Effects of respiratory stress on plasma prolactin concentration

Contents

1 Introduction

2 Methods
2.1 Subjects and protocols
2.1.1 Study A
2.1.2 Study B
2.1.3 Study C
2.2 Blood analysis and equipment
2.2.1 Prolactin
2.2.2 Lactate
2.2.3 Blood gases
2.2.4 Spirometer system
2.3 Data analysis

3 Result
3.1 Results study A
3.2 Results study B
3.3 Results study C

4 Discussion
4.1 Gas transport in the blood
4.2 Control of ventilation
4.2.1 Neurotransmitter control of respiration
4.2.2 Serotonergic influence on central respiratory activity
4.3 Acid-base equilibrium in brain .
4.4 Effects of hyperoxia on breathing
4.5 Ventilatory response to exercise
4.6 Prolactin .
4.6.1 Serotonin its implications on prolactin release .
4.6.2 Prolactin and stress

5 Summary

6 References .

7 Curriculum vitae

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1 Introduction

Biosynthesis of prolactin (PRL) occurs in anterior pituitary gland which releases PRL shortly after its formation into blood. The prominent target organ of PRL is the mamma with PRL dependent lactation. Long loop projections from suckle reflex and short-loop projection from pituitary gland to hypothalamus regulate PRL secretion. The synthesis and release of pituitary PRL are under a complex hypothalamic dual regulatory system that involves both prolactin-inhibiting factors (PIF) and prolactin-releasing factors (PRF) (Benker et al. 1990). Although hypothalamic control of PRL secretion is dominated by tonic inhibitory dopaminergic mechanism, a functional role of PRF appears to be necessary for acute secretory activities (Yen 1991). Hormones, such as e.g. estrogen, thyroid hormones and glucocorticoids are potent modulators affecting PRL synthesis, release or gene expression (Freeman et al. 2000). The most prominent excitatory neurotransmitter inducing PRL release is serotonin (5-HT) (Tuomisto and Manisto 1985; Van de Kar et al. 1989, Rittenhouse et al. 1993). Effects of 5-HT on PRL secretion were shown to be independent of DA. Especially serotonergic neurons originated in dorsal Raphe nuclei project to hypothalamus, thereby inducing 5-HT dependent PRL release from anterior pituitary (Clemens et al. 1978; De Meirleir et al. 1985).

PRL plays an important role for survival of the individual through its impact on reproduction and homeostasis in the organism. The role of PRL in reproduction has been widely investigated, whereas its homeostatic roles have not yet been examined systematically. PRL secretion is strongly affected by stress. As the response differs depending on the specific form of stress, one single mechanism of release cannot be defined. It has been shown before that stress induced by inhalation of high oxygen concentrations (>60 Vol.% O2) enhances PRL secretion (Str-der et al. 1999). It is also well established that exercise increases plasma PRL concentration depending on intensity and duration (DeMeirleir 1985, Fischer et al. 1991; 1992; Str-der et al. 1997). The underlying mechanisms regulating PRL secretion during these forms of stress are unknown. It appears that respiratory stress has an important impact on PRL secretion during oxygen inhalation as well as exercise. This assumption is based on findings showing that 5-HT regulates respiratory rhythm, possibly via serotonergic projections originating in the raphe nuclei (Bonham 1995).

The present study focuses predominantly on the relationship between neurosecretion of PRL and homeostasis. The aim of the present study is to expose healthy male humans to various forms of stress which induce different alterations in respiratory system and blood gases, and investigate changes in plasma PRL concentration. The forms of respiratory stress selected in this study were inhalation of increased oxygen concentration, inhalation of increased carbon dioxide concentration, voluntary hyperventilation as well as repeated high intensity exercise. It is hypothesized that hyperoxia (Haldane effect), short-term high intensive running exercise as well as inhalation of increased CO2 concentration (respiratory acidosis) cause an increase of CO2 partial pressure at the chemoreceptors in the brain. Thus compensatory mechanism ventilatory drive is augmented, based on the serotonergic system activation. This increase in serotonergic activity should also enhance PRL secretion. However, during augmented CO2 elemination caused by voluntary hyperventilation there should be a decrease in CO2 partial pressure without serotonergic system alterations. Consequently, plasma PRL concentration should not be affected.

Thus, primary purpose of the experiments was to investigate

- What alterations in blood gases and plasma PRL concentration occur during 45 min of inhalation of gas containing 80 Vol.% oxygen and 20 Vol.% nitrogen at rest?
- Does respiratory acidosis induced by four minutes of rebreathing 6 l of 7 Vol.% carbon dioxide in oxygen affect plasma PRL concentration?
- Does increased carbon dioxide removal induced by controlled voluntary hyperventilation over 6 minutes at rest increase plasma PRL concentration?
- Is the augmentation in plasma PRL concentration after repeated high intensity exercise related to carbon dioxide removal by compensatory respiratory mechanisms and lactate clearance?

2 METHODS

2.1 Subjects and protocols

2.1.1 Study A

Six healthy males (age: 24,7 ± 1,6 yr, weight: 80,8 ± 8,8 kg, height: 186,2 ± 7,2 cm) agreed to participate in this investigation. During examination, subjects reported at the laboratory at 8:30 a.m. after an overnight fast, and a catheter was placed into an antecubital vein. Baseline capillary and venous blood samples (0) were withdrawn after 30 min rest. Afterwards, subjects were exposed in resting state to 45 min of gas inhalation through a face mask. Gas concentration consisted of 80% O2/ 20% N2 at normal barometric pressure. Gas was inhaled directly out of a tank via a ZX3000 Nitrox regulator and octopus (SEAC SUB, Colombano Certolini, Italy). Further venous and capillary blood samples were drawn every 15 min during gas inhalation. Plasma PRL concentrations was measured in all venous blood samples. Arterial gases were analysed in all capillary blood samples.

Tab. 1: Individual values, mean and standard deviation of the anthropometrical data of the subjects participating in study A.

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The aim of the studies was to compare ventilatory response and PRL secretion induced by changes in arterial pCO2 during a hypercapnia hyperoxia test (B-1) and a voluntary hyperventilation test (B-2).

Test-Protocol B-1

The subjects reported to the laboratory at 10 a.m. A catheter was placed into an antecubital vene, followed by a rest period of 30 min during which subjects were breathing room air. Afterwards subjects were exposed to a rebreathing test according to Read (1967) to determine central chemoreflex responses to hyperoxia hypercapnia. The method by Read is based on the progressive rise of pCO2 leading to a mixed venous plateau followed by a gradually rise of PetCO2. During rebreathing, expired carbon dioxide is constantly returned to the lungs. Therefore carbon dioxide accumulates and the pCO2 rises progressively in blood, lungs and cerebrospinal fluid. As pCO2 rises at the chemoreceptors, the CO2 concentration develops a progressive carbon dioxide stimulus to ventilation. A high initial concentration of oxygen in the rebreathing bag provides oxygen for metabolism and prevents the development of any hypoxic stimulus to ventilation. The methode is used to maintain CO2 constant, thus alveolar CO2 tension is a good index of CO2 tension at medullary chemoreceptor.

In the present study, the rebreathing test consisted of three phases carried out in immediate order. The baseline period consisted of 5 min of stable ventilation while breathing room air through a spirometer mask. Venous blood samples were taken immediately before the beginning of the baseline period. The transition to the hypercapnia period was done by rapidly connecting the face mask to the 6 l reservoir bag. After a maximal expiration, the rebreathing was performed over 4 min through a face mask out of a bag containing initially 7% CO2 in 93% O2. Venous blood samples were drawn after 2 min and at the end of this period. The recovery period lasted 11 min during which subjects were breathing room air. Blood samples were taken each 2 min during this period.

Plasma PRL concentration was determinded in all venous blood samples. Heart rate was also measured at same time points with a puls tester (Polar, x-Trainer, Helsinki, Finland). Oxygen saturation was continuosly monitored with a pulse oxymeter (Nellcor Puritan Bennett, NPB 40, Ireland). Continuos records of respiratory rate, tidal volumen, end-tidal CO2 and minute ventilation were obtained from spirometric recording integrated in the ventil piece connecting the face mask with the bag.

Test-Protocol B-2

The subjects reported on the test day at the laboratory at 10 h. A catheter was placed in an antecubital vene. After a rest period of 30 minutes in which subjects were breathing room air, venous blood samples were drawn for baseline measurement of plasma PRL concentration and capillary blood samples for analysation of blood gases. The test consisted of two phases. First, voluntary hyperventilation was performed over 6 min at a given respiratory rate of 28-32 per minute. Afterwards subjects returned to normoventilation during a recovery period of 9 min. Venous blood samples for measurement of PRL as well as capillary blood gas measurements were take after 2 and 6 min of hyperventilation, as well as after 2, 4 and 9 min during recovery phase. Heart rate was recorded at same time points.

2.1.3 Study C

Ten males sprinters (age: 21,9 ± 4,7 yr, weight: 80,7 ± 6,9 kg, height: 185,4 ± 4,7 cm) were recruited to participate in the trial. The anthropometrical data is shown in table 3.

Tab. 3: Individual values, mean and standard deviation of the anthropometrical data of the subjects participating in study C.

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The runners were subjected on the treadmill to three 400 m sprints at an intensity corresponding to 95 % of maximal performance capacity (average speed: 6,2 m/s, 6,4 m/s, 6,7 m/s) with two minutes breaks in between. Blood samples were drawn for measurement of plasma PRL concentration before and inmediately after the last 400 m sprint. After 3 and 5 min of recovery phase further venous blood samples were taken. Capillary blood gas analysis was performed at same time points.

2.2 Blood analysis and equipment

2.2.1 Prolactin

In all experiments venous blood samples were drawn in pre-chilled EDTA-containing vacutainers, cooled for 10 min in ice water and then centrifuged with 3000 rpm for 10 min at 4°C. Plasma PRL was determined with the Enzyme-Imuno-Assay-Automate ES 300 instrument and corresponding kits from Boehringer (Mannheim, Germany)

2.2.2 Lactate

Lactate was analysed enzymatically (Gutman and Wahlefeld 1974) with lactate analyser 5060 (Eppendorf, Hamburg, Germany).

2.2.3 Blood gases

Capillary blood gases analysis was performed with a Blood-Gas-System 940 (AVL, Schaffhausen, Germany)

2.2.4 Spirometer system

In trial B-1 continuos records of tidal volumen, inspiratory and expiratory time, and breath-to-breath minute ventilation (Abbildung in dieser Leseprobe nicht enthaltene), breath frequency and end-tidal CO2 (PetCO2 ) were received from the spirometer recording of the Oxycon Alpha System (Jaeger, W-rzburg, Germany).

2.3 Data analysis

In all experiments multiple factorial analysis of variance with repeated measurements and Newmann-Keuls post-hoc test were used to assess differences between test units and with time.

Significance level for all analyses was set at p < 0.05. Data was presented as mean ± and standard deviation (SD).

3 Results

3.1 Results study A

In study A, pO2 significantly increased (p<0.01; table 1) after 15 min and remained elevated at 30 and 45 min of inhalation. At these time points, pH also significantly increased (p<0.05) towards alkalosis. Hyperoxia lead to decreased (p<0.05) arterial blood pCO2 after 15, 30 and 45 min, although an initial increase of pCO2 in cerebrospinal fluid (CSF) can be expected due to a physiological response to increased oxygen availability.

Tab. 4: Effect of 80% hyperoxia on arterial oxygen saturation (SaO2), partial pressure of oxygen (pO2), partial pressure of carbon dioxide (pCO2) and pH in healthy volunteers (n = 6) participating in study A. All values are means ± standard deviation. Significant difference to value at rest is indicated by ** for p<0.01 and by * for p<0.05.

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During recovery, minute ventilation and PetCO2 returned to baseline values. PetCO2 significantly decreased from 8,49 ± 0,72 to 4,68 ± 0,45 kPa (p<0.01). The respiratory rate increased significantly (p<0.01) from 14,15 ± 3,05 at the end of the HH period to 18,1 ± 4,9 breath/min, which caused a fall of PetCO2 (respiratory cleareance of CO2 ) (table 6). The Plasma PRL concentration increased from 11,59 ± 1,49 at the end of the HH to 13,63 ± 1,97 (p<0.01).

The heart rate did not change during the experiment. SO2 increased during hyperoxia hypercapnia exposition (p<0.01) and returned to basal value immediately during the recovery period.

Fig. 2: Effects of 4 min of CO2 rebreathing on plasma prolactin (PRL) concentration, minute ventilation (Abbildung in dieser Leseprobe nicht enthaltenE), breath frequency (BF) and partial pressure of carbon dioxide (pCO2) in healthy volunteers of study B-1 (n = 10).

In study B-2, during 6-minutes of voluntary hyperventilation significant increases in arterial pH and pCO2 occured (table 7). The respiration rate augmented (p<0.01) from 14,3 ± 3,03 to 30,1 ± 9,5 burst/min, thereby inducing an increase (p<0.01) of pH from 7,39 ± 0,04 to 7,58 ± 0.04 and an decrease (p<0.01) of pCO2 from 39,91 ± 2,62 mmHg to 21,73 ± 2,59 mmHg. With cessation of voluntary hyperventilation, pH and pCO2 returned to basal values. The arterial pH decreased (p<0.01) from 7,58 ± 0.04 to 7,4 ± 0,03 and the pCO2 increased (p<0.01) from 21,73 ± 2,59 mmHg to 36,90 ± 3,85 mmHg at end of the recovery period. SO2 increased (p<0.01) during hyperventilation and decreased during recovery below basal value. The respiration rate decreased (p<0.01) after hyperventilation to 13,7 ± 4 (burst/min). No significant changes in plasma PRL concentration were found throughout the trial.

Tab 7: Arterial blood gases and respiratory rate as well as plasma prolactin concentration at rest, after 2 (VH 2) and 6 (VH 6) min of voluntary hyperventilation (VH) and after 4 (RP 4) and 9 (RP 9) min of recovery period (RP) in healthy volunteers (n = 10). Partial pressure of carbon dioxid = pCO2, oxygen partial pressure = pO2, breathing frequency = BF, blood oxygen saturation = SO2, plasma prolactin concentration = PRL. All values are mean ± standard deviation.

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Fig. 3: Partial pressure of carbon dioxid (pCO2), breathing frequency (BF) and plasma prolactin concentration (PRL) at rest (0), during 6 min of voluntary hyperventilation as well as during 9 min of recovery in healthy volunteers (n = 10).

3.3 Results study C

During a treadmill test significant changes of the acid-base variables as well as plasma PRL concentration were found (table 8, figure 4). Plasma PRL concentration increased (p<0.01) during exercise from 9,4 ± 5,9 ng/dl to 29,0 ± 14,4 ng/dl. Capillary pH decreased of from 7,39 ± 0,02 to 7,06 ± 0,06 (p<0.01) while pCO2 decreased from 41,29 ± 2,19 mmHg to 34,38 ± 2,11 mmHg (p<0.01) at end of exercise. Capillary lactate increased (p<0.01) during exercise from 1,4 ± 0,58 mmol/l to 13,8 ± 2,06 mmol/l.

Further increases (p<0.01) in plasma PRL concentration were found during the recovery period. During this period, pCO2 significantly decreased (p<0.01).

Tab. 8: Partial pressure of carbon dioxid (pCO2), pH, capillary lactate (La), heart frequency (HF) and plasma prolactin (PRL) concentration at rest and during 5 min of recovery period (RP 0-5) immediately after exercise in healthy volunteers of study C (n = 10). All values are mean ± standard deviation.

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Fig. 4: Changes in plasma prolactin (PRL) concentration, pH and partial pressure of carbon dioxid (pCO2) after exercise in study C (n= 10).

4 Discussion

More than 300 separate biological actions of PRL have been reported. The most important functions of PRL are related to reproduction and homeostasis. The role of PRL in reproduction has been widely investigated, however, similar information is lacking about of the role of PRL in maintaining homeostasis. Homeostasis can be disturbed by ventilatory acid-base alterations. Homeostasis of blood pH and blood gas tensions depend on proper matching of ventilation to oxygen uptake and CO2 production. The effect of ventilatory acid-base alterations on PRL secretion has been investigated in the present study.

4.1 Gas transport in the blood

The hemoglobin in the red blood cells provides the major vehicle for oxygen transport in blood (97%). Oxygen dissolved in plasma is less important in terms of O2 transport (3%). Most carbon dioxide in the body is stored as bicarbonat (HCO3- ) in the bones (70%). The relative contribution of hemoglobin in carbon dioxide transport (carbaminohemoglobin) is less (23%). Free CO2 in plasma only amounts to about 7 %. A diagrammatic representation of CO2 transport in plasma and in the red blood cell is shown in figure 5. The hydration of carbon dioxide is much faster in red blood cells than in plasma because of the presence of carbonic anhydrase within the red blood cell. Hemoglobin is a weaker acid when deoxygenated, which facilitates the buffering of the hydrogen ion released during hydration. Bicarbonate ions leave the red cell, and chloride ions diffuse inward to maintain electrical neutrality (HCO3- /Cl- exchange) (Taylor et al. 1989).

The Christiansen-Douglas-Haldane effect (Haldane effect) was first described in 1914 (Christiansen et al. 1914). The Haldane effect is of physiological importance for carbon dioxide transport. It defines that the CO2 dissociation curve is affected by the degree of hemoglobin oxygenation. The reason for this effect is that the binding with oxygen makes hemoglobin acid, thereby reducing its affinity to carbon dioxide.

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