Distribution of Corticotrophin‐Releasing Factor Type 1 Receptor‐Like Immunoreactivity in the Rat Pituitary

Corticotrophin‐releasing factor (CRF) regulates the hypothalamic‐pituitary‐adrenal axis response to stress through its type 1 receptor (CRF₁) in the corticotrophs of the anterior pituitary. Although CRF₁ mRNA expression has been confirmed in the rat pituitary, the distribution pattern of CRF₁ protein in the pituitary has not been reported. Therefore, we generated an antiserum against the amino acid fragment corresponding to the 177–188 sequence of the first extracellular loop of the rat CRF₁. Using the antiserum, CRF₁‐like immunoreactivity (CRF₁‐LI) was detected in the anterior lobe cells of the rat pituitary where some of them expressed intense signals. CRF₁‐LI also appeared in the intermediate lobe cells and on the fibre‐like elements of the posterior lobe of the pituitary. Dual immunofluorescence labelling showed that corticotrophs exhibited the highest percentage of CRF₁ (male: 27.1 ± 3.0%, female: 18.0 ± 3.0%), followed by lactotrophs (male: 6.7 ± 3.0%, female: 12.1 ± 1.3%), gonadotrophs (male: 2.6 ± 1.0%, female: 7.5 ± 0.5%), thyrotrophs (male: 2.9 ± 0.1%, female: 5.3 ± 1.2%) and somatotrophs (male: 1.1 ± 0.3%, female: 1.2 ± 0.5%). The percentage of CRF₁‐LI‐positive cells that were corticotrophs was significantly higher in male rats than in female rats, whereas CRF₁‐LI‐positive lactotrophs and gonadotrophs were significantly higher in female rats than in male rats. Almost all of the melanotrophs were positive for CRF₁ in the intermediate lobe (98.9 ± 0.2%). CRF₁‐LI and the percentage of CRF₁‐LI in corticotrophs were decreased in the anterior pituitary, and the distribution patterns were altered from a diffuse to punctate one by adrenalectomy; the changes were restored by treatment with dexamethasone (100 μg/kg bw). These results suggest that CRF₁ is involved in the modulation of the functions of the pituitary; moreover, protein expression and the distribution patterns of CRF₁ are regulated by glucocorticoids in the rat anterior pituitary.     

Role of Pituitary Adenylate Cyclase‐Activating Polypeptide in Modulating Hypothalamus‐Pituitary Neuroendocrine Functions in Mouse Cell Models

Pituitary adenylate cyclase‐activating polypeptide (PACAP) was originally identified as a hypothalamic activator of cyclic adenosine monophosphate production in pituitary cells. PACAP and its receptor are expressed not only in the central nervous system, but also in peripheral organs, and function to stimulate pituitary hormone synthesis and secretion as both a hypothalamic‐pituitary‐releasing factor and an autocrine‐paracrine factor within the pituitary. PACAP stimulates the expression of the gonadotrophin α, luteinising hormone (LH) β and follicle‐stimulating hormone (FSH) β subunits, as well as the gonadotrophin‐releasing hormone (GnRH) receptor and its own PACAP type I receptor (PAC1R) in gonadotrophin‐secreting pituitary cells. In turn, GnRH, which is known to be a crucial component of gonadotrophin secretion, stimulates the expression of PACAP and PAC1R in gonadotrophs. In addition, PAC1R and PACAP modulate the functions of GnRH‐producing neurones in the hypothalamus. This review summarises the current understanding of the possible roles of PACAP and PAC1R in modulating hypothalamus and pituitary neuroendocrine cells in the mouse models.     

Signalling Pathways Mediating Secretory and Mitogenic Responses to Galanin and Pituitary Adenylate Cyclase-Activating Polypeptide in the 235-1 Clonal Rat Lactotroph Cell Line

The neuropeptides galanin and pituitary adenylate cyclase-activating peptide (PACAP) have been implicated in the physiological regulation of lactotroph function. Using the 235-1 clonal lactotroph rat cell line we have studied the signalling pathways mediating the secretory and mitogenic responses to galanin and PACAP. Both peptides stimulated prolactin release to a similar maximal extent. PACAP (100  nM) stimulated an increase in the proliferation rate of 235-1 cells, but was significantly less effective than 100  nM galanin (161.8±2.3% vs 296.1±9.1% of control). PACAP stimulated cAMP accumulation with an ED₅₀ of 3.2  nM, and a maximal effect of almost two-fold at a concentration of 100  nM. Galanin depleted cAMP, by 30% at a concentration of 100  nM. The aminosteroid phospholipase C (PLC) inhibitor U-73122 virtually abolished maximal peptide stimulated prolactin release. Depletion of inositol phosphates or downregulation of protein kinase C reduced maximal peptide stimulated prolactin release from about 260% to about 160% of unstimulated release. Both peptides at a concentration of 100  nM caused a sustained increase in intracellular calcium when incubated with cells for 30  min. These results demonstrate that both peptides stimulate prolactin release and the proliferation rate of 235-1 cells. The most important signalling pathway for prolactin release activated by both peptides is via PLC, although they also regulate cAMP levels, which are increased by PACAP and decreased by galanin. Despite maximal peptide stimulated prolactin release being equal, galanin has a greater mitogenic effect on 235-1 cells than PACAP, raising the possibility that it activates an additional mitogenic signalling pathway.     

Deciphering Brain Insulin Receptor and Insulin‐Like Growth Factor 1 Receptor Signalling

Insulin receptor (IR) and insulin‐like growth factor 1 receptor (IGF1R) are highly conserved receptor tyrosine kinases that share signalling proteins and are ubiquitously expressed in the brain. Central application of insulin or IGF1 exerts several similar physiological outcomes, varying in strength, whereas disruption of the corresponding receptors in the brain leads to remarkably different effects on brain size and physiology, thus highlighting the unique effects of the corresponding hormone receptors. Central insulin/IGF1 resistance impacts upon various levels of the IR/IGF1R signalling pathways and is a feature of the metabolic syndrome and neurodegenerative diseases such as Alzheimer's disease. The intricacy of brain insulin and IGF1 signalling represents a challenge for the identification of specific IR and IGF1R signalling differences in pathophysiological conditions. The present perspective sheds light on signalling differences and methodologies for specifically deciphering brain IR and IGF1R signalling.     

Effect of Deletion of Ghrelin‐O‐Acyltransferase on the Pulsatile Release of Growth Hormone in Mice

Ghrelin, a gut hormone originating from the post‐translational cleavage of preproghrelin, is the endogenous ligand of growth hormone secretagogue receptor 1a (GHS‐R1a). Within the growth hormone (GH) axis, the biological activity of ghrelin requires octanoylation by ghrelin‐O‐acyltransferase (GOAT), conferring selective binding to the GHS‐R1a receptor via acylated ghrelin. Complete loss of preproghrelin‐derived signalling (through deletion of the Ghrl gene) contributes to a decline in peak GH release; however, the selective contribution of endogenous acyl‐ghrelin to pulsatile GH release remains to be established. We assessed the pulsatile release of GH in ad lib. fed male germline goat^?/? mice, extending measures to include mRNA for key hypothalamic regulators of GH release, and peripheral factors that are modulated relative to GH release. The amount of GH released was reduced in young goat^?/? mice compared to age‐matched wild‐type mice, whereas pulse frequency and irregularity increased. Altered GH release did not coincide with alterations in hypothalamic Ghrh, Srif, Npy or Ghsr mRNA expression, or pituitary GH content, suggesting that loss of Goat does not compromise canonical mechanisms that contribute to pituitary GH production and release. Although loss of Goat resulted in an irregular pattern of GH release (characterised by an increase in the number of GH pulses observed during extended secretory events), this did not contribute to a change in the expression of sexually dimorphic GH‐dependent liver genes. Of interest, circulating levels of insulin‐like growth factor (IGF)‐1 were elevated in goat^?/? mice. This rise in circulating levels of IGF‐1 was correlated with an increase in GH pulse frequency, suggesting that sustained or increased IGF‐1 release in goat^?/? mice may occur in response to altered GH release patterning. Our observations demonstrate that germline loss of Goat alters GH release and patterning. Although the biological relevance of altered GH secretory patterning remains unclear, we propose that this may contribute to sustained IGF‐1 release and growth in goat^?/? mice.     

Multiple Ca2+ Channel‐Dependent Components in Growth Hormone Secretion from Rat Anterior Pituitary Somatotrophs

The involvement of L‐type Ca²⁺ channels in both ‘basal’ and ‘stimulated’ growth hormone (GH) secretion is well established; however, knowledge regarding the involvement of non‐L‐type Ca²⁺ channels is lacking. We investigated whether non‐L‐type Ca²⁺ channels regulate GH secretion from anterior pituitary (AP) cells. To this end, GH secretion was monitored from dissociated AP cells, which were incubated for 15 min with 2 mm K⁺ (‘basal’ secretion) or 60 mm K⁺ (‘stimulated’ secretion). The role of non‐L‐type Ca²⁺ influx was investigated using specific channel blockers, including ω‐agatoxin‐IVA, ω‐conotoxin GVIA or SNX‐482, to block P/Q‐, N‐ or R‐type Ca²⁺ channels, respectively. Our results demonstrate that P/Q‐, N‐ and R‐type Ca²⁺ channels contributed 21.2 ± 1.9%, 20.2 ± 7.6% and 11.4 ± 1.8%, respectively, to ‘basal’ GH secretion and 18.3 ± 1.0%, 24.4 ± 5.4% and 14.2 ± 4.8%, respectively, to ‘stimulated’ GH secretion. After treatment with a ‘cocktail’ that comprised the previously described non‐L‐type blockers, non‐L‐type Ca²⁺ channels contributed 50.9 ± 0.4% and 45.5 ± 2.0% to ‘basal’ and ‘stimulated’ GH secretion, respectively. Similarly, based on the effects of nifedipine (10 μM), L‐type Ca²⁺ channels contributed 34.2 ± 3.7% and 54.7 ± 4.1% to ‘basal’ and ‘stimulated’ GH secretion, respectively. Interestingly, the relative contributions of L‐type/non‐L‐type Ca²⁺ channels to ‘stimulated’ GH secretion were well correlated with the relative contributions of L‐type/non‐L‐type Ca²⁺ channels to voltage‐gated Ca²⁺ influx in AP cells. Finally, we demonstrated that compartmentalisation of Ca²⁺ channels is important for GH secretion. Lipid raft disruption (methyl‐β‐cyclodextrin, 10 mm ) abrogated the compartmentalisation of Ca²⁺ channels and substantially reduced ‘basal’ and ‘stimulated’ GH secretion by 43.2 ± 3.4% and 58.4 ± 4.0%, respectively. In summary, we have demonstrated that multiple Ca²⁺ channel‐dependent pathways regulate GH secretion. The proper function of these pathways depends on their compartmentalisation within AP cell membranes.     

Evidence for a Trophic Action of the Glycoprotein Hormone α-Subunit in Rat Pituitary

The effect of the α-subunit of luteinizing hormone (LHα) on lactotroph growth in 14-day-old rat pituitary was studied in vitro using a reaggregate pituitary cell culture system. LHα significantly expanded both the total population of cells expressing prolactin mRNA and the number of [³H]thymidine incorporating prolactin mRNA expressing cells. No such effect could be elicited by LH. Both effects were inhibited by simultaneous addition of an anti-LHα antiserum but not by normal rabbit serum. Anti-LHα antiserum added alone to the cultures caused a small decrease in the number of prolactin mRNA expressing cells and in [³H]thymidine labelling of the latter. It is concluded that LHα may be a trophic factor of lactotrophs not only during fetal development, as suggested by others previously, but also during the rapid expansion of this cell type during postnatal life in the rat.     

Rapid Transition of NESTIN‐Expressing Dividing Cells from PROP1‐Positive to PIT1‐Positive Advances Prenatal Pituitary Development

We recently reported that the quantitative and qualitative transition of stem/progenitor cells occurs by the acquisition of a novel mechanism in the terminal differentiation during postnatal development of the anterior pituitary. We hypothesised that this novel mechanism is an alteration of a cell supply system accompanying proliferation of the progenitor cells. In the present study, we examined the proliferation activities of progenitor cells by indication of the expression of Nestin, a marker of rapidly dividing progenitor cells, aiming to verify our hypothesis and to resolve another outstanding issue regarding whether the Nestin gene is expressed in the pituitary. We found that NESTIN‐positive dividing cells certainly exist in the pituitary through all stages of development. Almost all of the PROP1‐positive progenitor cells express Nestin in early embryonic pituitary development. Thereafter, Nestin‐expressing dividing cells involved in the cell supply system transfer from PROP1‐positive progenitor cells to committed progenitor cells, such as PIT1‐positive cells, on neonatal pituitary development. Furthermore, our data, together with the findings of previous studies on cell lineage tracing analyses using Nestin‐Cre mice derived by the central nervous system (CNS)‐specific Nestin promoter, suggest that at least two regulation systems for Nestin‐expression exist in the pituitary, with the majority of these not being CNS‐specific.     

Pituitary Gonadotrophic Hormone Synthesis,Secretion, Subunit Gene Expression and Cell Structure in Normal and Follicle‐Stimulating Hormone β Knockout,Follicle‐Stimulating Hormone Receptor Knockout,Luteinising Hormone Receptor Knockout,Hypogonadal and Ovariectomised Female Mice

To investigate the relationship between gonadotroph function and ultrastructure, we have compared, in parallel in female mice, the effects of several different mutations that perturb the hypothalamic‐pituitary‐gonadal axis. Specifically, serum and pituitary gonadotrophin concentrations, gonadotrophin gene expression, gonadotroph structure and number were measured. Follicle‐stimulating hormone β knockout (FSHβKO), follicle‐stimulating hormone receptor knockout (FSHRKO), luteinising hormone receptor knockout (LuRKO), hypogonadal (hpg) and ovariectomised mice were compared with control wild‐type or heterozygote female mice. Serum levels of LH were elevated in FSHβKO and FSHRKO compared to heterozygote females, reflecting the likely decreased oestrogen production in KO females, as demonstrated by the threadlike uteri and acyclicity. As expected, there was no detectable FSH in the serum or pituitary and an absence of expression of the FSHβ subunit gene in FSHβKO mice. However, there was a significant increase in expression of the FSHβ and LHβ subunit genes in FSHRKO female mice. The morphology of FSHβKO and FSHRKO gonadotrophs was not significantly different from the control, except that secretory granules in FSHRKO gonadotrophs were larger in diameter. In LuRKO and ovariectomised mice, stimulation of LHβ and FSHβ mRNA, as well as serum protein concentrations, were reflected in subcellular changes in gonadotroph morphology, including more dilated rough endoplasmic reticula and fewer, larger secretory granules. In the gonadotophin‐releasing hormone deficient hpg mouse, gonadotrophin mRNA and protein levels were significantly lower than in control mice and gonadotrophs were correspondingly smaller with less abundant endoplasmic reticula and reduced numbers of secretory granules. In summary, major differences in pituitary content and serum concentrations of the gonadotrophins LH and FSH were found between control and mutant female mice. These changes were associated with changes in expression of the gonadotrophin subunit genes and were reflected in the cellular structure and secretory granule appearance within the gonadotroph cells.     

Signalling Pathway Alterations in Pituitary Adenomas: Involvement of Gsα, cAMP and Mitogen-Activated Protein Kinases

Despite extensive research on sporadic pituitary adenomas, it is not yet possible to assign one protein alteration to one specific type of pituitary adenomas. Nevertheless, alterations of the cAMP pathway appear to be molecular hallmarks of most growth hormone (GH)-secreting adenomas. However, these alterations do not confer specific phenotypes to patients carrying these alterations. In this review, we summarise the literature regarding signalling alterations observed in GH-secreting adenomas. We focus on Gsα alterations and their possible cross-talk with the extracellular signal-related kinase (ERK)1/2 pathway. In the light of results obtained on human somatotroph adenoma cells in primary culture and on models of murine somatotroph cell lines, we postulate a crucial role for ERK1/2 in GH-secreting adenomas downstream of cAMP pathway alterations that might impact the tumoural phenotype.     

Seasonal Effect of Gonadotrophin Inhibitory Hormone on Gonadotrophin‐Releasing Hormone‐induced Gonadotroph Functions in the Goldfish Pituitary

We have shown that native goldfish gonadotrophin inhibitory hormone (gGnIH) differentially regulates luteinsing hormone (LH)‐β and follicle‐stimulating hormone (FSH)‐β expression. To further understand the functions of gGnIH, we examined its interactions with two native goldfish gonadotrophin‐releasing hormones, salmon gonadotrophin‐releasing hormone (sGnRH) and chicken (c)GnRH‐II in vivo and in vitro. Intraperitoneal injections of gGnIH alone reduced serum LH levels in fish in early and mid gonadal recrudescence; this inhibition was also seen in fish co‐injected with either sGnRH or cGnRH‐II during early recrudescence. Injection of gGnIH alone elevated pituitary LH‐β and FSH‐β mRNA levels at early and mid recrudescence, and FSH‐β mRNA at late recrudescence. Co‐injection of gGnIH attenuated the stimulatory influences of sGnRH on LH‐β in early recrudescence, and LH‐β and FSH‐β mRNA levels in mid and late recrudescence, as well as the cGnRH‐II‐elicited increase in LH‐β, but not FSH‐β, mRNA expression at mid and late recrudescence. sGnRH and cGnRH‐II injection increased pituitary gGnIH‐R mRNA expression in mid and late recrudescence but gGnIH reduced gGnIH‐R mRNA levels in late recrudescence. gGnIH did not affect basal LH release from perifused pituitary cells and continual exposure to gGnIH did not alter the LH responses to acute applications of GnRH. However, a short 5‐min GnIH treatment in the middle of a 60‐min GnRH perifusion selectively reduced the cGnRH‐II‐induced release of LH. These novel results indicate that, in goldfish, gGnIH and GnRH modulate pituitary GnIH‐R expression and gGnIH differentially affects sGnRH and cGnRH‐II regulation of LH secretion and gonadotrophin subunit mRNA levels. Furthermore, these actions are manifested in a reproductive stage‐dependent manner.     

High‐Cholesterol Diet Disrupts the Levels of Hormones Derived from Anterior Pituitary Basophilic Cells

Emerging evidence shows that elevated cholesterol levels are detrimental to health. However, it is unclear whether there is an association between cholesterol and the pituitary. We investigated the effects of a high‐cholesterol diet on pituitary hormones using in vivo animal studies and an epidemiological study. In the animal experiments, rats were fed a high‐cholesterol or control diet for 28 weeks. In rats fed the high‐cholesterol diet, serum levels of thyroid‐stimulating hormone (TSH; also known as thyrotrophin), luteinising hormone (LH) and follicle‐stimulating hormone (FSH) produced by the basophilic cells of the anterior pituitary were elevated in a time‐dependent manner. Among these hormones, TSH was the first to undergo a significant change, whereas adrenocorticotrophic hormone (ACTH), another hormone produced by basophilic cells, was not changed significantly. As the duration of cholesterol feeding increased, cholesterol deposition increased gradually in the pituitary. Histologically, basophilic cells, and especially thyrotrophs and gonadotrophs, showed an obvious increase in cell area, as well as a potential increase in their proportion of total pituitary cells. Expression of the β‐subunit of TSH, FSH and LH, which controls hormone specificity and activity, exhibited a corresponding increase. In the epidemiological study, we found a similar elevation of serum TSH, LH and FSH and a decrease in ACTH in patients with hypercholesterolaemia. Significant positive correlations existed between serum total cholesterol and TSH, FSH or LH, even after adjusting for confounding factors. Taken together, the results of the present study suggest that the high‐cholesterol diet affected the levels of hormones derived from anterior pituitary basophilic cells. This phenomenon might contribute to the pituitary functional disturbances described in hypercholesterolaemia.     

Cortical ablation induces time‐dependent changes in rat pituitary somatotrophs and upregulates growth hormone receptor expression in the injured cortex

The pituitary appears to be vulnerable to brain trauma, and its dysfunction is a common feature after traumatic brain injury. The role of pituitary growth hormone (GH) in brain repair after injury has been envisaged, but more studies must be performed to understand completely the importance of GH in these processes. Because some of the neuroprotective effects of GH are mediated directly through the GH receptor (GHR), we examined GHR expression in the rat cerebral cortex after sensorimotor cortex ablation. RT‐PCR, immunohistochemistry, and double immunofluorescence had been performed to analyze the correlation between GHR expression in the injured cortex and activity of GH cells in the pituitary. Our results showed that the volume of GH‐immunopositive cells was reduced at days 2 and 7 postsurgery (dps), and volume density of GH cells was significantly decreased at 14 dps, all compared with appropriate sham controls. At 30 dps all investigated parameters had returned to control level. In the injured cortex, GHR expression was transiently upregulated. Increased GHR immunoreactivity was observed in reactive astrocytes at 7 and particularly at 14 dps. In neuronal cells, an increase of GHR immunoreactivity was seen in neuronal cell bodies and well‐defined primary dendrites at 14 and especially at 30 dps. The results presented here suggest that, during recovery from brain injury, changes in activity of pituitary GH cells result in upregulation of GHR that may have a role in neuronal arborization and glial proliferation in the injured cortex. © 2014 Wiley Periodicals, Inc.     

Hypothalamic Galanin‐Like Peptide Rescues the Onset of Puberty in Food‐Restricted Weanling Rats

Galanin‐like peptide (GALP) is a known mediator of metabolism and reproduction; however, the role that GALP plays in the onset of puberty is unknown. First, we tested the hypothesis that central GALP administration could rescue puberty in food‐restricted weanling rats. GALP treatment in food‐restricted rats of both sexes rescued the timing of the onset of puberty to that seen in ad lib. fed controls. Second, we tested whether GALP translation knocked‐down in ad lib. fed, prepubertal rats would alter the timing of puberty. Knock‐down females, but not males, showed a significant (P < 0.01) delay in the onset of puberty compared to controls. Third, we sought evidence that the role of GALP in pubertal onset is mediated by the kisspeptin system. In situ hybridisation analyses showed a significant (P < 0.01) reduction in Kiss1 mRNA within the hypothalamic arcuate nucleus in food‐restricted rats compared to ad lib. fed controls and this reduction was prevented with i.c.v. GALP administration. Furthermore, analyses of Fos‐immunoreactivity (‐IR) after i.c.v. GALP treatment did not elicit Fos‐IR within any kisspeptin neurones, nor are GALP and kisspeptin peptides or mRNA colocalised. These data demonstrate that hypothalamic GALP infusion maintained the onset of puberty in food‐restricted weanling rats, although probably not via direct innervation of kisspeptin neurones.     

Signalling Pathway Mediated by CXCR7, an Alternative Chemokine Receptor for Stromal-Cell Derived Factor-1α, in AtT20 Mouse Adrenocorticotrophic Hormone-Secreting Pituitary Adenoma Cells

Stromal cell-derived factor (SDF)-1 and its receptor, CXCR4, have been identified in both neurones and glia of many brain areas. Previous studies have mainly focused on the role of SDF-1 and CXCR4 in modulating the hypothalamic-pituitary axis and their possible involvement in the development of pituitary adenomas. An alternative SDF-1 receptor, CXCR7, has recently been identified, but it has not been studied in the context of pituitary adenomas. The present study aimed to investigate the distribution and function of CXCR7 in pituitary adenomas. The expression of CXCR7, normalised to β-actin, was assessed by tissue microarray analysis of 62 adenomas, including 23 growth hormone (GH)-producing adenomas, 22 nonfunctioning adenomas, seven prolactin (PRL)-producing adenomas, six adrenocorticotrophic hormone-producing adenomas and four thyroid-stimulating hormone-producing adenomas. In vitro functional studies used RNA interference (RNAi) and cDNA microarray analysis to evaluate the CXCR7 signalling pathway in AtT-20 mouse pituitary adenoma cells treated with recombinant mouse SDF-1α and transfected with RNAi against Cxcr7 or control RNAi. In tissue microarray analysis, prominent expression of CXCR7 was observed in GH-producing adenomas and PRL-producing adenomas, and in macroadenomas (P < 0.05). Intracellular signalling via CXCR7 up-regulated Bub1, Cdc29 and Ccnb1, and down-regulated Asns, Gpt, Pycr1, Cars and Dars. The present study demonstrates that the SDF-1α/CXCR7 signalling pathway regulates genes involved in cell cycle control, amino acid metabolism and ligase activity, which comprise targets that are distinct from those of CXCR4.     

Activity‐Dependent Notch Signalling in the Hypothalamic‐Neurohypophysial System of Adult Mouse Brains

Notch signalling has a key role in cell fate specification in developing brains; however, recent studies have shown that Notch signalling also participates in the regulation of synaptic plasticity in adult brains. In the present study, we examined the expression of Notch3 and Delta‐like ligand 4 (DLL4) in the hypothalamic‐neurohypophysial system (HNS) of the adult mouse. The expression of DLL4 was higher in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) compared to adjacent hypothalamic regions. Double‐labelling immunohistochemistry using vesicular GABA transporter and glutamate transporter revealed that DLL4 was localised at a subpopulation of excitatory and inhibitory axonal boutons against somatodendrites of arginine vasopressin (AVP)‐ and oxytocin (OXT)‐containing magnocellular neurones. In the neurohypophysis (NH), the expression of DLL4 was seen at OXT‐ but not AVP‐containing axonal terminals. The expression of Notch3 was seen at somatodendrites of AVP‐ and OXT‐containing magnocellular neurones in the SON and PVN and at pituicytes in the NH. Chronic physiological stimulation by salt loading, which remarkably enhances the release of AVP and OXT, decreased the number of DLL4‐immunoreactive axonal boutons in the SON and PVN. Moreover, chronic and acute osmotic stimulation promoted proteolytic cleavage of Notch3 to yield the intracellular fragments of Notch3 in the HNS. Thus, the present study demonstrates activity‐dependent reduction of DLL4 expression and proteolytic cleavage of Notch3 in the HNS, suggesting that Notch signalling possibly participates in synaptic interaction in the hypothalamic nuclei and neuroglial interaction in the NH.     

Role of AMP‐Activated Protein Kinase Activators in Antiproliferative Multi‐Drug Pituitary Tumour Therapies: Effects of Combined Treatments with Compounds Affecting the mTOR‐p70S6 Kinase Axis in Cultured Pituitary Tumour Cells

AMP‐activated protein kinase (AMPK) is activated under conditions that deplete cellular ATP levels and elevate AMP levels. We have recently shown that AMPK can represent a valid target for improving the medical treatment of growth hormone (GH)‐secreting pituitary adenomas and the effects of its activation or inhibition in pituitary tumour cells are worthy of further characterisation. We aimed to determine whether AMPK may have a role in combined antiproliferative therapies based on multiple drugs targeting cell anabolic functions at different levels in pituitary tumour cells to overcome the risk of cell growth escape phenomena. Accordingly, we tried to determine whether a rationale exists in combining compounds activating AMPK with compounds targeting the phosphatidylinositol‐3‐kinase (PI3K)/Akt/mTOR/p70S6K signalling pathway. AMPK down‐regulation by specific small‐interfering RNAs confirmed that activated AMPK had a role in restraining growth of GH3 cells. Hence, we compared the effects of compounds directly targeting the mTOR‐p70S6K axis, namely the mTOR inhibitor rapamycin and the p70S6K inhibitor PF‐4708671, with the effects of the AMPK activator 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR) on cell signalling and cell growth, in rat pituitary GH3 cells. AICAR was able to reduce growth factor‐induced p70S6K activity, as shown by the decrease of phospho‐p70S6K levels. However, it was far less effective than rapamycin and PF‐4708671. We observed significant differences between the growth inhibitory effects of the three compounds in GH3 and GH1 cells. Interestingly, PF‐4708671 was devoid of any effect. AICAR was at least as effective as rapamycin and the co‐treatment was more effective than single treatments. AICAR induced apoptosis of GH3 cells, whereas rapamycin caused preferentially a decrease of cell proliferation. Finally, AICAR and rapamycin differed in their actions on growth factor‐induced extracellular signal regulated kinase 1/2 phosphorylation. In conclusion, the results of the present study suggest the increased efficacy of combined antiproliferative therapies, including rapamycin analogues and AMPK activators in GH‐secreting pituitary tumours, as a result of complementary and only partially overlapping mechanisms of action.