SRIF Receptor Research Articles

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

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.

A nitric oxide synthase inhibitor, L-NAME, prevents L-arginine-induced downregulation of the rat cortical somatostatinergic system.

Activation of NMDA receptors leads to nitric oxide (NO) synthesis by NO synthase (NOS) from L-arginine. Neuronal NOS colocalizes with somatostatinergic (SRIF) neurons and there is growing evidence of an interaction between NO and the cerebral SRIFergic system in several neurological diseases. Our aim was to study the effect of L-arginine on the regulation of the SRIFergic system in the frontoparietal cortex of male Sprague-Dawley rats. Intraperitoneal administration of L-arginine (150 mg/Kg), twice-daily during eight days, induced a decrease in SRIF receptor density, which was accompanied by a reduction in the capacity of SRIF to stimulate inositol-1,4,5-triphosphate (IP3) accumulation and SRIF-like immunoreactivity (SRIF-LI) levels. To determine if these changes were related to L-arginine-derived NO synthesis, a NOS inhibitor, Nω-nitro-L-arginine methyl ester was coadministered with L-arginine. Its coadministration prevented the reduction in the SRIF receptor density, accumulation of IP3 and SRIF-LI content. These findings indicate that L-arginine induces a deleterious effect on the cortical somatostatinergic system and that the inhibition of NOS could be helpful in some neurological disorders where this neurotransmitter system is affected.

The Protective Effects of IGF-I against β-Amyloid-related Downregulation of Hippocampal Somatostatinergic System Involve Activation of Akt and Protein Kinase A

Somatostatin (SRIF), a neuropeptide highly distributed in the hippocampus and involved in learning and memory, is markedly reduced in the brain of Alzheimer’s disease patients. The effects of insulin-like growth factor-I (IGF-I) against β amyloid (Aβ)-induced neuronal death and associated cognitive disorders have been extensively reported in experimental models of this disease. Here, we examined the effect of IGF-I on the hippocampal somatostatinergic system in Aβ-treated rats and the molecular mechanisms associated with changes in this peptidergic system. Intracerebroventricular Aβ25-35 administration during 14 days (300 pmol/day) to male rats increased Aβ25-35 levels and cell death and markedly reduced SRIF and SRIF receptor 2 levels in the hippocampus. These deleterious effects were associated with reduced Akt and cAMP response element-binding protein (CREB) phosphorylation and activation of c-Jun N-terminal kinase (JNK). Subcutaneous IGF-I co-administration (50 µg/kg/day) reduced hippocampal Aβ25-35 levels, cell death and JNK activation. In addition, IGF-I prevented the reduction in the components of the somatostatinergic system affected by Aβ infusion. Its co-administration also augmented protein kinase A (PKA) activity, as well as Akt and CREB phosphorylation. These results suggest that IGF-I co-administration may have protective effects on the hippocampal somatostatinergic system against Aβ insult through up-regulation of PKA activity and Akt and CREB phosphorylation.

Somatostatin system: molecular mechanisms regulating anterior pituitary hormones.

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.

Somatostatin Receptors Type 2 and 5 Expression and Localization During Human Pituitary Development

Somatostatin (SRIF), by acting mainly through sst2 and sst5 receptors, is a potent inhibitor of hormonal secretion by the human anterior pituitary gland. However, the pattern of protein expression of these SRIF receptors remains unknown during pituitary development. To get further insights into the physiological role of SRIF receptors in human development and pituitary function, the present study examined the developmental expression of the sst2 and sst5 receptors in the individual cell types of the anterior human pituitary. Thirteen fetal human pituitaries were investigated between 13 to 38 weeks of gestation (WG) by double-labeling immunofluorescence with antibodies raised against sst2 or sst5 receptors and GH, LH, FSH, TSH, or pro-opiomelanocortin proteins. SRIF immunoreactivity in the hypothalamus and median eminence was investigated at the same developmental ages. Immunoreactivity for the sst2 receptor was evident as early as 13 to 15 WG and onward mainly in TSH-, LH-, and FSH-expressing cells, whereas sst5 immunoreactivity was apparent at the late development stages (35-38 WG). GH-expressing cells mainly expressed sst5 immunoreactivity. SRIF-positive fibers and cells were detected as soon as 13 to 16 WG in the hypothalamus and median eminence and their densities increased with gestational age. The early appearance of hypothalamic SRIF cells and fibers suggests a physiological link between SRIF and its receptors during pituitary development. Whereas sst2 receptors might play a primary role in the differentiation and regulation of TSH, LH, and FSH cells, sst5 receptors appear to be mainly involved in GH regulation from birth onward.

Acute up-regulation of the rat brain somatostatin receptor-effector system by leptin is related to activation of insulin signaling and may counteract central leptin actions

Leptin and somatostatin (SRIF) have opposite effects on food seeking and ingestive behaviors, functions partially regulated by the frontoparietal cortex and hippocampus. Although it is known that the acute suppression of food intake mediated by leptin decreases with time, the counter-regulatory mechanisms remain unclear. Our aims were to analyze the effect of acute central leptin infusion on the SRIF receptor-effector system in these areas and the implication of related intracellular signaling mechanisms in this response. We studied 20 adult male Wister rats including controls and those treated intracerebroventricularly with a single dose of 5μg of leptin and sacrificed 1 or 6h later. Density of SRIF receptors was unchanged at 1h, whereas leptin increased the density of SRIF receptors at 6h, which was correlated with an elevated capacity of SRIF to inhibit forskolin-stimulated adenylyl cyclase activity in both areas. The functional capacity of SRIF receptors was unaltered as cell membrane levels of αi1 and αi2 subunits of G inhibitory proteins were unaffected in both brain areas. The increased density of SRIF receptors was due to enhanced SRIF receptor subtype 2 (sst2) protein levels that correlated with higher mRNA levels for this receptor. These changes in sst2 mRNA levels were concomitant with increased activation of the insulin signaling, c-Jun and cyclic AMP response element-binding protein (CREB); however, activation of signal transducer and activator of transcription 3 was reduced in the cortex and unchanged in the hippocampus and suppressor of cytokine signaling 3 remained unchanged in these areas. In addition, the leptin antagonist L39A/D40A/F41A blocked the leptin-induced changes in SRIF receptors, leptin signaling and CREB activation. In conclusion, increased activation of insulin signaling after leptin infusion is related to acute up-regulation of the SRIF receptor-effector system that may antagonize short-term leptin actions in the rat brain.

Folate Receptor-Mediated Drug Targeting: A Possible Strategy for Nonfunctioning Pituitary Adenomas?

Clinically nonfunctioning pituitary adenomas (NFPAs) mostly arise from gonadotroph cells and account for about one-third of all pituitary tumors. Because they are not associated with hormone hypersecretion, these tumors are often diagnosed late when they are large and locally invasive. Consequently, the complete surgical removal of NFPAs is hard to achieve and they tend to recur. Medical treatment of NFPAs is still challenging because they are usually resistant to mainstay peptide receptor therapy with somatostatin (SRIF) analogs (octreotide and lanreotide) or dopamine agonists (bromocriptine and cabergoline). Indeed, although these agents may be useful in some cases to prevent residual tumor growth (1), their efficacy has not been confirmed in larger studies. Recently, SOM230 (pasireotide), a broad-range SRIF analog with affinity for SRIF receptor subtypes 1 to 3 and 5 (2) and BIM-23A760 (dopastatin), a chimeric agent interacting with both SRIF receptor and dopamine receptors were developed (3). However, NFPAs only partially respond to these compounds, at least in vitro (4, 5). After the discovery that NFPAs display overactivation of phosphatidylinositol 3-kinase/protein kinase B (AKT)/mammalian target of rapamycin pathway (6, 7), compounds inhibiting this signaling cascade at various levels have been tested against these tumors. The results are very promising, especially for dual inhibitors such as BEZ235 and XL765, which inhibit both mammalian target of rapamycin and the upstream phosphatidylinositol 3-kinase and exert a potent antiproliferative activity against primary human NFPA cells (8), rodent pituitary cell lines (9), and primary rat gonadotroph adenoma cells in vitro (10). In vivo studies are now required to prove or disprove the efficacy of these agents. Conventional chemotherapy has been one of the major medical advances of the last decades, but it is usually associated with poor specificity for the cancer cells and high toxicity to the normal cells. To overcome these limitations, it would be ideal to identify ways to deliver a biologically effective concentration of anticancer agents to the tumor tissues with very high specificity. To reach this ultimate goal, extensive efforts have been undertaken to develop tumor-selective drugs by conjugating therapeutic agents to hormones, antibodies, and vitamin derivatives. Among them, folic acid holds great promise as a tumor-homing agent (11). Folate (or folic acid) is an essential B vitamin, which plays a pivotal role in cell survival by participating in the biosynthesis of nucleic and amino acids. Folate is internalized into the cells via a low-affinity reduced folate carrier protein or via high-affinity folate receptors. The best studied isoform of these receptors is folate receptor(FR ), a cell surface glycosyl phosphatidylinositol-anchored glycoprotein that can internalize bound folates and folate-conjugated compounds via receptor-mediated endocytosis (12). Whereas FR has a very restricted expression pattern in normal tissues, it is expressed at a high level in various human cancers, particularly epithelial carcinomas, including nonmucinous ovarian carcinoma, cervical carcinomas, and testicular choriocarcinomas, and less frequently in breast, endometrial colon, and renal cell carcinomas (13, 14). Due to its selective expression in tumor but not normal cells, FR has become one of the most investigated cellular surface antigens for targeted delivery of a variety of molecules, including imaging agents, chemotherapeutic agents, oligodeoxynucleotides, and macromolecules. Various types of drug carriers have been conjugated to folate such as liposomes, lipid nanoparticles, polymeric nanoparticles, polymers, and micelles filled with the molecule that needs to be delivered (11). FR -targeted liposomal

Somatostatin receptor pathophysiology in the neuroendocrine system

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.

Antiangiogenic role of miR-361 in human umbilical vein endothelial cells: functional interaction with the peptide somatostatin

Somatostatin (SRIF) acts as antiangiogenic factor, but its role in the regulation of microRNAs (miRNAs) targeting proangiogenic factors is unknown. We used human umbilical vein endothelial cells (HUVEC) to investigate whether (1) miRNAs targeting proangiogenic factors are influenced by hypoxia, (2) their expression is regulated by SRIF, and (3) SRIF-regulated miRNAs affect HUVEC angiogenic phenotype. The involvement of signal transducer and activator of transcription (STAT) 3 and hypoxia inducible factor (HIF)-1 in miRNA effects was studied. Quantitative real-time PCR, Western blot, cell proliferation assays, and enzyme-linked immunosorbent assay (ELISA) were used. Using specific algorithms, three miRNAs (miR-17, miR-18b, and miR-361) were predicted to bind angiogenesis-associated factors including STAT3, HIF-1α, and vascular endothelial growth factor (VEGF). Hypoxia downregulates miR-17 and miR-361 without affecting miR-18b. SRIF restored decreased levels of miR-361 acting at the SRIF receptor sst(1). Downregulated miR-361 was also restored by HIF-1α inhibition with YC-1. Combined application of SRIF did not influence YC-1-induced miR-361 downregulation, suggesting that YC-1 and SRIF modulate miR-361 through a common mechanism involving HIF-1α. This possibility was confirmed by the result that HIF-1α activation in normoxia-downregulated miR-361 and that this downregulation was prevented by SRIF. miR-361 overexpression reduced hypoxia-induced cell proliferation and VEGF release indicating miR-361 involvement in the acquisition of an angiogenic phenotype by HUVEC. miR-361 effects on VEGF were enhanced by the coadministration of SRIF. Our results suggest that (1) SRIF regulates miR-361 expression through a control on HIF-1, (2) miR-361 affects HUVEC angiogenic phenotype, and (3) SRIF and miR-361 act cooperatively in limiting hypoxia-induced VEGF release.

Leptin-induced downregulation of the rat hippocampal somatostatinergic system may potentiate its anorexigenic effects

The learning and memory mechanisms in the hippocampus translate hormonal signals of energy balance into behavioral outcomes involved in the regulation of food intake. As leptin and its receptors are expressed in the hippocampus and somatostatin (SRIF), an orexigenic neuropeptide, may inhibit leptin-mediated suppression of food intake in other brain areas, we asked whether chronic leptin infusion induces changes in the hippocampal somatostatinergic system and whether these modifications are involved in leptin-mediated effects. We studied 18 male Wistar rats divided into three groups: controls (C), treated intracerebroventricularly (icv) with leptin (12μg/day) for 14days (L) and a pair-fed group (PF) that received the same amount of food consumed by the L group. Food restriction increased whereas leptin decreased the hippocampal SRIF receptor density, due to changes in SRIF receptor 2 protein levels. These changes in the PF group were concurrent with an increase of hippocampal G protein-coupled receptor kinase 2 protein levels and activation of Akt and cyclic AMP response element binding protein. The inhibitory effect of SRIF on adenylyl cyclase (AC) activity, however, was decreased in L rats, coincident with lower G inhibitory α3 and higher AC-I levels as well as signal transducer and activator of transcription factor 3 activation. In addition, 20 male Wistar rats were included to analyze whether the leptin antagonist L39A/D40A/F41A and the SRIF receptor agonist SMS 201–995 modify SRIF signaling and food intake, respectively. Administration of L39A/D40A/F41A reversed changes in SRIF signaling, whereas SMS 201–995 ameliorated food consumption in L. Altogether, these results suggest that increased somatostatinergic tone in PF rats may be a mechanism to improve the hippocampal orexigenic effects in a situation of metabolic demand, whereas down-regulation of this system in L rats may represent a mechanism to enhance the anorexigenic effects of leptin.

Molecular mechanisms of somatostatin receptor trafficking

The neuropeptide somatostatin (SRIF) is an important modulator of neurotransmission in the central nervous system and acts as a potent inhibitor of hormone and exocrine secretion. In addition, SRIF regulates cell proliferation in normal and tumorous tissues. The six somatostatin receptor subtypes (sst1, sst2A, sst2B, sst3, sst4, and sst5), which belong to the G protein-coupled receptor (GPCR) family, share a common molecular topology: a hydrophobic core of seven transmembrane-spanning α-helices, three intracellular loops, three extracellular loops, an amino-terminus outside the cell, and a carboxyl-terminus inside the cell. For most of the GPCRs, intracytosolic sequences, and more particularly the C-terminus, are believed to interact with proteins that are mandatory for either exporting neosynthesized receptor, anchoring receptor at the plasma membrane, internalization, recycling, or degradation after ligand binding. Accordingly, most of the SRIF receptors can traffic not only in vitro within different cell types but also in vivo. A picture of the pathways and proteins involved in these processes is beginning to emerge.

Neuropeptidomics of mouse hypothalamus after imipramine treatment reveal somatostatin as a potential mediator of antidepressant effects

Excessive activation of the hypothalamic–pituitary–adrenal (HPA) axis has been associated with numerous diseases, including depression, and the tricyclic antidepressant imipramine has been shown to suppress activity of the HPA axis. Central hypothalamic control of the HPA axis is complex and involves a number of neuropeptides released from multiple hypothalamic subnuclei. The present study was therefore designed to determine the effects of imipramine administration on the mouse hypothalamus using a peptidomics approach. Among the factors found to be downregulated after acute (one day) or chronic (21 days) imipramine administration were peptides derived from secretogranin 1 (chromogranin B) as well as peptides derived from cerebellin precursors. In contrast, peptides SRIF-14 and SRIF-28 (1–11) derived from somatostatin (SRIF, somatotropin release inhibiting factor) were significantly upregulated by imipramine in the hypothalamus. Because diminished SRIF levels have long been known to occur in depression, a second part of the study investigated the roles of individual SRIF receptors in mediating potential antidepressant effects. SRA880, an antagonist of the somatostatin-1 autoreceptor (sst1) which positively modulates release of endogenous SRIF, was found to synergize with imipramine in causing antidepressant-like effects in the tail suspension test. Furthermore, chronic co-administration of SRA880 and imipramine synergistically increased BDNF mRNA expression in the cerebral cortex. Application of SRIF or L054264, an sst2 receptor agonist, but not L803807, an sst4 receptor agonist, increased phosphorylation of CaMKII and GluR1 in cerebrocortical slices. Our present experiments thus provide evidence for antidepressant-induced upregulation of SRIF in the brain, and strengthen the notion that augmented SRIF expression and signaling may counter depressive-like symptoms.This article is part of a Special Issue entitled ‘Anxiety and Depression’.

Immunohistochemical localization and quantitative expression of somatostatin receptors in normal human spleen and thymus: Implications for the in vivo visualization during somatostatin receptor scintigraphy.

[111In-DTPA-D-Phe1]-octreotide scintigraphy allows the visualization of SRIF receptor (SSR)-expressing tumors, including thymic tumors, and normal tissues. While the spleen is clearly visualized, the thymus is not depicted, although both contain SSR. We evaluated whether the heterogeneity, the type, and the amount of SSR might explain this contrasting finding. By ligand-binding the number of [125I-Tyr11]-SRIF- 14 binding sites resulted comparable between the two tissues, whereas the number of [125I-Tyr3]-octreotide sites was significantly higher in the spleen (p<0.001). Quantitative RTPCR showed a significantly higher expression of sst2A mRNA in the spleen, whereas a significantly higher expression of SRIF and sst3 in the thymus. The highest density of sst2A in the spleen is in line with the in vivo uptake of [111In-DTPA-D-Phe1]- octreotide, which is considered a sst2-preferring ligand. The specificity is confirmed by the evidence that in vivo [111In-DTPA- D-Phe1]-octreotide uptake can be abolished during chronic administration of "cold" octreotide. Immunohistochemistry confirmed a preferential expression of sst2A on microenvironmental cells and of sst3 on lymphoid cells. The heterogeneity of SSR expression and the higher SRIF content explain the lack of thymus visualization during scintigraphy, whereas thymic tumors, which do not express SRIF, are visualized. Apart from the affinity of the radioligand, also the efficacy of the internalization is crucial for the in vivo uptake, and both heterogeneity and SRIF content affect this process. These observations might have an important impact when interpretating in vivo visualization of SSR-positive lesions, and when treatment with novel SRIF analogs is considered.

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

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 μM 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.

Expression, Localization, and Functional Coupling of the Somatostatin Receptor Subtype 2 in a Mouse Model of Oxygen-Induced Retinopathy

Purpose. In the mouse model of oxygen-induced retinopathy (OIR), somatostatin-14 (SRIF) acting at the SRIF receptor subtype 2 (sst(2)) inhibits angiogenic responses to hypoxia through a downregulation of vascular endothelial growth factor. Information about where SRIF-sst(2) interactions take place is lacking, and downstream effectors mediating SRIF-sst(2) antiangiogenic actions are unknown. Methods. In the OIR model, retinal expression of SRIF was evaluated with RT-PCR and radioimmunoassay. The bindings of [(125)I]LTT-SRIF-28 and [(125)I]Tyr(3)-octreotide were measured in coronal sections of the eye. With Western blot analysis, the authors evaluated the levels of sst(2A) and the expression and activity of the signal transducer and activator of transcription (STAT)3. The analysis of STAT3 was performed in hypoxic mice treated with the sst(2) agonist octreotide or with the sst(2) antagonist D-Tyr(8) cyanamid 154806 (CYN). Retinal localization of sst(2A) was assessed by single and double immunohistochemistry with an endothelial cell marker. Results. In the hypoxic retina, both SRIF and sst(2) levels as well as [(125)I]Tyr(3)-octreotide binding were downregulated. In addition, sst(2A) immunostaining was decreased in the neuroretina but was increased in capillaries. Hypoxia increased both the expression and the activity of STAT3. This increase was inhibited by octreotide but was strengthened by CYN. Conclusions. These data suggest that sst(2) expressed by capillaries may be responsible for the antiangiogenic effects of SRIF and that downstream effectors in this action include the transcription factor STAT3. These results support the possibility of using sst(2)-selective ligands in the treatment of proliferative retinopathies and indicate STAT3 as an additional target for a novel therapeutic approach.

Regulation of prostate cancer cell proliferation by somatostatin receptor activation

Although some evidence supports the antitumoral effects of somatostatin (SRIF) and related agonists, the available data in prostate cancer (PCa) model systems and clinical studies are few, conflicting and not conclusive. This study investigated the effects of lanreotide and new mono- and bi-specific SRIF agonists on proliferation, ligand-driven SRIF receptor (sst) dimerization and secretory pattern of the IGF system in LNCaP cells, a model of androgen-dependent PCa. LNCaP expressed all sst(s), but sst(4). Among them, sst(1) and sst(3) were inversely regulated by serum concentration. sst(1)/sst(2) and sst(2)/sst(5) dimers were constitutively present and further stabilized by treatment with BIM-23704 (sst(1)/sst(2)) and BIM-23244 (sst(2)/sst(5)), respectively. Dose-response studies showed that lanreotide and BIM-23244 were significantly more potent in inhibiting LNCaP cell proliferation than BIM-23120 (sst(2)) and BIM-23206 (sst(5)) alone or in combination. Treatment with BIM-23926 [corrected] (sst(1)) markedly reduced cell proliferation, whereas exposure to BIM-23704 resulted in a lower cell growth inhibition. The antiproliferative effects of BIM-23244, lanreotide and BIM-23704 were unchanged, reduced and abolished by the sst(2) antagonist BIM-23627, respectively. All SRIF analogs caused a significant induction in p27(KipI) and p21 and down-regulation of protein expression of cyclin E, as well as reduced IGF-I and IGF-II secretion. In particular, the administration of exogenous IGF-I, at variance to IGF-II, counteracted the inhibitory effect on cell proliferation of these compounds. Moreover, SRIF agonists reduced endogenous IGFBP-3 proteolysis. These results show that, in LNCaP cells, activation of sst(1) and sst(2)/sst(5) results in relevant antiproliferative/antisecretive actions.

Functional effects of somatostatin receptor 1 activation on synaptic transmission in the mouse hippocampus

Somatostatin-14 (SRIF) co-localizes with GABA in the hippocampus and regulates neuronal excitability. A role of SRIF in the control of hippocampal activity has been proposed, although the exact contribution of each SRIF receptor (sst(1)-sst(5)) in mediating SRIF action requires some clarification. We used hippocampal slices of wild-type and sst(1) knockout (KO) mice and selective pharmacological tools to provide conclusive evidence for a role of sst(1) in mediating SRIF inhibition of synaptic transmission. With single- and double-label immunohistochemistry, we determined the distribution of sst(1) in hippocampal slices and we quantified sst(1) colocalization with SRIF. With electrophysiology, we found that sst(1) activation with CH-275 inhibited both the NMDA- and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-mediated responses. Results from sst(1) KO slices confirmed the specificity of CH-275 effects; sst(1) activation did not affect the inhibitory transmission which was in contrast increased by sst(4) activation with L-803,087 in both wild-type and sst(1) KO slices. The AMPA-mediated responses were increased by L-803,087. Functional interaction between sst(1) and sst(4) is suggested by the finding that their combined activation prevented the CH-275-induced inhibition of AMPA transmission. The involvement of pre-synaptic mechanisms in mediating inhibitory effects of sst(1) on excitatory transmission was demonstrated by the finding that CH-275 (i) increased the paired-pulse facilitation ratio, (ii) did not influence the AMPA depolarization in the presence of tetrodotoxin, and (iii) inhibited glutamate release induced by epileptiform treatment. We conclude that SRIF control of excitatory transmission through an action at sst(1) may represent an important contribution to the regulation of hippocampal activity.

Differential Ligand-Mediated Pituitary Somatostatin Receptor Subtype Signaling: Implications for Corticotroph Tumor Therapy

Pituitary targeted pharmacotherapy for Cushing's disease is challenging and ineffective. Unlike octreotide and lanreotide, the multisomatostatin receptor (SST) analog pasireotide that exhibits SST5 greater than SST2 binding affinity offers potential for treating Cushing's disease. Because corticotroph cells express SST5 more abundantly than SST2, pasireotide likely exerts superior corticotroph action mainly through SST5. However, there is no direct evidence for this assumption, and moreover, the ligand effect on corticotroph SST2 is not known. We used AtT20 mouse pituitary corticotroph tumor cells stably overexpressing SST2 or SST5 and TtT/GF mouse pituitary folliculostellate cells stably or transiently expressing SST receptors to examine ligand-receptor activation by SST2- and SST5-selective agonists. We show that pasireotide was more potent than either octreotide or somatostatin-14 in mouse corticotroph cells. Pasireotide potency is not affected by SST2 abundance, SST2 antagonist treatment, or octreotide cotreatment in SST2-overexpressing cells. Pasireotide also does not induce SST2 internalization and attenuates octreotide or SRIF14-induced SST2 internalization only at superphysiological dose ranges. In contrast, octreotide attenuates pasireotide potency in SST5-overexpressing cells. Moreover, short exposure to pasireotide causes prolonged inhibition of forskolin or CRH-induced cAMP accumulation, in contrast to somatostatin-14- and SST2-selective agonists that induced postwithdrawal cAMP rebound. Long-term pasireotide signaling effects are enhanced by SST5 overexpression. The results indicate that SST5 determines short- and long-term enhanced pasireotide action in corticotroph cells, whereas the ligand action on SST2 is negligible.

Reduced Somatostatin in Hypothalamus of Young Male Mouse Increases Local but not Circulatory GH

The release of growth hormone (GH) from the pituitary gland is primarily inhibited by somatostatin (SRIF) from the hypothalamus via interactions with five types of SRIF receptors (SSTRs). However, the inhibition mechanism of SRIF on GH has not been fully examined. In this study, we repressed the hypothalamic SRIF in young male mice by stereotaxic injection of the lentiviral-shRNA against SRIF to investigate the role of hypothalamic SRIF on hormone secretion in the GH/IGF-1 axis. We found that the reduction of SRIF in hypothalamus was associated with an increase in the protein, but not the mRNA level, of the GH in the pituitary where SSTR 2 and SSTR 5 act importantly. Interestingly, the level of blood circulatory SRIF, GH, IGF-1 and the body weight were not significantly influenced by the downregulation of hypothalamic SRIF. Our findings provide insights into the mechanisms underlying the inhibition of SRIF on GH secretion.

The β-d-Glucose Scaffold as a β-Turn Mimetic

Activity and selectivity are typically the first considerations when designing a drug. However, absorption, distribution, metabolism, excretion, and toxicity (ADMET) are equally important considerations. Peptides can provide a combination of potent binding and exquisite selectivity, as evidenced by their pervasive use as enzymes, hormones, and signaling agents within living systems. In particular, peptidic turn motifs are key elements of molecular recognition. They may be found at the exposed surfaces of globular proteins, where they are available for binding interactions with other peptides and small molecules. However, despite these advantages, peptides often make poor drugs. The amide backbone is subject to rapid enzymatic proteolysis, resulting in short half-lives. Furthermore, the ability of the amide backbone to hydrogen bond with water restricts its ability to cross membranes and, consequentially, results in poor oral bioavailability. Accordingly, the development of nonpeptidic scaffolds that mimic peptidic turn motifs represents a promising means of converting peptidic agents into more drugable molecules. In this Account, we describe the design and synthesis of beta-turn mimetics that use a beta-D-glucose scaffold, the first use of a sugar scaffold for this purpose. Somatostatin (SRIF) is a small protein (14 amino acid residues) human hormone; a shorter (6 amino acid residues) synthetic peptide, L-363,301, is a fully peptidal agonist. These two cyclic peptides share the beta-turn motif comprising Phe(7)-Trp(8)-Lys(9)-Thr(10) (d-Trp(8) in the case of L-363,301), of which the tryptophan and lysine residues in the i + 1 and i + 2 positions, respectively, are critical for binding. In 1988, we initiated a program that tested and validated the then-novel proposition that the beta-D-glucose scaffold can mimic the beta-turn in L-363,301. The beta-D-glucose scaffold proved to be an attractive mimic of a beta-turn in part because it permits the convenient attachment of amino acid side chains via facile etherification reactions, rather than carbon-carbon bond formations; it is also an inexpensive starting material with well-defined stereochemistry. From the beginning, biological assays were used alongside physical measurements to assess the relevance of the design. Our first two synthetic targets, compounds 6 and 7, bound the SRIF receptors on benchmark (AtT-20) cells, albeit weakly, consistent with the objective of the design. Subsequently, a better ligand (8) and two congeners were found to be agonists at the SRIF receptors, providing convincing evidence that the peptide backbone is not required for receptor binding or signal transduction. The unexpectedly high level of receptor affinity of selected analogs, as well as the fortuitous discovery that our peptidomimetics were active against several chemically distinct receptors, led us to hypothesize that these monosaccharides could access multiple potential binding modes. Our later studies of this sugar scaffold confirmed this property, which we termed pseudosymmetry, whereby multiple similar but nonidentical motifs are displayed within a single analog. We propose the presence of pseudosymmetry to be an element of privilege and an advantage for lead discovery.