Glucocorticoid Negative Feedback Research Articles

A role for epidermal growth factor in the morphological and functional maturation of the adrenal gland in the fetal rhesus monkey in vivo.

We determined the effects of epidermal growth factor (EGF) and beta-methasone on the growth and development of the adrenal gland of the fetal rhesus monkey in vivo between 121-128 days of gestation. The adrenal to body weight ratio was significantly greater (P < 0.05) in EGF-treated fetuses (0.988 +/- 0.046 x 10(-3) g/g) and significantly reduced (P < 0.05) in beta-methasone-treated fetuses (0.401 +/- 0.056 x 10(-3) g/g) compared with that in control fetuses (0.689 +/- 0.050 x 10(-3) g/g). The increase in adrenal weight with EGF administration was due to hypertrophy of definitive zone cells of the adrenal cortex, whereas the reduction in adrenal weight after beta-methasone treatment was due to a decrease in the size of definitive and fetal zone cells of the adrenal cortex. By Western analysis, EGF treatment induced a significant (P < 0.05) 2.8-fold increase in the amount of protein for 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD) in the fetal adrenal. EGF also stimulated the induction of immunocytochemical staining for 3 beta HSD in transitional zone cells of the adrenal cortex. In contrast, beta-methasone resulted in 2.6-, 4.5-, and 6.6-fold significant decreases (P < 0.05) in the amount of protein for cytochrome P450 cholesterol side-chain cleavage, cytochrome P450 17 alpha-hydroxylase/17,20-lyase, and 3 beta HSD in the fetal adrenal. After beta-methasone treatment. 3 beta HSD staining was detected in some of the definitive zone cells, with no 3 beta HSD staining in the transitional zone. In conclusion, growth and functional differentiation of fetal primate adrenal gland can be accelerated prematurely by EGF and inhibited by glucocorticoid negative feedback.

Acute administration of buspirone increases the escape of hypothalamic-pituitary-adrenal-axis hormones from suppression by dexamethasone in depression

Recently, our laboratory found a significant enhancing effect of L-5-hydroxy-tryptophan (L-5-HTP) on post-dexamethasone (DST) plasma adrenocorticotropic hormone (ACTH) and cortisol levels in major-but not in minor-depression. To further elucidate the effects of central serotonin (5-HT) activity on the negative feedback of glucocorticoids on hypothalamic-pituitary-adrenal (HPA)-axis function in depression, this study investigates the effects of buspirone, a 5-HT1A receptor agonist, on post-DST ACTH and cortisol levels in 75 depressed subjects. Plasma post-DST ACTH and cortisol concentrations were significantly increased by the acute administration of buspirone (30 mg PO) compared to placebo. There were no differences in buspirone-induced post-DST ACTH or cortisol responses between minor and major depression. There were significant correlations between post-DST ACTH and cortisol, and between post-DST-buspirone ACTH and cortisol. The buspirone-induced post-DST cortisol responses were significantly higher in depressed women than men. It is concluded that buspirone may augment ACTH and, consequently, cortisol escape from suppression by dexamethasone in major as well as in minor depression.

Nelson's syndrome associated with a somatic frame shift mutation in the glucocorticoid receptor gene.

Nelson's syndrome is the appearance and/or progression of ACTH-secreting pituitary macroadenomas in patients who had previously undergone bilateral adrenalectomy for Cushing's disease. Extremely high plasma ACTH levels and aggressive neoplastic growth might be explained by the lack of appropriate glucocorticoid negative feedback due to defective glucocorticoid signal transduction. To study the glucocorticoid receptor (GR) gene in Nelson's syndrome, DNA was extracted from pituitary adenomas and leukocytes of four patients with this condition and amplified by PCR for direct sequence analysis. In one of the tumors, a heterozygous mutation, consisting of an insertion of a thymine between complementary DNA nucleotides 1188 and 1189, was found in exon 2. This frame-shift mutation led to premature termination at amino acid residue 366 of the wild-type coding sequence, excluding the expression of a functioning receptor protein from the defective allele. The mutation was not detected in the sequence of the GR gene in the patient's leukocyte DNA, indicating a somatic origin. By lowering the receptor number in tumorous cells, this defect might have caused local resistance to negative glucocorticoid feedback similar to that caused by the presence of a null allele in a kindred with the generalized glucocorticoid resistance syndrome. P53 protein accumulation, previously reported in 60% of corticotropinomas, could not be detected in any of the four pituitary tumors examined by immunohistochemistry. We suggest that a somatic GR defect might have played a pathophysiological role in the tumorigenesis of the corticotropinoma bearing this mutation.

In situ hybridization analysis of vasopressin gene transcription in the paraventricular and supraoptic nuclei of the rat: regulation by stress and glucocorticoids.

Hypothalamic arginine vasopressin-containing neurons are prime elements in central circuits regulating the hypothalamo-pituitary-adrenocortical stress response. It is known that release and synthesis of vasopressin are cued by stressful stimuli. The present study was designed to assess effects of stress on vasopressin transcription and mRNA expression in defined populations of vasopressin neurons in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. Intron- and exon-directed in situ hybridization analyses were used to examine stress regulation of vasopressin heteronuclear (hn) gene transcription and mRNA levels. Actions of glucocorticoids on vasopressin induction were tested using adrenalectomized rats implanted with subcutaneous pellets delivering a constant, physiological dose of corticosterone. Pellet implantation into adrenalectomized rats allows for normal pituitary-adrenal tone in the absence of the ability to mount glucocorticoid stress responses. Elevation of vasopressin heteronuclear (hn) RNA in the medial parvicellular PVN was observed in both normal and adrenalectomized-corticosterone replaced rats as early as 30 minutes after stress initiation. In control rats, vasopressin hnRNA levels returned to baseline by 120 minutes. In contrast, vasopressin hnRNA remained elevated 120 minutes post-restraint in adrenalectomized-corticosterone replaced rats, indicating that the glucocorticoid stress response acts to rapidly inhibit vasopressin transcription. Significant changes in post-stress vasopressin mRNA levels were observed in the parvicellular PVN of control rats 90 minutes following restraint induction, returning to normal expression profiles by 120 minutes. Adrenalectomized-replaced rats showed elevated vasopressin mRNA expression at all time points examined. No changes were observed in magnocellular vasopressin-containing nuclei at any time point, suggesting that magnocellular vasopressin is not induced by this particular stress paradigm. Thus, in parvicellular paraventricular nucleus neurons the vasopressin gene is rapidly induced by stress. Restraint-induced up-regulation of vasopressin transcription is limited by glucocorticoid secretion, consistent with direct actions of glucocorticoid negative feedback on the vasopressin gene in parvicellular neurons.

Stress increases brain-derived neurotropic factor messenger ribonucleic acid in the hypothalamus and pituitary.

Brain-derived neurotropic factor (BDNF) is a member of the nerve growth factor family that is important for neuronal survival and plasticity. We recently demonstrated that stress decreases BDNF messenger RNA (mRNA) levels in the hippocampus, which raises the possibility that BDNF may play a role in regulation of the hypothalamic-pituitary-adrenal axis. The purpose of this study was to determine whether BDNF expression is present and influenced by stress in other brain areas relevant to control of the hypothalamic-pituitary-adrenal axis. Using in situ hybridization, we found that BDNF mRNA is present in the parvocellular portion of the hypothalamic paraventricular nucleus (PVN), the lateral hypothalamus, and the anterior and neurointermediate lobes of the pituitary in rats. Acute (2-h) or repeated immobilization stress increased BDNF mRNA in all of these areas. This was in distinct contrast to stress-induced decreases in extrahypothalamic areas, including the basolateral amygdala, claustrum, and cingulate cortex as well as the hippocampus. BDNF was expressed in both CRF and TRH neurons in the PVN. Reducing glucocorticoid or thyroid negative feedback increased BDNF mRNA in the PVN and anterior pituitary, but not in the neurointermediate lobe. These results suggest that BDNF is a stress-responsive intercellular messenger that may be an important component of the stress response.

Increased expression of corticotropin-releasing hormone and vasopressin messenger ribonucleic acid (mRNA) in the hypothalamic paraventricular nucleus during repeated stress: association with reduction in glucocorticoid receptor mRNA levels.

Hypothalamic-pituitary-adrenal (HPA) responses remain intact or increase after chronic or repeated stress despite robust levels of circulating glucocorticoids that would be expected to restrain the responsiveness of the axis. The purpose of this study was to determine whether chronic stress altered corticosteroid receptor messenger RNA (mRNA) levels at any locus known to mediate glucocorticoid feedback on HPA function (i.e. hippocampus or hypothalamus), whether such effects were glucocorticoid dependent, and whether changes in corticosteroid receptor function could potentially contribute to the putative shift from corticotropin-releasing hormone (CRH) to arginine vasopressin (AVP) in the hypothalamic paraventricular nucleus (PVN) in the modulation of pituitary adrenal function occurring during chronic stress. We compared the stress responsiveness of sham-operated rats to that of adrenalectomized rats using a moderate dose of corticosterone (CORT) pellet replacement (ADX + CORT group). Acute immobilization caused a significant increase in CRH, but not AVP, mRNA levels in the parvocellular PVN in sham rats. The ADX + CORT group showed significantly greater increases in both CRH and AVP mRNA levels in the PVN compared to sham rats. These data indicate that PVN AVP mRNA levels are more sensitive to glucocorticoid negative feedback than are the levels of CRH mRNA. In repeated stress, the sham groups showed robust increases in PVN CRH and AVP mRNA levels despite high levels of plasma CORT. The rise in AVP mRNA levels was greater than that in CRH mRNA. Type II glucocorticoid receptor mRNA in the hippocampus and PVN was decreased in the repeatedly stressed sham group. These data suggest a decrease in the CORT negative feedback restraint of PVN CRH and AVP mRNA levels repeated stress and a persistence of relatively greater responsiveness of AVP mRNA levels to CORT negative feedback. After repeated stress in ADX+CORT rats, both PVN CRH and AVP mRNA levels showed robust responses, with a relatively greater increase in AVP mRNA. These data indicate that a CORT-mediated decrease in hippocampal and hypothalamic glucocorticoid receptor mRNA levels is not the only mechanism contributing to the maintenance of a robust HPA response after repeated stress. Similarly, we postulate that the relative shift from CRH to AVP in the PVN after repeated stress is mediated by both a greater sensitivity of AVP to CORT negative feedback and CORT-independent mechanisms.

Corticosteroid-binding globulin (CBG) production by hepatic and extra-hepatic sites in the ovine fetus; effects of CBG on glucocorticoid negative feedback on pituitary cells in vitro

Plasma cortisol levels increase in fetal sheep during late gestation and this is associated with an increase in plasma corticosteroid-binding globulin (CBG) concentrations. However, the relative tissue sources of plasma CBG, the ontogeny of its biosynthesis and glycoform composition have not been established in the ovine fetus. Therefore we examined whether changes in plasma corticosteroid binding capacity (CBC) in fetal sheep during late gestation were associated with different patterns of glycosylation and reflected changes in tissue CBG expression. Since free cortisol is considered the bioactive fraction, we measured changes in the percent and absolute free cortisol in fetal plasma during late gestation. In order to examine whether CBG alters cortisol negative feedback at the level of the fetal pituitary, we also examined the effect of exogenous CBG in mediating the glucocorticoid-induced suppression of basal and corticotrophin-releasing hormone (CRH)-stimulated ACTH release from fetal pituitary cells in culture. The mean free cortisol concentration in plasma was not different between days 15 and 20 prior to parturition, and between 5 and 10 days prepartum, although it did rise between these times. Plasma CBC in chronically catheterized fetuses rose from 23.3 +/- 4.6 ng/ml at day 115 to 86.5 +/- 20.8 ng/ml at term and then decreased rapidly after birth. Between day 125 and day 140 of pregnancy approximately 10% of fetal plasma CBG was retarded by Concanavalin-A chromatography. This proportion increased at birth and attained adult values of > 70% by one month of age. By Northern blotting the relative levels of CBG mRNA in the fetal liver did not change between days 100 and 125, then increased significantly at day 140, but declined at term and in newborn lambs. CBG mRNA was undetectable in total RNA from lung, kidney, hypothalamus and placentomes, but was present in the fetal pituitary at days 125 and 140. Reverse transcription-PCR was used to confirm the presence of CBG mRNA in pituitary tissue from term fetuses. In cultures of term fetal pituitary cells, added CBG attenuated the cortisol- but not the dexamethasone-mediated suppression of basal and CRH-stimulated ACTH release. We conclude that in fetal sheep there is an increase in the corticosteroid binding capacity of plasma during late pregnancy which regulates, in part, free cortisol levels in the circulation. The liver is the major site of CBG biosynthesis in the fetus and at least until day 140 of gestation the rise in plasma CBC is associated with an increase in hepatic CBG mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Corticosteroid Regulation of Gene Expression and Binding Characteristics of Vasopressin Receptors in the Rat Brain

Arginine-vasopressin (AVP) plays significant roles in neuroendocrine and autonomic regulation, and in processing of cognitive information. Its synthesis and secretion are subject to control by circulating glucocorticoids. The lateral septum and subdivisions of the hippocampus are innervated by AVP-ergic fibres and, together with AVP-producing neurons in the hypothalamic paraventricular nucleus, are major neural targets of glucocorticoid negative feedback. In this study, we investigated the effects of chronic adrenalectomy (ADX) and subsequent treatment with supraphysiological doses of corticosterone (B) on the gene expression of AVP receptors of the V1a subtype in the septum, hippocampus and hypothalamic arcuate (ARC) nucleus using semiquantitative in situ hybridization histochemistry. Adrenalectomy did not alter AVP receptor expression in any of the structures studied. Supplementation with B significantly decreased AVP receptor expression in the lateral septum and hippocampus, whereas receptor mRNA levels in the ARC were indistinguishable from those measured in controls. In a complementary study, we investigated the binding characteristics of V1 AVP receptors in membrane preparations from the hippocampus. Adrenalectomy significantly decreased the number of AVP binding sites, and chronic corticosteroid treatment was associated with a further suppression of AVP receptor concentrations in this structure. These results indicate that the gene transcription of V1a AVP receptors in the brain is regulated by circulating glucocorticoids in a site-specific fashion that largely reflects the corticosteroid sensitivity of the corresponding structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogens decrease expression of the corticotropin-releasing factor gene in the hypothalamic paraventricular nucleus and of the proopiomelanocortin gene in the anterior pituitary of ovariectomized rats

It is known that estrogens modulate the hypothalamopituitary-adrenal (HPA) axis both under resting conditions and during exposure to stress. Nevertheless, the site of action of estrogens is not still fully elucidated. We sought to determine if estrogens could act on the major hypothalamic ACTH secretagogue: corticotropin-releasing factor (CRF). Mature rats were ovariectomized (OVX) and 2 weeks later implanted with silastic capsules containing 17β-estradiol (E(2)). Animals were sacrificed 7 days later. CRF mRNA in the hypothalamic paraventricular nucleus (PVN) and proopiomelanocortin (POMC) mRNA in the anterior pituitary were measured byin situ hybridization. CRF content in the median eminence was measured by semiquantitative immunocytochemistry. E(2) treatment induced a significant decrease of CRF mRNA levels in the PVN (3.70±0.14vs 4.79±0.15 copies of probe×10(-3)/μm(3) of tissue in OVX rats,P<0.05), an accumulation of immunoreactive CRF in the zona externa of the median eminence (207±36vs 100±15% in OVX rats,P<0.05), and a decrease of POMC mRNA levels in the anterior pituitary (4.6±0.6vs 6.9±0.6 copies of probe ×10(-2)/μm(3) of tissue in OVX rats,P<0.05). These results demonstrate that estrogens have a negative effect on CRF gene expression and secretion and on POMC gene expression. Whether estrogens modulate directly the CRF-synthesizing cells or act through an increase of the glucocorticoid negative feedback remains to be determined.

Mycoplasma fermentans activates the hypothalamo-pituitary adrenal axis in the rat.

Intracerebroventricular administration in rats of heat inactivated Mycoplasma fermentans caused a dose- and time-dependent increase in serum adrenocorticotrophin (ACTH) and corticosterone (CS). In rats with complete deafferentation of the mediobasal hypothalamus, which markedly depleted the median eminence CRF-41, the ACTH and CS responses to M. fermentans were completely inhibited. Pretreatment with dexamethasone abolished the adrenocortical response to M. fermentans. In lipopolysaccharide (LPS) unresponsive C3H/HeJ mice LPS failed to induce the adrenocortical response while administration of M. fermentans elicited a normal CS response. These results suggest that: M. fermentans can activate the hypothalamo-pituitary-adrenal axis via a central mechanism which involves hypothalamic ACTH secretagogue(s), and this effect is sensitive to the negative feedback of glucocorticoids. It is possible that the elevated glucocorticoid levels resulting from mycoplasma infection may be involved in the pathogenesis of mycoplasma-associated diseases.

Effects of serotonin precursors on the negative feedback effects of glucocorticoids on hypothalamic-pituitary-adrenal axis function in depression

In order to investigate the relationships between brain serotonergic turnover and hypothalamic-pituitary-adrenal (HPA) axis function in unipolar depression, the authors measured intact adrenocorticotropic hormone (ACTH) and cortisol levels in baseline conditions and after combined dexamethasone (1 mg PO) and L-5-hydroxytryptophan (L-5-HTP, 200 mg PO) administration in 13 minor, 17 simple major, and 17 melancholic subjects. L-5-HTP significantly enhanced post-DST ACTH and cortisol secretion in major--but not in minor--depressed subjects. Major depressed subjects with or without melancholia exhibited significantly higher post-DST ACTH and cortisol responses to L-5-HTP than minor depressed subjects. L-5-HTP administration converted some major depressed ACTH or cortisol suppressors into nonsuppressors. L-5-HTP stimulated ACTH or cortisol secretion to the same extent in major depressed HPA-axis suppressors and nonsuppressors. It is concluded that L-5-HTP loading may augment ACTH and, consequently, cortisol escape from suppression by dexamethasone in major but not in minor depressed subjects. The findings show that serotonergic mechanisms modulate the negative feedback of glucocorticoids on central HPA-axis regulation. It is hypothesized that the higher L-5-HTP-induced post-DST HPA-axis hormone responses in major depression reflect upregulated 5-HT2 receptor-driven breakthrough secretion of pituitary ACTH from suppression by dexamethasone.

Regulation of Pituitary ACTH Secretion during Chronic Stress

Maintenance of adequate levels of response of the hypothalamic-pituitary-adrenal axis during chronic stress is important for survival. Three basic patterns of response can be identified depending on the type of stress: (a) desensitization of ACTH responses to the sustained stimulus, but hyperresponsiveness to a novel stress despite elevated plasma glucocorticoid levels, as occurs in physical-psychological paradigms; (b) no desensitization of ACTH response to the repeated stimulus and hyperresponsiveness to a novel stress, as occurs during repeated painful stress and insulin hypoglycemia; and (c) small and transient increases in ACTH, but sustained elevations of plasma corticosterone and diminished ACTH responses. The level of response of the pituitary corticotroph is determined by differential regulation of the hypothalamic regulators, corticotropin-releasing hormone (CRH) and vasopressin (VP), and the sensitivity of the negative glucocorticoid feedback. While osmotic stimulation increases VP expression in magnocellular neurons of the paraventricular (PVN) and supraoptic nuclei of the hypothalamus, chronic stress paradigms with high pituitary responsiveness are associated with activation of CRH and CRH/VP parvicellular neurons of the PVN, predominantly of the VP-containing population. While moderate increase of CRH output is important for stimulation of POMC transcription, the increase of the VP:CRH secretion ratio appears to be important in maintaining the secretory capacity of the pituitary corticotroph during chronic stimulation. Decreased sensitivity of the glucocorticoid feedback, probably due to interaction of glucocorticoid receptors with transcription factors induced by CRH and VP, is critical for the maintenance of ACTH responses in the presence of elevated plasma glucocorticoid levels during chronic stress. Although both CRH and VP receptors are activated and undergo regulatory variations during chronic stress, only the changes in VP receptor levels are parallel to the changes in pituitary ACTH responsiveness. The inhibitory effect of chronic osmotic stimulation on ACTH secretion in spite of high circulating levels of VP is probably the result of diminished activity or parvicellular PVN neurons and downregulation of pituitary VP receptors. Although the exact interaction between regulatory factors and the molecular mechanisms controlling the sensitivity of the corticotroph during adaptation to chronic stress remain to be determined, it is clear that regulation of the proportional secretion of CRH and VP in the PVN, modulation of pituitary VP receptors, and the sensitivity to feedback inhibition play a critical role.

Glucocorticoid negative feedback in pituitary corticotropes. Pivotal role for calcineurin inhibition of adenylyl cyclase.

Glucocorticoid negative feedback in pituitary corticotropes. Pivotal role for calcineurin inhibition of adenylyl cyclase.

Early environmental programming hypothalamic-pituitary-adrenal responses to stress

This paper summarizes the effects of various early environmental manipulations (infantile handling, maternal separation, neonatal immune challenge) on the development of hypothalamic-pituitary-adrenal (HPA) responses to stressful stimuli. Each manipulation results in a permanent effect on HPA responsivity to stress, although the effects qualitatively are quite different. In each case the effects are apparent in terms of different levels of activity of the corticotrophin-releasing hormone/arginine vasopressin neurons in the paraventricular nucleus of the hypothalamus. These effects, in turn, seem to be derived from changes in forebrain corticosteroid receptor systems which determine glucocorticoid negative feedback sensitivity. We feel that these effects represent examples of early environmental 'programming' of CNS function and reflect naturally-occurring plasticity in neuroendocrine defense systems. This programming also seems to have important implications for health in later life by determining vulnerability to certain forms of pathology.

Studies of the secretion of corticotropin-releasing factor and arginine vasopressin into the hypophysial-portal circulation of the conscious sheep. II. The central noradrenergic and neuropeptide Y pathways cause immediate and prolonged hypothalamic-pituitary-adrenal activation. Potential involvement in the pseudo-Cushing's syndrome of endogenous depression and anorexia nervosa.

Studies were performed to determine the effects of intracerebroventricular norepinephrine (NE) or neuropeptide Y (NPY) on the ovine hypothalamic-pituitary-adrenal (HPA) axis. NE (50 micrograms) increased mean hypophysial-portal corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) levels (1 h, 1.3- and 2.9-fold; 4 h, 2.2- and 5.7-fold) and caused acute and sustained increases in mean plasma ACTH and cortisol. NPY (50 microgram) also increased mean CRF and AVP levels (1 h, 1.4- and 4.2-fold; 4 h, 1.1- and 1.9-fold), increased pituitary-adrenal activity at 1 h, and caused ACTH hypersecretion at 4 h. When added to cultured ovine anterior pituitary cells, NPY neither increased basal ACTH release nor augmented CRF- or AVP-induced ACTH release. We conclude that: (a) activation of either the central noradrenergic or NPY pathways causes an acute and sustained stimulation of the ovine HPA axis; (b) such activation increases the AVP/CRF ratio, suggesting a dominant role for AVP in the ovine stress response; and (c) the central noradrenergic or NPY systems may cause sustained HPA activation by attenuating or disrupting the glucocorticoid negative feedback on those brain areas concerned with regulation of the HPA axis. The possible roles of the central noradrenergic and NPY systems in the etiology of the hypercortisolemia of endogenous depression and anorexia nervosa are discussed.

Strain differences in hypothalamic-pituitary-adrenal activity and stress ulcer

Strain differences in stress responsiveness have been previously described, but specific components of the hypothalamic-pituitary-adrenal (HPA) axis responsible for stress hypo- or hyperactivity have not yet been characterized. This study proposed to analyze the effect of restraint stress on different measures of HPA function and stress ulcer in stress-ulcer prone Wistar-Kyoto (WKY) and Fisher 344 (F-344) rats and in the ulcer-resistant Wistar strain. Adult male rats of these strains were sham adrenalectomized, adrenalectomized, and adrenalectomized-replaced with corticosterone pellet. Ten days after surgery, animals were subjected to the 2-h ulcerogenic water-restraint stress and killed 2 h later. Intact WKY rats had dramatically more ulcers and higher anterior pituitary adrenocorticotropic hormone (ACTH) and proopiomelanocortin mRNA levels than the other two strains. In WKY rats, adrenalectomy increased ulcer incidence but did not affect thymus weight, ACTH content, or hypothalamic corticotropin-releasing factor mRNA levels, in contrast to the profound effects of adrenalectomy on these parameters in the other strains. Furthermore, corticosterone replacement was either without effect or enhanced the effect of adrenalectomy on these parameters in WKY rats, while it reversed the effects of adrenalectomy in the other strains. These data imply that WKY rats respond to stress with enhanced and prolonged changes in peripheral functions that are regulated by glucocorticoids, suggesting the presence of impaired efficacy of the glucocorticoid negative feedback on HPA function.

Fast glucocorticoid feedback inhibition of ACTH secretion in the ovariectomized rat: effect of chronic estrogen and progesterone.

The purpose of this study was to determine whether estrogen and progesterone influence fast glucocorticoid negative feedback regulation of the ACTH and corticosterone (CORT) responses to stress. Mature rats were ovariectomized and 6 weeks later implanted with 17 beta-estradiol (E2, 0.5 mg), E2 and progesterone (P, 100 mg; E2 + P group) or placebo pellets (OVX). Seven days later rats were subjected to a single or repeated intermittent footshock stress (0.2 mA, 15 s duration, 0.5 s on). The repeated stress was of the same intensity and duration, and was applied either during the time domain of the rate-sensitive fast glucocorticoid feedback when plasma CORT levels are rising (5 min after the onset of the first stress), or at the time of peak CORT response (15 min) to the initial stress. Plasma ACTH and CORT were measured from serial samples. Estrogen replacement alone or in combination with progesterone lowered the immediate (t = 5) ACTH and CORT response to a single stress in ovariectomized animals. The second stress applied 5 min after the initial stress produced net ACTH responses similar to those obtained after a single stress in the OVX and E2 + P-replaced hormone groups, while total ACTH responses were lower in the E2-treated group. In ovariectomized animals, a facilitation of ACTH response by a prior stress is apparent in response to a footshock 15 min later, when the integrated ACTH secretion is significantly greater than the response measured after a single shock, or after a repeated shock 5 min apart. Anterior pituitary proopiomelanocortin (POMC) mRNA levels were lower in groups with E2 or E2 + P replacement compared to OVX animals. In contrast, hypothalamic corticotropin-releasing factor (CRF) mRNA levels did not increase significantly. However, hypothalamic glucocorticoid receptor (GR) mRNA levels increased after 17 beta-estradiol treatment, and this increase was reversed by progesterone. These results suggest that prior stress leads to both a fast-feedback inhibition and a facilitation of the subsequent stress response. In the absence of gonadal hormones this facilitation is balanced by fast-feedback inhibition during the glucocorticoid fast-feedback time domain, and is unmasked outside of this time domain. Estrogen suppresses POMC mRNA synthesis leading to a decrease in the availability of releasable ACTH, thereby reducing the facilitation. Progesterone may counter this effect of estrogen by decreasing the efficacy of the fast rate-sensitive glucocorticoid negative feedback.

Rank-related differences in cardiovascular function among wild baboons: Role of sensitivity to glucocorticoids.

In a population of wild baboons living in East Africa, we have observed endocrine differences among individuals as a function of dominance rank. Among these differences, we previously observed indirect evidence for dominant males being more. responsive to sympathetic catecholamines than were subordinate males. The present report tests this explicitly. Male baboons of known rank were anesthetized, and sympathetic and parasympathetic activity was pharmacologically inhibited. In experiment I, males were challenged with epinephrine; over a wide dose range, dominant males had the largest and fastest rises in systolic pressure, the greatest peak systolic pressure, and the most rapid recovery from the epinephrine challenge. Similar rank-related differences in heart rate response to epinephrine also occurred. Experiment II showed that this phenomenon probably reflects rank-related differences at both the heart and vasculature. As evidence, the same rank differences in systolic blood pressure responsiveness occurred when males were challenged with the alpha receptor agonist phenylephrine (which predominantly constricts systemic veins and arterioles), while the same rank differences in heart rate responses occurred following stimulation with the beta receptor agonist isoproterenol (which selectively works at the heart). These data were obtained from animals stressed by anesthetization, known to cause considerable glucocorticoid secretion in this population. Such steroids are well known for their capacity to augment catecholamine action. In experiment III, we blocked endogenous glucocorticoid secretion with the steroidogenesis inhibitor metyrapone, and repeated the epinephrine challenge. Under these conditions, the rank differences in epinephrine responsiveness were eliminated. Thus, dominant males are not so much preferentially sensitive to catecholamine action, as much as to the potentiating effects of glucocorticoids upon such action. In agreement, we have observed previously that dominant males are also more sensitive to glucocorticoid negative feedback regulation. © Wiley-Liss, Inc.

Glucocorticoids do not affect intracellular calcium transients in corticotrophs: evidence supporting an effect distal to calcium influx.

The influx of extracellular calcium is a critical step involved in the stimulated release of adrenocorticotropin (ACTH) from pituitary corticotrophs. It has been proposed that the mechanism of early glucocorticoid feedback is mediated through inhibition of stimulus-evoked calcium transients. We tested this hypothesis using corticotrophic mouse pituitary AtT-20 cells by coevaluating secretory dynamics and cytosolic calcium transients. In static monolayer culture and in a dynamic microperifusion system, dexamethasone (DEX, 100 nM, 2 h) significantly inhibited ACTH secretion stimulated by corticotropin-releasing hormone (CRH, 100 nM). When ACTH was stimulated by KCl (56 mM) depolarization or the voltage-dependent calcium channel agonist, maitotoxin (MTX, 1 ng/ml), DEX did not inhibit secretion. In contrast, CRH-, KCl-, and MTX-stimulated ACTH secretion were significantly inhibited in static monolayer culture when cells were pretreated with the voltage-dependent calcium channel blocker, nifedipine (NIF, 1 microM, 15 min), confirming the requirement for the influx of extracellular calcium. Intracellular calcium was measured under similar culture conditions in populations of cells grown on coverslips, utilizing the fluorescent calcium indicator, fura-2. DEX had no effect on basal, spike, or plateau calcium levels in response to CRH, KCl or MTX stimulation. For example, CRH stimulation resulted in an increase in intracellular calcium from a basal concentration of 90 +/- 3.1 nM (mean +/- SE) to a plateau of 222 +/- 8.7 nM, whereas the plateau after DEX was 225 +/- 4.1 nM. In contrast, NIF significantly lowered the stimulated calcium response to each secretagogue (spike and plateau). These results do not support the hypothesis that glucocorticoids suppress stimulated secretion of ACTH from corticotrophs through an effect on intracellular calcium transients. Instead, the data suggest that early glucocorticoid negative feedback occurs at a step(s) distal to the influx of extracellular calcium.

Vasupressin and the Endocrine Response To Stress

This paper highlights the role of vasopressin in the endocrine response to stress at various levels of regulation in the hypothalamic-pituitary-adrenocortical system. Novel data arc presented regarding the interaction of vasopressin and corticotropin-releasing factor (CRF) in rat anterior pituitary cells. In this system CRF-induced cyclic AMP synthesis is under feedback inhibition by intracellular free calcium. Vasopressin enhances the cyclic AMP response to CRF apparently by reducing the efficiency of calcium inhibition. As corticosteroid mediated inhibition of ACTH release is also mediated by a calcium-dependent process, these findings show a mechanism whereby vasopressin may change the set-point of glucocorticoid negative feedback at the pituitary level.