Rat Anterior Pituitary Research Articles

Molecular characterisation and functional interrogation of a local natriuretic peptide system in rodent pituitaries, αT3-1 and LβT2 gonadotroph cells

In the pituitary, C-type natriuretic peptide (CNP) has been implicated as a gonadotroph-specific factor, yet expression of the CNP gene (Nppc) and CNP activity in gonadotrophs is poorly defined. Here, we examine the molecular expression and putative function of a local gonadotroph natriuretic peptide system. Nppc, along with all three natriuretic peptide receptors (Npr1, Npr2 and Npr3), was expressed in both alphaT3-1 and LbetaT2 cells and primary mouse pituitary tissue, yet the genes for atrial-(ANP) and B-type natriuretic peptides (Nppa and Nppb) were much less abundant. Putative processing enzymes of CNP were also expressed in alphaT3-1 cells and primary mouse pituitaries. Transcriptional analyses revealed that the proximal 50 bp of the murine Nppc promoter were sufficient for GNRH responsiveness, in an apparent protein kinase C and calcium-dependent manner. Electrophoretic mobility shift assays showed Sp1/Sp3 proteins form major complexes within this region of the Nppc promoter. CNP protein was detectable in rat anterior pituitaries, and electron microscopy detected CNP immunoreactivity in secretory granules of gonadotroph cells. Pharmacological analyses of natriuretic peptide receptor activity clearly showed ANP and CNP are potent activators of cGMP production. However, functional studies failed to reveal a role for CNP in regulating cell proliferation or LH secretion. Surprisingly, CNP potently stimulated the human glycoprotein hormone alpha-subunit promoter in LbetaT2 cells but not in alphaT3-1 cells. Collectively, these findings support a role for CNP as the major natriuretic peptide of the anterior pituitary, and for gonadotroph cells as the major source of CNP expression and site of action.

Sex differences in adrenocortical structure and function. XXV. Quantitative analysis of ACTH-immunoreactive cells in the anterior pituitary of gonadectomized and gonadal hormone replaced male and female rats.

Coupled immunocytochemical and stereologic studies of ACTH-immunoreactive cells were performed on anterior pituitaries of control male and female rats and on gonadectomized and testosterone or estradiol replaced animals. The average area and average volume of studied cells as well as their number were similar in anterior pituitary glands of control male and female rats. Orchiectomy increased average area of ACTH-immunoreactive cells, an effect reversed by testosterone while neither ovariectomy nor estradiol replacement had an effect on the area of studied cells. Average volume of these cells was similar in all experimental groups studied. Orchiectomy lowered the total number of ACTH-immunoreactive cells in the gland, an effect reversed by testosterone. Also ovariectomy lowered their number while after estradiol they were more numerous, even in comparison with control females. There is no significant correlation between the size and total number of ACTH-immunoreactive cells of the rat anterior pituitary gland and the size of the adrenal gland in both, male and female control, gonadectomized and testosterone or estradiol replaced rats.

Studies on hypothalamo-pituitary corticoliberin system. IV. Quantitative changes in ACTH-immunoreactive anterior pituitary cells evoked by long-term intraventricular CRF administration and adrenalectomy.

The aim of the study was to investigate, by coupled immunocytochemical and stereologic methods, the effects of long-term intraventricular administration of ovine CRF (oCRF) on ACTH-immunoreactive cells of the rat anterior pituitary and to compare this effect with that of adrenalectomy. CRF-treated rats received 100 ng of oCRF daily for 8 days while control rats were administered with bovine serum albumin (BSA). After BSA administration number of ACTH-immunoreactive cells/mm2 was greater than in intact rats and CRF lowered their number, however, they were still more numerous than in intact rats. Neither average area nor average volume of studied cells were changed in control and CRF-treated animals if compared with intact ones. The total number of ACTH-immunoreactive cells was higher in the glands of BSA and CRF-treated rats than in intact animals. On the other hand, 60 h after bilateral adrenalectomy a marked increase in both, average area and average volume of ACTH-immunoreactive cells was found. The different response of rat corticotropes to intraventricular CRF administration and adrenalectomy may depend on the presence of operating negative corticosterone feedback in animals with adrenal glands intact. Moreover, the present study revealed the lack of correlation between the average area and average volume of ACTH-immunoreactive cells and the weight of the adrenal gland.

Adiponectin Regulate Growth Hormone Secretion Via Adiponectin Receptor Mediated Ca2+ Signalling in Rat Somatotrophs In Vitro

Obesity is associated with reduced levels of growth hormone (GH) and the disruption of pulsatile GH secretion. This results in relative GH deficiency. It is likely that a regulatory relationship between GH secretion and adipose tissue exists as the secretion of GH recovers to normal levels after a reduction in body weight. This report characterise the expression and interaction of adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) and adiponectin, respectively, in regulating the activity of GH secreting cells. Polymerase chain reaction analysis of the GH3 cell line, rat anterior pituitary gland and isolated somatotroph cells from transgenic GFP expressing mice confirmed the expression of both AdipoR1 and AdipoR2 in GH secretory cells. Because GH cells expressed both receptors, it is likely that the measured increase in GH secretion, observed in primary cultured rat pituitary cells after 30 min of incubation with full-length murine adiponectin, was mediated by a direct receptor regulated process. Adiponectin induced an increase in intracellular Ca(2+) through both the influx of extracellular Ca(2+) and the release of intracellular Ca(2+) stores resulting in the secretion of GH. Furthermore, results confirm that this increase in GH secretion depended mainly on an increase in Ca(2+) influx through L-type Ca(2+) channels. It is concluded that adiponectin directly regulates GH secretion from somatotrophs by binding to either adiponectin receptor, and that this is mediated via a similar process observed after the stimulation of GH secretion by GH-releasing hormone.

The Effect of Castration and Androgen Replacement on Rat Pituitary Follicle Stimulating Hormone Investigated by Reversed-Phase High Performance Liquid Chromatography

Rat follicle stimulating hormone (rFSH) was analysed in extracts of rat anterior pituitary using a newly developed separation method of reversed-phase high performance liquid chromatography (RP-HPLC) and post-column detection of immunoreactivity (rFSH-IR) by radioimmunoassay (RIA) or immunoprecipitation with a specific anti-rFSH antibody. Good separation of rFSH was achieved on a Waters Radial-Peak 8MBC18 cartridge with a linear (60 min) gradient from 10-60% acetonitrile at constant 0.1% trifluoroacetic acid concentration. The elution profiles of radioiodinated purified rFSH [(125I)rFSH] as well as extracts of adult male and female rat pituitaries contained two main immunoreactive components (peaks 1 and 2) which eluted at 16-17 min and 21 min respectively. In the (125I)rFSH elution profile, the peaks of immunobinding corresponded to two major peaks of radioactivity. The male pituitary showed greater binding to anti-rFSH-serum of peak 1 and 2 eluate compared to female pituitary. At day 20 after orchidectomy, the amount of immunoreactive rFSH (rFSH-IR) increased in both peaks (by 30% in peak 1 and 180% in peak 2); however, the rFSH-IR level in peak 2 appeared to be more variable compared with that in peak 1. Subcutaneous implantation of 5 mg or 15 mg of dihydrotestosterone (DHT) at the time of orchidectomy prevented the post-castration elevation of rFSH-IR at 10 and 20 days after surgery. The precise nature and physiological significance of the two elution peaks are not clear at this time; however, only peak 2 showed immunoreactivity with anti-rLH-alpha-subunit antibody (AFP 87713681) and the elution time of peak 2 coincided with that of (125I) rLH-alpha subunit (AFP-7264 B).(ABSTRACT TRUNCATED AT 250 WORDS)

The transcriptional factor PREB mediates MCP-1 transcription induced by cytokines in human vascular endothelial cells

Objective The prolactin regulatory element binding (PREB) protein is a transcriptional factor that regulates prolactin promoter activity in rat anterior pituitary. It is expressed not only in the anterior pituitary but also in the cardiovascular system, including in human umbilical vascular endothelial cells (HUVECs). Monocyte chemoattractant protein-1 (MCP-1) is a major chemotactic factor for monocytes and a key factor initiating the inflammatory process of atherogenesis. MCP-1 is expressed in HUVECs in response to several different stimuli, including interleukin (IL)-1β and tumor necrosis factor (TNF)-α. Methods and results MCP-1 expression was regulated by IL-1β and TNF-α and cytokine-induced PREB expression. Conversely, over-expression of PREB using a PREB-expressing adenovirus increased MCP-1 expression in HUVECs. In addition, PREB induced the expression of the luciferase reporter protein under the MCP-1 promoter control. EMSA showed that the transcriptional effect of PREB was mediated by its binding to the PREB-responsive cis-element of the MCP-1 promoter. Finally, we used siRNA to inhibit PREB expression in HUVECs and demonstrated that knockdown of PREB expression attenuated the effects of IL-1β and TNF-α on MCP-1 expression. Conclusions In summary, our findings show that PREB can function as a transcriptional regulator of the MCP-1 promoter in response to cytokines.

Spatio-temporal relation between cadherin switching and cytogenesis of hormone-producing cells in the developing rat adenohypophysis

Cadherins are a family of transmembrane glycoproteins that mediate cell-to-cell adhesion in solid tissues and have been reported to regulate not only morphogenesis but also cell motility, proliferation, and function by activating intracellular signaling pathways. We recently found that primordial cells in the developing rat adenohypophysis co-expressed E- and N-cadherins, but endocrine cells lost E-cadherin to possess only N-cadherin at certain embryonic stages. In the present study, we aimed to elucidate the temporal relationships between cadherin expression and cell proliferation as well as between cadherin expression and the onset of hormone production in embryonic adenohypophyses. Adenohypophyses and their primordia from embryonic and postnatal rats were fixed in Bouin's fluid and paraffin sections were routinely prepared. Multiple fluorescence immunohistochemistry was performed for combinations of E-cadherin, N-cadherin, proliferating cell nuclear antigen (a marker of proliferating cells), cyclin D1, and pituitary hormones. In primordia from embryonic days 13 through 16, proliferative activities were seen in cells that co-expressed E- and N-cadherin. Cells arrested proliferation coincidentally when they lost E-cadherin after embryonic day 16. Possession of E-cadherin was closely related with expression of cyclin D1 at this stage. Moreover, hormone production was observed from embryonic day 16 only in cells that lost E-cadherin. In the developing adenohypophysis, proliferation and differentiation of hormone-producing cells have been reported to be regulated by a variety of external humoral factors. Our results raise the possibility that changes in cadherins are closely involved in these processes.

Nitric oxide sensitive-guanylyl cyclase subunit expression changes during estrous cycle in anterior pituitary glands

17beta-estradiol (E2) exerts inhibitory actions on the nitric oxide pathway in rat adult pituitary glands. Previously, we reported that in vivo E2 acute treatment had opposite effects on soluble guanylyl cyclase (sGC) subunits, increasing alpha1- and decreasing beta1-subunit protein and mRNA expression and decreasing sGC activity in immature rats. Here we studied the E2 effect on sGC protein and mRNA expression in anterior pituitary gland from adult female rats to address whether the maturation of the hypothalamus-pituitary axis influences its effects and to corroborate whether these effects occur in physiological conditions such as during estrous cycle. E2 administration causes the same effect on sGC as seen in immature rats, and these effects are estrogen receptor dependent. These results suggest that E2 is the main effector of these changes. Since the sGC alpha-subunit increases while the sGC activity decreases, we studied if other less active isoforms of the sGC alpha-subunit are expressed. Here we show for the first time that sGCalpha2 and sGCalpha2 inhibitory (alpha2i) isoforms are expressed in this gland, but only sGCalpha2i mRNA increased after E2 acute treatment. Finally, to test whether E2 effects take place under a physiological condition, sGC subunit expression was monitored over estrous cycle. sGCalpha1, -beta1, and -alpha2i fluctuate along estrous cycle, and these changes are directly related with E2 level fluctuations rather than to NO level variations. These findings show that E2 physiologically regulates sGC expression and highlight a novel mechanism by which E2 downregulates sGC activity in rat anterior pituitary gland.

Novel action of pituitary urocortin 2 in the regulation of expression and secretion of gonadotropins

Urocortins 2 (Ucn 2), one of the corticotropin releasing factor (CRF) peptide family, is thought to be an endogenous ligand for CRF type 2 receptor (CRF-R2). We previously demonstrated that Ucn 2 is expressed in the corticotrophs of rat pituitary, and the mRNA expression and secretion of Ucn 2 in corticotrophs of rat anterior pituitary are regulated by CRF and glucocorticoids. Since CRF-R2 has been reported to be expressed on gonadotrophs of the rat pituitary, we hypothesized that pituitary Ucn 2 may control the expression and secretion of gonadotropins. Monolayer culture of rat anterior pituitary cells showed that the secretion of gonadotropins was suppressed by Ucn 2. A CRF-R2 selective antagonist, adenoviral-mediated expression of short interfering RNA against CRF-R2, and anti-Ucn 2 rabbit IgG increased the secretion and mRNA expression of gonadotropins. intraperitoneal injection of anti-Ucn 2 IgG into immature male rats significantly increased the secretion and mRNA expression of gonadotropins compared with those in normal rabbit IgG-injected rats. Daily i.p. injection of anti-Ucn 2 IgG into immature female rats induced a tendency toward earlier occurrence of menarche compared with normal rabbit IgG-injected rats. These findings suggest that pituitary Ucn 2 is involved in the regulatory mechanism of the expression and secretion of gonadotropins through its tonic and inhibitory action on gonadotrophs in a paracrine manner.

Effects of dietary interventions on the anterior pituitary and hypothalamic transcriptome

Event Abstract Back to Event Effects of dietary interventions on the anterior pituitary and hypothalamic transcriptome Pierrette Gaudreau1, 2*, Karine Bedard1, Julie Bedard1 and Guylaine Ferland3 1 CHUM Research Center (CRCHUM), Laboratory of Neuroendocrinology of Aging, United Kingdom 2 University of Montreal, Department of Medicine, Canada 3 University of Montreal, Department of Nutrition, Canada Introduction: Several hypotheses have been proposed to explain the aging process. Long-term moderate caloric restriction (CR) is recognized as a powerful intervention to prevent or delay the decline of physiological functions, such as somatopause, and the appearance of agerelated diseases. Methods: The GeneChip rat genome 230 2.0 array (Affymetrix) was used to study the effects of aging, CR and/or substitution of casein for soy, as the source of dietary protein. Gene expression profiling was performed in the anterior pituitary and hypothalamus of 20-monthold male rats, fed ad-libitum (AL) with casein compared with age-matched animals submitted to a CR since 8 months of age and 2 month-old AL casein-fed rats. Results: In the rat anterior pituitary, 377 genes were regulated by aging (≥ 1.5- fold change, p < 0.05). They were mainly involved in transcription, intracellular signaling, proliferation, adhesion, G-protein coupled receptor signaling, apoptosis, cell cycle, oxidation/reduction and DNA repair. A major effect of CR was observed for 58 genes. Several of them were up regulated by aging and down regulated by CR, indicating a potent effect of CR on age-related gene expression. In addition, 14 genes regulated by aging were normalized by CR. In the hypothalamus, 73 genes were affected by aging, while 12 other were regulated by CR. The highest categories represented were related to intracellular signaling, cell adhesion, apoptosis and exocytosis. Conclusion: These results should help to identify mechanisms underlying the beneficial effects of CR on the hypothalamo-pituitary axis and design dietary/pharmacological interventions to develop novel models of healthy aging. Conference: 3rd Mediterranean Conference of Neuroscience , Alexandria, Egypt, 13 Dec - 16 Dec, 2009. Presentation Type: Oral Presentation Citation: Gaudreau P, Bedard K, Bedard J and Ferland G (2009). Effects of dietary interventions on the anterior pituitary and hypothalamic transcriptome. Front. Neurosci. Conference Abstract: 3rd Mediterranean Conference of Neuroscience . doi: 10.3389/conf.neuro.01.2009.16.100 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 23 Nov 2009; Published Online: 23 Nov 2009. * Correspondence: Pierrette Gaudreau, CHUM Research Center (CRCHUM), Laboratory of Neuroendocrinology of Aging, Angus, United Kingdom, pierrette.gaudreau@umontreal.ca Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Pierrette Gaudreau Karine Bedard Julie Bedard Guylaine Ferland Google Pierrette Gaudreau Karine Bedard Julie Bedard Guylaine Ferland Google Scholar Pierrette Gaudreau Karine Bedard Julie Bedard Guylaine Ferland PubMed Pierrette Gaudreau Karine Bedard Julie Bedard Guylaine Ferland Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Occurrence and phenotype of glutamatergic elements in the adenohypophysis of the rat

Event Abstract Back to Event Occurrence and phenotype of glutamatergic elements in the adenohypophysis of the rat Zsuzsa S. Kocsis1*, Imrem Kallo1, Gabor Wittmann1, Csaba Fekete1, Zsolt Liposits1 and Erik Hrabovszky1 1 Hungarian Academy of Sciences, Hungary These immunocytochemical studies of the rat adenohypophysis identified type-1 and type-2 vesicular glutamate transporters (VGLUT1 and VGLUT2), markers for glutamatergic neuronal phenotype, in adenohypophysial cells. Use of several different antibodies with similar staining results verified that VGLUT1 and VGLUT2 immunoreactivities were due to the local synthesis of the authentic transporter proteins. VGLUT1 immunoreactivity was observed in a relatively small population of adenohypophysial cells. Dual-immunofluorescent experiments revealed the occurrence of VGLUT1 in 45.9±1.8% of ACTH-immunoreactive corticotrope cells and 7.7±0.9% of LH-immunoreactive gonadotrope cells, whereas cells of the other peptide hormone phenotypes were devoid of the VGLUT1 signal. In contrast, VGLUT2 occurred in high percentages of LH- (93.3±1.3%), FSH- (44.7±3.9%), and TSH- (70.0±5.6%) immunoreactive cells and its much lower incidence in cells of the prolactin, GH and ACTH phenotypes. The presence of the glutamate markers VGLUT1 and VGLUT2 in distinct functional subsets of adenohypophysial cells provide proof for the putative involvement of adenohypophysial glutamate in autocrine/paracrine regulatory mechanisms that require clarification. Supported by OTKA (K69127, T73002) and the Sixth EU Research Framework Programme (contract LSHM-CT-2003-503041). Conference: 12th Meeting of the Hungarian Neuroscience Society, Budapest, Hungary, 22 Jan - 24 Jan, 2009. Presentation Type: Poster Presentation Topic: Homeostatic regulatory mechanisms Citation: Kocsis ZS, Kallo I, Wittmann G, Fekete C, Liposits Z and Hrabovszky E (2009). Occurrence and phenotype of glutamatergic elements in the adenohypophysis of the rat. Front. Syst. Neurosci. Conference Abstract: 12th Meeting of the Hungarian Neuroscience Society. doi: 10.3389/conf.neuro.01.2009.04.015 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 25 Feb 2009; Published Online: 25 Feb 2009. * Correspondence: Zsuzsa S Kocsis, Hungarian Academy of Sciences, Budapest, Hungary, srablue@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Zsuzsa S Kocsis Imrem Kallo Gabor Wittmann Csaba Fekete Zsolt Liposits Erik Hrabovszky Google Zsuzsa S Kocsis Imrem Kallo Gabor Wittmann Csaba Fekete Zsolt Liposits Erik Hrabovszky Google Scholar Zsuzsa S Kocsis Imrem Kallo Gabor Wittmann Csaba Fekete Zsolt Liposits Erik Hrabovszky PubMed Zsuzsa S Kocsis Imrem Kallo Gabor Wittmann Csaba Fekete Zsolt Liposits Erik Hrabovszky Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Effect of adlay (Coix lachryma‐jobi L. var. ma‐yuen Stapf.) hull extracts on testosterone release from rat Leydig cells

Adlay has been used as a traditional Chinese medicine for the treatment of many diseases. However, few studies have reported the effects of adlay seeds on the endocrine system. In the present study, the effects of methanol extracts of adlay hull (AHM) on testosterone synthesis were studied. Rat Leydig cells were incubated with different reagents including human chorionic gonadotropin, 8-bromo-adenosine-3',5'-cyclic monophosphate, forskolin, A23187, progesterone and androstenedione in the presence or absence of AHM. The rat anterior pituitary (AP) gland was treated with gonadotropin-releasing hormone (GnRH) in vitro in the presence or absence of AHM, and the concentrations of luteinizing hormone (LH) in the media were measured. AHM decreased testosterone release via the inhibition of (1) the PKA and PKC signal transduction pathways, (2) 17beta-HSD enzyme activity in rat Leydig cells, and (3) in vitro GnRH-induced LH secretion.

Complement C5a receptors in the pituitary gland: expression and function

Communication between the immune and endocrine system is important for the control of inflammation that is primarily mediated through the hypothalamic-pituitary-adrenal axis. The innate immune system rapidly responds to pathogens by releasing complement proteins that include the anaphylatoxins C3a and C5a. We previously reported the existence of C3a receptors in the anterior pituitary gland and now describe the presence of C5a receptors in the gland. C5a and its less active derivative (C5adR) can bind to its own receptor and to another receptor called C5L2. Using RT-PCR and immunocytochemistry, C5a receptors and C5L2 were demonstrated in the rat anterior pituitary gland and in several rodent anterior pituitary cell lines. Western blotting analysis showed that C5a stimulated the phosphorylation of MAPK and AKT but not p38; C5adR on the other hand, had no effect on any of the signal molecules investigated. The effects of C5a and C5adR on the secretion of the inflammatory molecule, macrophage migration inhibitory factor (MIF) were investigated by ELISA. Both compounds showed a dose-dependent inhibition of MIF release, 30-40% inhibition at around 35-70 nM agonist with IC50 values of around 20 nM. C5a and C5adR also stimulated ACTH secretion (up to 25%) from AtT-20DV16 cells. These data show that functional C5a receptors (C5a and C5L2) are present in the anterior pituitary gland and they may play a role in dampening down inflammation by inhibiting the release of MIF and stimulating the release of ACTH.

The Transcriptional Factor Prolactin Regulatory Element-Binding Protein Mediates the Gene Transcription of Adrenal Scavenger Receptor Class B Type I via 3′,5′-Cyclic Adenosine 5′-Monophosphate

Prolactin regulatory element-binding (PREB) protein is a transcription factor that regulates prolactin promoter activity in the rat anterior pituitary. The PREB protein is not only expressed in the anterior pituitary but also in the adrenal gland. However, the role of PREB in the adrenal gland is not clearly understood. Scavenger receptor class B type I (SR-BI) is a receptor for high-density lipoprotein that mediates the cellular uptake of high-density lipoprotein-cholesteryl ester and is a major route for cholesterol delivery to the steroidogenic pathway in the adrenal gland. In the present study, we have examined the role of PREB in regulating SR-BI. SR-BI expression was found to be regulated by cAMP, which stimulated the expression of PREB in a dose-dependent manner. Conversely, overexpression of PREB using a PREB-expressing adenovirus increased the expression of the SR-BI protein in the adrenocortical cell line Y-1. In addition, PREB induced the expression of the luciferase reporter protein that was under the control of the SR-BI promoter. EMSA showed that PREB mediates its transcriptional effect by binding to the PREB-responsive cis-element of the SR-BI promoter. Finally, we used small interfering RNA to inhibit PREB expression in the Y-1 cells and demonstrated that the knockdown of PREB expression attenuated the effects of cAMP on SR-BI expression. In summary, our data showed that in the adrenal gland, PREB regulates the transcription of the SR-BI gene via cAMP.

Functional expression of CXCR4 in somatotrophs: CXCL12 activates GH gene, GH production and secretion, and cellular proliferation

The interaction of chemokine (C-X-C motif) ligand 12 (CXCL12) and its receptor CXCR4 may play an important role in the regulation of anterior pituitary function. In this study, we investigated the expression of CXCL12 and CXCR4 and their role in normal rat pituitary and GH-producing GH3 tumor cell line. RT-PCR analysis and immunohistochemistry revealed that CXCR4 was expressed in normal rat anterior pituitary and GH3 tumor cells. Double immunofluorescent staining showed the complete colocalization of CXCR4 with GH in rat pituitary, indicating that CXCR4 is specifically expressed in rat somatotrophs. Using rat primary pituitary cell cultures and GH releasing hormone receptor expressing stable GH3 cells (GH3-GHRHR), we evaluated the function of CXCL12 compared with GHRH. CXCL12 stimulated GH gene activation in both primary rat anterior pituitary cells and GH3-GHRHR cells. CXCL12 also stimulated GH secretion from primary rat pituitary cells in a dose-dependant manner. BrdU incorporation was increased in response to CXCL12 addition in GH3 cell culture, indicating CXCL12-induced cell proliferation. CXCL12-dependent phosphorylation of ERK1/2 was also confirmed by western blot analysis, supporting the evidence that MAPK is an intracellular mediator of CXCL12/CXCR4 interaction in GH3 cell proliferation. In conclusion, these results indicate that CXCL12/CXCR4 interaction plays an important role in GH production, secretion, and the proliferation of somatotrophs.

PREB regulates transcription of pancreatic glucokinase in response to glucose and cAMP

Prolactin regulatory element binding (PREB) is a transcription factor that regulates prolactin promoter activity in rat anterior pituitary. The PREB protein is not only expressed in the anterior pituitary but also in the pancreas. We have recently reported that in pancreatic beta-cells, PREB regulates the transcription of the insulin gene in response to glucose stimulation. In the current study, we have examined the role of PREB in regulating glucokinase (GK) in pancreatic beta-cells. To analyse the effects of PREB on GK gene transcription, we employed a reporter gene assay. In the cells expressing or with knocked down PREB, GK expression was determined. GK expression was regulated by glucose and cAMP, and both glucose and cAMP stimulated the expression of PREB in a dose-dependent manner. Conversely, overexpression of PREB using a PREB-expressing adenovirus increased the expression of the GK protein. GK enzymatic activity was also significantly increased in the cells that stably expressed PREB. In addition, PREB induced GK promoter activity. Chromatin immunoprecipitation (ChIP) analyses showed that PREB mediated its transcriptional effect by binding to the PREB-responsive cis-element of the GK promoter. Finally, we used siRNA to inhibit PREB expression in cells and demonstrated that the knockdown of PREB attenuated the effects of glucose and cAMP on GK expression. Our data show that in pancreatic -cells, PREB regulates the transcription of the GK gene in response to glucose and cAMP.

The role of apoptosis in early embryonic development of the adenohypophysis in rats

BackgroundApoptosis is involved in fundamental processes of life, like embryonic development, tissue homeostasis, or immune defense. Defects in apoptosis cause or contribute to developmental malformation, cancer, and degenerative disorders.MethodsThe developing adenohypophysis area of rat fetuses was studied at the embryonic stage 13.5 (gestational day) for apoptotic and proliferative cell activities using histological serial sections.ResultsA high cell proliferation rate was observed throughout the adenohypophysis. In contrast, apoptotic cells visualized by evidence of active caspase-3, were detected only in the basal epithelial cones as an introducing event for fusion and closure of the pharyngeal roof.ConclusionWe can clearly show an increasing number of apoptotic events only at the basic fusion sides of the adenohypophysis as well as in the opening region of this organ. Apoptotic destruction of epithelial cells at the basal cones of the adenohypophysis begins even before differentiation of the adenohypophyseal cells and their contact with the neurohypophysis. In early stages of development, thus, apoptotic activity of the adenohypophysis is restricted to the basal areas mentioned. In our test animals, the adenohypophysis develops after closure of the anterior neuroporus.

A High Concentration of Resiniferatoxin Inhibits Ion Channel Function in Clonal Neuroendocrine Cells

Resiniferatoxin (RTX) is a potent agonist of the transient receptor potential vanilloid 1 channel (TRPV1) found in peripheral nociceptors. RTX causes cellular excitation first, followed by a long-lasting refractory state, which has suggested its therapeutic use for pain control. RTX's effect could result from specific actions on TRPV1 channels, but might also arise from previously reported TRPV1-independent effects. We have tested whether exposure to RTX compromises ion channels in a TRPV1-independent manner. Clonal rat anterior pituitary (GH(3)) cells, loaded with the Ca(+2)-sensitive fluorescent dye (fluo-4), were stimulated with the Na(+) channel activator veratridine (VTD) or directly depolarized by 60 mM K(+) solution. The physiological effects of exposure to RTX were evaluated by stimulated increases of fluorescence from raised intracellular [Ca(2+)]. The presence of 10 microM RTX acutely reduced the median fluorescence changes by VTD and 60 mM K(+) to 45% and 50%, respectively (P = 0.018 and 0.043). Prolonged exposure (24 h) of cells to 10 microM RTX, followed by a 2 h washout, reduced the median fluorescence changes by VTD and 60 mM K(+) to 5.6% and 42% of control changes, respectively (P = 0.027 and 0.011). Cell responses to VTD partially recovered, to 42% of control, after incubation in RTX-free medium for 24 h. RTX at 10 microM directly and acutely inhibited voltage-dependent Ca(2+) channels, in a TRPV1-independent manner. Prolonged exposure (24 h) to 10 microM RTX inhibited voltage-dependent Na(+) channels in addition to the Ca(2+) channels, in at least a partially reversible manner.

Effects of arecoline on testosterone release in rats

Arecoline is one of the major components of betel nuts, which have been consumed as chewing gum in Southeast Asia. In this study, the effects of arecoline on testosterone (T) secretion were explored. Male rats were injected with human chorionic gonadotropin (hCG, 5 IU/kg) or arecoline (1 microg/kg) plus hCG via a jugular catheter. Blood samples were collected at several time intervals subsequent to the challenge. Rat anterior pituitary was treated with gonadotropin-releasing hormone in vitro with or without arecoline, and then the concentrations of luteinizing hormone (LH) in the medium were measured. Rat Leydig cells were purified by Percoll density gradient centrifugation and incubated with arecoline, hCG, forskolin, 8-bromo-cAMP (8-Br-cAMP), nifedipine, nimodipine, or tetrandrine at 34 degrees C for 1 h. A single intravenous injection of arecoline resulted in an increase of the hCG-induced level of plasma T. Administration of arecoline (10(-8) to 10(-6) M) in vitro increased T production in Leydig cells. The stimulatory effect of arecoline on T release in vitro was enhanced by hCG (0.001 IU/ml), forskolin (10(-6) M), or 8-Br-cAMP (10(-5) M). By contrast, nifedipine, nimodipine, or tetrandrine inhibited the increased T concentrations induced by arecoline. Western blot showed that arecoline increases steroidogenic acute regulatory (StAR) protein expression compared with vehicle. These results suggested that arecoline stimulates testosterone production by acting directly on Leydig cells via mechanisms involving an activation of L-type calcium channels, increasing the activity of 17beta-hydroxysteroid dehydrogenase and enhancing the expression of StAR.

Electron microscopic observations of the anterior pituitary gland: Part I. The neurons in the “transitional zone” of the anterior pituitary gland

Since [Westlud, K.N., Chils, G.V., 1982. Localization of serotonin fibers in the rat adenohypophysis. Endocrinology 111, 1761–1763] initially identified the serotonin nerve fibers in the anterior pituitary gland, attention has been paid to the rostral zone of the anterior lobe into which nerve fibers enter and subsequently spread to deeper regions of the lobe. The rostral zone is the trifurcated junction of the partes tuberalis, intermedia and distalis, and has the important role(s) for hormone secretion via the “transitional zone” [Sato, G, Shirasawa, N, Sakuma, E, Sato, Y, Asai, Y, Wada, I, Horiuchi, O, Sakamoto, A, Herbert, DC, Soji, T, 2005a. Intercellular communications within the rat anterior pituitary. XI: An immunohistochemical study of distributions of S-100 positive cells in the anterior pituitary of the rat. Tissue and Cell 37, 269–280.]. The objective of this study was to focus on the ultrastructure of this “zone.” All of the animals studied were fixed by perfusion with glutaraldehyde via the left ventricle of the heart and examined by electron microscopy. In the “transitional zone,” a cluster of neuronal elements was observed between the folliculo–stellate cell-rich area and the anterior lobe. This cluster consisted of myelinated fibers, unmyelinated fibers, neuroendocrine fibers, large cells, and supporting cells. The large cells were perikarya of neurons which made a “ganglion-like” structure with associated satellite cells. Agranular, folliculo–stellate cells were intermingled among the elements. This is the first report that neuronal elements form clusters in the “transitional zone.” A relationship of the unmyelinated and neuroendocrine fibers in the basal layer and in the “transitional zone” is discussed.