It has been well established that serum prolactin (PRL) concentration increases after exhaustive exercise (5,14); however, the stimulatory mechanism and the physiological function remain unknown. PRL is synthesized in the lactotrope cells by the anterior pituitary. In humans, release of PRL is subject to the action of dopamine as the main inhibitor (1,19). Increased dopamine synthesis and metabolism were shown during and after exercise (17), suggesting that exercise-induced PRL release is not related to alterations in the dopaminergic system activity and PRL-releasing factors (PRF) are responsible for the acute secretory activities. Of the hypothalamic, systemic, and local factors acting as PRF, none has yet emerged as a primary releasing factor. Serotonin (5-HT) is one of the most prominent excitatory neurotransmitters for PRL release (15,28). Serotonergic neurons originating in the dorsal raphe nuclei project via pathways to the hypothalamus, inducing PRL release from the anterior pituitary (4). The physiological role of 5-HT on PRL secretion has been widely used as a hormonal probe for the 5-HT activity (24).
Abundant evidence exists that serotonergic mechanisms are involved in the control of pH homeostasis within the medulla (20), and the most well-established effect of 5-HT is the activation of respiratory control through an augmentation of phrenic motoneuronal activity (3). Serotonergic neurons send axons to many regions of the medulla and spinal cord. The axons terminate directly at or in close proximity to the phrenic motoneurons (18) and central respiratory neurons (11,32). Direct evidence of in vivo actions of 5-HT comes from studies in cats in which serotonergic dorsal raphe cells displayed increased firing in response to hypercapnia acidosis (31). Wang et al. (33) have shown that 5-HT in the medullary raphe within the ventromedial medulla plays a role in chemosensitivity. It was demonstrated that the acidosis-stimulated neurons in the medullary raphe are serotonergic (34). Recently, Rojas Vega et al. (21) reported that plasma PRL concentration increases after respiratory acidosis at rest and suggested that chemosensitivity-related 5-HT system activation might have had the collateral effect of causing PRL release.
As it was shown that 1) serotonergic neurons are sensors of pH and respond to acidosis with respiratory system activation, 2) 5-HT is also the main PRF, and 3) respiratory acidosis causes an augmentation of PRL secretion at rest, it can be hypothesized that via metabolic acidosis chemosensitivity-related 5-HT system activation might have the collateral effect of causing PRL release after exhaustive exercise. Thus, the aim of the present study was to investigate whether buffering by infusion of bicarbonate reduces postexercise serum PRL concentration.
Seven healthy males (age: 25.4 ± 1.6 yr; height: 177.8 ± 7.5 cm; weight: 74.6 ± 8.6 kg) were recruited for the study, which was approved by the local ethics committee. The subjects were recreational athletes performing the following activities: jogging, cycling, soccer, and basketball. They were informed about the aim of the study and received a written detailed explanation about all tests, potential discomforts, risks, and procedures employed in the investigation, after which the subjects gave their written consent. The subjects were medically screened by a physician before participation. The examination of the subjects included medical history, blood analysis, and resting ECG. All subjects were in good health and free of any evidence of renal dysfunction, cardiac abnormality, respiratory disease, or any other physical condition placing them at risk for participation in the study. During the preexamination, each subject also performed a cardiopulmonary exercise test (CPX) consisting of incremental exercise on a cycle ergometer (Ergoline 9500) with 40-W steps each 5 min. A 12-lead ECG and blood pressure were recorded on a ZAN 680 Ergospirometer system (Oberthulba, Germany) throughout the test. No evidence of cardiopulmonary limitations was found in any of the examined volunteers. The subjects reached a peak power output of 310 ± 43.6 W or 4.3 ± 0.5 W·kg−1, respectively. The power output at the anaerobic threshold at 4 mmol·L−1 blood lactate (LA) was 212.3 ± 52.5 W. To minimize exaggerated hormonal response due to novel stress condition in the main trials, the subjects were familiarized with all test procedures and all employed equipment before their arrival on the first test day.
The subjects refrained from strenuous physical exercise for 2 d before each experimental session. At the time that each of the main trials was scheduled, the subjects had followed an individually identical balanced diet for 1 wk, had received a standardized vegetarian meal with water ad libitum at 9 p.m. the night before test days, had fasted overnight, and had received a standardized breakfast (one roll with 25 g of cherry jam, 300 mL of decaffeinated fruit tea with 0.02 g of dextrose-saccharin sweetener) ≥ 2 h before arrival. The subjects were advised to avoid coffee as well as caffeinated and/or carbonated beverages for 24 h prior to testing. After arrival, one catheter was placed in a superficial antecubital vein in both the right and left arms. The catheters were kept patent by 1 mL of normal saline, and the subjects rested for 30 min before an initial sample (5 mL) was obtained. Additional capillary samples from the earlobe were collected for blood gas and LA analysis. A continuous infusion with the placebo (normal saline; PL) or the isotonic sodium bicarbonate (BIC) followed. Subjects were not informed about the order of the treatments. Normal saline was always administered in the first trial while in the second trial buffering was then achieved by the necessary amount of BIC in mEq calculated from (base excess in mEq·mL−1 at the end of the ramp exercise test with placebo) × (body weight in kilograms) × (0.2 L·kg−1 body weight of extracellular fluid). The administered dose was determined according to suggestions by Shapiro et al. (22) for treatment of metabolic acidosis. The mode was chosen on the basis of the predictable pharmacokinetic and to avoid acute gastrointestinal distress. The rate of administration was adjusted so that two thirds of the calculated volume was infused during 40 min preexercise and one third was administered during the ramp exercise test. With a time interval of 1 wk, all subjects underwent the main trials at the same time of day, with exercise beginning between 9 and 11 a.m. The venous blood samples were drawn from the catheter in the right arm while the infusion was administered in the left arm. The fixed times for the collection of capillary and venous blood samples were at baseline before start of infusion, immediately at the termination of infusion at rest, after a 10-min warm-up period on the cycle ergometer, at maximum exertion during the ramp exercise test, and at 3, 6, 10, and 15 min postexercise.
In the trials, the subjects were exposed to a ramp-fashioned exercise on a cycle ergometer with spirometric measures and a PL or BIC infusion. The exercise tests were identical in both ramp test sessions. Each subject's individual seat and handlebar heights were reproduced on a SRM cycle ergometer (Jülich, Germany) during the tests. After the subject was given instructions and the feet were secured in the pedal straps, a 10-min warm-up period followed at a power of 2 W·kg−1 body weight during which the pedal rate was 70 rpm. At the end of the warm-up period, the subjects proceeded directly with the ramp exercise test. This test consisted of an initial 2 min of cycling at 2 W·kg−1 body weight followed by incremental exercise to exhaustion with 25-W steps every 30 s. It was ensured that subjects had reached maximal aerobic capacity by fulfilling the following criteria: reaching HRmax (220 − age in years), RPE > 17, increase of RER > 1.1, ratio of respiratory minute volume in milliliters and the oxygen uptake during that same minute > 30-35, no further increase of the quotient of oxygen uptake in milliliters and pulse rate during the same minute, and increase of blood LA concentration > 8 mmol·L−1 (10). After exhaustion was reached, an active recovery period of 15 min followed during which the subjects pedaled in a free-wheel fashion solely against the intrinsic frictional resistance of the ergometer. In both trials, a 12-lead ECG and spirometric measurements were obtained by a ZAN 680 spirometer during the warm-up, ramp exercise test, and recovery period. Additionally, HR was continuously recorded with a HR monitor (Polar x-Trainer, Helsinki, Finland). Ventilatory measurements during exercise were obtained on breath-by-breath basis, averaged, and reported for 10-s intervals.
Blood BIC concentration (HCO)3−), base excess (BE), pH, carbon dioxide pressure (PCO2), and partial pressure of oxygen (PO2) were analyzed in arterialized blood of the earlobe using the blood gas analyzer AVL OPTI CCA. The earlobe was warmed by manual massage to obtain arterialized blood. A deep puncture was made with a lancet so that a free flow of blood exuded from the wound without squeezing the area. A preheparinized capillary tube (200 μL) was used to collect the blood and placed deep into the drop of blood. To maintain the anaerobic conditions, the sample was immediately inserted without air bubbles into the electrode chambers for the blood gas measurement. The acid-base status of arterialized capillary blood from the earlobe shows a consistent correlation with arterial blood in well-perfused individuals (22).
Capillary LA concentration was measured in duplicate using the LA analyzer Biosen 5130 EKF (Magdeburg, Germany). By means of calibrated 20-μL one-time micropipette (BRAND, Hagen, Germany), capillary blood samples were extracted from the earlobe, placed in 2-mL Safe-Lock vessels, and blended manually with its 1000-μL system solution. The analysis of the samples was based on the enzymatic-amperometric measuring method and was administered with the help of the technical device Biosen C line (EKF-diagnostic, Barleben, Germany). The technical device was equipped with EKF chip sensors that transform the sample concentration into evaluable electrical signals.
Venous blood samples (5 mL) were drawn in prechilled serum venipuncture tubes. Serum was separated by centrifugation (3000 rpm for 10 min at 4°C) and stored in Eppendorf tubes at -70°C. Serum PRL concentrations were analyzed in duplicate using the ES 300 analyzer and enzyme-linked immunosorbent assay (ELISA) kits by Boehringer (Germany). The detection limit of the ELISA for PRL was within a range of 0.47-470 ng·mL−1. For the respective concentrations, the intraassay coefficient of variation was 2.8% (mean 3.4 ng·mL−1) and 2.5% (mean 30.9 ng·mL−1).
Data are presented as mean ± SD. Multifactorial repeated-measures ANOVA was applied for statistical analysis of the data using the software program easystat 2.0 (Lüpsen, Cologne, Germany). This program automatically carries out sphericity test for homogeneity of variances. In the case of nonhomogeneity of variances, multiple comparison of means is done with Bonferroni adjustment. Significance levels for all analyses were set at P < 0.05.
Power output, maximal oxygen uptake, HR, and LA.
Peak power output during the ramp test did not differ between PL (442.9 ± 55.4 W; 5.9 ± 0.6 W·kg−1) and BIC (446.4 ± 66.8 W; 6.0 ± 0.5 W·kg−1) trial. Maximal oxygen uptake (PL: 53.4 ± 8.8 mL·kg−1·min−1; BIC: 55.5 ± 5.2 mL·kg−1·min−1) and exercise duration to exhaustion (PL: 7.5 ± 1.0 min; BIC: 7.5 ± 1.4 min) were not affected by infusions. HR and LA concentration (Fig. 1) did not differ between trials at any point in time. After warm-up, the HR was significantly (P < 0.01) augmented in PL to 134.3 ± 9.7 bpm and in BIC to 135.9 ± 7.1 bpm, whereas LA had not changed from concentration at rest. In both trials, HRmax (PL: 190.3 ± 8.5 bpm; BIC: 196.1 ± 11.3 bpm) was reached at exhaustion, whereas maximal LA concentrations were found 3 and 6 min postexercise.
Capillary HCO3−, BE, pH, pCO2, and pO2 are shown in Figure 1. Prior to the start of infusions, there were no significant differences between the trials in any of the blood gas parameters. Significantly increased (P < 0.01) capillary HCO3−, pH, and BE were found at the beginning of the warm-up in BIC. Exercise induced a significant (P < 0.01) decrease in capillary HCO3−, pH, BE, and pCO2 at exhaustion and during the recovery period in both trials. In both trials, the lowest values of HCO3− and BE were found between 3 and 10 min of the recovery period. Significantly higher HCO3−, pH, and BE were found during BIC infusion and postexercise in comparison with the PL trial. Capillary pO2 did not change from resting values, but an increase (P < 0.05) was found from the end of warm-up to 3 min of the recovery period. PCO2 and PO2 did not differ between trials at any point in time.
Serum PRL concentration (Fig. 2) did not change in BIC during the increase of HCO3−, pH, and BE at rest and during moderate exercise in the warm-up period. Serum PRL concentration was significantly increased 3 min after the ramp test until end of PL, whereas in BIC, serum PRL concentration did not change from values at rest. Significant (P < 0.01) differences between PL and BIC in serum PRL concentration were found 10 and 15 min postexercise (Fig. 2).
Several studies provide evidence for a physiological role of pH in hormonal responses to exercise, for instance, for β-endorphin (26) and growth hormone (9), suggesting an impact of acidosis on the pituitary. Anaerobic exercise is also associated with hyperprolactinemia. In many areas, PRL is released from the anterior pituitary from cells that are highly interspersed with growth hormone secretory cells. Also secretory stimuli were found to be similar. Although earlier studies showed a relationship between exercise intensity and acute PRL secretion and suggested that acidosis might play a role (5,14,24), the mechanisms responsible for the augmented PRL secretion are not well understood. During heavy exercise, anaerobic metabolism induces lactic acidosis, and blood pH regulation depends mainly on the HCO3− concentration (25). The buffering of hydrogen ions with BIC and the increased ventilatory effort minimizes the degree of acidemia. Therefore, the contribution of these regulatory mechanisms is considered to be of functional significance in maintaining homeostasis (6,27). However, blood pH is not only regulated by HCO3−, and the activity of LA/H+ cotransporter proteins such as the monocarboxylate transporters may also play an important role (12). The recovery phase up to about 3 min after exercise is characterized by the increase in blood LA concentration and the nadir of pH and BIC. Hyperventilation and the reaction catalyzed by the carbonic anhydrases then remove CO2 to restore the pH baseline value. It was demonstrated that serotonergic neurons are sensors of pH (20,33), and the primary serotonergic response to acidosis consists of an activation of respiratory function (3). However, 5-HT also plays a prominent role in the acute release of PRL through pathways that project from the medullar raphe to the hypothalamus (30), and thus chemosensitivity-related 5-HT system activation might have the collateral effect of causing PRL release.
BE is the best indicator of the actual metabolic situation, and the normal values are considered to be within 3 mmol·L−1. A BE deficit ≥ 10 mmol·L−1 denotes a clinically significant metabolic acid base imbalance, and the H+ concentration exceeds the normal buffering capacity of the body (22). The present data suggest that deviation from the normal buffer base status triggers PRL release in humans. Previously, Elias et al. (7) investigated the effect of pH on PRL release during an incremental exercise test to exhaustion. Contrary to our findings, the authors observed a higher PRL secretion after alkali administration in comparison with PL treatment. However, this study only showed augmented PRL concentrations 60 min postexercise did not include any measurements between cessation of exercise and 15 min postexercise, and no blood gas analysis was carried out at the time points when augmented PRL was found. Furthermore, the training status of the subjects was lower than in our study (V˙O2max: 3.3 ± 0.2 vs 3.9 ± 0.8 L·min−1), which might have affected the results. Some studies reported that the exercise-induced increase in PRL is higher in better-trained subjects, and that as a result of training, the release of PRL is enhanced or starts at lower exercise intensities (29).
The data of the present study are in accord with earlier findings by Rojas Vega et al. (21), which demonstrated that acute PRL increase can be induced by changes in acid-base status at rest. Plasma PRL response in a rebreathing test (subjects inhaled 6 L of a gas mixture comprising 7% by volume of carbon dioxide and 93% by volume of oxygen from a respiration bag through a face mask for 4 min; unpublished data, 2005) was also affected by the training status of the subjects. After a 10-wk endurance training program (3× wk−1), subjects showed an earlier increase of plasma PRL concentration and an earlier maximum value compared with pretraining. It can be speculated that these alterations are related to increased sensitivity of chemosensitivity receptors for CO2 and an improved responsiveness of the serotonergic system. The present investigation suggests that in humans exercise-induced metabolic acidosis also causes PRL secretion, implying that the stimulus for the PRL secretion is probably a decrease in pH. The differences in plasma PRL concentration between the trials of the present study are not related to an alteration in the blood oxygenation level, which could be expected because the affinity of hemoglobin to oxygen is dependent on the pH. However, PO2 did not differ between BIC and PL. Also, PO2 did not exceed 100 mm Hg at any point in time. It was shown earlier that a PO2 increase greater than 150 mm Hg seems to be necessary for an effect on plasma PRL concentration (23). Interestingly, a study assessing the involvement of a chemiosmotic mechanism in exocytotic PRL release of dispersed anterior pituitary cells from female rats revealed that BIC ions significantly increased the basal PRL release, and higher concentrations of BIC seemed to favor secretory granule lysis, indicating a possible interaction of this ion with the granule content (2). However, in this study PRL remained unaffected by pH changes within physiological ranges, and caution must be applied when transferring these findings to humans due to the differences in the physiology of PRL between rats and humans (1).
Whereas the physiological function of PRL in the induction of maternal lactogenesis is well established, the physiological function of PRL secretion in males and during exercise is not known. The present data indicate an involvement of PRL in compensation mechanisms of acidosis. Future studies should investigate whether PRL participates in the active transport of ions between cells and extracellular fluid following acidosis because ion exchange involves hormonal regulation in other parts of the body (8). Furthermore, PRL receptors have been identified in circumventricular organs of the brain and also in the area postrema, a chemosensitive area, supporting the notion that PRL may exert an effect on regulation of osmotic and electrolyte balance (13,16).
In summary, the new finding of the present study is that buffering of metabolic acidosis reduces the increase of serum PRL concentration induced by exercise to exhaustion in male recreational athletes. Serotonergic pathways are involved in maintaining homeostasis under metabolic acidosis due to the crucial role of this neurotransmitter in chemosensitivity. As 5-HT is also a PRF, it is suggested that acidosis might cause the increase in serum PRL concentration after exercise to exhaustion via serotonergic system activation.
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