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Understanding the molecular mechanisms underlying pituitary organogenesis and function is essential for improving therapeutics and molecular diagnoses for hypopituitarism. We previously found that deletion of the forkhead factor, Foxo1, in the pituitary gland early in development delays somatotrope differentiation. While these mice grow normally, they have reduced growth hormone expression and free serum insulin-like growth factor-1 (IGF1) levels, suggesting a defect in somatotrope function. FOXO factors show functional redundancy in other tissues, so we deleted both Foxo1 and its closely related family member, Foxo3, from the primordial pituitary. We find that this results in a significant reduction in growth. Consistent with this, male and female mice in which both genes have been deleted in the pituitary gland (dKO) exhibit reduced pituitary growth hormone expression and serum IGF1 levels. Expression of the somatotrope differentiation factor, Neurod4, is reduced in these mice. This suggests a mechanism underlying proper somatotrope function is the regulation of Neurod4 expression by FOXO factors. Additionally, dKO mice have reduced Lhb expression and females also have reduced Fshb and Prl expression. These studies reveal FOXO transcription factors as important regulators of pituitary gland function., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: The X-linked immunoglobulin superfamily, member 1 (IGSF1), gene is highly expressed in the hypothalamus and in pituitary cells of the POU1F1 lineage. Human loss-of-function mutations in IGSF1 cause central hypothyroidism, hypoprolactinemia, and macroorchidism. Additionally, most affected adults exhibit higher than average IGF-1 levels and anecdotal reports describe acromegaloid features in older subjects. However, somatotrope function has not yet been formally evaluated in this condition., Objective: We aimed to evaluate the role of IGSF1 in human and murine somatotrope function., Patients, Design, and Setting: We evaluated 21 adult males harboring hemizygous IGSF1 loss-of-function mutations for features of GH excess, in an academic clinical setting., Main Outcome Measures: We compared biochemical and tissue markers of GH excess in patients and controls, including 24-hour GH profile studies in 7 patients. Parallel studies were undertaken in male Igsf1-deficient mice and wild-type littermates., Results: IGSF1-deficient adult male patients demonstrated acromegaloid facial features with increased head circumference as well as increased finger soft-tissue thickness. Median serum IGF-1 concentrations were elevated, and 24-hour GH profile studies confirmed 2- to 3-fold increased median basal, pulsatile, and total GH secretion. Male Igsf1-deficient mice also demonstrated features of GH excess with increased lean mass, organ size, and skeletal dimensions and elevated mean circulating IGF-1 and pituitary GH levels., Conclusions: We demonstrate somatotrope neurosecretory hyperfunction in IGSF1-deficient humans and mice. These observations define a hitherto uncharacterized role for IGSF1 in somatotropes and indicate that patients with IGSF1 mutations should be evaluated for long-term consequences of increased GH exposure., (© Endocrine Society 2019.)

1. In this study, geese ( Anas cygnoides ) embryonic pituitary cells were cultured in vitro to determine if glucocorticoids could induce growth hormone (GH) expression and to investigate the molecular mechanisms involved in this process. 2. On embryonic day 15 (e15) and e20 the pituitary cells were treated with corticosterone (CORT), membrane impermeable bovine serum albumin-conjugate corticosterone (CORT-BSA), dexamethasone (DEX), and a glucocorticoid receptor (GR) antagonist (RU486) to detect responsiveness of somatotrophs to glucocorticoids. 3. Treatment with CORT, CORT-BSA, and DEX for as little as 6 h increased the percentage of GH-positive cells ( P < 0.01) and increased GH mRNA expression (P < 0.01) in e15 goose pituitary cells compared to the control. CORT significantly increased the level of GH protein secreted from cultured e15 goose embryonic pituitary cells, and CORT-BSA increased GH secretion from e20 goose embryonic pituitary cells. 4. A significant increase was observed in the glucocorticoid receptor in GR transcription levels (P < 0.01) with CORT, CORT-BSA, and DEX treatment. Furthermore, the CORT-stimulated GH mRNA expression was completely negated by pre-treatment with RU486. 5. These findings demonstrate that glucocorticoids can stimulate somatotroph differentiation in vitro , as characterised by enhanced GH protein secretion and mRNA expression in cultured geese embryonic pituitary cells. The membrane GR was involved in pituitary somatotroph differentiation induced by glucocorticoids during the embryonic development of geese.

Normal growth requires that pituitary-secreted growth hormone (GH) bind to its specific receptor and activate a complex signaling cascade, leaving to production of insulin-like growth factor-I (IGF-I), which, in turn, activates its own receptor (IGF1R). The GH receptor (GHR) is preformed as a dimer and is transported in a nonligand bound state to the cell surface. Binding of GH to the GHR dimer, results in a conformational change of the dimer, activation of the intracellular Janus Kinase 2 (JAK2) and phosphorylation of signal transducer and activator of transcription (STAT) 5B. Phosphorylated STAT5B dimers are then translocated to the nucleus, where they transcriptionally activate multiple genes, including those for IGF-I, IGF binding protein-3 and the acid-labile subunit (ALS)., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene predispose humans to pituitary adenomas, particularly of the somatotroph lineage. Mice with global heterozygous inactivation of Aip (Aip(+/-)) also develop pituitary adenomas but differ from AIP-mutated patients by the high penetrance of pituitary disease. The endocrine phenotype of these mice is unknown. The aim of this study was to determine the endocrine phenotype of Aip(+/-) mice by assessing the somatic growth, ultradian pattern of GH secretion and IGF1 concentrations of longitudinally followed male mice at 3 and 12 months of age. As the early stages of pituitary tumorigenesis are controversial, we also studied the pituitary histology and somatotroph cell proliferation in these mice. Aip(+/-) mice did not develop gigantism but exhibited a leaner phenotype than wild-type mice. Analysis of GH pulsatility by deconvolution in 12-month-old Aip(+/-) mice showed a mild increase in total GH secretion, a conserved GH pulsatility pattern, but a normal IGF1 concentration. No pituitary adenomas were detected up to 12 months of age. An increased ex vivo response to GHRH of pituitary explants from 3-month-old Aip(+/-) mice, together with areas of enlarged acini identified on reticulin staining in the pituitary of some Aip(+/-) mice, was suggestive of somatotroph hyperplasia. Global heterozygous Aip deficiency in mice is accompanied by subtle increase in GH secretion, which does not result in gigantism. The absence of pituitary adenomas in 12-month-old Aip(+/-) mice in our experimental conditions demonstrates the important phenotypic variability of this congenic mouse model., (© 2016 Society for Endocrinology.)

Several transgenic mouse models with disruption in the growth hormone (GH) axis support the role of GH in augmenting metabolic homeostasis. Specifically, interest has focused on GH's lipolytic properties and ability to affect adipose deposition. Furthermore, both GH and insulin growth factor 1 (IGF-1) may also play a direct or indirect role in adipose development. The somatotroph insulin-like growth factor-1 receptor knockout (SIGFRKO) mouse with only a modest increase in serum GH and IGF-1 demonstrates less adipose tissue than controls. In order to characterize the metabolic phenotype of SIGFRKO mice, histologic analysis of fat depots confirmed a smaller average diameter of adipocytes in the SIGFRKO mice compared to controls. These changes were accompanied by an increase in lipolytic gene expression in fat depots. Indirect calorimetry performed on 6-8week old male mice and again at 25weeks of age demonstrated that SIGFRKO mice, at both ages, had a higher VO2 and increased energy expenditure when compared with controls. The calculated respiratory exchange ratio (RER) was lower in the younger SIGFRKO mice compared to controls. No differences in food consumption or in either ambulatory or total activity were seen between SIGFRKO and control mice in either age group. These studies highlight the role of GH in adipose deposition and its influence on the expression of lipolytic genes resulting in an altered metabolic state, thus providing a mechanism for the decrease in weight gain seen in the SIGFRKO mouse model., (Copyright © 2016 Elsevier Inc. All rights reserved.)

To test the role of wtPIT-1 (PITWT) or PIT-1 (R271W) (PIT271) in somatolactotroph cells, we established, using inducible lentiviral vectors, sublines of GH4C1 somatotroph cells that allow the blockade of the expression of endogenous PIT-1 and/or the expression of PITWT or PIT271, a dominant negative mutant of PIT-1 responsible for Combined Pituitary Hormone Deficiency in patients. Blocking expression of endogenous PIT-1 induced a marked decrease of cell proliferation. Overexpressing PITWT twofold led also to a dose-dependent decrease of cell proliferation that was accompanied by cell death. Expression of PIT271 induced a strong dose-dependent decrease of cell proliferation accompanied by a very pronounced cell death. These actions of PIT271 are independent of its interaction/competition with endogenous PIT-1, as they were unchanged when expression of endogenous PIT-1 was blocked. All these actions are specific for somatolactotroph cells, and could not be observed in heterologous cells. Cell death induced by PITWT or by PIT271 was accompanied by DNA fragmentation, but was not inhibited by inhibitors of caspases, autophagy or necrosis, suggesting that this cell death is a caspase-independent apoptosis. Altogether, our results indicate that under normal conditions PIT-1 is important for the maintenance of cell proliferation, while when expressed at supra-normal levels it induces cell death. Through this dual action, PIT-1 may play a role in the expansion/regression cycles of pituitary lactotroph population during and after lactation. Our results also demonstrate that the so-called "dominant-negative" action of PIT271 is independent of its competition with PIT-1 or a blockade of the actions of the latter, and are actions specific to this mutant variant of PIT-1.

Melatonin (MT) is secreted by the pineal gland and exhibits a striking circadian rhythm in its release. Depending on the species studied, some pituitary hormones also display marked circadian/seasonal patterns and rhythms of secretion. However, the precise relationship between MT and pituitary function remains controversial, and studies focusing on the direct role of MT in normal pituitary cells are limited to nonprimate species. Here, adult normal primate (baboons) primary pituitary cell cultures were used to determine the direct impact of MT on the functioning of all pituitary cell types from the pars distalis. MT increased GH and prolactin (PRL) expression/release in a dose- and time-dependent fashion, a response that was blocked by somatostatin. However, MT did not significantly affect ACTH, FSH, LH, or TSH expression/release. MT did not alter GHRH- or ghrelin-induced GH and/or PRL secretions, suggesting that MT may activate similar signaling pathways as ghrelin/GHRH. The effects of MT on GH/PRL release, which are likely mediated through MT1 receptor, involve both common (adenylyl cyclase/protein kinase A/extracellular calcium-channels) and distinct (phospholipase C/intracellular calcium-channels) signaling pathways. Actions of MT on pituitary cells also included regulation of the expression of other key components for the control of somatotrope/lactotrope function (GHRH, ghrelin, and somatostatin receptors). These results show, for the first time in a primate model, that MT directly regulates somatotrope/lactotrope function, thereby lending support to the notion that the actions of MT on these cells might substantially contribute to the define daily patterns of GH and PRL observed in primates and perhaps in humans.

Leptin is a cytokine produced by white fat cells, skeletal muscle, the placenta, and the pituitary gland among other tissues. Best known for its role in regulating appetite and energy expenditure, leptin is produced largely by and in proportion to white fat cells. Leptin is also important to the maintenance and function of the GH cells of the pituitary. This was shown when the deletion of leptin receptors on somatotropes caused decreased numbers of GH cells, decreased circulating GH, and adult-onset obesity. To determine the source of leptin most vital to GH cells and other pituitary cell types, we compared two different leptin knockout models with Cre-lox technology. The global Lep-null model is like the ob/ob mouse, whereby only the entire exon 3 is deleted. The selective adipocyte-Lep-null model lacks adipocyte leptin but retains pituitary leptin, allowing us to investigate the pituitary as a potential source of circulating leptin. Male and female mice lacking adipocyte leptin (Adipocyte-lep-null) did not produce any detectable circulating leptin and were infertile, suggesting that the pituitary does not contribute to serum levels. In the presence of only pituitary leptin, however, these same mutants were able to maintain somatotrope numbers and GH mRNA levels. Serum GH trended low, but values were not significant. However, hypothalamic GHRH mRNA was significantly reduced in these animals. Other serum hormone and pituitary mRNA differences were observed, some of which varied from previous results reported in ob/ob animals. Whereas pituitary leptin is capable of maintaining somatotrope numbers and GH mRNA production, the decreased hypothalamic GHRH mRNA and low (but not significant) serum GH levels indicate an important role for adipocyte leptin in the regulation of GH secretion in the mouse. Thus, normal GH secretion may require the coordinated actions of both adipocyte and pituitary leptin.

To understand how gonadotropin-inhibitory hormone (GnIH) regulates goldfish GH cell functions, we monitored GH release and expression during early, mid-, and/or late gonadal recrudescence. In vivo and in vitro responses to goldfish (g) GnIH were different, indicating direct action at the level of pituitary, as well as interactions with other neuroendocrine factors involved in GH regulation. Injection of gGnIH consistently reduced basal serum GH levels but elevated pituitary gh mRNA levels, indicating potential dissociation of GH release and synthesis. Goldfish GnRH (sGnRH and cGnRHII) injection differentially stimulated serum GH and pituitary gh mRNA levels with some seasonal differences; these responses were reduced by gGnIH. In contrast, in vitro application of gGnIH during 24-h static incubation of goldfish pituitary cells generally elevated basal GH release and attenuated sGnRH-induced changes in gh mRNA, while suppressing basal gh mRNA levels at mid- and late recrudescence but elevating them at early recrudescence. gGnIH attenuated the GH release responses to sGnRH during static incubation at early, but not at mid- and late recrudescence. In cell column perifusion experiments examining short-term GH release, gGnIH reduced the cGnRHII- and sGnRH-stimulated secretion at late recrudescence but inhibited tha action of cGnRHII only during mid-recrudescence. Interestingly, a reduction of basal GH release upon perifusion with gGnIH during late recrudescence was followed by a rebound increase in GH release upon gGnIH removal. These results indicate that gGnIH exerts complex effects on basal and GnRH-stimulated goldfish GH cell functions and can differentially affect GH release and mRNA expression in a seasonal reproductive manner., (© 2014 Society for Endocrinology.)

Deletion of the signaling domain of leptin receptors selectively in somatotropes, with Cre-loxP technology, reduced the percentage of immunolabeled GH cells and serum GH. We hypothesized that the deficit occurred when leptin's postnatal surge failed to stimulate an expansion in the cell population. To learn more about the deficiency in GH cells, we tested their expression of GHRH receptors and GH mRNA and the restorative potential of secretagogue stimulation in vitro. In freshly plated dissociated pituitary cells from control male mice, GHRH alone (0.3 nM) increased the percentage of immunolabeled GH cells from 27 ± 0.05% (vehicle) to 42 ± 1.8% (P < .002) and the secretion of GH 1.8-3×. Deletion mutant pituitary cells showed a 40% reduction in percentages of immunolabeled GH cells (16.7 ± 0.4%), which correlated with a 47% reduction in basal GH levels (50 ng/mL control; 26.7 ng/mL mutants P = .01). A 50% reduction in the percentage of mutant cells expressing GHRH receptors (to 12%) correlated with no or reduced responses to GHRH. Ghrelin alone (10 nM) stimulated more GH cells in mutants (from 16.7-23%). When added with 1-3 nM GHRH, ghrelin restored GH cell percentages and GH secretion to levels similar to those of stimulated controls. Counts of somatotropes labeled for GH mRNA confirmed normal percentages of somatotropes in the population. These discoveries suggest that leptin may optimize somatotrope function by facilitating expression of membrane GHRH receptors and the production or maintenance of GH stores.

In a recent study, we showed that the adult pituitary gland is capable of regenerating transgenically ablated growth hormone-producing (GH(+)) somatotropes. Here, we investigated whether the gland's regenerative capacity is more general and also applies to the other major hormonal cell type, the prolactin-producing (PRL(+)) lactotropes. We set up the transgenic PRLCre/inducible diphtheria toxin receptor (iDTR) mouse model, in which the PRL promoter drives expression of Cre that induces DTR in lactotropes. Injection of female mice with DT for different periods causes a gradual ablation of PRL(+) cells, reaching a maximum of 70% after 10-day DT treatment. During the following weeks, lactotropes progressively reappear achieving a 60% restoration after 6 weeks. The Sox2(+) stem/progenitor cell compartment displays a prompt reaction to the DT-triggered cell ablation injury, including expansion of the marginal-zone niche and coexpression of PRL, the latter only very rarely observed in control pituitary. Throughout the regeneration period (2-6 weeks), Sox2(+) as well as double Sox2(+)/PRL(+) cells continue to be more abundant than in control pituitary. In addition to this stem cell reaction, surviving or newborn lactotropes increase their proliferative activity, and bihormonal PRL(+)/GH(+) cells become detectable suggesting somatotrope-to-lactotrope transdifferentiation. In conclusion, the adult pituitary gland is capable of restoring lactotrope cells after destruction, further confirming its regenerative competence. Repair of lactotropes appears to be driven by a combination of mechanisms, including recruitment from stem cells, proliferation of lactotropes, and transdifferentiation of somatotropes.

Growth hormone (GH) is a prominent metabolic factor that is targeted by glucocorticoids; however, their role in GH production remains controversial. This is explained in part by discrepancies between in vitro and in vivo, short-term versus long-term exposure and even species-specific effects. The prevailing view, however, is that glucocorticoids are negative modulators of growth and GH production. An examination of recent findings from elegant avian and gene ablation in mice studies as well as clinical case reports, suggests this is not the case. The evidence suggests that the effect of glucocorticoids on growth and GH production can be uncoupled, and reveals they play a crucial and positive role in maturation of functional somatotrophs, the GH-producing cells of the anterior pituitary. Here, we provide an overview and insights into the possible roles of glucocorticoids in the development of somatotrophs before birth as well as regulation of GH production in infancy (neonatal) and adulthood (postnatal). A fully functional glucocorticoid-signaling pathway appears to be required for establishment of somatotrophs before birth, and glucocorticoids continue to be required for maintenance of GH production in the newborn. There is evidence to suggest progenitor somatotrophs may persist after birth, and perhaps account for the ability of glucocorticoid therapy to correct some cases of GH deficiency as a result of compromised glucocorticoid signaling. Finally, there is support for positive regulation of avian, murine and human GH gene activation and/or expression by glucocorticoids, however, there appears to be no common mechanism and the contribution of direct versus indirect effects remains unclear. Thus, our observations reveal a largely hidden face of glucocorticoids, specifically, a positive role in somatotroph development and GH gene activation/expression, which may enable us to better understand the differential effect of glucocorticoids on growth and GH production in human studies., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The integrity of homeostasis can be affected by chronic stress, and hyposomatotropism is evident in chronic stress-associated illnesses. In the present study, we demonstrated that a continuous stress (CS) severely affected somatotrophs among hormone-secreting cells in the anterior lobe (AL) of the pituitary by using a rat CS model. Among AL cells, the proliferation of somatotrophs was almost entirely suppressed in rats that had 3-5 days of CS (5dCS), although other hormone-secreting cells continued to proliferate. The cell size of somatotrophs was reduced at 5dCS (P<0.01), the number of secretory granules was increased at 3dCS (P<0.01) and serum growth hormone (GH) was on declining trend during 1 to 5dCS, suggesting that GH release was inhibited. GH-releasing hormone (GHRH) mRNA level in the arcuate nucleus was transiently decreased, whereas its receptor expression in the AL was significantly increased in CS rats. When 5dCS rats were injected with GHRH, transient GH secretion was observed, whereas proliferation of somatotrophs did not occur. The GHRH administration failed to stimulate extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and the nuclear translocation of ERK in somatotrophs. These results suggest that somatotrophs of 5dCS rats expressed sufficient GHRH receptor, which could transfer a signal for GH release. However, the GHRH-induced proliferation signal was blocked somewhere between the receptor and ERK1/2. Because significant increase of corticosterone in the initial stage (the 1-3dCS) was observed in this model, the corticosterone may affect the signalling. Although the mechanism underlying the blockage of the proliferation signal in somatotrophs under CS remains unclear, these somatotrophic disorder, suggesting that the present animal model may be useful for understanding the molecular mechanisms of chronic stress-associated illnesses., (© 2012 The Authors. Journal of Neuroendocrinology © 2012 British Society for Neuroendocrinology.)

The local renin-angiotensin system (RAS) is present in the pituitary gland, and inhibitory effects of angiotensins on the lactosomatotroph (GH3) cell growth have been revealed. The aim of this study was to examine the influence of various angiotensin peptides and angiotensin AT1, AT2, and AT4 receptors antagonists on the cell proliferation, viability, and VEGF secretion in pituitary lactosomatotroph GH3 cell culture in order to identify receptors involved in antiproliferative effects of angiotensins on GH3 tumor cells. Cell viability and proliferation using Mosmann method and BrdU incorporation during DNA synthesis, and VEGF secretion using ELISA assay were estimated. The inhibitory effects of ang II, ang IV, and ang 5-8 on the cell viability and BrdU incorporation in GH3 culture were not abolished by AT1, AT2, and AT4 receptors antagonists. Ang II, as well as ang III and ang IV at lower concentrations stimulated the secretion of VEGF in GH3 cell culture. The secretion of VEGF was inhibited by ang III and ang IV at higher concentrations. AT1 and AT2 receptors antagonists prevented the proangiogenic effects of ang II. Ang II, ang IV, and ang 5-8 decrease the cell number and proliferation in GH3 cell culture independently of the AT1, AT2, and AT4 receptors. These peptides affect also secretion of VEGF in culture examined. Both the AT1 and AT2 receptors appear to mediate the proangiogenic effects of ang II.

Both endocrine and non-endocrine cells of the pituitary gland are organized into structural and functional networks which are formed during embryonic development but which may be modified throughout life. Structural mapping of the various endocrine cell types has highlighted the existence of distinct network motifs and relationships with the vasculature which may relate to temporal differences in their output. Functional characterization of the network activity of growth hormone and prolactin cells has revealed a role for cell organization in gene regulation, the plasticity of pituitary hormone output and remarkably the ability to memorize altered demand. As such, the description of these endocrine cell networks alters the concept of the pituitary from a gland which simply responds to external regulation to that of an oscillator which may memorize information and constantly adapt its coordinated networks' responses to the flow of hypothalamic inputs., (Copyright © 2012. Published by Elsevier Inc.)

Relative to mammals, the neuroendocrine control of pituitary growth hormone (GH) secretion and synthesis in teleost fish involves numerous stimulatory and inhibitory regulators, many of which are delivered to the somatotrophs via direct innervation. Among teleosts, how multifactorial regulation of somatotroph functions are mediated at the level of post-receptor signalling is best characterized in goldfish. Supplemented with recent findings, this review focuses on the known intracellular signal transduction mechanisms mediating the ligand- and function-specific actions in multifactorial control of GH release and synthesis, as well as basal GH secretion, in goldfish somatotrophs. These include membrane voltage-sensitive ion channels, Na(+)/H(+) antiport, Ca(2+) signalling, multiple pharmacologically distinct intracellular Ca(2+) stores, cAMP/PKA, PKC, nitric oxide, cGMP, MEK/ERK and PI3K. Signalling pathways mediating the major neuroendocrine regulators of mammalian somatotrophs, as well as those in other major teleost study model systems are also briefly highlighted. Interestingly, unlike mammals, spontaneous action potential firings are not observed in goldfish somatotrophs in culture. Furthermore, three goldfish brain somatostatin forms directly affect pituitary GH secretion via ligand-specific actions on membrane ion channels and intracellular Ca(2+) levels, as well as exert isoform-specific action on basal and stimulated GH mRNA expression, suggesting the importance of somatostatins other than somatostatin-14., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The secretion of growth hormone (GH) from somatotrophs located within the anterior pituitary gland is stimulated by endogenous hypothalamic growth hormone-releasing hormone (GHRH) and the GH secretagogue (GHS) ghrelin, and inhibited by somatotropin-releasing inhibitory factor (SRIF, also known as somatostatin). These factors bind to specific G-protein-coupled receptors on the cell membrane, and directly or indirectly modify the properties of ion channels and second messenger systems. Ultimately this results in a change in intracellular free Ca(2+) concentration ([Ca(2+)](i)) and the secretion of GH. Somatotrophs possess a variety of ion channels on their membranes, and modification of these ion channels, especially Ca(2+), K(+), and Na(+) channels, is tightly linked to intracellular Ca(2+) levels and therefore hormone secretion. Various issues regarding receptor distribution, role of ion channels, alteration of membrane potential, and involvement of intracellular signaling system in the control of GH secretion are discussed in this review. In particular, this work will focus on ion channels and [Ca(2+)](i) in somatotrophs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: Available data suggest that estrogens and leptin play a role in the control of the pubertal process. In humans and some mammal species the increase of the activity of gonadotropic axis accompanies the decrease in the rate of growth at puberty. The effect of 17β-estradiol and/or leptin administration on the somatotropic and gonadotropic axes was studied using prepubertal female rats as an animal model., Material and Methods: Prepubertal female rats received estradiol/saline, estradiol/leptin, oil/leptin or oil/saline (vehicles) respectively. The changes of growth rate, and serum 17β-estradiol, leptin, GH, IGF-I and gonadotropins levels as well as LHRH and estrogen receptor (ER) concentrations in the medial basal hypothalamus (MBH) and the pituitary were determined. All hormones concentrations were measured by radioimmunoassay and ER by radioligand methods ., Results: In estradiol and/or leptin treated animals noticeable reduction of rate of growth was found. The decrease of growth in response to estradiol treatment accompanied the increase GH level and the decrease of IGF-I concentration in the circulation. Both hormones operating together activated reproductive axis, what was manifested by a significant increase of LHRH abundant in the hypothalamus as well as elevated LH and FSH levels in the circulation. In these rats a significant decrease of the estrogen receptor concentrations in the pituitary was observed., Conclusion: The role of estradiol and leptin in the control of growth and reproduction seems to overlap only partially. Estradiol plays a significant role in the activation of the reproductive axis, and leptin takes part as a permissive factor in pubertal process.

During embryogenesis, the growth hormone-secreting cells (somatotrophs) and the prolactin-secreting cells (lactotrophs) develop from a common progenitor cell (somatomammotroph). Postnatally, in situations when one of these two hormones is oversecreted, often the second hormone is, too. The question posed is whether the increased secretion of growth hormone together with that of prolactin over long periods of time is a risk factor for malignancy.

Several receptors linked to the adenylyl cyclase signaling pathway stimulate electrical activity and calcium influx in endocrine pituitary cells, and a role for an unidentified sodium-conducting channel in this process has been proposed. Here we show that forskolin dose-dependently increases cAMP production and facilitates calcium influx in about 30% of rat and mouse pituitary cells at its maximal concentration. The stimulatory effect of forskolin on calcium influx was lost in cells with inhibited PKA (cAMP-dependent protein kinase) and in cells that were haploinsufficient for the main PKA regulatory subunit but was preserved in cells that were also haploinsufficient for the main PKA catalytic subunit. Spontaneous and forskolin-stimulated calcium influx was present in cells with inhibited voltage-gated sodium and hyperpolarization-activated cation channels but not in cells bathed in medium, in which sodium was replaced with organic cations. Consistent with the role of sodium-conducting nonselective cation channels in PKA-stimulated Ca(2+) influx, cAMP induced a slowly developing current with a reversal potential of about 0 mV. Two TRP (transient receptor potential) channel blockers, SKF96365 and 2-APB, as well as flufenamic acid, an inhibitor of nonselective cation channels, also inhibited spontaneous and forskolin-stimulated electrical activity and calcium influx. Quantitative RT-PCR analysis indicated the expression of mRNA transcripts for TRPC1 >> TRPC6 > TRPC4 > TRPC5 > TRPC3 in rat pituitary cells. These experiments suggest that in pituitary cells constitutively active cation channels are stimulated further by PKA and contribute to calcium signaling indirectly by controlling the pacemaking depolarization in a sodium-dependent manner and directly by conducting calcium.

Message encoding for three isoforms of somatostatin (SS) peptides, SS-14, goldfish brain (gb)SS-28 and [Pro²]SS-14, are expressed in goldfish hypothalamus and pituitary tissues. All three native goldfish SSs are active in reducing basal and stimulated growth hormone (GH) responses in cultured goldfish pituitary cells, although with different potencies and efficacies. In the present study, we examined the effects of these three endogenous SSs on electrophysiological properties of goldfish somatotrophs and their physiological relevance. Voltage-sensitive K+ , Ca²+ and Na+ channels in identified goldfish somatotrophs in primary culture were isolated using whole-cell, amphotericin B-perforated patch-clamp techniques. None of the three SSs affected Na+ currents but all three SSs increased maximal K+ current magnitude, with SS-14 being the most effective. [Pro²]SS14 did not affect Ba²+ currents through voltage-sensitive Ca²+ channels but SS14 decreased the magnitude of early and late Ba²+ currents, whereas gbSS-28 reduced that of the late Ba²+ current. Under current-clamp conditions, SS14 and gbSS28 attenuated evoked action potential magnitudes by 34% and 18%, respectively, although [Pro²]SS14 had no effects. However, all three SSs decreased basal intracellular Ca²+ levels ([Ca²+ ](i)) and suppressed basal GH release. These data suggest that, although the ability of SS-14 and gbSS-28 to decrease basal [Ca²+](i) and GH release can be explained, at least in part, by their attenuating effects on cell excitability and current flow through voltage-sensitive Ca²+ channels, [Pro²]SS14-induced reduction in GH responses and [Ca²+](i) cannot be explained by changes in Ca²+ channel properties., (© 2010 The Authors. Journal of Neuroendocrinology © 2010 Blackwell Publishing Ltd.)

Growth hormone release in goldfish is partly dependent on voltage-sensitive Ca(2+) channels but somatotrope electrophysiological events affecting such channel activities have not been elucidated in this system. The electrophysiological properties of goldfish somatotropes in primary culture were studied using the whole-cell and amphotericin B-perforated patch-clamp techniques. Intracellular Ca(2+) concentration ([Ca(2+)]i) of identified somatotropes was measured using Fura-2/AM dye. Goldfish somatotropes had an average resting membrane potential of -78.4 ± 4.6 mV and membrane input resistance of 6.2 ± 0.2 GΩ. Voltage steps from a holding potential of -90 mV elicited a non-inactivating outward current and transient inward currents at potentials more positive than 0 and -30 mV, respectively. Isolated current recordings indicate the presence of 4-aminopyridine- and tetraethylammonium (TEA)-sensitive K(+), tetrodotoxin (TTX)-sensitive Na(+), and nifedipine (L-type)- and ω-conotoxin GVIA (N-type)-sensitive Ca(2+) channels. Goldfish somatotropes rarely fire action potentials (APs) spontaneously, but single APs can be induced at the start of a depolarizing current step; this single AP was abolished by TTX and significantly reduced by nifedipine and ω-conotoxin GVIA. TEA increased AP duration and triggered repetitive AP firing resulting in an increase in [Ca(2+)]i, whereas TTX, nifedipine and ω-conotoxin GVIA inhibited TEA-induced [Ca(2+)]i pulses. These results indicate that in goldfish somatotropes, TEA-sensitive K(+) channels regulate excitability while TTX-sensitive Na(+) channels together with N- and L-type Ca channels mediates the depolarization phase of APs. Opening of voltage-sensitive Ca(2+) channels during AP firing leads to increases in [Ca(2+)]i., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Ghrelin stimulates growth hormone release and cell proliferation, which strongly supports a significant role for this peptide in the control of growth hormone-releasing adenomas function and growth. Nitric oxide can influence the stimulatory effects of ghrelin on growth hormone secretion in growth hormone-releasing adenomas. However, the effect of nitric oxide (NO) on ghrelin-induced cell proliferation and the mechanism of this effect in the adenoma were not clarified. In this study, we observed that ghrelin, at a concentration of 10⁻⁹ to 10⁻⁶ M, significantly increased BrdU incorporation into rat GH3 cells. A NO donor, S-nitroso-N-acetylpenicillamine (SNAP), blunted basal, and ghrelin-induced cell proliferation. A blocker of NO synthase, Nw-nitro-L-arginine methyl ester hydrochloride (NAME), had no influence on these actions. The activation of extracellular signal-regulated kinase (ERK) 1/2 was examined by western blotting. The results showed that SNAP reduced ghrelin-stimulated ERK1/2 activation but NAME had no influence on this activation. Together, this study indicates that NO inhibited ghrelin-induced cell proliferation by blocking ERK1/2 activation in GH3 cells.

Voltage-gated calcium channels (VGCCs) are osmosensitive. The hypothesis that this property of VGCCs stems from their susceptibility to alterations in the mechanical properties of the bilayer was tested on VGCCs in pituitary cells using cone-shaped lysophospholipids (LPLs) to perturb bilayer lipid stress. LPLs of different head group size and charge were used: lysophosphatidylcholine (LPC), lysophosphatidylinositol (LPI), lysophosphatidylserine (LPS) and lysophosphatidylethanolamine (LPE). Phosphatidylcholine (PC) and LPC (C6:0) were used as controls. We show that partition of both LPC and LPI into the membrane of pituitary cells suppressed L-type calcium channel currents (I(L)). This suppression of I(L) was slow in onset, reversible upon washout with BSA and associated with a depolarizing shift in activation ( approximately 8mV). In contrast to these effects of LPC and LPI on I(L), LPS, LPE, PC and LPC (C6:0) exerted minimal or insignificant effects. This difference may be attributed to the prominent conical shape of LPC and LPI compared to the shapes of LPS and LPE (which have smaller headgroups), and to PC (which is cylindrical). The similar effects of LPC and LPI on I(L), despite differences in the structure and charge of their headgroups suggest a common lipid stress dependent mechanism in their action on VGCCs., (2010 Elsevier Ltd. All rights reserved.)

Estrogens are crucial determinants in the regulation of anterior pituitary function and maintenance of tissue homeostasis. Estrogen actions in this gland are exerted through both classical and non-classical mechanisms of action. This review summarizes the expression of classical α- and β-estrogen receptors and variant isoforms of estrogen receptors in anterior pituitary cell subpopulations. We also analyze estrogen receptor signaling pathways involved in estrogenic actions in the anterior pituitary gland, especially in lactotropes and somatotropes. Complex interactions between multiple signaling pathways are involved in estrogen regulation of hormone secretion, cell proliferation and cell death in this gland. Insight into these pituitary responses to estrogens would help to understand pituitary function and tumorigenesis., (Copyright © 2010 S. Karger AG, Basel.)

Our view of anterior pituitary organization has been altered with the recognition that folliculo-stellate (FS) and somatotroph cell populations form large-scale three-dimensional homotypic networks. This morphological cellular organization may optimize communication within the pituitary gland promoting coordinated pulsatile secretion adapted to physiological needs. The aim of this study was to identify the molecules involved in the formation and potential functional organization and/or signaling within these cell-cell networks. Here, we have focused on one class of cell adhesion molecules, the cadherins, since beta-catenin has been detected in the GH cell network. We have characterized, by qPCR and immunohistochemistry, their cellular expression and distribution. We have also examined whether their expression could be modulated during pituitary tissue remodeling. The mouse anterior pituitary has a restricted and cell-type specific repertoire of cadherin expression: cadherin-11 is exclusively expressed in TSH cells; N-cadherin displays a ubiquitous expression pattern but with different levels of expression between endocrine cell types; E-cadherin is restricted to homotypic contacts between FS cells; while cadherin-18 is expressed both in somatotrophs and FS cells. Thus, each cell type presents a defined combinatorial expression of different subsets of cadherins. This cell-type specific cadherin expression profile emerges early during development and undergoes major changes during postnatal development. These results suggest the existence within the anterior pituitary of cell-cell contact signaling based on a defined pattern of cadherin expression, which may play a crucial role in cellular recognition during the formation and fate of pituitary cell homotypic networks.

There is a growing body of literature focusing on the somatotropic axis and regulation of aging and longevity. Many of these reports derive data from multiple endocrine mutants, those that exhibit both elevated growth hormone (GH) and insulin-like growth factor I (IGF-1) or deficiencies in one or both of these hormones. In general, both spontaneous and genetically engineered GH and IGF-1 deficiencies have lead to small body size, delayed development of sexual maturation and age-related pathology, and life span extension. In contrast, characteristics of high circulating GH included larger body sizes, early puberty and reproductive senescence, increased cancer incidence and reduced life span when compared to wild-type animals with normal plasma hormone concentrations. This information, along with that found in multiple other species, implicates this anabolic pathway as the major regulator of longevity in animals.

Heterozygous germline mutations in the gene encoding the aryl hydrocarbon receptor-interacting protein (AIP) were first described in two Finnish families with pituitary adenomas. The gene is involved in about 15% of familial isolated pituitary adenomas (FIPA), in about 50% of cases of familial acromegaly and in a small proportion of acromegalic patients with sporadic presentation. This review describes the genetic and clinical features of published patients with AIP, with either familial or sporadic pituitary tumors. A genotype-phenotype correlation is proposed: patients with AIP mutations resulting in a truncated protein are significantly younger than those bearing a mutation which preserves the structure of the C-terminal end of the protein (22.7 +/- 9.6 vs. 29.8 +/- 10.9 years). Pituitary tumors linked to AIP mutations are almost exclusively somatotropic (87.5%, n = 56/64) or lactotropic (9.4%, n = 6). Patients with AIP mutations are mostly men (70%, 44 M/19 F), suffer macroadenomas (97%) and are younger at diagnosis (24.4 +/- 10.5 years) than unselected patients with pituitary tumors. Thus, AIP is involved in the development of pituitary tumors, especially involving the somatomammotroph lineage. Genetic testing could be discussed for FIPAs and in young acromegalic patients with a sporadic presentation. Functional studies are needed to understand AIP-induced tumorigenesis., (Copyright 2009 S. Karger AG, Basel.)

Objective: To elucidate the effect of interleukin-1 beta (IL-1 beta) on human growth hormone (hGH) gene expression in a rat somatotropic pituitary cell line MtT/S., Methods: Stably transfected MtT/S cells were firstly established by transfecting 484-Luc1 plasmid which contained hGH gene promoter -484 to +30 bp and luciferase reporter gene. The effect of IL-1 beta on hGH gene expression was determined by assaying the luciferase activities. RT-PCR method was also used to determine whether IL-1 recepor mRNA was expressed in MtT/S cells., Results: The 10(3) U/mL IL-1 beta stimulated secretion and synthesis of GH, and promoted the 5'-promoter activity of GH gene in stably transfected MtT/SGL cells with the action of 1.38 times above the control. Among inhibitors of signaling transduction pathways, mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor PD98059 (40 micromol/L) and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (5 micromol/L) completely blocked the stimulatory effect of IL-1 beta, and phosphatidylinositol-3-kinase (PI3-K) inhibitor LY294002 partly abolished the effect of IL-1 beta. Western blot analysis further confirmed the activation of phosphorylated MEK and p38 MAPK in MtT/SGL cells. Neither over-expression of Pit-1 nor inhibition of Pit-1 expression affected induction of hGH promoter activity by IL-1 beta. A series of deletion constructs of hGH promoter were created to identify the DNA sequence that mediated the effect of IL-1 beta, and results showed that the stimulatory effect of IL-1 beta was abolished following deletion of the -196 to -132 bp fragment., Conclusions: IL-1 beta promotes GH secretion and synthesis in rat MtT/S somatotroph cells. The stimulatory effect of IL-1 beta on hGH gene promoter appears to require the activation of MEK, p38 MAPK, PI3-K, and a fragment of promoter sequence that spans the -196 to -132 bp of the gene, but it may be unlinked with Pit-1 protein.

The ease of in vivo experimental manipulation is one of the main factors that have made the chicken embryo an important animal model in developmental research, including developmental endocrinology. This review focuses on the development of the thyrotropic, corticotropic and somatotropic axes in the chicken, emphasizing the central role of the pituitary gland in these endocrine systems. Functional maturation of the endocrine axes entails the cellular differentiation and acquisition of cell function and responsiveness of the different glands involved, as well as the establishment of top-down and bottom-up anatomical and functional communication between the control levels. Extensive cross-talk between the above-mentioned axes accounts for the marked endocrine changes observed during the last third of embryonic development. In a final paragraph we shortly discuss how genomic resources and new transgenesis techniques can increase the power of the chicken embryo model in developmental endocrinology research.

GHRH depolarizes the membrane of somatotropes, leading to an increase in intracellular free Ca2+ concentration and GH secretion. Na+ channels mediate the rapid depolarization during the initial phase of the action potential, and this regulates Ca2+ influx and GH secretion. GHRH increases a tetrodotoxin-sensitive somatotrope Na+ current that is mediated by cAMP. TTX-resistant (TTX-R) Na+ channels are abundant in sensory neurons and cardiac myocytes, but their occurrence and/or function in somatotropes has not been investigated. Here we demonstrate expression of TTX-R Na+ channels and a TTX-R Na+ current, using patch-clamp method, in green fluorescent protein-GH transgenic mouse somatotropes. GHRH (100 nm) increased the TTX-R Na+ current in a reversible manner. The GHRH-induced increase in TTX-R Na+ current was not affected by the cAMP antagonist Rp-cAMP or protein kinase A inhibitors KT5720 or H89. The TTX-R current was increased by 8-bromoadenosine-cAMP (cAMP analog), forskolin (adenylyl-cyclase activator), and 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor), but the additional, GHRH-induced increase in TTX-R Na+ currents was not affected. U-73122 (phospholipase C inhibitor) and protein kinase C (PKC) inhibitors, Gö-6983 and chelerythrine, blocked the effect of GHRH. PKC activators, phorbol dibutyrate and phorbol myristate acetate, increased the TTX-R Na+ current, but GHRH had no further effect on the current. Na+-free extracellular medium significantly reduced GHRH-stimulated GH secretion. We conclude that GHRH-induced increase in the TTX-R Na+ current in mouse somatotropes is mediated by the PKC system. An increase in the TTX-R Na+ current may contribute to the GHRH-induced exocytosis of GH granules from mouse somatotropes.

The elevation in baseline circulating growth hormone (GH) that occurs in pregnant rats is thought to arise from increased pituitary GH secretion, but the underlying mechanism remains unclear. Distribution, Fourier and algorithmic analyses confirmed that the pregnancy-induced increase in circulating GH in 3-week pregnant rats was due to a 13-fold increase in baseline circulating GH (P < 0.01), without any significant alteration in the parameters of episodic secretion. Electron microscopy revealed that pregnancy resulted in a reduction in the proportion of mammosomatotrophs (P < 0.01) and an increase in type II lactotrophs (P < 0.05), without any significant change in the somatotroph population. However, the density of the secretory granules in somatotrophs from 3-week pregnant rats was reduced (P < 0.05), and their distribution markedly polarised; the granules being grouped nearest the vasculature. Pituitary GH content was not increased, but steady-state GH mRNA levels declined progressively during pregnancy (P < 0.05). In situ hybridisation revealed that pregnancy was accompanied by a suppression of GH-releasing hormone mRNA expression in the arcuate nuclei (P < 0.05) and enhanced somatostatin mRNA expression in the periventricular nuclei (P < 0.05), an expression pattern normally associated with increased GH feedback. Although gastric ghrelin mRNA expression was elevated by 50% in 3-week pregnant rats (P < 0.01), circulating ghrelin, GH-secretagogue receptor mRNA expression and the GH response to a bolus i.v. injection of exogenous ghrelin were all largely unaffected during pregnancy. Although trace amounts of 'pituitary' GH could be detected in the placenta with radioimmunoassay, significant GH-immunoreactivity could not be observed by immunohistochemistry, indicating that rat placenta itself does not produce 'pituitary' GH. Although not excluding the possibility that the pregnancy-associated elevation in baseline circulating GH could arise from alternative extra-pituitary sources (e.g. the ovary), our data indicate that this phenomenon is most likely to result from a direct alteration of somatotroph function.

Idiopathic Parkinson's disease and dopaminergic medication may influence pituitary hormone secretion. The present study aimed to reveal any abnormalities of the somatotrophic system induced by the disease itself and/or the dopaminergic therapy. Investigations of other pituitary hormones under basal and stimulated conditions, as well as an analysis of body composition, were also performed. This was a controlled diagnostic study in which luteinising hormone-releasing hormone, thyroid-releasing hormone, corticotrophin-releasing hormone and arginine hydrochloride were administered to ten patients with idiopathic Parkinson's disease under dopaminergic medication. Basal and stimulated hormone concentrations and bioelectrical impedance analyses were compared with those of healthy, age-matched controls. Basal growth hormone (GH) at -30 and 0 min was higher in Parkinsonian patients (2.74 +/- 3.79 ng/ml versus 0.53 +/- 0.10 ng/ml and 2.12 +/- 2.44 ng/ml versus 0.51 +/- 0.03 ng/ml; P < 0.05). The area under the GH curve after stimulation was greater in Parkinsonian patients (502.4 +/- 202.6 ng x min/ml versus 312.0 +/- 98.5 ng x min/ml; P < 0.05), depending on higher basal GH levels, rather than a greater arginine response. No differences in insulin growth factor (IGF)-1 or IGF-BP3 concentrations were detected. There were no differences between the groups in basal and stimulated gonadotrophic, corticotrophic and thyrotrophic function, or body composition. Prolactin was below the detection limit in the patients during the course of the study. Parkinsonian patients experience marked hypoprolactinaemia and repeated stimulation of GH secretion during chronic dopaminergic therapy. Our findings suggest a peripheral GH resistance in these chronically-treated patients.

Living organisms represent, in essence, dynamic interactions of high complexity between membrane-separated compartments that cannot exist on their own, but reach behaviour in co-ordination. In multicellular organisms, there must be communication and co-ordination between individual cells and cell groups to achieve appropriate behaviour of the system. Depending on the mode of signal transportation and the target, intercellular communication is neuronal, hormonal, paracrine or juxtacrine. Cell signalling can also be self-targeting or autocrine. Although the notion of paracrine and autocrine signalling was already suggested more than 100 years ago, it is only during the last 30 years that these mechanisms have been characterised. In the anterior pituitary, paracrine communication and autocrine loops that operate during fetal and postnatal development in mammals and lower vertebrates have been shown in all hormonal cell types and in folliculo-stellate cells. More than 100 compounds have been identified that have, or may have, paracrine or autocrine actions. They include the neurotransmitters acetylcholine and gamma-aminobutyric acid, peptides such as vasoactive intestinal peptide, galanin, endothelins, calcitonin, neuromedin B and melanocortins, growth factors of the epidermal growth factor, fibroblast growth factor, nerve growth factor and transforming growth factor-beta families, cytokines, tissue factors such as annexin-1 and follistatin, hormones, nitric oxide, purines, retinoids and fatty acid derivatives. In addition, connective tissue cells, endothelial cells and vascular pericytes may influence paracrinicity by delivering growth factors, cytokines, heparan sulphate proteoglycans and proteases. Basement membranes may influence paracrine signalling through the binding of signalling molecules to heparan sulphate proteoglycans. Paracrine/autocrine actions are highly context-dependent. They are turned on/off when hormonal outputs need to be adapted to changing demands of the organism, such as during reproduction, stress, inflammation, starvation and circadian rhythms. Specificity and selectivity in autocrine/paracrine interactions may rely on microanatomical specialisations, functional compartmentalisation in receptor-ligand distribution and the non-equilibrium dynamics of the receptor-ligand interactions in the loops.

Cultured pituitary somatotrophs release growth hormone in response to spontaneous Ca(2+) entry through voltage-gated calcium channels (VGCCs) that is governed by plateau-bursting electrical activity and is regulated by several neurohormones, including GH-releasing hormone (GHRH) and somatostatin. Here we combine experiments and theory to clarify the mechanisms underlying spontaneous and receptor-controlled electrical activity. Experiments support a role of a Na(+)-conducting and tetrodotoxin-insensitive channel in controlling spontaneous and GHRH-stimulated pacemaking, the latter in a cAMP-dependent manner; an opposing role of spontaneously active inwardly rectifying K(+) (K(ir)) channels and G-protein-regulated K(ir) channels in somatostatin-mediated inhibition of pacemaking; as well as a role of VGCCs in spiking and large conductance (BK-type) Ca(2+)-activated K(+) channels in plateau bursting. The mathematical model is compatible with a wide variety of experimental data involving pharmacology and extracellular ion substitution and supports the importance of constitutively active tetrodotoxin-insensitive Na(+) and K(ir) channels in maintaining spontaneous pacemaking in pituitary somatotrophs. The model also suggests that these channels are involved in the up- and downregulation of electrical activity by GHRH and somatostatin. In the model, the plateau bursting is controlled by two functional populations of BK channels, characterized by distance from the VGCCs. The rapid activation of the proximal BK channels is critical for the establishment of the plateau, whereas slow recruitment of the distal BK channels terminates the plateau.

It is well established that pituitary somatotropes fire spontaneous action potentials (SAP) which generate Ca(2+) signals of sufficient amplitude to trigger growth hormone (GH) release. It is also known that ghrelin and synthetic GH-releasing peptides (GHRPs) stimulate GH secretion, though the mechanisms involved remain unclear. In the current report, we show that the chronic (96h) treatment with ghrelin and GHRP-6 increases the firing frequency of SAP in the somatotrope GC cell line. This action is associated with a significant increase in whole-cell inward current density. In addition, long-term application of Na(+) or L-type Ca(2+) current antagonists decreases GHRP-6-induced release of GH, indicating that the ionic currents that give rise to SAP play important roles for hormone secretion in the GC cells. Together, our results suggest that ghrelin and GHPR-6 may increase whole-cell inward current density thereby enhancing SAP firing frequency and facilitating GH secretion from GC somatotropes.

This report describes the development and validation of the rGHp-Cre transgenic mouse that allows for selective Cre-mediated recombination of loxP-modified alleles in the GH-producing cells of the anterior pituitary. Initial screening of the rGHp-Cre parental line showed Cre mRNA was specifically expressed in the anterior pituitary gland of adult Cre+/- mice and cephalic extracts of e17 Cre+/- fetuses. Heterozygote rGHp-Cre transgenic mice were crossbred with Z/AP reporter mice to generate Cre+/-,Z/AP+/- offspring. In this model system, the GH promoter-driven, Cre-mediated recombination of the Z/AP reporter leads to human placental alkaline phosphatase (hPLAP) expression that serves to mark cells that currently produce GH, in addition to cells that would have differentiated from GH cells but currently do not express the GH gene. Double immunocytochemistry of adult male and female Cre+/-,Z/AP+/- pituitary cells revealed the majority (approximately 99%) of GH-producing cells of the anterior pituitary also expressed hPLAP, whereas ACTH-, TSH-, and LH-producing cells were negative for hPLAP, confirming previous reports that corticotropes, thyrotropes, and gonadotropes develop independently of the somatotrope lineage. A small subset (approximately 10%) of the prolactin-producing cells was positive for hPLAP, consistent with previous reports showing lactotropes can arise from somatotropes during pituitary development. However, the fact that 90% of prolactin-producing cells were negative for hPLAP suggests that the majority of lactotropes in the adult mouse pituitary gland develop independently of the somatotrope lineage. In addition to developmental studies, the rGHp-Cre transgenic mouse will provide a versatile tool to study the role of a variety of genes in somatotrope function and neoplastic transformation.

In humans, circulating GH levels are increased in catabolic states and suppressed in obesity. In both extremes, normalization of the metabolic environment normalizes GH release, leading to the conclusion that changes in metabolic hormones and/or metabolites promote changes in GH synthesis and release. Metabolic regulation of GH secretion can be mediated centrally by modulation of hypothalamic GHRH and somatostatin input to the pituitary and/or by direct regulation of pituitary somatotrope function. Although data are available showing glucocorticoids, free fatty acids (FFA), IGF-I, and insulin have direct effects on rat somatotrope function, little information is available regarding the direct pituitary effects of these metabolic factors in primates. Therefore, this study examined the effects of glucocorticoids (dexamethasone (0.1-100 nM) and hydrocortisone (10 nM)), FFA (oleic and linoleic acid, 100 and 400 microM each), IGF-I (0.5-50 nM), and insulin (0.5-50 nM) on GH release and GH, GHRH-receptor (GHRH-R) and ghrelin-receptor (GHS-R) mRNA levels, in primary pituitary cell cultures of baboons (Papio anubis) after 24 h treatment. A commercial ELISA kit was used to determine the amount of GH released into the media, while quantitative real-time reverse transcription-PCR was used to determine mRNA levels. To design species-specific primers for baboon GH, GHRH-R, GHS-R, insulin receptor (INSR), IGF-I receptor (IGF-IR), pituitary-specific transcription factor-1 (Pit-1), and cyclophilin A (used as a housekeeping gene) cDNA, sequence data for each baboon transcript were obtained and this data were submitted to Genbank. Glucocorticoids, FFA, insulin and IGF-I treatment did not significantly alter the expression of Pit-1, a transcription factor essential for normal somatotrope development and function. However, as previously reported in the rat, glucocorticoids increased, while FFA, IGF-I and insulin decreased GH release in baboon pituitary cell cultures, where changes in GH release were reflected by comparable changes in GH mRNA levels. In addition, glucocorticoids increased, while FFA, IGF-I and insulin decreased the expression of the GH stimulatory receptors, GHRH-R and GHS-R, without significantly altering cyclophilin A mRNA levels. A role of insulin/INSR pathway, independent of IGF-I, in regulating pituitary function is supported by the fact that (1) IGF-I and insulin significantly suppressed somatotrope function at doses (0.5 and 5 nM respectively) not anticipated to activate their respective receptors, and (2) the baboon pituitary expresses INSR mRNA at levels comparable to or greater than that of tissues commonly considered as insulin sensitive (i.e. liver, skeletal muscle, and fat). Taken together, these results demonstrate that metabolic factors can directly modulate primate somatotrope function through regulating GH synthesis and release, as well as mediating the expression of receptors important in central (GHRH) and systemic (ghrelin) regulation of GH secretion.

GH secretion is regulated by GHRH and somatostatin via actions on their specific receptors in pituitary somatotropes. Ghrelin and synthetic analogs, GHRPs, also stimulate GH release via GHS-receptors (GHS-R). To examine the long-term effect of GHRH and/or GHRP on somatotropes, primary cultured ovine somatotropes were treated with GHRH (10(-9) and 10(-8) M) and GHRP-2 (10(-8) and 10(-7) M) for up to 2 d. After treatment, culture medium was collected for GH assay, and total RNA was extracted for RT-PCR analysis. To evaluate cell cultures used in this report, somatotrope-enriched pituitary cells were challenged by 6 h GHRH and dexamethasone (DEX) treatment. As expected, GHRH significantly decreased, whereas DEX increased, the levels of GHRHR mRNA. Combined low doses of GHRH (10(-9) M) and GHRP-2 (10(-8) M) treatment for 24 h increased accumulated GH secretion, significantly more than that induced by high doses of GHRH (10(-8) M) and GHRP-2 (10(-7) M). While levels of GHRH-R mRNA increased, GHS-R mRNA levels were decreased by low doses of GHRH and GHRP-2 for 24 h. High doses of GHRH and/or GHRP-2 for 2 d did not increase GH secretion in the second day of treatment and reduced the level of GHRH-R mRNA. High doses of GHRP-2 treatment decreased the levels of both GHRH-R and GHS-R mRNA. Low doses of GHRH and/or GHRP-2 for 2 d increased the level of GHS-R mRNA without changing GHRH-R mRNA levels. Such treatment also increased ghrelin- (10(-9) M) or ghrelin/GHRH (10(-9) M)-induced GH secretion. These results suggest that low doses of GHRP-2 and GHRH prime somatotropes for stimulation by GHRH and ghrelin.

We have recently reported that EGF triggers an original form of cell death in pituitary cell line (GH4C1) with a phenotype sharing some characteristics of both apoptosis (internucleosomal DNA fragmentation) and paraptosis (caspase-independence and cytoplasmic vacuolization). However, the endonuclease involved in EGF-induced DNA fragmentation has not been assessed so far. In the present work we therefore further explored the putative paraptosis involvement in EGF-induced cell death and asked whether L-DNaseII might be involved. Indeed, this endonuclease is known to mediate internucleosomal DNA fragmentation in caspase independent manner. Our Western blot, immunocytochemistry and enzymatic measurement assays show that EGF triggers a cleavage of Leukocyte Elastase Inhibitor (LEI) precursor into L-DNaseII, its subsequent enzymatic activation and nuclear translocation thus pointing to the involvement of this endonuclease pathway in caspase-independent DNA fragmentation. In addition, EGF-induced cell death can be blocked by paraptosis inhibitor AIP-1/Alix, but not with its anti-apoptotic C-terminal fragment (Alix-CT). Altogether these data suggest that EGF-induced cell death defines a novel, L-DNaseII-mediated form of paraptosis.