Somatostatin

Primary Hepatic Neuroendocrine Tumor Treated With Lattice and External Beam Radiation: A Case Report.

Yeidaliz Barreto Cruz, Gordon L Grado

Neuroendocrine tumors (NETs) originate from neuroendocrine cells, which receive neural input and play a key role in transmitting signals by secreting bioactive substances such as monoamines (e.g., serotonin), peptides (e.g., somatostatin), and hormones (e.g., insulin) that regulate a wide range of physiological functions. While NETs in the liver are typically metastatic, often originating from other sites in the gastrointestinal (GI) tract, they can, in rare cases, manifest as primary tumors in the liver. These are referred to as primary hepatic neuroendocrine tumors (PHNT). We present the case of a 62-year-old male patient diagnosed with PHNT confirmed through imaging and immunohistochemistry analysis. Given the rarity of this diagnosis, there are no established treatment guidelines for PHNT. In this case, lattice radiotherapy (LRT) via TomoTherapy (TomoTherapy, Inc. in Madison, WI) was selected as the first-line treatment approach for the tumor.

Selective ligand recognition and activation of somatostatin receptors SSTR1 and SSTR3.

Yujue Wang, Youwei Xu, Yue Wang +11 more

Somatostatin receptors (SSTRs) exert critical biological functions such as negatively regulating hormone release and cell proliferation, making them popular targets for developing therapeutics to treat endocrine disorders, especially neuroendocrine tumors. Although several panagonists mimicking the endogenous ligand somatostatin are available, the development of more effective and safer somatostatinergic therapies is limited due to a lack of molecular understanding of the ligand recognition and regulation of divergent SSTR subtypes. Here, we report four cryoelectron microscopy structures of Gi-coupled SSTR1 and SSTR3 activated by distinct agonists, including the FDA-approved panagonist pasireotide as well as their selective small molecule agonists L-797591 and L-796778. Our structures reveal a conserved recognition pattern of pasireotide in SSTRs attributed to the binding with a conserved extended binding pocket, distinct from SST14, octreotide, and lanreotide. Together with mutagenesis analyses, our structures further reveal the dynamic feature of ligand binding pockets in SSTR1 and SSTR3 to accommodate divergent agonists, the key determinants of ligand selectivity lying across the orthosteric pocket of different SSTR subtypes, as well as the molecular mechanism underlying diversity and conservation of receptor activation. Our work provides a framework for rational design of subtype-selective SSTR ligands and may facilitate drug development efforts targeting SSTRs with improved therapeutic efficacy and reduced side effects.

The disrupting effect of chlormequat chloride on growth hormone is associated with pregnancy.

Zongzhen Wu, Long Ma, Deqi Su +1 more

Since chlormequat chloride is widely applied as a plant growth regulator in agriculture and horticulture, its exposure through food consumption is common. We demonstrated previously that chlormequat chloride exposure during pregnancy led to embryos with bigger sizes associated with higher levels of growth hormone (GH) on gestation day 11 (GD11). However, the dose-effect relationship of chlormequat chloride at a lower dose range was not established, and the underlying mechanisms of its promoting effects on embryonic growth and development were not fully elucidated. To address these, pregnant rats were orally exposed to chlormequat chloride at 0, 0.05, 0.5 and 5 mg/kg.bw from GD0 to 11 and the embryonic growth and growth related hormones were evaluated on GD11. We found that the growth and development of the embryos was significantly promoted in a dose dependent manner by chlormequat chloride. Chlormequat chloride also increased embryonic GH, GH releasing hormone (GHRH), and somatostatin (SRIF), and inhibited the embryonic cAMP dependent protein kinase A (PKA) signaling pathway. Chlormequat chloride increased GH synthesis modulated by GHRH/SRIF-PKA-Pituitary specific transcription factor 1 (Pit-1) in the maternal rats. Intriguingly, chlormequat chloride did not show any effects on GH and PKA signaling pathways in the non-pregnant female rats. These findings together suggest that the disrupting effect of chlormequat chloride on GH is associated with pregnancy.

Better Peptides via Chemical Glycosylation: Somatostatin Analogues Having a Human Complex-Type N-Glycan with Improved Drug Properties.

Hirofumi Ochiai, Taiji Shimoda, Kazuhiro Fukae +9 more

Somatostatin (somatotropin release-inhibiting factor, SRIF) is a growth hormone inhibitory factor in the form of a 14- or 28-amino acid peptide. SRIF affects several physiological functions through its action on five distinct SRIF receptor subtypes (sst1-5). Native SRIF has only limited clinical applications due to its rapid degradation in plasma. To overcome this obstacle, we have developed glycosylated SRIF analogues that possess not only metabolic stability but also high affinity to all five receptor subtypes by attaching human complex-type oligosaccharides. Such glycosylated SRIF analogues with improved pharmacokinetic profiles could be potent and novel therapeutic drugs for SRIF-related diseases in which several SRIF receptor subtypes are closely involved, and also shed light on new indications. Our results show that chemical glycosylation can be a powerful tool for the development of peptide and protein analogues superior to the original molecules with enhanced drug properties.

Gastrin: From Physiology to Gastrointestinal Malignancies.

Suzann Duan, Karen Rico, Juanita L Merchant

Abetted by widespread usage of acid-suppressing proton pump inhibitors (PPIs), the mitogenic actions of the peptide hormone gastrin are being revisited as a recurring theme in various gastrointestinal (GI) malignancies. While pathological gastrin levels are intricately linked to hyperplasia of enterochromaffin-like cells leading to carcinoid development, the signaling effects exerted by gastrin on distinct cell types of the gastric mucosa are more nuanced. Indeed, mounting evidence suggests dichotomous roles for gastrin in both promoting and suppressing tumorigenesis. Here, we review the major upstream mediators of gastrin gene regulation, including inflammation secondary to Helicobacter pylori infection and the use of PPIs. We further explore the molecular biology of gastrin in GI malignancies, with particular emphasis on the regulation of gastrin in neuroendocrine neoplasms. Finally, we highlight tissue-specific transcriptional targets as an avenue for targetable therapeutics.

Development of anti-somatostatin receptors CAR T cells for treatment of neuroendocrine tumors.

Barbara Mandriani, Eleonora Pellè, Francesco Mannavola +9 more

Neuroendocrine tumors (NETs) overexpress somatostatin receptors (SSTRs).

Evolution of Neuroendocrine Tumor Therapy.

Thomas M O'Dorisio, Alan G Harris, M Sue O'Dorisio

To better understand developments in treatment of neuroendocrine tumors of the gastroenteropancreatic system, and the pivotal roles of native somatostatin and its long-acting analogues play in normal peptide regulation and neuropeptide excess associated with neuroendocrine tumors (NETs), this article delineates and defines distinct eras in the history and discovery of gastrointestinal endocrinology. We highlight the collaboration between academia and industry in basic science and the clinical research that advanced Lu-177-DOTATATE to approval as standard of care therapy for low-grade NETs. Examples of new radioisotopes and therapy compounds currently in development for diagnosis and therapy for high-grade NETs are also discussed.

Regulation of Somatostatin Receptor 2 Trafficking by C-Tail Motifs and the Retromer.

Courtney Olsen, Kimiya Memarzadeh, Arzu Ulu +3 more

The Gi-coupled somatostatin receptor 2 (SST2) is a G protein-coupled receptor (GPCR) that mediates many of somatostatin's neuroendocrine actions. Upon stimulation, SST2 is rapidly internalized and transported to early endosomes before being recycled to the plasma membrane. However, little is known about the intracellular itinerary of SST2 after it moves to the early endosomal compartment or the cytoplasmic proteins that regulate its trafficking. As postsynaptic density protein/discs large 1/zonula occludens-1 (PDZ) domain interactions often regulate the trafficking and signaling potential of GPCRs, we examined the role of the SST2 PDZ ligand and additional C-terminal residues in controlling its intracellular trafficking. We determined that SST2 can recycle to the plasma membrane via multiple pathways, including a LAMP1/Rab7-positive late endosome to the trans-Golgi network (TGN) pathway. Trafficking from the late endosome to the TGN is often regulated by the retromer complex of endosomal coat proteins, and disrupting the retromer components sorting nexins 1/2 inhibits the budding of SST2 from late endosomes. Moreover, trafficking through the late endosomal/TGN pathway is dependent on an intact PDZ ligand and C-terminal tail, as truncating either the 3 or 10 C-terminal amino acids of SST2 alters the pathway through which it recycles to the plasma membrane. Moreover, addition of these amino acids to a heterologous receptor is sufficient to redirect it from a degradation pathway to a recycling itinerary. Our results demonstrate that endosomal trafficking of SST2 is dependent on numerous regulatory mechanisms controlled by its C terminus and the retromer machinery.

Somatostatin receptor pathophysiology in the neuroendocrine system.

Manuela Albertelli, Marica Arvigo, Mara Boschetti +3 more

The actions of somatostatin (SRIF) are mediated by specific G protein-coupled receptors, named SRIF receptor (SSTR) subtypes 1, 2, 3 and 5. SRIF binding to SSTR activates a series of second messenger systems, resulting in the inhibition of calcium channels and adenylate cyclase activity, ultimately leading to inhibition of hormone secretion, while stimulation of other second messengers, such as phosphotyrosine phosphatases play a role in the control of cell growth. The SSTR and dopamine receptor families share a 30% sequence homology and appear to be structurally related. The knowledge on the pathophysiology of these two families of G protein-coupled receptors in neuroendocrine tumors has progressively increased due to the new insights in receptor dimerization, internalization and trafficking. Depending on the expression of different SSTRs in tissues, their combinations and interactions affect the functionality of the subtypes expressed and the influence of the microenvironment, the response to ligands and, by consequence, the response to treatment can be very different.

Peptide Receptor Radionuclide Therapy With 177Lu-Octreotate in Patients With Somatostatin Receptor Expressing Neuroendocrine Tumors: Six Years' Assessment.

Mohammadali Hamiditabar, Muzammil Ali, Joseph Roys +6 more

Peptide receptor radionuclide therapy (PRRT) with radiolabeled somatostatin analogues is a promising treatment for patients with inoperable, well to moderately differentiated metastatic neuroendocrine tumors (NETs). In continuation of our novel study with the radionuclide lutetium Lu, we now present further results of Lu DOTATATE therapy in managing NETs and other somatostatin receptor-expressing tumors in a larger and more diverse patient group.

In vitro effects of somatostatin on the growth hormone-insulin-like growth factor axis in orange-spotted grouper (Epinephelus coioides).

Bin Wang, Jirong Jia, Guokun Yang +5 more

Growth in vertebrates is mainly mediated by the growth hormone (GH)-insulin-like growth factor (IGF) axis, and somatostatin (SRIF) inhibits growth by decreasing GH release at the pituitary level and antagonizing the release and action of GHRH in the hypothalamus. However, the effects of SRIF on the regulation of growth at levels other than GH release from the pituitary gland are less well known. In the present study, we comprehensively examined the pituitary and peripheral actions of SRIF on the GH-IGF axis in grouper using a primary pituitary and hepatocyte cell culture system. Our results showed that SRIF inhibited GH release at the pituitary level, but had no influence on GH mRNA expression. Basal hepatic GH receptor 1 (GHR1), IGF-I and IGF-II mRNA levels declined over time, whereas GHR2 mRNA levels remained stable throughout the culture period. GH stimulated the hepatic expression of GHR and IGF mRNAs in a dose-dependent manner, while SRIF suppressed both basal and GH-stimulated expression of GHR and IGF mRNAs in primary cultured hepatocytes. The inhibition of GHR and IGF mRNA levels by SRIF was not attributed to the rate of mRNA degradation. To the best of our knowledge, we demonstrated the effects of SRIF on basal and GH-stimulated IGF-II mRNA levels in teleosts for the first time. These results indicate that SRIF regulates growth at the level of the pituitary and peripheral liver.

Somatostatin system: molecular mechanisms regulating anterior pituitary hormones.

Tamar Eigler, Anat Ben-Shlomo

The somatostatin (SRIF) system, which includes the SRIF ligand and receptors, regulates anterior pituitary gland function, mainly inhibiting hormone secretion and to some extent pituitary tumor cell growth. SRIF-14 via its cognate G-protein-coupled receptors (subtypes 1-5) activates multiple cellular signaling pathways including adenylate cyclase/cAMP, MAPK, ion channel-dependent pathways, and others. In addition, recent data have suggested SRIF-independent constitutive SRIF receptor activity responsible for GH and ACTH inhibition in vitro. This review summarizes current knowledge on ligand-dependent and independent SRIF receptor molecular and functional effects on hormone-secreting cells in the anterior pituitary gland.

Reg IV is differently expressed in enteroendocrine cells of human small intestine and colon.

Kukka Heiskala, Leif C Andersson

Reg IV is a 17 kD secreted C-type lectin physiologically found in selected enteroendocrine cells (EEC). It is thought be involved in the regulation of normal and pathological intestinal and/or neuroendocrine differentiation and proliferation but its ultimate functional role(s) is still unclear. We used immunostaining and compared the cellular expression of Reg IV with a panel of neuroendocrine markers in human GI-tract tissue samples. Reg IV showed cellular co-distribution with serotonin and chromogranin A in all parts of GI-tract. Co-localization of Reg IV with somatostatin was seen in colon and with substance P in ileum. Subpopulations of cells expressing Reg IV overlapped with EECs containing GLP-1, GLP-2, secretin, PYY, and ghrelin, depending on the anatomical localization of the samples. The results further underscore the high degree of diversity among EECs and suggest that Reg IV may be involved in the finetuning of functions exerted by the neuroendocrine cells in the GI-tract.

Traditional Chinese medicine formulas for irritable bowel syndrome: from ancient wisdoms to scientific understandings.

Hai-Tao Xiao, Linda Zhong, Siu-Wai Tsang +2 more

Traditional Chinese Medicine (TCM) serves as the most common alternative therapeutic approach for Western medicine and benefits IBS patients globally. Due to the lack of scientific evidence in the past, TCM formulas were not internationally well recognized as promising IBS remedies. In this review, firstly, we present the etiology and therapy of IBS in terms of traditional Chinese medical theory. Secondly, we summarize the clinical randomized controlled trials (RCTs) of TCM formulas for IBS patients that are available in the literature (from 1998 to September 2013), in which 14 RCTs conducted of high quality were discussed in detail. Of the 14 selected trials, 12 of those concluded that TCM formulas provided superior improvement in the global symptoms of IBS patients over the placebo or conventional medicines. As well, all 14 RCTs suggested that TCM formulas have good safety and tolerability. Last but not least, we explore the pharmacological mechanisms of the anti-IBS TCM formulas available in the literature (from 1994 to September, 2013). Collectively, in combating IBS symptoms, most TCM formulas exert multi-targeting actions including the regulation of neurotransmitters and hormones in the enteric nervous system (ENS), modulation of smooth muscle motility in the gastrointestinal (GI) tract, modulation of the hypothalamic-pituitary-adrenal (HPA) axis, attenuation of intestinal inflammation and restoration of intestinal flora, etc. In conclusion, TCM formulas appear to be promising for IBS treatment. This review provides a useful reference for the public in furthering a better understanding and acceptance of TCM formulas as IBS remedies.

Actions of NPY, and its Y1 and Y2 receptors on pulsatile growth hormone secretion during the fed and fasted state.

Lili Huang, Hwee Y Tan, Matthew J Fogarty +5 more

The hypothalamic NPY system plays an important role in regulating food intake and energy expenditure. Different biological actions of NPY are assigned to NPY receptor subtypes. Recent studies demonstrated a close relationship between food intake and growth hormone (GH) secretion; however, the mechanism through which endogenous NPY modulates GH release remains unknown. Moreover, conclusive evidence demonstrating a role for NPY and Y-receptors in regulating the endogenous pulsatile release of GH does not exist. We used genetically modified mice (germline Npy, Y1, and Y2 receptor knock-out mice) to assess pulsatile GH secretion under both fed and fasting conditions. Deletion of NPY did not impact fed GH release; however, it reversed the fasting-induced suppression of pulsatile GH secretion. The recovery of GH secretion was associated with a reduction in hypothalamic somatotropin release inhibiting factor (Srif; somatostatin) mRNA expression. Moreover, observations revealed a differential role for Y1 and Y2 receptors, wherein the postsynaptic Y1 receptor suppresses GH secretion in fasting. In contrast, the presynaptic Y2 receptor maintains normal GH output under long-term ad libitum-fed conditions. These data demonstrate an integrated neural circuit that modulates GH release relative to food intake, and provide essential information to address the differential roles of Y1 and Y2 receptors in regulating the release of GH under fed and fasting states.

Long-term efficacy and safety of pegvisomant in combination with long-acting somatostatin analogs in acromegaly.

S J C M M Neggers, S E Franck, F W M de Rooij +8 more

Treatment for acromegaly patients with long-acting somatotropin release-inhibiting factor (LA-SRIF) often does not result in complete normalization of IGF-1. Addition of pegvisomant (PEGV), a GH receptor antagonist, could improve this; however, the literature has not described long-term follow-up.

Identification of the receptors for somatostatin (SST) and cortistatin (CST) in chickens and investigation of the roles of cSST28, cSST14, and cCST14 in inhibiting cGHRH1-27NH2-induced growth hormone secretion in cultured chicken pituitary cells.

Fengyan Meng, Guian Huang, Shunyu Gao +3 more

Somatostatin receptors (SSTRs) are proposed to mediate the actions of somatostatin (SST) and its related peptide, cortistatin (CST), in vertebrates. However, the identity, functionality, and tissue expression of these receptors remain largely unknown in most non-mammalian vertebrates including birds. In this study, five SSTRs (named cSSTR1, cSSTR2, cSSTR3, cSSTR4, cSSTR5) were cloned from chicken brain by RT-PCR. Using a pGL3-CRE-luciferase reporter system, we demonstrated that activation of each cSSTR expressed in CHO cells by cSST28, cSST14 and cCST14 treatment could inhibit forskolin-induced luciferase activity of CHO cells, indicating the functional coupling of all cSSTRs to Gi protein(s). Interestingly, cSSTR1-4 expressed in CHO cells could be activated by cSST28, cSST14 and cCST14 with high potencies, suggesting that they may function as the receptors common for these peptides. In contrast, cSSTR5 could be potently activated by cSST28 only, indicating that it is a cSST28-specific receptor. Using RT-PCR, wide expression of cSSTRs was detected in chicken tissues including pituitary. In accordance with their expression in pituitary, cSST28, cSST14, and cCST14 were demonstrated to inhibit basal and novel cGHRH1-27NH2-induced GH secretion in cultured chicken pituitary cells dose-dependently (0-10nM) by Western blot analysis, suggesting the involvement of cSSTR(s) common for these peptides in mediating their inhibitory actions. Collectively, our study establishes a molecular basis to elucidate the roles of SST/CST in birds and provide insights into the roles of SST/CST in vertebrates, such as their conserved actions on pituitary.

Differential involvement of signaling pathways in the regulation of growth hormone release by somatostatin and growth hormone-releasing hormone in orange-spotted grouper (Epinephelus coioides).

Bin Wang, Chaobin Qin, Cong Zhang +3 more

Somatostatin is the most effective inhibitor of GH release, and GHRH was recently identified as one of the primary GH-releasing factors in teleosts. In this study, we analyzed the possible intracellular transduction pathways that are involved in the mechanisms induced by SRIF and GHRH to regulate GH release. Using a pharmacological approach, the blockade of the PLC/IP/PKC pathway reversed the SRIF-induced inhibition of GH release but did not affect the GHRH-induced stimulation of GH release. Furthermore, SRIF reduced the GH release induced by two PKC activators. Inhibitors of the AC/cAMP/PKA pathway reversed both the SRIF- and GHRH-induced effects on GH release. Moreover, the GH release evoked by forskolin and 8-Br-cAMP were completely abolished by SRIF. The blockade of the NOS/NO pathway attenuated the GHRH-induced GH release but had minimal effects on the inhibitory actions of SRIF. In addition, inhibitors of the sGC/cGMP pathway did not modify the SRIF- or GHRH-induced regulation of GH release. Taken together, these findings indicate that the SRIF-induced inhibition of GH release is mediated by both the PLC/IP/PKC and the AC/cAMP/PKA pathways and not by the NOS/NO/sGC/cGMP pathway. In contrast, the GHRH-induced stimulation of GH secretion is mediated by both the AC/cAMP/PKA and the NOS/NO pathways and is independent of the sGC/cGMP pathway and the PLC/IP/PKC system.

A multi-institutional, phase II open-label study of ganitumab (AMG 479) in advanced carcinoid and pancreatic neuroendocrine tumors.

J R Strosberg, J A Chan, D P Ryan +9 more

The IGF pathway has been implicated in the regulation of neuroendocrine tumor (NET) growth, and preliminary studies suggested that ganitumab (AMG 479), a human MAB against IGF1R, may have antitumor activity in this setting. We performed a two-cohort phase II study of ganitumab in patients with metastatic progressive carcinoid or pancreatic NETs (pNETs). This open-label study enrolled patients (≥18 years) with metastatic low- and intermediate-grade carcinoid or pNETs. Inclusion criteria included evidence of progressive disease (by Response Evaluation Criteria in Solid Tumors (RECIST)) within 12 months of enrollment, ECOG PS 0-2, and fasting blood sugar <160  mg/dl. Prior treatments were allowed and concurrent somatostatin analog therapy was permitted. The primary endpoint was objective response. Secondary endpoints included overall survival (OS), progression-free survival (PFS), and safety. Sixty patients (30 carcinoid and 30 pNETs) were treated with ganitumab 18  mg/kg every 3 weeks, among whom 54 patients were evaluable for survival and 53 patients for response. There were no objective responders by RECIST. The median PFS duration was 6.3 months (95% CI, 4.2-12.6) for the entire cohort; 10.5 months for carcinoid patients, and 4.2 months for pNET patients. The OS rate at 12 months was 66% (95% CI, 52-77%) for the entire cohort. The median OS has not been reached. Grade 3/4 AEs were rare and consisted of hyperglycemia (4%), neutropenia (4%), thrombocytopenia (4%), and infusion reaction (1%). Although well tolerated, treatment with single-agent ganitumab failed to result in significant tumor responses among patients with metastatic well-differentiated carcinoid or pNET.

Somatostatin, somatostatin analogs and somatostatin receptor dynamics in the biology of cancer progression.

M Ruscica, M Arvigo, L Steffani +2 more

The pharmacological effects (i.e., inhibition of endocrine secretion and cell proliferation) mediated by the hormone somatostatin (SRIF) are derived from its universal high-affinity binding to five different G proteincoupled receptors (GPCRs), named sst1-5. However, SRIF has a half-life of less than 3 min, whereas the available mono- and bi-specific SRIF preferential analogs show prolonged half-life and increased potency. These compounds may control tumor development, cell proliferation and metastatization by direct actions, including cell division arrest in G0/G1 phase (i.e., induction of cyclin-dependent kinase inhibitor p27(kip1) or p21(Cip1)), induction of apoptosis (i.e., induction of p53 and Bax) and suppression of cell invasion. Along with these direct actions on the biology of cancer progression, in vivo SRIF analogs may also regulate tumor growth through indirect actions, by suppressing the secretion of growth-promoting hormones and growth factors and angiogenesis. Interestingly, when ssts are co-expressed, they may interact forming homo- or heterodimers, also with other GPCRs such as type 2 dopamine receptor and the μ-opioid receptor 1, altering their original pharmacological and functional properties. Dimers can be not only constitutive, but perhaps also ligandpromoted: hence, compounds with high affinity for different ssts isoforms may be used to achieve effects elicited by specific dimers. Future developments in the knowledge of ssts dynamics upon SRIF and SRIF analogs binding in neoplastic tissues may allow the full elucidation of the pathophysiological role of this system and the exploitation of the therapeutic potential of its modulation.

Somatostatin analogs and chimeric somatostatin-dopamine molecules differentially regulate human growth hormone and prolactin gene expression and secretion in vitro.

Anna Gruszka, Michael D Culler, Shlomo Melmed

We tested effects of selective somatostatin receptor 2 (SST2) agonist BIM-23120, SST5 agonist BIM-23206 and chimeric somatostatin-dopamine molecules (SRIF/DA) BIM-23A760 and BIM-23A761 on GH and PRL secretion and gene expression in human GH/PRL-secreting pituitary tumors in vitro. In "responders" group BIM-23120 suppressed GH levels by 26±4%, BIM-23206 by 31±5%, BIM-23A760 by 23±4%, BIM-23A761 by 39±8% and D(2)-dopamine agonist BIM-53097 by 31±5%. Using real-time PCR we demonstrated that GH inhibition was not accompanied by decreased GH mRNA levels. PRL secretion was inhibited by BIM-23A760 (29±5%), BIM-23A761 (34±4%), BIM-23206 (26±4%) and BIM-53097 (36±2%). SRIF/DA and BIM-53097 also suppressed PRL mRNA levels. Concluding, SST2 and SST5 agonists and SRIF/DA inhibit GH secretion, but do not suppress GH gene transcription. SRIF/DA and BIM-53097 inhibit both PRL secretion and PRL gene expression. SST5 agonist inhibits PRL secretion, but does not suppress PRL gene expression. D(2) affinity is crucial in SRIF/DA action on PRL gene expression.

Influence of membrane ion channel in pituitary somatotrophs by hypothalamic regulators.

Seung-Kwon Yang, Frederik Steyn, Chen Chen

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.

Nanobiology and physiology of growth hormone secretion.

Lloyd L Anderson, Colin G Scanes

Growth hormone (GH) secretion is controlled by hypothalamic releasing hormones from the median eminence together with hormones and neuropeptides produced by peripheral organs. Secretion of GH involves movement of secretory vesicles along microtubules, transient 'docking' with the porosome in the cell membrane and subsequent release of GH. Release of GH is stimulated by GH releasing hormone (GHRH) and inhibited by somatostatin (SRIF). Ghrelin may be functioning to stimulate GH release from somatotropes acting via the GH secretagogue (GHS) receptor (GHSR). However, recent physiological studies militate against this. In addition, ghrelin does influence GH release acting within the hypothalamus. Release of GH from the somatotropes involves the GH-containing secretory granules moving close to the cell surface followed by transitory fusion of the secretory granules with the porosomes located in multiple secretory pits in the cell membrane. Other peptides/proteins can influence GH secretion, particularly in species of non-mammalian vertebrates.

Effect of SOM230 (pasireotide) on corticotropic cells: action in dogs with Cushing's disease.

Victor Castillo, Marily Theodoropoulou, Johanna Stalla +7 more

SOM230 (pasireotide) is a multiligand somatostatin (SRIF) analog able to bind to somatostatin receptor (SSTR) subtypes 1, 2, 3 and 5, and trigger antisecretory and antiproliferative signaling cascades. Canines have become in vivo models to test the pharmacological treatment of corticotropinomas because they frequently develop Cushing's disease in a spontaneous manner, due to adrenocorticotropic hormone (ACTH)-producing pituitary adenomas. Different levels of expression of SSTR2 and SSTR5 have been shown in both mouse AtT20 cells and canine tumoral corticotropinoma cells. The objective of this study was to evaluate whether SOM230 controls both tumor cell growth and hormone synthesis, therefore controlling the disease. SOM230 was tested in dogs suffering from Cushing's disease (10 animals were treated continuously during 6 months, and another 10 were treated with 3 cycles consisting of 2 months of treatment followed by a 2-month rest period). A significant decrease in ACTH, urinary cortisol creatinine ratio, adenoma size (magnetic nuclear resonance) and improvement of clinical signs were obtained, without side effects. AtT20 cells treated with SOM230 suppressed pro-opiomelanocortin (POMC) promoter activity through SSTR2, via the G(i) α-subunit, and reduced Nur77/Nurr1 transcriptional activity. We conclude that SOM230, in addition to its well-described antisecretory effects, inhibits, as shown in AtT20 cells, ACTH synthesis at the POMC transcriptional level, an effect mediated mainly through SSTR2, and limits tumor growth. The controlled Cushing's disease in the dogs that received the treatment indicates that SOM230 has a potential therapeutic use in humans suffering from Cushing's disease.

The β isoenzyme of Ca(2+)/calmodulin-dependent kinase type II as possible mediator of somatostatin functions in pituitary tumour cells.

Davide Cervia

Somatostatin or somatostatin release inhibiting factor (SRIF) analogues are indicated for the treatment of somatotropinomas that hypersecrete growth hormone (GH). Indeed, SRIF inhibits intracellular Ca(2+) concentration ([Ca(2+)](i)), thus allowing the inhibition of GH secretion. In the present study, our hypothesis was that Ca(2+)/calmodulin-dependent kinase type II (CaMKII), a multifunctional serine/threonine protein kinase, is part of those signalling mechanisms mediating SRIF functions. All four CaMKII isoenzymes (termed α, β, γ and δ) are expressed in rat somatotroph GC cells, although only CaMKIIβ is inhibited by SRIF at both mRNA and protein levels. Similarly to SRIF, the specific knockdown of CaMKIIβ by RNA interference induces a decrease of [Ca(2+)](i). The effects of SRIF and those of CaMKIIβ knockdown are non-additive. These results are confirmed by the pharmacological blockade of CAMKII. We also observed that, similarly to SRIF, the specific knockdown of CaMKIIβ induces a decrease of both GH content/secretion. These results raise the hypothesis that CaMKIIβ may mediate, at least in part, the SRIF-induced control of [Ca(2+)](i). In addition, CaMKIIβ seems to play a positive role in maintaining the exocytosis of GH. Our data provide a framework for better elucidating the pathophysiological role of SRIF transduction network in somatotropinomas.

Differential temporal and spatial regulation of somatostatin receptor phosphorylation and dephosphorylation.

Madhumita Ghosh, Agnes Schonbrunn

The G(i)-coupled somatostatin 2A receptor (sst2A) mediates many of the neuromodulatory and neuroendocrine actions of somatostatin (SS) and is targeted by the SS analogs used to treat neuroendocrine tumors. As for other G protein-coupled receptors, agonists stimulate sst2A receptor phosphorylation on multiple residues, and phosphorylation at different sites has distinct effects on receptor internalization and uncoupling. To elucidate the spatial and temporal regulation of sst2A receptor phosphorylation, we examined agonist-stimulated phosphorylation of multiple receptor GPCR kinase sites using phospho-site-specific antibodies. SS increased receptor phosphorylation sequentially, first on Ser-341/343 and then on Thr-353/354, followed by receptor internalization. Reversal of receptor phosphorylation was determined by the duration of prior agonist exposure. In acutely stimulated cells, in which most receptors remained on the cell surface, dephosphorylation occurred only on Thr-353/354. In contrast, both Ser-341/343 and Thr-353/354 were rapidly dephosphorylated when cells were stimulated long enough to allow receptor internalization before agonist removal. Consistent with these observations, dephosphorylation of Thr-353/354 was not affected by either hypertonic sucrose or dynasore, which prevent receptor internalization, whereas dephosphorylation of Ser-341/343 was completely blocked. An okadaic acid- and fostriecin-sensitive phosphatase catalyzed the dephosphorylation of Thr-353/354 both intracellularly and at the cell surface. In contrast, dephosphorylation of Ser-341/343 was insensitive to these inhibitors. Our results show that the phosphorylation and dephosphorylation of neighboring GPCR kinase sites in the sst2A receptor are subject to differential spatial and temporal regulation. Thus, the pattern of receptor phosphorylation is determined by the duration of agonist stimulation and compartment-specific enzymatic activity.

Real-time monitoring of somatostatin receptor-cAMP signaling in live pituitary.

Stefan Jacobs, Davide Calebiro, Viacheslav O Nikolaev +2 more

Fluorescence resonance energy transfer using genetically encoded biosensors has proven to be a powerful technique to monitor the spatiotemporal dynamics of cAMP signals stimulated by G(s)-coupled receptors in living cells. In contrast, real-time imaging of G(i)-mediated cAMP signals under native conditions remains challenging. Here, we describe the use of transgenic mice for cAMP imaging in living pituitary slices and primary pituitary cells. This technique can be widely used to assess the contribution of various pituitary receptors, including individual G(i) protein-coupled somatostatin receptors, to the regulation of cAMP levels under physiologically relevant settings.

Electrophysiological effect of ghrelin and somatostatin on rat hypothalamic arcuate neurons in vitro.

Kyohei Mori, Juhyon Kim, Kazuo Sasaki

Growth hormone (GH) secretion from the pituitary gland is partly regulated by GH releasing hormone (GHRH)-containing neurons located in the hypothalamic arcuate nucleus (ARC). GHRH-containing neurons express the GH secretagogue (GHS) receptor (GHS-R) and the somatostatin (SRIF) receptor. Recently, an endogenous ligand for the GHS-R named ghrelin was found. Therefore, it seems that both ghrelin and SRIF are involved in the hypothalamic regulation of GH release via GHRH-containing neurons in the ARC. In extracellular single unit recordings from in vitro hypothalamic slice preparations from rats, application of 100 nM ghrelin substantially excited ARC neurons (82.5%), whereas 1 microM SRIF substantially inhibited them (81.8%). The ghrelin-induced excitatory and SRIF-induced inhibitory effects on ARC neurons were dose-dependent and persisted during synaptic blockade using low-Ca(2+)/high-Mg(2+) solution. In addition, the effects were antagonized by [D-Lys(3)]-GHRP-6, a GHS-R antagonist, and CYN154806, a SRIF receptor subtype sst2 antagonist, respectively. When ghrelin and SRIF were sequentially applied to ARC neurons, 95.2% were excited by ghrelin and inhibited by SRIF. Similarly, 85.0% of ARC neuroendocrine cells that project to the median eminence were excited by ghrelin and inhibited by SRIF. These results indicate that ARC neuroendocrine cells projecting to the median eminence are dose-dependently, postsynaptically and oppositely regulated by ghrelin through GHS-R and SRIF via the SRIF sst2 receptor subtype. Our results also suggest that most of these ARC neuroendocrine cells are presumably GHRH-containing neurons and are involved in the cellular processes through which ghrelin and SRIF participate in the hypothalamic regulation of GH release.

Pituitary somatostatin receptor signaling.

Anat Ben-Shlomo, Shlomo Melmed

Somatotropin-release inhibitory factor (SRIF) is a major regulator of pituitary function, mostly inhibiting hormone secretion and to a lesser extent pituitary cell growth. Five SRIF receptor subtypes (SSTR1-5) are ubiquitously expressed G-protein coupled receptors. In the pituitary, SSTR1, 2, 3 and 5 are expressed, with SSTR2 and SSTR5 predominating. As new SRIF analogs have recently been introduced for treatment of pituitary disease, we evaluate the current knowledge of cell-specific pituitary SRIF receptor signaling and highlight areas of future research for comprehensive understanding of these mechanisms. Elucidating pituitary SRIF receptor signaling enables understanding of pituitary hormone secretion and cell growth, and also encourages future therapeutic development for pituitary disorders.

Metabolic regulation of ghrelin O-acyl transferase (GOAT) expression in the mouse hypothalamus, pituitary, and stomach.

Manuel D Gahete, Jose Córdoba-Chacón, Roberto Salvatori +3 more

Ghrelin acts as an endocrine link connecting physiological processes regulating food intake, body composition, growth, and energy balance. Ghrelin is the only peptide known to undergo octanoylation. The enzyme mediating this process, ghrelin O-acyltransferase (GOAT), is expressed in the gastrointestinal tract (GI; primary source of circulating ghrelin) as well as other tissues. The present study demonstrates that stomach GOAT mRNA levels correlate with circulating acylated-ghrelin levels in fasted and diet-induced obese mice. In addition, GOAT was found to be expressed in both the pituitary and hypothalamus (two target tissues of ghrelin's actions), and regulated in response to metabolic status. Using primary pituitary cell cultures as a model system to study the regulation of GOAT expression, we found that acylated-ghrelin, but not desacyl-ghrelin, increased GOAT expression. In addition, growth-hormone-releasing hormone (GHRH) and leptin increased, while somatostatin (SST) decreased GOAT expression. The physiologic relevance of these later results is supported by the observation that pituitary GOAT expression in mice lacking GHRH, SST and leptin showed opposite changes to those observed after in vitro treatment with the corresponding peptides. Therefore, it seems plausible that these hormones directly contribute to the regulation of pituitary GOAT. Interestingly, in all the models studied, pituitary GOAT expression paralleled changes in the expression of a dominant spliced-variant of ghrelin (In2-ghrelin) and therefore this transcript may be a primary substrate for pituitary GOAT. Collectively, these observations support the notion that the GI tract is not the only source of acylated-ghrelin, but in fact locally produced des-acylated-ghrelin could be converted to acylated-ghrelin within target tissues by locally active GOAT, to mediate its tissue-specific effects.