Growth hormone response to different consecutive stress stimuli in healthy men: is there any difference?

The contribution of growth hormone (GH), released during acute and repeated stressful situations, to the development of stress-related disorders is often neglected. We have hypothesized that the modulation of the GH response to sequential stress exposure in humans depends mainly on the nature of the stressor. To test this hypothesis, we compared GH responses to different stressful situations, namely aerobic exercise, hypoglycemia and hyperthermia, which were applied in two sequential sessions separated by 80-150 min. In addition, administration of the dopaminergic drug apomorphine was used as a pharmacological stimulus. GH responses to submaximal exercise (bicycle ergometer, increasing work loads of 1.5, 2.0 and 2.5 W/kg, total duration 20 min) and hyperthermia in a sauna (80 degrees C, 30 min) were prevented when preceded by the same stress stimulus. Hypoglycemia induced by insulin (0.1 IU/kg intravenously) resulted in a significant GH response also during the second of the two consecutive insulin tests, though the response was reduced. Administration of apomorphine (0.75 mg subcutaneously) or insulin prevented the increase in GH release in response to a sequential bolus of apomorphine, while hypoglycemia induced a significant elevation in GH levels even if applied after a previous treatment with apomorphine. In conclusion, the feedback inhibition of the GH response to a sequential stress stimulus depends on the stimulus used. Unlike in the case of exercise and hyperthermia, mechanisms involved in the stress response to hypoglycemia appear to overcome the usual feedback mechanisms and to re-induce the GH response when applied after another stimulus.

Xylazine activates oxytocinergic but not vasopressinergic hypothalamic neurons under normal and hyperosmotic conditions in rats.

Role of central alpha2-adrenoceptors in the regulation of hypothalamic magnocellular cells was studied under hyperosmotic challenge elicited by hypertonic saline (HS). Rats pretreated with receptor agonist, xylazine (XYL), were injected intraperitoneally with different (low: 0.375, moderate: 0.75, high: 1.5 M) HS 30 min later. The activity of the paraventricular (PVN) and supraoptic (SON) vasopressin and oxytocin perikarya was established by Fos-dual-immunohistochemistry 60 min after HS administration. Results showed that 1/XYL is a potent stimulus for oxytocin but not vasopressin magnocellular cells under basal and weak hyperosmotic conditions 2/highHS completely overlaps the effect of XYL. In addition, XYL partially suppressed Fos expression in the parvocellular PVN cells activated by highHS. The data suggest that alpha2-adrenoceptors may play an important role in the regulation of oxytocinergic PVN and SON neurons under basal and weak hyperosmotic conditions and that alpha2-adrenoceptors may also participate in the control of PVN parvocellular cells under intense osmotic challenge.

Neuroendocrine changes in adult female rats prenatally exposed to phenytoin.

Neuroendocrine changes in fetal hydantoin syndrome have not been described yet. This study was aimed to verify the hypothesis that prenatal exposure to phenytoin influences the stress response of adult female offspring in an animal model. To study possible development of depression like state, hedonic behavior and long-term changes in neuropeptide gene expression in the hypothalamus were investigated. Treatment consisted of per os administration of 150 mg/kg of phenytoin or water daily, from day 7-18 of gestation. Adult female offspring (6 animals per group) were acutely stressed by 1 min handling. Blood was collected in conscious rats via tail artery cannulas before, 1, 15 and 30 min after the handling. Exposure to phenytoin in uterus resulted in increased catecholamine and corticosterone concentrations in response to a mild stressor of 1 min handling in adult offspring. The gestational treatment used in this study did not induce a depression like state nor long-term changes in neuropeptide gene expression in the adult offspring. In conclusion, prenatal exposure to phenytoin treatment enhanced the stress response of adult female offspring. Possible new component of fetal hydantoin syndrome is the increase in catecholamine release in response to a mild stressor in adulthood.

Involvement of glutamate neurotransmission in the development of excessive wheel running in Lewis rats.

Physical activities such as long-distance running can form a habit and might be related to drug-induced addictive behaviors. We investigated possible modulations of N-methyl-D-aspartate (NMDA) receptor subunits during voluntary wheel running in brain regions implicated in reward and addiction. It was observed that Lewis rats progressively increased their amount of daily running, reaching maximum levels of 4-6 km/day. After 3 weeks of running, mRNA levels coding for NR2A and NR2B subunits were increased in the ventral tegmental area, while only NR2A mRNA levels were found to be elevated in the frontal cortex. Long-term wheel running was also associated with increased binding of specific NMDA receptor antagonist [3H]CGP39653 in the frontal cortex. Moreover, pharmacological inhibition of glutamate release by repeated administration of phenytoin (20 mg/kg IP for 21 days) significantly suppressed daily running. These results suggest that glutamatergic neurotransmission might be related to neurobiological mechanisms underlying the compulsive character of voluntary wheel running.

Reduction of rise in blood pressure and cortisol release during stress by Ginkgo biloba extract (EGb 761) in healthy volunteers.

The standardized extract of Ginkgo biloba (EGb 761) was found not only to improve memory and aging associated cognitive deficits but also to exert beneficial effects on mood. An antistress action of the extract has been suggested but not directly proven. The present study was aimed to evaluate the effects of EGb 761 on salivary cortisol and blood pressure responses during stress in healthy young volunteers (n = 70) in a double blind placebo controlled design. A stress model involving a combination of static exercise (handgrip) and mental stimuli was used. Single treatment with EGb 761 (120 mg) reduced stress-induced rise in blood pressure without affecting the heart rate. Salivary cortisol responses showed differences with respect to the gender and the time of day of the stress exposure, with the activation only in male subjects in the afternoon. This activation was absent if they were treated with EGb 761. The performance in a short memory test with higher scores achieved by women remained unaffected by EGb 761 treatment. Thus, this study provides evidence that EGb 761 has an inhibitory action on blood pressure and it may influence cortisol release in response to some stress stimuli.

Nitric oxide synthase mRNA levels correlate with gene expression of angiotensin II type-1 but not type-2 receptors, renin or angiotensin converting enzyme in selected brain areas.

Recent data suggest that there is interaction between peripheral angiotensin II and nitric oxide. However, sparse information is available on the mutual interaction of these two compounds in the brain. The potential intercourse of nitric oxide with brain neuropeptides needs to be substantiated by assessing its local production and gene expression of the synthesizing enzymes involved. The aim of the present study was to evaluate whether the gene expression of brain nitric oxide synthase (bNOS) is related to the sites of gene expression of different components of the rat brain renin angiotensin system (renin, angiotensin converting enzyme (ACE) or angiotensin receptors of AT1 and AT2 subtypes). The levels of corresponding mRNAs were measured and correlated in nine structures of adult rat brain (hippocampus, amygdala, septum, thalamus, hypothalamus, cortex, pons, medulla and cerebellum). As was expected, positive correlation was observed between renin and angiotensin-converting enzyme mRNAs. Moreover, a significant correlation was found between brain NO synthase and AT1 receptor mRNAs, but not with mRNA of the AT2 receptor, ACE and renin. Parallel distribution of mRNAs coding for bNOS and AT1 receptors in several rat brain structures suggests a possible interaction between brain angiotensin 11 and nitric oxide, which remains to be definitely demonstrated by other approaches.

Changes in angiotensin AT1 receptor mRNA levels in the rat brain after immobilization stress and inhibition of central nitric oxide synthase.

OBJECTIVE: To study functional interactions between angiotensin II AT1 receptors and nitric oxide (NO) activity in different brain areas in rats exposed to immobilization stress. METHODS: Central inhibition of nitric oxide synthase (NOS) was provided by intracerebroventricular (i.c.v.) administration of (N-omega-nitro-L-arginine-methylester) L-NAME and analysis of AT1 receptor mRNA was performed using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. The immobilization in prone position lasted 2 hrs and the rats were sacrificed 24 hr later. The hypothalamus, hippocampus, thalamus, and cortex were isolated from fresh brains. RESULTS: In the cortex, gene expression of AT1 receptors was unaffected either by L-NAME treatment, or by a single exposure to immobilization stress for 2 hours followed by 24 hours of rest. In the hippocampus, the repeated treatment with L-NAME increased mRNA levels of AT1 receptors approximately 9-times compared to those in the control (untreated) group. Immobilization also increased AT1 receptor mRNA levels in the hippocampus which was similar to that induced by the L-NAME. The increase of AT1 receptor mRNA levels in the hippocampus of immobilized rats was not further altered when the animals were pretreated with L-NAME. In control rats, exposure to immobilization resulted in a significant rise in mRNA levels coding for AT1 receptors in the hypothalamus, but not in the thalamus. L-NAME treatment showed a tendency of increase in AT1 receptor mRNA levels in the hypothalamus. Moreover, when animals treated with L-NAME were subjected to immobilization, a further increase in AT1 receptor mRNA levels was observed in the hypothalamus in comparison with corresponding controls. CONCLUSIONS: The present data indicate that a single immobilization stress results in increased gene expression of AT1 receptors in the hypothalamus and hippocampus. The rise in AT1 mRNA levels in the same brain structures after repeated treatment with L-NAME allow to suggest an interaction between the central angiotensin II and nitric oxide.

Effect of nitric oxide inhibition on blood pressure and corticosterone responses in adult rats neonatally treated with glutamate.

The role of nitric oxide (NO) in the regulation of blood pressure and hypothalamic-pituitary-adrenal function of adult rats treated with monosodium glutamate (MSG) during the neonatal period was investigated. Blood pressure and the heart rate were registered by a computerized system of direct blood pressure measurement through an indwelling cannula in the femoral artery. The inhibition of the activity of NO synthase by acute injection of Nomega-nitro-L-argininemethylester (L-NAME, 30 mg/kg, i.v.) to control rats produced a rise of blood pressure and a fall of heart rate. Both responses were reduced in MSG-treated rats. Repeated administration of L-NAME (50 mg/kg, i.p, two times daily for 4 days) increased BP in both groups of animals. Corticosterone concentrations in the plasma were significantly increased in response to repeated L-NAME administration in MSG-treated rats, while ACTH levels were similar in both groups of animals. These data suggest that some of the cardiovascular and endocrine changes in rats treated with MSG may be due to the abnormal function of the NO system.

Studies on the physiological role of ANF in ACTH regulation.

To investigate the impact of changes in the level of the endogenous atrial natriuretic factor (ANF) on pituitary-adrenocortical activity, the secretion of corticotropin (ACTH) and corticosterone was studied under the conditions of enhanced and decreased circulating ANF levels in rats. Volume expansion (intravenous infusion of 5 ml of saline within 2 min) induced significant elevation in ANF levels 5 min after the infusion, whereas ACTH levels remained unchanged during the first 20 min and were elevated only at 40 min, i.e. at the time when ANF levels were again normal. Water deprivation for 48 h resulted in decreased ANF levels and increased corticosterone concentrations. ANF concentrations in peripheral blood obtained under thiopental anesthesia were lower than those in blood sampled in the same rats in conscious state. However, such changes were not observed in water deprived animals. In addition, ANF was found to be present in the hypophysial portal blood of anesthetized rats. In conscious sheep, portal ANF levels were significantly higher than those in peripheral blood. Our results support the suggestion of an inhibitory role of ANF in the control of ACTH release and indicate that this role of ANF is physiologically relevant.

Activity of the hypothalamic pituitary adrenal axis and sympathoadrenal system during food and water deprivation in the rat.

It has been previously shown that chronic water deprivation or hypertonic saline intake, osmotic stress models with concomitant decrease in food intake, decrease hypothalamic CRH mRNA levels and ACTH responses to acute stimulation. To determine the contribution of food restriction to the effects of osmotic stimulation, the function of the hypothalamic pituitary adrenal axis was analyzed in rats subjected to food deprivation, water deprivation or their combination for 60 h. In all three groups, basal levels of plasma corticosterone were increased, while ACTH and catecholamines were unchanged. Basal plasma vasopressin levels were normal in food deprived rats, but significantly increased in water deprived and simultaneously food and water deprived rats. In contrast to the 25% reduction of plasma ACTH responses to 30 min immobilization by water deprivation, food deprivation had no inhibitory effect and prevented the decreased ACTH responsiveness caused by water deprivation. In control rats, plasma corticosterone levels increased 22.5-fold 30 min after immobilization, and this response was significantly potentiated in the water deprived, food deprived and combined food and water deprived groups. The elevation in plasma catecholamines in response to acute immobilization was also enhanced in both water deprived and food deprived rats. In situ hybridization studies showed a 35% increase in VP mRNA levels in the PVN after water deprivation, whereas food deprivation caused a slight decrease and prevented the stimulatory effect of water deprivation. CRH mRNA in the PVN was reduced by 27% after food deprivation and by 67% after water deprivation, but simultaneous food and water deprivation caused a significantly smaller reduction similar to that in food deprivation alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex differences in endocrine response to hyperthermia in sauna.

Neuroendocrine response was investigated during and after a single 20 min bath in sauna (80 degrees C) in a group of 8 healthy men and 8 healthy women. In an additional group of 8 young men, the dynamics of plasma ACTH and cortisol levels were studied during a 30 min sauna exposure (90 degrees C). This dynamic study showed a biphasic response of plasma cortisol which decreased during the initial phase of sauna bath (15 min) and increased thereafter, reaching its maximum 15 min after the end of bathing. Maximal increase in plasma ACTH levels occurred 15 min earlier. In the first sauna exposed group the increase in body temperature was the same (about 2 degrees C) in both sexes. Nevertheless, the elevation in plasma ACTH concentration was significantly more pronounced in women than in men. In the plasma collected at the end of sauna bath inside the sauna room, a significant rise in both adrenaline and noradrenaline levels was found. Though the catecholamine responses were similar in both groups, the increase in heart rate during sauna bath was significantly higher in women. Sauna-induced prolactin release was also more pronounced in women compared with men. Thus hyperthermia induced by sauna exposure resulted in a more pronounced neuroendocrine activation in women compared with men. Moreover, it is evident that repeated blood sampling is necessary to reveal the sauna-induced activation of some hormonal systems.

Albumin content in the developing rat brain in relation to the blood-brain barrier.

The problem of functional development of the blood-brain barrier (BBB) has been the subject of intensive discussion for many years. Some morphological data indicate that the cerebral endothelium becomes impermeable to macromolecules already before birth. On the other hand, specific features in transport of macromolecular markers in fetuses and neonates were described supporting the hypothesis of an immature BBB. Part of the confusion seems to result from inadequately designed experiments, concerning frequently the administration of markers. In our study we decided not to use an exogenous marker and to follow up to changes of endogenous albumin content in selected brain regions of rats from the neonatal period to adulthood. Shortly after birth, the concentrations of albumin in all brain regions studied (hypothalamus, hippocampus, cortex, striatum, brainstem, cerebellum) were significantly higher compared to those of adult animals. Moreover, the distribution of albumin within these structures in newborns differed from that in adults. Several factors may contribute to the accumulation of albumin in the developing rat brain, including increased permeability of the BBB.

Vasopressin and 1-deamino-8-D-arginine-vasopressin (DDAVP) reduce elevated plasma catecholamine levels in rats with hypothalamic deafferentation.

1. Anterolateral cut (ALC) of the medial basal hypothalamus (MBH) in rats produces an elevation of plasma catecholamine levels, especially of norepinephrine (NE), in unstressed animals and a more pronounced rise of plasma NE levels in response to immobilization (IMO). Animals with ALC have a destroyed corresponding vasopressin (AVP) and other peptides containing innervation of the median eminence and the posterior pituitary, resulting in the prevention of increased AVP secretion during the early intervals of IMO. 2. The administration of AVP (Pitressin, 7 days, 1 IU per rat i.m.) or of 1-deamino-8-D-arginine-vasopressin (DDAVP), an AVP analogue without pressoric activity, taken in drinking water (about 100 micrograms per day) was almost equally potent in decreasing the elevated water consumption and plasma NE levels in unstressed rats with ALC. However, the stress-induced potentiation of plasma NE levels in rats with ALC was not influenced by AVP substitution and only partly reduced by DDAVP in the late IMO intervals. 3. The lack of circulating vasopressin is the main factor in the mechanism of increased activity of the sympathoadrenal system induced by ALC in unstressed rats. 4. The regulation of sympathoadrenal activity by vasopressin and DDAVP in rats with ALC seems to be mediated predominantly by V2-subtype receptors. 5. In stressed rats with ALC the potentiation of plasma NE levels was not reduced after AVP or DDAVP administration, suggesting that some addition regulatory mechanisms were involved.

Impaired glucose utilization in man during acute exposure to environmental heat.

In 6 healthy males the oral glucose tolerance test (OGTT) was performed after the administration of 100 g glucose during the hyperthermic Finnish sauna bath (85 degrees C) of 30 min duration. The lowered insulin response (P less than 0.001) to glucose challenge during heating and the subsequent prolonged hyperglycemia (P less than 0.001) after heating were observed, when compared to OGTT under thermoneutral conditions (23 degrees C). It is suggested that the heat-induced decrease in visceral blood flow and stimulation of sympathoadrenomedullary and pituitary activity may be responsible for this effect.