ACTH, alpha-MSH, and control of cortisol release: cloning, sequencing, and functional expression of the melanocortin-2 and melanocortin-5 receptor in Cyprinus carpio

Cortisol release from fish head kidney during the acute phase of the stress response is controlled by the adrenocorticotropic hormone (ACTH) from the pituitary pars distalis (PD). Alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin, from the pars intermedia (PI), have been implicated in cortisol release during the chronic phase. The present study addresses the regulation of cortisol release by ACTH and alpha-MSH in common carp (Cyprinus carpio) and includes characterization of their receptors, namely, the melanocortin-2 and melanocortin-5 receptors (MC2R and MC5R). We could not demonstrate corticotropic activity of alpha-MSH, beta-endorphin, and combinations of these. We do show a corticotrope in the PI, but its identity is as yet uncertain. Carp restrained for 1 and 7 days showed elevated plasma cortisol and alpha-MSH levels; cortisol is still elevated but lower at day 7 than day 1 of restraint. Interrenal response capacity is unaffected, as estimated by stimulation with a maximum dose ACTH in a superfusion setup. MC2R and MC5R appear phylogenetically well conserved. MC2R is predominantly expressed in head kidney; a low abundance was found in spleen and kidney. MC5R is expressed in brain, pituitary PD, kidney, and skin. Quantitative PCR analysis of MC2R and MC5R expression in the head kidney of restrained fish reveals MC2R mRNA downregulation after 7 days restraint, in line with lower plasma cortisol levels seen. We discuss regulation of corticosteroid production from a phylogenetic perspective. We propose that increased levels of alpha-MSH exert a positive feedback on hypothalamic corticotropin-releasing hormone release to sustain a mild stress axis activity.     

Corticotrophin-releasing factors in the hypothalamus of the developing fetal sheep.

Corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) are secreted from the hypothalamic median eminence to elicit the secretion of ACTH from the pituitary corticotrophs. During fetal development there is progressive maturation of the hypothalamic-pituitary-adrenal axis, manifest as increasing plasma ACTH and cortisol concentrations, which in species such as sheep culminates in the onset of birth. However, the precise nature of the hypothalamic signal controlling fetal pituitary ACTH secretion remains poorly understood. To investigate the ontogeny of this hypothalamic signal, the present study examined immunoreactive and bioactive ACTH-releasing factors in the developing fetal sheep hypothalamus. Immunoreactive CRH and AVP were measured by radioimmunoassay in extracts of hypothalami taken at day 70, day 100, and day 130 gestation (term = 145 days). There was a progressive and significant (P < 0.01) increase in hypothalamic CRH and AVP concentrations which was particularly marked between d100 and d130 gestation. AVP was always present in higher concentrations that CRH, although this difference was significantly reduced by day 130 gestation as the result of a large increase in the content of CRH relative to AVP. Sephadex G50 chromatography revealed that immunoreactive CRH and AVP in hypothalamic extracts existed as single molecular forms corresponding to synthetic peptides at each gestational age. In addition, these immunoreactive forms of CRH and AVP possessed significant ACTH-releasing bioactivity as measured in primary cultures of adult sheep anterior pituitary cells. Furthermore, significant bioactivity was present in high and low molecular weight fractions eluted after chromatography which did not contain any CRH or AVP immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corticosteroid-binding globulin (CBG) in fetal development

In fetal sheep the prepartum increase in plasma cortisol concentration is associated with an increase in high affinity corticosteroid binding activity in plasma. This appears to reflect an increase in corticosteroid-binding globulin (CBG) biosynthesis from the fetal liver, and evidence is presented that hepatic CBG gene expression is increased by exposure to glucocorticoids in the fetus. Immunoreactive CBG is found in other fetal tissues, and CBG mRNA is present in fetal pituitary. CBG reduces the ability of cortisol to exert negative feedback on basal or CRH-stimulated ACTH output by fetal sheep pituitary cells in culture. We suggest that CBG interacts with cortisol in a manner that maintains a low negative feedback on the pituitary, and perhaps hypothalamus. This constitutes a component of the cascade of events that is associated with hypothalamic-pituitary-adrenal activation in the late gestation fetus, and with the onset of parturition.     

Immunoreactive forms of ACTH released by the adenohypophysis of the rat during the perinatal period in vivo and in vitro studies

Immunoreactive ACTH levels were determined in whole plasma, eluate fractions of plasma and perfusates of anterior lobe extracts subjected to chromatography on Sephadex G-50 fine. In the fetal plasma, ACTH levels were higher on day 19 than on days 17, 18, 20 and 21. After birth, ACTH concentrations dropped to reach the lowest values in one week old newborns; thereafter they increased until weaning on day 21. They were then similar to those of non pregnant adult females. Three peaks of immunoreactive ACTH were present in all the chromatograms; the first one eluted near the void volume ("big" ACTH, PM approximately 44,000), the third one coeluted with human ACTH (1-39) ("little" ACTH, PM approximately 4,500) and the second one eluted midway between the 2 previous peaks ("intermediate" ACTH, PM approximately 13,000). During the last days of pregnancy, the proportion of the "little" form of ACTH in the fetal plasma showed a gradual increase whereas that of the "big" one decreased. On days 17, 19 and 21 of gestation the anterior lobes of fetal pituitary glands released in vitro these 3 forms of immunoreactive ACTH in the same proportions as those observed in the anterior lobes. In contrast, the proportions of the circulating forms of ACTH were quite different; the "little" one gradually increased and the "big" one decreased as gestation progressed. In vitro controlled tryptic digestion of the isolated "big" form led to the appearance of "intermediate" and "little" forms suggesting some transformations in the circulation of the ACTH forms released by the fetal hypophysis in vivo.     

Prenatal exposure to dexamethasone alters hippocampal drive on hypothalamic-pituitary-adrenal axis activity in adult male rats

Glucocorticoids are essential for normal hypothalamic-pituitary-adrenal (HPA) axis activity; however, recent studies warn that exposure to excess endogenous or synthetic glucocorticoid during a specific period of prenatal development adversely affects HPA axis stability. We administered dexamethasone (DEX) to pregnant rats during the last week of gestation and investigated subsequent HPA axis regulation in adult male offspring in unrestrained and restraint-stressed conditions. With the use of real-time PCR and RIA, we examined the expression of regulatory genes in the hippocampus, hypothalamus, and pituitary, including corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), glucocorticoid receptors (GR), mineralcorticoid receptors (MR), and 11-beta-hydroxysteroid dehydrogenase-1 (11beta-HSD-1), as well as the main HPA axis hormones, adrenal corticotropic hormone (ACTH) and corticosterone (CORT). Our results demonstrate that the DEX-exposed group exhibited an overall change in the pattern of gene expression and hormone levels in the unrestrained animals. These changes included an upregulation of CRH in the hypothalamus, a downregulation of MR with a concomitant upregulation of 11beta-HSD-1 in the hippocampus, and an increase in circulating levels of both ACTH and CORT relative to unrestrained control animals. Interestingly, both DEX-exposed and control rats exhibited an increase in pituitary GR mRNA levels following a 1-h recovery from restraint stress; however, the increased expression in DEX-exposed rats was significantly less and was associated with a slower return to baseline CORT compared with controls. In addition, circulating levels of ACTH and CORT as well as hypothalamic CRH and hippocampal 11beta-HSD-1 expression levels were significantly higher in the DEX-exposed group compared with controls following restraint stress. Taken together, these data demonstrate that late-gestation DEX exposure in rats is associated with persistent changes in both the modulation of HPA axis activity and the HPA axis-mediated response to stress.     

Developmental correlation between hypothalamic somatostatin and hypophysial growth hormone

Summary The objective of the present study was to determine, by means of immunocytochemistry, the age in fetal development at which GH is first detectable in the pituitary gland and somatostatin in the median eminence, and to correlate temporally the development of these two hormones throughout the remainder of pregnancy. Mice were studied at 15–19 days of gestation with the peroxidase-antiperoxidase (PAP) technique of Sternberger. Somatotropes in the pars distalis were initially detected at 16 days of gestation and by 17 days they were a prominent component of the parenchymal cell population of the hypophysis. These cells were ovoid and distributed uniformly throughout the pars distalis; many were located adjacent to sinusoidal capillaries. Their number and staining intensity increased by 19 days. Somatostatin was not consistently observed in the median eminence until 19 days of gestation. Reaction product indicative of the presence of somatostatin in presumptive nerve endings was located on the ventral surface of the median eminence and in the external lamina of the infundibulum in proximity to the superficial portal capillaries. Results of the present investigation support the concept that the potential for neuroendocrine control of GH secretion exists in the mouse by the end of fetal development. Several hypotheses concerning the temporal relationship between the appearance of somatostatin in the hypothalamus and of GH in the anterior pituitary gland are discussed.Supported by a Biomedical Research Support Grant (NIH RR 5417). Appreciation is extended to the National Pituitary Agency, NIAMDD for the following radioiodination-grade hormones: hGH, rPRL, rTSH, rFSH and hCG     

Corticotrophs and peptides

Corticotrophs were long thought to be a static, homogeneous population of cells that respond positively to hypothalamic stimulation, are inhibited by glucocorticoid feedback and secrete a single biologically active peptide, ACTH(1-39). Our current understanding is that this is an oversimplification and corticotrophs are a dynamic and more complex group of cells. The biosynthetic precursors of ACTH and other cleavage products of proopiomelanocortin (POMC) have been found to be secreted by anterior pituitary cells, to circulate and to have biological activity. POMC and the biosynthetic intermediate, pro-ACTH, exert activity antagonistic to ACTH(1-39) on glucocorticoid secretion by adrenal cells, and other derivatives of POMC are mitogenic to adrenocortical cells. In terms of responses to hypothalamic and peripheral factors, corticotrophs are functionally heterogeneous. This is reflected in the sensitivity of individual subtypes of corticotrophs to CRH, vasopressin and glucocorticoids. There is a functional plasticity amongst the various types of corticotrophs. During gestation, in fetal sheep, changes occur in the overall ACTH-secretory responses to CRH relative to vasopressin, the proportions of total corticotrophs that respond to the respective peptides and the average secretory response of individual cells. Corticotrophs also respond to locally produced pituitary factors. Local actions of leukaemia inhibitory factor are demonstrated by the effects of immunoneutralization of the peptide in pituitary cells. Urocortin and preproTRH(178-199) are locally produced peptides with potent stimulatory and inhibitory actions on corticotrophs, respectively. The specific roles of these peptides are under investigation.     

Developmental aspects of the hypothalamic-pituitary-adrenal axis

The ovine fetal adrenal cortex and pituitary are functional secretory organs by the end of the first third of gestation (term is 142-152 days). By half-way through gestation the zona glomerulosa is mature morphologically, more than 80% of the aldosterone in fetal blood is of fetal adrenal origin, but conventional stimuli, for example, increased plasma K+ or angiotensin II, do not increase aldosterone secretion until near term. The zona fasciculata is immature histologically, relatively unresponsive to ACTH, and contributes less than 10% of the cortisol in fetal blood between 100 and 120 days of gestation. After this time the zona fasciculata cells begin to mature, to respond to ACTH and to produce an increasing proportion of the cortisol in fetal blood. A functional relationship between hypothalamus-pituitary-adrenal cortex matures over the last fifth of gestation. It is hypothesized that cortisol exerts a local effect in maturation of fetal zona fasciculata cells, such that low concentrations of ACTH have increasingly larger effects on growth and secretion of the fasciculata and that the level of negative feedback by cortisol on the hypothalamic-pituitary axis is reset. The analogy is drawn between the changes in gonadotrophin and gonadal hormones which culminates in puberty in man and the changes in ACTH and cortisol which culminate in parturition in sheep.     

Cadmium induces changes in corticotropic and prolactin cells in the Podarcis sicula lizard pituitary gland

We analyzed the effect of cadmium on corticotropic (ACTH) and prolactin (PRL) cells in the pituitary gland of the Podarcis sicula (P. sicula) lizard under chronic exposure to this metal. Adult lizards were given CdCl₂ in drinking water at the dose of 10 µg/10 g body mass for 120 days. Light microscopy was performed after histological and immunohistochemical staining, and the effects were followed at regular time intervals up to 120 days post-treatment. We detected substantial variations in the general morphology of the pituitary: unlike the control lizards in which the gland appeared compact, the treated lizards showed a glandular tissue with dilated spaces that were more extensive at 90 and 120 days. PRL and ACTH cells showed an increase in occurrence and immunostaining intensity in treated lizards in comparison with the same cells of control animals. This cellular increase peaked for PRL at 30 days in the rostral, medial and also caudal pars distalis of the gland. ACTH cells appeared to increase markedly after 60 days of treatment in both the pars distalis and the pars intermedia. Again, at 60 days small, isolated ACTH cells were also found in the caudal pars distalis in which these cells were generally absent. However, at 120 days both these cellular types showed an occurrence, distribution and morphology similar to those observed in the control lizards. In lizards, protracted oral exposure to cadmium evidently involves an alteration of the normal morphology of the gland and an inhibitory effect of ACTH and PRL cells, since they increase in occurrence and immunostaining. Yet in time the inhibitory effect of cadmium on ACTH and PRL cells falls back and their occurrence appears similar to that of the control lizard.Key words: cadmium, corticotropic and prolactin cells, lizard.     

Estrogen and androgen influence hypothalamic AVP and CRF concentrations in fetal and adult sheep

The present study tested the hypothesis that action of sex steroids on the hypothalamus-pituitary-adrenal (HPA) axis is measurable in the hypothalamus. Late-gestation fetal sheep were treated (5 mg/21 days) with either estradiol, androstenedione, or tamoxifen and compared to age-matched control fetuses. Tamoxifen significantly increased hypothalamic corticotropin releasing factor (CRF) and arginine vasopressin (AVP) concentrations, and androstenedione significantly decreased hypothalamic CRF concentration. Adult sheep were treated with estrone (10 mg/21 days), and responded with significant increases in hypothalamic AVP concentration, but not in immunoreactive ACTH concentration or processing within the pituitary. The results demonstrate that the effect of estrogen on the HPA axis is measurable in the hypothalamus, and is therefore not primarily at the anterior pituitary.     

Immunohistochemical localization of ACTH in the adult and fetal sheep pituitary

Light microscope peroxidase-antiperoxidase immunohistochemistry has been applied to the pituitary of adult and fetal sheep from 40 to 145 days of gestation. In the adult, immunoreactive ACTH cells were darkly stained and angular with cytoplasmic processes surrounding neighbouring unstained cells. In the fetus, cells which stained for ACTH were observed in the pars distalis at 40 days. From approximately 90 days, two morphologically distinct ACTH-positive cell types were clearly discernible. The predominant type was large and variably stained. These cells usually occurred in clusters and were often arranged in palisades. The other type resembled ACTH-positive cells in the adult. After 130 days the population of large cells declined and completely disappeared before term in most fetuses. The pars intermedia showed a different pattern of staining. In the fetus, ACTH-positive cells were observed in this region after 60 days gestation and by 90 days almost all the pars intermedia cells were strongly stained. By contrast, the cells in the adult pars intermedia were only lightly stained.     

Pro-opiomelanocortin peptides in the human fetal pituitary

Levels of immunoreactive pro-opiomelanocortin (POMC) peptides (N- and C-terminal ACTH, N- and C-terminal LPH and α-MSH) have been measured in pituitary extracts from human fetuses of 12–22 weeks gestation. The levels of ACTH were 30–200 times higher than α-MSH in all fetuses studied. Sephadex G-75 and G-25 chromatography of 8 extracts showed peaks of 34 kilodaltons (K) POMC, 22K ACTH, β-LPH, γ-LPH, β-endorphin, approximately 8K ACTH, 1–39 ACTH, α-MSH and CLIP. The 8K and 22K forms of ACTH are both partly glycosylated.In vitro culture of pituitaries from 2 fetuses (22 and 26 weeks gestation) gave a detectable basal output of ACTH but not of α-MSH. Stimulation of these pituitary cells with human fetal and rat hypothalamic extracts and with synthetic ovine CRF-41 produced a significant increase in ACTH release, and either small or undetectable amounts of α-MSH.These results demonstrate the presence of POMC-related peptides in early gestation human fetal pituitaries and suggest that ACTH, and not α-MSH, is the major corticotrophic hormone at this stage of gestation.     

Ontogenesis of the α-MSH, β-MSH and ACTH cells in the foetal hypophysis of the rat. Correlation with the growth of the adrenals and adrenocortical activity

Pituitary sections from 15 to 21 day-old rat foetuses have been studied with the immunofluorescence technique, using antibodies anti alpha-MSH, anti beta-MSH and anti beta (1-24) ACTH. The first ACTH cells appear on day 17 of pregnancy in the pars distalis of the hypophysis and only on day 18 in the pars intermedia. beta-msh cells have been observed on day 16 in the pars anterior and on day 17 in the pars intermedia, while alpha-MSH cells appear only on day 18 and exclusively in the pars intermedia. The cytodifferentiation of the beta-MSH and ACTH cells occurs in the pars intermedia with about a 24 hours delay in comparison to that of the pars distalis. The first revealed cells are always located in the posterior half of the pituitary gland. The corticostimulating activity of the hypophysis has been tested with the fluorescence intensity of the corticotrophs, the adrenal weight, the adrenal content in corticosterone and the plasma corticosterone level. The fluorescence of the corticotrophs increases on day 18, shows a maximum on day 19 and decreases until term. The adrenal weight rises regularly between day 16 to day 20, thereafer the increase subsides. Adrenal and plasma corticosterone concentrations reach a peak on day 19 of pregnancy. These data suggest that hypophyseal corticostimulating activity is very high between days 18 and 19 and decreases between days 19 and 21.     

Multiple unit activity recording in the corticotropic area of the deafferented hypothalamus and corticosteronemia in the pigeon.

10 Diurnal variations in both multiple unit activity and plasma corticosterone level were suppressed after complete neural isolation of the basal hypothalamus in the pigeon. 20 It is suggested that the circadian activity of the hypothalamic pituitary corticotropic unit partially depends upon the inhibiting influence from the hippocampic-septal structures.     

Adrenocortical responses to adrenocorticotrophin in the hypophysectomized ovine fetus

Fetal adrenocortical responsiveness to ACTH declines during 90-120 days gestation and fetal pituitary peptides have been implicated in this refractoriness. In these studies the ACTH-induced cortisol responses were measured in 11 ovine fetuses of 114 days gestation. Five animals were hypophysectomized as evidenced by prolonged gestation, pituitary histology, TRH-testing, delayed maturation and decreasing fetal plasma prolactin concentrations (less than 1 ng.ml-1) (P less than 0.005). Resting cortisol concentrations decreased from 22.4 to 8.1 ng.ml-1 in the hypophysectomy group and were not different from the control group (19.6-14.9 ng.ml-1) over the 5 days of study. Responses measured as increments in plasma cortisol concentrations increased equally and successively in both groups. Since pituitary ablation fails to enhance fetal adrenal responsiveness to ACTH we conclude that refractoriness is unlikely to be caused by an inhibitor of pituitary origin.     

Regulation of the hypothalamic-pituitary-adrenal axis in birth

In sheep an increase in fetal pituitary-adrenal function, reflected in rising concentrations of plasma ACTH and cortisol, is important in relation to fetal organ maturation and the onset of parturition. This review presents evidence that implicates the hypothalamic-pituitary-adrenal axis in the control of parturition and describes recent experiments that explore in detail the maturation of the fetal hypothalamus and pituitary in relation to fetal adrenal function. Recent improvements for the measurement of ACTH in unextracted plasma and the ability to maintain vascular catheters in chronically catheterized fetal sheep have enabled subtle changes in fetal ACTH concentrations to be detected. As a result of these advances it has now been established that the terminal rise in cortisol, which is responsible for the onset of parturition in sheep, is preceded by an increase in fetal plasma ACTH concentrations. This has led to the hypothesis that birth results from the sequential development of the fetal hypothalamic-pituitary-adrenal axis with the signal originating from the fetal brain. This increase in trophic drive to the fetal adrenal may result from changes in the responsiveness of the fetal pituitary gland to factors that stimulate the release of ACTH. Corticotropin releasing factor (CRF) and arginine vasopressin are two such factors that stimulate the secretion of ACTH and cortisol secretion in the chronically catheterized fetal sheep. The response to these factors increases with gestational age and is sensitive to glucocorticoid feedback. Furthermore, repeated administration of CRF to immature fetal sheep results in pituitary and adrenal activation and in some cases may lead to premature parturition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urocortin: a mechanism for the sustained activation of the HPA axis in the late-gestation ovine fetus?

We hypothesized that urocortin might be produced in the pituitary of the late-gestation ovine fetus in a manner that could contribute to the regulation of ACTH output. We used in situ hybridization and immunohistochemistry to identify urocortin mRNA and protein in late-gestation fetal pituitary tissue. Levels of urocortin mRNA rose during late gestation and were associated temporally with rising concentrations of pituitary proopiomelanocortin (POMC) mRNA. Urocortin was localized both to cells expressing ACTH and to non-ACTH cells by use of dual immunofluorescence histochemistry. Transfection of pituitary cultures with urocortin antisense probe reduced ACTH output, whereas added urocortin stimulated ACTH output from cultured pituitary cells. Cortisol infusion for 96 h in chronically catheterized late-gestation fetal sheep significantly stimulated levels of pituitary urocortin mRNA. We conclude that urocortin is expressed in the ovine fetal pituitary and localizes with, and can stimulate output of, ACTH. Regulation of urocortin by cortisol suggests a mechanism to override negative feedback and sustain feedforward of fetal hypothalamic-pituitary-adrenal function, leading to birth.     

Stimulation of 17 beta-hydroxy steroid dehydrogenase in the rat anterior pituitary gland by progesterone

Anterior pituitary gland and hypothalamic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity was measured in the immature castrated estradiol primed rat to determine if differences in enzyme activity could explain the progesterone induced reduction of bound estradiol nuclear receptors of the anterior pituitary gland but not the hypothalamus. Higher levels of 17 beta-HSD activity were found in the anterior pituitary gland as compared to the hypothalamus. The enzyme activity in the anterior pituitary gland was stimulated by progesterone administered either in combination with estradiol for 4 days or as a single injection following 4 days of estradiol priming. No progesterone effects were found on hypothalamic 17 beta-HSD. Under the experimental conditions used, progesterone administration did not alter uterine 17 beta-HSD. An increase in anterior pituitary gland and uterine 17 beta-HSD was also induced by estrogen administration.     

Hypothalamic influence on differentiation of the immunoreactive ACTH beta-LPH, alpha- and beta-endorphin containing cells in adenohypophysial primordia of rat fetus in organ culture (author's transl)]

Adenohypophysial primordia were isolated in rat fetuses from day 12.5 to day 15.5 of gestation. The organ culture employed for maintenance of the primordia was made up according to Watanabe et al. (1973). The fixation of primordia in Bouin Hollande's solution was performed after 9, 8, 7 or 6 days of culture when the normal duration of pregnancy was achieved. The cultivated primordia were immunologically studied using different antisera: anti-alpha(17-39)ACTH, anti-beta(1-24)ACTH, anti-beta-LPH, anti-alpha and anti-beta-endorphins, with immunoperoxidase or immunofluorescence techniques, including control experiments of the specificity of the antisera. A similar study was performed on pituitaries removed from normal rat fetuses from day 16.5 of gestation and each day up to birth, and fixated immediately. In vivo the first cells reacting with all the antisera used in this study were observed on day 16.5 of gestation; their number increased during gestation (Fig. 1 A, B and C). Immunoreactive cells with the different antisera could be detected in primordia isolated on day 12.5 of gestation after 9 days of culture. Numerous groups of cells were observed in primordia of older fetuses (Fig. 2 A and B). These data indicate that the corticotropic cells in rat fetuses could start to be differentiated without stimuli from the hypothalamus since primordia were isolated before the appearance of this cell type in normal rat fetuses and before the differentiation of the hypothalamus. The presence of ACTH and other peptides such as beta-LPH or beta-endorphin would support the hypothesis of a common precursor in this cell type existing early in gestation. Similar results were obtained in human fetuses.     

Hypothalamic control of ACTH secretion

The hypothalamic regulation of ACTH secretion has been reviewed. Recent biochemical investigations on corticotropin-releasing factor (CRF) suggest that CRF is present in the hypothalamus under two or more different molecular weight forms, their structure being not elucidated yet. Vasopressin has a CRF-like activity. However, contradictory results have been reported on the role of AVP as a physiological CRF. The synthesis of CRF appears to occur in a large hypothalamic area outside the median eminence. CRF-carrying fibers are thought to pass through the lateral retrochiasmatic area and project on the hypophysial portal vessels at the junction between the pituitary stalk and the median eminence. Conflicting data have been published on the influence of monoamines on ACTH secretion. In the dog, ACTH release is inhibited by the alpha-adrenergic receptors, this effect being not as clearly demonstrated in other species. The stimulation of nicotinic and muscarinic receptors followed by increased ACTH secretion. Glucocorticoids appear to lower ACTH secretion through an action at both the hypothalamic and pituitary levels.