Institute, University of Copenhagen, Copenhagen, Denmark e Department of Neuroanaesthesia, The Neuroscience Centre, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark b
Received 22 April 2002; received in revised form 1 July 2002; accepted 3 July 2002
Abstract Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are potent vasorelaxant peptides. This study examined exerciseinduced changes in CGRP and AM levels in 12 healthy sea level natives at sea level (SL) and subsequently after 24 h (HA1) and 5 days (HA5) in high altitude hypoxia (4559 m). Plasma values of CGRP, AM, calcitonin, noradrenaline, adrenaline, lactate and heart rate were measured at rest and during maximal exercise (Wmax). On each study day, the dopamine D2-receptor antagonist, domperidone (30 mg; n = 6), or no medication (n = 6) was given 1 h before exercise. Wmax at SL, HA1 and HA5 increased CGRP and AM along with heart rate, lactate and catecholamines, whereas, calcitonin remained unchanged. The maximal CGRP levels at Wmax were significantly decreased at HA1 (74.3 F 6.1 pmol/l; p = 0.002) and HA5 (69.6 F 6.0 pmol/l; p < 0.001) compared to maximal CGRP at SL (85.1 F 4.9 pmol/l). A similar pattern was observed for lactate and the relation between CGRP and lactate release showed a close linear correlation (r2 = 0.63, P < 0.0001). Domperidone produced a marked increase in noradrenaline at Wmax, but had no affect on CGRP or AM. In conclusion, CGRP release during hypoxic exercise does not respond to domperidone-induced changes in circulating levels of noradrenaline, rather the release may be directly related to the production of lactate. D 2002 Elsevier Science B.V. All rights reserved. Keywords: CGRP; Calcitonin; Adrenalin; Noradrenaline; Lactic acid; Exercise; Hypoxia; Humans
1. Introduction Calcitonin gene-related peptide (CGRP), calcitonin and adrenomedullin are structurally related peptides belonging to the same peptide family [1]. CGRP is a 37-amino acid vasoactive neuropeptide produced by alternative splicing of the transcript of the calcitonin/aCGRP gene [2]. Whereas, calcitonin is expressed almost exclusively in the C cells of the thyroid gland, aCGRP is widely distributed in the central and peripheral nervous systems in mammals. A
*
Corresponding author. Department of Clinical Physiology and Nuclear Medicine, University Hospital of Copenhagen, Glostrup Hospital, Nordre Ringvej, DK-2600 Glostrup, Denmark. Tel.: +45-4323-2451, +454323-2429; fax: +45-4323-3928. E-mail address: [email protected] (P. Hasbak).
second CGRP isoform, hCGRP, was encoded by a different gene locus and was expressed in specific neuronal sites [3]. The a- and h-CGRP isoforms exhibit overlapping biological activities in most vascular beds [4]. The release of aCGRP from sensory pain fibers has been implicated in the perception of pain [5]. Being a very potent vasodilator [6], aCGRP causes a decrease in blood pressure and an increase in heart rate when administered intravenously to healthy volunteers [7,8]. In most studies, the total concentration of CGRP (a and h CGRP) is given, because no available assay can differentiate between human a and h forms. CGRP levels in plasma were elevated in response to exercise in healthy individuals [9] and has been proposed to counteract the vasoconstrictor response to enhanced release of adrenalin and noradrenaline [10,11]. Moreover, CGRP release can be induced by the release of lactate as described in animal studies [12,13]. Another possible determinant for CGRP
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release is tissue hypoxia. Hypoxia has marked effects on the artery caliber and modifies the release of different vasoactive mediators [14]. Plasma levels of CGRP have been shown to decrease during hypoxia [15]. Adrenomedullin is a 52-amino acid peptide originally extracted from a human pheochromocytoma [16]. Current evidence points to vascular tissue and especially endothelial cells as the major source of circulating adrenomedullin [17]. Adrenomedullin has strong hypotensive properties, and has been demonstrated to induce relaxation in different vascular beds [18]. Exercise has been reported by some studies [19], but not others [20,21], to increase plasma adrenomedullin in man, with a correlation between plasma adrenomedullin and blood pressure [19]. Exposure to high altitude has been reported to be associated with an increase in plasma adrenomedullin in man [22]. The aims of this study were to study the possible exercise- and hypoxia-induced release of CGRP, adrenomedullin and calcitonin in humans and the relationship between these peptides and circulating concentrations of lactate and catecholamines. Preganglionergic dopamine D2 receptor act as autoreceptors that reduce the amount of noradrenaline released from sympathetic nerve endings [23,24] and dopaminergic D2 receptor antagonists have been found to increase the plasma concentration of noradrenaline both at rest and during exercise. Thus, we used the dopaminergic D2 receptor blocker domperidone to further characterize the relation between preganglionic release of noradrenaline and circulating levels of CGRP.
2. Methods The study was carried out as part of an integrated protocol of which some of the results have already been published [25]. Twelve healthy subjects (five females and seven males), aged 26.1 F 1.4 years (mean F S.E.M.), a body mass of 73.5 F 0.8 kg and a height of 179.2 F 4.3 cm, entered the study after having given their written informed consent. The study was approved by the regional Ethical Committee of Copenhagen. The subjects were studied at sea level (SL) in Copenhagen, Denmark, and subsequently 24 h (HA1) and 5 days (HA5) after arrival to high altitude. The subjects were airlifted by helicopter to the Capanna Regina Margherita Hut, Monte Rosa, Italy (altitude = 4559 m). The high altitude experiments were carried out 1 week after the sea level experiments. Room temperature in both laboratories was 19 – 21 jC. At altitude physical activity was kept at a minimum. At sea level, strenuous physical activity was not allowed 72 h before the experiment. The protocols for the three study days were identical and were conducted at the same time of the day. A catheter was inserted in an antecubital vein, and heart rate and arterial blood pressure were recorded after 30 min of supine rest. Thereafter, blood samples were drawn for measurements of plasma concentrations of CGRP, calcito-
nin, adrenomedullin, catecholamines, lactate and hematocrit. Subjects were then divided randomly into two groups, one receiving 30 mg of domperidone orally (four males, two females) and a control group (three males, three females). After a light 5-min warm-up at 120 W and 80 rpm on a Monark 848 cycle ergometer (Monark, Sweden), the subjects started a maximal exercise test. The protocol was designed to exhaust the subjects within 3 –5 min. The start workload was estimated from the warm-up and was then increased by 40 W every 1.5 min until exhaustion. Maximal exercise (Wmax) was the maximal workload corresponding to peak VO2. At sea level, achievement of VO2max was accepted if two of the following criteria were fulfilled: (1) plateau in oxygen uptake ( < 100 ml/min/40 W increase), (2) plasma lactate concentration > 8.0 mmol/l, or (3) respiratory exchange ratio>1.15. Gas exchange was recorded every 15 s by a MedGraphic CPX/d system (St. Paul, MN, USA). Before use, the system was calibrated with calibration gases, which had previously been analysed by the micro-Scholander technique. Heart rate was measured beat by beat with a Polar Vantage XL monitor (Oy, Finland). Oxygen saturation was recorded continuously by a Nellcor pulse oxymeter (Hayward, USA) with the sensor attached to the first or second finger. Blood samples at maximal exercise were drawn immediately (within seconds) after the end of the exercise session. Plasma catecholamines were measured by high-performance liquid chromatography (Waters, Millipore, MA, USA) [26]. Five milliliters of blood were drawn into ice-chilled tubes containing 1.7 mg/ml EGTA and 1.1 mg/ml reduced glutathione. Blood samples for determination of CGRP and calcitonin were collected in tubes containing heparin (15 IU/ ml blood) and aprotinin (‘‘Trasylol’’, Bayer, Leverkusen, Germany, 100 KIU/ml blood). All blood samples were separated at 3000 g for 10 min and stored at 20 jC for subsequent analysis. At high altitude, samples were stored in liquid nitrogen until the return to the sea level laboratory. CGRP and calcitonin were determined by radioimmunoassay (RIA), as previously described [27,28]. As for other CGRP RIA assays, it was not possible to distinguish between a and h forms of CGRP. To determine adrenomedullin in plasma, samples (1.0 ml) were mixed with an equal volume of 1% trifluoroacetic acid (TFA) in H2O and extracted through C-18 Sep-Pak cartridges (Waters, Milford, MA, USA). The proteins were eluted with 60% acetonitrile in 1% TFA and the recovered volume was dried in a centrifugal concentrator. Extracts were reconstituted in 250 Al RIA assay buffer for a two-tube assay, spun at 14 000 rpm for 10 min at 4 jC to remove any solid matter and two 100 Al aliquots from each sample were separated for analysis. The RIA was performed using the Phoenix human adrenomedullin RIA kit (Phoenix Pharmaceuticals, USA) and following the manufacturer’s instructions. The Vitrosk 700 System (Johnson & Johnson Clinical Diagnostics) and Vitros LAC Slides were used for a
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Table 2 Changes in hematocrit and maximal workload at sea level (SL), high altitude day 1 (HA1) and day 5 (HA5)
Hematocrit (%) Workload (W)
rest Wmax
SL
HA1
HA5
41.4 F 0.8 312.2 F 10.2
42.8 F 1.0 246.8 F 8.2 *
45.4 F 1.2 * 253.3 F 6.2 * ,y
Maximal exercise (Wmax) is the maximal workload corresponding to peak VO2. Results are given as mean F S.E.M. * p < 0.05 compared to sea level data. y p < 0.05 compared to Mt. Rosa day 1 data.
Fig. 1. Changes in plasma CGRP concentrations at rest and at maximal exercise (Wmax) (n = 12). Results are given as mean F S.E.M. * p < 0.05 compared to rest.
quantitative measurement of the lactate concentration in plasma. Hematocrit was measured in triplicate by the use of a minicentrifuge at 2000 g for 5 min. 2.1. Statistics Results were given as mean F S.E.M. Statistical difference of variance between groups and study days was analysed by a two-way ANOVA for repeated measures. If allowed for, differences between groups and study days were assessed by unpaired and paired t-tests, respectively. Regression analysis was performed to examine the relationship between plasma concentrations of lactate and CGRP. p < 0.05 was considered significant. Data for noradrenaline were presented separately (n = 6 in each group) depending on whether the subjects received domperidone or not. The remaining data were pooled (n = 12), because no significant difference was found between the groups.
In contrast to adrenomedullin, the absolute CGRP response at Wmax decreased progressively at HA1 and HA5 compared with conditions at SL. No changes in the calcitonin levels were detected during exercise or hypoxia (Table 1). At resting conditions, CGRP, calcitonin and adrenomedullin levels were unaltered by hypoxia. 3.2. Workload, heart rate and lactate The obtained maximal workload was significantly higher at SL compared with HA1 and HA5. Furthermore, a significant workload difference was found between HA1 and HA5 (Table 2). Both the maximal values of heart rate and plasma lactate decreased successively on HA1 and HA5 compared with maximal values at SL (Figs. 2 and 3). Resting values of heart rate increased during acute hypoxia but on HA5 have returned to values not different from those obtained at SL (Fig. 2). 3.3. Catecholamines Exercise in both environments increased the plasma concentrations of noradrenaline and adrenaline (Figs. 4 and 5a,b). Domperidone had no effect on resting plasma
3. Results 3.1. CGRP, calcitonin and adrenomedullin In both environments, exercise increased circulating levels of CGRP and adrenomedullin (Fig. 1 and Table 1).
Table 1 Changes in plasma adrenomedullin and calcitonin concentrations at rest and at maximal exercise (Wmax) Adrenomedullin (pmol/l) Calcitonin (pmol/l)
Rest Wmax Rest Wmax
SL
HA1
HA5
6.9 F 0.7 8.0 F 0.8 * 13.1 F 2.0 14.9 F 1.6
6.8 F 0.6 7.4 F 0.6 * 12.9 F 1.5 15.4 F 1.6
6.9 F 0.7 8.3 F 0.7 * 14.1 F 2.0 15.6 F 2.7
Results are given as mean F S.E.M. * p < 0.05 compared to rest.
Fig. 2. Changes in heart rate at rest and at maximal exercise (Wmax) (n = 12). Results are given as mean F S.E.M. * p < 0.05 compared to rest.
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Fig. 3. Changes in plasma lactate at rest and at maximal exercise (Wmax) (n = 12). Results are given as mean F S.E.M. * p < 0.05 compared to rest.
concentrations of catecholamines. However, at Wmax plasma concentrations of noradrenaline were higher in the domperidone group than in the control group (Fig. 5a and b). 3.4. CGRP and lactate correlation Plasma concentrations of lactate matched the pattern of CGRP release at Wmax at sea level and during hypoxia. Furthermore, a linear correlation plot between the relative increase in CGRP and lactate levels during exercise showed a close linear correlation (r2 = 0.63, p < 0.0001) (Fig. 6). 3.5. Hematocrit levels during hypoxia Hematocrit levels increased progressively at high altitude (Table 2).
Fig. 4. Changes in plasma adrenaline concentrations at rest and at maximal exercise (Wmax) (n = 12). Results are given as mean F S.E.M. * p < 0.05 compared to rest.
Fig. 5. (a) The domperidone group (n = 6). Changes in plasma noradrenaline concentrations at rest and at maximal exercise (Wmax). * p < 0.05 compared to rest, #p < 0.05 compared with non-domperidone group. (b) The non-doperidone group (n = 6). Changes in plasma noradrenaline concentrations at rest and at maximal exercise (Wmax). * p < 0.05 compared to rest, #p < 0.05 compared with non-domperidone group.
Fig. 6. Linear correlation plot between the relative increase in CGRP and lactate levels during exercise (n = 12). The regression line, y = 0.42x + 3.5 ( p < 0.0001); r2 = 0.63. The 95% confidence interval of the regression line is shown with dotted line.
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4. Discussion CGRP is the most potent endogenous vasodilatatory peptide described, thus, far [29] and both CGRP and adrenomedullin play a role not only in the control of vascular tone but also has a direct effect on the heart [30 – 32]. Since the discovery of CGRP and adrenomedullin, the significance of various release factors has been investigated, primarily in animal studies. Hypoxia, exercise, sympathetic activity and lactate are such factors. 4.1. Hypoxia Hypoxia causes vasodilatation in most vascular beds, including the coronary, skeletal muscle and cerebral circulations, whereas, vasoconstriction occurs in the pulmonary circulation [33]. Keith and Ekman [15] have demonstrated a hypoxia-induced decrease in resting CGRP levels in hypoxic rats (FIO2 10%). In the present study, no significant decrease were found in resting CGRP levels during hypoxia, but the subjects were exposed to hypoxia for only 5 days compared to 17 – 21 days in the work of Keith and Ekman [15]. The present study showed that hypoxia in a timedependent manner decreased in the maximal CGRP levels at Wmax. This decrease in exercise-induced release of CGRP was not caused by a reduction in the absolute workload, as this was actually increased from HA1 to HA5. CGRP levels were measured in venous plasma. Hematocrit levels increase during exercise, where the increase may be up to 10%. Hematocrit levels were not determined during exercise in this study, but were probably increased by the same factor in all three Wmax bouts. Thus, changes in hematocrit cannot explain the increase in CGRP values during exercise. It may be hypothesized that the synthesis of CGRP receptors is stimulated by hypoxia causing a decrease in the need for CGRP release. Studies indicate that CGRP receptors are highly expressed especially by vascular smooth muscle cells within the pulmonary vasculature. These receptors have been shown to be up-regulated by hypoxia [34]. Whether CGRP receptors are generally up-regulated in humans during hypoxia is not known. Several studies have shown that hypoxia induce increase of both adrenomedullin mRNA and protein expression in different cell cultures [35 –39]. Plasma levels were raised in experimental pulmonary hypertension [40], a consequence of staying at high altitude. Furthermore, Toepfer et al. [22] reported elevated plasma adrenomedullin concentrations at high altitude compared to sea level conditions. In contrast to Toepfer et al. [22], we do not find any increase in circulating adrenomedullin concentrations during hypoxia, but the study conditions were not the same. In the Toepfer study, the subjects climbed to the summit in 72 h, whereas, our subjects were passively airlifted directly to high altitude within 30 min. The adrenomedullin elevation in the Toepfer study may therefore be explained by the physical strain rather than by the hypoxia per se. It cannot be excluded that
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hypoxia-induced changes on a cellular level in the production of adrenomedullin remained undetectable in plasma from a peripheral vein. Changes in hematocrit can, to some degree, explain the difference in exercise-induced adrenomedullin production, in contrast to CGRP. 4.2. Sympathetic activity Intense exercise in man produces an increased blood flow through contracting skeletal muscle and an increase in heart rate, cardiac output and arterial blood pressure together with an increase in sympathetic nervous activity [41]. Since CGRP is one of the most potent endogenous vasodilators known, several studies have hypothesized that CGRP counteracts the sympathetic vasoconstrictors adrenaline and noradrenaline [10,11,42,43]. Thus, adrenaline infusion in humans increased the plasma concentrations up to 6.4 nM and resulted in CGRP release, whereas, CGRP levels were not affected by noradrenaline infusion [44,45]. In the present study, no significant changes in adrenaline concentration were found when comparing the Wmax values during normoxia and hypoxia, and the release pattern of noradrenaline was not identical to that of CGRP at Wmax. The dopaminergic D2-antagonist domperidone was administered to six subjects. At SL, HA1 and HA5, the domperidone group had significantly higher maximal noradrenaline concentrations compared to the control group [25]. However, this domperidoneinduced increase in noradrenaline did not affect the CGRP release either. Thus, our data do not support that CGRP release was mediated by adrenaline/noradrenaline, and no correlation between changes in circulating levels of catecholamines and CGRP could be found. 4.3. Lactate Lactate is released by exercising skeletal muscle cells as the oxygen requirements of working muscle cells exceed adequate oxygen delivery to the cells. In the present study, the subjects produced high concentrations of lactate. At Wmax, the release of lactate showed a similar pattern as observed for the CGRP release, namely a relative decrease in maximal concentration which was accentuated with time in hypoxia. A linear correlation was found between the increase in CGRP and lactate release, suggesting a relationship between the lactate production and the CGRP release. This is supported by animal studies showing that lactate and low pH induce release of CGRP in rats [12]. In conclusion, the exercise-induced CGRP release is, in contrast to adrenomedullin, modified by hypoxia. The exact signal for the decrease in CGRP release cannot be stated from this study, but lactate and low pH are candidates. Our results do not support the hypothesis that CGRP solely acts as a counter-regulatory mechanism to the sympathetic vasoconstrictor noradrenaline. Lactate formation and low pH in muscle tissue may be mediators of sensory nerve fiber activation and CGRP release.
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