Pituitary Proopiomelanocortin mRNA Research Articles

Effect of Repeated Lipopolysaccharide Administration on Tissue Cytokine Expression and Hypothalamic‐Pituitary‐Adrenal Axis Activity in Rats

The effects of chronic immune challenge on cytokine expression and hypothalamic-pituitary-adrenal axis (HPA) axis responses to stress were studied in Wistar rats after administration of increasing doses of lipopolysaccharide (LPS). Repeated LPS (R-LPS) decreased body weight and increased adrenal weight and pituitary pro-opiomelanocortin mRNA levels. LPS injection increased plasma adrenocorticotropic hormone (ACTH) and corticosterone but the effect was attenuated in R-LPS. Plasma corticosterone but not ACTH responses to restraint were also reduced in R-LPS. Basal and restraint-stimulated corticotropin releasing hormone (CRH) mRNA levels were lower in R-LPS, but responses to a new LPS injection were similar to controls. In contrast, type 1 CRH receptor (CRH-R1) mRNA responses to both LPS and restraint were blunted in R-LPS. Vasopressin mRNA levels in parvocellular neurones were higher in R-LPS, and increased further after restraint but not after a new LPS injection. Glucocorticoid receptor (GR) levels in the paraventricular nucleus (PVN) increased after a single LPS or R-LPS (24 h after the last injection) but declined after a new injection in R-LPS. Interleukin (IL)-1beta and IL-6 mRNAs increased in the pituitary, spleen and circumventricular organs after single or R-LPS, suggesting that cytokines may contribute to the activation of the HPA axis though pathways from the circumventricular organs as well as paracrine effects in the pituitary. The data show that (i) adaptation of the HPA axis during repeated LPS injection involves increases in vasopressin : CRH expression ratios in parvocellular neurones; (ii) that hypothalamic CRH and vasopressin responses to acute stimulation are independent of CRH-R1 expression in the PVN; and (iii) there is a dissociation between pituitary and adrenal responses to acute stress suggesting a decrease of adrenal sensitivity to ACTH.

Hypothalamo-pituitary-adrenocortical dysregulation in aging F344/Brown-Norway F1 hybrid rats.

Hypothalamo-pituitary-adrenocortical (HPA) axis aging was studied in young (3 mo), middle aged (15 mo) and aged (30 mo) F344/Brown Norway hybrid rats. This strain was selected to obviate HPA-relevant pathologies found in other aging models. Aged, unstressed rats showed enhanced central HPA drive, marked by elevated ACTH release and decreased pituitary proopiomelanocortin and corticotropin-releasing factor receptor 1 (CRH-R1) mRNAs. Acute corticosterone responses to spatial novelty were exacerbated in aged rats; however, responses to restraint or hypoxia were not affected. Chronic stress exposure also differentially increased HPA drive in aged animals, marked by elevated paraventricular nucleus CRH peptide levels and pituitary proopiomelanocortin mRNA. Plasma ACTH and pituitary POMC and CRH-R1 mRNA expression in middle-aged rats were intermediate those of young and aged animals. Middle-aged animals responded to chronic stress with disproportionate increases in CRH mRNA levels, and increased corticosterone secretion following hypoxia but not novelty. The results suggest a gradual increase in HPA tone across the aging process, culminating in marked hyperresponsivity to both acute and chronic stress in senescence.

Effects of Cortisol and Estradiol on Pituitary Expression of Proopiomelanocortin, Prohormone Convertase-1, Prohormone Convertase-2, and Glucocorticoid Receptor mRNA in Fetal Sheep

We hypothesized that in the late-gestation sheep fetus there is an interaction between the prepartum rise in cortisol and the increase in placental estradiol production that allows expression of key components of the fetal hypothalamic-pituitary-adrenal (HPA) axis. Therefore, the goal of this study was to investigate the effects of cortisol on the fetal HPA axis in the presence and absence of increased placental estradiol production. We obtained fetal plasma samples and pituitary tissue from animals that had received an infusion of either cortisol, cortisol and 4-hydroxyandrostenedione (40HA, an aromatase inhibitor), saline, or saline + 40HA controls. Cortisol significantly decreased plasma adrenocorticotropic hormone concentrations, and in the presence of 40HA reduced pituitary proopiomelanocortin (POMC) mRNA levels in the pars distalis. There was no effect of any treatment on the expression of the key POMC processing enzymes, prohormone convertase-1 or -2 in the fetal pituitary. Conversely, levels of glucocorticoid receptor (GR) mRNA in the pituitary were increased with cortisol treatment in the absence of increased estradiol. We suggest that in the late-gestation sheep fetus, cortisol and estradiol have opposite effects on pituitary POMC and GR mRNA expression, and interact to regulate these key components of the fetal HPA axis.

Regulation of proopiomelanocortin messenger ribonucleic acid levels in the ovine fetal anterior pituitary in vitro

Parturition in sheep is dependent upon maturation of the fetal hypothalamo-pituitary-adrenocortical (HPA) axis. Anterior pituitary expression of the ACTH precursor, proopiomelanocortin (POMC), increases during the final days of gestation in spite of exponentially increasing fetal plasma cortisol levels. Lesion of the hypothalamic paraventricular nucleus prevents the late gestation increase in POMC mRNA. The purpose of this study was to examine glucocorticoid, corticotropin releasing factor (CRF) and arginine vasopressin (AVP) regulation of POMC mRNA levels in fetal anterior pituitary corticotropes in vitro and to address potential interactions between glucocorticoids and neuropeptides in regulating POMC. Anterior pituitaries from fetal sheep at two gestational ages (dGA; 118–125 dGA, n=9; 140–144 dGA, n=7) were enzymatically dispersed. POMC mRNA levels were determined at 24, 48 and 72 h post-dispersion. CRF, AVP and dexamethasone (DEX) regulation of POMC mRNA were determined at 24 and 72 h post-dispersion. The capacity of CRF and AVP to modulate DEX suppression of POMC mRNA levels was also examined. POMC mRNA was elevated at 24 h ( P<0.01) and 48 h ( P<0.05) post-dispersion compared to 0 h (immediately post-dispersion) in 140–144 dGA but not 118–125 dGA corticotropes. DEX suppressed POMC mRNA in a dose-dependent manner (when administered at 24 h post-dispersion) in the 140–144 dGA anterior pituitary cells but not 118–125 dGA anterior pituitary cells. Administration of DEX (10 nM) at 0 h prevented the increase in POMC mRNA levels observed at 24 h post dispersion in the 140–144 dGA group. Neither CRF nor AVP (administered at either 24 or 72 h post-dispersion) altered POMC mRNA levels in either 118–125 or 140–144 dGA anterior pituitary cells. Continuous exposure of anterior pituitary cells with either CRF or AVP (50 pM) through 96 h increased ( P<0.05) POMC mRNA. No synergistic or additive effects were observed with CRF and AVP. Four hour pretreatment with CRF but not AVP (100 nM at 24 h post-dispersion) attenuated ( P<0.05) DEX suppression of POMC mRNA levels in 140–144 dGA corticotropes. In conclusion, our results indicate that direct glucocorticoid suppression of POMC expression in fetal sheep initiates between ∼120 and ∼140 dGA, coincident with the period of gestation when fetal plasma cortisol is exponentially rising. Further, while short duration exposure of fetal corticotropes to either CRF or AVP had no effect on POMC mRNA, CRF appears capable of interfering with glucocorticoid suppression of POMC mRNA. The latter observation provides a potential mechanism via which the fetal PVN may counter rising fetal plasma cortisol concentrations resulting in the previously observed late gestation increase in anterior pituitary POMC mRNA.

Chronic Daily Ethanol and Withdrawal: 1. Long‐Term Changes in the Hypothalamo‐Pituitary‐Adrenal Axis

Hypothalamo-pituitary-adrenal (HPA) function has been demonstrated to be compromised for weeks and even months after alcoholics cease ethanol consumption. Because nonalcoholic subjects with family history-associated increased risk for alcoholism also exhibit compromised HPA function, it is not clear whether defects in the HPA axis of abstinent alcoholics reflect a preexisting condition that may be responsible for increased risk for alcohol abuse versus a persisting adaptational change in response to prolonged alcohol abuse. Consequently, we investigated whether chronic daily ethanol consumption and withdrawal by male Sprague Dawley rats would induce persistent HPA changes consistent with those demonstrated in abstinent alcoholics. In an initial experiment in which ethanol (5%, w/v) was incrementally introduced to liquid diet over a 1 week period followed by 4 weeks of chronic ethanol consumption, not only ethanol-treated rats but also pair-fed control rats exhibited decreased (p < 0.05 vs. ad-libitum-fed controls) anterior pituitary pro-opiomelanocortin (POMC) mRNA concentrations and associated decreases in plasma corticosterone and adrenocorticotropin (ACTH) levels for at least 3 weeks after gradual withdrawal of ethanol from the diet. Pair-feeding-induced decreases (p < 0.05) in thymus and spleen weights suggested that the pair-fed controls were likely stressed in this model, probably in response to the marked and irregular suppression of liquid diet consumption immediately after introduction of ethanol. Consequently, a second model was developed in which ethanol was introduced to the liquid diet much more gradually (i.e., over 3 weeks). In contrast with the rapid ethanol-introduction model, this more prolonged ethanol introduction followed by 4 weeks of chronic daily ethanol consumption increased plasma corticosterone levels (p < 0.05), increased adrenal gland weight (p < 0.05), and decreased thymus and spleen weights (both p < 0.01) without altering any of these parameters in the pair-fed controls. Three weeks after gradual withdrawal of ethanol from the diet, anterior pituitary POMC mRNA concentrations were suppressed (p < 0.05) and thymus and spleen weights were increased (p < 0.05) versus both pair-fed and ad-libitum-fed controls, accompanied by trends for decreased basal plasma corticosterone and adrenal weights. Chronic daily ethanol treatment induced changes in the HPA axis that persisted for at least 3 weeks after complete cessation of ethanol consumption. These persistent alterations in the HPA axis are similar to the aberrant HPA regulation of abstinent alcoholics, sons of alcoholics, Lewis rats, and individuals who suffer from posttraumatic stress disorder and some types of depression, that is, categories of individuals who all exhibit increased risk for high ethanol consumption. Thus, these chronic daily ethanol-induced persistent changes in the HPA axis may have significant roles in alcohol abstinence syndrome and may increase vulnerability to relapse.

Activation of the hypothalamic-pituitary-adrenal axis with repetitive umbilical cord occlusion in the preterm ovine fetus

To determine whether repeated hypoxic insults with umbilical cord occlusion over 4 days will lead to activation of the hypothalamic-pituitary-adrenal (HPA) axis altered adrenocortical responsiveness in the preterm ovine fetus. Umbilical cord occlusions of 90 seconds duration were performed every 30 minutes for 3 to 5 hours each day (experimental group n = 7, control group n = 7; at 112-116 days' gestation, term = 147 days). Arterial blood was sampled at predetermined times for blood gases and pH, plasma ACTH, and cortisol. Pituitary proopiomelanocortin (POMC) and glucocorticoid receptor (GR) mRNA also were localized and quantified by in situ hybridization. During umbilical cord occlusions fetal arterial oxygen pressure (approximately 17 mmHg) and pH (approximately 0.05) decreased, and carbon dioxide pressure increased (approximately 8 mmHg) as measured on days 1 and 4, but with no cumulative blood gas or pH change over successive occlusions for any of the 4 study days. Plasma ACTH increased, as measured after cord occlusion and over the course of successive cord occlusions on days 1 and 4, and returned to control values by the next day. The cumulative increase in ACTH was much less on day 4 than day 1 (15 +/- 3 compared with 101 +/- 25 pg/mL, P <.05). Plasma cortisol increased, as measured after cord occlusion and over the course of successive cord occlusions on day 4 only (2.7 +/- 0.4 to 4. 7 +/- 0.3 ng/mL, P <.05). POMC mRNA increased 2.5-fold in the pars distalis of the pituitaries from cord occlusion compared to control fetuses, but was unchanged in the pars intermedia. GR mRNA, which was detected in the pars distalis only, was unaltered. Repetitive umbilical cord occlusion in the preterm ovine fetus resulted in the activation of the HPA axis, with increased adrenocortical responsiveness over time, and involved differential regulation of POMC mRNA expression in the pars distalis and pars intermedia of the pituitary, but with no change in GR.

Corticotropin-releasing hormone links pituitary adrenocorticotropin gene expression and release during adrenal insufficiency.

Corticotropin-releasing hormone (CRH)-deficient (KO) mice provide a unique system to define the role of CRH in regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Despite several manifestations of chronic glucocorticoid insufficiency, basal pituitary proopiomelanocortin (POMC) mRNA, adrenocorticotrophic hormone (ACTH) peptide content within the pituitary, and plasma ACTH concentrations are not elevated in CRH KO mice. The normal POMC mRNA content in KO mice is dependent upon residual glucocorticoid secretion, as it increases in both KO and WT mice after adrenalectomy; this increase is reversed by glucocorticoid, but not aldosterone, replacement. However, the normal plasma levels of ACTH in CRH KO mice are not dependent upon residual glucocorticoid secretion, because, after adrenalectomy, these levels do not undergo the normal increase seen in KO mice despite the increase in POMC mRNA content. Administration of CRH restores ACTH secretion to its expected high level in adrenalectomized CRH KO mice. Thus, in adrenal insufficiency, loss of glucocorticoid feedback by itself can increase POMC gene expression in the pituitary; but CRH action is essential for this to result in increased secretion of ACTH. This may explain why, after withdrawal of chronic glucocorticoid treatment, reactivation of CRH secretion is a necessary prerequisite for recovery from suppression of the HPA axis.

Pituitary-Adrenal Axis Regulation in Crh-Deficient Mice

Corticotropin-releasing hormone (CRH)-deficient (knockout (KO)) mice demonstrate severely impaired adrenal responses to restraint, ether, and fasting, and lack the normal diurnal glucocorticoid (GC) rhythm. Here, we summarize recent studies determining the role of CRH in augmenting plasma adrenocorticotrophic hormone (ACTH) concentration after glucocorticoid withdrawal and pituitary-adrenal axis stimulation in the context of inflammation. Even though GC insufficient, basal pituitary proopiomelanocortin (POMC) mRNA, ACTH peptide content within the pituitary, and plasma ACTH concentrations are not elevated in CRH KO mice. POMC mRNA content in CRH KO mice increases following adrenalectomy, and this increase is reversed by GC, but not aldosterone, replacement. In marked contrast to the increase in POMC mRNA, plasma ACTH does not increase in the CRH KO mice following adrenalectomy. Administration of CRH to adrenalectomized CRH KO mice results in acute, robust ACTH secretion. Thus, loss of GC feedback can increase POMC gene expression in the pituitary, but CRH action is essential for increased secretion of ACTH into the circulation. While GC secretion is impaired in CRH KO mice after most stimuli, we have found near-normal GC responses to inflammation and systemic immune challenge. Studies in mice with CRH and IL-6 deficiency reveal that IL-6 is essential for activation of the pituitary-adrenal axis during inflammatory and other stressors in the absence of CRH.

Regulation of prohormone convertase 1 (PC1) by gp130-related cytokines

The processing of pro-opiomelanocortin (POMC) to generate bioactive ACTH in the anterior pituitary is mediated by prohormone convertase 1 (PC1). Leukemia inhibitory factor (LIF) and interleukin 6 (IL-6), two cytokines sharing the common gp130 receptor subunit and functioning through activation of the intracellular JAK/STAT pathway, induce POMC synthesis and ACTH release. We investigated the effects of LIF and IL-6 on PC1 expression and its subsequent processing of POMC. A significant time-dependent up-regulation of both PC1 protein and mRNA by LIF and IL-6 was seen in mouse corticotroph AtT-20 cells. IL-6 or LIF increased the synthesis of ACTH-related products with a concomitant increase in bioactive 5 and 13 kDa ACTH indicating coordinated regulation of substrate and processing enzyme. AtT-20 cells transiently transfected with a human PC1-promoter-luciferase reporter construct and treated with LIF or IL-6 showed significantly increased luciferase activity. Additionally, lipopolysaccharide (LPS) administration to rats resulted in an increase in both pituitary PC1 and POMC mRNA. These findings suggest that the ACTH increase induced by LIF and IL-6 is due to both increased POMC synthesis as well as increased POMC processing by up-regulation of PC1. These two coordinately regulated processing events probably exert central roles in the pathophysiological response to some stresses, such as inflammatory stress.

The influence of sex and gonadectomy on the hypothalamo-pituitary-adrenal axis of the sheep.

There is a sex difference in the hypothalamo-pituitary-adrenal (HPA) axis of many species, although there are sparse data on the sheep. In the present study we have compared the HPA axes of intact and gonadectomised adult male and female sheep at the level of the median eminence, pituitary and adrenal glands using a variety of in vitro approaches. The concentration of arginine vasopressin (AVP) was higher (P<0.01) in the median eminence of male than female sheep, and was also elevated by gonadectomy of either sex (P<0.01). The concentration of corticotrophin-releasing factor (CRF) in the median eminence did not differ between the sexes, but was also elevated in both sexes following gonadectomy (P<0.01). Anterior pituitary pro-opiomelanocortin mRNA concentrations were higher (P<0.05) in intact male sheep than in intact females, with the levels in gonadectomised animals of both sexes being intermediate. In contrast to this finding, basal ACTH secretion from anterior pituitary cells was higher (P<0.05) in cultures derived from female sheep than those from males, but gonadectomy was without effect. There was no effect of sex or gonadectomy on in vitro ACTH secretion in response to AVP, CRF or the combination of AVP and CRF, and in all cases the combination of AVP and CRF generated greater (P<0.0001) ACTH secretion than AVP alone. AVP alone was more effective (P<0.01) than CRF alone as an ACTH secretagogue. The adrenal glands were larger (P<0.05) in female than male sheep, with no effect of gonadectomy. Basal cortisol production was greatest (P<0.05) in cultures of adrenal cells from intact male sheep, though ACTH- and 8BrcAMP-induced cortisol production was greater in the cultures of cells from females (P=0.05); there were no effects of gonadectomy. Cultures of adrenocortical cells from male sheep had greater (P<0.05) basal cAMP production, but ACTH-stimulated cAMP production did not differ between any of the groups of animals. These findings show a range of differences in the HPA axis of male and female sheep. Furthermore, they suggest that the heightened activity of the axis in the female occurs primarily due to differences at the level of the adrenal gland, and that greater adrenal responsiveness of female animals is due to differences in the latter stages of steroidogenesis, rather than an effect on ACTH signal transduction at its receptor.

Differential involvement of adrenal and gonadal steroids in anterior and intermediate pituitary pro-opiomelanocortin mRNA expression induced by the endogenous benzodiazepine, octadecaneuropeptide, in adult male rats.

The involvement of the endogenous benzodiazepine, octadecaneuropeptide (ODN), in the regulation of proopiomelanocortin (POMC) mRNA expression at the pituitary level, and the influence of adrenal and gonadal steroids, have been studied using a quantitative in situ hybridization technique. I.c.v. injection of ODN (4 micrograms/kg) in sham-operated rats induced a 17 and 7% decrease in the POMC mRNA expression in anterior and intermediate pituitary lobes respectively. To determine the reciprocal involvement of adrenal and gonadal steroids in this regulation, animals were adrenalectomized and/or castrated. Adrenalectomy significantly increased POMC mRNA expression by 48% at the anterior pituitary level, but induced a 10% decrease of hybridization signal at the intermediate pituitary lobe (vs control sham-operated). Adrenal ablation reversed the effect induced by ODN and increased POMC mRNA expression at the anterior and intermediate pituitary levels by 60 and 10% respectively, compared with control sham-operated. By contrast, castration, which produced a decrease in POMC mRNA in the anterior pituitary and an increase in the intermediate lobe, did not modify the negative influence of ODN observed in sham-operated animals. When rats were adrenalectomized and castrated, the adrenalectomy influence was predominant at the anterior pituitary level, since ODN increased significantly the hybridization signal (+68% vs control sham-operated), while the castration influence was predominant at the intermediate pituitary level, since ODN induced an 11% decrease in POMC mRNA signal compared with control sham-operated. These studies indicate that, in vivo, the decrease in POMC mRNA expression in the anterior and intermediate pituitary induced by an endogenous benzodiazepine is differently modulated by adrenal and gonadal steroids, with a predominant influence of adrenal steroids at the anterior pituitary level and gonadal steroids at the intermediate pituitary level.

Dissociation of changes in hypothalamic corticotropin-releasing hormone and pituitary proopiomelanocortin mRNA levels after prolonged stress exposure

Repeated immobilization stress for 7 days caused marked increase in corticotropin-releasing hormone (CRH) mRNA in the paraventricular nucleus (PVN, 90% above the control) and proopiomelanocortin (POMC) mRNA in the anterior pituitary (73% above the control) in rats. Prolongation of the stress exposure to 42 days led to a decline of CRH mRNA response (42% above the control) in comparison with that after 7 days, while POMC mRNA persisted at the same level. Observed adaptation of CRH but not POMC responses suggests that other factor(s) next to CRH participate in sustained rise in POMC expression during long-term stress.

Changes of Pituitary Proopiomelanocortin mRNA Levels during Metamorphosis of the Bullfrog Larvae

Adrenal corticoids accelerate metamorphosis of amphibians by potentiating the action of thyroid hormone. Adrenal corticoid secretion is considered to be controlled mainly by adrenocorticotropic hormone generated from proopiomelanocortin (POMC) in the anterior lobe of the pituitary. In order to assess the changes in POMC mRNA levels during metamorphosis, a cDNA for POMC was isolated from a cDNA library constructed from bullfrog (Rana catesbeiana) pituitary polyadenylated RNA. Northern blot analysis using the POMC cDNA as a probe revealed that POMC mRNA levels in the anterior lobe were relatively low during premetamorphosis, rose during prometamorphosis, reached the maximum at the end of prometamorphosis and remained very high during climax. The POMC mRNA levels of the intermediate lobe, where α-melanophore-stimulating hormone is generated from POMC, were also determined in metamorphosing tadpoles. The POMC mRNA levels of the intermediate lobe increased as metamorphosis progressed and were maximal at mid-climax. High POMC mRNA levels were observed even in larvae that had adapted to a white background. The significance of these findings and their relationships to the hormonal requirements during metamorphosis are discussed.

Elevation of brain 5-HT activity, POMC expression, and plasma cortisol in socially subordinate rainbow trout.

Agonistic behavior, brain concentrations of serotonin (5-hydroxytryptamine, 5-HT), and 5-hydroxyindoleacetic acid (5-HIAA, the main 5-HT metabolite), plasma cortisol levels, and the pituitary expression of pro-opiomelanocortin (POMC) A and B mRNA were determined in socially dominant and subordinate rainbow trout after 1 or 7 days of social interaction. Telencephalic and brain stem 5-HIAA/5-HT ratios, plasma cortisol levels, and pituitary POMC mRNA concentrations were elevated in fish being subordinate for 1 day. Furthermore, neither telencephalic 5-HIAA/5-HT ratios nor pituitary POMC A or POMC B mRNA expression showed any decline after 7 days of social interaction. By contrast, plasma cortisol concentrations of subordinate fish declined after 7 days but were still significantly higher than in dominant fish. Furthermore, in subordinate fish, hypothalamic 5-HIAA/5-HT ratios and plasma cortisol levels were highly correlated, suggesting an important role of hypothalamic 5-HT in the regulation of the teleost hypothalamic-pituitary-interrenal (HPI) axis. The number of aggressive acts received and plasma cortisol levels were highly correlated in 1-day subordinates, a relationship not seen in fish subjected to 1 wk of subordination. Thus the chronic stress experienced by subordinates in established dominance hierarchies appears to be more closely related to the threat imposed by the presence of the dominant fish than to actual aggressive encounters. The sustained elevation of pituitary POMC mRNA expression, an effect mainly related to an increase of melanotropic POMC expression, in subordinates could be a mechanism serving to maintain HPI axis excitability and promote acclimation in these individuals.

Divergent changes in plasma ACTH and pituitary POMC mRNA after cortisol administration to late-gestation ovine fetus.

Plasma concentrations of cortisol and adrenocorticotropic hormone (ACTH) rise in the late-gestation sheep fetus at approximately the same time as there is an increase in the plasma levels of corticosteroid-binding globulin (CBG). We hypothesized that intrafetal cortisol infusion during late pregnancy would stimulate an increase in fetal plasma CBG, which in turn would bind cortisol and diminish glucocorticoid negative-feedback regulation of the fetal pituitary, leading to an increase in plasma ACTH concentrations. Cortisol was infused into chronically catheterized fetal sheep beginning at 126.1 +/- 0.5 days of gestation and continued for 96 h. Control fetuses were infused with saline. In cortisol-infused fetuses, the plasma cortisol concentrations rose significantly from control levels (4.4 +/- 0.6 ng/ml) to 19.3 +/- 3.1 ng/ml within 24 h and remained significantly elevated throughout the infusion period. Plasma immunoreactive (i.r.) ACTH concentrations were significantly elevated in cortisol-infused fetuses within 24-48 h and remained significantly higher than in controls throughout the 96-h experimental period. Plasma free cortisol concentrations increased 10-fold and remained significantly elevated in cortisol-infused animals, despite a rise in plasma corticosteroid-binding capacity. Levels of pituitary proopiomelanocortin (POMC) mRNA in the fetal pars distalis and pars intermedia were 96 and 38% lower, respectively, after 96 h of cortisol infusion. Therefore physiological elevations of plasma cortisol, in the late-gestation ovine fetus, lead to increases in mean plasma irACTH concentrations, but this is not associated with increases in fetal pituitary POMC mRNA levels.

Quantification of proopiomelanocortin mRNA in peripheral lymphocytes of alcoholics

Recent studies indicate the production of adrenocorticotropic hormone (ACTH) by human mononuclear leucocytes constitutively or in response to hypothalamic factors and interferon inducers. These results have led to the proposal that the control mechanisms for the proopiomelanocortin (POMC) gene in lymphocytes may be similar to that of pituitary cells. After chronic ethanol treatment, changes in the pituitary POMC mRNA in rats have been reported, but so far nothing is known about changes in the POMC gene expression in lymphocytes under these conditions. Therefore, the expression of the POMC gene was investigated in human peripheral lymphocytes in both volunteers and alcoholics. POMC mRNA levels in these cells were significantly increased in alcoholics, with one alcoholic showing an even more than five times higher POMC mRNA level compared to the other patients. After detoxification, a significant decrease in the POMC mRNA of alcoholic patients was observed, except in the one with the extremely elevated POMC mRNA level, where the opposite occurred. An influence of chronic ethanol ingestion on POMC gene expression in lymphocytes could be assumed. The observed changes seem to be correlated with ethanol intake, because the elevated POMC mRNA amounts found in lymphocytes of alcoholics after admission to the hospital were already declining after the detoxification period.

Ontogeny of Hypothalamic Corticotropin‐Releasing Factor and Anterior Pituitary Pro‐opiomelanocortin Expression in Male and Female Offspring of Alcohol‐Exposed and Adrenalectomized Dams

This study was designed to determine whether the previously described sexually dimorphic changes in rat hypothalamic corticotropin-releasing factor (CRF) and anterior pituitary pro-opiomelanocortin (POMC) mRNA expression in response to fetal alcohol exposure (FAE) are present prepubertally and whether they are altered by maternal adrenalectomy. Hypothalamic CRF and anterior pituitary POMC mRNA levels were determined in male and female offspring of adrenalectomized (ADX) and sham-adrenalectomized (Sham) dams exposed to alcohol (FAE) or a pair-fed (PF) control diet during the last 2 weeks of gestation. CRF and POMC mRNA levels were measured by Northern blotting at 1, 7, 14, and 21 days of age. In offspring of control PF dams, CRF mRNA levels increased faster in females, increasing by day 7, followed by a decrease at days 14 and 21, whereas in males there was a gradual increase from days 1 to 21. FAE altered the ontogenic profile of CRF mRNA in female offspring by delaying and exaggerating the rise of CRF expression to day 14, but produced no effect in males. Maternal adrenalectomy, combined with FAE, resulted in an early rise of CRF mRNA on day 14 in male offspring. In females, the combined ADX/FAE treatment resulted in significantly increased CRF mRNA levels, compared with those of ADX/PF offspring, on days 7 and 14. By day 21, these differences in CRF mRNA levels between the ADX/FAE and ADX/PF offspring had disappeared. POMC mRNA levels generally increased by day 7, followed by a dramatic decrease by day 14 and another increase by day 21. FAE male offspring showed decreased levels of POMC mRNA, whereas females were not affected. Maternal adrenalectomy reversed this inhibition in male offspring, resulting in POMC mRNA levels similar to those measured in male offspring of PF control animals. In contrast, POMC mRNA levels of female offspring of ADX dams decreased in response to FAE. These data suggest that the previously observed switch from suppressed to enhanced POMC expression in FAE males is the result of developmental events beyond weaning. Because this sexually dimorphic regulation of CRF and POMC expression by prenatal alcohol exposure and maternal adrenalectomy occurs before the presence of adult levels of sex steroids, this suggests an organizational effect on the developing hypothalamic-pituitary-adrenal function.

Fetal grafts containing suprachiasmatic nuclei restore the diurnal rhythm of CRH and POMC mRNA in aging rats.

We assessed whether fetal tissue containing the suprachiasmatic nuclei (SCN) can restore age-related changes in the diurnal rhythm of hypothalamic corticotropin-releasing hormone (CRH) and anterior pituitary proopiomelanocortin (POMC) mRNA. Young, middle-aged, and middle-aged SCN-transplanted rats were killed at seven times of day. In young rats, CRH mRNA exhibited a diurnal rhythm in the dorsomedial paraventricular nuclei but not in other subdivisions of the nuclei. No rhythm was detected in aging rats. SCN transplants restored a rhythm in CRH mRNA, but the timing was not precisely the same as in young animals. POMC mRNA exhibited a daily rhythm in young rats. Aging abolished the rhythm and decreased the average mRNA level; fetal transplants restored the rhythm, but the amplitude remained attenuated. These data are the first demonstration that fetal tissue can restore the diurnal rhythm of a neuroendocrine axis that is driven by the SCN. We conclude that the neuroendocrine substrate from the aging host remains capable of responding to diurnal cues to express diurnal rhythmicity in CRH/POMC mRNA when fetal SCN transplants confer the appropriate signals.

Sexually dimorphic effects of maternal adrenalectomy on hypothalamic corticotrophin-releasing factor, glucocorticoid receptor and anterior pituitary POMC mRNA levels in rat neonates

The stress hyporesponsive period (SHRP) occurs during the first two weeks of life (from day 4 to day 14) in the rat. SHRP may occur due to immature hypothalamic-pituitary-adrenal (HPA) regulatory mechanisms of the neonate. Decreased expression of corticotropin-releasing factor (CRF) has been observed during this period, and this decreased hypothalamic `drive' may contribute to the manifestation of SHRP in the rat neonate. Since maternal corticosteroids are elevated toward the end of gestation they may suppress fetal CRF expression leading to a less than adequate activation of the neonatal hypothalamus in response to stress. Therefore, the purpose of the present study was to clarify the contribution of maternal glucocorticoids to the decreased CRF expression observed during SHRP in the neonatal rat. We investigated the effects of maternal adrenalectomy on hypothalamic CRF, glucocorticoid receptor (GR) and anterior pituitary proopiomelanocortin (POMC) mRNA levels in male and female neonates. Maternal adrenalectomy, or sham surgery, was performed on day 8 of gestation and the mRNA levels of CRF, GR and POMC were measured by Northern blotting in the offspring on their postnatal days 1, 7, 14, and 21. The observed changes in the mRNA levels of these genes suggests that an important developmental event occurs in the regulation of these HPA genes between neonatal days 7 and 14 corresponding to the termination of SHRP. Female offspring had significantly higher levels of CRF mRNA than males throughout this period. The lack of maternal corticosteroids evoked a gender-specific response in the neonates. In female offspring, maternal adrenalectomy resulted in a dramatic and correlated increase in mRNA levels of hypothalamic CRF and GR on day 14, with pituitary POMC expression not following this increase. There was no significant effect of maternal adrenalectomy on the expression of these genes in males. These results suggest a sex difference in response to maternal glucocorticoids in the fetus. However, the role of maternal corticosteroids in the low expression of CRF during SHRP could not be established from this study, since their removal by adrenalectomy did not advance the expression profile of CRF toward an earlier increase in the neonatal hypothalamus.

Inhibition of the hypothalamic-pituitary-thyroid axis in response to lipopolysaccharide is independent of changes in circulating corticosteroids.

We have investigated the role of circulating glucocorticoids in the suppression of the hypothalamic-pituitary-thyroid (HPT) axis following lipopolysaccharide (LPS) injection in rats. Intraperitoneal injection of LPS (2.5 mg/kg) suppressed paraventricular nucleus thyrotropin-releasing hormone (TRH) mRNA, pituitary thyroid-stimulating hormone (TSH) mRNA and plasma triiodothyronine. In these animals LPS also increased paraventricular nucleus corticotropin-releasing hormone (CRH) mRNA, pituitary proopiomelanocortin (POMC) mRNA and plasma corticosterone levels. To investigate the role of plasma corticosterone in the suppression of the HPT axis, we clamped the plasma corticosterone level at morning baseline level by bilateral adrenalectomy and corticosterone pellet implantation. Ten days after surgery, LPS injection evoked a dramatic increase in CRH mRNA and POMC mRNA. Despite the lack of change in plasma corticosterone in the corticosterone-clamped rats, LPS was still able to suppress TRH and TSH mRNA levels in both corticosterone-clamped and sham-operated rats. These data indicate that in response to LPS, suppression of the HPT axis occurs and is independent of the LPS-induced increase in plasma corticosterone.