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)     

Vasopressinergic regulation of the hypothalamic-pituitary-adrenal axis: implications for stress adaptation

In addition to its role on water conservation, vasopressin (VP) regulates pituitary ACTH secretion by potentiating the stimulatory effects of corticotropin releasing hormone (CRH). The pituitary actions of VP are mediated by plasma membrane receptors of the V1b subtype, coupled to calcium-phospholipid signaling systems. VP is critical for adaptation of the hypothalamic-pituitary-adrenal (HPA) axis to stress as indicated by preferential expression of VP over CRH in parvocellular neurons of the hypothalamic paraventricular nucleus, and the upregulation of pituitary VP receptors during stress paradigms associated with corticotroph hyperresponsiveness. V1b receptor mRNA levels and coupling of the receptor to phospolipase C are stimulated by glucocorticoids, effects which may contribute to the refractoriness of VP-stimulated ACTH secretion to glucocorticoid feedback. The data suggest that vasopressinergic regulation of the HPA axis is critical for sustaining corticotroph responsiveness in the presence of high circulating glucocorticoid levels during chronic stress.     

In vivo correlation between c-Fos expression and corticotroph stimulation by adrenocorticotrophic hormone secretagogues in rat anterior pituitary gland

In the anterior pituitary gland, c-Fos expression is evoked by various stimuli. However, whether c-Fos expression is directly related to the stimulation of anterior pituitary cells by hypothalamic secretagogues is unclear. To confirm whether the reception of hormone-releasing stimuli evokes c-Fos expression in anterior pituitary cells, we have examined c-Fos expression of anterior pituitary glands in rats administered with synthetic corticotrophin-releasing hormone (CRH) intravenously or subjected to restraint stress. Single intravenous administration of CRH increases the number of c-Fos-expressing cells, and this number does not change even if the dose is increased. Double-immunostaining has revealed that most of the c-Fos-expressing cells contain adrenocorticotrophic hormone (ACTH); corticotrophs that do not express c-Fos in response to CRH have also been found. However, restraint stress evokes c-Fos expression in most of the corticotrophs and in a partial population of lactotrophs. These results suggest that c-Fos expression increases in corticotrophs stimulated by ACTH secretagogues, including CRH. Furthermore, we have found restricted numbers of corticotrophs expressing c-Fos in response to CRH. Although the mechanism underlying the different responses to CRH is not apparent, c-Fos is probably a useful immunohistochemical marker for corticotrophs stimulated by ACTH secretagogues. This work was supported by the Jichi Medical University young investigator award.     

Evidence that endogenous SST inhibits ACTH and ghrelin expression by independent pathways

Corticosterone and total ghrelin levels are increased in somatostatin (SST) knockout mice (Sst-/-) compared with SST-intact controls (Sst+/+). Because exogenous ghrelin can increase glucocorticoids, the question arises whether elevated levels of ghrelin contribute to elevated corticosterone levels in Sst-/- mice. We report that Sst-/- mice had elevated mRNA levels for pituitary proopiomelanocortin (POMC), the precursor of adrenocorticotropic hormone (ACTH), whereas mRNA levels for hypothalamic corticotropin-releasing hormone (CRH) did not differ from Sst+/+ mice. Furthermore, SST suppressed pituitary POMC mRNA levels and ACTH release in vitro independently of CRH actions. In contrast, it has been reported that ghrelin increases glucocorticoids via a central effect on CRH secretion and that n-octanoyl ghrelin is the form of ghrelin that activates the GHS-R1a and modulates CRH neuronal activity. Consistent with elevations in total ghrelin levels, Sst-/- mice displayed an increase in stomach ghrelin mRNA levels, whereas hypothalamic and pituitary expression of ghrelin was not altered. Despite the increase in total ghrelin levels, circulating levels of n-octanoyl ghrelin were not altered in Sst-/- mice. Because glucocorticoids and ghrelin increase in response to fasting, we examined the impact of fasting on the adrenal axis and ghrelin in Sst+/+ and Sst-/- mice and found that endogenous SST does not significantly contribute to this adaptive response. We conclude that endogenous SST inhibits basal ghrelin gene expression in a tissue specific manner and independently and directly inhibits pituitary ACTH synthesis and release. Thus endogenous SST exerts an inhibitory effect on ghrelin synthesis and on the adrenal axis through independent pathways.     

Attenuation of corticotropin-releasing hormone and arginine vasopressin responsiveness during late-gestation pregnancy in sheep

Responsiveness of the hypothalamo-pituitary-adrenal axis is decreased during pregnancy. Therefore, the objective of the present study was to determine if responsiveness at the level of individual corticotrophs to corticotropin-releasing hormone (CRH) or arginine vasopressin (AVP) is decreased during pregnancy in sheep. Anterior pituitaries (APs) were collected from pregnant and nonpregnant ewes. Half of the APs were dispersed, and cells were placed on immobilon and treated with vehicle, CRH (10 nM), or AVP (100 nM) for 2 h. Cells were then fixed and incubated with ACTH or pro-opiomelanocortin (POMC) antibodies. The percentage of cells staining positive for immunoreactive (ir) ACTH or POMC, the percentage of cells secreting irACTH or POMC, and the area of irACTH or POMC secretion were measured. RNA was extracted from the other half of the APs to quantify CRH type 1 (CRH-R1) and vasopressin type 1b (V1b) receptor mRNA by ribonuclease protection assay. CRH treatment increased the percentage of corticotrophs with relatively large areas of irACTH and POMC secretion in nonpregnant, but not in pregnant, ewes. AVP treatment significantly increased the percentage of irACTH- and POMC-secreting cells in nonpregnant, but not in pregnant, ewes. V1b receptor mRNA, but not CRH-R1 receptor mRNA, was significantly decreased during pregnancy. These results suggest that corticotroph responsiveness to CRH and AVP is decreased during pregnancy in sheep. Therefore, reduced corticotroph responsiveness may contribute to stress hyporesponsivity during pregnancy.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates cyclic AMP formation as well as peptide output of cultured pituitary melanotrophs and AtT-20 corticotrophs.

The present study was aimed at investigating whether PACAP stimulates accumulation of cAMP, as well as hormonal secretion of homogeneous populations of pituitary proopiomelanocortin (POMC) cells, namely melanotrophs and AtT-20 corticotrophs. PACAP was shown to enhance cAMP accumulation in a dose-dependent fashion in both cell types (with EC50 values of approx. 10(-10) M) and elicited additive increases of cAMP production with CRF in melanotrophs, but not in corticotrophs. PACAP also stimulated dose-dependently the secretion of alpha-MSH and ACTH, with EC50 concentrations of about 10(-9) M. In melanotrophs, bromocriptine significantly depressed PACAP-induced cAMP formation and blunted by more than 90% stimulated alpha-MSH release. This study shows that (1) pituitary POMC cells did respond to PACAP by enhancing cAMP accumulation and elevating hormone secretion as well; (2) the effect of PACAP was additive with CRF on cAMP production in melanotrophs, but not in corticotrophs, while there was no additivity on peptide output from both cell types; (3) activation of dopamine receptors in melanotrophs dampened both cAMP formation and peptide secretion. These findings are consistent with PACAP playing a possible hypophysiotropic role in the regulation of pituitary POMC cell activity.     

Mechanisms Underlying the Increased Plasma ACTH Levels in Chronic Psychosocially Stressed Male Mice

Mice exposed to chronic subordinate colony housing (CSC, 19 days), an established paradigm for chronic psychosocial stress, show unaffected basal morning plasma corticosterone (CORT) concentrations, despite enlarged adrenal glands and an increased CORT response to an acute heterotypic stressor. In the present study we investigate the mechanisms underlying these phenomena at the level of the pituitary. We show that both basal and acute stressor-induced (forced swim (FS), 6 min) plasma adrenocorticotropic hormone (ACTH) concentrations, the number of total and corticotroph pituitary cells, and relative protein expression of pituitary mineralocorticoid receptor and FK506-binding protein 51 was increased in CSC compared with single-housed control (SHC) mice, while relative corticotropin releasing hormone (CRH) receptor 1 (CRH-R1) and glucocorticoid receptor protein expression was down-regulated. Relative pituitary pro-opiomelanocortin and arginine vasopressin (AVP) receptor 1b (AVPR-1b) protein expression, FS (6 min)-induced ACTH secretion in dexamethasone-blocked mice, and the number of AVP positive magnocellular and parvocellular neurons in the paraventricular hypothalamic nucleus (PVN) was unaffected following CSC. Taken together, the data of the present study indicate that 19 days of CSC result in pituitary hyperactivity, under both basal and acute heterotypic stress conditions. Although further studies have to assess this in detail, an increased number of pituitary corticotrophs together with unaffected relative pituitary AVPR-1b and decreased CRH-R1 protein expression following CSC suggests that pituitary hyperdrive is mediated by newly formed corticotrophs that are more sensitive to AVP than CRH. Moreover, our data indicate that changes in PVN AVP and negative feedback inhibition seem not to play a major role in pituitary hyperactivity following CSC.     

Pituitary and hypothalamic hormones in normal and neoplastic adrenal medullae: biologically active corticotropin-releasing hormone and corticotropin

Six normal and 8 neoplastic adrenal medullae were assayed for several immunoreactive (IR) proopiomelanocortin (POMC) and hypothalamic peptides. IR-POMC peptides were found in normal and tumor tissue in concentrations ranging from 0.0003 to 0.1% of those in pituitary. Their molecular sizes resembled those of pituitary intermediate lobe POMC peptides. No intact POMC was found. One pheochromocytoma contained fully bioactive IR-adrenocorticotropic hormone (IR-ACTH; Mr approximately 4,500) and an intermediate-sized (Mr approximately 10,000) IR-ACTH with approximately 69% bioactivity. Normal and tumorous medullae contained IR-corticotropin-releasing hormone (CRH) in concentrations ranging from 0.6 to 4% of those in hypothalamus except for one pheochromocytoma that contained 40 times that amount of IR-CRH, which was chromatographically indistinguishable from hypothalamic CRH and fully bioactive. IR-somatostatin and IR-growth hormone-releasing hormone were found in both tissue types, but IR-gonadotropin-releasing hormone and IR-thyrotropin-releasing hormone (TRH) were not, although IR-histidyl-proline diketopiperazine, a putative TRH metabolite, was found. IR-arginine vasopressin was found in two normal medullae, but not in pheochromocytomas.     

Elevated corticosterone and inhibition of ACTH responses to CRH and ether in the neonatal rat: effect of hypoxia from birth

Hypoxia is a common cause of neonatal morbidity and mortality. We have previously demonstrated a dramatic ACTH-independent activation of adrenal steroidogenesis in hypoxic neonatal rats, leading to increases in circulating corticosterone levels. The purpose of the present study was to determine if this ACTH-independent increase in corticosterone inhibits the ACTH response to acute stimuli. Neonatal rats were exposed to normoxia (control) or hypoxia from birth to 5 or 7 days of age. At the end of the exposure, plasma ACTH and corticosterone were measured before and after either ether vapors were administered for 3 min or CRH (10 microg/kg) was given intraperitoneally. Thyroid function, pituitary pro-opiomelanocortin (POMC) mRNA and ACTH content, and hypothalamic corticotropin-releasing hormone (CRH), neuropeptide Y (NPY), and AVP mRNA were also assessed. Hypoxia led to a significant increase in corticosterone without a large increase in ACTH, confirming previous studies. The ACTH responses to ether or CRH administration were almost completely inhibited in hypoxic pups. Hypoxia did not affect the established regulators of the neonatal hypothalamic-pituitary-adrenal axis, including pituitary POMC or ACTH content, hypothalamic CRH, NPY, or AVP mRNA (parvo- or magnocellular), or thyroid function. We conclude that hypoxia from birth to 5 or 7 days of age leads to an attenuated ACTH response to acute stimuli, most likely due to glucocorticoid negative feedback. The neural and biochemical mechanism of this effect has yet to be elucidated.     

Pituitary corticotroph ontogeny and regulation in transgenic zebrafish

We characterized zebrafish proopiomelanocortin (POMC) gene promoter, and sequence analysis revealed that the promoter contains regulatory elements conserved among vertebrate species. To monitor the ontogeny of the pituitary POMC lineage in living vertebrates, we generated transgenic zebrafish expressing green fluorescent protein (GFP) driven by the POMC promoter. Zebrafish POMC-GFP is first expressed asymmetrically as two bilateral groups of cells most anterior to the neural ridge midline at 18-20 h post fertilization (hpf). POMC-GFP-positive cells then fuse into a single-cell mass within the pituitary anlage after 24 hpf and subsequently organize as distinct anterior and posterior domains between 48 and 64 hpf. Immunohistochemical studies with ACTH and alphaMSH antisera showed that POMC-GFP was mainly targeted to both anterior and posterior pituitary corticotrophs, whereas posterior pituitary region melanotrophs did not express GFP. To determine in vivo zebrafish corticotroph responses, dexamethasone (10(-5) m) was added to live embryos, which selectively suppressed POMC-GFP expression in the anterior group of corticotrophs, suggesting a distinct domain that is responsive to glucocorticoid feedback. Transgenic zebrafish with specific POMC-GFP expression in pituitary corticotrophs offers a powerful genetic system for in vivo study of vertebrate corticotroph lineage development.     

Lesion of central part of the dorsomedial nucleus alters vasopressin but not corticotropin releasing hormone mRNA levels in rat hypothalamic paraventricular nucleus

Functional significance of neural projections from the hypothalamic dorsomedial nucleus (DMN) to the paraventricular nucleus (PVN) was investigated using surgical lesion of the central part of the DMN. Under basal conditions, DMN lesion resulted in a decrease in magnocellular vasopressin (AVP) mRNA levels in the PVN, rise in pituitary proopiomelancortin (POMC) mRNA concentrations and elevated plasma corticosterone levels. Corticotropin-releasing hormone (CRH) mRNA levels remained unaffected. In sham operated animals, osmotic stress induced by hypertonic saline injection failed to modify AVP mRNA, but increased CRH and POMC mRNA levels and peripheral hormone release. The rise in CRH mRNA levels after osmotic stress was potentiated in DMN lesioned animals. Thus, the DMN participates in the control of hypothalamic peptide gene expression and pituitary adrenocorticotropic function.     

Vasopressin pressor receptor-mediated activation of HPA axis by acute ethanol stress in rats

The plasma arginine vasopressin (AVP), ACTH, and corticosterone levels and the hypothalamic corticotropin-releasing hormone (CRH) content were measured after oral administration of 1 ml of 75% ethanol to rats, a model known to induce acute gastric erosions and stress. Elevated plasma AVP, ACTH, and corticosterone levels were detected 1 h after ethanol administration. Treatment with the vasopressin pressor (V(1)) receptor antagonist [d(CH(2))(5)Tyr(Me)-AVP] before ethanol administration significantly reduced the ACTH and corticosterone level increases. A higher hypothalamic CRH content was measured at 30 or 60 min after ethanol administration. V(1) receptor antagonist injection, 5 min before ethanol administration, inhibited the rise in hypothalamic CRH content. The protein synthesis blocker cycloheximide prevented the hypothalamic CRH content elevation after stress. The AVP-, CRH-, and AVP + CRH-induced in vitro ACTH release in normal anterior pituitary tissue cultures was also prevented by pretreatment with the V(1) receptor antagonist. The results support the hypothesis that stress-induced AVP may not only act directly on the ACTH producing anterior pituitary cells but also indirectly at the hypothalamic level via the synthesis and release of CRH.     

Acute and chronic regulation of pituitary receptors for vasopressin and corticotropin releasing hormone

At least two hypothalamic peptides, corticotropin releasing hormone (CRH) and vasopressin (VP), are important in regulating adrenocorticotropin (ACTH) release from the anterior pituitary. Both are secreted in a pulsatile manner and stimulate ACTH secretion by interacting with G protein-coupled receptors (GPCRs), namely the type 1 CRH receptor and V1b receptor, respectively. Repeated or prolonged stimulation with either peptide can cause reduced ACTH responsiveness or desensitisation, both in vivo and in vitro. Desensitisation of perifused sheep anterior pituitary cells to VP was found to be rapid and occurred following treatment with 5 nM VP for 5 min. This is within the range of concentrations and durations of VP pulses seen in sheep portal blood during acute stress. In contrast, significant desensitisation of the ACTH response to CRH required pre-treatment for longer than 25 min with a CRH concentration of 1 nM, suggesting that endogenous pulses may not elicit desensitisation. Although rapid GPCR desensitisation involves uncoupling of receptors from their G proteins, commonly mediated by receptor phosphorylation, and internalisation of receptors, desensitisation of neither the CRH nor VP receptor was mediated by PKA or PKC, respectively. Desensitisation of the response to VP was found to be dependent upon receptor internalisation, and resensitisation could be delayed by treatment with a protein phosphatase 2B inhibitor. The rapid kinetics of desensitisation of the ACTH response to VP suggest that this process is important in regulating the response to acute rather than chronic stress. If, as has been suggested, CRH acts in a permissive way to set corticotrope gain, desensitisation to CRH could also be important in long term regulation of ACTH secretion.     

The role of ether-a-go-go-related gene K(+) channels in glucocorticoid inhibition of adrenocorticotropin release by rat pituitary cells

The present study investigated the role of K(+) channels in the inhibitory effect of glucocorticoid on adrenocorticotropin (ACTH) release induced by corticotropin-releasing hormone (CRH) using cultured rat anterior pituitary cells. Apamin and charybdotoxin (CTX) did not have a significant effect on ACTH release induced by CRH (1 nM). Tetraethylammonium (TEA), a broad spectrum K(+) channel blocker, increased the ACTH response to CRH only at the highest concentration (10 mM). The exposure to 100 nM corticosterone for 60 min inhibited the CRH-induced ACTH release. Neither TEA, apamin, nor CTX affected the inhibitory effect of corticosterone. In contrast, astemizole (Ast) and E-4031, ether-a-go-go-related gene (erg) K(+) channel blockers, abolished the inhibitory effect of corticosterone on CRH-induced ACTH release (1.25+/-0.10 vs. 1.45+/-0.11 ng/well at 10 microM Ast, p>0.05, 1.71+/-0.16 vs. 1.91+/-0.32 ng/well at 10 microM E-4031, p>0.05, vehicle vs. corticosterone). RT-PCR demonstrated all three subtypes of rat-erg mRNAs in the pituitary and corticosterone increased only erg1 mRNA up to 2.47+/-0.54 fold. In conclusion, erg K(+) channels were up-regulated by glucocorticoid, and have indispensable roles in delayed glucocorticoid inhibition of CRH-induced ACTH release by rat pituitary cells.