Somatostatin receptors, an expanding gene family: cloning and functional characterization of human SSTR3, a protein coupled to adenylyl cyclase.

We previously reported the cloning of two distinct somatostatin receptor (SSTR) subtypes, SSTR1 and SSTR2. Although both SSTR1 and SSTR2 bound somatostatin specifically and with high affinity, neither was coupled to adenylyl cyclase, a major cellular effector of somatostatin's actions. Here we report the cloning and functional characterization of a third member of the SSTR family. Human SSTR3 is a protein of 418 amino acids and has 45% and 46% identity with human SSTR1 and SSTR2, respectively. RNA blotting studies showed that SSTR3 mRNA could be readily detected in brain and pancreatic islets. The pharmacological properties of human SSTR3 were characterized by transiently expressing the human SSTR3 gene in COS-1 cells. Membranes from cells expressing human SSTR3 bound the somatostatin agonist [125I]CGP 23996 specifically and with high affinity, with a rank order of potency of somatostatin-28 = CGP 23996 > somatostatin-14 > SMS-201-995. Studies using cells transiently coexpressing the human dopamine D1 receptor and human SSTR3 showed that somatostatin was able to inhibit dopamine-stimulated cAMP formation in a dose-dependent manner, indicating that SSTR3 was functionally coupled to adenylyl cyclase. These results indicate that the diverse biological effects of somatostatin are mediated by a family of receptor with distinct, but overlapping, tissue distributions, unique pharmacological properties, and potentially different functions.     

Somatostatin-28 regulates GLP-1 secretion via somatostatin receptor subtype 5 in rat intestinal cultures

Five somatostatin receptors (SSTRs) bind somatostatin-14 (S-14) and somatostatin-28 (S-28), but SSTR5 has the highest affinity for S-28. To determine whether S-28 acting through SSTR5 mediates inhibition of glucagon-like peptide-1 (GLP-1), fetal rat intestinal cell cultures were treated with somatostatin analogs with relatively high specificity for SSTRs 2-5. S-28 dose-dependently inhibited GLP-1 secretion stimulated by gastrin-releasing peptide more potently than S-14 (EC(50) 0.01 vs. 5.8 nM). GLP-1 secretion was inhibited by an SSTR5 analog, BIM-23268, more potently than S-14 and nearly as effectively as S-28. The SSTR5 analog L-372,588 also suppressed GLP-1 secretion equivalent to S-28, but a structurally similar peptide, L-362,855 (Tyr to Phe at position 7), was ineffective. An SSTR2-selective analog was less effective than S-28, and an SSTR3 analog was inactive. Separate treatment with GLP-1-(7-36)-NH(2) increased S-28 and S-14 secretion by three- and fivefold; BIM-23268 abolished S-28 without altering S-14, whereas the SSTR2 analog was inactive. The results indicate that somatostatin regulation of GLP-1 secretion occurs via S-28 through activation of SSTR5. GLP-1-stimulated S-28 secretion is also autoregulated by SSTR5 activation, suggesting a feedback loop between GLP-1 and S-28 modulated by SSTR5.     

Cortistatin radioligand binding in wild-type and somatostatin receptor-deficient mouse brain

Cortistatin-14 (CST-14) is a recently discovered member of the somatostatin family of neuropeptides. It shares 11 of its 14 amino acids with somatostatin-14 (SRIF-14). In the present study, binding sites for cortistatin-14 in the mouse brain were examined and compared to those for somatostatin using iodinated cortistatin-14 and iodinated somatostatin-14. By in vitro receptor autoradiography, high densities of cortistatin-14 and somatostatin-14 specific binding sites were detected in the cortex, hippocampal formation, basolateral amygdala and medial habenula. Unlabeled 100 nM cortistatin-14 inhibited iodinated somatostatin-14 binding in the hippocampus, but not in the cortex or amygdaloid nuclei. In somatostatin receptor subtype-2 knock-out (KO) mice, autoradiographic iodinated somatostatin-14 binding was observed in the hippocampus and habenula but was removed in the cortex and amygdaloid nuclei, specific iodinated cortistatin-14 binding sites were found in the hippocampus, habenula and throughout the cortex. We conclude that the somatostatin receptor subtype-2 is responsible for somatostatin binding in cortical and amygdaloid regions and that cortistatin predominantly interacts with the same receptors as somatostatin.     

Effects of ammonia,chloroquine, and monensin on the vacuolar apparatus of an absorptive epithelium

Summary The distribution of two major immunoreactive forms of somatostatin, somatostatin-14 and somatostatin-34, within the brain, pancreas and intestine of adult lampreys, Petromyzon marinus, was identified using antisera raised against these peptides. Immunostaining of the brain is similar in juveniles and upstream migrants, and somatostatin-14 is the major somatostatin form demonstrated. A few somatostatin-34-containing cells are localized within the olfactory bulbs, thalamus and hypothalamus, but cells immunoreactive to anti-somatostatin-34 in the hypothalamus and thalamus do not co-localize somatostatin-14. Immunostaining of pinealocytes within the pineal pellucida with anti-somatostatin-14 may infer a novel function for this structure. Somatostatin-14 and somatostatin-34 are co-localized within D-cells of the cranial pancreas and caudal pancreas of juveniles and upstream migrants. Numerous somatostatin-34-immunoreactive cells are distributed within the epithelial mucosa of the anterior intestine but not all of these cells cross-react with anti-somatostatin-14. It appears that somatostatin-34 is the major somatostatin in the pancreo-gastrointestinal system of adult lampreys.     

Synthesis,radiolabeling, and preclinical evaluation of a new octreotide analog for somatostatin receptor‐positive tumor scintigraphy

Radiolabeled somatostatin analogs have become powerful tools in the diagnosis and staging of neuroendocrine tumors, which express somatostatin receptors. The aim of this study was to evaluate a new somatostatin analog, 6‐hydrazinopyridine‐3‐carboxylic acid‐Ser³‐octreotate (HYNIC‐SATE) radiolabeled with ^99mTc, using ethylenediamine‐N,N′‐diacetic acid and tricine as coligands, to be used as a radiopharmaceutical for the in vivo imaging of somatostatin receptor subtype 2 (SSTR2)‐positive tumor. Synthesis of the peptide was carried out on a solid phase using a standard Fmoc strategy. Peptide conjugate affinities for SSTR2 were determined by receptor binding affinity on rat brain cortex and C6 cell membranes. Internalization rate of ^99mTc‐HYNIC‐SATE was studied in SSTR2‐expressing C6 cells that were used for intracranial tumor studies in rat brain. A reproducible in vivo C6 glioma model was developed in Sprague–Dawley rat and confirmed by histopathology and immunohistochemical analysis. Biodistribution and imaging properties of this new radiopeptide were also studied in C6 tumor‐bearing rats. Radiolabeling was performed at high specific activities, with a radiochemical purity of >96%. Peptide conjugate showed high affinity binding for SSTR2 (HYNIC‐SATE IC₅₀ = 1.60 ± 0.05 n m ) and specific internalization into rat C6 cells. After administration of ^99mTc‐HYNIC‐SATE in C6 glioma‐bearing rats, a receptor specific uptake of radioactivity was observed in SSTR‐positive organs and in the implanted intracranial tumor and rapid excretion from nontarget tissues via kidneys. ^99mTc‐HYNIC‐SATE is a new receptor‐specific radiopeptide for targeting SSTR2‐positive brain tumor and might be of great promise in the scintigraphy of SSTR2‐positive tumors. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Evidence for the presence of dihydro (reduced) somatostatin-14 in the guinea pig brain

Analysis of somatostatin-like immunoreactivity (SLI) in guinea pig brain by HPLC and radioimmunoassay revealed an unexpected peak of SLI eluting at a retention time slightly later than standard somatostatin-14. The following evidence argues that this peak represents dihydro (H2) somatostatin-14. (1) The peak had the same retention time as standard [H2]somatostatin. (2) The possibility of a reduction artefact due to tissue processing was excluded by adding exogenous somatostatin-14 or 125I-labeled N-Tyr-somatostatin-14 to tissue and observing that no corresponding reduced peptides were generated. (3) Mild oxidation of brain extracts with H2O2 decreased, whereas mild reduction with dithiothreitol increased, the proposed peak of [H2]somatostatin. (4) Reaction of tissue extracts with iodoacetamide decreased the size of the proposed [H2]somatostatin peak but resulted in generation of a new peak co-eluting with standard carboxymethylated somatostatin-14. The proportion of the [H2]somatostatin peak in five brain regions, the hypothalamus, amygdala, cerebral cortex, brainstem and cerebellum, ranged from 6 to 20% of total SLI. The probability of somatostatin-14 existing endogenously in reduced or oxidized forms may have implications for its biological function in the guinea pig.     

Structure,function, and expression pattern of a novel sodium-coupled citrate transporter (NaCT) cloned from mammalian brain

Citrate plays a pivotal role not only in the generation of metabolic energy but also in the synthesis of fatty acids, isoprenoids, and cholesterol in mammalian cells. Plasma levels of citrate are the highest ( approximately 135 microm) among the intermediates of the tricarboxylic acid cycle. Here we report on the cloning and functional characterization of a plasma membrane transporter (NaCT for Na+ -coupled citrate transporter) from rat brain that mediates uphill cellular uptake of citrate coupled to an electrochemical Na+ gradient. NaCT consists of 572 amino acids and exhibits structural similarity to the members of the Na+-dicarboxylate cotransporter/Na+ -sulfate cotransporter (NaDC/NaSi) gene family including the recently identified Drosophila Indy. In rat, the expression of NaCT is restricted to liver, testis, and brain. When expressed heterologously in mammalian cells, rat NaCT mediates the transport of citrate with high affinity (Michaelis-Menten constant, approximately 20 microm) and with a Na+:citrate stoichiometry of 4:1. The transporter does interact with other dicarboxylates and tricarboxylates but with considerably lower affinity. In mouse brain, the expression of NaCT mRNA is evident in the cerebral cortex, cerebellum, hippocampus, and olfactory bulb. NaCT represents the first transporter to be identified in mammalian cells that shows preference for citrate over dicarboxylates. This transporter is likely to play an important role in the cellular utilization of citrate in blood for the synthesis of fatty acids and cholesterol (liver) and for the generation of energy (liver and brain). NaCT thus constitutes a potential therapeutic target for the control of body weight, cholesterol levels, and energy homeostasis.     

Identification and characterization of subtype selective somatostatin receptor agonists.

High affinity, subtype selective non-peptide agonists of somatostatin receptor subtypes 1-5 were identified in combinatorial libraries constructed based on molecular modeling of known peptide agonists. Simultaneous traditional chemical synthesis yielded an additional series of somatostatin subtype-2 receptor (SSTR2) selective agonists. These compounds have been used to further define the physiological functions of the individual somatostatin receptor subtypes. In vitro experiments demonstrated the role of the SSTR2 in inhibition of glucagon release from mouse pancreatic alpha-cells and the somatostatin subtype-5 receptor (SSTR5) as a mediator of insulin secretion from pancreatic beta-cells. Both SSTR2 and SSTR5 regulated growth hormone release from the rat anterior pituitary gland. In vivo studies performed with SSTR2 receptor selective compounds demonstrated effective inhibition of pulsatile growth hormone release in rats. The SSTR2 selective compounds also lowered plasma glucose levels in normal and diabetic animal models. The availability of high affinity, subtype selective non-peptide agonists for each of the somatostatin receptors provides a direct approach to defining their physiological function both peripherally and in the central nervous system.     

Effects of somatostatin and its analogues on tyrosine kinase activity in rodent tumors

The effects of native somatostatin-14 (SS-14) and of its two analogues, octreotide and CH-275, on the activity of tyrosine kinases (TK) in two rodent tumors (rat pituitary tumor and murine colonic cancer) were studied in vitro. The activity of TK was measured in tissue homogenates using gamma[(32)P]ATP as the donor of the phosphoryl group and poly(Glu(80), Tyr(20)) as a substrate. It was found that native SS-14 inhibited TK activity in both investigated tumors. Octreotide, which acts preferentially via somatostatin receptor subtype 2 (SSTR2), was very effective in inhibiting TK activity in the rat pituitary tumor, but it is a rather weak inhibitor of TK activity in murine colonic cancer. CH-275, a selective ligand of the SSTR1 subtype of SS receptors, suppressed TK activity in the pituitary tumor but was ineffective in the colonic cancer. It is hypothesized that the effect of neuropeptide somatostatin (SS-14) on murine colonic cancer is exerted via the subtype of receptor which does not interact with CH-275 and has no or low affinity for octreotide (SSTR 4, 3 or 5?).     

Agonist-dependent interaction of the rat somatostatin receptor subtype 2 with cortactin-binding protein 1.

We report here an interaction between the C terminus of the rat somatostatin receptor subtype 2 (SSTR2) and a protein that has recently been identified as cortactin-binding protein 1 (CortBP1). Interaction is mediated by the PDZ (PSD-95/discs large/ZO-1) domain of CortBP1. As shown by in situ hybridization, SSTR2 and cortactin-binding protein are coexpressed in the rat brain. The association between SSTR2 and the PDZ-domain of CortBP1 was verified by overlay assays and by coprecipitation after transfection in human embryonic kidney (HEK) cells. Analysis by confocal microscopy indicates that CortBP1 is distributed diffusely throughout the cytosol in transfected cells and that it becomes concentrated at the plasma membrane when SSTR2 is present. This process is largely increased when the receptor is stimulated by somatostatin; as CortBP1 interacts with the C terminus of SSTR2, our data suggest that the binding of agonist to the receptor increase the accessibility of the receptor C terminus to the PDZ domain of CortBP1. Our data for the first time establish a link between a G-protein coupled receptor and constituents of the cytoskeleton.     

Feeding responses to central administration of several somatostatin analogs in chicks

Somatostatin is well known as an inhibitor of growth hormone release from the anterior pituitary. Its effects are exerted via 5 subtypes of receptors, which are named SSTR1 through 5. We recently reported that intracerebroventricular (ICV) injection of somatostatin stimulates feeding behavior in chicks. However, the specific receptors which mediate this orexigenic effect have not been identified in chicks. Thus, the purpose of the present study was to identify the receptor subtypes involved in somatostatin-induced feeding using 5 somatostatin analogs. Chicks that received vapreotide and octreotide (less than 3 nmol), which are agonist of SSTR2 and SSTR5, increased their food intake. Additionally, chicks ICV injected with BIM23056 or L-817,818 (SSTR3 and SSTR5 agonists, respectively) also had increased food intake. However, ICV injection of the SSTR4 agonist L-803,087 did not cause an orexigenic effect, suggesting that SSTR4 might not be important in somatostatin-induced feeding behavior. In summary, results from this study may be interpreted as SSTR2, SSTR3 and SSTR5 are related to somatostatin-associated feeding behavior in chicks.     

Immunological Detection of Isoforms of the Somatostatin Receptor Subtype, SSTR2

Abstract: Somatostatin (SRIF) induces its diverse physiological actions through interactions with different receptor subtypes. Multiple SRIF receptor subtypes have recently been cloned. To analyze the physical properties of receptor subtype SSTR2, two different peptide-directed antibodies were generated against SSTR2. Antibody “2e3,” directed against the peptide SSCTINWPGESGAWYT (residues 191–206), corresponding to a region in the predicted third extracellular domain of mouse SSTR2, and antibody “2i4,” directed against the peptide SGTEDGERSDS (residues 333–343) from the predicted cytoplasmic tail of mouse SSTR2, were developed. In Chinese hamster ovary (CHO) cells stably expressing the mouse SSTR2 gene (CHOB), the antibody 2e3 recognized specifically a protein of 93-kDa protein by immunoblotting. No specific immunoreactivity was detected by 2e3 in nontransfected CHO cells or CHO cells stably expressing vector alone or human SSTR1 or mouse SSTR3 genes. The antibody 2i4 specifically immunoprecipitated SSTR2 solubilized from CHOB cells that could be labeled with the SSTR2-specific ligand ¹²⁵I-MK-678. Furthermore, both 2e3 and 2i4 specifically immunoprecipitated 93-kDa [³⁵S]methionine-labeled proteins from CHOB cells, indicating that they recognize the same proteins. In contrast to studies in CHOB cells, immunoblotting studies showed that 2e3 detected specifically a single 148-kDa protein from different regions of the rat brain that have previously been shown to express high levels of SSTR2 mRNA and SRIF receptors with high affinity for ¹²⁵I-MK-678. In contrast, no immunoreactivity was detected in rat kidney, liver, or lung, which do not express SSTR2. No 93-kDa protein was detected specifically in the rat brain. The 148-kDa protein detected by 2e3 is an SRIF receptor because 2e3 and 2i4 specifically immunoprecipitated solubilized rat brain SRIF receptors that could be reversibly labeled with ¹²⁵I-MK-678. As in rat brain, 2e3 interacted specifically with a single 148-kDa protein in rat pituitary, in the rat pancreatic cell line AR42J, and in the HEK 293 cell line derived from human kidney, all of which express SSTR2 mRNA and SRIF receptors with high affinity for ¹²⁵I-MK-678. These findings indicate that rat brain and pituitary, as well as a pancreatic and a kidney cell line, express primarily a form of SSTR2 different from CHOB cells. The multiple forms of SSTR2 may result from differential post-translational processing of SSTR2 because 2e3 immunoprecipitated 41-kDa in vitro translation products generated from mRNA extracted from CHOB and AR42J cells. This 41-kDa protein has the predicted size of unprocessed SSTR2. These results demonstrate that 2e3 and 2i4 antibodies interact specifically with SSTR2. Detection of two different size proteins by the SSTR2 peptide-directed antibodies suggests the existence of multiple forms of SSTR2.     

Isolation of [Pro2,Met13]somatostatin-14 and somatostatin-14 from the frog brain reveals the existence of a somatostatin gene family in a tetrapod.

Two somatostatin-related peptides were isolated in pure form from an extract of the brain of the European green frog, Rana ridibunda. The primary structure of the most abundant component was identical to that of mammalian somatostatin-14. The primary structure of the second component, present in approximately 5% of the abundance of somatostatin-14, was established as Ala-Pro-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Met-Cys. This sequence shows two substitutions (Pro for Gly2 and Met for Ser13) compared with mammalian somatostatin-14. The data provide evidence for a somatostatin gene family in tetrapods as well as in teleost fish.     

Suppression of ANP secretion by somatostatin through somatostatin receptor type 2

Somatostatin is a cyclic-14 amino acid peptide which mainly distributed in digestive system and brain. Somatostatin receptor (SSTR) is a G-protein coupled receptor and all five SSTR subtypes are expressed in cardiomyocytes. The aim of this study was to investigate the effect of somatostatin on atrial natriuretic peptide (ANP) secretion and its signaling pathway. Somatostatin (0.01 and 0.1 nM) decreased ANP secretion in isolated beating rat atrium in a dose-dependent manner. But atrial contractility and translocation of extracellular fluid were not changed. Somatostatin-induced decrease in ANP secretion was significantly attenuated by the pretreatment with CYN 154806 (SSTR type 2 antagonist; 0.1 μM), but not by BIM 23056 (SSTR type 5 antagonist; 0.1 μM) and urantide (urotensin II receptor antagonist; 0.1 μM). When pretreated with an agonist for SSTR type 2 (Seglitide, 0.1 nM) and SSTR type 5 (L 817818, 0.1 nM), only Seglitide reduced ANP secretion similar to that of somatostatin. The suppressive effect of somatostatin on ANP secretion was attenuated by the pretreatment with an inhibitor for adenylyl cyclase (MDL-12330A, 5 μM) or protein kinase A (KT 5720, 0.1 μM). In diabetic rat atria, the suppressive effect of somatostatin on ANP secretion and concentration was attenuated. Real time-PCR and western blot shows the decreased level of SSTR type 2 mRNA and protein in diabetic rat atria. These data suggest that somatostatin decreased ANP secretion through SSTR type 2 and an attenuation of suppressive effect of somatostatin on ANP secretion in diabetic rat atria is due to a down-regulation of SSTR type 2.     

A monoclonal antibody to somatostatin with potent in vivo immunoneutralizing activity

The spleen from a Robertsonian mouse with high titer and affinity antiserum after being immunized with somatostatin-14 conjugated to keyhole limpet hemocyanin was fused with FOX-NY cells. Hybridomas were cloned by limiting dilution, subcloned, and ascites was produced from the highest affinity close in pristine-primed Balb/c mice. Ascites fluid contained approximately 20 mg/ml IgG and bound 50% of 1 fmol 125I-[Tyr1]-somatostatin at a final dilution of 1:10,000,000. Binding of this IgG1 antibody, CURE.S6, was inhibited by 50% at 40 pM concentrations of either somatostatin-14 or somatostatin-28, but was not inhibited by [D-Trp8 -somatostatin at 1000-fold higher concentrations. The antibody produced very intense specific immunohistochemical staining of somatostatin endocrine cells in the stomach and pancreas and of intestinal somatostatin neurons with extremely low background staining. Intravenous injection of 2 mg purified antibody in urethane-anesthetized rats resulted in 300-fold increase in plasma GH within 15 min. CURE.S6 is a high affinity monoclonal antibody directed at the biologically active somatostatin ring structure. This antibody is useful for in vivo immunoneutralization of exogenous and endogenous somatostatin in the rat and also is an excellent reagent for immunohistochemical localization of somatostatin.     

Antibody to synthetic somatostatin-28(1-12): immunoreactivity with somatostatin in brain is dependent on orientation of immunizing peptide

Rabbits were immunized with synthetic peptides representing the neurotransmitter dodecapeptide somatostatin-28(1-12) (SANSNPAMAPRE) coupled to the carrier protein keyhole limpet hemocyanin (KLH) at either the amino or the carboxyl terminus. Although all rabbits produced high-titer antisera to immunizing peptide, as assayed by ELISA, only rabbits immunized with peptide coupled to carrier at the amino terminus yielded antibodies that bound to native somatostatin in mouse brain slices. This effect of peptide coupling orientation on epitope specificity of peptide antisera is likely to be significant to other investigators who use predetermined peptide sequences to generate immunohistochemical reagents.     

Cloning and expression of a novel mouse somatostatin receptor (SSTR2B).

A mouse somatostatin (SS) receptor cDNA was cloned from neuroblastoma x glioma (NG108-15) cells. The sequence is almost identical to that of the mouse SSTR2 receptor [(1992) Proc. Natl. Acad. Sci. USA 89, 251)] but lacks about 300 nucleotides between transmembrane domain VII and the C-terminus. This spliced variant of SSTR2 (designated SSTR2B) encodes a protein which is 23 residues shorter than that predicted from the SSTR2 sequence, and differs in 15 amino acids at the C-terminus. mRNA corresponding to SSTR2B occurs in mouse tissues in higher abundance than that of SSTR2. SSTR2B binds SS peptides with high affinity when expressed in mammalian cells.     

High affinity binding sites for a somatostatin-28 analog in rat brain

Using an iodinated analog of a large (28 residues) and biologically active form of somatostatin, ¹²⁵I[Leu⁸,D-Trp²²,Tyr²⁵]SS-28, it was possible to demonstrate saturable and high affinity binding sites (dissociation constant = 0.46 ± 0.04 nM) in rat cortical membranes. Somatostatin, somatostatin-28, as well as two potent analogs, [D-Trp⁸] somatostatin and [D-Trp²²] somatostatin-28, could completely displace the radiogland in the nanomolar range whereas the inactive analog Des-Trp⁸-somatostatin and the unrelated peptide GnRH showed no affinity for these binding sites; octa- and nona-peptide analogs of somatostatin were inactive. High binding was found in hippocampus, amygdala, tuberculum olfactorium, caudate-putamen and cortex; moderate binding in midbrain and hypothalamus, and no binding in the cerebellum. These results suggest that specific somatostatin receptors can be measured within the brain with ¹²⁵I[Leu⁸,D-Trp²²,Tyr²⁵] SS-28 as radioligand.