ACTH Secretagogues Research Articles

FRI187 Increased Prolactin Secretion In Inferior Petrosal Sinus Sampling Following Desmopressin Administration In Patients With ACTH-Dependent Cushing’s Syndrome

Abstract Disclosure: M. Guay-Gagnon: None. N. Younes: None. A. La Fontaine: None. S. Larose: None. É. Thérasse: None. A. Lacroix: None. Background: Previous studies have described an increase in central prolactin (PRL) secretion in response to corticotropin-releasing hormone (CRH) during inferior petrosal sinus sampling (IPSS) in a proportion of patients with Cushing’s disease (CD). Desmopressin is used during IPSS instead of CRH because of its wider availability, lower cost, and similar accuracy. It is unknown whether central PRL secretion can also increase after desmopressin as it does after CRH. Objective: Evaluate the central PRL response to desmopressin administration in patients with ACTH-dependent Cushing’s syndrome (CS) undergoing IPSS. Method: We conducted a retrospective study of patients who underwent IPSS in our center between January 2015 and May 2022. IPSSs using desmopressin were collected, as well as those using either CRH (1 mcg/kg IV bolus) or CRH + desmopressin for comparison. ACTH and PRL were measured in bilateral petrosal sinuses at −5, 0, and 3, 5, 10 minutes after a 10 mcg IV desmopressin bolus; simultaneous peripheral ACTH, PRL and cortisol were measured at -5, 0, 3, 5, 10, 30, 45 and 60 minutes. A >50% central PRL increase from the average of −5 and 0 minutes values to peak value was considered a positive response. Results: 30 IPSSs performed with desmopressin were included: 24 patients with CD and 6 patients with ectopic ACTH secretion (EAS). 73.3% were women and mean age was 48.2 ± 14.8 y.o. 12 patients with CD (50%) had a >50% PRL increase on at least one side. The peak PRL value and highest central/peripheral (C/P) ACTH ratio after desmopressin were ipsilateral in 10 out of 12 patients (83%). Among the 9 patients whose pathology was available, only one showed co-expression of ACTH and PRL on immunohistochemistry (IHC), along with GH. In contrast, among the 12 patients with no PRL response, 2 had ACTH and PRL co-expression, and 1 had PRL expression only. No somatic genetic USP8 analyses were performed. Among the 6 patients with EAS, none had a central or peripheral PRL response after desmopressin. 21 IPSSs with either CRH or CRH + desmopressin were collected. 14 out of 18 patients (77.8%) with CD had a >50% PRL increase after stimulation. For 10 out of these 14 patients (71.4%), peak PRL value and C/P ACTH ratio were ipsilateral. 2 out of 3 IPSSs with CRH or CRH + desmopressin in patients with EAS (one resected pulmonary neuroendocrine tumor and one occult EAS) had a >50% PRL increase after stimulation. Conclusion: In our study, 50% of IPSSs showed a significant central PRL increase after desmopressin in patients with CD, but none in patients with EAS. In IPSSs with CRH or CRH + desmopressin, a significant PRL increase was observed in 77.8% of patients with CD, and 2 out of 3 patients with EAS. On pathology, there was no correlation with PRL expression on IHC. Further research is needed to better understand the mechanisms for prolactin secretion following ACTH secretagogue administration and its potential clinical implications. Presentation: Friday, June 16, 2023

The vasotocinergic system and its role in the regulation of stress in birds.

The regulation of stress in birds includes a complex interaction of neural systems affecting the hypothalamic-pituitary-adrenal (HPA) axis. In addition to the hypothalamic paraventricular nucleus, a structure called the nucleus of the hippocampal commissure likewise affects the output of pituitary stress hormones and appears to be unique to avian species. Within the anterior pituitary, the avian V1a and V1b receptors were found in corticotropes. Based on our studies with central administration of hormones in the chicken, corticotropic releasing hormone (CRH) is a more potent ACTH secretagogue than arginine vasotocin (AVT). In contrast, when applied peripherally, AVT is more efficacious. Co-administration of AVT and CRH peripherally, resulted in a synergistic stimulation of corticosterone release. Data suggest receptor oligomerization as one possible mechanism. In birds, vasotocin receptors associated with stress responses include the V1a and V1b receptors. Three-dimensional, homology-based structural models of the avian V1aR were built to test agonists and antagonists for each receptor that were screened by molecular docking to map their binding sites on each receptor. Additionally, binding affinity values for each available peptide antagonist to the V1aR and V1bR were determined. An anterior pituitary primary culture system was developed to determine how effective each antagonist blocked the function of each receptor in culture when stimulated by a combination of AVT/CRH administration. Use of an antagonist in subsequent in vivo studies identified the V1aR in regulating food intake in birds. The V1aR was likewise found in circumventricular organs of the brain, suggesting a possible function in stress.

Differential delayed responses of arginine vasotocin and its receptors in septo-hypothalamic brain structures and anterior pituitary that sustain hypothalamic–pituitary-adrenal (HPA) axis functions during acute stress

Recently, we proposed that corticotropin releasing hormone (CRH) neurons in the nucleus of hippocampal commissure (NHpC), located in the septum, function as a part of the traditional hypothalamic–pituitary-adrenal (HPA) axis in avian species. CRH and its receptor, CRHR1, are regulated differently in the NHpC compared to the paraventricular nucleus (PVN) following feed deprivation (FD). Therefore, we followed up our work by examining arginine vasotocin (AVT), the other major ACTH secretagogue, and its receptors, V1aR and V1bR, gene expression during FD stress in the NHpC, PVN, and ventral mediobasal hypothalamus/median eminence (MBHv/ME). The objectives were to 1) identify AVT perikarya, fibers and its two major receptors, V1aR and V1bR, in the NHpC, PVN, and MBHv/ME using immunohistochemistry, 2) determine the effect of stress on AVT, V1aR and V1bR mRNA expression in the same three brain structures, NHpC, PVN, and MBHv/ME; and, 3) ascertain the expression pattern of V1aR and V1bR mRNA in the anterior pituitary and measure plasma stress hormone, corticosterone (CORT), concentration following FD stress. Male chicks (Cobb 500), 14 days of age, were divided into six groups (10 birds/treatment) and subjected to different times of FD stress: (Control, 1 h, 2 h, 3 h, 4 h, and 8 h). For each bird, blood, brain, and anterior pituitary were sampled and frozen immediately. The NHpC, PVN, and MBHv/ME were micro-dissected for RT-PCR. Data were analyzed using one-way ANOVA followed by Tukey Kramer HSD test using a significance level of p < 0.05. Perikarya of AVT neurons were identified in the PVN but not in the NHpC nor MBHv/ME, and only V1aR-immunoreactivity (ir) was observed in the three structures, however, gene expression data for AVT and its two receptors were obtained in all structures. Both AVT and V1aR mRNA are expressed and increased significantly in the PVN following FD stress (p < 0.01). For the first time, V1bR mRNA was documented in the avian brain and specifically shown upregulated in the NHpC and PVN (p < 0.01) following stress. Additionally, delayed significant gene expression of AVT and its receptors in the PVN showed a positive feedback relationship responsible for maintaining CORT release. In contrast, a significant downregulation of AVT mRNA and upregulation of V1aR mRNA occurred in the NHpC (p < 0.01) during FD showing a negative feedback relationship between AVT and its receptors, V1aR and V1bR. Within the MBHv/ME and anterior pituitary, a gradual increase of AVT mRNA in PVN as well as MBHv/ME was associated with significant upregulation of V1bR (p < 0. 01) and downregulation of V1aR (p < 0.01) in both MBHv/ME and anterior pituitary indicating AVT regulates its receptors differentially to sustain CORT release and control overstimulation of the anterior pituitary during a stress response.

DIAGNOSIS OF ENDOCRINE DISEASE: The role of the desmopressin test in the diagnosis and follow-up of Cushing's syndrome.

Desmopressin is a vasopressin analogue selective for type 2 vasopressin receptors that mediate renal water retention. In contrast to the native hormone arginine vasopressin, a well-known ACTH secretagogue, desmopressin, exerts minimal or no activity on ACTH excretion. However, in a substantial proportion of patients with ACTH-dependent Cushing's syndrome (CS), desmopressin elicits an ACTH and cortisol response, which contrasts with the minimal responses obtained in healthy subjects. The mechanism underlying this paradoxical response involves upregulation of vasopressin type 3 and/or the aberrant expression of type 2 receptors by neoplastic ACTH-producing cells. This makes desmopressin administration a suitable test enabling the distinction between neoplastic from functional (formerly termed 'pseudo-Cushing syndrome') ACTH-dependent cortisol excess. Several studies have now established an adjunctive role of desmopressin in the initial diagnostic workup of CS. Despite some early data indicating that this test may also have a role in distinguishing between Cushing's disease (CD) and ectopic ACTH secretion, subsequent studies failed to confirm this observation. The ability of the paradoxical response to desmopressin to depict the presence of neoplastic ACTH-secreting cells was also exploited in the follow-up of patients with CD undergoing surgery. Loss of the desmopressin response, performed in the early postoperative period, was a good predictor for a favorable long-term outcome. Moreover, during follow-up, reappearance of desmopressin paradoxical response was an early indicator for recurrence. In conclusion, the desmopressin test is a valid tool in both the diagnosis and follow-up of patients with CD and should be more widely applied in the workup of these patients.

Gestational hypoxia modulates expression of corticotropin-releasing hormone and arginine vasopressin in the paraventricular nucleus in the ovine fetus.

Maturation of the fetal hypothalamo-pituitary-adrenocortical (HPA) axis is critical for organ maturation necessary for the fetus to transition to the ex-utero environment. Intrauterine stressors can hasten maturation of the HPA axis leading to fetal growth restriction and in sheep, premature birth. We have previously reported that high-altitude mediated, long-term-moderate gestational hypoxia (LTH) during gestation has a significant impact on the fetal HPA axis. Significant effects were observed at the level of both the anterior pituitary and adrenal cortex resulting in elevated plasma ACTH during late gestation with decreased adrenocortical expression of enzymes rate limiting for cortisol synthesis. As such, these fetuses exhibited the normal ontogenic rise in fetal plasma cortisol but an exaggerated cortisol response to acute stress. This study extended these findings to ACTH secretagogue expression in the PVN using insitu hybridization. We report that the expression of AVP but not CRH was increased in the medial parvocellular PVN (mpPVN) in the LTH fetus. This represented an increase in both AVP mRNA per neuron as well as an increase in AVP hybridizing neurons with no increase in mpPVN CRH neurons. LTH had no effect on PVN volume, area of CRH or AVP hybridization, thus LTH did not have a trophic effect on the size of the nucleus. In conclusion, there appears to be a switch from CRH to AVP as a primary ACTH secretagogue in response to LTH, supporting our previous findings of increased anterior pituitary sensitivity to AVP over CRH in the LTH fetus.

The effects of ghrelin/GHSs on AVP mRNA expression and release in cultured hypothalamic cells in rats

Ghrelin, the endogenous ligand for growth hormone secretagogues (GHSs) receptor (GHS-R), increases adrenocorticotropin (ACTH) and cortisol (corticosterone) as well as GH secretion in humans and animals. However, the site of GHSs action to induce ACTH secretion is not fully understood. To clarify the mechanisms of the action of ghrelin/GHSs on ACTH secretion, we analyzed the effects of KP-102 and ghrelin on the mRNA expression and release of corticotropin releasing factor (CRF) and arginine vasopressin (AVP), ACTH secretagogues, in monolayer-cultured hypothalamic cells of rats. Incubation of cells with KP-102 for 4 h and 8 h and with ghrelin for 4 h significantly increased AVP mRNA expression and release without changing CRF mRNA expression. CRF levels in culture media were undetectable. Suppression of GHS-R expression by siRNA blocked ghrelin- and KP-102-induced AVP mRNA expression and release. NPY significantly increased AVP mRNA expression and release. Furthermore, treatment of cells with anti-NPY IgG blocked KP-102-induced AVP mRNA expression and release. We previously reported that KP-102 significantly increases NPY mRNA expression in cultured hypothalamic cells. Taken together, these results suggest that ACTH secretion by ghrelin/GHSs is induced mainly through hypothalamic AVP, and that NPY mediates the action of ghrelin/GHSs.

Reciprocal Regulation of TREK-1 Channels by Arachidonic Acid and CRH in Mouse Corticotropes

Arachidonic acid (AA) is generated in the anterior pituitary gland upon stimulation by the ACTH secretagogue, CRH. Using the patch clamp technique, we examined the action of AA on the excitability of single pituitary corticotropes obtained from a transgenic mouse strain that expresses the enhanced green fluorescent protein driven by the proopiomelanocortin promoter. CRH evoked depolarization, but AA caused hyperpolarization. Under voltage clamp condition, AA caused a rapid inhibition of the delayed rectifier K(+) current and then increased a background K(+) current. Inhibition of AA metabolism did not prevent the activation of the K(+) current by AA, suggesting a direct action of AA. The sensitivity of the AA-activated K(+) current to fluoxetine, chlorpromazine, extracellular acidification, diphenylbutylpiperidine antipsychotics, and the membrane permeable cAMP analog [8-(4-chlorophenylthio)-cAMP] suggest that the current is mediated via TWIK-related K(+) channel (TREK)-1 channels. Activation of the CRH receptors that are coupled to the adenylate cyclase pathway suppressed the activation of TREK-1 current by AA and reversed the AA-mediated hyperpolarization. Intracellular acidification (pH 7.0) increased the basal amplitude of TREK-1 current and resulted in hyperpolarizaton. CRH suppressed the basal TREK-1 current in cells with intracellular acidification and caused depolarization. Our finding indicates that TREK-1 channels are important in setting the resting potential in corticotropes. The opposing actions of CRH and AA on the excitability of corticotropes raise the possibility that AA may act as a negative feedback regulator to reduce the stimulatory action of CRH and thus prevent excessive ACTH release during chronic stress.

Long-term hypoxia enhances ACTH response to arginine vasopressin but not corticotropin-releasing hormone in the near-term ovine fetus

This study tested the hypothesis that long-term hypoxia (LTH) results in enhanced fetal corticotrope sensitivity to the ACTH secretagogues, corticotropin-releasing hormone (CRH), and AVP. Ewes were maintained at high altitude (3,820 m) from 40 to 130-131 days of gestation. Upon return to the laboratory, hypoxia was maintained by maternal nitrogen infusion. Vascular catheters were placed in both LTH (n = 4) and normoxic controls (n = 4). Each fetus received a 15-min infusion of either saline, 100 ng/kg of ovine CRH, or 20 ng/kg of AVP/min over 3 consecutive days in a randomized order. Fetal blood samples were collected at 0, 15, 30, 60, and 90 min after the start of infusion and analyzed for ACTH(1-39), ACTH precursors, and cortisol. Anterior pituitaries were collected from additional noninstrumented fetuses for analysis of vasopressin receptor 1b (V1b) mRNA and protein. Basal plasma concentrations of both ACTH(1-39) and ACTH precursors were higher in LTH fetuses and were not altered by saline infusion. In response to CRH, ACTH(1-39) increased in both groups and was higher in the LTH group compared with control (P < 0.05). When analyzed as sum of ACTH(1-39) released (Delta0-90 min) above basal, CRH released equal amounts of ACTH(1-39) in both groups. In LTH fetuses, AVP evoked a greater ACTH(1-39) release (P < 0.05) when analyzed as an increased sum of ACTH(1-39) (Delta0-90 min) above basal. Both CRH and AVP elicited a release of ACTH precursors with no differences observed between LTH and control. AVP and CRH elicited significant increases in cortisol, which were higher in response to AVP than CRH. V1b mRNA and protein were elevated in the anterior pituitary of LTH fetuses compared with control. LTH significantly increases pituitary sensitivity to AVP. This enhanced sensitivity may be a mechanism of our previously observed enhanced corticotrope function.

Euthanasia in Endocrinology: The Choices Get More Complex

In 1927, the German physicist Werner Heisenberg published a paper that developed the principle of imprecision or uncertainty in measuring the position and momentum of subatomic particles; simply stated, “the more precisely the position is determined, the less precisely the momentum is known, . . . and vice versa.” This principle reflects a fact of life that most scientists learn at an early age:preciseexperimentalmeasurementsmayonlybepossibleunder conditions in which the properties of the system can no longer be completely defined. For endocrinologists, the problems are particularly acute because of the sensitivity of endocrine systems to metabolic, sensory, or pharmacologically induced perturbation. This makes itdifficult tostudythese systems invivo,without introducing the potential for experimentally induced error. The challenge is to develop procedures that induce as little disturbance of normal hormonal interactions as possible. Experiments involving euthanasia present a particular problem because of the need to balance ethical concerns in the treatment of animals with avoidance of procedures that might alter the experimental results. Most countries have now adopted guidelines for research that stress the principle that animals should not be unnecessarily exposed to pain and suffering. In the euthanasia guidelines of the American Veterinary Medical Association, for example, euthanasia isdefinedastheactof inducinghumanedeathinananimal, “with an emphasis on making the death as painless and distress free as possible” (1). Ensuring that death is distress free, however, may be possible only by depressing consciousness, through modulation of neuronal activity in the central nervous system. This is hard to do without also affecting the hypothalamic and limbic brain centers that normally regulate neuroendocrine function. Measures to achieve painless euthanasia may, therefore, themselves alter the endocrine endpoints under study, particularly if those endpoints are sensitive to changes in neuroendocrine hormone secretion. The demonstration in the mid-1970s that neural activity may continue in the brain for 13–14 sec after decapitation (2) resulted in adoption of policies encouraging sedation or complete anesthesia beforephysicalmethodsofeuthanasia,basedonthehypothesis that continued brain activity might reflect a perception of pain. Because many inhalant and noninhalant anesthetic drugs themselves induce stress responses and changes in hormone levels, their usefulness is limited (3,4). Several studies, however, indicated that sedationwith CO2 gas might provide a nonstressful alternative. Exposure to CO2 concentrations as low as 7.5% in air increases pain thresholds, whereas30–100%CO2 canproducerapid lossofconsciousness (5, 6). Importantly, brief exposure to CO2 ( 30 sec) did not seem to increase the levelsofACTH,ahormone that isnotoriously sensitive tostress (7).Forthisreason,CO2sedationhasbecomeawidelyused method to limit pain and distress before euthanasia. In the current issue of Endocrinology, however, Reed et al. (8) report that only 20–25 sec exposure of male rats to CO2 gas, sufficient to render them recumbent and unresponsive but still alive, results in a dramatic elevation in the levels of vasopressin and oxytocin in the circulation. Using methods based on a combination of RIA with liquid capillary chromatography-mass spectrometry to achieve definitive identification of the peptides, Reed et al. (8) show that plasma levels of vasopressin and oxytocin increase at least 10fold after brief CO2 exposure. The authors speculate that these findings may explain previous observations of CO2 induced analgesia, because of the reported ability of iv vasopressin to induce non-opioidergic analgesic effects (9) as well as observations on induction of ACTH release after longer periods (2 min) of CO2 exposure (7). The mechanisms of these effects remain unclear. Reed et al. (8) speculate that it may involve changes in a number of neurotransmitter systems in the brain, perhaps secondary to changes in blood pressure triggered by CO2 exposure, but additional work will be required to define the underlying mechanisms, given that the systems in the brain that control the posterior pituitary may also influence other aspects of endocrine function. The data also have ramifications for studies that may not appear to have a direct neuroendocrine connection, because of the powerful effects of the posteriorpituitaryhormonesaroundthebody. Inadditionto itsactions as an ACTH secretagogue, vasopressin increases blood pressure by increasing peripheral vascular resistance (10), stimulates water resorption in the kidney (11), and influences aggressive and social

Adrenalectomy enhances endotoxemia-induced hypophagia: higher activation of corticotrophin-releasing-factor and proopiomelanocortin hypothalamic neurons

Inflammatory and infectious processes evoke neuroendocrine and behavioral changes known as acute-phase response that includes activation of the hypothalamo-pituitary-adrenal (HPA) axis and reduction of food intake. Besides its action as the most important ACTH secretagogue, corticotrophin-releasing factor (CRF), synthesized in the paraventricular nucleus (PVN), is also involved in the control of food intake. Alpha-melanocyte stimulating hormone (α-MSH) in the arcuate nucleus also plays a role in the energy homeostasis, possessing anorexigenic effects. To investigate the participation of neuropeptides involved in the regulation of food intake during endotoxemia, we administrated lipopolysaccharide (LPS) in sham-operated and adrenalectomized (ADX) male Wistar rats to evaluate food intake, hormone responses and Fos-CRF and Fos-α-MSH immunoreactivity in the PVN and arcuate nucleus, as well as CRF and POMC mRNA expression in these hypothalamic nuclei. In sham-operated rats, treatment with LPS (100 µg/kg) showed lower food intake, higher plasma ACTH and corticosterone levels, as well as an increase in Fos-CRF double labeled neurons and CRF mRNA expression in the PVN, with no changes in Fos-α-MSH immunoreactivity and POMC mRNA expression in the arcuate nucleus, compared to saline treated rats. After LPS treatment, ADX rats showed further increase in plasma ACTH levels, marked decrease of food intake, higher Fos-CRF immunoreactive neurons in the PVN and CRF mRNA expression, as well as an increase in Fos-α-MSH immunoreactivity and POMC mRNA expression in the arcuate nucleus, compared to sham-operated rats treated with LPS. In conclusion, the present data indicate that the marked hypophagia during endotoxemia following ADX is associated with an increased activation of CRF and POMC neurons in the hypothalamus and an increased mRNA expression of these neuropeptides.

The hypothalamic-pituitary-adrenal axis response to stress in mice lacking functional vasopressin V1b receptors.

The role of arginine vasopressin (Avp) as an ACTH secretagogue is mediated by the Avp 1b receptor (Avpr1b) found on anterior pituitary corticotropes. Avp also potentiates the actions of CRH (Crh) and appears to be an important mediator of the hypothalamic-pituitary-adrenal axis response to chronic stress. To investigate the role of Avp in the hypothalamic-pituitary-adrenal axis response to stress, we measured plasma ACTH and corticosterone (CORT) levels in Avpr1b knockout (KO) mice and wild-type controls in response to two acute (restraint and insulin administration) and one form of chronic (daily restraint for 14 d) stress. No significant difference was found in the basal plasma levels of ACTH and CORT between the two genotypes. Acute restraint (30 min) increased plasma ACTH and CORT to a similar level in both the Avpr1b mutant and wild-type mice. In contrast, plasma ACTH and CORT levels induced by hypoglycemia were significantly decreased in the Avpr1b KO mice when compared with wild-type littermates. There was no difference in the ACTH response to acute and chronic restraint in wild-type mice. In the Avpr1b KO group subjected to 14 sessions of daily restraint, plasma ACTH was decreased when compared with wild-type mice. On the other hand, the CORT elevations induced by restraint did not adapt in the Avpr1b KO or wild-type mice. The data suggest that the Avpr1b is required for the normal pituitary and adrenal response to some acute stressful stimuli and is necessary only for a normal ACTH response during chronic stress.

The role of desmopressin in bilateral and simultaneous inferior petrosal sinus sampling for differential diagnosis of ACTH‐dependent Cushing's syndrome

Bilateral inferior petrosal sinus sampling (BIPSS) with corticotrophin-releasing hormone (CRH) stimulation is currently the gold standard test for the differential diagnosis of ACTH-dependent Cushing's syndrome. Reports on the use of desmopressin in this approach are limited. The aim of this study was to evaluate the use of desmopressin during BIPSS in a cohort of patients with ACTH-dependent Cushing's syndrome. A retrospective case-record study. Fifty-six patients with confirmed ACTH-dependent Cushing's syndrome underwent BIPSS with desmopressin stimulation when presenting negative pituitary tumour imaging. Central to peripheral (CEN:PER) ACTH gradient, lateralization of the ACTH source and surgical tumour confirmation were evaluated. A CEN:PER ACTH gradient was found in 40 patients under basal conditions (CEN:PER >or= 2) and in 47 patients after desmopressin stimulation (CEN:PER >or= 3). Ectopic ACTH-producing tumours (three lung carcinoid tumour, one thymus carcinoid tumour and one thymus hyperplasia) were confirmed in five out of nine patients without the CEN:PER ACTH gradient, and four cases were false negative for Cushing's disease. Lateralization (IPS:IPS >or= 1.4) was observed in 80.8% of patients under basal conditions (38/47) and in 97.8% after desmopressin (46/47), and it was surgically confirmed in 78.7%. There were no false-positive cases. Sensitivity and specificity were 92.1% and 100%, respectively. Desmopressin improves the differential diagnosis of ACTH-dependent Cushing's syndrome by amplifying the CEN:PER and IPS:IPS ACTH gradients, and is therefore a useful ACTH secretagogue in BIPSS.

The Hypothalamic-Pituitary-Adrenal Axis: What can it Tell us About Stressors?

The hypothalamic-pituitary-adrenal (HPA) axis is an extremely sensitive physiological system whose activation, with the consequent release of ACTH and glucocorticoids, is triggered by a wide range of psychological experiences and physiological perturbations (stressors). The HPA axis is also activated by a high number of pharmacological agents that markedly differ in structure and function, although the precise mechanisms remain in most cases unknown. Activation of the HPA axis is the consequence of the convergence of stimulatory inputs from different brain regions into the paraventricular nucleus of the hypothalamus (PVN), where the most important ACTH secretagogues (corticotrophin releasing factor, CRF, and arginin-vasopressin, AVP) are formed. Plasma levels of ACTH and corticosterone (the latter under more restricted conditions), are considered as good markers of stress for three main reasons: (a) their plasma levels are proportional to the intensity of emotional and systemic stressors, (b) daily repeated exposure to a stressor usually resulted in reduced ACTH response to the same stressor, that is termed adaptation or habituation; and (c) chronic exposure to stressful situations results in tonic changes in the HPA axis that can be used as indices of the accumulative impact of these situations. These changes can be evaluated under resting conditions (i.e. adrenal weight, CRF and AVP gene expression in the PVN) or after some challenges (administration of CRF, ACTH or dexamethasone) that are classical endocrinological tests. There is also evidence that the activation of the HPA axis may also reflect subtle changes in the characteristics of the stressful situations (unpredictability, lack of control, omission of expected rewards, presence of conspecifics), although this is a topic that requires further studies.

Temazepam Triggers the Release of Vasopressin into the Rat Hypothalamic Paraventricular Nucleus: Novel Insight into Benzodiazepine Action on Hypothalamic–Pituitary–Adrenocortical System Activity During Stress

We investigated the influence of a representative classical benzodiazepine on the regulation of the hypothalamic-pituitary-adrenocortical (HPA) axis activity both under basal conditions and stress. Adult male Wistar rats were intravenously administered with temazepam (0.5, 1, and 3 mg/kg body weight) and plasma concentrations of corticotropin (ACTH) and vasopressin (AVP) were measured in blood samples collected via chronically implanted jugular venous catheters. Simultaneously, the release of AVP within the hypothalamic paraventricular nucleus (PVN) was monitored via microdialysis. Plasma AVP levels remained unaffected by the different treatment conditions. Temazepam blunted the stressor exposure-induced secretion of ACTH in a dose-dependent manner. Concurrently, and also in a dose-dependent manner temazepam enhanced the intra-PVN release of AVP, known to originate from magnocellular neurons of the hypothalamic neurohypophyseal system. Furthermore, temazepam did not affect the in vitro secretion of ACTH from the adenohypophyseal cells. Taken together, the results of this study suggest that temazepam modulates the central nervous regulation of the HPA axis by altering intra-PVN AVP release. An increasingly released AVP of magnocellular origin seems to provide a negative tonus on ACTH secretion most probably via inhibiting the release of ACTH secretagogues from the median eminence into hypophyseal portal blood.

Effects of chronic stress and gender on HPA axis responsivity in a pharmacologic CRH-AVP challenge test

Findings in rodents document an increased central AVP release after repeated stress suggesting that AVP may be the predominant ACTH secretagogue under chronic stress conditions. We intended to test whether this altered AVP regulation can be demonstrated via a pharmacological challenge in chronically stressed humans. Two groups of school teachers (21 men, 21 women) reporting either high or low levels of perceived chronic stress received a single injection of 50µg hCRH compared to the same injection during concomitant infusion of 100ng/min AVP. While the synergistic effect of the combined CRH-AVP application normally results in an augmented cortisol response compared to CRH alone, we postulated that higher pre-existing levels of central AVP in stressed subjects should result in a blunted response to the combined CRH-AVP injection compared to the non stressed group. The postulated attenuation of HPA responses to the CRH-AVP administration in stressed subjects was observed only as a marginally significant effect resp. at a trend level. However, these effects disappeared when gender was included as a covariate. A significant interaction gender x challenge test (p<.05 for salivary cortisol; p<.01 for plasma cortisol and ACTH) indicated a lower response to single CRH administration but higher responses to the combined CRH-AVP challenge in women compared to men. Also, CBG levels were significantly higher in women compared to men during both stimulation tests (p<.05). We speculate that the distinct impact of gender may have masked a possibly existing stress effect. Our results contribute further data to the ongoing discussion about the role of gonadal steroids and CBG on the HPA axis activity.

Measuring stress responses in postpartum mothers: Perspectives from studies in human and animal populations

Reduced hypothalamic–pituitary–adrenal (HPA) responses to stress during the last week of pregnancy and lactation have been consistently observed in rat studies. Several contributing factors have been proposed for this phenomenon in lactation, including the suckling stimulus from the pups, hormones (oxytocin and prolactin) and opioids, a decrease in the ability of noradrenaline to potentiate hypothalamic responses and changes in pituitary responsiveness to ACTH secretagogues (AVP and CRF). In contrast to this vast literature using the rat model, only few studies have addressed this issue in the human population. The consensus is that women engaging in breastfeeding activities exhibit reduced anxiety, although the reductions in neuroendocrine and autonomic responses to stressors are variable, in part because of the different nature of the stressors used. Further work is required to investigate how additional factors, such as maternal parity or emotional salience of the stressor can affect stress responsiveness in postpartum women. Here, we review first the findings regarding stress responsiveness during lactation in both rat and human studies, and then discuss potential research avenues and methodological issues that could be the lead to future research protocols in human subjects. Knowing the reciprocal relationship in the mother–infant dyad, it is clear that investigation of the mechanisms regulating stress responses and mental health in postpartum mothers can only be beneficial to the development of the infant.

Chronic Treatment with the Monoamine Oxidase Inhibitor Phenelzine Increases Hypothalamic-Pituitary-Adrenocortical Activity in Male C57BL/6 Mice: Relevance to Atypical Depression

Atypical depression has been linked to low hypothalamic-pituitary-adrenocortical axis activity and exhibits physical and affective symptoms resembling those of glucocorticoid deficiency. Because atypical depression has also been defined by preferential responsiveness to monoamine oxidase inhibitors (MAO-I), we hypothesized that MAO-I reverse these abnormalities by interfering with glucocorticoid feedback and increasing hypothalamic-pituitary-adrenocortical activity. To test this hypothesis, we measured plasma hormones and ACTH secretagogue gene expression in male C57BL/6 mice treated chronically with saline vehicle or phenelzine, a representative MAO-I. Changes in glucocorticoid feedback were evaluated using adrenalectomized (ADX) mice with and without corticosterone replacement. Antidepressant efficacy was confirmed by decreased immobility during forced swim testing. Phenelzine significantly increased circadian nadir and postrestraint plasma corticosterone levels in sham-operated mice, an effect that correlated with increased adrenocortical sensitivity to ACTH. Phenelzine increased circadian nadir, but not poststress ACTH in ADX mice, suggesting that phenelzine augmented corticosterone secretion in sham-operated mice by increasing stimulation and decreasing feedback inhibition of hypothalamic-pituitary activity. Consistent with the latter possibility, phenelzine significantly increased plasma ACTH and paraventricular hypothalamus CRH mRNA in ADX, corticosterone-replaced mice. Phenelzine did not increase paraventricular hypothalamus CRH or vasopressin mRNA in ADX mice lacking corticosterone replacement. We conclude that chronic phenelzine treatment induces sustained increases in glucocorticoids by impairing glucocorticoid feedback, increasing adrenocortical responsiveness to ACTH, and increasing glucocorticoid-independent stimulation of hypothalamic-pituitary activity. The resulting drive for adrenocortical activity could account for the ability of MAO-I to reverse endocrine and psychiatric symptoms of glucocorticoid deficiency in atypical depression.

Involvement of Endogeneous Glutamate in the Stimulatory Effect of Norepinephrine and Serotonin on the Hypothalamo-Pituitary-Adrenocortical Axis

The effects of ionotropic glutamate receptor antagonists on the pituitary adrenal responses following injections of norepinephrine (NE) and serotonin (5-HT) receptor agonists into the hypothalamic paraventricular nucleus (PVN) or electrical stimulation of central NE and 5-HT pathways were studied in anesthetized male rats. PVN injections of an α₁-adrenergic receptor agonist or a serotonergic 5-HT_1A receptor agonist markedly increased both adrenocorticotropin (ACTH) and corticosterone (CS) serum levels. These responses were significantly inhibited by separate pre-injection of the selective non-NMDA and NMDA glutamate receptor subtype antagonists into the PVN in a dose-dependent manner. Electrical stimulation of either the ventral noradrenergic bundle or the dorsal raphe nucleus markedly increased serum ACTH and CS. These responses were also significantly attenuated by pre-injection of the above glutamate ionotropic receptor antagonists in a dose-dependent manner. These results suggest that glutamatergic interneurons in the PVN, acting via non-NMDA and NMDA receptors, may act as an excitatory mechanism in the NE and 5-HT control of hypothalamic ACTH secretagogues.

Corticotropin-releasing hormone and arginine vasopressin gene transcription in the hypothalamic paraventricular nucleus of unstressed rats: daily rhythms and their interactions with corticosterone.

To drive the daily ACTH secretory rhythm from anterior pituitary corticotropes signals from the circadian clock schedule CRH and, to lesser extent, arginine vasopressin (AVP) release from neuroendocrine terminals. In turn, releasable pools of CRH and AVP in neuroendocrine terminals are sustained by synthetic mechanisms in the medial parvicellular paraventricular nucleus, a critical component of which involves transcribing primary (heteronuclear (hn)) RNA transcripts from their cognate genes. To determine the fundamental daily patterns of ACTH secretagogue gene transcription in unstressed rats, we measured CRH and AVP hnRNA levels at 1- to 4-h intervals throughout the day using in situ hybridization. Crh gene transcription is readily detectable throughout the day, and shows a pronounced rhythm that is temporally correlated with CRH mRNA levels, but is uncoupled from ACTH release. However, avp gene transcription is barely detectable and shows no discernable rhythm. We then performed similar experiments in adrenalectomized rats with or without corticosterone replacement. Corticosterone-dependent mechanisms regulate CRH hnRNA levels at the nadir and peak as well as the onset of nocturnal crh gene transcription. A prominent rhythm of avp hnRNA seen in adrenalectomized animals was dampened by corticosterone. This study shows, first, CRH synthesis in intact animals is maintained by a nocturnal episode of crh gene transcription, parameters of which are modulated by corticosterone-dependent mechanisms; second, circulating corticosterone is sufficient to completely inhibit a daily rhythm of avp gene transcription present in adrenalectomized rats; third, the neural systems that activate crh gene transcription can be uncoupled from those driving ACTH release.

Effect of Interleukin 1β on the HPA Axis in H1-Receptor Knockout Mice

Objectives and Methods: Circulating cytokines such as interleukin-1 (IL-1), and tumor necrosis factor-alpha as well as lipopolysaccharide (LPS) are potent ACTH secretagogues, acting via stimulation of corticotropin-releasing hormone (CRH) and vasopressinergic neurons in the paraventricular nucleus of the hypothalamus (PVN). Histamine (HA) has been shown to stimulate ACTH secretion in rats, an effect in part mediated by CRH and arginine vasopressin (AVP). We have previously shown that inhibition of neuronal HA synthesis or central blockade of H₁ receptors (H₁R) decreased the ACTH response to LPS in male rats. To further elucidate the role of neuronal HA in cytokine-induced activation of the HPA axis, we compared the effect of H₁R knockout on IL-1β-induced ACTH secretion in adult male mice. Results: In H₁R knockout mice, ACTH secretion increased from basal levels of 261 to 492 pmol/l in response to IL-1β whereas the cytokine-induced ACTH secretion increased from 140 to 406 pmol/l in wild-type mice. Plasma corticosterone (CORT) rose from basal levels of 99 to 831 nmol/l in knockout mice upon IL-1β stimulation, whereas in wild-type mice CORT levels rose from 112 to 841 nmol/l. There was no significant difference in IL-1β-stimulated plasma ACTH or CORT levels between wild-type and knockout mice. Furthermore, there was no significant difference in basal or IL-1β-stimulated hypothalamic levels of histamine and tele-methyl-histamine between wild-type and knockout mice. HDC gene expression was significantly lower in knockout mice than in wild-type mice both under basal and IL-1β-stimulated conditions, while there were no significant differences in CRH gene expression in the PVN in knockout mice under basal and IL-1β-stimulated conditions. Increased basal expression of AVP in the PVN of knockout mice was observed in this study compared to wild-type mice. Conclusion: We conclude that the lack of the gene for histamine H₁R does not seem to be crucial for the ACTH and CORT response to IL-1β, either due to possible functional compensation in the H₁R knockout mouse or due to activation of pathways other than the neuronal histaminergic system.