Regulation of prolactin in mice with altered hypothalamic melanocortin activity

This study used two mouse models with genetic manipulation of the melanocortin system to investigate prolactin regulation. Mice with overexpression of the melanocortin receptor (MC-R) agonist, α-melanocyte-stimulating hormone (Tg-MSH) or deletion of the MC-R antagonist agouti-related protein (AgRP KO) were studied. Male Tg-MSH mice had lower blood prolactin levels at baseline (2.9±0.3 vs. 4.7±0.7ng/ml) and after restraint stress (68±6.5 vs. 117±22ng/ml) vs. WT (p<0.05); however, pituitary prolactin content was not different. Blood prolactin was also decreased in male AgRP KO mice at baseline (4.2±0.5 vs. 7.6±1.3ng/ml) and after stress (60±4.5 vs. 86.1±5.7ng/ml) vs. WT (p<0.001). Pituitary prolactin content was lower in male AgRP KO mice (4.3±0.3 vs. 6.7±0.5μg/pituitary, p<0.001) vs. WT. No differences in blood or pituitary prolactin levels were observed in female AgRP KO mice vs. WT. Hypothalamic dopamine activity was assessed as the potential mechanism responsible for changes in prolactin levels. Hypothalamic tyrosine hydroxylase mRNA was measured in both genetic models vs. WT mice and hypothalamic dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) content were measured in male AgRP KO and WT mice but neither were significantly different. However, these results do not preclude changes in dopamine activity as dopamine turnover was not directly investigated. This is the first study to show that baseline and stress-induced prolactin release and pituitary prolactin content are reduced in mice with genetic alterations of the melanocortin system and suggests that changes in hypothalamic melanocortin activity may be reflected in measurements of serum prolactin levels.     

Prolactin receptor antagonism in mouse anterior pituitary: effects on cell turnover and prolactin receptor expression

Since anterior pituitary expresses prolactin receptors, prolactin secreted by lactotropes could exert autocrine or paracrine actions on anterior pituitary cells. In fact, it has been observed that prolactin inhibits its own expression by lactotropes. Our hypothesis is that prolactin participates in the control of anterior pituitary cell turnover. In the present study, we explored the action of prolactin on proliferation and apoptosis of anterior pituitary cells and its effect on the expression of the prolactin receptor. To determine the activity of endogenous prolactin, we evaluated the effect of the competitive prolactin receptor antagonist Δ1-9-G129R-hPRL in vivo, using transgenic mice that constitutively and systemically express this antagonist. The weight of the pituitary gland and the anterior pituitary proliferation index, determined by BrdU incorporation, were higher in transgenic mice expressing the antagonist than in wild-type littermates. In addition, blockade of prolactin receptor in vitro by Δ1-9-G129R-hPRL increased proliferation and inhibited apoptosis of somatolactotrope GH3 cells and of primary cultures of male rat anterior pituitary cells, including lactotropes. These results suggest that prolactin acts as an autocrine/paracrine antiproliferative and proapoptotic factor in the anterior pituitary gland. In addition, anterior pituitary expression of the long isoform of the prolactin receptor, measured by real-time PCR, increased about 10-fold in transgenic mice expressing the prolactin receptor antagonist, whereas only a modest increase in the S3 short-isoform expression was observed. These results suggest that endogenous prolactin may regulate its own biological actions in the anterior pituitary by inhibiting the expression of the long isoform of the prolactin receptor. In conclusion, our observations suggest that prolactin is involved in the maintenance of physiological cell renewal in the anterior pituitary. Alterations in this physiological role of prolactin could contribute to pituitary tumor development.     

Agouti-related protein has an inhibitory paracrine role in the rat adrenal gland

alpha-Melanocyte-stimulating-hormone (alpha-MSH) is an agonist at the melanocortin 3 receptor (MC3-R) and melanocortin 4 receptor (MC4-R). alpha-MSH stimulates corticosterone release from rat adrenal glomerulosa cells in vitro. Agouti-related protein (AgRP) an endogenous antagonist at the MC3-R and MC4-R, is expressed in the adrenal gland. We investigated the expression of the MC3-R and MC4-R and the role of AgRP in the adrenal gland. MC3-R and MC4-R expression was detected in rat adrenal gland using RT-PCR. The effect of AgRP on alpha-MSH-induced corticosterone release was investigated using dispersed rat adrenal glomerulosa cells. AgRP administered alone did not affect corticosterone release, but co-administration of AgRP and alpha-MSH attenuated alpha-MSH-induced corticosterone release. To investigate glucocorticoid feedback, adrenal AgRP expression was compared in rats treated with dexamethasone to controls. AgRP mRNA was increased in rats treated with dexamethasone treatment compared to controls. Our findings demonstrate that adrenal AgRP mRNA is regulated by glucocorticoids. AgRP acting via the MC3-R or MC4-R may have an inhibitory paracrine role, blocking alpha-MSH-induced corticosterone secretion.     

Direct stimulation of pituitary prolactin release by glutamate

The ability of glutamate and other excitatory amino acids to stimulate prolactin secretion when administered to adult animals is hypothesized to depend on a central site of action in the brain, but there are no data to support this position. An alternative hypothesis was tested that glutamate would stimulate prolactin release when applied directly to primary cultures of dispersed adult female rat anterior pituitary cells studied in a perifusion protocol. Glutamate increased the rate of prolactin release within two minutes in a self-limited manner. Glutamate-stimulated prolactin release was augmented about 4-fold by elimination of magnesium from the perfusate and was associated with stimulation of pituitary calcium flux. Ketamine and MK-801 both reduced the basal rate of prolactin release and abolished the effects of glutamate. Pituitary cells of 10-day-old rats responded similarly to glutamate. Exposure to glutamate did not influence subsequent responses to physiological hypothalamic secretagogues, thus the likelihood of toxicity was minimized. These results suggest that the N-methyl-D-aspartate (NMDA) subclass of the glutamate receptor complex is involved. Prolactin secretion may be regulated physiologically through a functional glutamate receptor on pituitary cells.     

Tachykinins Induce Secretion of Prolactin from Perifused Rat Anterior Pituitary Cells by Interactions with Two Different Binding Sites

Abstract

Substance P and the two other mammalian tachykinins, neurokinin A and B, are accepted to have direct regulating effects at the anterior pituitary level. We have examined the effects of substance P (SP) and neurokinin B (NKB), alone and in combination, on prolactin release from cultured anterior pituitary cells grown on collagen-coated micro beads and placed in a perfusion system. Prolactin (Prl) secretion was observed within 25 s after exposure to either secretagogue and reached a maximum within 60-80 s. Furthermore, the prolactin response induced by SP and NKB was dose-dependent. Prl secretion remained constant for up to 4 h when SP or NKB were perifused and then fell gradually towards basal levels. Simultaneous addition of submaximal concentrations of SP and NKB resulted in an additive response compared with the responses of either secretagogue alone. Continuous (8 h) perifusion with SP did not prevent a normal prolactin response by NKB or TRH. These results indicate that the tachykinins, substance P and neurokinin B, release Prl from perifused female rat anterior pituitary cells by interaction with two different receptors, possibly the NK1 and NK3 tachykinin receptor subtypes.     

In vitro induction of CD25+ CD4+ regulatory T cells by the neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH)

Recently, we have found that the neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) not only suppresses IFN-gamma production, but also induces TGF-beta1 production by activated effector T cells. These alpha-MSH- treated effector T cells function as regulatory T cells in that they suppress IFN-gamma production and hypersensitivity mediated by other effector T cells. Experimental autoimmune uveoretinitis (EAU) was suppressed in its severity and incidence in mice that were injected with primed T cells activated in vitro by APC and antigen in the presence of alpha-MSH. Moreover, it appeared that alpha-MSH had converted a population of effector T cells polarized to mediate hypersensitivity into a population of T cells that now mediated immunoregulation. To characterize these alpha-MSH- treated T cells, primed T cells were TCR-stimulated in the presence of alpha-MSH in vitro and their lymphokine profile was examined. Such effector T cells displayed enhanced levels of TGF-beta1 production and no IFN-gamma or IL-10, with IL-4 levels remaining unchanged in comparison with inactivated T cells. In addition, if soluble TGF-beta receptor II was added to cocultures of alpha-MSH-treated T cells and activated Th1 cells, the alpha-MSH-treated T cells could not suppress IFN-gamma production by the Th1 cells. These results suggest that alpha-MSH induces T cells with a regulatory lymphokine pattern, and that through their production of TGF-beta1 these cells suppress other effector T cells. Examination of the alpha-MSH-treated T cells showed that alpha-MSH did not alter the phosphorylation of CD3 molecules following TCR engagement. Primed T cells express the melanocortin 5 receptor (MC5r), a receptor that is linked to an intracellular signalling pathway shared by other cytokine receptors. Blocking the receptor with antibody prevented alpha-MSH from suppressing IFN-gamma production by the activated regulatory T cells, suggesting that alpha-MSH immunoregulation is through the MC5r on primed T cells. Surface staining and cell sorting of the alpha-MSH- treated primed T cells showed that the regulatory T cells are CD25+ CD4+ T cells. From these results we find that alpha-MSH can mediate the induction of CD25+ CD4+ regulatory T cells. These regulatory T cells require specific antigen for activation, but through non-specific TGF-beta1-mediated mechanisms they can suppress other effector T cells.     

Melanocyte function and its control by melanocortin peptides.

Melanocytes are cells of neural crest origin. In the human epidermis, they form a close association with keratinocytes via their dendrites. Melanocytes are well known for their role in skin pigmentation, and their ability to produce and distribute melanin has been studied extensively. One of the factors that regulates melanocytes and skin pigmentation is the locally produced melanocortin peptide alpha-MSH. The effects of alpha-MSH on melanogenesis are mediated via the MC-1R and tyrosinase, the rate-limiting enzyme in the melanogenesis pathway. Binding of alpha-MSH to its receptor increases tyrosinase activity and eumelanin production, which accounts for the skin-darkening effect of alpha-MSH. Other alpha-MSH-related melanocortin peptides, such as ACTH1-17 and desacetylated alpha-MSH, are also agonists at the MC-1R and could regulate melanocyte function. Recent evidence shows that melanocytes have other functions in the skin in addition to their ability to produce melanin. They are able to secrete a wide range of signal molecules, including cytokines, POMC peptides, catecholamines, and NO in response to UV irradiation and other stimuli. Potential targets of these secretory products are keratinocytes, lymphocytes, fibroblasts, mast cells, and endothelial cells, all of which express receptors for these signal molecules. Melanocytes may therefore act as important local regulators of a range of skin cells. It has been shown that alpha-MSH regulates NO production from melanocytes, and it is possible that the melanocortins regulate the release of other signalling molecules from melanocytes. Therefore, the melanocortin signaling system is one of the important regulators of skin homeostasis.     

On the mechanism by which dopamine inhibits prolactin release in the anterior pituitary

An $\text{in}$$\text{vitro}$ perfusion system was used to assess the effects of chloride channel blockers, dopamine (DA) receptor agonists and antagonists, and GABA receptor agonists and antagonists on prolactin release from the mouse anterior pituitary. Dopamine and muscimol inhibited prolactin release ($\text{IC}{}_{50}{}^1\text{= 6 × 10}{}^{\mathrm{?8}}\text{M and}\text{10}{}^{\mathrm{?5}}\text{M}$ respectively). The GABA receptor antagonist bicuculline blocked the inhibition of prolactin release by muscimol but not dopamine. The dopamine receptor antagonist chlorpromazine blocked the dopamine- but not muscimol-induced inhibition of prolactin release. Haloperidol, however, reversed both the muscimol and dopamine induced inhibition of prolactin release. Furthermore, the chloride channel blocker picrotoxinin blocked the inhibition of prolactin release elicited by both dopamine and muscimol. These later results suggest that the anterior pituitary dopamine receptor which mediates the inhibition of prolactin release may be coupled to a picrotoxinin sensitive chloride ionophore and that haloperidol may affect the function of both DA and GABA receptors in the anterior pituitary.     

ACTH1-17 is a more potent agonist at the human MC1 receptor than alpha-MSH.

The melanocortin receptor MC1 is expressed on melanocytes and is an important control point for melanogenesis and other responses. Alpha-MSH, which is considered to be the major ligand at the human melanocortin (MC)1 receptor (hMC1R), is produced from proopiomelanocortin (POMC) in the pituitary and in the skin by melanocytes and keratinocytes. Other POMC peptides are also produced in the skin and their concentrations exceed those of alpha-MSH by several fold. One of the most abundant is ACTH1-17. We have shown that adrenocorticotrophic hormone (ACTH)1-17 is more potent than alpha-MSH in stimulating melanogenesis in human melanocytes and unlike alpha-MSH produces a biphasic dose response curve. In this study we have examined the ability of ACTH1-17 to function as a ligand at the hMC1R. Competitive binding assays with [125I]Nle4 DPhe7 alpha-MSH as labelled ligand were carried out in HEK 293 cells transfected with the hMC1R. ACTH1-17 showed high affinity for the hMC1R with a Ki value of 0.21 +/- 0.03 nM which was slightly higher than that of 0.13 +/- 0.005 nM for alpha-MSH. ACTH1-17 was, however, more potent than alpha-MSH in increasing cAMP and IP3 production in the transfected cells. Our results demonstrate that ACTH1-17 is a potent agonist at the hMC1R. It is therefore possible that ACTH1-17, which is found in the skin in greater concentrations than alpha-MSH, has an important role in the regulation of human melanocytes and other cell types that express the hMC1R.     

Cadmium Mimics Estrogen-Driven Cell Proliferation and Prolactin Secretion from Anterior Pituitary Cells

Cadmium (Cd) is a heavy metal of considerable occupational and environmental concern affecting wildlife and human health. Recent studies indicate that Cd, like other heavy metals, can mimic effects of 17β-estradiol (E2) involving E2 receptor (ER) activation. Lactotrophs, the most abundant cell type in anterior pituitary gland, are the main target of E2, which stimulates cell proliferation and increases prolactin secretion through ERα. The aim of this work was to examine whether Cd at nanomolar concentrations can induce cell proliferation and prolactin release in anterior pituitary cells in culture and whether these effects are mediated through ERs. Here we show that 10 nM Cd was able to stimulate lactotroph proliferation in anterior pituitary cell cultures from female Wistar rats and also in GH3 lactosomatotroph cell line. Proliferation of somatotrophs and gonadotrophs were not affected by Cd exposure. Cd promoted cell cycle progression by increasing cyclins D1, D3 and c-fos expression. Cd enhanced prolactin synthesis and secretion. Cd E2-like effects were blocked by the pure ERs antagonist ICI 182,780 supporting that Cd acts through ERs. Further, both Cd and E2 augmented full-length ERαexpression and its 46 kDa-splicing variant. In addition, when co-incubated Cd was shown to interact with E2 by inducing ERα mRNA expression which indicates an additive effect between them. This study shows for the first time that Cd at nanomolar concentration displays xenoestrogenic activities by inducing cell growth and stimulating prolactin secretion from anterior pituitary cells in an ERs-dependent manner. Cd acting as a potent xenoestrogen can play a key role in the aetiology of different pathologies of the anterior pituitary and in estrogen-responsive tissues which represent considerable risk to human health.     

alpha-MSH and gamma-MSH inhibit IL-1beta induced activation of the hypothalamic-pituitary-adrenal axis through central melanocortin receptors

Alpha-melanocyte-stimulating hormone (alpha-MSH) is a neuroimmunomodulatory peptide that is involved in the control of host responses trough modulation of production and action of proinflammatory cytokines in inflammatory cells in the periphery and within the central nervous system (CNS). However, little is known about the receptors that mediate the modulatory effects of alpha-MSH in the CNS. The objective of the present study was to establish the specific melanocortin receptors involved in the inhibition by MSH peptides of IL-1beta-induced activation of the HPA. i.c.v. injection of 12.5 ng of IL-1beta caused significant changes in plasma corticosterone, as compared to basal levels. The treatment with gamma-MSH (1 microg), an MC3 receptor agonist, resulted in significant reduction of the IL-1beta-induced plasma corticosterone levels. Administration of the MC3/MC4 receptor antagonist SHU9119 blocked this effect. Besides, treatment with a high dose of alpha-MSH (1 microg) increased plasma corticosterone. When alpha-MSH was given at a lower dose (0.1 microg), it did not modify corticosterone levels but caused an inhibitory effect on the corticosterone release induced by IL-1beta. The administration of SHU9119 or a more selective MC4 receptor antagonist like HS014 blocked the effects of alpha-MSH. In conclusion, our results suggest that both alpha-MSH and gamma-MSH are capable of inhibiting the effect of the IL-1beta on the activation of HPA axis acting at the CNS, and that this effect is mediated by specific central melanocortin receptors.     

Anterior pituitary pyroglutamyl peptidase II activity controls TRH-induced prolactin release

Ecto-peptidases modulate the action of peptides in the extracellular space. The relationship between peptide receptor and ecto-peptidase localization, and the physiological role of peptidases is poorly understood. Current evidence suggests that pyroglutamyl peptidase II (PPII) inactivates neuronally released thyrotropin-releasing hormone (TRH). The impact of PPII localization in the anterior pituitary on the endocrine activities of TRH is unknown. We have studied whether PPII influences TRH signaling in anterior pituitary cells in primary culture. In situ hybridization (ISH) experiments showed that PPII mRNA was expressed only in 5-6% of cells. ISH for PPII mRNA combined with immunocytochemistry for prolactin, beta-thyrotropin, or growth hormone, showed that 66% of PPII mRNA expressing cells are lactotrophs, 34% somatotrophs while none are thyrotrophs. PPII activity was reduced using a specific phosphorothioate antisense oligodeoxynucleotide or inhibitors. Compared with mock or scrambled oligodeoxynucleotide-treated controls, knock-down of PPII expression by antisense targeting increased TRH-induced release of prolactin, but not of thyrotropin. Similar data were obtained with either a transition-state or a tight binding inhibitor. These results demonstrate that PPII expression in lactotrophs coincides with its ability to control prolactin release. It may play a specialized role in TRH signaling in the anterior pituitary. Anterior pituitary ecto-peptidases may fulfill unique functions associated with their restricted cell-specific expression.     

Abnormalities of the hypothalamo-pituitary-thyroid axis in the pro-opiomelanocortin deficient mouse

The melanocortin system is an important regulator of body weight and the hypothalamo-pituitary-thyroid (HPT) axis. The pro-opiomelanocortin (POMC)-null mouse, deficient in all POMC-derived peptides, including alpha-melanocyte stimulating hormone (alpha-MSH), has an obese phenotype. We studied the HPT axis of POMC-null mice, which has not been previously investigated. Because alpha-MSH has a stimulatory effect on the HPT axis, we hypothesised that these mice would have a down-regulated thyroid axis, consistent with a recent study of POMC-null humans. The activity of the HPT axis was studied by collecting blood, pituitaries and hypothalami from ad libitum fed, adult POMC-null, heterozygous and wild-type mice. POMC-null mice had significantly elevated plasma total T(4) (TT(4)) and free T(3) (fT(3)) with reduced plasma thyroid stimulating hormone (TSH), pituitary TSH content and hypothalamic thyrotrophin stimulating hormone (TRH) content compared to wild-type mice. No significant differences between heterozygous and wild-type mice were observed. POMC-null mice have an abnormal HPT axis, which may contribute to their hyperphagia and obesity. These abnormalities are in contrast to those observed in POMC-null humans. These findings support a role for the melanocortin system in the regulation of the HPT axis.     

Gene expression and the physiological role of transforming growth factor-alpha in the mouse pituitary

Transforming growth factor-alpha (TGF-alpha), a member of the epidermal growth factor (EGF) family, is produced within the mouse anterior pituitaries. However, the cell types of TGF-alpha-expressing cells and the physiological roles of TGF-alpha within mouse pituitary glands remain unclear. The aim of the present study was to localize TGF-alpha mRNA-expressing cells, and to clarify the involvement of TGF-alpha in estrogen-induced DNA replication in mouse anterior pituitary cells. Northern blot analysis demonstrated TGF-alpha mRNA expression in adult male and female mouse anterior pituitaries. In situ hybridization analysis of the pituitaries in these mice showed that TGF-alpha mRNA-expressing cells in the anterior pituitary are round, oval, and medium-sized. TGF-alpha mRNA was colocalized in most of the growth hormone (GH) mRNA-expressing cells, while only some of the prolactin (PRL) mRNA-expressing cells. DNA replication in the anterior pituitary cells was detected by monitoring the cellular uptake of a thymidine analogue, bromodeoxyuridine (BrdU) in a primary serum-free culture system. Estradiol-17beta (E2) and TGF-alpha treatment increased the number of BrdU-labelled mammotrophs, indicating that E2 and TGF-alpha treatment stimulates the DNA replication in mammotrophs. Immunoneutralization of TGF-alpha with anti-TGF-alpha-antibodies nullified the E2-induced increase in DNA replication. RT-PCR analysis of TGF-alpha mRNA expression in ovariectomized female mice revealed that E2 increases TGF-alpha mRNA levels. These results indicate that the TGF-alpha produced primarily in the somatotrophs mediates the stimulatory effects of estrogen on the DNA replication of pituitary cells in a paracrine or autocrine manner.     

Pituitary site of action of endothelin: selective inhibition of prolactin release in vitro

Endothelin-3 (ET-3) inhibited in a dose-dependent, significant fashion prolactin release from cultured anterior pituitary cells (ovariectomized female and intact male rat donors, ED50 = 5 X 10(-9) M). ET-3 in log doses ranging from 10(-11) to 10(-6) M did not alter significantly the release of luteinizing hormone, growth hormone or thyroid stimulating hormone. The inhibitory effect of ET-3 (rat, human) was specific for that molecule since ET-1 (porcine, human) was ineffective and was not due to an action on the dopamine receptor since the inhibitory action was still expressed in the presence of 100 nM domperidone. These data further suggest a role for neuropeptides of the posterior lobe in the control of lactotroph function.     

Agouti: from mouse to man, from skin to fat

The agouti protein regulates pigmentation in the mouse hair follicle producing a black hair with a subapical yellow band. Its effect on pigmentation is achieved by antagonizing the binding of alpha-melanocyte stimulating hormone (alpha-MSH) to melanocortin 1 receptor (Mc1r), switching melanin synthesis from eumelanin (black/brown) to phaeomelanin (red/yellow). Dominant mutations in the non-coding region of mouse agouti cause yellow coat colour and ectopic expression also results in obesity, type 11 diabetes, increased somatic growth and tumourigenesis. At least some of these pleiotropic effects can be explained by antagonism of other members of the melanocortin receptor family by agouti protein. The yellow coat colour is the result of agouti chronically antagonizing the binding of alpha-MSH to Mc1r and the obese phenotype results from agouti protein antagonizing the binding of alpha-MSH to Mc3r and/or Mc4r. Despite the existence of a highly homologous agouti protein in humans, agouti signal protein (ASIP), its role has yet to be defined. However it is known that human ASIP is expressed at highest levels in adipose tissue where it may antagonize one of the melanocortin receptors. The conserved nature of the agouti protein combined with the diverse phenotypic effects of agouti mutations in mouse and the different expression patterns of human and mouse agouti, suggest ASIP may play a role in human energy homeostasis and possibly human pigmentation.     

Effect of the GABAA agonist muscimol on prolactin secretion from human prolactin-secreting adenomas and GH3 rat pituitary tumour cells.

The effect of muscimol, a specific potent GABAA receptor agonist, on prolactin release from human prolactin-secreting tissue was investigated using a perifusion system. Perifusion studies on normal rat anterior pituitary tissue, which has identical GABA receptors to those found in normal human pituitary glands, show that muscimol has a specific biphasic effect on prolactin release. This is characterized by an initial transient stimulation (222.3 +/- 21.6% of basal) lasting for 5-10 min followed by a more prolonged inhibitory phase (63.9 +/- 3.1% inhibition of basal). Five human prolactin-secreting adenomas were studied, and in none of the tumours could a biphasic response be demonstrated. One of the prolactin-secreting adenomas had a blunted inhibitory response, but the other 4 showed no inhibitory effect of muscimol on prolactin release. Muscimol had no significant effect on basal or thyrotropin-releasing-hormone (TRH)-stimulated prolactin secretion from GH3 rat pituitary tumour cells. These studies suggest that the GABAergic effect on prolactin secretion is absent or altered in both rat and human prolactin-secreting tumour cells.     

Suppression of basal and stress-induced prolactin release and stimulation of luteinizing hormone secretion by alpha-melanocyte-stimulating hormone

alpha-MSH and beta-endorphin, both synthesized from a common precursor, have opposite behavioral actions. In order to determine if these peptides have opposite effects on pituitary function, basal LH secretion and basal and stress-induced prolactin release were studied in adult male rats after intraventricular injection of alpha-MSH. Each rat also received intraventricular saline in order to serve as its own control. 18 micrograms alpha-MSH stimulated plasma LH from 16.5 +/- 2.5 (SEM) ng/ml to a peak of 27.2 +/- 4.0 and 26.0 +/- 4.9 ng/ml at 5 and 10 min, and suppressed prolactin from 3.5 +/- 0.7 ng/ml to 1.3 +/- 0.1 and 1.2 +/- 0.1 ng/ml at 15 and 30 min. Intraventricular alpha-MSH also significantly blunted the prolactin rise associated with the stress of swimming. 10 and 20 min after the onset of swimming, prolactin levels in rats pretreated with alpha-MSH were significantly diminished: 7.4 +/- 1.5 and 6.5 +/- 2.0 ng/ml vs 23.8 +/- 3.6 and 15.2 +/- 2.8 after normal saline. Similarly, des-acetyl alpha-MSH which is the predominant form of alpha-MSH in the hypothalamus, diminished the stress-induced prolactin rise from 18.4 +/- 5.3 and 11.2 +/- 3.4 ng/ml at 10 and 20 min to 10.0 +/- 2.4 and 5.5 +/- 1.6 ng/ml. We conclude that centrally administered alpha-MSH stimulates LH and suppresses basal and stress-induced prolactin release in male rats. These actions are opposite to those previously shown for beta-endorphin and suggest that alpha-MSH may antagonize the effects of beta-endorphin on pituitary function.     

Gonadotropin-releasing hormone (GnRH) positively regulates corticotropin-releasing hormone-binding protein expression via multiple intracellular signaling pathways and a multipartite GnRH response element in alphaT3-1 cells

CRH-binding protein (CRH-BP) binds CRH with high affinity and inhibits CRH-mediated ACTH release from anterior pituitary corticotrope-like cells in vitro. In female mouse pituitary, CRH-BP is localized not only in corticotropes, but is also expressed in gonadotropes and lactotropes. To investigate the functional significance of gonadotrope CRH-BP, we examined the molecular mechanisms underlying GnRH-regulated CRH-BP expression in alphaT3-1 gonadotrope-like cells. CRH-BP is endogenously expressed in alphaT3-1 cells, and quantitative real-time RT-PCR and ribonuclease protection assays demonstrate that GnRH induces a 3.7-fold increase in CRH-BP mRNA levels. GnRH also induces intracellular CRH-BP (2.0-fold) and secreted CRH-BP (5.3-fold) levels, as measured by [125I]CRH:CRH-BP chemical cross-linking. Transient transfection assays using CRH-BP promoter-luciferase constructs indicate that GnRH regulation involves protein kinase C-, ERK- and calcium-dependent signaling pathways and is mediated via a multipartite GnRH response element that includes activator protein 1 and cAMP response element (CRE) sites. The CRE site significantly contributes to GnRH responsiveness, independent of protein kinase A, representing a unique form of multipartite GnRH regulation in alphaT3-1 cells. Furthermore, EMSAs indicate that alphaT3-1 nuclear proteins specifically bind at activator protein 1 and CRE sites. These data demonstrate novel regulation of pituitary CRH-BP, highlighting the importance of the pituitary gonadotrope as a potential interface between the stress and reproductive axes.