Increased Plasma ACTH Concentrations Research Articles

Characterization of CRF1 receptor antagonists with differential peripheral vs central actions in CRF challenge in rats

The aim of this study was to investigate peripheral and central roles of corticotropin-releasing factor (CRF) in endocrinological and behavioral changes. Plasma adrenocorticotropin (ACTH) concentration was measured as an activity of hypothalamic-pituitary-adrenal (HPA) axis. As behavioral changes, locomotion and anxiety behavior were measured after CRF challenge intravenously (i.v.) for the peripheral administration or intracerebroventricularly (i.c.v.) for the central administration. Plasma ACTH concentration was significantly increased by both administration routes of CRF; however, hyperlocomotion and anxiety behavior were induced only by the i.c.v. administration. In the drug discovery of CRF1 receptor antagonists, we identified two types of compounds, Compound A and Compound B, which antagonized peripheral CRF-induced HPA axis activation to the same extent, but showed different effects on the central CRF signal. These had similar in vitro CRF1 receptor binding affinities (15 and 10nM) and functional activities in reporter gene assay (15 and 9.5nM). In the ex vivo binding assays using tissues of the pituitary, oral treatment with Compound A and Compound B at 10mg/kg inhibited [125I]-CRF binding, whereas in the assay using tissues of the frontal cortex, treatment of Compound A but not Compound B inhibited [125I]-CRF binding, indicating that only Compound A inhibited central [125I]-CRF binding. In the peripheral CRF challenge, increase in plasma ACTH concentration was significantly suppressed by both Compound A and Compound B. In contrast, Compound A inhibited the increase in locomotion induced by the central CRF challenge while Compound B did not. Compound A also reduced central CRF challenge-induced anxiety behavior and c-fos immunoreactivity in the cortex and the hypothalamic paraventricular nucleus. These results indicate that the central CRF signal, rather than the peripheral CRF signal would be related to anxiety and other behavioral changes, and CRF1 receptor antagonism in the central nervous system may be critical for identifying drug candidates for anxiety and mood disorders.

Adiponectin Depolarizes Parvocellular Paraventricular Nucleus Neurons Controlling Neuroendocrine and Autonomic Function

Adiponectin plays important roles in the control of energy homeostasis and autonomic function through peripheral and central nervous system actions. The paraventricular nucleus (PVN) of the hypothalamus is a primary site of neuroendocrine (NE) and autonomic integration, and, thus, a potential target for adiponectin actions. Here, we investigate actions of adiponectin on parvocellular PVN neurons. Adiponectin influenced the majority (65%) of parvocellular PVN neurons, depolarizing 47%, whereas hyperpolarizing 18% of neurons tested. Post hoc identification (single-cell RT-PCR) after recordings revealed that adiponectin depolarizes NE-CRH neurons, whereas intracerebroventricular injections of adiponectin in vivo caused increased plasma ACTH concentrations. Adiponectin also depolarized the majority of TRH neurons, however, NE-TRH neurons were unaffected, in accordance with in vivo experiments showing that intracerebroventricular adiponectin was without effect on plasma TSH. In addition, bath administration of adiponectin also depolarized both preautonomic TRH and oxytocin neurons. These results show that adiponectin acts in the central nervous system to coordinate NE and autonomic function through actions on specific functional groups of PVN neurons.

Correlation of Pituitary Histomorphometry with Adrenocorticotrophic Hormone Response to Domperidone Administration in the Diagnosis of Equine Pituitary Pars Intermedia Dysfunction

Functional evaluation of the pars intermedia (PI) is required for the early diagnosis of equine pituitary PI dysfunction (PPID), yet most assays target the hypothalamic-pituitary-adrenal axis, which regulates the pars anterior. In contrast, the PI is regulated by dopaminergic tone from hypothalamic neurons. Loss of dopaminergic inhibition is hypothesized to cause the PI hypertrophy and hyperplasia that result in the clinical manifestations of PPID. Domperidone, a dopamine receptor antagonist, should exacerbate the loss of dopaminergic inhibition in horses with PPID and increase the release of endogenous adrenocorticotrophic hormone (eACTH) by PI melanotrophs. To test this, plasma eACTH concentration was determined in horses with or without clinical signs of PPID at 0, 4, and 8 hours after oral administration of 3.3 mg domperidone/kg. Pituitary glands were evaluated postmortem by histologic grading and morphometry. In the 33 horses, median age, plasma ACTH concentration 8 hours after domperidone, and PI area in median sagittal sections were associated with histologic grade as follows: pituitary grade 1 (normal), n = 3, 7.5 years, 20.0 pg/ml, 0.16 cm(2); grade 2 (focal hypertrophy or hyperplasia), n = 9, 14.5 years, 27.1 pg/ml, 0.27 cm(2); grade 3 (diffuse adenomatous hyperplasia), n = 5, 21.0 years, 64.4 pg/ml, 0.48 cm(2); grade 4 (microadenomas), n = 12, 23.3 years, 128.0 pg/ml, 0.87 cm(2); grade 5 (adenoma), n = 4, 24.9 years, 720.5 pg/ml, 2.1 cm(2). Results suggest that horses with pituitary histologic grade > or =3 respond to domperidone with increased plasma ACTH concentration.

Reference ranges and factors affecting the human corticotropin-releasing hormone test in preterm, very low birth weight infants.

This prospective study aims to investigate the factors that influence the human CRH (hCRH) test and to provide reference ranges for plasma corticotropin (ACTH) and serum cortisol concentrations of the stimulation test in preterm, very low birth weight (VLBW) infants. Two hundred twenty-six hCRH tests were performed on 137 VLBW infants at d 7 and 14 of life. Plasma ACTH did not differ significantly between infants whose mothers did not receive antenatal corticosteroids (group 1) and those whose mothers received one or two doses (group 2) or more than two doses (group 3) of the drug. However, plasma ACTH levels at d 7 were found to be significantly higher in infants with severe lung disease who required intermittent positive-pressure ventilation (IPPV) or high-frequency oscillation ventilation (HFOV), compared with those who had milder pulmonary disease and did not require mechanical ventilation or needed only continuous positive airway pressure (CPAP) support (P < 0.011). A significantly higher rate of increase in plasma ACTH concentration at d 7 was also observed in infants whose mothers suffered from antepartum hemorrhage (P < 0.016). In contrast, infants in group 2 had significantly lower serum cortisol, compared with group 1 infants (P < 0.05), whereas group 3 infants did not have serum cortisol levels significantly different from those of patients in group 1 or 2. Significant positive correlation between serum cortisol at d 7 and the time interval between the last dose of antenatal dexamethasone and delivery was also observed in group 3 infants (r > 0.33, P < 0.045). In addition, infants who required IPPV or HFOV had significantly lower serum cortisol at d 7 (P < 0.0001), but this pattern of cortisol response was reversed on d 14, with infants requiring IPPV or HFOV having significantly higher serum cortisol (P < 0.036). The reference ranges for plasma ACTH and serum cortisol concentrations of the hCRH test at d 7 and 14 were also provided for group 1 and group 2 infants. This study demonstrates that even one or two doses of antenatal corticosteroids cause adrenal suppression in VLBW infants. Maternal antepartum hemorrhage also influences the pituitary response of preterm newborns in the first week of life. The change in the pattern of cortisol response in sick ventilated (IPPV or HFOV) infants during the first 2 wk of life suggests that a proportion of preterm infants may have inadequate adrenal response to stress in early postnatal life, but it is likely that rapid adaptation of the hypothalamic-pituitary-adrenal axis results in enhanced and more appropriate cortisol response by d 14. The percentile distribution of plasma ACTH and serum cortisol responses provides useful statistical reference data for interpretation of the hCRH test in VLBW infants and may also assist in facilitating the use of corticosteroids replacement therapy in cases with clinical manifestations suggestive of adrenal insufficiency.

Corticotropin-releasing factor but not urocortin is involved in adrenalectomy-induced adrenocorticotropin release.

Urocortin, a new corticotropin-releasing factor (CRF)-related peptide, has been reported to have the ability to bind to CRF receptors and to stimulate adrenocorticotropin (ACTH) secretion from the rat anterior pituitary in vivo and in vitro. In this study, we examined the effect of intravenous administration of urocortin-antiserum to investigate the role of endogenous urocortin on ACTH secretion from rat anterior pituitary after adrenalectomy. Male Sprague-Dawley rats, which were maintained in a conscious and undisturbed condition, were administered non-immunized rabbit serum (NRS), CRF-antiserum or urocortin-antiserum at a volume of 1 ml/kg b.w. 15 min before the injection of secretagogues. Synthetic rat urocortin (2 microg/kg B.W.) increased plasma ACTH concentrations by about sixfold the basal concentration. The pretreatment with urocortin-antiserum but not CRF-antiserum abolished the urocortin-induced increase in plasma ACTH concentrations. In adrenalectomized rats, plasma ACTH concentrations were markedly increased at basal conditions, and rapidly reduced after the administration of CRF-antiserum. By contrast, administration of urocortin-antiserum did not alter ACTH secretion induced by adrenalectomy. Our results suggest that endogenous urocortin is unlikely to be involved in ACTH release in adrenalectomized rats.

A regulatory loop between the hypothalamo-pituitary-adrenal (HPA) axis and circulating leptin: a physiological role of ACTH.

The product of the ob/ob gene, leptin, is known to be able to exert a modulator, role on HPA axis function. The aim of the present study was to determine whether endogenous ACTH and glucocorticoids exert any regulatory effect on leptin secretion. For this purpose bilaterally adrenalectomized (ADX) or sham operated (Sham) adult male rats were implanted with an indwelling i.v. catheter. A subgroup of ADX animals received, at the same time of surgery, a s.c. corticosterone (B) pellet (75 mg) (ADX+B). All animals were subjected to experimental designs 7 days after surgery. Our results indicate, as expected, that 7-day ADX animals have several fold increased basal ACTH plasma levels and non detectable circulating B, whereas ADX+B rats showed basal plasma ACTH levels in the range of Sham values and plasma B concentrations of about 5 microg/dl. Interestingly, basal plasma leptin levels were significantly (P < 0.05) decreased by 7 days post ADX, and B replacement therapy (ADX+B) restored circulating leptin to Sham levels. Acute dexamethasone (Dxmn, 30 microg/kg body weight, i.v.) treatment induced a very rapid decrease in plasma ACTH concentrations in both Sham and ADX rats, as well as a decrease in plasma B levels in Sham rats. Interestingly, Dxm test had no effect on plasma leptin levels in Sham animals; however, in ADX rats, the synthetic glucocorticoid increased plasma leptin concentrations, restoring the levels observed in Sham rats. This effect occurred at the same time when plasma ACTH levels were decreasing toward basal Sham values. These results clearly indicate that, beside the known effects of leptin on HPA axis function, circulating ACTH and glucocorticoid are able to modulate leptin secretion in plasma. The lack of circulating glucocorticoid and/or increased plasma ACTH concentrations, are responsible for decreasing leptin output, whereas decreased plasma ACTH concentrations allow an increase of leptin secretion in blood. Our data strongly support the existence of a closed, bi-directional, circuit between HPA axis function and adipose tissue metabolism. They further indicate the physiological relevance of different types of stress associated with many phenotypes of obesity.

Biochemical assessment of Cushing's disease in patients with corticotroph macroadenomas.

The majority of cases of Cushing's disease are due to an underlying pituitary corticotroph microadenoma (< or = 10 mm). Corticotroph macroadenomas (> 10 mm) are a less common cause of Cushing's disease, and little is known about specific clinical and biochemical findings in such patients. To define further the clinical characteristics of patients with corticotroph macroadenomas, we performed a retrospective review of Cushing's disease due to macroadenomas seen at Massachusetts General Hospital between 1979 and 1995. Of 531 patients identified with a diagnostic code of Cushing's syndrome, 20 were determined to have Cushing's disease due to a macroadenoma based on radiographic evidence of pituitary adenoma greater than 10 mm and pathological confirmation of a pituitary adenoma. A comparison review of charts of 24 patients with Cushing's disease due to corticotroph microadenomas identified on the basis of radiographic evidence of a normal pituitary gland or a pituitary adenoma 10 mm or less in diameter was also performed. The mean ages of the patients (+/- SD) with macroadenomas and microadenomas were similar (39 +/- 12 and 38 +/- 14 yr, respectively). The baseline median 24-h urine free cortisol (UFC) excretion was 1341 nmol/day (range, 304-69,033 nmol/day) and 877 nmol/day (range, 293-2,558 nmol/day) for macroadenoma and microadenoma patients, respectively (P = 0.058). After the 48-h high dose dexamethasone suppression test, UFC decreased by 77 +/- 19% (mean +/- SD) and 91 +/- 7% in macroadenoma and microadenoma subjects, respectively (P = 0.04). Fifty-six percent of macroadenoma patients and 92% of microadenoma patients had greater than 80% suppression of UFC after high dose dexamethasone administration (P = 0.03). The baseline median 24-h urinary 17-hydroxysteroid (17-OHCS) excretion was 52 mumol/day (range, 25-786 mumol/day) and 44 mumol/day (range, 17-86 mumol/day) for macroadenoma and microadenoma subjects, respectively (P = 0.09). After the standard high dose dexamethasone suppression test, 17-OHCS excretion decreased by 46 +/- 33% and 72 +/- 22% for macroadenoma and microadenoma subjects, respectively (P = 0.02). Fifty-three percent of patients with macroadenomas and 86% of patients with microadenomas had greater than 50% suppression of 17-OHCS after high dose dexamethasone administration (P = 0.02). Baseline plasma ACTH values were above the normal range in 83.3% of macroadenoma patients and in 45% of microadenoma subjects (P = 0.05). Tumors were immunostained with the MIB-1 antibody for Ki-67 to investigate proliferation in the adenomas. There was a trend for a higher Ki-67 labeling index in corticotroph macroadenomas, and seven (44%) macroadenomas vs. three (18%) microadenomas had labeling indexes greater than 3%, but this was not statistically significant. In summary, corticotroph macroadenomas are often associated with less glucocorticoid suppressibility than the more frequently occurring microadenomas. Therefore, the lack of suppression of UFC or 17-OHCS after the administration of high dose dexamethasone in a patient with Cushing's disease does not necessarily imply the presence of ACTH-independent Cushing's syndrome and is more commonly seen in patients with corticotroph macroadenomas than in those with microadenomas. Increased plasma ACTH concentrations are typical of patients with corticotroph macroadenomas and may be a more sensitive indicator of neoplastic corticotrophs than the UFC or 17-OHCS response to standard high dose dexamethasone testing.

Major role of 3',5'-cyclic adenosine monophosphate-dependent protein kinase A pathway in corticotropin-releasing factor gene expression in the rat hypothalamus in vivo.

To assess whether the cAMP-dependent protein kinase-A and/or the diacylglycerol-dependent protein kinase C (PKC) pathways play important roles in the activation of CRF neurons in vivo under physiological conditions, we tested the effect of microinjection of 8-bromo-cAMP (8-Br-cAMP) or 12-O-tetradecanoyl phorbol 13-acetate (TPA) into both paraventricular nuclei (PVN) of the hypothalamus in conscious rats. Both 8-Br-cAMP and TPA increased plasma ACTH concentrations and the POMC messenger RNA (mRNA) concentrations in the anterior pituitary. While injection of 8-Br-cAMP also increased CRF mRNA concentrations in hypothalamic tissue containing the PVN, TPA injection had no effect on CRF mRNA concentrations there. During insulin-induced hypoglycemia, which stimulates CRF gene expression and release, c-fos and c-jun mRNA increases in the hypothalamic tissue preceded the increase in the CRF mRNA level after insulin-induced hypoglycemia. Antisense oligodeoxyribonucleotides (oligos) directed against c-fos, c-jun, or the cAMP response element binding protein (CREB) mRNA were injected into both PVN before insulin-induced hypoglycemia to assess whether activator protein-1 or CREB mediates transcriptional activation of CRF during hypoglycemia. Only antisense oligo against CREB mRNA reduced the CRF mRNA level after insulin-induced hypoglycemia. These results suggest that protein kinase A may transduce intracellular signals in CRF neurons under physiological conditions and raises the possibility that CREB may be involved in stress-induced CRF gene expression.

Major role of 3',5'-cyclic adenosine monophosphate-dependent protein kinase A pathway in corticotropin-releasing factor gene expression in the rat hypothalamus in vivo

To assess whether the cAMP-dependent protein kinase-A and/or the diacylglycerol-dependent protein kinase C (PKC) pathways play important roles in the activation of CRF neurons in vivo under physiological conditions, we tested the effect of microinjection of 8-bromo-cAMP (8-Br-cAMP) or 12-O-tetradecanoyl phorbol 13-acetate (TPA) into both paraventricular nuclei (PVN) of the hypothalamus in conscious rats. Both 8-Br-cAMP and TPA increased plasma ACTH concentrations and the POMC messenger RNA (mRNA) concentrations in the anterior pituitary. While injection of 8-Br-cAMP also increased CRF mRNA concentrations in hypothalamic tissue containing the PVN, TPA injection had no effect on CRF mRNA concentrations there. During insulin-induced hypoglycemia, which stimulates CRF gene expression and release, c-fos and c-jun mRNA increases in the hypothalamic tissue preceded the increase in the CRF mRNA level after insulin-induced hypoglycemia. Antisense oligodeoxyribonucleotides (oligos) directed against c-fos, c-jun, or the cAMP response element binding protein (CREB) mRNA were injected into both PVN before insulin-induced hypoglycemia to assess whether activator protein-1 or CREB mediates transcriptional activation of CRF during hypoglycemia. Only antisense oligo against CREB mRNA reduced the CRF mRNA level after insulin-induced hypoglycemia. These results suggest that protein kinase A may transduce intracellular signals in CRF neurons under physiological conditions and raises the possibility that CREB may be involved in stress-induced CRF gene expression.

Intracerebroventricular administration of β-endorphin increases the expression of c- fos and of corticotropin-releasing factor messenger ribonucleic acid in the paraventricular nucleus of the rat

We evaluated the effects of intracerebroventricular (i.c.v.) administration of β-endorphin and naloxone, an opioid antagonist, on the induction of c- fos and corticotropin-releasing factor (CRF) mRNA to clarify the effects of β-endorphin on cellular activity and CRF gene expression in the paraventricular nucleus (PVN) of the rat using in situ hybridization. A significant induction of c- fos mRNA was noted in the PVN after i.c.v. injection of β-endorphin, compared to control. This induction was inhibited by the administration of naloxone. A significant increase in CRF mRNA levels in the PVN was observed 120 min after the i.c.v. injection of β-endorphin. This increase was partially, but significantly, inhibited by naloxone administration. In addition, i.c.v. administration of β-endorphin increased plasma ACTH concentration in freely moving rats, which was inhibited by intravenous injection of CRF antiserum. These results suggest that the i.c.v. injection of β-endorphin increases the neuronal activity and the biosynthesis of CRF in the PVN, and stimulates the secretion of ACTH by increasing CRF secretion. This effect on the PVN was mediated, at least in part, via the opioid receptor.

Effect of acetylcholine on corticotropin-releasing factor gene expression in the hypothalamic paraventricular nucleus of conscious rats.

To examine the physiological role of cholinergic input in the regulation of CRF neurons in the paraventricular nucleus (PVN) of the hypothalamus, acetylcholine (ACh) was microinjected bilaterally into the dorsolateral border of the PVN of conscious rats. Changes in the levels of POMC messenger RNA (mRNA) in the anterior pituitary, CRF mRNA in hypothalamic tissue containing the PVN, and plasma ACTH were assessed. Plasma ACTH concentrations increased in a dose-dependent manner after ACh injection (1-100 pmol/side), reaching a peak 30 min after ACh injection and returning to baseline within 120 min. The POMC mRNA level in the anterior pituitary and the hypothalamic CRF mRNA level increased in a dose-dependent manner 120 min after ACh (0.1-10 pmol/side) injection. Intracerebroventricular pretreatment with atropine completely abolished the ACh-induced increase in plasma ACTH concentrations, whereas pretreatment with hexamethonium was without significant effect. The intracerebroventricular injection of ACh also increased plasma ACTH concentrations in a dose-dependent manner in conscious rats, but not in pentobarbital-anesthetized animals. Thus, cholinergic hypothalamic input stimulates CRF gene expression in the PVN and CRF secretion into the portal circulation under physiological conditions. The use of conscious animals is essential in elucidating the physiological roles of neurotransmitters and other modulators regulating CRF neurons.

Effect of acetylcholine on corticotropin-releasing factor gene expression in the hypothalamic paraventricular nucleus of conscious rats

To examine the physiological role of cholinergic input in the regulation of CRF neurons in the paraventricular nucleus (PVN) of the hypothalamus, acetylcholine (ACh) was microinjected bilaterally into the dorsolateral border of the PVN of conscious rats. Changes in the levels of POMC messenger RNA (mRNA) in the anterior pituitary, CRF mRNA in hypothalamic tissue containing the PVN, and plasma ACTH were assessed. Plasma ACTH concentrations increased in a dose-dependent manner after ACh injection (1-100 pmol/side), reaching a peak 30 min after ACh injection and returning to baseline within 120 min. The POMC mRNA level in the anterior pituitary and the hypothalamic CRF mRNA level increased in a dose-dependent manner 120 min after ACh (0.1-10 pmol/side) injection. Intracerebroventricular pretreatment with atropine completely abolished the ACh-induced increase in plasma ACTH concentrations, whereas pretreatment with hexamethonium was without significant effect. The intracerebroventricular injection of ACh also increased plasma ACTH concentrations in a dose-dependent manner in conscious rats, but not in pentobarbital-anesthetized animals. Thus, cholinergic hypothalamic input stimulates CRF gene expression in the PVN and CRF secretion into the portal circulation under physiological conditions. The use of conscious animals is essential in elucidating the physiological roles of neurotransmitters and other modulators regulating CRF neurons.

Elevated plasma adrenocorticotropin concentration as evidence of limited adrenocortical reserve in patients with adrenomyeloneuropathy.

Adrenal insufficiency occurs in approximately two thirds of patients with adrenomyeloneuropathy. Its development may precede or follow the onset of neurological disease. To define the sensitivity of various tests of adrenal cortical function, we reviewed adrenocortical function tests in 28 patients with adrenomyeloneuropathy who had normal ACTH stimulation tests at the time of enrollment into an ongoing dietary study. Endocrine studies performed at 6-month intervals included a conventional ACTH stimulation test, plasma ACTH and cortisol concentrations, and 24-h urinary cortisol and aldosterone excretion rates. Eleven patients (39%) developed an elevated plasma ACTH concentration after a median follow-up of 3 yr. Their 24-h urinary cortisol and plasma basal and ACTH-stimulated cortisol concentrations were all normal. The mean basal cortisol level tended to be lower in patients who had increased plasma ACTH levels than in patients who maintained a normal plasma ACTH concentration during the study period (408 +/- 22 vs. 491 +/- 33 nmol/L; P = 0.05). Patients who had an increased plasma ACTH concentration and symptoms consistent with adrenal insufficiency had a lower mean 24-h urinary free cortisol level than those with normal plasma ACTH (196 +/- 22 vs. 281 +/- 30 nmol/day; P < 0.05). Plasma ACTH concentrations were persistently elevated in six patients in whom subsequent values were available. One individual later developed a subnormal ACTH-stimulated cortisol concentration consistent with overt adrenal insufficiency. Our results suggest that conventional provocative and integrative tests of adrenocortical function may not be sufficient to identify patients with adrenomyeloneuropathy who have compensated adrenal hypofunction. An elevated plasma ACTH concentration may represent an early marker for adrenocortical dysfunction and incipient adrenal insufficiency in AMN patients.

Surgical disconnection of the hypothalamus from the fetal pituitary abolishes the corticotrophic response to intrauterine hypoglycemia or hypoxemia in the sheep during late gestation.

We have investigated the ACTH and cortisol responses to acute episodes of hypoxemia or hypoglycemia in fetal sheep in which the hypothalamus and pituitary were surgically disconnected at between 112 and 123 days gestation. Before 130 days gestation, basal plasma concentrations of ACTH were significantly greater in the hypothalamo-pituitary disconnected (HPD) fetuses than in the intact fetal sheep (126-130 days; 105.0 +/- 11.4 ng/liter, HPD group; 64.0 +/- 9.5 ng/liter, intact group). After 130 days, however, there was no difference between plasma ACTH concentrations in the HPD (136-140 days; 154.7 +/- 16.7 ng/liter HPD group; 113.6 +/- 19.1 ng/liter, intact group) and intact fetal sheep, and in both groups the mean ACTH concentrations were significantly greater after 136 days gestation than before 130 days. In the HPD group, however, while the plasma ACTH concentrations were elevated there was no prepartum increase in the plasma concentrations of cortisol. A decrease in the fetal arterial blood PO2 by approximately 50% for 30 min between 123 and 132 days, stimulated a significant increase in fetal ACTH and cortisol concentrations in the intact but not in the HPD fetuses. In the intact group, plasma ACTH concentrations were also significantly increased (P less than 0.001) above control values (98.2 +/- 21.2 ng/liter) at 120 min after the start of an iv infusion of insulin (1 IU/60 min) (517.2 +/- 160.5 ng/liter) and were still elevated at 60 min after the end of the infusion period (1248.1 +/- 643.2 ng/liter). In the HPD fetuses, however, there was no significant change in plasma ACTH concentrations during or after the insulin infusion. In both the HPD and intact groups, there was a significant increase in plasma ACTH concentrations above control values (62.5 +/- 8.5 ng/liter intact; 135.0 +/- 30.6 ng/liter, HPD) after intrafetal administration of CRF (+10 min; 117.5 +/- 8.1 ng/liter, intact; 225.3 +/- 33.1 ng/liter, HPD) indicating that the secretory capacity of the pituitary corticotrophs was not reduced by the HPD procedure. Our results demonstrate that an intact hypothalamic-pituitary connection is required to generate a normal prepartum increase in fetal cortisol concentrations and is essential for an appropriate fetal pituitary-adrenal response to intrauterine hypoxemia and hypoglycemia.

Neuroendocrine and immunological mechanisms in stress-induced immunomodulation

Here, we report that emotional stressors (restraint, footshock) can affect humoral immune responses as well as the capacity of immune and accessory cells to secrete interleukins. Acute restraint stress (5 min) caused a 4- to 6-fold enhancement of splenic antibody responses to sheep red blood cells. In an attempt to study endocrine mechanisms, we administered antibodies raised in rats to corticotropin releasing factor (CRF). Intravenous administration of these antibodies prior to stress-exposure and immunization prevented the stress-induced increase in the humoral response. In a parallel experiment, we observed that CRF-immunoneutralization prevented the restraint stress-induced increase in plasma ACTH concentrations, but was without effect on plasma prolactin, melanocyte stimulating hormone, adrenaline and noradrenaline responses. These data suggest the presence of an indirect pathway involving ACTH and related peptides by which CRF controls humoral responses to stress. A pathway involving a direct mechanism of CRF at the level of the immune cells will be discussed. In a set of other experiments, we addressed the question of whether interleukin-1 and interleukin-6 plasma levels induced by injection of endotoxin could be modulated by emotional stress. Exposure to prolonged footshock stress (20 min) prior to endotoxin injection resulted in a blunted plasma ACTH and interleukin-1 response, without affecting the endotoxin-induced plasma interleukin-6 respose. These data suggest that at least one level at which emotional stress may influence immune function is by changing the capacity of immune cells to produce and/or secrete immune regulatory interleukins.

Atriopeptin Inhibits Stimulated Secretion of Adrenocorticotropin in Rats: Evidence for a Pituitary Site of Action*

The aim of this study was to resolve previous controversies regarding the effect of atriopeptin on the secretion of ACTH in vivo. Male Wistar rats were used throughout. The animals were subjected to lesioning of the hypothalamic paraventricular nucleus (PVN) or sham operation and implanted with indwelling jugular cannulae 5 days later for blood sampling and drug infusion. Two days after the insertion of the cannulae the animals were treated with saline or 103-126 amino acid residue atriopeptin iv: a bolus injection was given (200 or 40 pmol/rat) followed by an infusion (40 or 8 pmol/min) which was maintained for the entire duration of the experiment (70 min). Ten minutes after the bolus of atriopeptin the animals received iv a combination of 1 pmol 41-residue CRF and 10 pmol arginine vasopressin (CRF/AVP) to stimulate ACTH secretion. Serial blood samples (0.1 ml) were obtained at -10 min and immediately before the injection of CRF/AVP and at 5, 10, 20, 30, and 60 min afterwards. Plasma ACTH concentration was measured by RIA. In sham-operated rats CRF/AVP caused a 4-fold increase in plasma ACTH which peaked at 5 min and returned to baseline by 60 min. In sham-operated rats the higher dose of atriopeptin (200 pmol bolus, 40 pmol/min infusion) did not alter the effect of the stimulus between 5 and 30 min, and augmented plasma ACTH at 60 min. The smaller dose of atriopeptin reduced plasma ACTH at 10 and 20 min by 54% and 48%, respectively, and also decreased by 48% the net amount of ACTH released over 30 min in response to CRF/AVP. When given alone, the higher dose of atriopeptin caused a persistent (60 min) 10-13% reduction of mean arterial blood pressure, while the lower dose decreased blood pressure by about 9% for less than 10 min. In parallel, the higher dose of atriopeptin increased plasma ACTH concentration while the lower dose produced no change. In PVN-lesioned rats the CRF/AVP induced ACTH response was similar to that seen in sham-operated controls. Only the higher dose of atriopeptin was tested, and this markedly reduced CRF/AVP stimulated ACTH secretion at 5-60 min after CRF/AVP. Given alone, atriopeptin had no marked effect on plasma ACTH in PVN-lesioned rats, while its hypotensive action was similar to that in sham-operated animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Galaninergic Mechanisms Are Involved in the Regulation of Corticotropin and Thyrotropin Secretion in the Rat*

Galanin (GAL), a 29-amino acid peptide, affects the secretion of several anterior pituitary hormones, including PRL and GH. Since GAL coexists with vasopressin and CRH in the hypothalamic paraventricular nucleus (PVN), we have studied the pharmacological and physiological actions of GAL on ACTH and TSH secretion in freely moving male rats. Cannulae were surgically implanted in the right atria and brain, intraventricular or adjacent to the PVN, of adult Sprague-Dawley rats. Seven days later, GAL (500 or 1000 ng) or saline was infused into the PVN, and serial blood samples were obtained 5, 10, 20, and 40 min after the infusion. Some animals were also stressed by the inhalation of ether vapors for 2 min after the PVN infusion. Basal ACTH concentrations were increased 2-fold in saline-treated rats; however, plasma ACTH levels were unchanged after GAL infusion. The exposure of rats to ether vapors for 2 min after the infusion of saline into the PVN increased plasma ACTH concentrations from 22.8 +/- 6.0 to 596.6 +/- 59.9 pg/ml 10 min later. However, the infusion of GAL into the PVN attenuated stress-induced ACTH secretion. After GAL infusion, peak ACTH levels (332.7 +/- 84.0 pg/ml) were attained 5 min after ether exposure, followed by a rapid decline at 10 min (P less than 0.001) and 20 min (P less than 0.05). Plasma TSH concentrations were unchanged by GAL or saline infusion and were not affected by ether vapor inhalation. To determine the physiological significance of GAL in the control of ACTH and TSH secretion, endogenous GAL was immunoneutralized by the infusion of 3 microliters GAL antiserum (GAL-AS) into the third cerebral ventricle 25 and 1 h before withdrawing blood samples every 15 min for 6 h. Animals treated with normal rabbit serum (NRS) served as controls. Plasma ACTH concentrations were unchanged by NRS during the 6-h period. However, infusion of GAL-AS raised plasma ACTH concentrations to over 400 pg/ml 75 min after infusion in some animals. In general, plasma ACTH concentrations were increased 4 h of the 6-h sampling period compared to levels in NRS-treated controls. In contrast, GAL-AS reduced TSH concentrations by 50% compared to control values. In contrast to these marked actions of centrally administered GAL, ACTH secretion from dispersed anterior pituitary cells in vitro was unaffected by GAL in concentrations up to 10(-6) M. Furthermore, GAL did not alter CRH (1 nM)-induced ACTH secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of Cortisol Infusion on Basal and Corticotropin-eleasing Factor (CRF)-Stimulated Plasma ACTH Concentrations in the Sheep Fetus after Surgical Isolation of the Pituitary*

Although it has been demonstrated that glucocorticoids can inhibit basal and stimulus-induced increase in fetal plasma ACTH concentrations, the site(s) of action of the glucocorticoids in the fetal hypothalamo-pituitary axis are unclear. We have investigated the ontogeny of the negative feedback effect of cortisol on basal and CRF-stimulated ACTH secretion in intact fetal sheep and in fetal sheep after surgical disconnection of the hypothalamus and pituitary (HPD). In saline-pretreated fetal sheep there was a significant increase in plasma ACTH concentrations in response to an ovine CRF (oCRF) bolus (1 microgram) in HPD and intact fetuses at 120-128 days and at 138-149 days gestation. In the intact group the ACTH response at 120-240 min after oCRF was significantly lower (P less than 0.01) after 138 days compared with before 128 days. In this group the cortisol response during the first hour after oCRF was significantly greater (P less than 0.005) after 138 days than in the younger fetuses. In the HPD group there was no significant difference between the two gestational age ranges in either the ACTH or cortisol responses to oCRF. Between 120 and 128 days gestation, increasing plasma cortisol concentrations (110-170 nmol/liter above baseline values) by exogenous infusion had no significant effect on the basal fetal plasma ACTH concentrations in HPD or in intact fetuses. In contrast, after 138 days gestation, the basal plasma ACTH concentrations were significantly decreased by 50% during the cortisol infusion period in the intact but not in the HPD group. It would appear therefore, that the development of the negative feedback action of cortisol to suppress basal fetal plasma concentrations of ACTH is dependent on the presence of a functional fetal hypothalamus. In HPD and intact fetuses cortisol infusion abolished the fetal ACTH response to the oCRF bolus from as early as 120 days gestation. Thus the negative feedback action of cortisol on the CRF-induced increases in fetal plasma ACTH concentrations can occur directly at the fetal pituitary.

Corticotropin and Cortisol Secretion after Central 5-Hydroxytryptamine-1A (5-HT1A) Receptor Activation: Effects of 5-HT Receptor andβ-Adrenoceptor Antagonists

To explore the involvement of 5-hydroxytryptamine-1A (5-HT1A) receptors in hypothalamic-pituitary-adrenal (HPA) axis regulation, various doses of ipsapirone (IPS), a centrally acting pyrimidinylpiperazine with considerable affinity and selectivity for 5-HT1A recognition sites, were administered to normal subjects. IPS dose-dependently increased plasma ACTH concentrations from -78 +/- 63 to 614 +/- 250 pmol.min/L (P less than 0.01) and plasma cortisol concentrations from -10.8 +/- 2.9 x 10(3) to 21.3 +/- 8.2 x 10(3) nmol.min/L (P less than 0.01) at a dose of 0.3 mg/kg in six men. The nonselective 5-HT receptor antagonist metergoline which acts at both 5-HT1 and 5-HT2 receptors partially blocked the HPA response to IPS in six women and three men. The mean maximal integrated ACTH response decreased from 746 +/- 297 to 40 +/- 146 pmol/min.L (P less than 0.05), and the increase in cortisol was attenuated from 22.9 +/- 9.9 x 10(3) to 11.9 +/- 6.3 x 10(3) nmol.min/L (P less than 0.05). The nonselective beta-adrenergic and selective 5-HT1A/1B receptor antagonist (+/-)pindolol was without effect on basal HPA activity, but completely antagonized the IPS-induced plasma ACTH and cortisol responses. The mean maximal integrated ACTH response decreased to 8.0 +/- 78 pmol.min/L (P less than 0.05), and the cortisol response was reduced to -2.3 +/- 6.5 x 10(3) nmol.min/L (P less than 0.05). The selective beta 1-adrenoceptor antagonist betaxolol did not significantly alter the IPS-induced HPA response.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin Agonists Cause Parallel Activation of the Sympathoadrenomedullary System and the Hypothalamo-Pituitary-Adrenocortical Axis in Conscious Rats

The effects of three potent serotonin (5-HT) agonists with different structures and 5-HT receptor binding profiles on sympathoadrenomedullary and hypothalamo-pituitary-adrenocortical axis functions were assessed in conscious, freely moving male Sprague-Dawley rats. The 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5-HT1C agonist, m-chlorophenylpiperazine (m-CPP), and the 5-HT2/5-HT1C agonist, 1-(2,5-dimethoxy-4-iodophenyl)2-amino-propane (DOI), all produced marked dose-dependent increases in plasma epinephrine and ACTH concentrations. Both epinephrine and ACTH responses peaked at 10 min and showed strong positive correlations across all drugs and doses studied. Corticosterone increases showed a saturable response pattern and were close to the maximum level with a relatively small (approximately 2.5-fold) increase in plasma ACTH concentrations. Norepinephrine levels showed small dose-dependent increases after 8-OH-DPAT and m-CPP and decreases after DOI treatment. These results suggest that both the sympathoadrenomedullary system and the hypothalamo-pituitary-adrenocortical axis can be activated via 5-HT1 and 5-HT2 receptors and that these two systems may have common or similar regulatory mechanisms triggered by these stimuli.