Phosphorylation of TNF-alpha converting enzyme by gastrin-releasing peptide induces amphiregulin release and EGF receptor activation.

PubMed

Zhang, Qing; Thomas, Sufi M; Lui, Vivian Wai Yan; Xi, Sichuan; Siegfried, Jill M; Fan, Huizhou; Smithgall, Thomas E; Mills, Gordon B; Grandis, Jennifer Rubin

2006-05-02

G protein-coupled receptors induce EGF receptor (EGFR) signaling, leading to the proliferation and invasion of cancer cells. Elucidation of the mechanism of EGFR activation by G protein-coupled receptors may identify new signaling paradigms. A gastrin-releasing peptide (GRP)/GRP receptor-mediated autocrine pathway was previously described in squamous cell carcinoma of head and neck. In the present study, we demonstrate that TNF-alpha converting enzyme (TACE), a disintegrin and metalloproteinse-17, undergoes a Src-dependent phosphorylation that regulates release of the EGFR ligand amphiregulin upon GRP treatment. Further investigation reveals the phosphatidylinositol 3-kinase (PI3-K) as the intermediate of c-Src and TACE, contributing to their association and TACE phosphorylation. Phosphoinositide-dependent kinase 1 (PDK1), a downstream target of PI3-K, has been identified as the previously undescribed kinase to directly phosphorylate TACE upon GRP treatment. These findings suggest a signaling cascade of GRP-Src-PI3-K-PDK1-TACE-amphiregulin-EGFR with multiple points of interaction, translocation, and phosphorylation. Furthermore, knockdown of PDK1 augmented the antitumor effects of the EGFR inhibitor erlotinib, indicating PDK1 as a therapeutic target to improve the clinical response to EGFR inhibitors.

Expression of gastrin-releasing peptide by excitatory interneurons in the mouse superficial dorsal horn.

PubMed

Gutierrez-Mecinas, Maria; Watanabe, Masahiko; Todd, Andrew J

2014-12-11

Gastrin-releasing peptide (GRP) and its receptor have been shown to play an important role in the sensation of itch. However, although GRP immunoreactivity has been detected in the spinal dorsal horn, there is debate about whether this originates from primary afferents or local excitatory interneurons. We therefore examined the relation of GRP immunoreactivity to that seen with antibodies that label primary afferent or excitatory interneuron terminals. We tested the specificity of the GRP antibody by preincubating with peptides with which it could potentially cross-react. We also examined tissue from a mouse line in which enhanced green fluorescent protein (EGFP) is expressed under control of the GRP promoter. GRP immunoreactivity was seen in both primary afferent and non-primary glutamatergic axon terminals in the superficial dorsal horn. However, immunostaining was blocked by pre-incubation of the antibody with substance P, which is present at high levels in many nociceptive primary afferents. EGFP+ cells in the GRP-EGFP mouse did not express Pax2, and their axons contained the vesicular glutamate transporter 2 (VGLUT2), indicating that they are excitatory interneurons. In most cases, their axons were also GRP-immunoreactive. Multiple-labelling immunocytochemical studies indicated that these cells did not express either of the preprotachykinin peptides, and that they generally lacked protein kinase Cγ, which is expressed by a subset of the excitatory interneurons in this region. These results show that GRP is expressed by a distinct population of excitatory interneurons in laminae I-II that are likely to be involved in the itch pathway. They also suggest that the GRP immunoreactivity seen in primary afferents in previous studies may have resulted from cross-reaction of the GRP antibody with substance P or the closely related peptide neurokinin A.

Improvement of autism spectrum disorder symptoms in three children by using gastrin-releasing peptide.

PubMed

Becker, Michele Michelin; Bosa, Cleonice; Oliveira-Freitas, Vera Lorentz; Goldim, José Roberto; Ohlweiler, Lygia; Roesler, Rafael; Schwartsmann, Gilberto; Riesgo, Rudimar Dos Santos

2016-01-01

To evaluate the safety, tolerability and potential therapeutic effects of gastrin-releasing peptide in three children with autistic spectrum disorder. Case series study with the intravenous administration of gastrin-releasing peptide in the dose of 160pmol/kg for four consecutive days. To evaluate the results, parental impressions the Childhood Autism Rating Scale (CARS) and the Clinical Global Impression (CGI) Scale. Each child underwent a new peptide cycle after two weeks. The children were followed for four weeks after the end of the infusions. The gastrin-releasing peptide was well tolerated and no child had adverse effects. Two children had improved social interaction, with a slight improvement in joint attention and the interaction initiatives. Two showed reduction of stereotypes and improvement in verbal language. One child lost his compulsion to bathe, an effect that lasted two weeks after each infusion cycle. Average reduction in CARS score was 2.8 points. CGI was "minimally better" in two children and "much better" in one. This study suggests that the gastrin-releasing peptide is safe and may be effective in improving key symptoms of autism spectrum disorder, but its results should be interpreted with caution. Controlled clinical trials-randomized, double-blinded, and with more children-are needed to better evaluate the possible therapeutic effects of gastrin-releasing peptide in autism. Copyright © 2016 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

How gastrin-releasing peptide receptor (GRPR) and αvβ3 integrin expression reflect reorganization features of tumors after hyperthermia treatments.

PubMed

Hallasch, Sandra; Frick, Sindy; Jung, Maximilian; Hilger, Ingrid

2017-07-31

The outcome of tumor treatment via hyperthermia in the clinic has been reported to be heterogeneous. Here, we assessed how the presence of gastrin-releasing peptide receptor (GRPR) and α v β 3 integrin together with the morphology of the vascularization reflects the growth behavior of tumors after hyperthermia treatment. MDA-MB-231 tumor bearing mice were treated either with high (46 °C) or low dose (42 °C) water hyperthermia for 60 min. Changes of GRPR and α v β 3 integrin expression were assessed via multiplexed optical imaging. Vascularization was reconstructed and quantified by µCT imaging after contrast agent injection. We found that high dose hyperthermia is capable of increasing the expression of GRPR, α v β 3 integrin, CD31, and Ki67 in tumors. Also the morphology of tumor vasculature changed (increased relative blood volume and small-diameter vessel density, decreased expression of α-SMA). Low dose hyperthermia induced comparatively moderate effects on the investigated protein expression pattern and vascular remodeling. We conclude that under defined circumstances, specific temperature doses affect the reorganization of tumor regrowth, which is triggered by residual "dormant" cells even though tumor volumes are transiently decreasing. Further on, GRPR, α v β 3 integrin expression are versatile tools to surveil potential tumor regrow during therapy, beyond the conventional determination of tumor volumes.

Pre-clinical evaluation of eight DOTA coupled gastrin-releasing peptide receptor (GRP-R) ligands for in vivo targeting of receptor-expressing tumors.

PubMed

Accardo, Antonella; Galli, Filippo; Mansi, Rosalba; Del Pozzo, Luigi; Aurilio, Michela; Morisco, Anna; Ringhieri, Paola; Signore, Alberto; Morelli, Giancarlo; Aloj, Luigi

2016-12-01

Overexpression of the gastrin-releasing peptide receptor (GRP-R) has been documented in several human neoplasms such as breast, prostate, and ovarian cancer. There is growing interest in developing radiolabeled peptide-based ligands toward these receptors for the purpose of in vivo imaging and radionuclide therapy of GRP-R-overexpressing tumors. A number of different peptide sequences, isotopes, and labeling methods have been proposed for this purpose. The aim of this work is to perform a direct side-by-side comparison of different GRP-R binding peptides utilizing a single labeling strategy to identify the most suitable peptide sequence. Solid-phase synthesis of eight derivatives (BN1-8) designed based on literature analysis was carried out. Peptides were coupled to the DOTA chelator through a PEG4 spacer at the N-terminus. Derivatives were characterized for serum stability, binding affinity on PC-3 human prostate cancer cells, biodistribution in tumor-bearing mice, and gamma camera imaging at 1, 6, and 24 h after injection. Serum stability was quite variable among the different compounds with half-lives ranging from 16 to 400 min at 37 °C. All compounds tested showed K d values in the nanomolar range with the exception of BN3 that showed no binding. Biodistribution and imaging studies carried out for compounds BN1, BN4, BN7, and BN8 showed targeting of the GRP-R-positive tumors and the pancreas. The BN8 compound (DOTA-PEG-DPhe-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH2) showed high affinity, the longest serum stability, and the highest target-to-background ratios in biodistribution and imaging experiments among the compounds tested. Our results indicate that the NMeGly for Gly substitution and the Sta-Leu substitution at the C-terminus confer high serum stability while maintaining high receptor affinity, resulting in biodistribution properties that outperform those of the other peptides.

Gastrin-releasing peptide stimulates glycoconjugate release from feline trachea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lundgren, J.D.; Baraniuk, J.N.; Ostrowski, N.L.

1990-02-01

The effect of gastrin-releasing peptide (GRP) on respiratory glycoconjugate (RGC) secretion was investigated in a feline tracheal organ culture model. RGC secretion was stimulated by GRP in a dose-dependent fashion at concentrations from 10(-8) to 10(-5) M (range 15-38% increase above control) with a peak effect within 0.5-1 h of incubation. GRP-(14-27), the receptor binding portion of GRP, and the related molecule, bombesin, also stimulated RGC secretion by approximately 20% above control. Acetyl-GRP-(20-27) stimulated RGC release by 10%, whereas GRP-(1-16) was inactive. Autoradiographic studies with 125I-GRP revealed that specific binding was restricted to the submucosal glands and the surface epithelium.more » A specific radioimmunoassay showed the content of GRP in feline trachea after extraction with ethanol-acetic acid to be 156 +/- 91 fmol/g wet wt. Indirect immunohistochemistry indicated that ganglion cells located just outside the cartilage contained GRP-immunoreactive materials. GRP is a novel mucus secretagogue that may participate in regulating airway mucosal gland secretion.« less

In vitro and in vivo evaluation of a 64Cu-labeled NOTA-Bn-SCN-Aoc-bombesin analogue in gastrin-releasing peptide receptor expressing prostate cancer.

PubMed

Craft, Jeffrey M; De Silva, Ravindra A; Lears, Kimberly A; Andrews, Rebecca; Liang, Kexian; Achilefu, Samuel; Rogers, Buck E

2012-07-01

Bombesin (BN) is an amphibian peptide that binds to the gastrin-releasing peptide receptor (GRPR). It has been demonstrated that BN analogues can be radiolabeled for potential diagnosis and treatment of GRPR-expressing malignancies. Previous studies have conjugated various chelators to the eight C-terminal amino acids of BN [BN(7-14)] for radiolabeling with 64Cu. Recently, (1,4,7-triazacyclononane-1,4,7-triacetic acid) (NOTA) has been evaluated as the five-coordinate 64Cu complex, with results indicating GRPR-specific tumor uptake. This study aimed to conjugate S-2-(4-isothiocyanatobenzyl)-NOTA (p-SCN-Bn-NOTA) to BN(7-14) such that it could form a six-coordinate complex with 64Cu and to evaluate the resulting peptide. p-SCN-NOTA was conjugated to 8-aminooctanoic acid (Aoc)-BN(7-14) in solution to yield NOTA-Bn-SCN-Aoc-BN(7-14). The unlabeled peptide was evaluated in a cell binding assay using PC-3 prostate cancer cells and 125I-Tyr4-BN to determine the IC50 value. The peptide was radiolabeled with 64Cu and evaluated for internalization into PC-3 cells and for tumor uptake in mice bearing PC-3 xenografts using biodistribution and micro-positron emission tomography imaging studies. The binding assay demonstrated that NOTA-Bn-SCN-Aoc-BN(7-14) bound with high affinity to GRPR with an IC50 of 1.4 nM. The radiolabeled peptide demonstrated time-dependent internalization into PC-3 cells. In vivo, the peptide demonstrated tumor-specific uptake and imaging that were comparable to those of previously reported 64Cu-labeled BN analogues. These studies demonstrate that 64Cu-NOTA-Bn-SCN-Aoc-BN(7-14) binds to GRPR-expressing cells and that it can be used for imaging of GRPR-expressing prostate cancer. Copyright © 2012 Elsevier Inc. All rights reserved.

In vitro evaluation of (99m)Tc-EDDA/tricine-HYNIC-Q-Litorin in gastrin-releasing peptide receptor positive tumor cell lines.

PubMed

Yurt Lambrecht, Fatma; Durkan, Kübra; Ozgür, Aykut; Gündüz, Cumhur; Avcı, Cığır Biray; Susluer, Sunde Yılmaz

2013-05-01

Bombesin and its derivatives exhibit a high affinity for gastrin-releasing peptide receptor (GRPr), which is over-expressed in a variety of human cancers (prostate, pancreatic, lung, etc.). The aim of this study was to investigate the in vitro potential of the hydrazinonicotinamide (HYNIC)-Q-Litorin. (99m)Tc labeling was performed by using different co-ligands: tricine and ethylenediamine diacetic acid (EDDA). The radiochemical stability of radiolabeled peptide conjugates was checked at room temperature and in cysteine solution up to 24 h. The in vitro cell uptake of (99m)Tc-EDDA-HYNIC-Q-Litorin and (99m)Tc-tricine-HYNIC-Q-Litorin were evaluated on pancreatic tumor and control cell lines. Optimum specific activity and incubation time were determined for all the cell lines. The results showed that the cell uptake of the radiolabeled peptide conjugates in tumor cell lines were higher than in the control cell line. The findings of this study indicated the need for further development of in vivo study as a radiopharmaceutical for pancreatic tumor imaging.

Expression of cholecystokinin2-receptor in rat and human L cells and the stimulation of glucagon-like peptide-1 secretion by gastrin treatment.

PubMed

Cao, Yang; Cao, Xun; Liu, Xiao-Min

2015-03-01

Gastrin is a gastrointestinal hormone secreted by G cells. Hypergastrinemia can improve blood glucose and glycosylated hemoglobin levels. These positive effects are primarily due to the trophic effects of gastrin on β-cells. In recent years, many receptors that regulate secretion of glucagon-like peptide 1 (GLP-1) have been identified in enteroendocrine L cell lines. This led us to hypothesize that, in addition to the trophic effects of gastrin on β-cells, L cells also express cholecystokinin2-receptor (CCK2R), which may regulate GLP-1 secretion and have synergistic effects on glucose homeostasis. Our research provides a preliminary analysis of CCK2R expression and the stimulating effect of gastrin treatment on GLP-1 secretion in a human endocrine L cell line, using RT-PCR, Western blot, immunocytochemistry, and ELISA analyses. The expression of proglucagon and prohormone convertase 3, which regulate GLP-1 biosynthesis, were also analyzed by real-time PCR. Double immunofluorescence labeling was utilized to assess the intracellular localization of CCK2R and GLP-1 in L cells harvested from rat colon tissue. Our results showed that CCK2R was expressed in both the human L cell line and the rat L cells. We also showed that treatment with gastrin, a CCK2R agonist, stimulated the secretion of GLP-1, and that this effect was likely due to increased expression of proglucagon and PCSK1 (also known as prohormone convertase 3 (PC3 gene)). These results not only provide a basis for the role gastrin may play in intestinal L cells, and may also provide the basis for the development of a method of gastrin-mediated glycemic regulation. Copyright © 2014 Elsevier GmbH. All rights reserved.

Characterization of ligand binding and processing by gastrin-releasing peptide receptors in a small-cell lung cancer cell line.

PubMed Central

Cardona, C; Bleehen, N M; Reeve, J G

1992-01-01

The ligand-binding properties of the gastrin-releasing peptide (GRP) receptor and the cellular processing of GRP have been studied in the small-cell lung cancer (SCLC) cell line COR-L42. Scatchard analysis of GRP receptor expression indicated a single class of high-affinity receptors (Kd 1.5 nM) and approx. 6700 receptors/cell. GRP bound to its receptor with a Ki of 2.4 nM. The bombesin-related peptides neuromedin B (NMB) and phyllolitorin also bound to GRP receptors with Ki values of 22.7 and 59.1 nM respectively. Binding of 125I-GRP to COR-L42 cells increased rapidly at 37 degrees, achieved a maximum at 10 min and declined rapidly thereafter. At 4 degrees C, maximum binding was achieved at 30 min and the subsequent decline in cell-associated radioactivity was slower than that seen at 37 degrees C. Acid/salt extraction, to separate surface-bound ligand from internalized GRP, indicated that after receptor binding 125I-GRP was rapidly internalized. To determine the pathway of 125I-GRP degradation, binding studies were carried out with the lysosomotropic agent chloroquine (5 mM), and with phosphoramidon (10 microM), an inhibitor of the membrane-bound enzyme (EC 3.4.24.11). Both agents markedly inhibited the degradation of GRP, indicating that this process involves a lysosomal pathway and a phosphoramidon-sensitive pathway, possibly involving the EC 3.4.24.11 enzyme. GRP receptor down-regulation was observed following a 10 min exposure to 100 nM-GRP. With longer pretreatment times the number of binding sites recovered to 80% of control values. Treatment with 5 mM-chloroquine plus GRP or cycloheximide (10 micrograms/ml) plus GRP demonstrated that the majority of GRP receptors are recycled. NMB and phyllolitorin pretreatment did not influence the subsequent binding of 125I-GRP, suggesting that these peptides do not down-regulate GRP receptors. PMID:1310003

Cloning and characterization of cDNAs encoding human gastrin-releasing peptide.

PubMed Central

Spindel, E R; Chin, W W; Price, J; Rees, L H; Besser, G M; Habener, J F

1984-01-01

We have prepared and cloned cDNAs derived from poly(A)+ RNA from a human pulmonary carcinoid tumor rich in immunoreactivity to gastrin-releasing peptide, a peptide closely related in structure to amphibian bombesin. Mixtures of synthetic oligodeoxyribonucleotides corresponding to amphibian bombesin were used as hybridization probes to screen a cDNA library prepared from the tumor RNA. Sequencing of the recombinant plasmids shows that human gastrin-releasing peptide (hGRP) mRNA encodes a precursor of 148 amino acids containing a typical signal sequence, hGRP consisting of 27 or 28 amino acids, and a carboxyl-terminal extension peptide. hGRP is flanked at its carboxyl terminus by two basic amino acids, following a glycine used for amidation of the carboxyl-terminal methionine. RNA blot analyses of tumor RNA show a major mRNA of 900 bases and a minor mRNA of 850 bases. Blot hybridization analyses using human genomic DNA are consistent with a single hGRP-encoding gene. The presence of two mRNAs encoding the hGRP precursor protein in the face of a single hGRP gene raises the possibility of alternative processing of the single RNA transcript. Images PMID:6207529

Preparation and evaluation of 99mTc-EDDA/HYNIC-[Lys 3]-bombesin for imaging gastrin-releasing peptide receptor-positive tumours.

PubMed

Ferro-Flores, Guillermina; Arteaga de Murphy, Consuelo; Rodriguez-Cortés, Jeanette; Pedraza-López, Martha; Ramírez-Iglesias, María Teresa

2006-04-01

Bombesin is a peptide that was initially isolated from frog skin and which belongs to a large group of neuropeptides with many biological functions. The human equivalent is gastrin-releasing peptide (GRP), whose receptors are over-expressed in a variety of malignant tumours. To prepare a HYNIC-[Lys 3]-bombesin analogue that could be easily labelled with 99mTc from lyophilized kit formulations and to evaluate its potential as an imaging agent for GRP receptor-positive tumours. HYNIC was conjugated to the epsilon-amino group of Lys 3 residue at the N-terminal region of bombesin via succinimidyl-N-Boc-HYNIC at pH 9.0. 99mTc labelling was performed by addition of sodium pertechnetate solution and 0.2 M phosphate buffer pH 7.0 to a lyophilized formulation. Stability studies were carried out by reversed phase HPLC and ITLC-SG analyses in serum and cysteine solutions. In-vitro internalization was tested using human prostate cancer PC-3 cells with blocked and non-blocked receptors. Biodistribution and tumour uptake were determined in PC-3 tumour-bearing nude mice. 99mTc-EDDA/HYNIC-[Lys 3]-bombesin was obtained with radiochemical purities >93% and high specific activity ( approximately 0.1 GBq.nmol). Results of in-vitro studies demonstrated a high stability in serum and cysteine solutions, specific cell receptor binding and rapid internalization. Biodistribution data showed a rapid blood clearance, with predominantly renal excretion and specific binding towards GRP receptor-positive tissues such as pancreas and PC-3 tumours. 99mTc-EDDA/HYNIC-[Lys 3]-bombesin obtained from lyophilized kit formulations has promising characteristics for the diagnosis of malignant tumours that over-express the GRP receptor.

Bombesin-like peptides stimulate growth hormone secretion mediated by the gastrin-releasing peptide receptor in cattle.

PubMed

Zhao, Hongqiong; Matsuda, Seinosuke; Thanthan, Sint; Yannaing, Swe; Kuwayama, Hideto

2012-10-01

This study was designed to determine the effects of bombesin-like peptides (BLPs) on the secretion of growth hormone (GH) and to characterize the receptor subtypes mediating these effects in cattle. Four experiments were conducted: (1) six steers were randomly assigned to receive intravenous (IV) bolus injections of 0, 0.2, 1.0, 12.5 and 50.0 μg/kg neuromedin C (NMC); (2) seven pre-weaned calves were IV injected with 1.0 μg/kg NMC; (3) six steers were IV injected with 2.5μg/kg bovine gastrin-releasing peptide (GRP), 1.0 μg/kg NMC combined with 20.0 μg/kg [d-Lys(3)]-GHRP-6 (an antagonist for the GH secretagogue receptor type 1a [GHS-R1a]), 1.0 μg/kg NMC combined with 20.0 μg/kg N-acetyl-GRP(20-26)-OCH(2)CH(3) (N-GRP-EE, an antagonist for the GRP receptor), 20.0 μg/kg N-GRP-EE alone, 1.0 μg/kg neuromedin B (NMB); and (4) four rats were IV injected 1.0 μg/kg NMC. A serial blood sample was collected before and after injection. Plasma GH levels dose-dependently increased at 5 min after NMC injection and the minimal effective dose was 1.0 μg/kg. Plasma GH level was elevated by GRP, but not by NMB. The NMC-induced elevation of GH was completely blocked by N-GRP-EE. The administration of NMC elevated GH level in pre-weaned calves but not in rats. Ghrelin level was unaffected by any treatments; and [d-Lys(3)]-GHRP-6 did not block the NMC-induced elevation of GH. The results indicate BLP-induced elevation of GH levels is mediated by the GRP receptor but not through a ghrelin/GHS-R1a pathway in cattle. Copyright © 2012 Elsevier Inc. All rights reserved.

Gastrin-releasing peptide receptor (GRPr) promotes EMT, growth, and invasion in canine prostate cancer.

PubMed

Elshafae, Said M; Hassan, Bardes B; Supsavhad, Wachiraphan; Dirksen, Wessel P; Camiener, Rachael Y; Ding, Haiming; Tweedle, Michael F; Rosol, Thomas J

2016-06-01

The gastrin-releasing peptide receptor (GRPr) is upregulated in early and late-stage human prostate cancer (PCa) and other solid tumors of the mammary gland, lung, head and neck, colon, uterus, ovary, and kidney. However, little is known about its role in prostate cancer. This study examined the effects of a heterologous GRPr agonist, bombesin (BBN), on growth, motility, morphology, gene expression, and tumor phenotype of an osteoblastic canine prostate cancer cell line (Ace-1) in vitro and in vivo. The Ace-1 cells were stably transfected with the human GRPr and tumor cells were grown in vitro and as subcutaneous and intratibial tumors in nude mice. The effect of BBN was measured on cell proliferation, cell migration, tumor growth (using bioluminescence), tumor cell morphology, bone tumor phenotype, and epithelial-mesenchymal transition (EMT) and metastasis gene expression (quantitative RT-PCR). GRPr mRNA expression was measured in primary canine prostate cancers and normal prostate glands. Bombesin (BBN) increased tumor cell proliferation and migration in vitro and tumor growth and invasion in vivo. BBN upregulated epithelial-to-mesenchymal transition (EMT) markers (TWIST, SNAIL, and SLUG mRNA) and downregulated epithelial markers (E-cadherin and β-catenin mRNA), and modified tumor cell morphology to a spindle cell phenotype. Blockade of GRPr upregulated E-cadherin and downregulated VIMENTIN and SNAIL mRNA. BBN altered the in vivo tumor phenotype in bone from an osteoblastic to osteolytic phenotype. Primary canine prostate cancers had increased GRPr mRNA expression compared to normal prostates. These data demonstrated that the GRPr is important in prostate cancer growth and progression and targeting GRPr may be a promising strategy for treatment of prostate cancer. Prostate 76:796-809, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Expression of the gastrin-releasing peptide receptor, the prostate stem cell antigen and the prostate-specific membrane antigen in lymph node and bone metastases of prostate cancer.

PubMed

Ananias, Hildo J K; van den Heuvel, Marius C; Helfrich, Wijnand; de Jong, Igle J

2009-07-01

Cell membrane antigens like the gastrin-releasing peptide receptor (GRPR), the prostate stem cell antigen (PSCA), and the prostate-specific membrane antigen (PSMA), expressed in prostate cancer, are attractive targets for new therapeutic and diagnostic applications. Therefore, we investigated in this study whether these antigens are expressed in metastasized prostate cancer. Formalin-fixed, paraffin-embedded specimens of 15 patients with uni- or bilateral lymph node metastases of prostate cancer (totaling 21 cases) and 17 patient-cases of bone metastases were processed for immunohistochemistry with anti-GRPR, anti-PSCA, and anti-PSMA antibodies. A pathologist blinded to clinical and pathological data scored the immunoreactivity for these antibodies on a four-point scale (0 = no staining; 1+ = weak staining; 2+ = moderate staining; 3+ = strong staining) and documented the distribution pattern. GRPR staining in lymph node metastases was seen in 85.7% of cases (18 of 21 cases), PSCA in 95.2% (20/21), and PSMA in 100% (21/21). GRPR in bone metastases was seen in 52.9% of cases (9/17), PSCA in 94.1% (16/17), and PSMA in 100% (17/17). We have shown for the first time that GRPR is expressed in the vast majority of lymph node metastases and in 52.9% of bone metastases of prostate cancer. PSCA and PSMA are both highly expressed in lymph node and bone metastases. Although PSCA and PSMA are mostly expressed in prostate cancer metastases, GRPR offers an interesting alternative target as it can be targeted relatively easy with peptide-based (radio)pharmaceuticals. (c) 2009 Wiley-Liss, Inc.

Gastrin-Releasing Peptide (GRP) in the Ovine Uterus: Regulation by Interferon Tau and Progesterone1

PubMed Central

Song, Gwonhwa; Satterfield, M. Carey; Kim, Jinyoung; Bazer, Fuller W.; Spencer, Thomas E.

2008-01-01

Gastrin-releasing peptide (GRP) is abundantly expressed by endometrial glands of the ovine uterus and processed into different bioactive peptides, including GRP1-27, GRP18-27, and a C-terminus, that affect cell proliferation and migration. However, little information is available concerning the hormonal regulation of endometrial GRP and expression of GRP receptors in the ovine endometrium and conceptus. These studies determined the effects of pregnancy, progesterone (P4), interferon tau (IFNT), placental lactogen (CSH1), and growth hormone (GH) on expression of GRP in the endometrium and GRP receptors (GRPR, NMBR, BRS3) in the endometrium, conceptus, and placenta. In pregnant ewes, GRP mRNA and protein were first detected predominantly in endometrial glands after Day 10 and were abundant from Days 18 through 120 of gestation. Treatment with IFNT and progesterone but not CSH1 or GH stimulated GRP expression in the endometrial glands. Western blot analyses identified proGRP in uterine luminal fluid and allantoic fluid from Day 80 unilateral pregnant ewes but not in uterine luminal fluid of either cyclic or early pregnant ewes. GRPR mRNA was very low in the Day 18 conceptus and undetectable in the endometrium and placenta; NMBR and BRS3 mRNAs were undetectable in ovine uteroplacental tissues. Collectively, the present studies validate GRP as a novel IFNT-stimulated gene in the glands of the ovine uterus, revealed that IFNT induction of GRP is dependent on P4, and found that exposure of the ovine uterus to P4 for 20 days induces GRP expression in endometrial glands. PMID:18448839

New Gastrin Releasing Peptide Receptor-Directed [99mTc]Demobesin 1 Mimics: Synthesis and Comparative Evaluation.

PubMed

Nock, Berthold A; Charalambidis, David; Sallegger, Werner; Waser, Beatrice; Mansi, Rosalba; Nicolas, Guillaume P; Ketani, Eleni; Nikolopoulou, Anastasia; Fani, Melpomeni; Reubi, Jean-Claude; Maina, Theodosia

2018-04-12

We have previously reported on the gastrin releasing peptide receptor (GRPR) antagonist [ 99m Tc]1, ([ 99m Tc]demobesin 1, 99m Tc-[N 4 '-diglycolate-dPhe 6 ,Leu-NHEt 13 ]BBN(6-13)). [ 99m Tc]1 has shown superior biological profile compared to analogous agonist-based 99m Tc-radioligands. We herein present a small library of [ 99m Tc]1 mimics generated after structural modifications in (a) the linker ([ 99m Tc]2, [ 99m Tc]3, [ 99m Tc]4), (b) the peptide chain ([ 99m Tc]5, [ 99m Tc]6), and (c) the C-terminus ([ 99m Tc]7 or [ 99m Tc]8). The effects of above modifications on the biological properties of analogs were studied in PC-3 cells and tumor-bearing SCID mice. All analogs showed subnanomolar affinity for the human GRPR, while most receptor-affine 4 and 8 behaved as potent GRPR antagonists in a functional internalization assay. In mice bearing PC-3 tumors, [ 99m Tc]1-[ 99m Tc]6 exhibited GRPR-specific tumor uptake, rapidly clearing from normal tissues. [ 99m Tc]4 displayed the highest tumor uptake (28.8 ± 4.1%ID/g at 1 h pi), which remained high even after 24 h pi (16.3 ± 1.8%ID/g), well surpassing that of [ 99m Tc]1 (5.4 ± 0.7%ID/g at 24 h pi).

Gastrin-Releasing Peptide and Glucose Metabolism Following Pancreatitis.

PubMed

Pendharkar, Sayali A; Drury, Marie; Walia, Monika; Korc, Murray; Petrov, Maxim S

2017-08-01

Gastrin-releasing peptide (GRP) is a pluripotent peptide that has been implicated in both gastrointestinal inflammatory states and classical chronic metabolic diseases such as diabetes. Abnormal glucose metabolism (AGM) after pancreatitis, an exemplar inflammatory disease involving the gastrointestinal tract, is associated with persistent low-grade inflammation and altered secretion of pancreatic and gut hormones as well as cytokines. While GRP is involved in secretion of many of them, it is not known whether GRP has a role in AGM. Therefore, we aimed to investigate the association between GRP and AGM following pancreatitis. Fasting blood samples were collected to measure GRP, blood glucose, insulin, amylin, glucagon, pancreatic polypeptide (PP), somatostatin, cholecystokinin, gastric-inhibitory peptide (GIP), gastrin, ghrelin, glicentin, glucagon-like peptide-1 and 2, oxyntomodulin, peptide YY (PYY), secretin, vasoactive intestinal peptide, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein (MCP)-1, and interleukin-6. Modified Poisson regression analysis and linear regression analyses were conducted. Four statistical models were used to adjust for demographic, metabolic, and pancreatitis-related risk factors. A total of 83 individuals after an episode of pancreatitis were recruited. GRP was significantly associated with AGM, consistently in all four models (P -trend < 0.05), and fasting blood glucose contributed 17% to the variance of GRP. Further, GRP was significantly associated with glucagon (P < 0.003), MCP-1 (P < 0.025), and TNF-α (P < 0.025) - consistently in all four models. GRP was also significantly associated with PP and PYY in three models (P < 0.030 for both), and with GIP and glicentin in one model (P = 0.001 and 0.024, respectively). Associations between GRP and other pancreatic and gut hormones were not significant. GRP is significantly increased in patients with AGM after pancreatitis and is associated with increased levels of pro

Gastrin-Releasing Peptide and Glucose Metabolism Following Pancreatitis

PubMed Central

Pendharkar, Sayali A.; Drury, Marie; Walia, Monika; Korc, Murray; Petrov, Maxim S.

2017-01-01

Background Gastrin-releasing peptide (GRP) is a pluripotent peptide that has been implicated in both gastrointestinal inflammatory states and classical chronic metabolic diseases such as diabetes. Abnormal glucose metabolism (AGM) after pancreatitis, an exemplar inflammatory disease involving the gastrointestinal tract, is associated with persistent low-grade inflammation and altered secretion of pancreatic and gut hormones as well as cytokines. While GRP is involved in secretion of many of them, it is not known whether GRP has a role in AGM. Therefore, we aimed to investigate the association between GRP and AGM following pancreatitis. Methods Fasting blood samples were collected to measure GRP, blood glucose, insulin, amylin, glucagon, pancreatic polypeptide (PP), somatostatin, cholecystokinin, gastric-inhibitory peptide (GIP), gastrin, ghrelin, glicentin, glucagon-like peptide-1 and 2, oxyntomodulin, peptide YY (PYY), secretin, vasoactive intestinal peptide, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein (MCP)-1, and interleukin-6. Modified Poisson regression analysis and linear regression analyses were conducted. Four statistical models were used to adjust for demographic, metabolic, and pancreatitis-related risk factors. Results A total of 83 individuals after an episode of pancreatitis were recruited. GRP was significantly associated with AGM, consistently in all four models (P -trend < 0.05), and fasting blood glucose contributed 17% to the variance of GRP. Further, GRP was significantly associated with glucagon (P < 0.003), MCP-1 (P < 0.025), and TNF-α (P < 0.025) - consistently in all four models. GRP was also significantly associated with PP and PYY in three models (P < 0.030 for both), and with GIP and glicentin in one model (P = 0.001 and 0.024, respectively). Associations between GRP and other pancreatic and gut hormones were not significant. Conclusion GRP is significantly increased in patients with AGM after pancreatitis and is

Gastrin-releasing peptide receptor-induced internalization, down-regulation, desensitization, and growth: possible role for cyclic AMP.

PubMed

Benya, R V; Fathi, Z; Kusui, T; Pradhan, T; Battey, J F; Jensen, R T

1994-08-01

Stimulation of the gastrin-releasing peptide receptor (GRP-R) in Swiss 3T3 cells resembles that of a number of other recently described G protein-coupled receptors, insofar as both the phospholipase C and adenylyl cyclase signal transduction pathways are activated. GRP-R activation induces numerous alterations in both the cell and the receptor, but because two signal transduction pathways are activated it is difficult to determine the specific contributions of either pathway. We have found that BALB/3T3 fibroblasts transfected with the coding sequence for the GRP-R are pharmacologically indistinguishable from native receptor-expressing cells and activate phospholipase C in a manner similar to that of the native receptor but fail to increase cAMP in response to bombesin; thus, they may be useful cells to explore the role of activation of each pathway in altering cell and receptor function. Swiss 3T3 cells and GRP-R-transfected BALB/3T3 cells expressed identically glycosylated receptors that bound various agonists and antagonists similarly. G protein activation, as determined by evaluation of agonist-induced activation of phospholipase C and by analysis of the effect of guanosine-5'-(beta,gamma-imido)triphosphate on GRP-R binding affinity, was indistinguishable. Agonist stimulation of GRP-R caused similar receptor changes (internalization and down-regulation) and homologous desensitization in both cell types. Bombesin stimulation of Swiss 3T3 cells that had been preincubated with forskolin increased cAMP levels 9-fold, but no bombesin-specific increase in cAMP levels was detected in transfected cells, even though forskolin and cholera toxin increased cAMP levels in these cells. Quiescent Swiss 3T3 cells treated with bombesin rapidly increased c-fos mRNA levels and [3H]thymidine incorporation, whereas both effects were potentiated by forskolin. The specific protein kinase A inhibitor H-89 blocked increases in c-fos levels and [3H]thymidine incorporation induced by low

Distinct functions of opioid-related peptides and gastrin-releasing peptide in regulating itch and pain in the spinal cord of primates.

PubMed

Lee, Heeseung; Ko, Mei-Chuan

2015-06-29

How neuropeptides in the primate spinal cord regulate itch and pain is largely unknown. Here we elucidate the sensory functions of spinal opioid-related peptides and gastrin-releasing peptide (GRP) in awake, behaving monkeys. Following intrathecal administration, β-endorphin (10-100 nmol) and GRP (1-10 nmol) dose-dependently elicit the same degree of robust itch scratching, which can be inhibited by mu-opioid peptide (MOP) receptor and GRP receptor (BB2) antagonists, respectively. Unlike β-endorphin, which produces itch and attenuates inflammatory pain, GRP only elicits itch without affecting pain. In contrast, enkephalins (100-1000 nmol) and nociceptin-orphanin FQ (3-30 nmol) only inhibit pain without eliciting itch. More intriguingly, dynorphin A(1-17) (10-100 nmol) dose-dependently attenuates both β-endorphin- and GRP-elicited robust scratching without affecting pain processing. The anti-itch effects of dynorphin A can be reversed by a kappa-opioid peptide (KOP) receptor antagonist nor-binaltorphimine. These nonhuman primate behavioral models with spinal delivery of ligands advance our understanding of distinct functions of neuropeptides for modulating itch and pain. In particular, we demonstrate causal links for itch-eliciting effects by β-endorphin-MOP receptor and GRP-BB2 receptor systems and itch-inhibiting effects by the dynorphin A-KOP receptor system. These studies will facilitate transforming discoveries of novel ligand-receptor systems into future therapies as antipruritics and/or analgesics in humans.

Bombesin-like peptide receptors in human bronchial epithelial cells.

PubMed

Kane, M A; Toi-Scott, M; Johnson, G L; Kelley, K K; Boose, D; Escobedo-Morse, A

1996-01-01

Northern blot and RNAse protection assays previously failed to detect bombesin-like peptide (BLP) receptors in normal human lung tissue, but by RT/PCR cultured human bronchial epithelial (HBE) cells expressed all three BLP receptor subtypes, predominantly neuromedin B (NMB) receptor. By RT/PCR, we found expression of all three BLP receptor subtypes by human lung tissue and confirmed NMB receptor expression in six out of six HBE samples. However, transformed HBE BEAS B2B cells expressed only gastrin-releasing peptide (GRP) receptors; saturable, high-affinity (Kd = 3.5 nM) specific [125I]GRP binding confirmed functional GRP receptor, with M(r) = 75 kDa and immunologic cross-reactivity with GRP receptor from human small-cell lung carcinoma (SCLC) NCI-H345 cells. Altered regulation of BLP receptors may accompany transformation of normal lung cells to cancer.

The effect of the mammalian neuropeptide, gastrin-releasing peptide (GRP), on gastrointestinal and pancreatic hormone secretion in man.

PubMed

Wood, S M; Jung, R T; Webster, J D; Ghatei, M A; Adrian, T E; Yanaihara, N; Yanaihara, C; Bloom, S R

1983-10-01

Gastrin-releasing peptide, a newly isolated mammalian peptide similar in its structure and actions to the amphibian peptide, bombesin, has recently been localized to nerves in the brain, gut and pancreas. The present study investigates its effects on gut and pancreatic peptides in man. Intravenous infusion of 0.7 and 2.9 pmol min-1 kg-1 produced significant elevation of plasma gastrin, cholecystokinin-like immunoreactivity and neurotensin. It was found also to potentiate glucose-dependent insulin secretion. Its specific location in nerve fibres in the proximal gut and pancreas and its selective effect on gastroenteropancreatic peptides may favour its role as a physiological regulatory neuropeptide.

Neuromedin B and gastrin releasing peptide excite arcuate nucleus neuropeptide Y neurons in a novel transgenic mouse expressing strong renilla GFP in NPY neurons

PubMed Central

van den Pol, Anthony N.; Yao, Yang; Fu, Li-Ying; Foo, Kylie; Huang, Hao; Coppari, Roberto; Lowell, Brad; Broberger, Christian

2009-01-01

Neuropeptide Y (NPY) is one of the most widespread neuropeptides in the brain. Transgenic mice were generated that expressed bright renilla GFP in most or all of the known NPY cells in the brain, which otherwise were not identifiable. GFP expression in NPY cells was confirmed with immunocytochemistry and single cell RT-PCR. NPY neurons in the hypothalamic arcuate nucleus play an important role in energy homeostasis and endocrine control. Whole cell patch clamp recording was used to study identified arcuate NPY cells. Primary agents that regulate energy balance include melanocortin receptor agonists, AgRP, and cannabinoids; none of these substances substantially influenced electrical properties of NPY neurons. In striking contrast, neuropeptides of the bombesin family, including gastrin releasing peptide and neuromedin B which are found in axons in the arcuate nucleus and may also be released from the gut to signal the brain, showed strong direct excitatory actions at nanomolar levels on the NPY neurons, stronger than the actions of ghrelin and hypocretin/orexin. Bombesin-related peptides reduced input resistance and depolarized the membrane potential. The depolarization was attenuated by several factors: substitution of choline for sodium, extracellular Ni2+, inclusion of BAPTA in the pipette, KB-R7943 and SKF96365. Reduced extracellular calcium enhanced the current, which reversed around − 20 mV. Together, these data suggest two mechanisms, activation of non-selective cation channels and the sodium/calcium exchanger. Since both NPY and POMC neurons, which we also studied, are similarly directly excited by bombesin-like peptides, the peptides may function to initiate broad activation, rather than the cell-type selective activation or inhibition reported for many other compounds that modulate energy homeostasis. PMID:19357287

Effect of porcine gastrin releasing peptide on gastric secretion and motility and the release of hormonal peptides in conscious cats.

PubMed

Vagne, M; Collinet, M; Cuber, J C; Bernard, C; Chayvialle, J A; McDonald, T J; Mutt, V

1987-01-01

The effect of porcine gastrin releasing peptide (GRP) was compared to those of bombesin (BBS) and pentagastrin (PG) in conscious cats. GRP and BBS augmented acid and pepsin secretions, as well as antral motility with an early effect comparable to that produced by pentagastrin with an elevation of low amplitude contractions and a diminution of high amplitude contractions. BBS and GRP increased plasma gastrin and pancreatic polypeptide (PP) levels and decreased motilin levels measured by a C terminus-directed antiserum. In all cases, BBS and GRP displayed parallel dose-response curves. PG showed slight differences in the slopes of the dose-response curves slopes of the dose-response curves except for acid secretion stimulation where no difference was noted (PG was the most effective) and for pepsin stimulation where the difference was large (PG was much less effective). According to the different targets studied, BBS was 4 to 9 times more potent than GRP, 6 to 200 times more than PG. Gastrin release, elicited by the lowest ED50 of both BBS and GRP, should be considered as their primary effect in the cat.

Histone deacetylase inhibition prevents the impairing effects of hippocampal gastrin-releasing peptide receptor antagonism on memory consolidation and extinction.

PubMed

Petry, Fernanda S; Dornelles, Arethuza S; Lichtenfels, Martina; Valiati, Fernanda E; de Farias, Caroline Brunetto; Schwartsmann, Gilberto; Parent, Marise B; Roesler, Rafael

2016-07-01

Hippocampal gastrin-releasing peptide receptors (GRPR) regulate memory formation and extinction, and disturbances in GRPR signaling may contribute to cognitive impairment associated with neurodevelopmental disorders. Histone acetylation is an important epigenetic mechanism that regulates gene expression involved in memory formation, and histone deacetylase inhibitors (HDACis) rescue memory deficits in several models. The present study determined whether inhibiting histone deacetylation would prevent memory impairments produced by GRPR blockade in the hippocampus. Male Wistar rats were given an intrahippocampal infusion of saline (SAL) or the HDACi sodium butyrate (NaB) shortly before inhibitory avoidance (IA) training, followed by an infusion of either SAL or the selective GRPR antagonist RC-3095 immediately after training. In a second experiment, the infusions were administered before and after a retention test trial that served as extinction training. As expected, RC-3095 significantly impaired consolidation and extinction of IA memory. More importantly, pretraining administration of NaB, at a dose that had no effect when given alone, prevented the effects of RC-3095. In addition, the combination of NaB and RC-3095 increased hippocampal levels of the brain-derived neurotrophic factor (BDNF). These findings indicate that HDAC inhibition can protect against memory impairment caused by GRPR blockade. Copyright © 2016 Elsevier B.V. All rights reserved.

Gastrin-releasing peptide and its receptor increase arthritis fibroblast-like synoviocytes invasiveness through activating the PI3K/AKT pathway.

PubMed

Clarimundo, Vanessa Schuck; Farinon, Mirian; Pedó, Renata Ternus; Teixeira, Vivian Oliveira Nunes; Nör, Carolina; Gulko, Percio S; Xavier, Ricardo Machado; de Oliveira, Patricia Gnieslaw

2017-09-01

Rheumatoid arthritis (RA) is an autoimmune disease that leads to joint destruction. The fibroblast-like synoviocytes (FLS) has a central role on the disease pathophysiology. The present study aimed to examine the role of gastrin-releasing peptide (GRP) and its receptor (GRPR) on invasive behavior of mice fibroblast-like synoviocytes (FLS), as well as to evaluate GRP-induced signaling on PI3K/AKT pathway. The expression of GRPR in FLS was investigated by immunocytochemistry, western blot (WB) and qRT-PCR. The proliferation and invasion were assessed by SRB and matrigel-transwell assay after treatment with GRP and/or RC-3095 (GRPR antagonist), and/or Ly294002 (inhibitor of PI3K/AKT pathway). Finally, AKT phosphorylation was assessed by WB. GRPR protein was detected in FLS and the exposure to GRP increased FLS invasion by nearly two-fold, compared with untreated cells (p<0.05), while RC-3095 reversed that effect (p<0.001). GRP also increased phosphorylated AKT expression in FLS. When Ly294002 was added with GRP, it prevented the GRP-induced increased cell invasiveness (p<0.001). These data suggest that GRPR expression in FLS and that exogenous GRP are able to activate FLS invasion. This effect occurs at least in part through the AKT activation. Therefore, understanding of the GRP/GRPR pathway could be relevant in the development of FLS-targeted therapy for RA. Copyright © 2017 Elsevier Inc. All rights reserved.

Neuromedin B and gastrin-releasing peptide excite arcuate nucleus neuropeptide Y neurons in a novel transgenic mouse expressing strong Renilla green fluorescent protein in NPY neurons.

PubMed

van den Pol, Anthony N; Yao, Yang; Fu, Li-Ying; Foo, Kylie; Huang, Hao; Coppari, Roberto; Lowell, Bradford B; Broberger, Christian

2009-04-08

Neuropeptide Y (NPY) is one of the most widespread neuropeptides in the brain. Transgenic mice were generated that expressed bright Renilla green fluorescent protein (GFP) in most or all of the known NPY cells in the brain, which otherwise were not identifiable. GFP expression in NPY cells was confirmed with immunocytochemistry and single-cell reverse transcription-PCR. NPY neurons in the hypothalamic arcuate nucleus play an important role in energy homeostasis and endocrine control. Whole-cell patch clamp recording was used to study identified arcuate NPY cells. Primary agents that regulate energy balance include melanocortin receptor agonists, AgRP, and cannabinoids; none of these substances substantially influenced electrical properties of NPY neurons. In striking contrast, neuropeptides of the bombesin family, including gastrin-releasing peptide and neuromedin B, which are found in axons in the mediobasal hypothalamus and may also be released from the gut to signal the brain, showed strong direct excitatory actions at nanomolar levels on the NPY neurons, stronger than the actions of ghrelin and hypocretin/orexin. Bombesin-related peptides reduced input resistance and depolarized the membrane potential. The depolarization was attenuated by several factors: substitution of choline for sodium, extracellular Ni(2+), inclusion of BAPTA in the pipette, KB-R7943, and SKF96365. Reduced extracellular calcium enhanced the current, which reversed around -20 mV. Together, these data suggest two mechanisms, activation of nonselective cation channels and the sodium/calcium exchanger. Since both NPY and POMC neurons, which we also studied, are similarly directly excited by bombesin-like peptides, the peptides may function to initiate broad activation, rather than the cell-type selective activation or inhibition reported for many other compounds that modulate energy homeostasis.

Metabolism and inactivation of gastrin releasing peptide by endopeptidase-24.11 in the dog.

PubMed

Bunnett, N W; Turner, A J; Debas, H T

1989-09-01

The purpose of this investigation was to examine the metabolism and inactivation of gastrin releasing peptide 10 (GRP10) by endopeptidase-24.11 prepared from the stomach wall. GRP10 was metabolized in vitro by gastric endopeptidase-24.11. The metabolites were purified by high-pressure liquid chromatography and identified as (1-8) GRP10 and (9-10) GRP10 by amino acid analysis, indicating hydrolysis of the His8-Leu9 bond. The intravenous administration of GRP10 to conscious dogs stimulated gastrin release, gastric acid secretion, pancreatic protein secretion and pancreatic bicarbonate secretion. Incubation of GRP10 with endopeptidase-24.11 significantly diminished the biological activity of the digests compared to control digests containing heat-inactivated enzyme. This effect was abolished by the enzyme inhibitor phosphoramidon. It is concluded that endopeptidase-24.11 from the stomach metabolizes and inactivates GRP10.

Expression of matrix metalloproteinase-9 and bombesin/gastrin-releasing peptide in human prostate cancers and their lymph node metastases.

PubMed

Ishimaru, Hisashi; Kageyama, Yukio; Hayashi, Tetsuo; Nemoto, Tetsuo; Eishi, Yoshinobu; Kihara, Kazunori

2002-01-01

Neuroendocrine differentiation and subsequent excretion of neuropeptides have been demonstrated to be associated with progression of human prostate cancer. Among neuropeptides found to exist in the prostate, bombesin/gastrin-releasing peptide has been shown to upregulate matrix metalloproteinase-9 (MMP-9) in human prostate cancer cell lines. Expression levels of bombesin, MMP-9, and neuron-specific enolase were examined by immunohistochemistry in 41 cases of clinically organ-confined prostate cancers including 9 with microscopic lymph node metastases. Twenty-seven (64%) of the 41 radical prostatectomy specimens were positive for both MMP-9 and bombesin. Expression of these molecules was observed in almost the same population of the cancer cells. The remaining 14 cases were negative for both MMP-9 and bombesin. High-grade tumors (Gleason sum > or = 7) were more likely to express MMP-9 and bombesin (21/24:88%) than low-grade tumors (Gleason sum > or = 6) (7/17:41%). In eight of the nine cases with pathological lymph node metastases, expression of MMP-9 and bombesin was also noted in metastatic sites. Neuron-specific enolase was positive in 16 cases (39%) and not always associated with the expression of bombesin. Expression of bombesin and expression of MMP-9 are common in human prostate cancers and may be related to an aggressive phenotype.

Radiometal-Dependent Biological Profile of the Radiolabeled Gastrin-Releasing Peptide Receptor Antagonist SB3 in Cancer Theranostics: Metabolic and Biodistribution Patterns Defined by Neprilysin.

PubMed

Lymperis, Emmanouil; Kaloudi, Aikaterini; Sallegger, Werner; Bakker, Ingrid L; Krenning, Eric P; de Jong, Marion; Maina, Theodosia; Nock, Berthold A

2018-05-16

Recent advances in oncology involve the use of diagnostic/therapeutic radionuclide-carrier pairs that target cancer cells, offering exciting opportunities for personalized patient treatment. Theranostic gastrin-releasing peptide receptor (GRPR)-directed radiopeptides have been proposed for the management of GRPR-expressing prostate and breast cancers. We have recently introduced the PET tracer 68 Ga-SB3 (SB3, DOTA- p-aminomethylaniline-diglycolic acid-DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt), a receptor-radioantagonist that enables the visualization of GRPR-positive lesions in humans. Aiming to fully assess the theranostic potential of SB3, we herein report on the impact of switching 68 Ga to 111 In/ 177 Lu-label on the biological properties of resulting radiopeptides. Notably, the bioavailability of 111 In/ 177 Lu-SB3 in mice drastically deteriorated compared with metabolically robust 68 Ga-SB3, and as a result led to poorer 111 In/ 177 Lu-SB3 uptake in GRPR-positive PC-3 xenografts. The peptide cleavage sites were identified by chromatographic comparison of blood samples from mice intravenously receiving 111 In/ 177 Lu-SB3 with each of newly synthesized 111 In/ 177 Lu-SB3-fragments. Coinjection of the radioconjugates with the neprilysin (NEP)-inhibitor phosphoramidon led to full stabilization of 111 In/ 177 Lu-SB3 in peripheral mouse blood and resulted in markedly enhanced radiolabel uptake in the PC-3 tumors. In conclusion, in situ NEP-inhibition led to indistinguishable 68 Ga/ 111 In/ 177 Lu-SB3 profiles in mice emphasizing the theranostic prospects of SB3 for clinical use.

Bcl-2-independent induction of apoptosis by neuropeptide receptor antagonist in human small cell lung carcinoma cells.

PubMed

Matsumoto, Y; Kawatani, M; Simizu, S; Tanaka, T; Takada, M; Imoto, M

2000-01-01

The broad-spectrum antagonist of neuropeptide receptor, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P, induced apoptosis selectively in human small cell lung carcinoma (SCLC) cells, which express gastrin-releasing peptide receptor, but not in other types of tumor cells as well as normal cells. The addition of gastrin-releasing peptide or bombesin and the inhibitor of caspase-3 suppressed [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P-induced apoptosis. Moreover, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P-induced apoptosis was not suppressed by Bcl-2 over-expression. Thus, blockage of gastrin-releasing peptide receptor-mediated signaling may provide a novel therapeutic option in SCLC which has become resistant to conventional chemotherapeutic agents.

Processing of mammalian preprogastrin-releasing peptide.

PubMed

Reeve, J R; Cuttitta, F; Vigna, S R; Shively, J E; Walsh, J H

1988-01-01

The processing of preprogastrin-releasing peptide in mammalian tissues and in cultured cells takes place at discrete sites (Figure 6). Signal peptidase cleaves away the signal peptide from the amino terminus of gastrin-releasing peptide. An exopeptidase activity may remove dipeptides from the amino terminus. The amidation site (not shown in Fig. 6; see Fig. 2) has the same general sequence (Gly-Lys-Lys) seen for other amidated peptides. Cleavage after single basic residues yields gene-related products from Form I or II preproGRP. A unique non-basic cleavage yields a gene-related product from Form III preproGRP. The processing that occurs to form GRP, GRP, and GRP gene-related peptides is shown in Figure 7. ProGRP is cleaved by a series of enzymes to form GRP with an amidated carboxyl-terminal methionine (indicated by an asterisk in Fig. 7). GRP is cleaved to form the decapeptide GRP. The carboxyl-terminal flanking peptides of all three mRNA translation products are cleaved to form several gastrin-releasing peptide gene-related products. Knowledge of the processing of gastrin-releasing peptide and its gene-related products will allow synthesis of duplicates of the stored forms of these peptides, which can then be used for biological testing.

Targeting gastrin-releasing peptide as a new approach to treat aggressive refractory neuroblastomas.

PubMed

Paul, Pritha; Gillory, Lauren A; Kang, JungHee; Qiao, Jingbo; Chung, Dai H

2011-03-01

The overall survival for neuroblastoma remains dismal, in part due to the emergence of resistance to chemotherapeutic drugs. We have demonstrated that gastrin-releasing peptide (GRP), a gut peptide secreted by neuroblastoma, acts as an autocrine growth factor. We hypothesized that knockdown of GRP will induce apoptosis in neuroblastoma cells and potentiate the cytotoxic effects of chemotherapeutic agents. The human neuroblastoma cell lines (JF, SK-N-SH) were transfected with small interfering (si) RNA targeted at GRP. Apoptosis was assessed by DNA fragmentation assay. Immunoblotting was used to confirm molecular markers of apoptosis, and flow cytometry was performed to determine cell cycle arrest after GRP knockdown. siGRP resulted in an increase in apoptosis in the absence of chemotherapeutic interventions. A combination of GRP silencing and chemotherapeutic drugs resulted in enhanced apoptosis when compared to either of the treatments alone. GRP silencing led to increased expression of proapoptotic proteins, p53 and p21. Silencing of GRP induces apoptosis in neuroblastoma cells; it acts synergistically with chemotherapeutic effects of etoposide and vincristine. GRP knockdown-mediated apoptosis appears to be associated with upregulation of p53 in neuroblastoma cells. Targeting GRP may be postulated as a potential novel agent for combinational treatment to treat aggressive neuroblastomas. Copyright © 2011 Mosby, Inc. All rights reserved.

Regulation and signaling of human bombesin receptors and their biological effects.

PubMed

Weber, H Christian

2009-02-01

This review will highlight recent advances in the understanding of molecular mechanisms by which mammalian bombesin receptors are regulated and which intracellular signaling pathways have been characterized to mediate agonist-dependent receptor biological effects. Mammalian bombesin receptors have been demonstrated to be involved in a larger array of physiological and pathophysiological conditions than previously reported. Pharmacological experiments in vitro and in vivo as well as utilization of animals genetically deficient of the gastrin-releasing peptide receptor demonstrated roles in memory and fear behavior, lung development and injury, small intestinal cell repair, autocrine tumor growth, and mediating signals for pruritus and penile reflexes. Intracellular signaling studies predominantly of the gastrin-releasing peptide receptor owing to its frequent overexpression in some human malignancies showed that PI3 kinase activation is an important mechanism of cell proliferation. Tumor cell treatment including gastrin-releasing peptide receptor antagonists combined with inhibition of epidermal growth factor receptor resulted in an additive effect on blocking cell proliferation. Novel molecular mechanisms of the orphan bombesin receptor subtype-3 and gastrin-releasing peptide receptor gene regulation have been elucidated. Inhibition of gastrin-releasing peptide receptor signaling in human malignancies represents an attractive target for pharmacological treatment. Novel functions of bombesin related peptides have been identified including processes in the central nervous system, lung and intestinal tract.

GROWTH OF HUMAN PANCREATIC CANCER IS INHIBITED BY DOWN-REGULATION OF GASTRIN GENE EXPRESSION

PubMed Central

Matters, Gail L.; Harms, John F.; McGovern, Christopher O.; Jayakumar, Calpurnia; Crepin, Keisha; Smith, Zachary P.; Nelson, Melissa C.; Stock, Heather; Fenn, Craig W.; Kaiser, James; Kester, Mark; Smith, Jill P.

2009-01-01

Objectives This study evaluated the effects of gastrin mRNA down-regulation on growth of human pancreatic cancer. Methods Gastrin expression was examined in human pancreatic cancer cell lines by RT-PCR and peptide expression was assessed by immunocytochemistry. Gastrin was down-regulated using either stable transfection of an antisense gastrin cDNA or one of three shRNA (short hairpin RNA) constructs. Tumor formation was evaluated following either subcutaneous or orthotopic injections into nude mice. The effect of nanoliposomes loaded with gastrin siRNA was tested in mice bearing pancreatic tumors. Results Stable transfection of gastrin antisense or shRNAs into BxPC-3 cells resulted in clones with >90% reduction in gastrin mRNA. Tumor growth rate and incidence of metastases in both wild type and transfected pancreatic cancer cells was directly proportional to the degrees of gastrin mRNA expression. Immunofluoresence analysis confirmed that gastrin peptide levels were decreased in antisense and shRNA tumors. Gastrin knockdown clones had lower Ki-67 and increased cleaved caspase-3 staining, consistent with known effects of gastrin on proliferation and apoptosis. Tumors in mice treated with gastrin siRNA were smaller than controls. Conclusions These results suggest that RNAi targeting of gastrin could serve as an effective treatment for pancreatic cancer. PMID:19465883

The gastrin/cholecystokinin-B receptor on prostate cells--a novel target for bifunctional prostate cancer imaging.

PubMed

Sturzu, Alexander; Klose, Uwe; Sheikh, Sumbla; Echner, Hartmut; Kalbacher, Hubert; Deeg, Martin; Nägele, Thomas; Schwentner, Christian; Ernemann, Ulrike; Heckl, Stefan

2014-02-14

The means of identifying prostate carcinoma and its metastases are limited. The contrast agents used in magnetic resonance imaging clinical diagnostics are not taken up into the tumor cells, but only accumulate in the interstitial space of the highly vasculated tumor. We examined the gastrin/cholecystokinin-B receptor as a possible target for prostate-specific detection using the C-terminal seven amino acid sequence of the gastrin peptide hormone. The correct sequence and a scrambled control sequence were coupled to the fluorescent dye rhodamine and the magnetic resonance imaging contrast agent gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Expression analysis of the gastrin receptor mRNA was performed by reverse transcriptase polymerase chain reaction on PC3 prostate carcinoma cells, U373 glioma, U2OS osteosarcoma and Colo205 colon carcinoma cells. After having confirmed elevated expression of gastrin receptor in PC3 cells and very low expression of the receptor in Colo205 cells, these two cell lines were used to create tumor xenografts on nude mice for in vivo experiments. Confocal lasers scanning microscopy and magnetic resonance imaging showed a high specificity of the correct conjugate for the PC3 xenografts. Staining of the PC3 xenografts was much weaker with the scrambled conjugate while the Colo205 xenografts showed no marked staining with any of the conjugates. In vitro experiments comparing the correct and scrambled conjugates on PC3 cells by magnetic resonance relaxometry and fluorescence-activated cell sorting confirmed markedly higher specificity of the correct conjugate. The investigations show that the gastrin receptor is a promising tumor cell surface target for future prostate-cancer-specific imaging applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Positron emission tomography (PET) imaging of prostate cancer with a gastrin releasing peptide receptor antagonist--from mice to men.

PubMed

Wieser, Gesche; Mansi, Rosalba; Grosu, Anca L; Schultze-Seemann, Wolfgang; Dumont-Walter, Rebecca A; Meyer, Philipp T; Maecke, Helmut R; Reubi, Jean Claude; Weber, Wolfgang A

2014-01-01

Ex vivo studies have shown that the gastrin releasing peptide receptor (GRPr) is overexpressed on almost all primary prostate cancers, making it a promising target for prostate cancer imaging and targeted radiotherapy. Biodistribution, dosimetry and tumor uptake of the GRPr antagonist ⁶⁴Cu-CB-TE2A-AR06 [(⁶⁴Cu-4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo(6.6.2)hexadecane)-PEG₄-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-LeuNH₂] were studied by PET/CT in four patients with newly diagnosed prostate cancer (T1c-T2b, Gleason 6-7). No adverse events were observed after injection of ⁶⁴Cu-CB-TE2A-AR06. Three of four tumors were visualized with high contrast [tumor-to-prostate ratio > 4 at 4 hours (h) post injection (p.i.)], one small tumor (T1c, < 5% tumor on biopsy specimens) showed moderate contrast (tumor-to-prostate ratio at 4 h: 1.9). Radioactivity was cleared by the kidneys and only the pancreas demonstrated significant accumulation of radioactivity, which rapidly decreased over time. ⁶⁴Cu-CB-TE2A-AR06 shows very favorable characteristics for imaging prostate cancer. Future studies evaluating ⁶⁴Cu-CB-TE2A-AR06 PET/CT for prostate cancer detection, staging, active surveillance, and radiation treatment planning are necessary.

Gastrin-releasing peptide induces monocyte adhesion to vascular endothelium by upregulating endothelial adhesion molecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Mi-Kyoung; Park, Hyun-Joo; Department of Dental Pharmacology, BK21 PLUS Project, School of Dentistry, Pusan National University, Yangsan 626-870

Gastrin-releasing peptide (GRP) is a neuropeptide that plays roles in various pathophysiological conditions including inflammatory diseases in peripheral tissues; however, little is known about whether GRP can directly regulate endothelial inflammatory processes. In this study, we showed that GRP promotes the adhesion of leukocytes to human umbilical vein endothelial cells (HUVECs) and the aortic endothelium. GRP increased the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by activating nuclear factor-κB (NF-κB) in endothelial cells. In addition, GRP activated extracellular signal-regulated kinase 1/2 (ERK1/2), p38MAPK, and AKT, and the inhibition of these signaling pathways significantly reduced GRP-inducedmore » monocyte adhesion to the endothelium. Overall, our results suggested that GRP may cause endothelial dysfunction, which could be of particular relevance in the development of vascular inflammatory disorders. - Highlights: • GRP induces adhesion of monocytes to vascular endothelium. • GRP increases the expression of endothelial adhesion molecules through the activation of NF-κB. • ERK1/2, p38MAPK, and Akt pathways are involved in the GRP-induced leukocyte adhesiveness to endothelium.« less

Relation of gastric acid and pepsin secretion to serum gastrin levels in dogs given bombesin and gastrin-17.

PubMed

Hirschowitz, B I; Molina, E

1983-05-01

To quantitate bombesin stimulation of gastric acid and pepsin via release of gastrin, five gastric fistula dogs were given graded doses (60-1,250 pmol X kg-1 X h-1) of bombesin tetradecapeptide and 40-2,000 pmol X kg-1 X h-1 of synthetic gastrin-17 (G-17). Acid and pepsin output and serum gastrin were proportional to the dose of stimulant. The half-maximal dose of bombesin for gastrin release was 200 pmol X kg-1 X h-1. Bombesin-stimulated acid secretion related to serum gastrin concentrations was congruent with the G-17 curve, but with a maximum of only 62% of the G-17 maximum before declining by 27% despite higher serum gastrin levels. This suggested that bombesin stimulates acid secretion only via gastrin release and inhibits at higher doses by releasing another inhibitory peptide, most likely somatostatin, which is also released by bombesin. The same mechanism could apply to supramaximal inhibition of acid and pepsin seen with high doses of G-17. Because the pepsin curve related to serum gastrin was to the left of the G-17 curve, we concluded that another secretagogue released by bombesin acts synergistically with gastrin on pepsin secretion. Therefore, bombesin stimulates gastric secretion through gastrin release, but its effects are modified by peptides coreleased to a) increase pepsin output at low doses and b) limit the output of acid and pepsin to 50-60% of the G-17 maximum.

N-terminal modifications improve the receptor affinity and pharmacokinetics of radiolabeled peptidic gastrin-releasing peptide receptor antagonists: examples of 68Ga- and 64Cu-labeled peptides for PET imaging.

PubMed

Gourni, Eleni; Mansi, Rosalba; Jamous, Mazen; Waser, Beatrice; Smerling, Christiane; Burian, Antje; Buchegger, Franz; Reubi, Jean Claude; Maecke, Helmut R

2014-10-01

Gastrin-releasing peptide receptors (GRPrs) are overexpressed on a variety of human cancers, providing the opportunity for peptide receptor targeting via radiolabeled bombesin-based peptides. As part of our ongoing investigations into the development of improved GRPr antagonists, this study aimed at verifying whether and how N-terminal modulations improve the affinity and pharmacokinetics of radiolabeled GRPr antagonists. The potent GRPr antagonist MJ9, Pip-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH(2) (Pip, 4-amino-1-carboxymethyl-piperidine), was conjugated to 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and radiolabeled with (68)Ga and (64)Cu. The GRPr affinity of the corresponding metalloconjugates was determined using (125)I-Tyr(4)-BN as a radioligand. The labeling efficiency of (68)Ga(3+) was compared between NODAGA-MJ9 and NOTA-MJ9 in acetate buffer, at room temperature and at 95°C. The (68)Ga and (64)Cu conjugates were further evaluated in vivo in PC3 tumor xenografts by biodistribution and PET imaging studies. The half maximum inhibitory concentrations of all the metalloconjugates are in the high picomolar-low nanomolar range, and these are the most affine-radiolabeled GRPr antagonists we have studied so far in our laboratory. NODAGA-MJ9 incorporates (68)Ga(3+) nearly quantitatively (>98%) at room temperature within 10 min and at much lower peptide concentrations (1.4 × 10(-6) M) than NOTA-MJ9, for which the labeling yield was approximately 45% under the same conditions and increased to 75% at 95°C for 5 min. Biodistribution studies showed high and specific tumor uptake, with a maximum of 23.3 ± 2.0 percentage injected activity per gram of tissue (%IA/g) for (68)Ga-NOTA-MJ9 and 16.7 ± 2.0 %IA/g for (68)Ga-NODAGA-MJ9 at 1 h after injection. The acquisition of PET images with the (64)Cu-MJ9 conjugates at later time points clearly showed the efficient clearance of the accumulated

Powerful inhibition of in-vivo growth of experimental hepatic cancers by bombesin/gastrin-releasing peptide antagonist RC-3940-II.

PubMed

Szepeshazi, Karoly; Schally, Andrew V; Rick, Ferenc G; Block, Norman L; Vidaurre, Irving; Halmos, Gabor; Szalontay, Luca

2012-10-01

Hepatic carcinoma is a major health problem worldwide. Its incidence is increasing in Western countries and there is currently no effective systemic therapy against it. Targeted treatment modalities developed in the past few years have provided very limited success. Development of new treatment strategies is therefore essential. We investigated the effects of bombesin/gastrin-releasing peptide (BN/GRP) antagonist RC-3940-II on experimental human liver cancers in nude mice. SK-Hep-1 and Hep-G2 cancers transplanted subcutaneously into nude mice were treated daily with 10 or 20 µg of RC-3940-II. Tumor growth was monitored for 50-184 days in five experiments. Tumor gene expression was analyzed with PCR array and protein expression by immunoblotting. Characteristics of BN/GRP receptors in the tumors were analyzed by binding assays. Effects of RC-3940-II on cell proliferation were investigated in vitro. RC-3940-II inhibited the growth of SK-Hep-1 cancers in nude mice by 65-98%, with total regression in 9 of 36 tumors in three experiments. The BN/GRP antagonist inhibited the growth of Hep-G2 cancers as well by 73-82% in two experiments, being effective even on originally large tumors. Gene expression analysis showed an increase in several angiogenesis inhibitors and decrease in proangiogenic genes after RC-3940-II treatment. Receptor assays demonstrated high-affinity binding sites for BN/GRP in both tumor lines. BN/GRP antagonist RC-3940-II powerfully inhibits growth of SK-Hep-1 and Hep-G2 cancers in nude mice. Its effect may be linked to changes in expression of those cancer genes important in angiogenesis, invasion, and metastasis. RC-3940-II may be considered for further investigations in treatment of liver cancers.

Effects of bolus doses of fat on small intestinal structure and on release of gastrin, cholecystokinin, peptide tyrosine-tyrosine, and enteroglucagon.

PubMed Central

Jenkins, A P; Ghatei, M A; Bloom, S R; Thompson, R P

1992-01-01

To investigate the enterotrophic effects of bolus doses of long chain triglycerides, two groups of eight female Wistar rats were fed identical diets with 48.2% total calories as the essential fatty acid rich oil Efamol. To one group the oil was given in twice daily bolus doses by gavage, while for the other group the oil was mixed with the remainder of the feed and thus consumed over 24 hours. The animals were killed after 20 to 22 days. Bolus dosing significantly increased parameters of mucosal mass along the length of the small intestine in association with an increase in two hour accumulation of vincristine arrested metaphases in small intestinal crypts. In a second experiment, four replicate studies were carried out, each involving two groups of 12 rats respectively fed as described above. After 21 days one animal from each group was killed every two hours, providing regular plasma samples over 24 hours for measurement of gastrin, cholecystokinin, peptide tyrosine-tyrosine and enteroglucagon. Bolus dosing markedly enhanced release of peptide tyrosine-tyrosine and enteroglucagon, but not of gastrin or cholecystokinin. Thus, the enhanced enterotrophic effects of bolus doses of long chain triglycerides could be mediated by release of a distally located gut peptide, perhaps enteroglucagon. PMID:1541417

Gastrin-releasing peptide-induced down-regulation of tumor suppressor protein PTEN (phosphatase and tensin homolog deleted on chromosome ten) in neuroblastomas.

PubMed

Qiao, Jingbo; Kang, Junghee; Cree, Jeremy; Evers, B Mark; Chung, Dai H

2005-05-01

To evaluate whether aggressive, undifferentiated neuroblastomas express tumor suppressor protein PTEN (phosphatase and tensin homolog deleted on chromosome ten) and to examine the effects of gastrin-releasing peptide (GRP) on PTEN gene and protein expression. We have previously shown that neuroblastomas secrete GRP, which binds to its cell surface receptor (GRP-R) to stimulate cell growth in an autocrine fashion. However, the effects of GRP on expression of the tumor suppressor gene PTEN have not been elucidated in neuroblastomas. Paraffin-embedded sections from human neuroblastomas were analyzed for PTEN and phospho-Akt protein expression by immunohistochemistry. Human neuroblastoma cell lines (SK-N-SH and SH-SY5Y) were stably transfected with the plasmid pEGFP-GRP-R to establish GRP-R overexpression cell lines, and the effects of GRP on PTEN gene and protein expression were determined. A decrease in the ratio of PTEN to phospho-Akt protein expression was identified in poorly differentiated neuroblastomas. An increase in GRP binding capacity was confirmed in GRP-R overexpressing cells, which demonstrated an accelerated constitutive cell growth rate. PTEN gene and protein expression was significantly decreased in GRP-R overexpressing cells when compared with controls. Our findings demonstrate decreased expression of the tumor suppressor protein PTEN in more aggressive undifferentiated neuroblastomas. An increase in GRP binding capacity, as a result of GRP-R overexpression, down-regulates PTEN expression. These findings suggest that an inhibition of the tumor suppressor gene PTEN may be an important regulatory mechanism involved in GRP-induced cell proliferation in neuroblastomas.

Roux-en-Y gastric bypass augments the feeding responses evoked by gastrin releasing peptides

PubMed Central

Washington, Martha C.; Mhalhal, Thaer R.; Berger, Tanisha Johnson-Rouse Jose; Heath, John; Seeley, Randy; Sayegh, Ayman I.

2016-01-01

Background Roux-en-Y gastric bypass (RYGB) is the most effective method for the treatment of obesity and metabolic disease Roux-en-Y gastric bypass (RYGB) may reduce body weight by altering the feeding responses evoked by the short term satiety peptides. Materials and Methods Here, we measured meal size (MS, chow), intermeal interval (IMI) length and satiety ratio (SR, IMI/MS; food consumed per a unit of time) by the small and the large forms of gastrin releasing peptide (GRP) in rats, GRP-10 and GRP-29 (0, 0.1, 0.5 nmol/kg) infused in the celiac artery (CA, supplies stomach and upper duodenum) and the cranial mesenteric artery (CMA, supplies small and large intestine) in a RYGB rat model. Results GRP-10 reduced MS, prolonged the IMI and increased the SR only in the RYGB group, whereas GRP-29 evoked these responses by both routes and in both groups. Conclusion The RYGB procedure augments the feeding responses evoked by exogenous GRP, possibly by decreasing total food intake, increasing latency to the first meal, decreasing number of meals or altering the sites of action regulating MS and IMI length by the two peptides. PMID:27884350

In Vitro Mouse and Human Serum Stability of a Heterobivalent Dual-Target Probe That Has Strong Affinity to Gastrin-Releasing Peptide and Neuropeptide Y1 Receptors on Tumor Cells.

PubMed

Ghosh, Arijit; Raju, Natarajan; Tweedle, Michael; Kumar, Krishan

2017-02-01

Receptor-targeting radiolabeled molecular probes with high affinity and specificity are useful in studying and monitoring biological processes and responses. Dual- or multiple-targeting probes, using radiolabeled metal chelates conjugated to peptides, have potential advantages over single-targeting probes as they can recognize multiple targets leading to better sensitivity for imaging and radiotherapy when target heterogeneity is present. Two natural hormone peptide receptors, gastrin-releasing peptide (GRP) and Y1, are specifically interesting as their expression is upregulated in most breast and prostate cancers. One of our goals has been to develop a dual-target probe that can bind both GRP and Y1 receptors. Consequently, a heterobivalent dual-target probe, t-BBN/BVD15-DO3A (where a GRP targeting ligand J-G-Abz4-QWAVGHLM-NH 2 and Y1 targeting ligand INP-K [ɛ-J-(α-DO3A-ɛ-DGa)-K] YRLRY-NH 2 were coupled), that recognizes both GRP and Y1 receptors was synthesized, purified, and characterized in the past. Competitive displacement cell binding assay studies with the probe demonstrated strong affinity (IC 50 values given in parentheses) for GRP receptors in T-47D cells (18 ± 0.7 nM) and for Y1 receptors in MCF7 cells (80 ± 11 nM). As a further evaluation of the heterobivalent dual-target probe t-BBN/BVD15-DO3A, the objective of this study was to determine its mouse and human serum stability at 37°C. The in vitro metabolic degradation of the dual-target probe in mouse and human serum was studied by using a 153 Gd-labeled t-BBN/BVD15-DO3A and a high-performance liquid chromatography/radioisotope detector analytical method. The half-life (t 1/2 ) of degradation of the dual-target probe in mouse serum was calculated as 7 hours and only ∼20% degradation was seen after 6 hours incubation in human serum. The slow in vitro metabolic degradation of the dual-target probe can be compared with the degradation t 1/2 of the corresponding monomeric probes, BVD15

CCK receptors-related signaling involved in nitric oxide production caused by gastrin 17 in porcine coronary endothelial cells.

PubMed

Grossini, Elena; Caimmi, Philippe; Molinari, Claudio; Uberti, Francesca; Mary, David; Vacca, Giovanni

2012-03-05

In anesthetized pigs gastrin-17 increased coronary blood flow through CCK1/CCK2 receptors and β(2)-adrenoceptors-related nitric oxide (NO) release. Since the intracellular pathway has not been investigated the purpose of this study was to examine in coronary endothelial cells the CCK1/CCK2 receptors-related signaling involved in the effects of gastrin-17 on NO release. Gastrin-17 caused a concentration-dependent increase of NO production (17.3-62.6%; p<0.05), which was augmented by CCK1/CCK2 receptors agonists (p<0.05). The effect of gastrin-17 was amplified by the adenylyl-cyclase activator and β(2)-adrenoceptors agonist (p<0.05), abolished by cAMP/PKA and β(2)-adrenoceptors and CCK1/CCK2 receptors blockers, and reduced by PLC/PKC inhibitor. Finally, Western-blot revealed the preferential involvement of PKA vs. PKC as downstream effectors of CCK1/CCK2 receptors activation leading to Akt, ERK, p38 and endothelial NOS (eNOS) phosphorylation. In conclusion, in coronary endothelial cells, gastrin-17 induced eNOS-dependent NO production through CCK1/CCK2 receptors- and β(2)-adrenoceptors-related pathway. The intracellular signaling involved a preferential PKA pathway over PKC. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Radiolabeled gastrin/CCK analogs in tumor diagnosis: towards higher stability and improved tumor targeting.

PubMed

Kaloudi, A; Nock, B A; Krenning, E P; Maina, T; De Jong, M

2015-09-01

Cholecystokinin subtype 2 receptors (CCK2R) are overexpressed in several human cancers, including medullary thyroid carcinoma. Gastrin and cholecystokinin (CCK) peptides that bind with high affinity and specificity to CCK2R can be used as carriers of radioactivity to CCK2R-expressing tumor sites. Several gastrin and CCK related peptides have been proposed for diagnostic imaging and radionuclide therapy of primary and metastatic CCK2R-positive human tumors. Their clinical application has been restricted to a great extent by their fast in vivo degradation that eventually compromises tumor uptake. This problem has been addressed by structural modifications of gastrin and CCK motifs, which, however, often lead to suboptimal pharmacokinetic profiles. A major enzyme implicated in the catabolism of gastrin and CCK based peptides is neutral endopeptidase (NEP), which is widely distributed in the body. Coinjection of the NEP inhibitor phosphoramidon (PA) with radiolabeled gastrin and other peptide analogs has been recently proposed as a new promising strategy to increase bioavailability and tumor-localization of radiopeptides in tumor sites. Specifically, co-administration of PA with the truncated gastrin analog [(111)In-DOTA]MG11 ([((111)In-DOTA)DGlu(10)]gastrin(10-17)) impressively enhanced the levels of intact radiopeptide in mouse circulation and has led to an 8-fold increase of CCK2R-positive tumor uptake in SCID mice. This increased tumor uptake, visualized also by SPECT/CT imaging, is expected to eventually translate into higher diagnostic sensitivity and improved therapeutic efficacy of radiolabeled gastrin analogs in CCK2R-expressing cancer patients.

Catabolism of gastrin releasing peptide and substance P by gastric membrane-bound peptidases.

PubMed

Bunnett, N W; Kobayashi, R; Orloff, M S; Reeve, J R; Turner, A J; Walsh, J H

1985-01-01

The catabolism of two gastric neuropeptides, the C-terminal decapeptide of gastrin releasing peptide-27 (GRP10) and substance P (SP), by membrane-bound peptidases of the porcine gastric corpus and by porcine endopeptidase-24.11 ("enkephalinase") has been investigated. GRP10 was catabolized by gastric muscle peptidases (specific activity 1.8 nmol min-1 mg-1 protein) by hydrolysis of the His8-Leu9 bond and catabolism was inhibited by phosphoramidon (I50 approx. 10(-8) M), a specific inhibitor of endopeptidase-24.11. The same bond in GRP10 was cleaved by purified endopeptidase-24.11, and hydrolysis was equally sensitive to inhibition by phosphoramidon. SP was catabolized by gastric muscle peptidases (specific activity 1.7 nmol min-1 mg-1 protein) by hydrolysis of the Gln6-Phe7, Phe7-Phe8 and Gly9-Leu10 bonds, which is identical to the cleavage of SP by purified endopeptidase-24.11. The C-terminal cleavage of GRP10 and SP would inactivate the peptides. It is concluded that a membrane-bound peptidase in the stomach wall catabolizes and inactivates GRP10 and SP and that, in its specificity and sensitivity to phosphoramidon, this peptidase resembles endopeptidase-24.11.

Eradication of Helicobacter pylori restores the inhibitory effect of cholecystokinin on postprandial gastrin release in duodenal ulcer patients.

PubMed Central

Konturek, J W; Gillessen, A; Konturek, S J; Domschke, W

1995-01-01

Helicobacter pylori infection may be associated with duodenal ulcer (DU) and accompanied by enhanced gastrin release but the mechanism of this H pylori related hypergastrinaemia in DU patients is unclear. Cholecystokinin (CCK) has been implicated in the feedback control of gastrin release and gastric acid secretion in healthy subjects. This study therefore investigated if CCK participates in the impairment of postprandial gastrin release and gastric secretion in six DU patients. Tests were undertaken with and without elimination of endogenous CCK by loxiglumide, a selective CCK-A receptors antagonist, before and after eradication of H pylori with triple therapy (omeprazole, amoxicyllin, bismuth). In H pylori positive DU patients, the post-prandial decline in pH (with median pH 3.5) was accompanied by a pronounced increment in plasma gastrin but the administration of loxiglumide did not affect significantly this postprandial rise in plasma gastrin and gastric pH profile. After eradication of H pylori, the plasma gastrin concentration was reduced while the median postprandial pH was significantly increased (median pH 4.3). The administration of loxiglumide resulted in significantly greater increase in postprandial plasma gastrin and greater decrease in pH (median pH 3.1) in these patients. This study shows that (a) infection with H pylori is accompanied by an enhanced gastrin release and gastric acidity in DU patients, (b) the failure of loxiglumide to affect plasma gastrin or gastric acid secretion in H pylori infected DU patients could be attributed, at least in part, to the failure of endogenous CCK to control gastrin release and gastric secretion by releasing somatostatin, and (c) the test with loxiglumide may be useful in the identification of patients with impaired feedback control of gastrin release and gastric secretion resulting from infection with H pylori. PMID:7489932

Extraintestinal roles of bombesin-like peptides and their receptors: lung.

PubMed

Qin, Xiao-Qun; Qu, Xiangping

2013-02-01

Description of the recent findings of the biological roles of bombesin-like peptides and their receptors in lungs. Gastrin-releasing peptide (GRP) was involved in the airway inflammation in murine models of airway hyperreactivity. The circulating proGRP could serve as a valuable tumor marker for small-cell lung cancers, and the plasma level of proGRP is more stable compared with that of serum proGRP. Recent studies also shed light on the intracellular signaling pathways of bombesin receptor subtype-3 (BRS-3) activation in cultured human lung cancer cells. The relevant biology of BLPs and their receptors in lung cancers and other lung diseases still remains largely unknown. With the development of several highly specific BRS-3 agonists, recent studies provided some insights into the biological effects of BRS-3 in lungs.

Role of gastrin-peptides in Barrett's and colorectal carcinogenesis

PubMed Central

Chueca, Eduardo; Lanas, Angel; Piazuelo, Elena

2012-01-01

Gastrin is the main hormone responsible for the stimulation of gastric acid secretion; in addition, gastrin and its derivatives exert proliferative and antiapoptotic effects on several cell types. Gastrin synthesis and secretion are increased in certain situations, for example, when proton pump inhibitors are used. The impact of sustained hypergastrinemia is currently being investigated. In vitro experiments and animal models have shown that prolonged hypergastrinemia may be related with higher cancer rates; although, this relationship is less clear in human beings. Higher gastrin levels have been shown to cause hyperplasia of several cell types; yet, the risk for developing cancer seems to be the same in normo- and hypergastrinemic patients. Some tumors also produce their own gastrin, which can act in an autocrine manner promoting tumor growth. Certain cancers are extremely dependent on gastrin to proliferate. Initial research focused only on the effects of amidated gastrins, but there has been an interest in intermediates of gastrin in the last few decades. These intermediates aren’t biologically inactive; in fact, they may exert greater effects on proliferation and apoptosis than the completely processed forms. In certain gastrin overproduction states, they are the most abundant gastrin peptides secreted. The purpose of this review is to examine the gastrin biosynthesis process and to summarize the results from different studies evaluating the production, levels, and effects of the main forms of gastrin in different overexpression states and their possible relationship with Barrett’s and colorectal carcinogenesis. PMID:23236230

Novel activity of angiotensin-converting enzyme. Hydrolysis of cholecystokinin and gastrin analogues with release of the amidated C-terminal dipeptide.

PubMed Central

Dubreuil, P; Fulcrand, P; Rodriguez, M; Fulcrand, H; Laur, J; Martinez, J

1989-01-01

ACE (angiotensin-converting enzyme; peptidyl dipeptidase A; EC 3.4.15.1), cleaves C-terminal dipeptides from active peptides containing a free C-terminus. We investigated the hydrolysis of cholecystokinin-8 [CCK-8; Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] and of various gastrin analogues by purified rabbit lung ACE. Although these peptides are amidated at their C-terminal end, they were metabolized by ACE to several peptide fragments. These fragments were analysed by h.p.l.c., isolated and identified by comparison with synthetic fragments, and by amino acid analysis. The initial and major site of hydrolysis was the penultimate peptide bond, which generated a major product, the C-terminal amidated dipeptide Asp-Phe-NH2. As a secondary cleavage, ACE subsequently released di- or tri-peptides from the C-terminal end of the remaining N-terminal fragments. The cleavage of CCK-8 and gastrin analogues was inhibited by ACE inhibitors (Captopril and EDTA), but not by other enzyme inhibitors (phosphoramidon, thiorphan, bestatin etc.). Hydrolysis of [Leu15]gastrin-(14-17)-peptide [Boc (t-butoxycarbonyl)-Trp-Leu-Asp-Phe-NH2] in the presence of ACE was found to be dependent on the chloride-ion concentration. Km values for the hydrolysis of CCK-8, [Leu15]gastrin-(11-17)-peptide and Boc-[Leu15]gastrin-(14-17)-peptide at an NaCl concentration of 300 mM were respectively 115, 420 and 3280 microM, and the catalytic constants were about 33, 115 and 885 min-1. The kcat/Km for the reactions at 37 degrees C was approx. 0.28 microM-1.min-1, which is approx. 35 times less than that reported for the cleavage of angiotensin I. These results suggest that ACE might be involved in the metabolism in vivo of CCK and gastrin short fragments. PMID:2554881

Preclinical evaluation of 68Ga-DOTA-minigastrin for the detection of cholecystokinin-2/gastrin receptor-positive tumors.

PubMed

Brom, Maarten; Joosten, Lieke; Laverman, Peter; Oyen, Wim J G; Béhé, Martin; Gotthardt, Martin; Boerman, Otto C

2011-04-01

In comparison to somatostatin receptor scintigraphy, gastrin receptor scintigraphy using 111In-DTPA-minigastrin (MG0) showed added value in diagnosing neuroendocrine tumors. We investigated whether the 68Ga-labeled gastrin analogue DOTA-MG0 is suited for positron emission tomography (PET), which could improve image quality. Targeting of cholecystokinin-2 (CCK2)/gastrin receptor-positive tumor cells with DOTA-MG0 labeled with either 111In or 68Ga in vitro was investigated using the AR42J rat tumor cell line. Biodistribution was examined in BALB/c nude mice with a subcutaneous AR42J tumor. In vivo PET imaging was performed using a preclinical PET-computed tomographic scanner. DOTA-MG0 showed high receptor affinity in vitro. Biodistribution studies revealed high tumor uptake of 68Ga-DOTA-MG0: 4.4 ± 1.3 %ID/g at 1 hour postinjection. Coadministration of an excess unlabeled peptide blocked the tumor uptake (0.7 ± 0.1 %ID/g), indicating CCK2/gastrin receptor-mediated uptake (p  =  .0005). The biodistribution of 68Ga-DOTA-MG0 was similar to that of 111In-DOTA-MG0. Subcutaneous and intraperitoneal tumors were clearly visualized by small-animal PET imaging with 5 MBq 68Ga-DOTA-MG0. 111In- and 68Ga-labeled DOTA-MG0 specifically accumulate in CCK2/gastrin receptor-positive AR42J tumors with similar biodistribution apart from the kidneys. AR42J tumors were clearly visualized by microPET. Therefore, 68Ga-DOTA-MG0 is a promising tracer for PET imaging of CCK2/gastrin receptor-positive tumors in humans.

Exploiting cancer’s phenotypic guise against itself: targeting ectopically expressed peptide G-protein coupled receptors for lung cancer therapy

PubMed Central

Khan, Mahjabin; Huang, Tao; Lin, Cheng-Yuan; Wu, Jiang; Fan, Bao-Min; Bian, Zhao-Xiang

2017-01-01

Lung cancer, claiming millions of lives annually, has the highest mortality rate worldwide. This advocates the development of novel cancer therapies that are highly toxic for cancer cells but negligibly toxic for healthy cells. One of the effective treatments is targeting overexpressed surface receptors of cancer cells with receptor-specific drugs. The receptors-in-focus in the current review are the G-protein coupled receptors (GPCRs), which are often overexpressed in various types of tumors. The peptide subfamily of GPCRs is the pivot of the current article owing to the high affinity and specificity to and of their cognate peptide ligands, and the proven efficacy of peptide-based therapeutics. The article summarizes various ectopically expressed peptide GPCRs in lung cancer, namely, Cholecystokinin-B/Gastrin receptor, the Bombesin receptor family, Bradykinin B1 and B2 receptors, Arginine vasopressin receptors 1a, 1b and 2, and the Somatostatin receptor type 2. The autocrine growth and pro-proliferative pathways they mediate, and the distinct tumor-inhibitory effects of somatostatin receptors are then discussed. The next section covers how these pathways may be influenced or ‘corrected’ through therapeutics (involving agonists and antagonists) targeting the overexpressed peptide GPCRs. The review proceeds on to Nano-scaled delivery platforms, which enclose chemotherapeutic agents and are decorated with peptide ligands on their external surface, as an effective means of targeting cancer cells. We conclude that targeting these overexpressed peptide GPCRs is potentially evolving as a highly promising form of lung cancer therapy. PMID:29262666

Comparative effect of chronic bombesin, gastrin-releasing peptide and caerulein on the rat pancreas.

PubMed

Damgé, C; Hajri, A; Lhoste, E; Aprahamian, M

1988-02-01

This study was designed to compare, on a molar basis, the effect of chronic bombesin, gastrin-releasing peptide (GRP) and caerulein on pancreatic growth in the rat. These 3 peptides were administered s.c. 3 times daily for 4 days at the following concentrations: 0.036, 0.36, 3.6 and 7.2 nmol/kg of body weight. Bombesin and GRP induced pancreatic growth in a dose-dependent manner from 3.6 nmol/kg. This growth was characterized by an increase in pancreatic weight, its protein and RNA contents but not in DNA content suggesting cellular hypertrophy. Caerulein exerted a biphasic effect on pancreatic growth, inducing cellular hypertrophy at low doses since 0.36 nmol/kg and atrophy with the highest dose (7.2 nmol/kg). Bombesin and caerulein (until 3.6 nmol/kg) increased the pancreatic content in chymotrypsin more than in amylase. The 7.2 nmol/kg caerulein treatment depressed all enzyme activities while the same dose of GRP increased pancreatic lipase content. It is concluded that (1) bombesin and GRP are equipotent trophic factors for the pancreas; (2) caerulein is the most potent factor and exerts a biphasic effect on pancreatic growth; (3) pancreatic growth and synthesis and/or secretion of enzymes are not regulated through the same mechanism.

Calcitonin gene-related peptide and somatostatin releases correlated with the area under the lafutidine concentration-time curve in human plasma.

PubMed

Ikawa, K; Shimatani, T; Azuma, Y; Inoue, M; Morikawa, N

2006-08-01

To examine the effects of the histamine H(2)-receptor antagonist, lafutidine, at clinical dosage (10 mg tablet after a standardized meal) on plasma levels of the gastrointestinal peptides, calcitonin gene-related peptide (CGRP), somatostatin and gastrin. Six healthy male volunteers ate a standardized meal, and received either lafutidine orally at a dose of 10 mg or water only (control). Blood samples were taken before and up to 4 h after the drug administration. Plasma lafutidine concentrations were determined by high pressure liquid chromatography. Pharmacokinetic analysis of lafutidine was performed using one-compartmental model. The levels of immunoreactive substances of plasma CGRP, somatostatin and gastrin were measured by enzyme immunoassay, and the amount of peptide release was calculated by the trapezoidal method. Lafutidine significantly increased plasma CGRP levels at 1, 1.5, 2.5 and 4 h and the total amount of CGRP release (192 +/- 14.0 pg.h/mL) compared with the control group (128 +/- 21.5 pg.h/mL). Lafutidine significantly increased the plasma somatostatin levels at 1 and 1.5 h, and the total amount of somatostatin released (107 +/- 18.2 pg.h/mL) compared with the control (78.4 +/- 7.70 pg.h/mL). The area under the drug concentration-time curve (AUC) from 0 to 4 h after administration correlated well with the Delta-CGRP and Delta-somatostatin release but not with total amount of gastrin released. However, plasma gastrin levels were significantly elevated at 1.5 h after drug administration. Lafutidine at clinical dosage increases plasma CGRP and the somatostatin. The amounts released correlated with the AUC of lafutidine in humans. These results suggest that the increased release of CGRP and somatostatin may contribute to its gastroprotective and anti-acid secretory effect.

Stability of pro-gastrin-releasing peptide in serum versus plasma.

PubMed

Yoshimura, Toru; Fujita, Kenju; Kawakami, Satoshi; Takeda, Katsumichi; Chan, Sabrina; Beligere, Gangamani; Dowell, Barry

2008-01-01

Although serum assays for pro-gastrin-releasing peptide (ProGRP) assays have been commercially available in Japan for several years, the stability of ProGRP in serum and plasma has not been well documented. We investigated the stability of ProGRP in serum and plasma with fresh and stored (frozen) specimens, as well as the cause of the observed instability in serum. ProGRP concentrations in fresh serum were decreased by 6-28% after room temperature storage for 2 h and by 8-32% after 2-8 degrees C storage for 24 h. The average change in ProGRP concentrations in fresh plasma was within +/-10% of baseline for more than 4 h at room temperature and for more than 24 h at 2-8 degrees C. The incubation of a serine protease, thrombin (activated blood coagulation factor II), in a buffer solution containing ProGRP caused decreases in ProGRP concentrations. Following the addition of phenylmethylsulfonyl fluoride, a serine protease inhibitor, to serum, the serum stability for ProGRP was similar to that in plasma. ProGRP is significantly more stable in plasma than in serum. We speculate that thrombin in serum is one of the factors that inactivate ProGRP in serum by proteolysis of the ProGRP antigen. The use of plasma samples for ProGRP may improve the clinical reliability of this marker by minimizing preanalytical changes in ProGRP concentrations. (c) 2008 S. Karger AG, Basel.

Stimulation of gastrin release and gastric secretion: effect of bombesin and a nonapeptide in fistula dogs with and without fundic vagotomy.

PubMed

Hirschowitz, B I; Gibson, R G

1978-01-01

Bombesin and a synthetic bombesin nonapeptide were studied by intravenous infusion at a dose of 0.5 microgram.kg-1.h-1 for 4 h in 7 dogs with esophagostomy and gastric fistula. In 3 of the dogs who had highly selective (fundic) vagotomy, mean integrated gastrin output over 4 h was double that in the 4 dogs with vagi intact during both nonapeptide (1,554 vs. 700 pg.ml-1.4 h-1) and bombesin infusion (2,442 vs. 1,440 pg.ml-1.4 h-1). Peak concentrations of serum gastrin reached during bombesin (490 +/- 100 vs. 320 +/- 90) were higher than those during nonapeptide infusion (270 +/- 40 vs. 160 +/- 28 pg/ml) in the vagotomized and intact dogs, respectively. The difference between vagotomized and vagally intact dogs suggests that the fundic vagotomy removed an inhibitor of gastrin release from the innervated antrum. Despite these differences in gastrin release, gastric acid output with the two peptides was the same (49--52 mEq/4 h) whether the fundus was denervated or innervated. This suggests that bombesin may stimulate gastric acid secretion by the release of an additional secretagogue which is not measured by the gastrin assay. Neither of the two inhibitors of gastrin release--antral acidification to pH 1.4 or less or atropine (100 microgram/kg)-- inhibited gastrin release by bombesin, even though the atropine reduced acid output by 80%. Bombesin is a potent gastric stimulus whose action is only partly explained by the measured gastrin release.

Circulating levels of cholecystokinin and gastrin-releasing peptide in rainbow trout fed different diets.

PubMed

Jönsson, Elisabeth; Forsman, Antti; Einarsdottir, Ingibjörg E; Egnér, Barbro; Ruohonen, Kari; Björnsson, Björn Thrandur

2006-09-01

Cholecystokinin (CCK) and gastrin-releasing peptide (GRP) are gastrointestinal peptides thought to be important regulators of intake and digestion of food in vertebrates. In this study, pre- and postprandial plasma levels of CCK and GRP were measured in rainbow trout (Oncorhynchus mykiss) by the establishment of homologous radioimmunoassays, and the hormonal levels assessed in relation to dietary lipid:protein ratio and food intake. Fish were acclimated to either a high protein/low lipid diet (HP/LL diet; 14.1% lipids) or a normal protein/high lipid diet (NP/HL diet; 31.4% lipids). On three consecutive sampling days, radio-dense lead-glass beads were included in the diets for assessment of feed intake. Fish were terminally sampled for blood and stomach contents prior to feeding at time 0, and at 0.3, 1, 2, 4, 6, and 24 h after feeding. There was a postprandial elevation of plasma CCK levels, which was most evident after 4 and 6 h. Fish fed the NP/HL diet had higher plasma CCK levels compared with those fed the HP/LL diet. Plasma CCK levels were not affected by the amount of food ingested. GRP levels in plasma were not influenced by sampling time, diet, or feed intake. The results indicate that the endocrine release of gastrointestinal CCK is increased during feeding and may be further influenced by the dietary lipid:protein ratio in rainbow trout. Plasma GRP levels, on the other hand, appear not to be influenced by feeding or diet composition.

Theranostic Perspectives in Prostate Cancer with the Gastrin-Releasing Peptide Receptor Antagonist NeoBOMB1: Preclinical and First Clinical Results.

PubMed

Nock, Berthold A; Kaloudi, Aikaterini; Lymperis, Emmanouil; Giarika, Athina; Kulkarni, Harshad R; Klette, Ingo; Singh, Aviral; Krenning, Eric P; de Jong, Marion; Maina, Theodosia; Baum, Richard P

2017-01-01

We recently introduced the potent gastrin-releasing peptide receptor (GRPR) antagonist 68 Ga-SB3 ( 68 Ga-DOTA-p-aminomethylaniline-diglycolic acid-DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt), showing excellent tumor localizing efficacy in animal models and in patients. By replacement of the C-terminal Leu 13 -Met 14 -NH 2 dipeptide of SB3 by Sta 13 -Leu 14 -NH 2 , the novel GRPR antagonist NeoBOMB1 was generated and labeled with different radiometals for theranostic use. We herein report on the biologic profile of resulting 67/68 Ga-, 111 In-, and 177 Lu-NeoBOMB1 radioligands in GRPR-expressing cells and mouse models. The first evidence of prostate cancer lesion visualization in men using 68 Ga-NeoBOMB1 and PET/CT is also presented. NeoBOMB1 was radiolabeled with 67/68 Ga, 111 In, and 177 Lu according to published protocols. The respective metalated species nat Ga-, nat In-, and nat Lu-NeoBOMB1 were also synthesized and used in competition binding experiments against [ 125 I-Tyr 4 ]BBN in GRPR-positive PC-3 cell membranes. Internalization of 67 Ga-, 111 In-, and 177 Lu-NeoBOMB1 radioligands was studied in PC-3 cells at 37°C, and their metabolic stability in peripheral mouse blood was determined by high-performance liquid chromatography analysis of blood samples. Biodistribution was performed by injecting a 67 Ga-, 111 In-, or 177 Lu-NeoBOMB1 bolus (74, 74, or 370 kBq, respectively, 100 μL, 10 pmol total peptide ± 40 nmol Tyr 4 -BBN: for in vivo GRPR blockade) in severe combined immunodeficiency mice bearing PC-3 xenografts. PET/CT images with 68 Ga-NeoBOMB1 were acquired in prostate cancer patients. NeoBOMB1 and nat Ga-, nat In-, and nat Lu-NeoBOMB1 bound to GRPR with high affinity (half maximal inhibitory concentration, 1-2 nM). 67 Ga-, 111 In-, and 177 Lu-NeoBOMB1 specifically and strongly bound on the cell membrane of PC-3 cells displaying low internalization, as expected for receptor antagonists. They showed excellent metabolic stability in peripheral mouse blood

Identification of the sexually dimorphic gastrin-releasing peptide system in the lumbosacral spinal cord that controls male reproductive function in the mouse and Asian house musk shrew (Suncus murinus).

PubMed

Tamura, Kei; Kobayashi, Yasuhisa; Hirooka, Asuka; Takanami, Keiko; Oti, Takumi; Jogahara, Takamichi; Oda, Sen-Ichi; Sakamoto, Tatsuya; Sakamoto, Hirotaka

2017-05-01

Several regions of the brain and spinal cord control male reproductive function. We previously demonstrated that the gastrin-releasing peptide (GRP) system, located in the lumbosacral spinal cord of rats, controls spinal centers to promote penile reflexes during male copulatory behavior. However, little information exists on the male-specific spinal GRP system in animals other than rats. The objective of this study was to examine the functional generality of the spinal GRP system in mammals using the Asian house musk shrew (Suncus murinus; suncus named as the laboratory strain), a specialized placental mammal model. Mice are also used for a representative model of small laboratory animals. We first isolated complementary DNA encoding GRP in suncus. Phylogenetic analysis revealed that suncus preproGRP was clustered to an independent branch. Reverse transcription-PCR showed that GRP and its receptor mRNAs were both expressed in the lumbar spinal cord of suncus and mice. Immunohistochemistry for GRP demonstrated that the sexually dimorphic GRP system and male-specific expression/distribution patterns of GRP in the lumbosacral spinal cord in suncus are similar to those of mice. In suncus, we further found that most GRP-expressing neurons in males also express androgen receptors, suggesting that this male-dominant system in suncus is also androgen-dependent. Taken together, these results indicate that the sexually dimorphic spinal GRP system exists not only in mice but also in suncus, suggesting that this system is a conserved property in mammals. J. Comp. Neurol. 525:1586-1598, 2017. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Serum pro-gastrin-releasing peptide (31-98) in benign prostatic hyperplasia and prostatic carcinoma.

PubMed

Nagakawa, Osamu; Furuya, Yuzo; Fujiuchi, Yasuyoshi; Fuse, Hideki

2002-09-01

To clarify whether serum levels of pro-gastrin-releasing peptide (ProGRP) (31-98) could be a useful marker in patients with prostatic carcinoma. GRP is produced and secreted by prostatic neuroendocrine cells. Serum levels of ProGRP(31-98) were measured by enzyme-linked immunosorbent assay in 20 patients with benign prostatic hyperplasia and 107 patients with prostatic carcinoma. The mean serum levels of ProGRP(31-98) in patients with distant metastasis and hormone-resistant prostate cancer were significantly elevated compared with those in patients with organ-confined disease. Significantly elevated levels of ProGRP(31-98) were detected in 9 patients with prostatic carcinoma before any treatment. During hormone-resistant prostate cancer progression, ProGRP(31-98) levels were elevated in 9 patients (23%). Of the 9 patients with Stage D3 and elevated serum ProGRP, 4 had a normal serum prostate-specific antigen level. ProGRP may be a potential tumor marker for prostate cancer. Additional studies in large groups of patients are needed to define the clinical value of ProGRP.

CCKB/gastrin receptors mediate changes in sodium and potassium absorption in the isolated perfused rat kidney.

PubMed

von Schrenck, T; Ahrens, M; de Weerth, A; Bobrowski, C; Wolf, G; Jonas, L; Jocks, T; Schulz, M; Bläker, M; Neumaier, M; Stahl, R A

2000-09-01

To evaluate the function of cholecystokinin B (CCKB)/gastrin receptors in the rat kidney, we identified the receptors by Northern blot and localized the receptors by immunohistochemistry. The functional effects of gastrin were studied under standardized in vitro conditions using the isolated perfused kidney. Rat kidneys were mounted in an organ bath by attaching the renal artery to a perfusion system. A catheter was inserted into the renal vein and the ureter to collect samples that were analyzed for the concentrations of electrolytes. After a preperfusion period, gastrin-17-I was given via the renal artery (10-8 to 10-6 mol/L). Subsequently, hemodynamic parameters (for example, perfusate flow) and changes in sodium and potassium absorption were determined. All data were subjected to a nonparametric analysis of variance and, in case of significant results, to subsequent paired comparisons by the a posteriori Wilcoxon test. Northern blot analysis detected CCKB receptor transcripts in total RNA isolated from kidneys. Immunohistochemistry localized CCKB receptors on tubules and collecting duct cells. Compared with controls, gastrin (10-6 mol/L) caused a decrease in the fractional sodium reabsorption (basal 80%, 10 minutes after application of gastrin 71%, after 20 minutes 62%, P < 0.05). This effect was inhibited by the CCKB receptor antagonist L-365,260. Gastrin decreased urinary potassium excretion at 10-8 and 10-6 mol/L [maximal decrease at 10-6 mol/L from baseline values (100%) to 49% after 10 minutes and to 69% after 20 minutes, P < 0.05, N = 6]. This effect was also abolished by the CCKB receptor antagonist L-365,260. Gastrin (10-6 mol/L) reduced perfusate flow by 31% (P < 0.05). CCKB receptors are expressed in the rat kidney on tubules and collecting ducts. These receptors mediate changes in renal potassium and sodium absorption. In addition, gastrin causes a decrease in perfusate flow, indicating that CCKB receptors might also modulate vascular resistance in

Bombesin-like peptides stimulate somatostatin release from rat fundic D cells in primary culture.

PubMed

Schaffer, K; Herrmuth, H; Mueller, J; Coy, D H; Wong, H C; Walsh, J H; Classen, M; Schusdziarra, V; Schepp, W

1997-09-01

In several species, bombesin-like neuropeptides stimulate somatostatin release in in vitro preparations of gastric mucosa. We sought to determine if this response is due to a direct effect on fundic D cells. Rat fundic mucosal cells were isolated by pronase E (1% D cells). D cells were separated by counterflow elutriation and subsequent density-gradient centrifugation (Nycodenz) (15% D cells) and grown in primary culture for 48 h (46% D cells). Cultured cells were double stained with affinity-purified rabbit-anti-gastrin-releasing peptide (GRP) receptor antibody and mouse monoclonal antibody to human somatostatin. After incubation with rhodamine-labeled anti-rabbit and fluorescein isothiocyanate-labeled anti-mouse antibodies, reactions were visualized by fluorescence microscopy. All cells positive for somatostatin had GRP receptors, whereas all non-D cells showed no expression in this G cell-free culture system. Somatostatin release from cultured cells was stimulated by sulfated cholecystokinin octapeptide (CCK-8; EC50 3 X 10(-10) M) and epinephrine (EC50 4 X 10(-8) M), which are established stimuli for canine fundic D cells. Bombesin (EC50 6 X 10(-11) M), its mammalian analog GRP-27, and neuromedin C (GRP-10) (EC50 1 X 10(-10) M, for both) were almost equally potent stimuli of somatostatin release, eliciting maximal response at 10(-9) M (400-550% above basal). Neuromedin B was less potent and effective (maximal response at 10(-8) M, 230% above basal). [D-Phe6]bombesin-(6-13)-OMe, a specific bombesin receptor antagonist, inhibited bombesin-stimulated somatostatin release in a competitive manner (IC50 9 X 10(-8) M). Potentiating interactions were observed between bombesin and dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP) or epinephrine, but not between bombesin and CCK-8. We conclude that bombesin-like peptides directly stimulate somatostatin release by interacting with specific receptors on rat fundic D cells. Bombesin-like peptides appear to induce Ca(2

Pro-Gastrin Releasing Peptide: A New Serum Marker for Endometrioid Adenocarcinoma.

PubMed

Kiseli, Mine; Caglar, Gamze Sinem; Yarci Gursoy, Asli; Tasci, Tolga; Candar, Tuba; Akincioglu, Egemen; Pabuccu, Emre Goksan; Boran, Nurettin; Tulunay, Gokhan; Umudum, Haldun

2018-06-13

Gastrin-releasing peptide (GRP) is thought to play a role in the metastatic process of various malignancies. The more stable precursor of GRP, pro-GRP (ProGRP), has been shown to be secreted by neuroendocrine tumors. This study was designed to assess the validity of ProGRP as a diagnostic marker in endometrioid adenocarcinomas (EAs) of the endometrium. Thirty-seven patients with a diagnosis of EA, 23 patients with endometrial hyperplasia, and 32 age-matched controls with normal endometrial histology were recruited for this study. Serum ProGRP and cancer antigen 125 (CA125) values were compared between groups. Median serum ProGRP levels were significantly higher in the cancer group compared to corresponding levels in both the hyperplasia and control groups (p = 0.008 and p < 0.001 respectively; endometrial cancer: 27.5 pg/mL; hyperplasia: 16.1 pg/mL; controls: 12.9 pg/mL). Age and endometrial thickness were positively correlated with ProGRP levels (r = 0.322, p = 0.006 and r = 0.269, p = 0.023, respectively). Receiver Operating Characteristic curve analyses for EA revealed a threshold of 20.81 pg/mL, with a sensitivity of 60.7% and specificity of 81.4%, positive predictive value of 68% and negative predictive value of 76.1%. Significantly higher ProGRP levels were observed in patients with EA than in controls. Serum ProGRP has good diagnostic sensitivity and specificity for EA. © 2018 S. Karger AG, Basel.

Effects of intranasal and peripheral oxytocin or gastrin-releasing peptide administration on social interaction and corticosterone levels in rats.

PubMed

Kent, Pamela; Awadia, Alisha; Zhao, Leah; Ensan, Donna; Silva, Dinuka; Cayer, Christian; James, Jonathan S; Anisman, Hymie; Merali, Zul

2016-02-01

The intranasal route of drug administration has gained increased popularity as it is thought to allow large molecules, such as peptide hormones, more direct access to the brain, while limiting systemic exposure. Several studies have investigated the effects of intranasal oxytocin administration in humans as this peptide is associated with prosocial behavior. There are, however, few preclinical studies investigating the effects of intranasal oxytocin administration in rodents. Oxytocin modulates hypothalamic-pituitary-adrenal (HPA) axis functioning and it has been suggested that oxytocin's ability to increase sociability may occur through a reduction in stress reactivity. Another peptide that appears to influence both social behavior and HPA axis activity is gastrin-releasing peptide (GRP), but it is not known if these GRP-induced effects are related. With this in mind, in the present study, we assessed the effects of intranasal and intraperitoneal oxytocin and GRP administration on social interaction and release of corticosterone in rats. Intranasal and intraperitoneal administration of 20, but not 5 μg, of oxytocin significantly increased social interaction, whereas intranasal and peripheral administration of GRP (20 but not 5 μg) significantly decreased levels of social interaction. In addition, while intranasal oxytocin (20 μg) had no effect on blood corticosterone levels, a marked increase in blood corticosterone levels was observed following intraperitoneal oxytocin administration. With GRP, intranasal (20 μg) but not peripheral administration increased corticosterone levels. These findings provide further evidence that intranasal peptide delivery can induce behavioral alterations in rodents which is consistent with findings from human studies. In addition, the peptide-induced changes in social interaction were not linked to fluctuations in corticosterone levels. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bombesin-like peptides and receptors in normal fetal baboon lung: roles in lung growth and maturation.

PubMed

Emanuel, R L; Torday, J S; Mu, Q; Asokananthan, N; Sikorski, K A; Sunday, M E

1999-11-01

Previously, we have shown that bombesin-like peptide (BLP) promotes fetal lung development in rodents and humans but mediates postnatal lung injury in hyperoxic baboons. The present study analyzed the normal ontogeny of BLP and BLP receptors as well as the effects of BLP on cultured normal fetal baboon lungs. Transcripts encoding gastrin-releasing peptide (GRP), a pulmonary BLP, were detectable on gestational day 60 (ED60), peaked on approximately ED90, and then declined before term (ED180). Numbers of BLP-immunopositive neuroendocrine cells peaked from ED80 to ED125 and declined by ED160, preceding GRP-receptor mRNAs detected from ED125 until birth. BLP (0.1-10 nM) stimulated type II cell differentiation in organ cultures as assessed by [(3)H]choline incorporation into surfactant phospholipids, electron microscopy, and increased surfactant protein (SP) A- and/or SP-C-immunopositive cells and SP-A mRNA. BLP also induced neuroendocrine differentiation on ED60. Cell proliferation was induced by GRP, peaking on ED90. Similarly, blocking BLP degradation stimulated lung growth and maturation, which was completely reversed by a BLP-specific antagonist. The dissociation between GRP and GRP-receptor gene expression during ontogeny suggests that novel BLP receptors and/or peptides might be implicated in these responses.

Targeted imaging of gastrin-releasing peptide receptors with 99mTc-EDDA/HYNIC-[Lys3]-bombesin: biokinetics and dosimetry in women.

PubMed

Santos-Cuevas, Clara L; Ferro-Flores, Guillermina; Arteaga de Murphy, Consuelo; Pichardo-Romero, Pablo A

2008-08-01

The gastrin-releasing peptide receptor (GRP-R) is expressed in several normal human tissues and is overexpressed in various human tumors including breast, prostate, small-cell lung cancer and pancreatic cancer. Recently, 99mTc-EDDA/HYNIC-[Lys]-bombesin (99mTc-HYNIC-BN) was reported as a radiopharmaceutical with high stability in human serum, specific cell GRP-R binding and rapid cell internalization. The aim of this study was to determine the biokinetics and dosimetry of 99mTc-HYNIC-BN and the feasibility of using this radiopharmaceutical to image GRP-R in four early breast cancer patients and seven healthy women. Whole-body images were acquired at 20, 90, 180 min, and 24 h after 99mTc-HYNIC-BN administration. The same regions of interest were drawn around source organs on each time frame and regions of interest were converted to activity (conjugate view counting method). The image sequence was used to extrapolate 99mTc-HYNIC-BN time-activity curves in each organ to calculate the total number of disintegrations (N) that occurred in the source regions. N data were the input for the OLINDA/EXM code to calculate internal radiation dose estimates. 99mTc-HYNIC-BN had a rapid blood clearance with mainly renal excretion. No statistically significant differences (P>0.05) in the radiation-absorbed doses among cancer patients and healthy women were observed. The average equivalent doses (n=11) were 24.8+/-8.8 mSv (kidneys), 7.3+/-1.8 mSv (lungs), 6.5+/-4.0 mSv (breast), 2.0+/-0.3 mSv (pancreas), 1.6+/-0.3 mSv (liver), 1.2+/-0.2 mSv (ovaries), and 1.0+/-0.2 mSv (red marrow). The effective dose was 3.3+/-0.6 mSv. The images showed well-differentiated concentration of 99mTc-HYNIC-BN in cancer mammary tissue. All the absorbed doses were comparable with those known for most of the 99mTc studies. 99mTc-HYNIC-BN shows high tumor uptake in breasts with malignant tumors so it is a promising imaging radiopharmaceutical to target site-specific early breast cancer. The results obtained

ACE-like hydrolysis of gastrin analogs and CCK-8 by fundic mucosal cells of different species with release of the amidated C-terminal dipeptide.

PubMed

Dubreuil, P; Fulcrand, P; Rodriguez, M; Laur, J; Bali, J P; Martinez, J

1990-06-19

Various gastrin analogues and CCK-8 (Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2) are hydrolyzed in vitro by angiotensin-converting enzyme (ACE), the main and initial cleavage occurring at the Met-Asp (or Leu-Asp) bond, releasing the C-terminal dipeptide amide Asp-Phe-NH2. Tetragastrin analogues (e.g., Boc-Trp-Leu-Asp-Phe-NH2) are degraded by a vesicular membrane fraction from rat gastric mucosa, yielding the C-terminal dipeptide Asp-Phe-NH2. We report here on the degradation of gastrin analogues and CCK-8 by a gastric mucosal cell preparation containing specific gastrin receptors. We have shown that gastrin analogues were specifically degraded by gastric mucosal cells from different species (e.g., rabbit and dog) at 37 degrees C (pH 7.4), releasing the C-terminal dipeptide Asp-Phe-NH2, similarly to ACE. This cleavage was found to be temperature and pH sensitive, and was inhibited by metalloproteinase inhibitors and by captopril, strongly suggesting that this enzymatic system closely resembles ACE. We have also demonstrated that a close correlation seems to exist between the apparent affinity of the gastrin analogues for gastrin receptors on gastric mucosal cells, and their ability of being hydrolyzed by this cell preparation. Moreover, all gastrin analogues which have been demonstrated to act as gastrin antagonists remained unaffected in the incubation conditions.

Cloning of cDNAs encoding amphibian bombesin: evidence for the relationship between bombesin and gastrin-releasing peptide.

PubMed Central

Spindel, E R; Gibson, B W; Reeve, J R; Kelly, M

1990-01-01

Bombesin is a tetradecapeptide originally isolated from frog skin; its mammalian homologue is the 27-amino acid peptide gastrin-releasing peptide (GRP). cDNAs encoding GRP have been cloned from diverse species, but little is yet known about the amphibian bombesin precursor. Mass spectrometry of HPLC-separated skin exudate from Bombina orientalis was performed to demonstrate the existence of authentic bombesin in the skin of this frog. A cDNA library was prepared from the skin of B. orientalis and mixed oligonucleotide probes were used to isolate cDNAs encoding amphibian bombesin. Sequence analysis revealed that bombesin is encoded in a 119-amino acid prohormone. The carboxyl terminus of bombesin is flanked by two basic amino acids; the amino terminus is not flanked by basic amino acids but is flanked by a chymotryptic-like cleavage site. Northern blot analysis demonstrated similarly sized bombesin mRNAs in frog skin, brain, and stomach. Polymerase chain reaction was used to show that the skin and gut bombesin mRNAs encoded the identical prohormones. Prohormone processing, however, differed between skin and gut. Chromatography showed the presence of only authentic bombesin in skin whereas gut extracts contained two peaks of bombesin immunoreactivity, one consistent in size with bombesin and one closer in size to mammalian GRP. Thus the same bombesin prohormone is processed solely to bombesin in skin but is processed to a peptide similar in size to bombesin and to a peptide similar in size to mammalian GRP in stomach. Images PMID:2263631

Identification of the bombesin receptor on murine and human cells by cross-linking experiments.

PubMed

Kris, R M; Hazan, R; Villines, J; Moody, T W; Schlessinger, J

1987-08-15

The bombesin receptor present on the surface of murine and human cells was identified using 125I-labeled gastrin-releasing peptide as a probe, the cross-linking agent disuccinimidyl suberate, and sodium dodecyl sulfate gels. A clone of NIH-3T3 cells which possesses approximately 80,000 bombesin receptors/cell with a single binding constant of approximately 1.9 X 10(-9) M was used in these studies. In addition, we used Swiss 3T3 cells and a human glioma cell line which possesses approximately 100,000 and approximately 55,000 bombesin receptors/cell, respectively. Under conditions found optimal for binding, it is demonstrated that 125I-labeled gastrin-releasing peptide can be cross-linked specifically to a glycoprotein of apparent molecular mass of 65,000 daltons on the surface of the NIH-3T3 cells. Similar results were obtained when the cross-linked product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or non-reducing conditions. Moreover, the cross-linking reaction is specific and saturable and the 65,000-dalton polypeptide is not observed when the cross-linking experiments were performed with a NIH-3T3 cell line which is devoid of bombesin receptors. Interestingly, glycoproteins with apparent molecular weights of 75,000 were labeled specifically by 125I-labeled gastrin-releasing peptide when similar experiments were performed with Swiss 3T3 cells and with human glioma cell line GM-340. These different molecular weights may indicate differential glycosylation as treatment with the enzyme N-glycanase reduced the apparent molecular weight of the cross-linked polypeptide to 45,000. On the basis of these results it is concluded that the cross-linked polypeptides represent the bombesin receptor or the ligand-binding subunit of a putative larger bombesin receptor expressed on the surface of these cells.

The gastrin-releasing peptide analog bombesin preserves exocrine and endocrine pancreas morphology and function during parenteral nutrition

PubMed Central

Pierre, Joseph F.; Neuman, Joshua C.; Brill, Allison L.; Brar, Harpreet K.; Thompson, Mary F.; Cadena, Mark T.; Connors, Kelsey M.; Busch, Rebecca A.; Heneghan, Aaron F.; Cham, Candace M.; Jones, Elaina K.; Kibbe, Carly R.; Davis, Dawn B.; Groblewski, Guy E.; Kudsk, Kenneth A.

2015-01-01

Stimulation of digestive organs by enteric peptides is lost during total parental nutrition (PN). Here we examine the role of the enteric peptide bombesin (BBS) in stimulation of the exocrine and endocrine pancreas during PN. BBS protects against exocrine pancreas atrophy and dysfunction caused by PN. BBS also augments circulating insulin levels, suggesting an endocrine pancreas phenotype. While no significant changes in gross endocrine pancreas morphology were observed, pancreatic islets isolated from BBS-treated PN mice showed a significantly enhanced insulin secretion response to the glucagon-like peptide-1 (GLP-1) agonist exendin-4, correlating with enhanced GLP-1 receptor expression. BBS itself had no effect on islet function, as reflected in low expression of BBS receptors in islet samples. Intestinal BBS receptor expression was enhanced in PN with BBS, and circulating active GLP-1 levels were significantly enhanced in BBS-treated PN mice. We hypothesized that BBS preserved islet function indirectly, through the enteroendocrine cell-pancreas axis. We confirmed the ability of BBS to directly stimulate intestinal enteroid cells to express the GLP-1 precursor preproglucagon. In conclusion, BBS preserves the exocrine and endocrine pancreas functions during PN; however, the endocrine stimulation is likely indirect, through the enteroendocrine cell-pancreas axis. PMID:26185331

Analysis of the 2-deoxy-D-glucose-induced vagal stimulation of gastric secretion and gastrin release in dogs using methionine-enkephalin, morphine and naloxone.

PubMed

Anderson, W; Molina, E; Rentz, J; Hirschowitz, B I

1982-09-01

Gastric acid and pepsin secreted in 3 hr and antral gastrin released in response to vagal excitation induced by 2-deoxy-D-glucose (2DG), 625 mumol/kg i.v., were studied in six conscious trained gastric fistula dogs. During a 2-hr infusion, Met-enkephalin (96 nmol/kg/hr; delta receptor) reduced the 2DG response by 50%; when the enkephalin was stopped there was a rapid rebound to peak values. Met-enkephalin also blocked the release of gastrin in the first 15 min. By itself, Met-enkephalin did not stimulate secretion and slightly depressed gastrin. By contrast, morphine (96 nmol/kg/hr; mu receptor) augmented and sustained the 2DG gastric acid secretory response. This effect was blocked by naloxone. Morphine alone caused a small rise in serum gastrin after 90 min, followed by a delayed gastric acid secretion of about 30% of the peak 2DG response. Naloxone, a mu opiate antagonist (mu/delta, 27:1), also inhibited the 2DG gastric secretory response by about 50% and augmented the Met-enkephalin inhibition of secretion without blocking either the secretory rebound or the effect on gastrin release. None of the three opiates changed the direct cholinergic gastric secretory or gastrin-releasing effects of bethanechol. Thus, vagal stimulation of the stomach involves pathways which can be influenced by both mu and delta opiates, with apparently opposite effects, proximal to the level of acetylcholine action on the gastric mucosa. The central and peripheral control points in the activation of the stomach via the vagus which are sensitive to opiates have yet to be located and explained.

Preclinical and first clinical experience with the gastrin-releasing peptide receptor-antagonist [⁶⁸Ga]SB3 and PET/CT.

PubMed

Maina, Theodosia; Bergsma, Hendrik; Kulkarni, Harshad R; Mueller, Dirk; Charalambidis, David; Krenning, Eric P; Nock, Berthold A; de Jong, Marion; Baum, Richard P

2016-05-01

Gastrin-releasing peptide receptors (GRPR) represent attractive targets for tumor diagnosis and therapy because of their overexpression in major human cancers. Internalizing GRPR agonists were initially proposed for prolonged lesion retention, but a shift of paradigm to GRPR antagonists has recently been made. Surprisingly, radioantagonists, such as [(99m)Tc]DB1 ((99m)Tc-N4'-DPhe(6),Leu-NHEt(13)]BBN(6-13)), displayed better pharmacokinetics than radioagonists, in addition to their higher inherent biosafety. We introduce here [(68)Ga]SB3, a [(99m)Tc]DB1 mimic-carrying, instead of the (99m)Tc-binding tetraamine, the chelator DOTA for labeling with the PET radiometal (68)Ga. Competition binding assays of SB3 and [(nat)Ga]SB3 were conducted against [(125)I-Tyr(4)]BBN in PC-3 cell membranes. Blood samples collected 5 min postinjection (pi) of the [(67)Ga]SB3 surrogate in mice were analyzed using high-performance liquid chromatography (HPLC) for degradation products. Likewise, biodistribution was performed after injection of [(67)Ga]SB3 (37 kBq, 100 μL, 10 pmol peptide) in severe combined immunodeficiency (SCID) mice bearing PC-3 xenografts. Eventually, [(68)Ga]SB3 (283 ± 91 MBq, 23 ± 7 nmol) was injected into 17 patients with breast (8) and prostate (9) cancer. All patients had disseminated disease and had received previous therapies. PET/CT fusion images were acquired 60-115 min pi. SB3 and [(nat)Ga]SB3 bound to the human GRPR with high affinity (IC50: 4.6 ± 0.5 nM and 1.5 ± 0.3 nM, respectively). [(67)Ga]SB3 displayed good in vivo stability (>85 % intact at 5 min pi). [(67)Ga]SB3 showed high, GRPR-specific and prolonged retention in PC-3 xenografts (33.1 ± 3.9%ID/g at 1 h pi - 27.0 ± 0.9%ID/g at 24 h pi), but much faster clearance from the GRPR-rich pancreas (≈160%ID/g at 1 h pi to <17%ID/g at 24 h pi) in mice. In patients, [(68)Ga]SB3 elicited no adverse effects and clearly visualized cancer lesions. Thus, 4 out of 8 (50 %) breast

Eradicating Helicobacter pylori infection lowers gastrin mediated acid secretion by two thirds in patients with duodenal ulcer.

PubMed

el-Omar, E; Penman, I; Dorrian, C A; Ardill, J E; McColl, K E

1993-08-01

Helicobacter pylori (H pylori) raises serum gastrin but it is unclear whether this stimulates increased acid secretion. Gastrin mediated acid secretion and plasma gastrin after the intravenous infusion of gastrin releasing peptide was studied in nine H pylori negative and nine H pylori positive healthy volunteers, and in 11 duodenal ulcer patients. Nine of the last group were re-examined one month after eradication of H pylori. The median acid output (mmol/h) to gastrin releasing peptide (40 pmol/kg/h) in the H pylori positive healthy volunteers was 15.1 (range 3.3-38.3), which was three times that of the H pylori negative healthy volunteers (median = 5.5, range 1.0-9.0) (p < 0.02). The median acid output in the duodenal ulcer patients with H pylori was 37 (range 8.5-57), which was > six times that of the H pylori negative healthy volunteers. Eradication of H pylori in the duodenal ulcer patients lowered their acid secretion by a median of 66% (range 30%-80%) (p < 0.01) and to values equivalent to the H pylori positive healthy volunteers. The pepsin output in response to gastrin releasing peptide followed the same pattern as the acid output. The median plasma gastrin concentrations during gastrin releasing peptide were similar in the H pylori positive duodenal ulcer patients (150 ng/l, range 95-400) and H pylori positive healthy volunteers (129 ng/l, range 23-420) and both were appreciably higher than H pylori negative healthy volunteers (60 ng/l, range 28-135) (p < 0.005 for each). Eradication of H pylori lowered the plasma gastrin in the duodenal ulcer patients to values equivalent to the H pylori negative healthy volunteers. These findings show a threefold increase in acid secretion in H pylori positive healthy volunteers that is explained by H pylori induced hypergastrinaemia and a sixfold increase in acid secretion in the duodenal ulcer patients that is explained by the combination of H pylori induced hypergastrinaemia and an exaggerated acid response to

Eradicating Helicobacter pylori infection lowers gastrin mediated acid secretion by two thirds in patients with duodenal ulcer.

PubMed Central

el-Omar, E; Penman, I; Dorrian, C A; Ardill, J E; McColl, K E

1993-01-01

Helicobacter pylori (H pylori) raises serum gastrin but it is unclear whether this stimulates increased acid secretion. Gastrin mediated acid secretion and plasma gastrin after the intravenous infusion of gastrin releasing peptide was studied in nine H pylori negative and nine H pylori positive healthy volunteers, and in 11 duodenal ulcer patients. Nine of the last group were re-examined one month after eradication of H pylori. The median acid output (mmol/h) to gastrin releasing peptide (40 pmol/kg/h) in the H pylori positive healthy volunteers was 15.1 (range 3.3-38.3), which was three times that of the H pylori negative healthy volunteers (median = 5.5, range 1.0-9.0) (p < 0.02). The median acid output in the duodenal ulcer patients with H pylori was 37 (range 8.5-57), which was > six times that of the H pylori negative healthy volunteers. Eradication of H pylori in the duodenal ulcer patients lowered their acid secretion by a median of 66% (range 30%-80%) (p < 0.01) and to values equivalent to the H pylori positive healthy volunteers. The pepsin output in response to gastrin releasing peptide followed the same pattern as the acid output. The median plasma gastrin concentrations during gastrin releasing peptide were similar in the H pylori positive duodenal ulcer patients (150 ng/l, range 95-400) and H pylori positive healthy volunteers (129 ng/l, range 23-420) and both were appreciably higher than H pylori negative healthy volunteers (60 ng/l, range 28-135) (p < 0.005 for each). Eradication of H pylori lowered the plasma gastrin in the duodenal ulcer patients to values equivalent to the H pylori negative healthy volunteers. These findings show a threefold increase in acid secretion in H pylori positive healthy volunteers that is explained by H pylori induced hypergastrinaemia and a sixfold increase in acid secretion in the duodenal ulcer patients that is explained by the combination of H pylori induced hypergastrinaemia and an exaggerated acid response to

Retro-inverso forms of gastrin5-12 are as biologically active as glycine-extended gastrin in vitro but not in vivo.

PubMed

Marshall, Kathryn M; Laval, Marie; Sims, Ioulia; Shulkes, Arthur; Baldwin, Graham S

2015-12-01

Non-amidated gastrin peptides such as glycine-extended gastrin (Ggly) are biologically active in vitro and in vivo and have been implicated in the development of gastric and colonic cancers. Previous studies have shown that the truncated form of Ggly, the octapeptide LE5AY, was still biologically active in vitro, and that activity was dependent on ferric ion binding but independent of binding to the cholecystokinin 2 (CCK2) receptor. The present work was aimed at creating more stable gastrin-derived 'super agonists' using retro-inverso technology. The truncated LE5AY peptide was synthesized using end protecting groups in three forms with l-amino acids (GL), d-amino acids (GD) or retro-inverso (reverse order with d-amino acids; GRI). All of these peptides bound ferric ions with a 2:1 (Fe: peptide) ratio. As predicted, Ggly, GL and GRI were biologically active in vitro and increased cell proliferation in mouse gastric epithelial (IMGE-5) and human colorectal cancer (DLD-1) cell lines, and increased cell migration in DLD-1 cells. These activities were likely via the same mechanism as Ggly since no CCK1 or CCK2 binding was identified, and GD remained inactive in all assays. Surprisingly, unlike Ggly, GL and GRI were not active in vivo. While Ggly stimulated colonic crypt height and proliferation rates in gastrin knockout mice, GL and GRI did not. The apparent lack of activity may be due to rapid clearance of these smaller peptides. Nevertheless further work designing and testing retro-inverso gastrins is warranted, as it may lead to the generation of super agonists that could potentially be used to treat patients with gastrointestinal disorders with reduced mucosal function. Copyright © 2015 Elsevier Inc. All rights reserved.

Pancreatic β-Cells Express the Fetal Islet Hormone Gastrin in Rodent and Human Diabetes.

PubMed

Dahan, Tehila; Ziv, Oren; Horwitz, Elad; Zemmour, Hai; Lavi, Judith; Swisa, Avital; Leibowitz, Gil; Ashcroft, Frances M; In't Veld, Peter; Glaser, Benjamin; Dor, Yuval

2017-02-01

β-Cell failure in type 2 diabetes (T2D) was recently proposed to involve dedifferentiation of β-cells and ectopic expression of other islet hormones, including somatostatin and glucagon. Here we show that gastrin, a stomach hormone typically expressed in the pancreas only during embryogenesis, is expressed in islets of diabetic rodents and humans with T2D. Although gastrin in mice is expressed in insulin + cells, gastrin expression in humans with T2D occurs in both insulin + and somatostatin + cells. Genetic lineage tracing in mice indicates that gastrin expression is turned on in a subset of differentiated β-cells after exposure to severe hyperglycemia. Gastrin expression in adult β-cells does not involve the endocrine progenitor cell regulator neurogenin3 but requires membrane depolarization, calcium influx, and calcineurin signaling. In vivo and in vitro experiments show that gastrin expression is rapidly eliminated upon exposure of β-cells to normal glucose levels. These results reveal the fetal hormone gastrin as a novel marker for reversible human β-cell reprogramming in diabetes. © 2017 by the American Diabetes Association.

Pancreatic β-Cells Express the Fetal Islet Hormone Gastrin in Rodent and Human Diabetes

PubMed Central

Dahan, Tehila; Ziv, Oren; Horwitz, Elad; Zemmour, Hai; Lavi, Judith; Swisa, Avital; Leibowitz, Gil; Ashcroft, Frances M.; In’t Veld, Peter

2017-01-01

β-Cell failure in type 2 diabetes (T2D) was recently proposed to involve dedifferentiation of β-cells and ectopic expression of other islet hormones, including somatostatin and glucagon. Here we show that gastrin, a stomach hormone typically expressed in the pancreas only during embryogenesis, is expressed in islets of diabetic rodents and humans with T2D. Although gastrin in mice is expressed in insulin+ cells, gastrin expression in humans with T2D occurs in both insulin+ and somatostatin+ cells. Genetic lineage tracing in mice indicates that gastrin expression is turned on in a subset of differentiated β-cells after exposure to severe hyperglycemia. Gastrin expression in adult β-cells does not involve the endocrine progenitor cell regulator neurogenin3 but requires membrane depolarization, calcium influx, and calcineurin signaling. In vivo and in vitro experiments show that gastrin expression is rapidly eliminated upon exposure of β-cells to normal glucose levels. These results reveal the fetal hormone gastrin as a novel marker for reversible human β-cell reprogramming in diabetes. PMID:27864307

Infusion of exogenous cholecystokinin-8, gastrin releasing peptide-29 and their combination reduce body weight in diet-induced obese male rats.

PubMed

Mhalhal, Thaer R; Washington, Martha C; Newman, Kayla; Heath, John C; Sayegh, Ayman I

2017-02-01

We hypothesized that exogenous gastrin releasing peptide-29 (GRP-29), cholecystokinin-8 (CCK-8) and their combination reduce body weight (BW). To test this hypothesis, BW was measured in four groups of diet-induced obese (DIO) male rats infused in the aorta (close to the junctions of the celiac and cranial mesenteric arteries) with saline, CCK-8 (0.5 nmol/kg), GRP-29 (0.5 nmol/kg) and CCK-8+GRP-29 (0.5 nmol/kg each) once daily for a total of 23 days. We found that CCK-8, GRP-29 and CCK-8+GRP-29 reduce BW relative to saline control. In conclusion, CCK-8, GRP-29 and their combination reduce BW in the DIO rat model. If infused near their gastrointestinal sites of action CCK-8, GRP-29 and their combination may have a role in regulating BW. Published by Elsevier Ltd.

Bombesin receptor-mediated imaging and cytotoxicity: review and current status

PubMed Central

Sancho, Veronica; Di Florio, Alessia; Moody, Terry W.; Jensen, Robert T.

2010-01-01

The three mammalian bombesin (Bn) receptors (gastrin-releasing peptide [GRP] receptor, neuromedin B [NMB] receptor, BRS-3) are one of the classes of G protein-coupled receptors that are most frequently over-express/ectopically expressed by common, important malignancies. Because of the clinical success of somatostatin receptor-mediated imaging and cytotoxicity with neuroendocrine tumors, there is now increasing interest in pursuing a similar approach with Bn receptors. In the last few years then have been more than 200 studies in this area. In the present paper, the in vitro and in vivo results, as well as results of human studies from many of these studies are reviewed and the current state of Bn receptor-mediated imaging or cytotoxicity is discussed. Both Bn receptor-mediated imaging studies as well as Bn receptor-mediated tumoral cytotoxic studies using radioactive and non-radioactive Bn-based ligands are covered. PMID:21034419

Suppression of gastrin release and gastric secretion by gastric inhibitory polypeptide (GIP) and vasoactive intestinal polypeptide (VIP).

PubMed Central

Villar, H V; Fender, H R; Rayford, P L; Bloom, S R; Ramus, N I; Thompson, J C

1976-01-01

Five dogs prepared with Heidenhain pouches received infusions of saline, GIP and VIP before and after a standard meat meal. Blood samples were obtained under basal conditions and at subsequent intervals for measurement of gastrin, insulin, GIP and VIP by radioimmunoassay. GIP and VIP infusions had no effect on basal levels of gastrin. GIP and VIP (in common with secretin and glucagon) were found to suppress food-stimulated release of gastrin and gastrin-stimulated acid secretion from the Heidenhain pouch. Insulin levels were significantly elevated during GIP and VIP infusions. Food released GIP (and perhaps VIP. PMID:938120

Overexpression of pro-gastrin releasing peptide promotes the cell proliferation and progression in small cell lung cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gong, Zhiyun; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032; Lu, Renquan

Pro-gastrin releasing peptide (ProGRP) plays the role of oncogene in small cell lung cancer (SCLC). In this study, we aim to explore the biological function of ProGRP in SCLC cells and its potential mechanism. Expression of ProGRP in SCLC tissues and cell lines were detected by immunohistochemistry and western blot analysis, respectively. The transduced cell lines with ProGRP down-regulation were established using RNA interference technology. Cell viability, cologenic, apoptosis-associated assay and the biomarker levels determination for cell supernatant were performed in the transduced cells to elucidate the biological functions and mechanisms of ProGRP in SCLC cells. Our data showed thatmore » ProGRP protein was demonstrated a higher level in SCLC tissues and cells compared with the control, and its diagnostic efficiency was better than NSE, further, the higher levels of ProGRP were detected in the patients with extensive disease stage (P < 0.05), were also the unfavorable factor to the prognosis of SCLC patients. Additionally, the concentration of serum ProGRP is a useful biomarker in disease-monitoring of the patients with SCLC. Down-regulation of ProGRP significantly reduced SCLC cell growth, repressed colony formation, but increased cancer cell apoptosis. Additionally, repression of ProGRP also induced change in the cell cycle and output of NSE. Our data indicated that ProGRP serve as the useful biomarker in the management of SCLC and might be a potential therapeutic target. - Highlights: • ProGRP is overexpressed in the tissues and sera of the patients with SCLC. • Down-regulation of ProGRP inhibited cell proliferation. • Inhibition of ProGRP altered cell cycle distribution and triggers the apoptosis of lung cancer cells.« less

Two peptide receptor ligands (99m)Tc-EDDA/HYNIC-Tyr(3)-octreotide and (99m)Tc-EDDA/HYNIC-(D)Glu-octagastrin for scintigraphy of medullary thyroid carcinoma.

PubMed

Kosowicz, Jerzy; Mikołajczak, Renata; Czepczyński, Rafał; Ziemnicka, Katarzyna; Gryczyńska, Maria; Sowiński, Jerzy

2007-10-01

Somatostatin and gastrin receptors are overexpressed in medullary thyroid carcinoma (MTC) cells; hence, both of them are potential targets for peptide receptor scintigraphy and radiotherapy. Therefore, the aim of our study was to assess the clinical value of two technetium-99m-labeled peptides, a new gastrin analog, the EDDA/HYNIC-(D)Glu-octagastrin and a somatostatin analog, EDDA/HYNIC-Tyr(3)-octreotide (EDDA/HYNIC-TOC) for scintigraphy in patients with MTC to detect recurrences and metastases and select patients for peptide receptor radiotherapy. Thirty (30) patients, 20 females and 10 males, 22-83 years of age (mean, 52.7) with the diagnosis of MTC in different stages of the disease (preoperative, postsurgery, remission, recurrence, or metastatic disease) were included in this study. Before surgery, in all patients serum calcitonin concentrations were elevated. The diagnosis of MTC was confirmed in all cases by histopathology of the removed tumor and immunohistochemical staining giving positive reactions for calcitonin and chromogranin A. Imaging studies using (99m)Tc-EDDA/HYNIC-TOC and a new minigastrin analog, (99m)Tc-EDDA/HYNIC-(D)Glu-octagastrin, were performed in each patient and the results compared with each other and with other imaging methods. Scans of the whole body, head, neck, and chest were performed 2 and 4 hours after injections of the tracer, 500-600 MBq in each case, using a double-head Varicam (Elscint, Israel) gamma camera. (99m)Tc-EDDA/HYNIC-TOC detected somatostatin receptor-positive lesions in 20 patients with MTC, whereas (99m)Tc-EDDA/HYNIC-(D)Glu-octagastrin displayed gastrin receptors in 11 patients. In 9 cases, the scans were positive in both methods, although in 2 cases different pathologic foci were visualized. In 12 cases, only (99m)Tc-EDDA/HYNIC-TOC scintigraphy was positive, whereas in 3 other cases only (99m)Tc-EDDA/HYNIC-(D)Glu-octagastrin revealed pathologic lesions. Scintigraphy using (99m)Tc-HYNIC-TOC permits the visualization

Gut-expressed gustducin and taste receptors regulate secretion of glucagon-like peptide-1

PubMed Central

Jang, Hyeung-Jin; Kokrashvili, Zaza; Theodorakis, Michael J.; Carlson, Olga D.; Kim, Byung-Joon; Zhou, Jie; Kim, Hyeon Ho; Xu, Xiangru; Chan, Sic L.; Juhaszova, Magdalena; Bernier, Michel; Mosinger, Bedrich; Margolskee, Robert F.; Egan, Josephine M.

2007-01-01

Glucagon-like peptide-1 (GLP-1), released from gut endocrine L cells in response to glucose, regulates appetite, insulin secretion, and gut motility. How glucose given orally, but not systemically, induces GLP-1 secretion is unknown. We show that human duodenal L cells express sweet taste receptors, the taste G protein gustducin, and several other taste transduction elements. Mouse intestinal L cells also express α-gustducin. Ingestion of glucose by α-gustducin null mice revealed deficiencies in secretion of GLP-1 and the regulation of plasma insulin and glucose. Isolated small bowel and intestinal villi from α-gustducin null mice showed markedly defective GLP-1 secretion in response to glucose. The human L cell line NCI-H716 expresses α-gustducin, taste receptors, and several other taste signaling elements. GLP-1 release from NCI-H716 cells was promoted by sugars and the noncaloric sweetener sucralose, and blocked by the sweet receptor antagonist lactisole or siRNA for α-gustducin. We conclude that L cells of the gut “taste” glucose through the same mechanisms used by taste cells of the tongue. Modulating GLP-1 secretion in gut “taste cells” may provide an important treatment for obesity, diabetes and abnormal gut motility. PMID:17724330

Transplantations and Cloning of an Immortal Cell Line from Rat SCN

DTIC Science & Technology

1994-05-31

of SCN peptides in colonies was examined using rabbit polyclonal antisera against arginine vasopressin (AVP; Arnel Products), gastrin releasing ...examined for expression of arginine vasopressin (AVP), gastrin releasing peptide (GRP), somatostatin (SMT) and vasoactive intestinal polypeptide (VIP...neuronal markers and peptides found within SCN neurons in situ. Concordant with immunostaining data, content, release and mRNA expression of SCN

Effects of gonadotropin-releasing hormone (GnRH) on gastric secretion and gastrin release in the dog.

PubMed

Soldani, G; Del Tacca, M; Bambini, G; Polloni, A; Bernardini, C; Martinotti, E; Martino, E

1982-01-01

The effects of GnRH on gastric secretion and gastrin release from dogs provided with gastric fistulae and Heidenhain pouches have been investigated. A transient yet significant inhibition of pentagastrin-stimulated secretion from gastric fistulae was observed, while secretion from Heidenhain pouches was unchanged. The maximal inhibitory effect of GnRH on both acid and pepsin secretion stimulated by 2-deoxy-D-glucose was obtained from gastric fistulae. On the contrary, GnRH failed to affect either acid secretion stimulated by bethanechol or acid secretion and gastrin release induced by bombesin. The present results indicate that GnRH possesses an inhibitory action on gastric secretion from the vagally innervated stomach of the dog. The most likely inhibitory mechanism seems to be represented by a decrease of the vagal activity.

Peptide hormones and lung cancer.

PubMed

Moody, T W

2006-03-01

Several peptide hormones have been identified which alter the proliferation of lung cancer. Small cell lung cancer (SCLC), which is a neuroendocrine cancer, produces and secretes gastrin releasing peptide (GRP), neurotensin (NT) and adrenomedullin (AM) as autocrine growth factors. GRP, NT and AM bind to G-protein coupled receptors causing phosphatidylinositol turnover or elevated cAMP in SCLC cells. Addition of GRP, NT or AM to SCLC cells causes altered expression of nuclear oncogenes, such as c-fos, and stimulation of growth. Antagonists have been developed for GRP, NT and AM receptors which function as cytostatic agents and inhibit SCLC growth. Growth factor antagonists, such as the NT1 receptor antagonist SR48692, facilitate the ability of chemotherapeutic drugs to kill lung cancer cells. It remains to be determined if GRP, NT and AM receptors will served as molecular targets, for development of new therapies for the treatment of SCLC patients. Non-small cell lung cancer (NSCLC) cells also have a high density of GRP, NT, AM and epidermal growth factor (EGF) receptors. Several NSCLC patients with EGF receptor mutations respond to gefitinib, a tyrosine kinase inhibitor. Gefitinib relieves NSCLC symptoms, maintaining stable disease in patients who are not eligible for systemic chemotherapy. It is important to develop new therapeutic approaches using translational research techniques for the treatment of lung cancer patients.

Invertebrate Gonadotropin-Releasing Hormone-Related Peptides and Their Receptors: An Update

PubMed Central

Sakai, Tsubasa; Shiraishi, Akira; Kawada, Tsuyoshi; Matsubara, Shin; Aoyama, Masato; Satake, Honoo

2017-01-01

Gonadotropin-releasing hormones (GnRHs) play pivotal roles in reproductive functions via the hypothalamus, pituitary, and gonad axis, namely, HPG axis in vertebrates. GnRHs and their receptors (GnRHRs) are likely to be conserved in invertebrate deuterostomes and lophotrochozoans. All vertebrate and urochordate GnRHs are composed of 10 amino acids, whereas protostome, echinoderm, and amphioxus GnRH-like peptides are 11- or 12-residue peptide containing two amino acids after an N-terminal pyro-Glu. In urochordates, Halocynthia roretzi GnRH gene encodes two GnRH peptide sequences, whereas two GnRH genes encode three different GnRH peptides in Ciona intestinalis. These findings indicate the species-specific diversification of GnRHs. Intriguingly, the major signaling pathway for GnRHRs is intracellular Ca2+ mobilization in chordates, echinoderms, and protostomes, whereas Ciona GnRHRs (Ci-GnRHRs) are endowed with multiple GnRHergic cAMP production pathways in a ligand-selective manner. Moreover, the ligand-specific modulation of signal transduction via heterodimerization among Ci-GnRHR paralogs suggests the species-specific development of fine-tuning of gonadal functions in ascidians. Echinoderm GnRH-like peptides show high sequence differences compared to those of protostome counterparts, leading to the difficulty in classification of peptides and receptors. These findings also show both the diversity and conservation of GnRH signaling systems in invertebrates. The lack of the HPG axis in invertebrates indicates that biological functions of GnRHs are not release of gonadotropins in current invertebrates and common ancestors of vertebrates and invertebrates. To date, authentic or putative GnRHRs have been characterized from various echinoderms and protostomes as well as chordates and the mRNAs have been found to be distributed not only reproductive organs but also other tissues. Collectively, these findings further support the notion that invertebrate GnRHs have

Isolation of endopeptidase-24.11 (EC 3.4.24.11, "enkephalinase") from the pig stomach. Hydrolysis of substance P, gastrin-releasing peptide 10, [Leu5] enkephalin, and [Met5] enkephalin.

PubMed

Bunnett, N W; Turner, A J; Hryszko, J; Kobayashi, R; Walsh, J H

1988-10-01

The purpose of this investigation was to isolate the cell-surface enzyme endopeptidase-24.11 from the stomach wall of the pig and to examine the hydrolysis of the gastric neuropeptides. Endopeptidase-24.11 was isolated from gastric membranes by immunoadsorbent chromatography using a monoclonal antibody to porcine kidney endopeptidase-24.11. The enzyme was purified with a yield of 1.2 micrograms/g wet wt of fundic muscle. A single polypeptide chain of apparent subunit molecular weight of 90,000 was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gastric endopeptidase-24.11 hydrolyzed substance P, gastrin-releasing peptide 10, [Leu5] enkephalin, and [Met5] enkephalin by cleavage of peptide bonds on the N-terminal side of hydrophobic amino acids. The enzymatic activity was inhibited completely by phosphoramidon (10(-6) M) and strongly by 1,10-phenanthroline (10(-3) M), but was unaffected by captopril (10(-5) M).

Signal Peptide and Denaturing Temperature are Critical Factors for Efficient Mammalian Expression and Immunoblotting of Cannabinoid Receptors*

PubMed Central

WANG, Chenyun; WANG, Yingying; WANG, Miao; CHEN, Jiankui; YU, Nong; SONG, Shiping; KAMINSKI, Norbert E.; ZHANG, Wei

2013-01-01

Summary Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e., ≥95°C), forming a large molecular weight band when analyzed by immunoblotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immunoblot analysis. These findings provide very useful information for efficient mammalian expression and immunoblotting of membrane receptors. PMID:22528237

Antral G-cell in gastrin and gastrin-cholecystokinin knockout animals.

PubMed

Friis-Hansen, Lennart; Wierup, Nils; Rehfeld, Jens F; Sundler, Frank

2005-07-01

The antral hormone gastrin is the key regulator of gastric acid secretion, mucosal growth and differentiation. Gastrin is synthesized in the endocrine G-cells in the antroduodenal mucosa. We have now examined the way in which the loss of gastrin alone or gastrin plus cholecystokinin (CCK) affects the antral G-cell. Immunohistochemistry, radioimmunoassay and quantitative real-time polymerase chain reaction techniques were employed to examine the expression of genes belonging to the G-cell secretory pathway in gastrin and gastrin-CCK knockout mice. Transmission electron microscopy was used to examine the ultrastructure of the G-cells. The number of G-cells increased but the secretory granules were few and abnormally small in the G-cells of both mouse models compared with wildtypes. Thus, gastrin is not necessary for the formation of G-cells as such but the lack of gastrin reduces the number and size of their secretory granules suggesting that gastrin is vital for the formation and/or maintenance of secretory granules in G-cells.

Synthesis and exploration of novel radiolabeled bombesin peptides for targeting receptor positive tumor.

PubMed

De, Kakali; Banerjee, Indranil; Sinha, Samarendu; Ganguly, Shantanu

2017-03-01

Increasing evidence of peptide receptor overexpression in various cancer cells, warrant the development of receptor specific radiolabeled peptides for molecular imaging and therapy in nuclear medicine. Gastrin-releasing-peptide (GRP) receptor, are overexpressed in a variety of human cancer cells. The present study report the synthesis and biological evaluation of new bombesin (BBN) analogs, HYNIC-Asp-[Phe 13 ]BBN(7-13)-NH-CH 2 -CH 2 -CH3:BA1, HYNIC-Pro-[Tyr 13 Met 14 ]BBN(7-14)NH 2 :BA2 as prospective tumor imaging agent with compare to BBN(7-14)NH 2 :BS as standard. The pharmacophores were radiolabeled in high yields with 99m Tc, characterized for their stability in serum and saline, cysteine/histidine and were found to be substantially stable. Internalization/externalization and receptor binding studies were assessed using MDA-MB-231 cells and showed high receptor binding-affinity and favourable internalization. Fluorescence studies revealed that BA1 changed the morphology of the cells and could localize in the nucleus more effectively than BA2/BS. Cell-viability studies displayed substantial antagonistic and nuclear-internalization effect of BA1. BA1 also exhibited antiproliferative effect on MDA-MB-231 cell by inducing apoptosis. In vivo behaviour of the radiopeptides was evaluated in GRP receptor positive tumor bearing mice. The 99m Tc-BA1/ 99m Tc-BA2 demonstrated rapid blood/urinary clearance through the renal pathway and comparatively more significant tumor uptake image and favourable tumor-to-non-target ratios provided by 99m Tc-BA1. The specificity of the in vivo uptake was confirmed by co-injection with BS. Moreover, 99m Tc-BA1 provided a much clearer tumor image in scintigraphic studies than others. Thus the combination of favourable in vitro and in vivo properties renders BA1 as more potential antagonist bombesin-peptide for targeting GRP-receptor positive tumor. These properties are encouraging to carry out further experiments for non-invasive receptor

Design, synthesis and in vitro evaluation of heterobivalent peptidic radioligands targeting both GRP- and VPAC1-Receptors concomitantly overexpressed on various malignancies - Is the concept feasible?

PubMed

Lindner, Simon; Fiedler, Luise; Wängler, Björn; Bartenstein, Peter; Schirrmacher, Ralf; Wängler, Carmen

2018-05-29

Radiolabeled heterobivalent peptidic ligands (HBPLs), being able to address different receptors, are highly interesting tumor imaging agents as they can offer multiple advantages over monovalent peptide receptor ligands. However, few examples of radiolabeled HBPLs have been described so far. One promising approach is the combination of gastrin-releasing peptide receptor (GRPR)- and vasoactive intestinal peptide receptor subtype 1 (VPAC 1 R)-targeting peptides into one single radioligand since gastrinomas, prostate and breast cancer have been shown to concomitantly or complementarily overexpress both receptors. Here we report the design and synthesis of different HBPLs, comprising a GRPR-binding (BBN 7-14 ) and a VPAC 1 R-targeting (PACAP-27) peptide. The heterodimers were varied with regard to the distance between the peptide binders and the steric rigidity of the systems. We radiolabeled the HBPLs 19-23 as well as their monomeric reference standards 26 and 27 with 68 Ga, achieving radiochemical yields and purities of 95-99% and non-optimized molar activities of 25-61 GBq/μmol. We tested the stability of the radioligands and further evaluated them in vitro regarding their uptake in different prostate carcinoma cell lines (PC-3, DU-145 and VCaP cells). We found that the heterobivalent substances [ 68 Ga]19 - [ 68 Ga]23 showed comparable uptakes into the tumor cells to those of the respective monomers [ 68 Ga]26 and [ 68 Ga]27, indicating that both peptides are still able to address their target receptors. Furthermore, the obtained results indicate that in case of overall low receptor densities, heterobivalent peptides surpass peptide monomers in tumor cell uptake. Most importantly, it could be shown by blocking studies that both peptide parts of the HBPL [ 68 Ga]19 contributed to tumor cell uptake in VCaP cells, expressing both receptor types. Thus, we describe here the first examples of HBPLs being able to address the GRPR as well as the VPAC 1 R and have the

Involvement of TRPV1 in the expression and release of calcitonin gene-related peptide induced by rutaecarpine.

PubMed

Yang, Yongmei; Chen, Qingquan; Jia, Sujie; He, Limei; Wang, Aiping; Li, Dai; Li, Yuanjian; Li, Xiaohui

2018-04-01

The traditional Chinese herb Wu-Chu-Yu has been used to treat hypertension for hundreds of years. A previous study indicated that rutaecarpine was the effective component of Wu‑Chu‑Yu, which lowered blood pressure by elevating the expression level of calcitonin gene‑related peptide (CGRP). The present study was performed to investigate the role of transient receptor potential cation channel subfamily V member 1 (TRPV1) in CGRP expression and release induced by rutaecarpine. Dorsal root ganglia (DRG) obtained from Sprague‑Dawley rats were cultured to analyze the mRNA expression and release of CGRP. Calcium influx, as an indicator of TRPV1 activation, was measured in 293 cells with stable overexpression of TRPV1. The results demonstrated that the amount of CGRP in the cell culture supernatant and the mRNA expression of CGRPα and CGRPβ in DRG was upregulated by rutaecarpine in a concentration‑dependent manner, and was inhibited by the TRPV1 receptor antagonist capsazepine. In addition, intracellular Ca2+ levels were increased by Rut in the aforementioned 293 cell line, indicating the activation of TRPV1 by Rut. Therefore, it was concluded that TRPV1 was involved in the expression and release of CGRP stimulated by rutaecarpine, which provided novel mechanistic understanding of the treatment of hypertension using the Chinese herb Wu-Chu-Yu.

Effect of single dose of omeprazole on the gastrointestinal peptide response to food.

PubMed

Allen, J M; Adrian, T E; Webster, J; Howe, A; Bloom, S R

1984-02-01

The gastrointestinal peptide response to food was assessed in 6 healthy subjects following oral administration of 40 mg omeprazole. There was a small but statistically significant increase in basal plasma gastrin six hours after the dose of omeprazole, but the post-prandial plasma gastrin was not significantly increased. There was no significant effect on basal or post-prandial levels of somatostatin, insulin, pancreatic glucagon, enteroglucagon, gastric inhibitory polypeptide, pancreatic polypeptide, motilin, neurotensin, cholecystokinin, secretin, vasoactive intestinal peptide and gastrin-releasing peptide or blood glucose concentration.

Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry.

PubMed

Papac, D I; Hoyes, J; Tomer, K B

1994-09-01

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.

Somatostatin sst2 receptor-mediated inhibition of parietal cell function in rat isolated gastric mucosa.

PubMed Central

Wyatt, M. A.; Jarvie, E.; Feniuk, W.; Humphrey, P. P.

1996-01-01

1. The aim of this study was to determine the location and functional characteristics of the somatostatin (SRIF) receptor type(s) which mediate inhibition of acid secretion in rat isolated gastric mucosa. 2. Gastrin (1 nM-1 microM), dimaprit (10 microM-300 microM) and isobutyl methylxanthine (IBMX, 1 microM-100 microM) all caused concentration-dependent increases in acid output. Responses to gastrin were almost completely inhibited by ranitidine (10 microM) at a concentration which abolished the secretory response to dimaprit. In contrast, responses to IBMX were not changed by ranitidine suggesting that IBMX acts directly on the parietal cell and not indirectly by releasing histamine from enterochromaffin-like (ECL) cells. 3. SRIF-14 (1 nM-1 microM) had no effect on basal acid output, but inhibited acid output produced by gastrin, dimaprit and IBMX in a concentration-dependent manner with respective EC50 values of 46, 54 and 167 nM. The peptidase inhibitors, amastatin (10 microM) and phosphoramidon (1 microM), had no effect on SRIF-induced inhibition of dimaprit stimulated gastric acid secretion. 4. The inhibitory effect of a range of SRIF analogues on gastrin-, dimaprit- and IBMX-induced acid secretion was also studied. Irrespective of the secretagogue used to increase acid output, the rank order of potencies was similar (BIM-23027 = seglitide = octreotide > SRIF-14 = SRIF-28 > L-362,855). The linear peptide BIM-23056 was devoid of agonist or antagonist activity in concentrations up to 1 microM. 5. The sst2 receptor selective peptides, BIM-23027, seglitide and octreotide were the most potent inhibitors of gastrin-, dimaprit- and IBMX-induced acid secretion suggesting that SRIF receptors resembling the recombinant sst2 receptors are involved. Furthermore, since dimaprit and IBMX stimulate gastric acid secretion independently of histamine release, sst2 receptor-mediated inhibition must occur at the level of the parietal cell itself. PMID:8922739

Somatostatin sst2 receptor-mediated inhibition of parietal cell function in rat isolated gastric mucosa.

PubMed

Wyatt, M A; Jarvie, E; Feniuk, W; Humphrey, P P

1996-11-01

1. The aim of this study was to determine the location and functional characteristics of the somatostatin (SRIF) receptor type(s) which mediate inhibition of acid secretion in rat isolated gastric mucosa. 2. Gastrin (1 nM-1 microM), dimaprit (10 microM-300 microM) and isobutyl methylxanthine (IBMX, 1 microM-100 microM) all caused concentration-dependent increases in acid output. Responses to gastrin were almost completely inhibited by ranitidine (10 microM) at a concentration which abolished the secretory response to dimaprit. In contrast, responses to IBMX were not changed by ranitidine suggesting that IBMX acts directly on the parietal cell and not indirectly by releasing histamine from enterochromaffin-like (ECL) cells. 3. SRIF-14 (1 nM-1 microM) had no effect on basal acid output, but inhibited acid output produced by gastrin, dimaprit and IBMX in a concentration-dependent manner with respective EC50 values of 46, 54 and 167 nM. The peptidase inhibitors, amastatin (10 microM) and phosphoramidon (1 microM), had no effect on SRIF-induced inhibition of dimaprit stimulated gastric acid secretion. 4. The inhibitory effect of a range of SRIF analogues on gastrin-, dimaprit- and IBMX-induced acid secretion was also studied. Irrespective of the secretagogue used to increase acid output, the rank order of potencies was similar (BIM-23027 = seglitide = octreotide > SRIF-14 = SRIF-28 > L-362,855). The linear peptide BIM-23056 was devoid of agonist or antagonist activity in concentrations up to 1 microM. 5. The sst2 receptor selective peptides, BIM-23027, seglitide and octreotide were the most potent inhibitors of gastrin-, dimaprit- and IBMX-induced acid secretion suggesting that SRIF receptors resembling the recombinant sst2 receptors are involved. Furthermore, since dimaprit and IBMX stimulate gastric acid secretion independently of histamine release, sst2 receptor-mediated inhibition must occur at the level of the parietal cell itself.

Connective tissue growth factor is activated by gastrin and involved in gastrin-induced migration and invasion.

PubMed

Bhandari, Sabin; Bakke, Ingunn; Kumar, J; Beisvag, Vidar; Sandvik, Arne K; Thommesen, Liv; Varro, Andrea; Nørsett, Kristin G

2016-06-17

Connective tissue growth factor (CTGF) has been reported in gastric adenocarcinoma and in carcinoid tumors. The aim of this study was to explore a possible link between CTGF and gastrin in gastric epithelial cells and to study the role of CTGF in gastrin induced migration and invasion of AGS-GR cells. The effects of gastrin were studied using RT-qPCR, Western blot and assays for migration and invasion. We report an association between serum gastrin concentrations and CTGF abundancy in the gastric corpus mucosa of hypergastrinemic subjects and mice. We found a higher expression of CTGF in gastric mucosa tissue adjacent to tumor compared to normal control tissue. We showed that gastrin induced expression of CTGF in gastric epithelial AGS-GR cells via MEK, PKC and PKB/AKT pathways. CTGF inhibited gastrin induced migration and invasion of AGS-GR cells. We conclude that CTGF expression is stimulated by gastrin and involved in remodeling of the gastric epithelium. Copyright © 2016 Elsevier Inc. All rights reserved.

The non-peptide GLP-1 receptor agonist WB4-24 blocks inflammatory nociception by stimulating β-endorphin release from spinal microglia

PubMed Central

Fan, Hui; Gong, Nian; Li, Teng-Fei; Ma, Ai-Niu; Wu, Xiao-Yan; Wang, Ming-Wei; Wang, Yong-Xiang

2015-01-01

BACKGROUND AND PURPOSE Two peptide agonists of the glucagon-like peptide-1 (GLP-1) receptor, exenatide and GLP-1 itself, exert anti-hypersensitive effects in neuropathic, cancer and diabetic pain. In this study, we have assessed the anti-allodynic and anti-hyperalgesic effects of the non-peptide agonist WB4-24 in inflammatory nociception and the possible involvement of microglial β-endorphin and pro-inflammatory cytokines. EXPERIMENTAL APPROACH We used rat models of inflammatory nociception induced by formalin, carrageenan or complete Freund's adjuvant (CFA), to test mechanical allodynia and thermal hyperalgesia. Expression of β-endorphin and pro-inflammatory cytokines was measured using real-time quantitative PCR and fluorescent immunoassays. KEY RESULTS WB4-24 displaced the specific binding of exendin (9–39) in microglia. Single intrathecal injection of WB4-24 (0.3, 1, 3, 10, 30 and 100 μg) exerted dose-dependent, specific, anti-hypersensitive effects in acute and chronic inflammatory nociception induced by formalin, carrageenan and CFA, with a maximal inhibition of 60–80%. Spinal WB4-24 was not effective in altering nociceptive pain. Subcutaneous injection of WB4-24 was also antinociceptive in CFA-treated rats. WB4-24 evoked β-endorphin release but did not inhibit expression of pro-inflammatory cytokines in either the spinal cord of CFA-treated rats or cultured microglia stimulated by LPS. WB4-24 anti-allodynia was prevented by a microglial inhibitor, β-endorphin antiserum and a μ-opioid receptor antagonist. CONCLUSIONS AND IMPLICATIONS Our results suggest that WB4-24 inhibits inflammatory nociception by releasing analgesic β-endorphin rather than inhibiting the expression of proalgesic pro-inflammatory cytokines in spinal microglia, and that the spinal GLP-1 receptor is a potential target molecule for the treatment of pain hypersensitivity including inflammatory nociception. PMID:25176008

Inhibition of dipeptidyl peptidase-4 augments insulin secretion in response to exogenously administered glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, pituitary adenylate cyclase-activating polypeptide, and gastrin-releasing peptide in mice.

PubMed

Ahrén, Bo; Hughes, Thomas E

2005-04-01

Inhibition of dipeptidyl peptidase-4 (DPP-4) is currently being explored as a new approach to the treatment of type 2 diabetes. This concept has emerged from the powerful and rapid action of the enzyme to inactivate glucagon-like peptide-1 (GLP-1). However, other bioactive peptides with potential influence of islet function are also substrates of DPP-4. Whether this inactivation may add to the beneficial effects of DPP-4 inhibition is not known. In this study, we explored whether DPP-4 inhibition by valine-pyrrolidide (val-pyr; 100 micromol/kg administered through gastric gavage at t = -30 min) affects the insulin and glucose responses to iv glucose (1 g/kg) together with GLP-1 (10 nmol/kg), glucose-dependent insulinotropic polypeptide (GIP; 10 nmol/kg), pituitary adenylate cyclase-activating polypeptide 38 (PACAP38; 1.3 nmol/kg), or gastrin-releasing peptide (GRP; 20 nmol/kg) given at t = 0 in anesthetized C57BL/6J mice. It was found that the acute (1-5 min) insulin response to GLP-1 was augmented by val-pyr by 80% (4.2 +/- 0.4 vs. 7.6 +/- 0.8 nmol/liter), that to GIP by 40% (2.7 +/- 0.3 vs. 3.8 +/- 0.4 nmol/liter), that to PACAP38 by 75% (4.6 +/- 0.5 vs. 8.1 +/- 0.6 nmol/liter), and that to GRP by 25% (1.8 +/- 0.2 vs. 2.3 +/- 0.3 nmol/liter; all P < 0.05 or less). This was associated with enhanced glucose elimination rate after GLP-1 [glucose elimination constant (K(G)) 2.1 +/- 0.2 vs. 3.1 +/- 0.3%/min] and PACAP38 (2.1 +/- 0.3 vs. 3.2 +/- 0.3%/min; both P < 0.01), but not after GIP or GRP. The augmented insulin response to GRP by val-pyr was prevented by the GLP-1 receptor antagonist, exendin(3) (9-39), raising the possibility that GRP effects may occur secondary to stimulation of GLP-1 secretion. We conclude that DPP-4 inhibition augments the insulin response not only to GLP-1 but also to GIP, PACAP38, and GRP.

Transient receptor potential ankyrin 1 channels are involved in spontaneous peptide hormone release from astrocytes.

PubMed

Takizawa, Mai; Harada, Kazuki; Nakamura, Kazuaki; Tsuboi, Takashi

2018-07-02

Astrocytes, a large population of glial cells, detect neurotransmitters and respond by increasing intracellular Ca 2+ concentration ([Ca 2+ ] i ) and releasing chemical molecules called gliotransmitters. Recently discovered Ca 2+ influx through transient receptor potential ankyrin 1 (TRPA1) channels is reported to cause spontaneous [Ca 2+ ] i increase in astrocytes. While several physiological functions of TRPA1-mediated spontaneous Ca 2+ signal have been revealed, relation with gliotransmitter release, especially peptide hormone exocytosis is largely unknown. We therefore explored the [Ca 2+ ] i and exocytosis dynamics in rat astrocyte cell line C6 cells and primary astrocytes. TRPA1-mediated spontaneous [Ca 2+ ] i transients were observed in both C6 cells and primary astrocytes. Total internal reflection fluorescence microscopy revealed that Venus-tagged brain-derived neurotrophic factor and neuropeptide Y were released spontaneously from astrocytes. Activation of TRPA1 channels enhanced the frequency of peptide hormone exocytosis, and inhibition of TRPA1 channels decreased the number of peptide hormone exocytosis. These results suggest that TRPA1-mediated spontaneous [Ca 2+ ] i increase modulates the spontaneous release of peptide hormones from astrocytes. Copyright © 2018 Elsevier Inc. All rights reserved.

Pro-gastrin-releasing peptide (ProGRP) as a biomarker in small-cell lung cancer diagnosis, monitoring and evaluation of treatment response.

PubMed

Wojcik, Ewa; Kulpa, Jan Kanty

2017-01-01

Lung cancer belongs to malignant tumors that possess the highest rates of morbidity and mortality in the world. A number of morphological, biological and clinical features justify the distinction of small-cell carcinoma with respect to the other histological types of lung cancer. The predominant neuroendocrine phenotype is critical for the selection of biomarkers used in diagnostics, monitoring and evaluation of treatment response; early onset relapses in patients with small-cell lung cancer (SCLC) and the evaluation of their prognosis. Although for a long time the neuron-specific enolase (NSE) was considered to be the marker of choice for this tumor, it is now increasingly important to pay attention to concentrations of pro-gastrin-releasing peptide (ProGRP). The results of this marker have been implicated in the differential diagnosis of non-small lung cancer and SCLC, chemotherapy and radiotherapy monitoring as well as evaluation of treatment response. The subject of this series of studies is to determine the usefulness of ProGRP in the evaluation of patients' prognosis and its predictive value. The current aim for the optimization of the effectiveness of biochemical diagnostics of SCLC is recommended by complementary ProGRP and NSE studies. The present work is a summary of the latest reports regarding diagnostic utility of these markers in SCLC.

Preclinical comparison of Al18F- and 68Ga-labeled gastrin-releasing peptide receptor antagonists for PET imaging of prostate cancer.

PubMed

Chatalic, Kristell L S; Franssen, Gerben M; van Weerden, Wytske M; McBride, William J; Laverman, Peter; de Blois, Erik; Hajjaj, Bouchra; Brunel, Luc; Goldenberg, David M; Fehrentz, Jean-Alain; Martinez, Jean; Boerman, Otto C; de Jong, Marion

2014-12-01

Gastrin-releasing peptide receptor (GRPR) is overexpressed in human prostate cancer and is being used as a target for molecular imaging. In this study, we report on the direct comparison of 3 novel GRPR-targeted radiolabeled tracers: Al(18)F-JMV5132, (68)Ga-JMV5132, and (68)Ga-JMV4168 (JMV5132 is NODA-MPAA-βAla-βAla-[H-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2], JMV4168 is DOTA-βAla-βAla-[H-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2], and NODA-MPAA is 2-[4-(carboxymethyl)-7-{[4-(carboxymethyl)phenyl]methyl}-1,4,7-triazacyclononan-1-yl]acetic acid). The GRPR antagonist JMV594 (H-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) was conjugated to NODA-MPAA for labeling with Al(18)F. JMV5132 was radiolabeled with (68)Ga and (18)F, and JMV4168 was labeled with (68)Ga for comparison. The inhibitory concentration of 50% values for binding GRPR of JMV4168, JMV5132, (nat)Ga-JMV4168, and (nat)Ga-JMV5132 were determined in a competition-binding assay using GRPR-overexpressing PC-3 tumors. The tumor-targeting characteristics of the compounds were assessed in mice bearing subcutaneous PC-3 xenografts. Small-animal PET/CT images were acquired, and tracer biodistribution was determined by ex vivo measurements. JMV5132 was labeled with (18)F in a novel 1-pot, 1-step procedure within 20 min, without need for further purification and resulting in a specific activity of 35 MBq/nmol. Inhibitory concentration of 50% values (in nM) for GRPR binding of JMV5132, JMV4168, (nat)Ga-JMV5132, (nat)Ga-JMV4168, and Al(nat)F-JMV5132 were 6.8 (95% confidence intervals [CIs], 4.6-10.0), 13.2 (95% CIs, 5.9-29.3), 3.0 (95% CIs, 1.5-6.0), 3.2 (95% CIs, 1.8-5.9), and 10.0 (95% CIs, 6.3-16.0), respectively. In mice with subcutaneous PC-3 xenografts, all tracers cleared rapidly from the blood, exclusively via the kidneys for (68)Ga-JMV4168 and partially hepatobiliary for (68)Ga-JMV5132 and Al(18)F-JMV5132. Two hours after injection, the uptake of (68)Ga-JMV4168, (68)Ga-JMV5132, and Al(18)F-JMV5132 in PC-3

Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry.

PubMed Central

Papac, D. I.; Hoyes, J.; Tomer, K. B.

1994-01-01

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP. PMID:7530543

Bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by protein kinase C, and is enhanced by disruption of rho/cytoskeletal pathways.

PubMed Central

Seensalu, R; Avedian, D; Barbuti, R; Song, M; Slice, L; Walsh, J H

1997-01-01

Isolated canine G cells in primary culture have been used to study calcium, protein kinase C (PKC), and rho/cytoskeletal-dependent intracellular pathways involved in bombesin- stimulated gastrin release. A method to obtain highly purified G cells by culture (64% G cells) after flow cytometry on elutriated fractions of cells from digested canine gastric antral mucosa has been developed. Pretreatment of G cells with thapsigargin (10(-8)-10(-6) M) and release experiments in Ca2+-containing or -depleted media showed that influx of Ca2+ into the cells and not acute release from intracellular stores plays an important role in bombesin-stimulated gastrin release. Inhibition of PKC by the specific inhibitor GF 109 203X did not affect bombesin-stimulated release. Rho, a small GTP-binding protein that regulates the actin cytoskeleton, is specifically antagonized by Clostridium botulinum C3 exoenzyme. C3 (10 microg/ml) enhanced basal and bombesin-stimulated gastrin release by 315 and 266%, respectively. The importance of the cytoskeleton for regulation of gastrin release was emphasized by a more pronounced release of gastrin when the organization of the actin cytoskeleton was disrupted by cytochalasin D (5 x 10(-)7 and 10(-)6 M). Wortmannin, a potent inhibitor of phosphoinositide-3-kinase, did not alter bombesin-stimulated gastrin release. Thus, it is concluded that bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by PKC, and is enhanced by disruption of rho/cytoskeletal pathways. PMID:9276720

Bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by protein kinase C, and is enhanced by disruption of rho/cytoskeletal pathways.

PubMed

Seensalu, R; Avedian, D; Barbuti, R; Song, M; Slice, L; Walsh, J H

1997-09-01

Isolated canine G cells in primary culture have been used to study calcium, protein kinase C (PKC), and rho/cytoskeletal-dependent intracellular pathways involved in bombesin- stimulated gastrin release. A method to obtain highly purified G cells by culture (64% G cells) after flow cytometry on elutriated fractions of cells from digested canine gastric antral mucosa has been developed. Pretreatment of G cells with thapsigargin (10(-8)-10(-6) M) and release experiments in Ca2+-containing or -depleted media showed that influx of Ca2+ into the cells and not acute release from intracellular stores plays an important role in bombesin-stimulated gastrin release. Inhibition of PKC by the specific inhibitor GF 109 203X did not affect bombesin-stimulated release. Rho, a small GTP-binding protein that regulates the actin cytoskeleton, is specifically antagonized by Clostridium botulinum C3 exoenzyme. C3 (10 microg/ml) enhanced basal and bombesin-stimulated gastrin release by 315 and 266%, respectively. The importance of the cytoskeleton for regulation of gastrin release was emphasized by a more pronounced release of gastrin when the organization of the actin cytoskeleton was disrupted by cytochalasin D (5 x 10(-)7 and 10(-)6 M). Wortmannin, a potent inhibitor of phosphoinositide-3-kinase, did not alter bombesin-stimulated gastrin release. Thus, it is concluded that bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by PKC, and is enhanced by disruption of rho/cytoskeletal pathways.

Short communication: expression of peptide YY, proglucagon, neuropeptide Y receptor Y2, and glucagon-like peptide-1 receptor in bovine peripheral tissues.

PubMed

Pezeshki, A; Muench, G P; Chelikani, P K

2012-09-01

The role of distal gut signals in control of feed intake and metabolism in cattle has received scant attention. Peptide YY (PYY) and glucagon-like peptide-1, which are secreted from enteroendocrine cells of the distal gut in monogastrics have several functions, including regulation of energy balance. However, little is known of the tissue expression of these peptides and their receptors in cattle. The aim of the current study was to characterize the tissue distribution of PYY, neuropeptide Y receptor Y2 (Y2), proglucagon (GCG), and glucagon-like peptide-1 receptor (GLP1R) in various peripheral tissues of cattle. Four male 7-wk-old dairy calves were euthanized and 16 peripheral tissues were collected. Conventional PCR and quantitative real-time PCR were performed to confirm tissue expression and quantify the transcript abundance in various tissues. The results of conventional PCR revealed that mRNA for both PYY and Y2 was detectable in the rumen, abomasum, duodenum, jejunum, ileum, and colon but not in other tissues. Quantitative real-time PCR data demonstrated that PYY mRNA was 2- to 3-fold greater in the pancreas, kidney, and heart relative to the liver. By conventional PCR, GCG mRNA was detected in the abomasum, duodenum, jejunum, ileum, and colon and GLP1R mRNA was expressed in all gut segments, pancreas, spleen, and kidney. Quantitative real-time PCR data demonstrated that, relative to transcript abundance in the liver, GCG mRNA was 4- to 40-fold higher from abomasum to colon, and GLP1R mRNA was 50- to 300-fold higher from the rumen to colon, 14-fold greater in the pancreas, 18-fold higher in the spleen, and 166-fold greater in the kidney. The tissue distribution of PYY, GCG, and their receptors observed in the current study is, in general, consistent with expression patterns in monogastrics. The predominant expression of PYY, Y2, and GCG in the gut, and the presence of GLP1R in multiple peripheral tissues suggest a role for PYY in controlling gut functions and

Ultrastructural characterization of atrial natriuretic peptide receptors (ANP-R) mRNA expression in rat kidney cortex.

PubMed

Grandclément, B; Morel, G

1998-06-01

Atrial natriuretic peptide (ANP) and two complementary peptides named brain natriuretic peptide and C-type natriuretic peptide are involved in diuresis, natriuresis, hypotension and vasorelaxation. Their actions are mediated by highly selective and specific ANP receptors. Three subtypes have been characterized and cloned: ANP receptor A, -B and -C. In the present study, the mRNA for each subtype was detected by ultrastructural in situ hybridization on ultrathin sections of Lowicryl-embedded tissue and frozen tissue. The distribution of mRNA (visualized by gold particles) for each subtype was found to differ in different cells of the nephron. The three subtypes of this receptor family were expressed in all the parts of the nephron, but their expression levels were different. The ANPR-A mRNA was the most abundant in cells of glomerulus, proximal and distal tubules. The subtype C was the least expressed mRNA in glomerulus. In contrast, the subcellular localization of the three mRNAs was similar; they were found in the cytoplasmic matrix and the euchromatin of the nucleus. In conclusion, the differential expression of these mRNAs in kidney cortex indicates that these three peptides act directly in differing parts of nephron regions which are the glomerulus, the proximal and distal tubules.

HMC-1 human mast cells synthesize neurotensin (NT) precursor, secrete bioactive NT-like peptide(s) and express NT receptor NTS1.

PubMed

Cochrane, David E; Carraway, Robert E; Harrington, Kimberly; Laudano, Melissa; Rawlings, Stephen; Feldberg, Ross S

2011-12-01

To determine if mast cells synthesize the inflammatory peptide, neurotensin (NT), secrete immunoreactive and bioactive NT, and express the NT receptor NTS1. HMC-1 cells, pleural mast cells from Sprague-Dawley rats, LAD2 mast cells, and human cord blood mast cells were used. HMC-1 cells were stimulated with NT, C48/80, mastoparan, or PGE(2). For changes in cutaneous vascular permeability, anesthetized rats were injected intravenously with Evans Blue dye and intradermally with saline, NT, histamine, diphenhydramine, and C48/80. RT-PCR was used to identify RNA transcripts. Histamine was measured by fluorometric assay. In vivo cutaneous vascular permeability assays, radio-immunoassays for NT, Western blotting for the NT precursor protein and NTS1 protein from HMC-1 cells and tissues from rats were used. Immunohistochemistry was used to identify NT precursor-like proteins in HMC-1 mast cells. HMC-1 cells express mRNAs for NT precursor, PC5A processing enzyme and NTS1 receptor. Human cord blood mast cells and LAD2 mast cells express mRNA transcripts for NT precursor and NTS1. Western blotting showed NT precursor and NTS1 receptor in HMC1. Rat tissues with high numbers of mast cells contained NT precursor proteins. NT-like peptides from HMC-1 displayed NT-like bioactivity. HMC-1 mast cells synthesize and secrete immunoreactive and bioactive NT-like peptide(s) and express the NT receptor, suggesting that NT from mast cells might serve autocrine and paracrine roles.

Peptide processing and biology in human disease

PubMed Central

Kovac, Suzana; Shulkes, Arthur; Baldwin, Graham S.

2008-01-01

Purpose of review To describe recent advances in the processing of gastrointestinal hormones, and the consequences for human disease of mutations in the enzymes involved. Recent findings Although gastrointestinal prohormones were long regarded as devoid of biological activity, recent data indicates that the prohormones for both gastrin and gastrin-releasing peptide are bioactive, through different receptors from the mature hormones. Mutations in the family of prohormone convertases responsible for the initial steps in the processing of gastrointestinal hormones are associated with several different pathophysiological conditions in humans. Summary Human mutational studies, when taken together with the phenotypes observed in mice deficient in the prohormone convertases, emphasize the crucial importance of the processing enzymes in mammalian biology. Although the phenotypes may often be ascribed to defective production of a mature hormone or growth factor, the recognition that the precursors are independently bioactive suggests that the increased precursor concentrations may also contribute to the symptoms. The observation that the precursors often act through different receptors from the mature hormones may permit the development of precursor-selective antagonists for therapeutic use. PMID:19104240

Peptide processing and biology in human disease.

PubMed

Kovac, Suzana; Shulkes, Arthur; Baldwin, Graham S

2009-02-01

To describe recent advances in the processing of gastrointestinal hormones, and the consequences for human disease of mutations in the enzymes involved. Although gastrointestinal prohormones were long regarded as devoid of biological activity, recent data indicate that the prohormones for both gastrin and gastrin-releasing peptide are bioactive, through different receptors from the mature hormones. Mutations in the family of prohormone convertases responsible for the initial steps in the processing of gastrointestinal hormones are associated with several different pathophysiological conditions in humans. Human mutational studies, when taken together with the phenotypes observed in mice deficient in the prohormone convertases, emphasize the crucial importance of the processing enzymes in mammalian biology. Although the phenotypes may often be ascribed to defective production of a mature hormone or growth factor, the recognition that the precursors are independently bioactive suggests that the increased precursor concentrations may also contribute to the symptoms. The observation that the precursors often act through different receptors from the mature hormones may permit the development of precursor-selective antagonists for therapeutic use.

Glucagon Like Peptide-1 Receptor Expression in the Human Thyroid Gland

PubMed Central

Gier, Belinda; Butler, Peter C.; Lai, Chi K.; Kirakossian, David; DeNicola, Matthew M.

2012-01-01

Background: Glucagon like peptide-1 (GLP-1) mimetic therapy induces medullary thyroid neoplasia in rodents. We sought to establish whether C cells in human medullary thyroid carcinoma, C cell hyperplasia, and normal human thyroid express the GLP-1 receptor. Methods: Thyroid tissue samples with medullary thyroid carcinoma (n = 12), C cell hyperplasia (n = 9), papillary thyroid carcinoma (n = 17), and normal human thyroid (n = 15) were evaluated by immunofluorescence for expression of calcitonin and GLP-1 receptors. Results: Coincident immunoreactivity for calcitonin and GLP-1 receptor was consistently observed in both medullary thyroid carcinoma and C cell hyperplasia. GLP-1 receptor immunoreactivity was also detected in 18% of papillary thyroid carcinoma (three of 17 cases). Within normal human thyroid tissue, GLP-1 receptor immunoreactivity was found in five of 15 of the examined cases in about 35% of the total C cells assessed. Conclusions: In humans, neoplastic and hyperplastic lesions of thyroid C cells express the GLP-1 receptor. GLP-1 receptor expression is detected in 18% papillary thyroid carcinomas and in C cells in 33% of control thyroid lobes. The consequence of long-term pharmacologically increased GLP-1 signaling on these GLP-1 receptor-expressing cells in the thyroid gland in humans remains unknown, but appropriately powered prospective studies to exclude an increase in medullary or papillary carcinomas of the thyroid are warranted. PMID:22031513

Preclinical Evaluation of 68Ga-DOTA-Minigastrin for the Detection of Cholecystokinin-2/Gastrin Receptor–Positive Tumors

PubMed Central

Brom, Maarten; Joosten, Lieke; Laverman, Peter; Oyen, Wim J.G.; Béhé, Martin; Gotthardt, Martin; Boerman, Otto C.

2011-01-01

In comparison to somatostatin receptor scintigraphy, gastrin receptor scintigraphy using 111In-DTPA-minigastrin (MG0) showed added value in diagnosing neuroendocrine tumors. We investigated whether the 68Ga-labeled gastrin analogue DOTA-MG0 is suited for positron emission tomography (PET), which could improve image quality. Targeting of cholecystokinin-2 (CCK2)/gastrin receptor–positive tumor cells with DOTA-MG0 labeled with either 111In or 68Ga in vitro was investigated using the AR42J rat tumor cell line. Biodistribution was examined in BALB/c nude mice with a subcutaneous AR42J tumor. In vivo PET imaging was performed using a preclinical PET–computed tomographic scanner. DOTA-MG0 showed high receptor affinity in vitro. Biodistribution studies revealed high tumor uptake of 68Ga-DOTA-MG0: 4.4 ± 1.3 %ID/g at 1 hour postinjection. Coadministration of an excess unlabeled peptide blocked the tumor uptake (0.7 ± 0.1 %ID/g), indicating CCK2/gastrin receptor–mediated uptake (p = .0005). The biodistribution of 68Ga-DOTA-MG0 was similar to that of 111In-DOTA-MG0. Subcutaneous and intraperitoneal tumors were clearly visualized by small-animal PET imaging with 5 MBq 68Ga-DOTA-MG0. 111In- and 68Ga-labeled DOTA-MG0 specifically accumulate in CCK2/gastrin receptor–positive AR42J tumors with similar biodistribution apart from the kidneys. AR42J tumors were clearly visualized by microPET. Therefore, 68Ga-DOTA-MG0 is a promising tracer for PET imaging of CCK2/gastrin receptor–positive tumors in humans. PMID:21439259

SELF ADMINISTRATION OF OXYCODONE BY ADOLESCENT AND ADULT MICE AFFECTS STRIATAL NEUROTRANSMITTER RECEPTOR GENE EXPRESSION

PubMed Central

Mayer-Blackwell, B.; Schlussman, S. D.; Butelman, E. R.; Ho, A.; Ott, J.; Kreek, M. J.; Zhang, Y.

2014-01-01

Illicit use of prescription opioid analgesics (e.g., oxycodone) in adolescence is a pressing public health issue. Our goal was to determine whether oxycodone self administration differentially affects striatal neurotransmitter receptor gene expression in the dorsal striatum of adolescent compared to adult C57BL/6J mice. Groups of adolescent mice (4 weeks old, n= 12) and of adult mice (11 weeks old, n= 11) underwent surgery during which a catheter was implanted into their jugular veins. After recovering from surgery, mice self administered oxycodone (0.25 mg/kg/infusion) 2 h/day for 14 consecutive days or served as yoked saline controls. Mice were sacrificed within 1 h after the last self-administration session and the dorsal striatum was isolated for mRNA analysis. Gene expression was analyzed with real time PCR using a commercially available neurotransmitter receptor PCR array containing 84 genes. We found that adolescent mice self administered less oxycodone than adult mice over the 14 days. Monoamine oxidase A (Maoa) and neuropeptide Y receptor 5 mRNA levels were lower in adolescent mice than in adult mice without oxycodone exposure. Oxycodone self administration increased Maoa mRNA levels compared to controls in both age groups. There was a positive correlation of the amount of oxycodone self administered in the last session or across 14 sessions with Maoa mRNA levels. Gastrin-releasing peptide receptor mRNA showed a significant Drug × Age interaction, with point-wise significance. More genes in the dorsal striatum of adolescents (19) changed in response to oxycodone self administration compared to controls than in adult (4) mice. Overall, this study demonstrates that repeated oxycodone self administration alters neurotransmitter receptors gene expression in the dorsal striatum of adolescent and adult mice. PMID:24220688

Trophic effect of bombesin on the rat pancreas: is it mediated by the release of gastrin or cholecystokinin?

PubMed

Lhoste, E; Aprahamian, M; Pousse, A; Hoeltzel, A; Stock-Damge, C

1985-01-01

This work investigates the effect, on the rat pancreas, of a chronic administration of bombesin in function of the dose and duration of treatment and examines whether this effect may be mediated by the release of endogenous gastrin or cholecystokinin. Bombesin, administered three times daily for 5 or 15 days, induced a marked increase in pancreatic weight, its protein, RNA and enzyme contents with the dose of 10 micrograms/kg body weight; the ratios of pancreatic weight, protein and RNA contents to DNA contents increased significantly after a 5 day treatment, suggesting cellular hypertrophy. Pancreatic DNA content was markedly enhanced after a 15 day treatment, suggesting cellular hyperplasia. Antrectomy decreased plasma gastrin levels, but did not alter the pancreatico-trophic action of a 10 micrograms/kg bombesin treatment for 5 days. Proglumide, an inhibitor of cholecystokinin and gastrin in the pancreas, did not affect the growth of the pancreas induced by a 10 micrograms/kg bombesin treatment for 5 days. It is concluded that chronic bombesin induces, in the rat pancreas, cellular hypertrophy or hyperplasia depending on the duration of treatment. Pancreatic hypertrophy is not mediated by the release of endogenous gastrin or cholecystokinin.

Differential expression patterns of PQRFamide peptide and its two receptor genes in the brain and pituitary of grass puffer during the reproductive cycle.

PubMed

Shahjahan, Md; Doi, Hiroyuki; Ando, Hironori

2015-01-01

Pain-modulatory neuropeptides, PQRFamide (PQRFa) peptides, have recently been implicated in the regulation of reproduction in fish. As a first step toward investigating the role of PQRFa peptides on reproductive function in the grass puffer Takifugu niphobles, which is a semilunar spawner, we cloned genes encoding PQRFa peptide precursor (pqrfa) and its two types of receptors (pqrfa-r1 and pqrfa-r2), and examined changes in their expression levels in the brain and pituitary over several months during the reproductive cycle. The grass puffer PQRFa peptide precursor of 126 amino acid residues contains two putative PQRFa peptides, PQRFa-1 and PQRFa-2, which correspond to NPFF and NPAF in other vertebrates, respectively. The grass puffer PQRFa-R1 and PQRFa-R2 consist of 426 and 453 amino acid residues, respectively, and contain distinct characteristics of G-protein coupled receptors. These three genes were exclusively expressed in the brain and pituitary. The expression levels of pqrfa and pqrfa-r1 were significantly increased during the late stage of sexual maturation, but low in the spawning fish just after releasing sperms and eggs. Therefore, the grass puffer PQRFa peptide may have a role in the late stage of sexual maturation before spawning via PQRFa-R1. In contrast, the pqrfa-r2 expression showed maximum levels in the spawning fish and in the post-spawning period. The present results provide fundamental data suggesting that the grass puffer PQRFa peptide may have multiple roles in the control of reproduction that are dependent on the reproductive stages. Copyright © 2014 Elsevier Inc. All rights reserved.

Facile solid-phase synthesis of sulfated tyrosine-containing peptides: total synthesis of human big gastrin-II and cholecystokinin (CCK)-39.

PubMed

Kitagawa, K; Aida, C; Fujiwara, H; Yagami, T; Futaki, S; Kogire, M; Ida, J; Inoue, K

2001-01-12

Chemical synthesis of tyrosine O-sulfated peptides is still a laborious task for peptide chemists because of the intrinsic acid-lability of the sulfate moiety. An efficient cleavage/deprotection procedure without loss of the sulfate is the critical difficulty remaining to be solved for fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid-phase synthesis of sulfated peptides. To overcome the difficulty, TFA-mediated solvolysis rates of a tyrosine O-sulfate [Tyr(SO3H)] residue and two protecting groups, tBu for the hydroxyl group of Ser and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for the guanidino group of Arg, were examined in detail. The desulfation obeyed first-order kinetics with a large entropy (59.6 J.K-1.mol-1) and enthalpy (110.5 kJ.mol-1) of activation. These values substantiated that the desulfation rate of the rigidly solvated Tyr(SO3H) residue was strongly temperature-dependent. By contrast, the SN1-type deprotections were less temperature-dependent and proceeded smoothly in TFA of a high ionizing power. Based on the large rate difference between the desulfation and the SN1-type deprotections in cold TFA, an efficient deprotection protocol for the sulfated peptides was developed. Our synthetic strategy for Tyr(SO3H)-containing peptides with this effective deprotection protocol is as follows: (i) a sulfated peptide chain is directly constructed on 2-chlorotrityl resin with Fmoc-based solid-phase chemistry using Fmoc-Tyr(SO3Na)-OH as a building block; (ii) the protected peptide-resin is treated with 90% aqueous TFA at 0 degree C for an appropriate period of time for the cleavage and deprotection. Human cholecystokinin (CCK)-12, mini gastrin-II (14 residues), and little gastrin-II (17 residues) were synthesized with this method in 26-38% yields without any difficulties. This method was further applied to the stepwise synthesis of human big gastrin-II (34 residues), CCK-33 and -39. Despite the prolonged acid treatment (15-18 h at 0 degree C), the

A fast tumor-targeting near-infrared fluorescent probe based on bombesin analog for in vivo tumor imaging.

PubMed

Chen, Haiyan; Wan, Shunan; Zhu, Fenxia; Wang, Chuan; Cui, Sisi; Du, Changli; Ma, Yuxiang; Gu, Yueqing

2014-01-01

Bombesin (BBN), an analog of gastrin-releasing peptide (GRP), of which the receptors are over-expressed on various tumor cells, is able to bind to GRP receptor specifically. In this study, a near-infrared fluorescent dye (MPA) and polyethylene glycol (PEG) were conjugated to BBN analog to form BBN[7-14]-MPA and BBN[7-14]-SA-PEG-MPA. The successful synthesis of the two probes was proved by the characterization via sodium dodecylsulfate-polyacrylamide gel electrophoresis, infrared and optical spectra. Cellular uptakes studies indicated that BBN-based probes were mediated by gastrin-releasing peptide receptors (GRPR) on tumor cells and the PEG modified probe had higher affinity. The dynamic distribution and clearance investigations showed that the BBN-based probes were eliminated by the liver-kidney pathway. Furthermore, both of the BBN-based probes displayed tumor-targeting ability in GRPR over-expressed tumor-bearing mice. The PEG modified probe exhibited faster and higher tumor targeting capability than BBN[7-14]-MPA. The results implied that BBN[7-14]-SA-PEG-MPA could act as an effective fluorescence probe for tumor imaging. Copyright © 2014 John Wiley & Sons, Ltd.

Glucagon-like peptide-1 receptor expression on human eosinophils and its regulation of eosinophil activation.

PubMed

Mitchell, P D; Salter, B M; Oliveria, J P; El-Gammal, A; Tworek, D; Smith, S G; Sehmi, R; Gauvreau, G M; Butler, M; O'Byrne, P M

2017-03-01

Glucagon-like peptide-1 (GLP-1) and its receptor are part of the incretin family of hormones that regulate glucose metabolism. GLP-1 also has immune modulatory roles. To measure the expression of the GLP-1 receptor (GLP-1R) on eosinophils and neutrophils in normal and asthmatic subjects and evaluate effects of a GLP-1 analog on eosinophil function. Peripheral blood samples were taken from 10 normal and 10 allergic asthmatic subjects. GLP-1R expression was measured on eosinophils and neutrophils. Subsequently, the asthmatic subjects underwent allergen and diluent inhalation challenges, and GLP-1R expression was measured. Purified eosinophils, collected from mild asthmatic subjects, were stimulated with lipopolysaccharide (LPS) and a GLP-1 analog to evaluate eosinophil cell activation markers CD11b and CD69 and cytokine (IL-4, IL-5, IL-8 and IL-13) production. Glucagon-like peptide-1 receptor is expressed on human eosinophils and neutrophils. Eosinophil, but not neutrophil, expression of GLP-1R is significantly higher in normal controls compared to allergic asthmatics. The expression of GLP-1R did not change on either eosinophils or neutrophils following allergen challenge. A GLP-1 analog significantly decreased the expression of eosinophil-surface activation markers following LPS stimulation and decreased eosinophil production of IL-4, IL-8 and IL-13, but not IL-5. Glucagon-like peptide-1 receptor is expressed on human eosinophils and neutrophils. A GLP-1 analog attenuates LPS-stimulated eosinophil activation. GLP-1 agonists may have additional adjunctive indications in treating persons with concomitant type 2 diabetes mellitus and asthma. © 2016 John Wiley & Sons Ltd.

Dual integrin and gastrin-releasing peptide receptor targeted tumor imaging using 18F-labeled PEGylated RGD-bombesin heterodimer 18F-FB-PEG3-Glu-RGD-BBN.

PubMed

Liu, Zhaofei; Yan, Yongjun; Chin, Frederic T; Wang, Fan; Chen, Xiaoyuan

2009-01-22

Radiolabeled RGD and bombesin peptides have been extensively investigated for tumor integrin alpha(v)beta(3) and GRPR imaging, respectively. Due to the fact that many tumors are both integrin and GRPR positive, we designed and synthesized a heterodimeric peptide Glu-RGD-BBN, which is expected to be advantageous over the monomeric peptides for dual-receptor targeting. A PEG(3) spacer was attached to the glutamate alpha-amino group of Glu-RGD-BBN to enhance the (18)F labeling yield and to improve the in vivo kinetics. PEG(3)-Glu-RGD-BBN possesses the comparable GRPR and integrin alpha(v)beta(3) receptor-binding affinities as the corresponding monomers, respectively. The dual-receptor targeting properties of (18)F-FB-PEG(3)-Glu-RGD-BBN were observed in PC-3 tumor model. (18)F-FB-PEG(3)-Glu-RGD-BBN with high tumor contrast and favorable pharmacokinetics is a promising PET tracer for dual integrin and GRPR positive tumor imaging. This heterodimer strategy may also be an applicable method to develop other molecules with improved in vitro and in vivo characterizations for tumor diagnosis and therapy.

Small-molecule agonists for the glucagon-like peptide 1 receptor

PubMed Central

Knudsen, Lotte Bjerre; Kiel, Dan; Teng, Min; Behrens, Carsten; Bhumralkar, Dilip; Kodra, János T.; Holst, Jens J.; Jeppesen, Claus B.; Johnson, Michael D.; de Jong, Johannes Cornelis; Jorgensen, Anker Steen; Kercher, Tim; Kostrowicki, Jarek; Madsen, Peter; Olesen, Preben H.; Petersen, Jacob S.; Poulsen, Fritz; Sidelmann, Ulla G.; Sturis, Jeppe; Truesdale, Larry; May, John; Lau, Jesper

2007-01-01

The peptide hormone glucagon-like peptide (GLP)-1 has important actions resulting in glucose lowering along with weight loss in patients with type 2 diabetes. As a peptide hormone, GLP-1 has to be administered by injection. Only a few small-molecule agonists to peptide hormone receptors have been described and none in the B family of the G protein coupled receptors to which the GLP-1 receptor belongs. We have discovered a series of small molecules known as ago-allosteric modulators selective for the human GLP-1 receptor. These compounds act as both allosteric activators of the receptor and independent agonists. Potency of GLP-1 was not changed by the allosteric agonists, but affinity of GLP-1 for the receptor was increased. The most potent compound identified stimulates glucose-dependent insulin release from normal mouse islets but, importantly, not from GLP-1 receptor knockout mice. Also, the compound stimulates insulin release from perfused rat pancreas in a manner additive with GLP-1 itself. These compounds may lead to the identification or design of orally active GLP-1 agonists. PMID:17213325

Joseph Rudinger memorial lecture: Unexpected functions of angiotensin converting enzyme, beyond its enzymatic activity.

PubMed

Martinez, Jean

2017-10-01

Angiotensin converting enzyme (ACE) is a well-known enzyme, largely studied for its action on hypertension, as it produces angiotensin II from angiotensin I. This paper describes two original behaviours of ACE. We showed that ACE could hydrolyse gastrin, a neuropeptide from the gastrointestinal tract, releasing the C-terminal amidated dipeptide H-Asp-Phe-NH 2 . This dipeptide is believed to be involved in the gastrin-induced acid secretion in the stomach. This hypothetic mechanism of action of gastrin resulted in a strategy to rationally design gastrin receptor antagonists. Beyond, we showed that the brain renin angiotensin system (RAS) could be activated by a new characterized peptide named acein, resulting in stimulation of dopamine release within the striatum. This new and original 'receptor-like' activity for brain membrane-bound ACE is quite significant taking into account the role of dopamine in the brain, particularly in neurodegenerative diseases. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

PubMed

Ikeda, Yuichi; Kumagai, Hidetoshi; Okazaki, Hiroaki; Fujishiro, Mitsuhiro; Motozawa, Yoshihiro; Nomura, Seitaro; Takeda, Norifumi; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Komuro, Issei; Yanagisawa, Masashi

2015-01-01

Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.

Cardiovascular actions of the ghrelin gene-derived peptides and growth hormone-releasing hormone.

PubMed

Granata, Riccarda; Isgaard, Jörgen; Alloatti, Giuseppe; Ghigo, Ezio

2011-05-01

In 1976, small peptide growth hormone secretagogues (GHSs) were discovered and found to promote growth hormone (GH) release from the pituitary. The GHS receptor (GHS-R) was subsequently cloned, and its endogenous ligand ghrelin was later isolated from the stomach. Ghrelin is a 28-amino acid peptide, whose acylation is essential for binding to GHS-R type 1a and for the endocrine functions, including stimulation of GH secretion and subsequent food intake. Unacylated ghrelin, the other ghrelin form, although devoid of GHS-R binding is an active peptide, sharing many peripheral effects with acylated ghrelin (AG). The ghrelin system is broadly expressed in myocardial tissues, where it exerts different functions. Indeed, ghrelin inhibits cardiomyocyte and endothelial cell apoptosis, and improves left ventricular (LV) function during ischemia-reperfusion (I/R) injury. In rats with heart failure (HF), ghrelin improves LV dysfunction and attenuates the development of cardiac cachexia. Similarly, ghrelin exerts vasodilatory effects in humans, improves cardiac function and decreases systemic vascular resistance in patients with chronic HF. Obestatin is a recently identified ghrelin gene peptide. The physiological role of obestatin and its binding to the putative GPR39 receptor are still unclear, although protective effects have been demonstrated in the pancreas and heart. Similarly to AG, the hypothalamic peptide growth hormone-releasing hormone (GHRH) stimulates GH release from the pituitary, through binding to the GHRH-receptor. Besides its proliferative effects in different cell types, at the cardiovascular level GHRH inhibits cardiomyocyte apoptosis, and reduces infarct size in both isolated rat heart after I/R and in vivo after myocardial infarction. Therefore, both ghrelin and GHRH exert cardioprotective effects, which make them candidate targets for therapeutic intervention in cardiovascular dysfunctions.

Myoinhibiting peptides are the ancestral ligands of the promiscuous Drosophila sex peptide receptor

USDA-ARS?s Scientific Manuscript database

Male insects change behaviors of female partners by co-transferring accessory gland proteins (Acps) like sex peptide (SP), with their sperm. The Drosophila sex peptide receptor (SPR) is a G protein-coupled receptor expressed in the female’s nervous system and genital tract. While most Acps show a fa...

Expression of receptors for atrial natriuretic peptide on the murine bone marrow-derived stromal cells.

PubMed

Agui, T; Yamada, T; Legros, G; Nakajima, T; Clark, M; Peschel, C; Matsumoto, K

1992-05-01

Atrial natriuretic peptide (ANP) receptors were identified on both murine bone marrow-derived stromal cell lines A-3 and ALC and primary cultured cells using [125I]ANP binding assays and Northern blot analyses. The binding of [125I] ANP to the stromal cells was rapid, saturable, and of high affinity. The dissociation constants between ANP and its receptors on these cells showed no difference among cell types, while maximal binding capacity values were different among cell types. Competitive inhibition of [125I]ANP binding with C-atrial natriuretic factor, specific for ANP clearance receptor (ANPR-C), revealed that most of [125I]ANP-binding sites corresponded to ANPR-C. Northern blotting data corroborated that bone marrow-derived stromal cells expressed ANPR-C. However, in ALC cells, ANP biological receptors (either ANPR-A or ANPR-B), the mol wt of which is approximately 130K, were detected, and cGMP was accumulated after stimulation with ANP. On the other hand, in another stromal cell clone, A-3 cells, the expression of biological receptor was not detected in the affinity cross-linking and competitive inhibition experiments using [125I]ANP. However, A-3 cells accumulated cGMP by responding to ANPR-B-specific ligand, C-type natriuretic peptide. These results suggest that ALC cells equally express ANPR-A and ANPR-B, while A-3 cells express ANPR-B dominantly. Although the physiological roles of these receptors in the bone marrow is still not resolved, ANP is expected to play a role in the regulation of stromal cell functions in bone marrow.

Inhibition by somatostatin (growth-hormone release-inhibiting hormone, GH-RIH) of gastric acid and pepsin and G-cell release of gastrin.

PubMed Central

Barros D'sa, A A; Bloom, S R; Baron, J H

1978-01-01

Somatostatin (cyclic growth-hormone release-inhibiting hormone--GH-RIH) was infused into dogs with gastric fistulae. Somatostatin inhibited gastric acid response to four gastric stimulants--insulin, food, histamine, and pentagastrin. Histamine- and pentagastrin-stimulated pepsins were inhibited similarly to inhibition of acid. Somatostatin inhibited the gastrin response to insulin and food. PMID:348581

Design and structure of stapled peptides binding to estrogen receptors.

PubMed

Phillips, Chris; Roberts, Lee R; Schade, Markus; Bazin, Richard; Bent, Andrew; Davies, Nichola L; Moore, Rob; Pannifer, Andrew D; Pickford, Andrew R; Prior, Stephen H; Read, Christopher M; Scott, Andrew; Brown, David G; Xu, Bin; Irving, Stephen L

2011-06-29

Synthetic peptides that specifically bind nuclear hormone receptors offer an alternative approach to small molecules for the modulation of receptor signaling and subsequent gene expression. Here we describe the design of a series of novel stapled peptides that bind the coactivator peptide site of estrogen receptors. Using a number of biophysical techniques, including crystal structure analysis of receptor-stapled peptide complexes, we describe in detail the molecular interactions and demonstrate that all-hydrocarbon staples modulate molecular recognition events. The findings have implications for the design of stapled peptides in general.

Anti-tumor effects of peptide analogs targeting neuropeptide hormone receptors on mouse pheochromocytoma cells.

PubMed

Ziegler, C G; Ullrich, M; Schally, A V; Bergmann, R; Pietzsch, J; Gebauer, L; Gondek, K; Qin, N; Pacak, K; Ehrhart-Bornstein, M; Eisenhofer, G; Bornstein, S R

2013-05-22

Pheochromocytoma is a rare but potentially lethal chromaffin cell tumor with currently no effective treatment. Peptide hormone receptors are frequently overexpressed on endocrine tumor cells and can be specifically targeted by various anti-tumor peptide analogs. The present study carried out on mouse pheochromocytoma cells (MPCs) and a more aggressive mouse tumor tissue-derived (MTT) cell line revealed that these cells are characterized by pronounced expression of the somatostatin receptor 2 (sst2), growth hormone-releasing hormone (GHRH) receptor and the luteinizing hormone-releasing hormone (LHRH) receptor. We further demonstrated significant anti-tumor effects mediated by cytotoxic somatostatin analogs, AN-162 and AN-238, by LHRH antagonist, Cetrorelix, by the cytotoxic LHRH analog, AN-152, and by recently developed GHRH antagonist, MIA-602, on MPC and for AN-152 and MIA-602 on MTT cells. Studies of novel anti-tumor compounds on these mouse cell lines serve as an important basis for mouse models of metastatic pheochromocytoma, which we are currently establishing. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Gastrin and the growth of the gastrointestinal tract.

PubMed Central

Ekundayo, A A; Lee, C Y; Goodlad, R A

1995-01-01

While the proliferative effects of gastrin in the gastric fundus are well established, there is a considerable degree of confusion regarding the role of gastrin on the growth of the small intestine and colon. The hypothesis that gastrin is trophic throughout the gut was tested by giving three doses of pentagastrin and one of gastrin 17 to rats maintained by total parenteral nutrition (TPN). The rats were fed intravenously for one week, with the various peptides added to the TPN diet. The number of vincristine arrested metaphases per gland or crypt was then scored to determine the proliferative state. Both gastrin 17 and pentagastrin were found to be trophic in the gastric fundus, but not to the gastric antrum. A proliferative response was also seen in the duodenum, but with little evidence of a dose response element. No effect on small bowel weight was seen, and no proliferative effect was noted in the mid small bowel, thus the duodenal effect could be attributed to a local action of increased acid output on the duodenum, not a general role throughout the small intestine. No proliferative effects of pentagastrin or gastrin were seen in the colon. It is therefore concluded that the trophic role of gastrin is restricted to the gastric fundus and the proximal duodenum. PMID:7883218

Natriuretic peptide receptors regulate cytoprotective effects in a human ex vivo 3D/bioreactor model.

PubMed

Peake, Nicholas; Su, Nyan; Ramachandran, Manoj; Achan, Pramod; Salter, Donald M; Bader, Dan L; Moyes, Amie J; Hobbs, Adrian J; Chowdhury, Tina T

2013-07-24

The present study examined the effect of C-type natriuretic peptide (CNP) and biomechanical signals on anabolic and catabolic activities in chondrocyte/agarose constructs. Natriuretic peptide (Npr) 2 and 3 expression were compared in non-diseased (grade 0/1) and diseased (grade IV) human cartilage by immunofluoresence microscopy and western blotting. In separate experiments, constructs were cultured under free-swelling conditions or subjected to dynamic compression with CNP, interleukin-1β (IL-1β), the Npr2 antagonist P19 or the Npr3 agonist cANF⁴⁻²³. Nitric oxide (NO) production, prostaglandin E₂ (PGE₂) release, glycosaminoglycan (GAG) synthesis and CNP concentration were quantified using biochemical assays. Gene expression of Npr2, Npr3, CNP, aggrecan and collagen type II were assessed by real-time qPCR. Two-way ANOVA and a post hoc Bonferroni-corrected t-test were used to analyse the data. The present study demonstrates increased expression of natriuretic peptide receptors in diseased or older cartilage (age 70) when compared to non-diseased tissue (age 60) which showed minimal expression. There was strong parallelism in the actions of CNP on cGMP induction resulting in enhanced GAG synthesis and reduction of NO and PGE₂ release induced by IL-1β. Inhibition of Npr2 with P19 maintained catabolic activities whilst specific agonism of Npr3 with cANF⁴⁻²³ had the opposite effect and reduced NO and PGE₂ release. Co-stimulation with CNP and dynamic compression enhanced anabolic activities and inhibited catabolic effects induced by IL-1β. The presence of CNP and the Npr2 antagonist abolished the anabolic response to mechanical loading and prevented loading-induced inhibition of NO and PGE₂ release. In contrast, the presence of the Npr3 agonist had the opposite effect and increased GAG synthesis and cGMP levels in response to mechanical loading and reduced NO and PGE₂ release comparable to control samples. In addition, CNP concentration and

Effects of differently composed feeds and physical stress on plasma gastrin concentration in horses.

PubMed

Sandin, A; Girma, K; Sjöholm, B; Lindholm, A; Nilsson, G

1998-01-01

Plasma gastrin concentrations were determined in 6 Standardbreds (4 geldings and 2 mares) after 3 different meals consisting of unlimited amounts of hay (8-9 kg per horse), a restricted amount of hay (0.6 kg/100 kg body-weight) and grain (0.2 kg/100 kg body-weight) in combination or of grain alone (0.2 kg/100 kg body-weight). In another series of experiments the possible role of gastrin as a stress hormone was investigated. Plasma gastrin and cortisol concentrations were determined during fasting and compared with concentrations during hay feeding. In addition, gastrin and cortisol concentrations were determined before, during and after 2 kinds of physical exercise on a treadmill. Meal stimulation significantly increased the plasma gastrin concentration, irrespective of the meal composition. An immediate and large increase in plasma gastrin concentration was found when voluminous meals were given, whereas a small meal evoked a later onset of gastrin release, suggesting that gastric distention plays an important role in inducing gastrin release during a meal. Meals consisting of grain seem to evoke a slower onset and then a more prolonged gastrin response than a hay meal, possibly due to different emptying rates of the stomach. Nervous excitation may play a minor role in the activation of gastrin release in horses. No experimental support was obtained for the idea that gastrin acts as a stress hormone in the horse.

Multimodal use of calcitonin gene-related peptide and substance P in itch and acute pain uncovered by the elimination of vesicular glutamate transporter 2 from transient receptor potential cation channel subfamily V member 1 neurons.

PubMed

Rogoz, Katarzyna; Andersen, Helena H; Lagerström, Malin C; Kullander, Klas

2014-10-15

Primary afferents are known to use glutamate as their principal fast neurotransmitter. However, it has become increasingly clear that peptides have an influential role in both mediating and modulating sensory transmission. Here we describe the transmission accounting for different acute pain states and itch transmitted via the transient receptor potential cation channel subfamily V member 1 (TRPV1) population by either ablating Trpv1-Cre-expressing neurons or inducing vesicular glutamate transporter 2 (VGLUT2) deficiency in Trpv1-Cre-expressing neurons. Furthermore, by pharmacological inhibition of substance P or calcitonin gene-related peptide (CGRP) signaling in Vglut2-deficient mice, we evaluated the contribution of substance P or CGRP to these sensory modulations, with or without the presence of VGLUT2-mediated glutamatergic transmission in Trpv1-Cre neurons. This examination, together with c-Fos analyses, showed that glutamate via VGLUT2 in the Trpv1-Cre population together with substance P mediate acute cold pain, whereas glutamate together with CGRP mediate noxious heat. Moreover, we demonstrate that glutamate together with both substance P and CGRP mediate tissue-injury associated pain. We further show that itch, regulated by the VGLUT2-mediated transmission via the Trpv1-Cre population, depends on CGRP and gastrin-releasing peptide receptor (GRPR) transmission because pharmacological blockade of the CGRP or GRPR pathway, or genetic ablation of Grpr, led to a drastically attenuated itch. Our study reveals how different neurotransmitters combined can cooperate with each other to transmit or regulate various acute sensations, including itch. Copyright © 2014 the authors 0270-6474/14/3414055-14$15.00/0.

Targeting of peptide conjugated magnetic nanoparticles to urokinase plasminogen activator receptor (uPAR) expressing cells

NASA Astrophysics Data System (ADS)

Hansen, Line; Unmack Larsen, Esben Kjær; Nielsen, Erik Holm; Iversen, Frank; Liu, Zhuo; Thomsen, Karen; Pedersen, Michael; Skrydstrup, Troels; Nielsen, Niels Chr.; Ploug, Michael; Kjems, Jørgen

2013-08-01

Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific targeting peptide onto polyethylene glycol (PEG) coated USPIO nanoparticles by click chemistry resulted in a five times higher uptake in vitro in a uPAR positive cell line compared to nanoparticles carrying a non-binding control peptide. In accordance with specific receptor-mediated recognition, a low uptake was observed in the presence of an excess of ATF, a natural ligand for uPAR. The uPAR specific magnetic nanoparticles can potentially provide a useful supplement for tumor patient management when combined with MRI and drug delivery.Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific

High Affinity Binding of Indium and Ruthenium Ions by Gastrins

PubMed Central

Baldwin, Graham S.; George, Graham N.; Pushie, M. Jake

2015-01-01

The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10−7 and 1.1 x 10−6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10−15 and 1.7 x 10−7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10−13 and 1.2 x 10−5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0–3.3 Å, the Ru complex clearly demonstrated a short range Ru—Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy. PMID:26457677

Gastrin Induces Nuclear Export and Proteasome Degradation of Menin in Enteric Glial Cells.

PubMed

Sundaresan, Sinju; Meininger, Cameron A; Kang, Anthony J; Photenhauer, Amanda L; Hayes, Michael M; Sahoo, Nirakar; Grembecka, Jolanta; Cierpicki, Tomasz; Ding, Lin; Giordano, Thomas J; Else, Tobias; Madrigal, David J; Low, Malcolm J; Campbell, Fiona; Baker, Ann-Marie; Xu, Haoxing; Wright, Nicholas A; Merchant, Juanita L

2017-12-01

The multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner's glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia. Primary enteric glial cultures were generated from the VillinCre:Men1 FL/FL :Sst -/- mice or C57BL/6 mice (controls), with or without inhibition of gastric acid by omeprazole. Primary enteric glial cells from C57BL/6 mice were incubated with gastrin and separated into nuclear and cytoplasmic fractions. Cells were incubated with forskolin and H89 to activate or inhibit protein kinase A (a family of enzymes whose activity depends on cellular levels of cyclic AMP). Gastrin was measured in blood, tissue, and cell cultures using an ELISA. Immunoprecipitation with menin or ubiquitin was used to demonstrate post-translational modification of menin. Primary glial cells were incubated with leptomycin b and MG132 to block nuclear export and proteasome activity, respectively. We obtained human duodenal, lymph node, and pancreatic gastrinoma samples, collected from patients who underwent surgery from 1996 through 2007 in the United States or the United Kingdom. Enteric glial cells that stained positive for glial fibrillary acidic protein (GFAP+) expressed gastrin de novo through a mechanism that required PKA. Gastrin-induced nuclear export of menin via cholecystokinin B receptor (CCKBR)-mediated activation of PKA. Once exported from the nucleus, menin was ubiquitinated and degraded by the proteasome. GFAP and other markers of enteric glial cells (eg, p75 and S100B), colocalized with gastrin in human duodenal gastrinomas. MEN1-associated

(99m)Tc-labeled gastrins of varying peptide chain length: Distinct impact of NEP/ACE-inhibition on stability and tumor uptake in mice.

PubMed

Kaloudi, Aikaterini; Nock, Berthold A; Lymperis, Emmanouil; Krenning, Eric P; de Jong, Marion; Maina, Theodosia

2016-06-01

In situ inhibition of neutral endopeptidase (NEP) has been recently shown to impressively increase the bioavailability and tumor uptake of biodegradable gastrin radioligands. Furthermore, angiotensin converting enzyme (ACE) has been previously shown to cleave gastrin analogs in vitro. In the present study, we have assessed the effects induced by single or dual NEP/ACE-inhibition on the pharmacokinetic profile of three (99m)Tc-labeled gastrins of varying peptide chain length: [(99m)Tc]SG6 ([(99m)Tc-N4-Gln(1)]gastrin(1-17)), [(99m)Tc]DG2 ([(99m)Tc-N4-Gly(4),DGlu(5)]gastrin(4-17)) and [(99m)Tc]DG4 ([(99m)Tc-N4-DGlu(10)]gastrin(10-17)). Mouse blood samples were collected 5min after injection of each of [(99m)Tc]SG6/DG2/DG4 together with: a) vehicle, b) the NEP-inhibitor phosphoramidon (PA), c) the ACE-inhibitor lisinopril (Lis), or d) PA plus Lis and were analyzed by RP-HPLC for radiometabolite detection. Biodistribution was studied in SCID mice bearing A431-CCK2R(+/-) xenografts at 4h postinjection (pi). [(99m)Tc]SG6 or [(99m)Tc]DG4 was coinjected with either vehicle or the above described NEP/ACE-inhibitor regimens; for [(99m)Tc]DG2 control and PA animal groups were only included. Treatment of mice with PA induced significant stabilization of (99m)Tc-radiotracers in peripheral blood, while treatment with Lis or Lis+PA affected the stability of des(Glu)5 [(99m)Tc]DG4 only. In line with these findings, PA coinjection led to notable amplification of tumor uptake of radiopeptides compared to controls (P<0.01). Only [(99m)Tc]DG4 profited by single Lis (2.06±0.39%ID/g vs 0.99±0.13%ID/g in controls) or combined Lis+PA coinjection (8.91±1.61%ID/g vs 4.89±1.33%ID/g in PA-group). Furthermore, kidney uptake remained favourably low and unaffected by PA and/or Lis coinjection only in the case of [(99m)Tc]DG4 (<1.9%ID/g) resulting in the most optimal tumor-to-kidney ratios. In situ NEP/ACE-inhibition diversely affected the in vivo profile of (99m)Tc-radioligands based on

A2A-D2 receptor-receptor interaction modulates gliotransmitter release from striatal astrocyte processes.

PubMed

Cervetto, Chiara; Venturini, Arianna; Passalacqua, Mario; Guidolin, Diego; Genedani, Susanna; Fuxe, Kjell; Borroto-Esquela, Dasiel O; Cortelli, Pietro; Woods, Amina; Maura, Guido; Marcoli, Manuela; Agnati, Luigi F

2017-01-01

Evidence for striatal A2A-D2 heterodimers has led to a new perspective on molecular mechanisms involved in schizophrenia and Parkinson's disease. Despite the increasing recognition of astrocytes' participation in neuropsychiatric disease vulnerability, involvement of striatal astrocytes in A2A and D2 receptor signal transmission has never been explored. Here, we investigated the presence of D2 and A2A receptors in isolated astrocyte processes prepared from adult rat striatum by confocal imaging; the effects of receptor activation were measured on the 4-aminopyridine-evoked release of glutamate from the processes. Confocal analysis showed that A2A and D2 receptors were co-expressed on the same astrocyte processes. Evidence for A2A-D2 receptor-receptor interactions was obtained by measuring the release of the gliotransmitter glutamate: D2 receptors inhibited the glutamate release, while activation of A2A receptors, per se ineffective, abolished the effect of D2 receptor activation. The synthetic D2 peptide VLRRRRKRVN corresponding to the receptor region involved in electrostatic interaction underlying A2A-D2 heteromerization abolished the ability of the A2A receptor to antagonize the D2 receptor-mediated effect. Together, the findings are consistent with heteromerization of native striatal astrocytic A2A-D2 receptors that via allosteric receptor-receptor interactions could play a role in the control of striatal glutamatergic transmission. These new findings suggest possible new pathogenic mechanisms and/or therapeutic approaches to neuropsychiatric disorders. © 2016 International Society for Neurochemistry.

Growth hormone-releasing peptides.

PubMed

Ghigo, E; Arvat, E; Muccioli, G; Camanni, F

1997-05-01

Growth hormone-releasing peptides (GHRPs) are synthetic, non-natural peptides endowed with potent stimulatory effects on somatotrope secretion in animals and humans. They have no structural homology with GHRH and act via specific receptors present either at the pituitary or the hypothalamic level both in animals and in humans. The GHRP receptor has recently been cloned and, interestingly, it does not show sequence homology with other G-protein-coupled receptors known so far. This evidence strongly suggests the existence of a natural GHRP-like ligand which, however, has not yet been found. The mechanisms underlying the GHRP effect are still unclear. At present, several data favor the hypothesis that GHRPs could act by counteracting somatostatinergic activity both at the pituitary and the hypothalamic level and/or, at least partially, via a GHRH-mediated mechanism. However, the possibility that GHRPs act via an unknown hypothalamic factor (U factor) is still open. GHRP-6 was the first hexapeptide to be extensively studied in humans. More recently, a heptapeptide, GHRP-1, and two other hexapeptides, GHRP-2 and Hexarelin, have been synthesized and are now available for human studies. Moreover, non-peptidyl GHRP mimetics have been developed which act via GHRP receptors and their effects have been clearly demonstrated in animals and in humans in vivo. Among non-peptidyl GHRPs, MK-0677 seems the most interesting molecule. The GH-releasing activity of GHRPs is marked and dose-related after intravenous, subcutaneous, intranasal and even oral administration. The effect of GHRPs is reproducible and undergoes partial desensitization, more during continuous infusion, less during intermittent administration: in fact, prolonged administration of GHRPs increases IGF-1 levels both in animals and in humans. The GH-releasing effect of GHRPs does not depend on sex but undergoes age-related variations. It increases from birth to puberty, persists at a similar level in adulthood and

Marked differences between metalloproteases meprin A and B in substrate and peptide bond specificity.

PubMed

Bertenshaw, G P; Turk, B E; Hubbard, S J; Matters, G L; Bylander, J E; Crisman, J M; Cantley, L C; Bond, J S

2001-04-20

Meprin A and B are highly regulated, secreted, and cell-surface metalloendopeptidases that are abundantly expressed in the kidney and intestine. Meprin oligomers consist of evolutionarily related alpha and/or beta subunits. The work herein was carried out to identify bioactive peptides and proteins that are susceptible to hydrolysis by mouse meprins and kinetically characterize the hydrolysis. Gastrin-releasing peptide fragment 14-27 and gastrin 17, regulatory molecules of the gastrointestinal tract, were found to be the best peptide substrates for meprin A and B, respectively. Peptide libraries and a variety of naturally occurring peptides revealed that the meprin beta subunit has a clear preference for acidic amino acids in the P1 and P1' sites of substrates. The meprin alpha subunit selected for small (e.g. serine, alanine) or hydrophobic (e.g. phenylalanine) residues in the P1 and P1' sites, and proline was the most preferred amino acid at the P2' position. Thus, although the meprin alpha and beta subunits share 55% amino acid identity within the protease domain and are normally localized at the same tissue cell surfaces, they have very different substrate and peptide bond specificities indicating different functions. Homology models of the mouse meprin alpha and beta protease domains, based on the astacin crystal structure, revealed active site differences that can account for the marked differences in substrate specificity of the two subunits.

Interrelation between ghrelin and gastrin in patients with combination of chronic gastritis and type 2 diabetes mellitus.

PubMed

Sirchak, Elizaveta S; Patskun, Silviya V

2018-01-01

Introduction: Ghrelin is 28-amino-acid peptide that is produced by X/A-like cells present in the stomach. Gastrin is a hormone that stimulates gastric acid secretion and mucosal cell growth. The aim: to study the interrelation between ghrelin and gastrin levels in patients with combination of chronic gastritis and type 2 diabetes mellitus. Materials and methods: 60 Helicobacter pylori positive patients with a combination of chronic gastritis and type 2 diabetes mellitus were examined. The diagnosis of type 2 diabetes mellitus is based on the recommendations of the International Diabetes Federation (IDF, 2005). Gastric acid secretion function was studied by intra-stomach express-pH-metry (method of prof. V.N. Chernobrov). Serum gastrin was determined using ELISA using Gastrin-EIA test kit Cat. No. CS 001 030. Serum ghrelin was determined by immunoassay analysis using the Human Ghrelin ELISA Kit from RayBiotech No. 1.03930005306. Results: The obtained data testify to the existence of a feedback between the level of ghrelin and gastrin in the blood of patients with chronic gastritis and type 2 diabetes mellitus. That is, with increasing levels of gastrin in the blood, the level of ghrelin in the blood decreases and vice versa with a decrease in the level of gastrin in the blood, the level of ghrelin - increases. Conclusions: A significantly higher level of ghrelin was found in patients with type 2 diabetes mellitus and chronic gastritis compared with control group. The reverse association between gastrin and ghrelin levels in patients with combination of chronic gastritis and type 2 diabetes mellitus has been obtained.

Pharmacological lineage analysis revealed the binding affinity of broad-spectrum substance P antagonists to receptors for gonadotropin-releasing peptide.

PubMed

Arai, Kazune; Kashiwazaki, Aki; Fujiwara, Yoko; Tsuchiya, Hiroyoshi; Sakai, Nobuya; Shibata, Katsushi; Koshimizu, Taka-aki

2015-02-15

A group of synthetic substance P (SP) antagonists, such as [Arg(6),D-Trp(7,9),N(Me)Phe(8)]-substance P(6-11) and [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]-substance P, bind to a range of distinct G-protein-coupled receptor (GPCR) family members, including V1a vasopressin receptors, and they competitively inhibit agonist binding. This extended accessibility enabled us to identify a GPCR subset with a partially conserved binding site structure. By combining pharmacological data and amino acid sequence homology matrices, a pharmacological lineage of GPCRs that are sensitive to these two SP antagonists was constructed. We found that sensitivity to the SP antagonists was not limited to the Gq-protein-coupled V1a and V1b receptors; Gs-coupled V2 receptors and oxytocin receptors, which couple with both Gq and Gi, also demonstrated sensitivity. Unexpectedly, a dendrogram based on the amino acid sequences of 222 known GPCRs showed that a group of receptors sensitive to the SP antagonists are located in close proximity to vasopressin/oxytocin receptors. Gonadotropin-releasing peptide receptors, located near the vasopressin receptors in the dendrogram, were also sensitive to the SP analogs, whereas α1B adrenergic receptors, located more distantly from the vasopressin receptors, were not sensitive. Our finding suggests that pharmacological lineage analysis is useful in selecting subsets of candidate receptors that contain a conserved binding site for a ligand with broad-spectrum binding abilities. The knowledge that the binding site of the two broad-spectrum SP analogs partially overlaps with that of distinct peptide agonists is valuable for understanding the specificity/broadness of peptide ligands. Copyright © 2015 Elsevier B.V. All rights reserved.

Expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in control of GnRH secretion.

PubMed

Yang, Ying; Zhou, Li-bin; Liu, Shang-quan; Tang, Jing-feng; Li, Feng-yin; Li, Rong-ying; Song, Huai-dong; Chen, Ming-dao

2005-08-01

To investigate the expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in the control of GnRH secretion. Receptors of bombesin3, cholecystokinin (CCK)-A, CCK-B, glucagon-like peptide (GLP)1, melanin-concentrating hormone (MCH)1, orexin1, orexin2, neuromedin-B, neuropeptide Y (NPY)1 and NPY5, neurotensin (NT)1, NT2, NT3, and leptin receptor long form mRNA in GT1-7 cells were detected by reversed transcriptase-polymerase chain reaction. GT1-7 cells were treated with leptin, orexin A and orexin B at a cohort of concentrations for different lengths of time, and GnRH in medium was determined by radioimmunoassay (RIA). Receptors of bombesin 3, CCK-B, GLP1, MCH1, orexin1, neuromedin-B, NPY1, NPY5, NT1, NT3, and leptin receptor long form mRNA were expressed in GT1-7 cells, of which, receptors of GLP1, neuromedin-B, NPY1, and NT3 were highly expressed. No amplified fragments of orexin2, NT2, and CCK-A receptor cDNA were generated with GT1-7 RNA, indicating that the GT1-7 cells did not express mRNA of them. Leptin induced a significant stimulation of GnRH release, the results being most significant at 0.1 nmol/L for 15 min. In contrast to other studies in hypothalamic explants, neither orexin A nor orexin B affected basal GnRH secretion over a wide range of concentrations ranging from 1 nmol/L to 500 nmol/Lat 15, 30, and 60 min. Feeding and reproductive function are closely linked. Many orexigenic and anorexigenic signals may control feeding behavior as well as alter GnRH secretion through their receptors on GnRH neurons.

Neutrophil formyl-peptide receptors. Relationship to peptide-induced responses and emphysema.

PubMed

Stockley, R A; Grant, R A; Llewellyn-Jones, C G; Hill, S L; Burnett, D

1994-02-01

A reproducible assay was established to assess the number of formyl-peptide receptors expressed on the surface of human polymorphonuclear leukocytes (PMN). Using this assay the number of receptors was shown to demonstrate wide within- and between-subject variability. However, the receptor numbers were related to the chemotactic response (r = 0.572) and degranulation response (r = 0.512) to the peptide formyl-methionyl-leucyl-phenylalanine. Subsequent studies showed increased receptor numbers on PMN from patients with emphysema (median, 459 x 10(3)/cell; range, 207 to 1,080) as compared with age-matched control subjects (median, 288; range, 168 to 519; p < 0.02), which may explain the increased chemotactic response of the PMN to formyl peptides. This difference was not observed in patients with bronchiectasis, suggesting that the increased receptor number is a feature of emphysema. Furthermore, the increase was largely a feature of smokers with emphysema (median, 463; range, 362 to 1,080), whereas age-matched smokers without emphysema had lower numbers of receptors (p < 0.001; median, 332; range, 243 to 411). This observation suggests a mechanism that may explain the susceptibility of some smokers to the development of emphysema.

Gastrin blood test

MedlinePlus

Peptic ulcer - gastrin blood test ... to an abnormal amount of gastrin. This includes peptic ulcer disease . ... Too much gastrin can causes severe peptic ulcer disease. A higher ... kidney disease Long-term gastritis Over-activity of the gastrin- ...

Peptide conjugated polymeric nanoparticles as a carrier for targeted delivery of docetaxel.

PubMed

Kulhari, Hitesh; Pooja, Deep; Shrivastava, Shweta; V G M, Naidu; Sistla, Ramakrishna

2014-05-01

The aim of this research work was to develop Bombesin peptide (BBN) conjugated, docetaxel loaded nanocarrier for the treatment of breast cancer. Docetaxel loaded nanoparticles (DNP) were prepared by solvent evaporation method using sodium cholate as surfactant. BBN was conjugated to DNP surface through covalent bonding. Both DNP and BBN conjugated DNP (BDNP) were characterized by various techniques such as dynamic light scattering, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC) and thermogravimetric analysis. The particle diameter and zeta potential of BDNP were 136±3.95 nm and -10.8±2.7 mV, respectively. The change in surface charge and FTIR studies confirmed the formation of amide linkage between BBN and DNP. AFM analysis showed that nanoparticles were spherical in shapes. In nanoparticles, docetaxel was present in its amorphous form as confirmed by DSC and PXRD analysis and was stable during the thermal studies. The formulations showed the sustained release of DTX over the period of 120 h. During cellular toxicity assay in gastrin releasing peptide receptor positive breast cancer cells (MDA-MB-231), BDNP were found to be 12 times more toxic than pure DTX and Taxotere. The IC50 value for DTX, Taxotere, DNP and BDNP was >375, >375, 142.23 and 35.53 ng/ml, respectively. The above studies showed that Bombesin conjugated nanocarrier system could be a promising carrier for active targeting of anticancer drugs in GRP receptor over expressing cancer cells. Copyright © 2014 Elsevier B.V. All rights reserved.

A Cleavable N-Terminal Signal Peptide Promotes Widespread Olfactory Receptor Surface Expression in HEK293T Cells

PubMed Central

Shepard, Blythe D.; Natarajan, Niranjana; Protzko, Ryan J.; Acres, Omar W.; Pluznick, Jennifer L.

2013-01-01

Olfactory receptors (ORs) are G protein-coupled receptors that detect odorants in the olfactory epithelium, and comprise the largest gene family in the genome. Identification of OR ligands typically requires OR surface expression in heterologous cells; however, ORs rarely traffic to the cell surface when exogenously expressed. Therefore, most ORs are orphan receptors with no known ligands. To date, studies have utilized non-cleavable rhodopsin (Rho) tags and/or chaperones (i.e. Receptor Transporting Protein, RTP1S, Ric8b and Gαolf) to improve surface expression. However, even with these tools, many ORs still fail to reach the cell surface. We used a test set of fifteen ORs to examine the effect of a cleavable leucine-rich signal peptide sequence (Lucy tag) on OR surface expression in HEK293T cells. We report here that the addition of the Lucy tag to the N-terminus increases the number of ORs reaching the cell surface to 7 of the 15 ORs (as compared to 3/15 without Rho or Lucy tags). Moreover, when ORs tagged with both Lucy and Rho were co-expressed with previously reported chaperones (RTP1S, Ric8b and Gαolf), we observed surface expression for all 15 receptors examined. In fact, two-thirds of Lucy-tagged ORs are able to reach the cell surface synergistically with chaperones even when the Rho tag is removed (10/15 ORs), allowing for the potential assessment of OR function with only an 8-amino acid Flag tag on the mature protein. As expected for a signal peptide, the Lucy tag was cleaved from the mature protein and did not alter OR-ligand binding and signaling. Our studies demonstrate that widespread surface expression of ORs can be achieved in HEK293T cells, providing promise for future large-scale deorphanization studies. PMID:23840901

Morphine enhances nitric oxide release in the mammalian gastrointestinal tract via the micro(3) opiate receptor subtype: a hormonal role for endogenous morphine.

PubMed

Stefano, G B; Zhu, W; Cadet, P; Bilfinger, T V; Mantione, K

2004-03-01

Studies from our laboratory have revealed a novel micro opiate receptor, micro(3), which is expressed in both human vascular tissues and leukocytes. The micro(3) receptor is selective for opiate alkaloids, insensitive to opioid peptides and is coupled to constitutive nitric oxide (cNO) release. We now identify the micro(3) receptor characteristics in mammalian gut tissues. It appears that the various regions of the mouse gut release low levels of NO (0.02 to 4.6 nM ) in a pulsatile manner. We demonstrate that morphine stimulates cNO release (peak level 17 nM) in the mouse stomach, small intestine and large intestine in a naloxone and L-NAME antagonizable manner. Opioid peptides do not exhibit cNO-stimulating capabilities in these tissues. Taken together, we surmise morphine acts as a hormone to limit gut activity via micro(3) coupled to NO release since micro opiate receptors are found in the gut and endogenous morphine is not but is found in blood.

Potent and long-acting corticotropin releasing factor (CRF) receptor 2 selective peptide competitive antagonists.

PubMed

Rivier, J; Gulyas, J; Kirby, D; Low, W; Perrin, M H; Kunitake, K; DiGruccio, M; Vaughan, J; Reubi, J C; Waser, B; Koerber, S C; Martinez, V; Wang, L; Taché, Y; Vale, W

2002-10-10

We present evidence that members of the corticotropin releasing factor (CRF) family assume distinct structures when interacting with the CRF(1) and CRF(2) receptors. Predictive methods, physicochemical measurements, and structure-activity relationship studies have suggested that CRF, its family members, and competitive antagonists such as astressin [cyclo(30-33)[DPhe(12),Nle(21),Glu(30),Lys(33),Nle(38)]hCRF((12-41))] assume an alpha-helical conformation when interacting with their receptors. We had shown that alpha-helical CRF((9-41)) and sauvagine showed some selectivity for CRF receptors other than that responsible for ACTH secretion(1) and later for CRF2.(2) More recently, we suggested the possibility of a helix-turn-helix motif around a turn encompassing residues 30-33(3) that would confer high affinity for both CRF(1) and CRF(2)(2,4) in agonists and antagonists of all members of the CRF family.(3) On the other hand, the substitutions that conferred ca. 100-fold CRF(2) selectivity to the antagonist antisauvagine-30 [[DPhe(11),His(12)]sauvagine((11-40))] did not confer such property to the corresponding N-terminally extended agonists. We find here that a Glu(32)-Lys(35) side chain to side chain covalent lactam constraint in hCRF and the corresponding Glu(31)-Lys(34) side chain to side chain covalent lactam constraint in sauvagine yield potent ligands that are selective for CRF(2). Additionally, we introduced deletions and substitutions known to increase duration of action to yield antagonists such as cyclo(31-34)[DPhe(11),His(12),C(alpha)MeLeu(13,39),Nle(17),Glu(31),Lys(34)]Ac-sauvagine((8-40)) (astressin(2)-B) with CRF(2) selectivities greater than 100-fold. CRF receptor autoradiography was performed in rat tissue known to express CRF(2) and CRF(1) in order to confirm that astressin(2)-B could indeed bind to established CRF(2) but not CRF(1) receptor-expressing tissues. Extended duration of action of astressin(2)-B vs that of antisauvagine-30 is demonstrated in

Islet Cells Serve as Cells of Origin of Pancreatic Gastrin-Positive Endocrine Tumors.

PubMed

Bonnavion, Rémy; Teinturier, Romain; Jaafar, Rami; Ripoche, Doriane; Leteurtre, Emmanuelle; Chen, Yuan-Jia; Rehfeld, Jens F; Lepinasse, Florian; Hervieu, Valérie; Pattou, François; Vantyghem, Marie-Christine; Scoazec, Jean-Yves; Bertolino, Philippe; Zhang, Chang Xian

2015-10-01

The cells of origin of pancreatic gastrinomas remain an enigma, since no gastrin-expressing cells are found in the normal adult pancreas. It was proposed that the cellular origin of pancreatic gastrinomas may come from either the pancreatic cells themselves or gastrin-expressing cells which have migrated from the duodenum. In the current study, we further characterized previously described transient pancreatic gastrin-expressing cells using cell lineage tracing in a pan-pancreatic progenitor and a pancreatic endocrine progenitor model. We provide evidence showing that pancreatic gastrin-expressing cells, found from embryonic day 12.5 until postnatal day 7, are derived from pancreatic Ptf1a(+) and neurogenin 3-expressing (Ngn3(+)) progenitors. Importantly, the majority of them coexpress glucagon, with 4% coexpressing insulin, indicating that they are a temporary subpopulation of both alpha and beta cells. Interestingly, Men1 disruption in both Ngn3 progenitors and beta and alpha cells resulted in the development of pancreatic gastrin-expressing tumors, suggesting that the latter developed from islet cells. Finally, we detected gastrin expression using three human cohorts with pancreatic endocrine tumors (pNETs) that have not been diagnosed as gastrinomas (in 9/34 pNETs from 6/14 patients with multiple endocrine neoplasia type 1, in 5/35 sporadic nonfunctioning pNETs, and in 2/20 sporadic insulinomas), consistent with observations made in mouse models. Our work provides insight into the histogenesis of pancreatic gastrin-expressing tumors. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Islet Cells Serve as Cells of Origin of Pancreatic Gastrin-Positive Endocrine Tumors

PubMed Central

Bonnavion, Rémy; Teinturier, Romain; Jaafar, Rami; Ripoche, Doriane; Leteurtre, Emmanuelle; Chen, Yuan-Jia; Rehfeld, Jens F.; Lepinasse, Florian; Hervieu, Valérie; Pattou, François; Vantyghem, Marie-Christine; Scoazec, Jean-Yves; Bertolino, Philippe

2015-01-01

The cells of origin of pancreatic gastrinomas remain an enigma, since no gastrin-expressing cells are found in the normal adult pancreas. It was proposed that the cellular origin of pancreatic gastrinomas may come from either the pancreatic cells themselves or gastrin-expressing cells which have migrated from the duodenum. In the current study, we further characterized previously described transient pancreatic gastrin-expressing cells using cell lineage tracing in a pan-pancreatic progenitor and a pancreatic endocrine progenitor model. We provide evidence showing that pancreatic gastrin-expressing cells, found from embryonic day 12.5 until postnatal day 7, are derived from pancreatic Ptf1a+ and neurogenin 3-expressing (Ngn3+) progenitors. Importantly, the majority of them coexpress glucagon, with 4% coexpressing insulin, indicating that they are a temporary subpopulation of both alpha and beta cells. Interestingly, Men1 disruption in both Ngn3 progenitors and beta and alpha cells resulted in the development of pancreatic gastrin-expressing tumors, suggesting that the latter developed from islet cells. Finally, we detected gastrin expression using three human cohorts with pancreatic endocrine tumors (pNETs) that have not been diagnosed as gastrinomas (in 9/34 pNETs from 6/14 patients with multiple endocrine neoplasia type 1, in 5/35 sporadic nonfunctioning pNETs, and in 2/20 sporadic insulinomas), consistent with observations made in mouse models. Our work provides insight into the histogenesis of pancreatic gastrin-expressing tumors. PMID:26169832

Gastrin stimulates renal dopamine production by increasing the renal tubular uptake of l-DOPA.

PubMed

Jiang, Xiaoliang; Zhang, Yanrong; Yang, Yu; Yang, Jian; Asico, Laureano D; Chen, Wei; Felder, Robin A; Armando, Ines; Jose, Pedro A; Yang, Zhiwei

2017-01-01

Gastrin is a peptide hormone that is involved in the regulation of sodium balance and blood pressure. Dopamine, which is also involved in the regulation of sodium balance and blood pressure, directly or indirectly interacts with other blood pressure-regulating hormones, including gastrin. This study aimed to determine the mechanisms of the interaction between gastrin and dopamine and tested the hypothesis that gastrin produced in the kidney increases renal dopamine production to keep blood pressure within the normal range. We show that in human and mouse renal proximal tubule cells (hRPTCs and mRPTCs, respectively), gastrin stimulates renal dopamine production by increasing the cellular uptake of l-DOPA via the l-type amino acid transporter (LAT) at the plasma membrane. The uptake of l-DOPA in RPTCs from C57Bl/6J mice is lower than in RPTCs from normotensive humans. l-DOPA uptake in renal cortical slices is also lower in salt-sensitive C57Bl/6J than in salt-resistant BALB/c mice. The deficient renal cortical uptake of l-DOPA in C57Bl/6J mice may be due to decreased LAT-1 activity that is related to its decreased expression at the plasma membrane, relative to BALB/c mice. We also show that renal-selective silencing of Gast by the renal subcapsular injection of Gast siRNA in BALB/c mice decreases renal dopamine production and increases blood pressure. These results highlight the importance of renal gastrin in stimulating renal dopamine production, which may give a new perspective in the prevention and treatment of hypertension. Copyright © 2017 the American Physiological Society.

Region-Specific Onset of Handling-Induced Changes in Corticotropin-Releasing Factor and Glucocorticoid Receptor Expression

PubMed Central

Fenoglio, Kristina A.; Brunson, Kristen L.; Avishai-Eliner, Sarit; Chen, Yuncai; Baram, Tallie Z.

2011-01-01

Early-life experience including maternal care profoundly influences hormonal stress responses during adulthood. Daily handling on postnatal day (P) 2–9, eliciting augmented maternal care upon returning pups to their cage, permanently modifies the expression of the stress neuromodulators corticotropin-releasing factor (CRF) and glucocorticoid receptor (GR). We have previously demonstrated reduced hypothalamic CRF expression already at the end of the handling period, followed by enhanced hippocampal GR mRNA levels (by P45). However, the initial site(s) and time of onset of these enduring changes have remained unclear. Therefore, we used semiquantitative in situ hybridization to delineate the spatiotemporal evolution of CRF and GR expression throughout stress-regulatory brain regions in handled (compared with undisturbed) pups. Enhanced CRF mRNA expression was apparent in the amygdaloid central nucleus (ACe) of handled pups already by P6. By P9, the augmented CRF mRNA levels persisted in ACe, accompanied by increased peptide expression in the bed nucleus of the stria terminalis and reduced expression in the paraventricular nucleus. The earliest change in GR consisted of reduced expression in the ACe of handled pups on P9, a time point when hippocampal GR expression was not yet affected. Thus, altered gene expression in ACe, bed nucleus of the stria terminalis as well as paraventricular nucleus may contribute to the molecular cascade by which handling (and increased maternal care) influences the stress response long term. PMID:15044366

Molecular identification and functional expression of mu 3, a novel alternatively spliced variant of the human mu opiate receptor gene.

PubMed

Cadet, Patrick; Mantione, Kirk J; Stefano, George B

2003-05-15

Studies from our laboratory have revealed a novel mu opiate receptor, mu 3, which is expressed in both vascular tissues and leukocytes. The mu 3 receptor is selective for opiate alkaloids and is insensitive to opioid peptides. We now identify the mu 3 receptor at the molecular level using a 441-bp conserved region of the mu 1 receptor. Sequence analysis of the isolated cDNA suggests that it is a novel, alternatively spliced variant of the mu opiate receptor gene. To determine whether protein expressed from this cDNA exhibits the biochemical characteristics expected of the mu 3 receptor, the cDNA clone was expressed in a heterologous system. At the functional level, COS-1 cells transfected with the mu 3 receptor cDNA exhibited dose-dependent release of NO following treatment with morphine, but not opioid peptides (i.e., Met-enkephalin). Naloxone was able to block the effect of morphine on COS-1 transfected cells. Nontransfected COS-1 cells did not produce NO in the presence of morphine or the opioid peptides at similar concentrations. Receptor binding analysis with [(3)H]dihydromorphine further supports the opiate alkaloid selectivity and opioid peptide insensitivity of this receptor. These data suggest that this new mu opiate receptor cDNA encodes the mu 3 opiate receptor, since it exhibits biochemical characteristics known to be unique to this receptor (opiate alkaloid selective and opioid peptide insensitive). Furthermore, using Northern blot, RT-PCR, and sequence analysis, we have demonstrated the expression of this new mu variant in human vascular tissue, mononuclear cells, polymorphonuclear cells, and human neuroblastoma cells.

AM-37 and ST-36 Are Small Molecule Bombesin Receptor Antagonists.

PubMed

Moody, Terry W; Tashakkori, Nicole; Mantey, Samuel A; Moreno, Paola; Ramos-Alvarez, Irene; Leopoldo, Marcello; Jensen, Robert T

2017-01-01

While peptide antagonists for the gastrin-releasing peptide receptor (BB 2 R), neuromedin B receptor (BB 1 R), and bombesin (BB) receptor subtype-3 (BRS-3) exist, there is a need to develop non-peptide small molecule inhibitors for all three BBR. The BB agonist (BA)1 binds with high affinity to the BB 1 R, BB 2 R, and BRS-3. In this communication, small molecule BBR antagonists were evaluated using human lung cancer cells. AM-37 and ST-36 inhibited binding to human BB 1 R, BB 2 R, and BRS-3 with similar affinity ( K i = 1.4-10.8 µM). AM-13 and AM-14 were approximately an order of magnitude less potent than AM-37 and ST-36. The ability of BA1 to elevate cytosolic Ca 2+ in human lung cancer cells transfected with BB 1 R, BB 2 R, and BRS-3 was antagonized by AM-37 and ST-36. BA1 increased tyrosine phosphorylation of the EGFR and ERK in lung cancer cells, which was blocked by AM-37 and ST-36. AM-37 and ST-36 reduced the growth of lung cancer cells that have BBR. The results indicate that AM-37 and ST-36 function as small molecule BB receptor antagonists.

Growth hormone (GH)-releasing activity of chicken GH-releasing hormone (GHRH) in chickens.

PubMed

Harvey, S; Gineste, C; Gaylinn, B D

2014-08-01

Two peptides with sequence similarities to growth hormone releasing hormone (GHRH) have been identified by analysis of the chicken genome. One of these peptides, chicken (c) GHRH-LP (like peptide) was previously found to poorly bind to chicken pituitary membranes or to cloned and expressed chicken GHRH receptors and had little, if any, growth hormone (GH)-releasing activity in vivo or in vitro. In contrast, a second more recently discovered peptide, cGHRH, does bind to cloned and expressed cGHRH receptors and increases cAMP activity in transfected cells. The possibility that this peptide may have in vivo GH-releasing activity was therefore assessed. The intravenous (i.v.) administration of cGHRH to immature chickens, at doses of 3-100 μg/kg, significantly increased circulating GH concentrations within 10 min of injection and the plasma GH levels remained elevated for at least 30 min after the injection of maximally effective doses. The plasma GH responses to cGHRH were comparable with those induced by human (h) or porcine (p) GHRH preparations and to that induced by thyrotropin releasing hormone (TRH). In marked contrast, the i.v. injection of cGHRH-LP had no significant effect on circulating GH concentrations in immature chicks. GH release was also increased from slaughterhouse chicken pituitary glands perifused for 5 min with cGHRH at doses of 0.1 μg/ml or 1.0 μg/ml, comparable with GH responses to hGHRH1-44. In contrast, the perifusion of chicken pituitary glands with cGHRH-LP had no significant effect on GH release. In summary, these results demonstrate that cGHRH has GH-releasing activity in chickens and support the possibility that it is the endogenous ligand of the cGHRH receptor. Copyright © 2014 Elsevier Inc. All rights reserved.

Expression of CB2 cannabinoid receptor in Pichia pastoris.

PubMed

Feng, Wenke; Cai, Jian; Pierce, William M; Song, Zhao-Hui

2002-12-01

To facilitate purification and structural characterization, the CB2 cannabinoid receptor is expressed in methylotrophic yeast Pichia pastoris. The expression plasmids were constructed in which the CB2 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase 1 gene. A c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB2 to permit easy detection and purification. In membrane preparations of CB2 gene transformed yeast cells, Western blot analysis detected the expression of CB2 proteins. Radioligand binding assays demonstrated that the CB2 receptors expressed in P. pastoris have a pharmacological profile similar to that of the receptors expressed in mammalian systems. Furthermore, the epitope-tagged receptor was purified by metal chelating chromatography and the purified CB2 preparations were subjected to digestion by trypsin. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions detected 14 peptide fragments derived from the CB2 receptor. ESI mass spectrometry was used to sequence one of these peptide fragments, thus, further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope-tagged, functional CB2 cannabinoid receptor can be expressed in P. pastoris for purification.

Expression and processing of human preprogastrin in murine medullary thyroid carcinoma cells.

PubMed

Daugherty, D F; Dickinson, C J; Takeuchi, T; Bachwich, D; Yamada, T

1991-05-01

Gastrin, the primary hormonal mediator of postprandial gastric acid secretion, is produced from its precursor progastrin by a series of posttranslational processing reactions including dibasic residue cleavage and carboxyl-terminal alpha-amidation. Progastrin contains three dibasic cleavage signals, Arg57Arg58, Lys74Lys75, and Arg94Arg95, that appear to be cleaved differently in different tissues. Differential processing is a potential means by which the production of biologically active peptides may be regulated in a tissue-specific manner. To study these reactions further, we used the pZipNeo SV(X) retroviral vector to express human gastrin cDNA in a heterologous cell line (MTC 6-23) known to be capable of processing other peptide precursors. The psi 2 packaging cell line transfected with the gastrin cDNA-retroviral construct (pSVXgas) produced progastrin, but no substantial amounts of processed amidated gastrin were detected. amounts of processed amidated gastrin were detected. In contrast, MTC 6-23 cells infected with the viral stock obtained from the supernatant of pSVXgas-transfected psi 2 cells produced carboxyl-terminally amidated gastrin in all of its standard molecular forms, including sulfated and nonsulfated forms of tetratriacontagastrin (G-34), heptadecagastrin (G-17), and tetradecagastrin (G-14). These studies indicate that heterologous endocrine cell lines infected with a retroviral-peptide cDNA construct can serve as useful models for peptide hormone posttranslational processing.

Positive correlation between symptoms and circulating motilin, pancreatic polypeptide and gastrin concentrations in functional bowel disorders.

PubMed Central

Preston, D M; Adrian, T E; Christofides, N D; Lennard-Jones, J E; Bloom, S R

1985-01-01

Motilin, pancreatic polypeptide and gastrin blood concentrations in response to drinking water have been studied in 40 patients with functional bowel disease and compared with results in two groups of healthy control subjects. Patients with slow transit constipation and idiopathic megacolon showed impaired motilin release. Pancreatic polypeptide release was reduced in patients with slow transit constipation, but increased in those with functional diarrhoea. Gastrin release was impaired in all groups complaining of chronic constipation. Circulating motilin, pancreatic polypeptide and gastrin concentrations appear to bear some relationship to intestinal transit time in patients with functional bowel disorders. PMID:4054704

Positive correlation between symptoms and circulating motilin, pancreatic polypeptide and gastrin concentrations in functional bowel disorders.

PubMed

Preston, D M; Adrian, T E; Christofides, N D; Lennard-Jones, J E; Bloom, S R

1985-10-01

Motilin, pancreatic polypeptide and gastrin blood concentrations in response to drinking water have been studied in 40 patients with functional bowel disease and compared with results in two groups of healthy control subjects. Patients with slow transit constipation and idiopathic megacolon showed impaired motilin release. Pancreatic polypeptide release was reduced in patients with slow transit constipation, but increased in those with functional diarrhoea. Gastrin release was impaired in all groups complaining of chronic constipation. Circulating motilin, pancreatic polypeptide and gastrin concentrations appear to bear some relationship to intestinal transit time in patients with functional bowel disorders.

Bombesin functionalized gold nanoparticles show in vitro and in vivo cancer receptor specificity.

PubMed

Chanda, Nripen; Kattumuri, Vijaya; Shukla, Ravi; Zambre, Ajit; Katti, Kavita; Upendran, Anandhi; Kulkarni, Rajesh R; Kan, Para; Fent, Genevieve M; Casteel, Stan W; Smith, C Jeffrey; Boote, Evan; Robertson, J David; Cutler, Cathy; Lever, John R; Katti, Kattesh V; Kannan, Raghuraman

2010-05-11

Development of cancer receptor-specific gold nanoparticles will allow efficient targeting/optimum retention of engineered gold nanoparticles within tumors and thus provide synergistic advantages in oncology as it relates to molecular imaging and therapy. Bombesin (BBN) peptides have demonstrated high affinity toward gastrin-releasing peptide (GRP) receptors in vivo that are overexpressed in prostate, breast, and small-cell lung carcinoma. We have synthesized a library of GRP receptor-avid nanoplatforms by conjugating gold nanoparticles (AuNPs) with BBN peptides. Cellular interactions and binding affinities (IC(50)) of AuNP-BBN conjugates toward GRP receptors on human prostate cancer cells have been investigated in detail. In vivo studies using AuNP-BBN and its radiolabeled surrogate (198)AuNP-BBN, exhibiting high binding affinity (IC(50) in microgram ranges), provide unequivocal evidence that AuNP-BBN constructs are GRP-receptor-specific showing accumulation with high selectivity in GRP-receptor-rich pancreatic acne in normal mice and also in tumors in prostate-tumor-bearing, severe combined immunodeficient mice. The i.p. mode of delivery has been found to be efficient as AuNP-BBN conjugates showed reduced RES organ uptake with concomitant increase in uptake at tumor targets. The selective uptake of this new generation of GRP-receptor-specific AuNP-BBN peptide analogs has demonstrated realistic clinical potential in molecular imaging via x-ray computed tomography techniques as the contrast numbers in prostate tumor sites are severalfold higher as compared to the pretreatment group (Hounsfield unit = 150).

Mechanosensitive ion channel Piezo1 is expressed in antral G cells of murine stomach.

PubMed

Lang, Kerstin; Breer, Heinz; Frick, Claudia

2018-02-01

G cells in the antrum region of the murine stomach produce gastrin, the central hormone for controlling gastric activities. Secretion of gastrin is induced mainly by protein breakdown products but also by distensions of the stomach wall. Although G cells respond to protein fragments via distinct chemosensory receptor types, the mechanism underlying G cell activation upon distention is entirely ambiguous. Mechanosensitive ion channels are considered as potential candidates for such a task. Therefore, we explore the possibility of whether Piezo1, a polymodal sensor for diverse mechanical forces, is expressed in antral G cells. The experimental analyses revealed that the vast majority of G cells indeed expressed Piezo1. Within flask-like G cells at the base of the antral invaginations, the Piezo1 protein was primarily located at the basolateral portion, which is thought to be the release site for the exocytic secretion of gastrin. In the spindle-like G cells, which are oriented parallel to the invaginations, Piezo1 protein was restricted to the cell body where the hormone was also located, whereas the long processes appeared to be devoid of Piezo1 protein. Our results suggest that mechanosensitive channels such as Piezo1, located in close proximity to hormone-release sites, enable G cells to respond directly to antrum distensions with gastrin secretion.

Functional characterization and quantitative expression analysis of two GnRH-related peptide receptors in the mosquito, Aedes aegypti.

PubMed

Oryan, Alireza; Wahedi, Azizia; Paluzzi, Jean-Paul V

2018-03-04

To cope with stressful events such as flight, organisms have evolved various regulatory mechanisms, often involving control by endocrine-derived factors. In insects, two stress-related factors include the gonadotropin-releasing hormone-related peptides adipokinetic hormone (AKH) and corazonin (CRZ). AKH is a pleiotropic hormone best known as a substrate liberator of proteins, lipids, and carbohydrates. Although a universal function has not yet been elucidated, CRZ has been shown to have roles in pigmentation, ecdysis or act as a cardiostimulatory factor. While both these neuropeptides and their respective receptors (AKHR and CRZR) have been characterized in several organisms, details on their specific roles within the disease vector, Aedes aegypti, remain largely unexplored. Here, we obtained three A. aegypti AKHR transcript variants and further identified the A. aegypti CRZR receptor. Receptor expression using a heterologous functional assay revealed that these receptors exhibit a highly specific response for their native ligands. Developmental quantitative expression analysis of CRZR revealed enrichment during the pupal and adult stages. In adults, quantitative spatial expression analysis revealed CRZR transcript in a variety of organs including head, thoracic ganglia, primary reproductive organs (ovary and testis), as well as male carcass. This suggest CRZ may play a role in ecdysis, and neuronal expression of CRZR indicates a possible role for CRZ within the nervous system. Quantitative developmental expression analysis of AKHR identified significant transcript enrichment in early adult stages. AKHR transcript was observed in the head, thoracic ganglia, accessory reproductive tissues and the carcass of adult females, while it was detected in the abdominal ganglia and enriched significantly in the carcass of adult males, which supports the known function of AKH in energy metabolism. Collectively, given the enrichment of CRZR and AKHR in the primary and

Selective activation of the B natriuretic peptide receptor by C-type natriuretic peptide (CNP).

PubMed

Koller, K J; Lowe, D G; Bennett, G L; Minamino, N; Kangawa, K; Matsuo, H; Goeddel, D V

1991-04-05

The natriuretic peptides are hormones that can stimulate natriuretic, diuretic, and vasorelaxant activity in vivo, presumably through the activation of two known cell surface receptor guanylyl cyclases (ANPR-A and ANPR-B). Although atrial natriuretic peptide (ANP) and, to a lesser extent, brain natriuretic peptide (BNP) are efficient activators of the ANPR-A guanylyl cyclase, neither hormone can significantly stimulate ANPR-B. A member of this hormone family, C-type natriuretic peptide (CNP), potently and selectively activated the human ANPR-B guanylyl cyclase. CNP does not increase guanosine 3',5'-monophosphate accumulation in cells expressing human ANPR-A. The affinity of CNP for ANPR-B is 50- or 500-fold higher than ANP or BNP, respectively. This ligand-receptor pair may be involved in the regulation of fluid homeostasis by the central nervous system.

Cholecystokinin type B receptor antagonist PD-136,450 is a partial secretory agonist in the stomach and a full agonist in the pancreas of the rat.

PubMed Central

Schmassmann, A; Garner, A; Flogerzi, B; Hasan, M Y; Sanner, M; Varga, L; Halter, F

1994-01-01

Gastrin (cholecystokinin type B (CCK-B)) receptor antagonists may help to elucidate the physiological role of gastrin, have therapeutic potential as acid antisecretory drugs, and may be of use as adjuvant therapy for gastrin sensitive tumours. In binding studies, the gastrin receptor antagonist PD-136,450 had at least 1000 fold greater affinity for gastrin (CCK-B) than CCK-A receptors. In this study the biological activity of PD-136,450 was evaluated in conscious and anaesthetised rats. PD-136,450 antagonised gastrin stimulated acid secretion after subcutaneous (IC50: 0.28 mumol/kg; conscious rats) and intravenous (IC50: 0.17 mumol/kg; anaesthetised rats) administration. In basal secreting fistula animals, the compound stimulated acid output to 30 (5)% of the maximal response to gastrin. Stimulant activity was not caused by gastrin release. As an agonist PD-136,450 was about 350 times less potent than gastrin-17 on a molar basis. In addition, PD-136,450 was a powerful agonist of pancreatic secretion in anaesthetised rats. The specific gastrin antagonist L-365,260 inhibited the (partial) agonist activity of PD-136,450 in the stomach and the specific CCK-A receptor antagonist L-364,718 inhibited the agonist activity of PD-136,450 in the pancreas. It is concluded that the agonist effect of PD-136,450 is mediated via interaction with the gastrin (CCK-B) receptor in the stomach and the CCK-A receptor in the pancreas. PMID:8307482

Neurotensin expression and release in human colon cancers.

PubMed Central

Evers, B M; Ishizuka, J; Chung, D H; Townsend, C M; Thompson, J C

1992-01-01

Neurotensin (NT), a distal gut peptide released by intraluminal fats, is trophic for normal small bowel and colonic mucosa. In addition, NT stimulates growth of certain colon cancers; the mechanism for this effect is not known. The purpose of this study was to determine whether human colon cancers (HCC) (1) express the mRNA for NT/neuromedin N (N), (2) produce NT peptide, and (3) express the mRNA for a functional NT receptor (NTR). RNA was extracted from four HCC cell lines in culture, nine HCC lines established in athymic nude mice, and from six HCC and adjacent normal mucosa from freshly resected operative specimens; the RNA was analyzed for NT/N mRNA by Northern hybridization with a complementary DNA probe. Neurotensin peptide content, NTR expression, and intracellular Ca++ ([Ca++]i) mobilization in response to NT were evaluated in three HCC cell lines (LoVo, HT29, HCT116). Neurotensin/N mRNA transcripts were identified in all four of the HCC cell lines and in one of nine HCC in nude mice. Neurotensin expression was found in two of six freshly resected HCC and in none of the six corresponding samples of normal mucosa. Neurotensin peptide was identified by RIA in LoVo, HT29, and HCT116. In addition, NTR mRNA was found in HT29 and HCT116. Neurotensin stimulated [Ca++]i mobilization in HCT116 (without serum) and in LoVo (with 0.25% serum). These findings demonstrate the presence of NT/N mRNA and NT peptide and the presence of a functional NTR in certain HCC. Neurotensin, a potent trophic factor for normal gut mucosa, may function as an autocrine growth factor in certain human colon cancers. Images FIG. 1. FIG. 4. PMID:1329682

Somatostatin receptors as markers for endocrine tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reubi, J.C.

1987-06-19

Endocrine tumors of the gastrointestinal tract are relatively rare neoplasias that secrete large amounts of peptide hormones such as insulin, glucagon, gastrin, or vasoactive intestinal peptide (VIP). These substances are usually responsible for the distinct clinical features observed in patients with such tumors. Although most are relatively slow growing tumors, they may lead in early stages to dramatic symptoms such as hypoglycemia, gastric ulcerations, or watery diarrhea. Unfortunately they are often difficult to localize precisely at that stage. Somatostatin, a tetradecapeptide that inhibits peptide hormone release in various sites such as the pituitary, the pancreas, and the gastrointestinal tract, hasmore » been shown recently to have beneficial effects when given chronically in the form of a stable non-degradable octapeptide analogue (SMS 201-995) in such gastrointestinal endocrine tumors. This essay demonstrates with autoradiographic techniques the very high density of somatostatin receptors in one case of human gastrinoma. A hematoxylineosin-stained histologic section reveals a well-defined, 2-mm-long tumor surrounded by normal tissue. After incubation of the section with an iodinated somatostatin analogue (/sup 125/I-(Leu, D-Trp, Tyr)-somatostatin-28), the distribution of somatostatin receptors was visualized on tritium-sensitive films after a one-week exposure of the section in x-ray cassettes.« less

Bioactive peptides derived from natural proteins with respect to diversity of their receptors and physiological effects.

PubMed

Yoshikawa, Masaaki

2015-10-01

We have found various bioactive peptides derived from animal and plant proteins, which interact with receptors for endogenous bioactive peptides such as opioids, neurotensin, complements C3a and C5a, oxytocin, and formyl peptides etc. Among them, rubiscolin, a δ opioid peptide derived from plant RuBisCO, showed memory-consolidating, anxiolytic-like, and food intake-modulating effects. Soymorphin, a μ opioid peptide derived from β-conglycinin showed anxiolytic-like, anorexigenic, hypoglycemic, and hypotriglyceridemic effects. β-Lactotensin derived from β-lactoglobulin, the first natural ligand for the NTS2 receptor, showed memory-consolidating, anxiolytic-like, and hypocholesterolemic effects. Weak agonist peptides for the complements C3a and C5a receptors were released from many proteins and exerted various central effects. Peptides showing anxiolytic-like antihypertensive and anti-alopecia effects via different types of receptors such as OT, FPR and AT2 were also obtained. Based on these study, new functions and post-receptor mechanisms of receptor commom to endogenous and exogenous bioactive peptides have been clarified. Copyright © 2015 Elsevier Inc. All rights reserved.

Myristoylated alanine-rich C kinase substrate-mediated neurotensin release via protein kinase C-delta downstream of the Rho/ROK pathway.

PubMed

Li, Jing; O'Connor, Kathleen L; Greeley, George H; Blackshear, Perry J; Townsend, Courtney M; Evers, B Mark

2005-03-04

Myristoylated alanine-rich protein kinase C substrate (MARCKS) is a cellular substrate for protein kinase C (PKC). Recently, we have shown that PKC isoforms-alpha and -delta, as well as the Rho/Rho kinase (ROK) pathway, play a role in phorbol 12-myristate 13-acetate (PMA)-mediated secretion of the gut peptide neurotensin (NT) in the BON human endocrine cell line. Here, we demonstrate that activation of MARCKS protein is important for PMA- and bombesin (BBS)-mediated NT secretion in BON cells. Small interfering RNA (siRNA) to MARCKS significantly inhibited, whereas overexpression of wild-type MARCKS significantly increased PMA-mediated NT secretion. Endogenous MARCKS and green fluorescent protein-tagged wild-type MARCKS were translocated from membrane to cytosol upon PMA treatment, further confirming MARCKS activation. MARCKS phosphorylation was inhibited by PKC-delta siRNA, ROKalpha siRNA, and C3 toxin (a Rho protein inhibitor), suggesting that the PKC-delta and the Rho/ROK pathways are necessary for MARCKS activation. The phosphorylation of PKC-delta was inhibited by C3 toxin, demonstrating that the role of MARCKS in NT secretion was regulated by PKC-delta downstream of the Rho/ROK pathway. BON cell clones stably transfected with the receptor for gastrin releasing peptide, a physiologic stimulant of NT, and treated with BBS, the amphibian equivalent of gastrin releasing peptide, demonstrated a similar MARCKS phosphorylation as noted with PMA. BBS-mediated NT secretion was attenuated by MARCKS siRNA. Collectively, these findings provide evidence for novel signaling pathways, including the sequential regulation of MARCKS activity by Rho/ROK and PKC-delta proteins, in stimulated gut peptide secretion.

GRP-induced up-regulation of Hsp72 promotes CD16+/94+ natural killer cell binding to colon cancer cells causing tumor cell cytolysis.

PubMed

Taglia, Lauren; Matusiak, Damien; Benya, Richard V

2008-01-01

Gastrin-releasing peptide (GRP) and its receptor (GRPR) are not normally expressed by epithelial cells lining the adult human colon. However post malignant transformation both GRP and its receptor are aberrantly expressed in the colon where we have previously shown they act to retard metastasis by enhancing tumor cell attachment to the extracellular matrix. In the present study, we show that GRP signaling via its cognate receptor when both are aberrantly expressed in human colon cancer cells causes heat shock protein 72 (Hsp72) to be expressed. We show that GRP/GRPR induces expression of Hsp72 by signaling via focal adhesion kinase. When expressed, Hsp72 promotes the binding of CD16+ and CD94+ natural killer cells, resulting in tumor cell cytolysis. These findings demonstrate the presence of a novel mechanism whereby aberrantly expressed GRP/GRPR in human colorectal cancer attenuates tumor progression and may promote a favorable outcome.

Upregulation of α7 Nicotinic Receptors by Acetylcholinesterase C-Terminal Peptides

PubMed Central

Bond, Cherie E.; Zimmermann, Martina; Greenfield, Susan A.

2009-01-01

Background The alpha-7 nicotinic acetylcholine receptor (α7-nAChR) is well known as a potent calcium ionophore that, in the brain, has been implicated in excitotoxicity and hence in the underlying mechanisms of neurodegenerative disorders such as Alzheimer's disease. Previous research implied that the activity of this receptor may be modified by exposure to a peptide fragment derived from the C-terminal region of the enzyme acetylcholinesterase. This investigation was undertaken to determine if the functional changes observed could be attributed to peptide binding interaction with the α7-nAChR, or peptide modulation of receptor expression. Methodology/Principal Findings This study provides evidence that two peptides derived from the C-terminus of acetylcholinesterase, not only selectively displace specific bungarotoxin binding at the α7-nAChR, but also alter receptor binding properties for its familiar ligands, including the alternative endogenous agonist choline. Of more long-term significance, these peptides also induce upregulation of α7-nAChR mRNA and protein expression, as well as enhancing receptor trafficking to the plasma membrane. Conclusions/Significance The results reported here demonstrate a hitherto unknown relationship between the α7-nAChR and the non-enzymatic functions of acetylcholinesterase, mediated independently by its C-terminal domain. Such an interaction may prove valuable as a pharmacological tool, prompting new approaches for understanding, and combating, the process of neurodegeneration. PMID:19287501

AM-37 and ST-36 Are Small Molecule Bombesin Receptor Antagonists

PubMed Central

Moody, Terry W.; Tashakkori, Nicole; Mantey, Samuel A.; Moreno, Paola; Ramos-Alvarez, Irene; Leopoldo, Marcello; Jensen, Robert T.

2017-01-01

While peptide antagonists for the gastrin-releasing peptide receptor (BB2R), neuromedin B receptor (BB1R), and bombesin (BB) receptor subtype-3 (BRS-3) exist, there is a need to develop non-peptide small molecule inhibitors for all three BBR. The BB agonist (BA)1 binds with high affinity to the BB1R, BB2R, and BRS-3. In this communication, small molecule BBR antagonists were evaluated using human lung cancer cells. AM-37 and ST-36 inhibited binding to human BB1R, BB2R, and BRS-3 with similar affinity (Ki = 1.4–10.8 µM). AM-13 and AM-14 were approximately an order of magnitude less potent than AM-37 and ST-36. The ability of BA1 to elevate cytosolic Ca2+ in human lung cancer cells transfected with BB1R, BB2R, and BRS-3 was antagonized by AM-37 and ST-36. BA1 increased tyrosine phosphorylation of the EGFR and ERK in lung cancer cells, which was blocked by AM-37 and ST-36. AM-37 and ST-36 reduced the growth of lung cancer cells that have BBR. The results indicate that AM-37 and ST-36 function as small molecule BB receptor antagonists. PMID:28785244

Involvement of Prolactin-Releasing Peptide in the Activation of Oxytocin Neurones in Response to Food Intake

PubMed Central

Yamashita, M; Takayanagi, Y; Yoshida, M; Nishimori, K; Kusama, M; Onaka, T

2013-01-01

Food intake activates neurones expressing prolactin-releasing peptide (PrRP) in the medulla oblongata and oxytocin neurones in the hypothalamus. Both PrRP and oxytocin have been shown to have an anorexic action. In the present study, we investigated whether the activation of oxytocin neurones following food intake is mediated by PrRP. We first examined the expression of PrRP receptors (also known as GPR10) in rats. Immunoreactivity of PrRP receptors was observed in oxytocin neurones and in vasopressin neurones in the paraventricular and supraoptic nuclei of the hypothalamus and in the bed nucleus of the stria terminalis. Application of PrRP to isolated supraoptic nuclei facilitated the release of oxytocin and vasopressin. In mice, re-feeding increased the expression of Fos protein in oxytocin neurones of the hypothalamus and bed nucleus of the stria terminalis. The increased expression of Fos protein in oxytocin neurones following re-feeding or i.p. administration of cholecystokinin octapeptide (CCK), a peripheral satiety factor, was impaired in PrRP-deficient mice. CCK-induced oxytocin increase in plasma was also impaired in PrRP-deficient mice. Furthermore, oxytocin receptor-deficient mice showed an increased meal size, as reported in PrRP-deficient mice and in CCKA receptor-deficient mice. These findings suggest that PrRP mediates, at least in part, the activation of oxytocin neurones in response to food intake, and that the CCK–PrRP–oxytocin pathway plays an important role in the control of the termination of each meal. PMID:23363338

Release of digestive enzymes from the crustacean hepatopancreas: effect of vertebrate gastrointestinal hormones.

PubMed

Resch-Sedlmeier, G; Sedlmeier, D

1999-06-01

Vertebrate gastrointestinal hormones were tested on their ability to liberate digestive enzymes from the crustacean midgut gland. CCK-8 (desulfated form), gastrin, bombesin, secretin, and substance P were detected to release enzymes. Maximal concentrations observed were 5 nM CCK for protease release, 1 nM gastrin for protease and 100 nM for amylase release, 100 nM bombesin for protease release, 10 nM secretin for amylase and protease release, and 100 nM substance P for protease release. Unlike in vertebrates, glucagon was unable to stimulate enzyme release in crustaceans, this also applies to the counterpart insulin. These results may support the assumption that Crustacea possess endogenous factors resembling the above mentioned vertebrate hormones, at least in such a way that the appropriate receptors have the capacity to accept these hormones.

[Ala12]MCD peptide: a lead peptide to inhibitors of immunoglobulin E binding to mast cell receptors.

PubMed

Buku, A; Condie, B A; Price, J A; Mezei, M

2005-09-01

An effort was made to discover mast cell degranulating (MCD) peptide analogs that bind with high affinity to mast cell receptors without triggering secretion of histamine or other mediators of the allergic reaction initiated by immunoglobulin E (IgE) after mast cell activation. Such compounds could serve as inhibitors of IgE binding to mast cell receptors. An alanine scan of MCD peptide reported previously showed that the analog [Ala12]MCD was 120-fold less potent in histamine-releasing activity and fivefold more potent in binding affinity to mast cell receptors than the parent MCD peptide. Because this analog showed marginal intrinsic activity and good binding affinity it was subsequently tested in the present study as an IgE inhibitor. In contrast to MCD peptide, [Ala12]MCD showed a 50% inhibition of IgE binding to the Fc epsilon RI alpha mast cell receptor by using rat basophilic leukemia (RBL-2H3) mast cells and fluorescence polarization. Furthermore, in a beta-hexosaminidase secretory assay, the peptide also showed a 50% inhibition of the secretion of this enzyme caused by IgE. An attempt was made to relate structural changes and biologic differences between the [Ala12]MCD analog and the parent MCD peptide. The present results show that [Ala12]MCD may provide a base for designing agents to prevent IgE/Fc epsilon RI alpha interactions and, consequently, allergic conditions.

Cholecystokinin in the control of gastric acid and plasma gastrin and somatostatin secretion in healthy subjects and duodenal ulcer patients before and after eradication of Helicobacter pylori.

PubMed

Konturek, J W

1994-12-01

Exogenous cholecystokinin (CCK) is known to effect gastric secretory and motor functions but its physiological role in the control of these functions in healthy subjects and duodenal ulcer (DU) patients is unknown. In this study involving four series of young healthy normal and DU subjects, the gastric secretory tests were performed under basal conditions and following stimulation by modified sham-feeding (MSF), i.v. infusion of caerulein, gastrin releasing peptide (GRP) or pentagastrin (p-gastrin) (series A), after 500 ml of standard meal without or with addition of 15% soybean oil (series B) or acidification of meal to pH 2.5 (series C), and finally after eradication of Helicobacter pylori (HP) (series D). Studies were carried out without or with the pretreatment with placebo or loxiglumide, a specific antagonist of type A CCK receptors. In series A, the gastric secretion obtained by aspiration technique was measured after secretagogues (MSF, caerulein, GRP or p-gastrin), whereas in series B, C, and D intragastric pH was measured before and after test meal and plasma gastrin, CCK and somatostatin were assayed by specific radioimmunoassays. In healthy subjects, MSF increased gastric acid outputs to about 36% of p-gastrin maximum and treatment with loxiglumide failed to affect this secretion. Standard meal enhanced acid output to about 50% of p-gastrin maximum and raised plasma levels of gastrin, CCK but not somatostatin. The pretreatment with loxiglumide resulted in further increase both in gastric acid secretion and plasma gastrin and CCK, while somatostatin level was significantly reduced. Infusion of graded doses of caerulein or GRP resulted in dose-dependent stimulation of gastric acid secretion reaching, respectively, 35% and 25% of p-gastrin maximum. When loxiglumide was added, the acid responses to caerulein and GRP were further increased by 2-3 folds, attaining a peak similar to the p-gastrin maximum. Administration of loxiglumide resulted in a significant

Expression profiles of relaxin family peptides and their receptors indicate their influence on spermatogenesis in the domestic cat (Felis catus).

PubMed

Braun, B C; Müller, K; Jewgenow, K

2015-07-01

Disturbed spermatogenesis is a common problem in felines. Studying spermatogenesis in the domestic cat can improve the understanding of the biological background and help to counteract fertility problems in other feline species. Here, we analyzed 3 relaxin family peptides (relaxin, relaxin-3, and INSL3) and their receptors (RXFP1, RXFP2, and RXFP3) as potential spermatogenic factors involving their expression in the testis at different stages of its development. It may be concluded from its stage-dependent expression that relaxin, together with RXFP1, appears to be involved in the first stage of spermatogenesis, whereas relaxin-3 via binding to RXFP3 influences spermiogenesis. Furthermore, correlations were observed between relaxin, relaxin-3, RXFP1, RXFP2 and RXFP3 messenger RNA expression, and the relative numbers of haploid cells in testes. The peptide INSL3 was highly expressed at all testis development stages. Because of the low and stage-independent expression of its receptor RXFP2, an auto- and/or paracrine function of INSL3 in spermatogenesis seems unlikely. In the adult testis, messenger RNA expression of relaxin, RXFP1, and RXFP3 predominantly occurs in the tubular testis compartment, whereas INLS3 is mainly expressed in the interstitium. Copyright © 2015 Elsevier Inc. All rights reserved.

Gastrin regulates ABCG2 to promote the migration, invasion and side populations in pancreatic cancer cells via activation of NF-κB signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Juan; Xin, Beibei; Wang, Hui

Gastrin is absent in most normal adult pancreatic tissues but is highly expressed in pancreatic cancer tissues. Although Gastrin expression was reported to be associated with tumor proliferation in human pancreatic cancer, studies on the relationship between Gastrin and tumor metastasis in pancreatic cancer are rare. In this study, we performed an analysis to determine the effects of Gastrin on modulating the side populations, cell proportion and tumor cell metastatic potential and invasion activity and explored its mechanisms in pancreatic cancer. We indicated that Gastrin and ABCG2 were widely expressed in pancreatic cancer cell lines and overexpressed in cancer tissues.more » Gastrin induced ABCG2 expression, and this effect was mediated by NF-κB activation. Gastrin regulated the SP proportion of BxPC-3 cells via modulating ABCG2 expression. Through the regulation of the functions of NF-κB/ABCG2, Gastrin functionally promoted the migration and invasion in pancreatic cancer cell. The present study indicated that Gastrin induced ABCG2 expression by activating NF-κB and thereby modulated the SP proportion, tumor cell metastatic potential and invasion activity in pancreatic cancer. Gastrin could serve as an effective therapeutic target for the metastasis of pancreatic cancer. - Highlights: • Gastrin induces ABCG2 expression mediated by NF-κB activation. • Gastrin regulates NF-κB's function that binds to the ABCG2 promoter in BxPC-3 cells. • Gastrin promotes the SP proportion in BxPC-3 cells by modulating ABCG2 expression via activation of NF-κB molecule. • Gastrin induces an increase in migration and invasion potential in pancreatic cancer cell by regulating NF-κB/ABCG2 signaling.« less

Nutrient-induced glucagon like peptide-1 release is modulated by serotonin.

PubMed

Ripken, Dina; van der Wielen, Nikkie; Wortelboer, Heleen M; Meijerink, Jocelijn; Witkamp, Renger F; Hendriks, Henk F J

2016-06-01

Glucagon like peptide-1 (GLP-1) and serotonin are both involved in food intake regulation. GLP-1 release is stimulated upon nutrient interaction with G-protein coupled receptors by enteroendocrine cells (EEC), whereas serotonin is released from enterochromaffin cells (ECC). The central hypothesis for the current study was that nutrient-induced GLP-1 release from EECs is modulated by serotonin through a process involving serotonin receptor interaction. This was studied by assessing the effects of serotonin reuptake inhibition by fluoxetine on nutrient-induced GLP-1, PYY and CCK release from isolated pig intestinal segments. Next, serotonin-induced GLP-1 release was studied in enteroendocrine STC-1 cells, where effects of serotonin receptor inhibition were studied using specific and non-specific antagonists. Casein (1% w/v), safflower oil (3.35% w/v), sucrose (50mM) and rebaudioside A (12.5mM) stimulated GLP-1 release from intestinal segments, whereas casein only stimulated PYY and CCK release. Combining nutrients with fluoxetine further increased nutrient-induced GLP-1, PYY and CCK release. Serotonin release from intestinal tissue segments was stimulated by casein and safflower oil while sucrose and rebaudioside A had no effect. The combination with fluoxetine (0.155μM) further enhanced casein and safflower oil induced-serotonin release. Exposure of ileal tissue segments to serotonin (30μM) stimulated GLP-1 release whereas it did not induce PYY and CCK release. Serotonin (30 and 100μM) also stimulated GLP-1 release from STC-1 cells, which was inhibited by the non-specific 5HT receptor antagonist asenapine (1 and 10μM). These data suggest that nutrient-induced GLP-1 release is modulated by serotonin through a receptor mediated process. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Octopus gonadotrophin-releasing hormone: a multifunctional peptide in the endocrine and nervous systems of the cephalopod.

PubMed

Minakata, H; Shigeno, S; Kano, N; Haraguchi, S; Osugi, T; Tsutsui, K

2009-03-01

The optic gland, which is analogous to the anterior pituitary in the context of gonadal maturation, is found on the upper posterior edge of the optic tract of the octopus Octopus vulgaris. In mature octopus, the optic glands enlarge and secrete a gonadotrophic hormone. A peptide with structural features similar to that of vertebrate gonadotrophin-releasing hormone (GnRH) was isolated from the brain of octopus and was named oct-GnRH. Oct-GnRH showed luteinising hormone-releasing activity in the anterior pituitary cells of the Japanese quail Coturnix coturnix. Oct-GnRH immunoreactive signals were observed in the glandular cells of the mature optic gland. Oct-GnRH stimulated the synthesis and release of sex steroids from the ovary and testis, and elicited contractions of the oviduct. Oct-GnRH receptor was expressed in the gonads and accessory organs, such as the oviduct and oviducal gland. These results suggest that oct-GnRH induces the gonadal maturation and oviposition by regulating sex steroidogenesis and a series of egg-laying behaviours via the oct-GnRH receptor. The distribution and expression of oct-GnRH in the central and peripheral nervous systems suggest that oct-GnRH acts as a multifunctional modulatory factor in feeding, memory processing, sensory, movement and autonomic functions.

GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis

PubMed Central

Wrzaczek, Michael; Vainonen, Julia P; Stael, Simon; Tsiatsiani, Liana; Help-Rinta-Rahko, Hanna; Gauthier, Adrien; Kaufholdt, David; Bollhöner, Benjamin; Lamminmäki, Airi; Staes, An; Gevaert, Kris; Tuominen, Hannele; Van Breusegem, Frank; Helariutta, Ykä; Kangasjärvi, Jaakko

2015-01-01

Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor. PMID:25398910

GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis.

PubMed

Wrzaczek, Michael; Vainonen, Julia P; Stael, Simon; Tsiatsiani, Liana; Help-Rinta-Rahko, Hanna; Gauthier, Adrien; Kaufholdt, David; Bollhöner, Benjamin; Lamminmäki, Airi; Staes, An; Gevaert, Kris; Tuominen, Hannele; Van Breusegem, Frank; Helariutta, Ykä; Kangasjärvi, Jaakko

2015-01-02

Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor. © 2014 The Authors.

The chromatography-free release, isolation and purification of recombinant peptide for fibril self-assembly.

PubMed

Hartmann, B M; Kaar, W; Yoo, I K; Lua, L H L; Falconer, R J; Middelberg, A P J

2009-12-01

One of the major expenses associated with recombinant peptide production is the use of chromatography in the isolation and purification stages of a bioprocess. Here we report a chromatography-free isolation and purification process for recombinant peptide expressed in Escherichia coli (E. coli). Initial peptide release is by homogenization and then by enzymatic cleavage of the peptide-containing fusion protein, directly in the E. coli homogenate. Release is followed by selective solvent precipitation (SSP) to isolate and purify the peptide away from larger cell contaminants. Specifically, we expressed in E. coli the self-assembling beta-sheet forming peptide P(11)-2 in fusion to thioredoxin. Homogenate was heat treated (55 degrees C, 15 min) and then incubated with tobacco etch virus protease (TEVp) to release P(11)-2 having a native N-terminus. SSP with ethanol at room temperature then removed contaminating proteins in an integrated isolation-purification step; it proved necessary to add 250 mM NaCl to homogenate to prevent P(11)-2 from partitioning to the precipitate. This process structure gave recombinant P(11)-2 peptide at 97% polypeptide purity and 40% overall yield, without a single chromatography step. Following buffer-exchange of the 97% pure product by bind-elute chromatography into defined chemical conditions, the resulting peptide was shown to be functionally active and able to form self-assembled fibrils. To the best of our knowledge, this manuscript reports the first published process for chromatography-free recombinant peptide release, isolation and purification. The process proved able to deliver functional recombinant peptide at high purity and potentially low cost, opening cost-sensitive materials applications for peptide-based materials.

Characterization of three peptides derived from prosomatostatin [prosomatostatin-(1-63)-, -(65-76)- and -(79-92)-peptides] in a human pancreatic tumour.

PubMed

Conlon, J M; Eriksson, B; Grimelius, L; Oberg, K; Thim, L

1987-11-15

By using only reverse-phase h.p.l.c., three fragments of prosomatostatin were isolated from an extract of a human pancreatic neuroendocrine tumour that produced somatostatin, vasoactive intestinal polypeptide and gastrin-releasing peptide. The amino acid composition of the peptides indicated that they represented prosomatostatin-(1-63)-peptide, prosomatostain-(65-76)-peptide and prosomatostatin-(79-92)-peptide (somatostatin-14). The identity of prosomatostatin-(1-63)-peptide was confirmed by characterization of the products of digestion with Armillaria mellea (honey fungus) proteinase. Partial micro-sequencing of prosomatostatin-(1-63)-peptide showed that the Gly24-Ala25 bond of preprosomatostatin was the site of cleavage of the signal peptide. Thus human prosomatostatin is a protein of 92 amino acid residues that is proteolytically cleaved in a pancreatic tumour at the site of a dibasic-residue (arginine-lysine) processing site and at a single-monobasic-residue (arginine) processing site.

Vasoactive intestinal peptide-null mice demonstrate enhanced sweet taste preference, dysglycemia, and reduced taste bud leptin receptor expression.

PubMed

Martin, Bronwen; Shin, Yu-Kyong; White, Caitlin M; Ji, Sunggoan; Kim, Wook; Carlson, Olga D; Napora, Joshua K; Chadwick, Wayne; Chapter, Megan; Waschek, James A; Mattson, Mark P; Maudsley, Stuart; Egan, Josephine M

2010-05-01

It is becoming apparent that there is a strong link between taste perception and energy homeostasis. Recent evidence implicates gut-related hormones in taste perception, including glucagon-like peptide 1 and vasoactive intestinal peptide (VIP). We used VIP knockout mice to investigate VIP's specific role in taste perception and connection to energy regulation. Body weight, food intake, and plasma levels of multiple energy-regulating hormones were measured and pancreatic morphology was determined. In addition, the immunocytochemical profile of taste cells and gustatory behavior were examined in wild-type and VIP knockout mice. VIP knockout mice demonstrate elevated plasma glucose, insulin, and leptin levels, with no islet beta-cell number/topography alteration. VIP and its receptors (VPAC1, VPAC2) were identified in type II taste cells of the taste bud, and VIP knockout mice exhibit enhanced taste preference to sweet tastants. VIP knockout mouse taste cells show a significant decrease in leptin receptor expression and elevated expression of glucagon-like peptide 1, which may explain sweet taste preference of VIP knockout mice. This study suggests that the tongue can play a direct role in modulating energy intake to correct peripheral glycemic imbalances. In this way, we could view the tongue as a sensory mechanism that is bidirectionally regulated and thus forms a bridge between available foodstuffs and the intricate hormonal balance in the animal itself.

The degradation of bioactive peptides and proteins by dipeptidyl peptidase IV from human placenta.

PubMed

Nausch, I; Mentlein, R; Heymann, E

1990-11-01

The degradation of several bioactive peptides and proteins by purified human dipeptidyl peptidase IV is reported. It was hitherto unknown that human gastrin-releasing peptide, human chorionic gonadotropin, human pancreatic polypeptide, sheep prolactin, aprotinin, corticotropin-like intermediate lobe peptide and (Tyr-)melanostatin are substrates of this peptidase. Kinetic constants were determined for the degradation of a number of other natural peptides, including substance P, the degradation of which has been described earlier in a qualitative manner. Generally, small peptides are degraded much more rapidly than proteins. However, the Km-values seem to be independent of the peptide chain length. The influence of the action of dipeptidyl peptidase IV on the biological function of peptides and proteins is discussed.

An intrinsic agonist mechanism for activation of glucagon-like peptide-1 receptor by its extracellular domain

PubMed Central

Yin, Yanting; Zhou, X Edward; Hou, Li; Zhao, Li-Hua; Liu, Bo; Wang, Gaihong; Jiang, Yi; Melcher, Karsten; Xu, H Eric

2016-01-01

The glucagon-like peptide-1 receptor is a class B G protein coupled receptor (GPCR) that plays key roles in glucose metabolism and is a major therapeutic target for diabetes. The classic two-domain model for class B GPCR activation proposes that the apo-state receptor is auto-inhibited by its extracellular domain, which physically interacts with the transmembrane domain. The binding of the C-terminus of the peptide hormone to the extracellular domain allows the N-terminus of the hormone to insert into the transmembrane domain to induce receptor activation. In contrast to this model, here we demonstrate that glucagon-like peptide-1 receptor can be activated by N-terminally truncated glucagon-like peptide-1 or exendin-4 when fused to the receptor, raising the question regarding the role of N-terminal residues of peptide hormone in glucagon-like peptide-1 receptor activation. Mutations of cysteine 347 to lysine or arginine in intracellular loop 3 transform the receptor into a G protein-biased receptor and allow it to be activated by a nonspecific five-residue linker that is completely devoid of exendin-4 or glucagon-like peptide-1 sequence but still requires the presence of an intact extracellular domain. Moreover, the extracellular domain can activate the receptor in trans in the presence of an intact peptide hormone, and specific mutations in three extracellular loops abolished this extracellular domain trans-activation. Together, our data reveal a dominant role of the extracellular domain in glucagon-like peptide-1 receptor activation and support an intrinsic agonist model of the extracellular domain, in which peptide binding switches the receptor from the auto-inhibited state to the auto-activated state by releasing the intrinsic agonist activity of the extracellular domain. PMID:27917297

Regulatory effect and mechanism of gastrin and its antagonists on colorectal carcinoma

PubMed Central

He, Shuang-Wu; Shen, Kang-Qiang; He, Yu-Jun; Xie, Bin; Zhao, Yan-Ming

1999-01-01

AIM: To explore the effect and mechanism of gastrin and its an tagonists proglumide and somatostatin on colorectal carcinoma and their clinical significance. METHODS: A model of transplanted human colonic carcinoma was established from SW480 cell line in gymnomouse body. The volume and weight of transplanted carcinoma was observed under the effect of pentagatrin (PG), proglumide (PGL) and octapeptide somotostatin (SMS201-995, SMS). The cAMP content of carcinoma cell was determined by radioimmunoassay and the DNA, protein content and cell cycle were determined by flow-cytometry. The amount of viable cells was determined by MTT colorimetric analysis, IP3 content was determined by radioimmuno assay, Ca2+ concentration in cell by fluorometry and PKC activity by isotopic enzymolysis. The expression of gastrin, c-myc, c-fos and rasP21 in 48 case s of colorectal carcinoma tissue was detected by the immuno-cytochemistry SP method. Argyrophilia nucleolar organizer regions was determined with argyrophilia stain. RESULTS: The volume, weight, cAMP, DNA and protein content in carcinoma cell, cell amount and proliferation index of S and G2M phase in PG group were all significantly higher than those of control group. When PG was at the concentration of 25 mg/L, the amount of viable cells, IP3 content and Ca2+ concentration in cell and membrane PKC activity in PG group were significantly higher than those in control group; when PGL was at a concentration of 32 mg/L, they dropped to the lowest level in PG (25 mg/L) + PGL group, but without significant difference from the control group. The positive expression rate of gastrin, c-myc, c-fos and rasP21 in carcinoma tissue was 39.6%, 54.2%, 47.9% and 54.2% respectively and significantly higher than that in mucosa 3 cm and 6 cm adjacent to carcinoma tissue and normal colorectal mucosa. The positive expression rate of gastrin of highly-differentiated adenocarcinoma group was significantly higher than that of poorly-differentiated and

Gonadotropin-Releasing Hormone (GnRH) Receptor Structure and GnRH Binding

PubMed Central

Flanagan, Colleen A.; Manilall, Ashmeetha

2017-01-01

Gonadotropin-releasing hormone (GnRH) regulates reproduction. The human GnRH receptor lacks a cytoplasmic carboxy-terminal tail but has amino acid sequence motifs characteristic of rhodopsin-like, class A, G protein-coupled receptors (GPCRs). This review will consider how recent descriptions of X-ray crystallographic structures of GPCRs in inactive and active conformations may contribute to understanding GnRH receptor structure, mechanism of activation and ligand binding. The structures confirmed that ligands bind to variable extracellular surfaces, whereas the seven membrane-spanning α-helices convey the activation signal to the cytoplasmic receptor surface, which binds and activates heterotrimeric G proteins. Forty non-covalent interactions that bridge topologically equivalent residues in different transmembrane (TM) helices are conserved in class A GPCR structures, regardless of activation state. Conformation-independent interhelical contacts account for a conserved receptor protein structure and their importance in the GnRH receptor structure is supported by decreased expression of receptors with mutations of residues in the network. Many of the GnRH receptor mutations associated with congenital hypogonadotropic hypogonadism, including the Glu2.53(90) Lys mutation, involve amino acids that constitute the conserved network. Half of the ~250 intramolecular interactions in GPCRs differ between inactive and active structures. Conformation-specific interhelical contacts depend on amino acids changing partners during activation. Conserved inactive conformation-specific contacts prevent receptor activation by stabilizing proximity of TM helices 3 and 6 and a closed G protein-binding site. Mutations of GnRH receptor residues involved in these interactions, such as Arg3.50(139) of the DRY/S motif or Tyr7.53(323) of the N/DPxxY motif, increase or decrease receptor expression and efficiency of receptor coupling to G protein signaling, consistent with the native residues

Resveratrol inhibits proteinase-activated receptor-2-induced release of soluble vascular endothelial growth factor receptor-1 from human endothelial cells

PubMed Central

Al-Ani, Bahjat

2013-01-01

We recently reported that (i) activation of the proinflammatory receptor, proteinase-activated receptor-2 (PAR-2) caused the release of an important biomarker in preeclampsia, soluble vascular endothelial growth factor receptor-1 (sVEGFR-1, also known as sFlt-1) from human umbilical vein endothelial cells (HUVECs), and (ii) that the anti-oxidant and anti-inflammatory agent, resveratrol, is capable of inhibiting the proinflammatory cytokine-induced sVEGFR-1 release from human placenta. Based on these findings and because PAR-2 is upregulated by proinflammatory cytokines, we sought to determine whether resveratrol can inhibit PAR-2-induced sVEGFR-1 release. PAR-2 expressing cells, HUVECs and human embryonic kidney cells (HEK-293) transfected with a human VEGFR-1 promoter-luciferase reporter construct were incubated with PAR-2-activating peptide and/or resveratrol. Cell supernatants were assayed for sVEGFR-1 by enzyme-linked immunosorbent assay (ELISA), and VEGFR-1 promoter-luciferase assay was performed on the harvested cell lysates. Preincubation of HEK-293 cells with resveratrol significantly inhibited PAR-2-induced VEGFR-1 promoter activity without affecting cell viability as assessed by MTT assay. The addition of resveratrol also blocked PAR-2-mediated sVEGFR-1 release from HUVECs. The present study demonstrates that resveratrol suppressed both VEGFR-1 promoter activity and sVEGFR-1 protein release induced by PAR-2 activation, which further endorses our recent findings of a potential therapeutic role for resveratrol in preeclampsia. PMID:26933402

Bombesin related peptides/receptors and their promising therapeutic roles in cancer imaging, targeting and treatment

PubMed Central

Moreno, Paola; Ramos-Álvarez, Irene; Moody, Terry W.; Jensen, Robert T.

2016-01-01

Introduction Despite remarkable advances in tumor treatment, many patients still die from common tumors (breast, prostate, lung, CNS, colon, and pancreas), and thus, new approaches are needed. Many of these tumors synthesize bombesin (Bn)-related peptides and over-express their receptors (BnRs), hence functioning as autocrine-growth-factors. Recent studies support the conclusion that Bn-peptides/BnRs are well-positioned for numerous novel antitumor treatments, including interrupting autocrine-growth via the use of over-expressed receptors for imaging and targeting cytotoxic-compounds, either by direct-coupling or combined with nanoparticle-technology. Areas covered The unique ability of common neoplasms to synthesize, secrete, and show a growth/proliferative/differentiating response due to BnR over-expression, is reviewed, both in general and with regard to the most frequently investigated neoplasms (breast, prostate, lung, and CNS). Particular attention is paid to advances in the recent years. Also considered are the possible therapeutic approaches to the growth/differentiation effect of Bn-peptides, as well as the therapeutic implication of the frequent BnR over-expression for tumor-imaging and/or targeted-delivery. Expert opinion Given that Bn-related-peptides/BnRs are so frequently ectopically-expressed by common tumors, which are often malignant and become refractory to conventional treatments, therapeutic interventions using novel approaches to Bn-peptides and receptors are being explored. Of particular interest is the potential of reproducing BnRs in common tumors, such as the recent success of utilizing overexpression of somatostatin-receptors by neuroendocrine-tumors to provide the most sensitive imaging methods and targeted delivery of cytotoxic-compounds. PMID:26981612

Elevated serum progastrin-releasing peptide (31-98) in metastatic and androgen-independent prostate cancer patients.

PubMed

Yashi, Masahiro; Muraishi, Osamu; Kobayashi, Yutaka; Tokue, Akihiko; Nanjo, Hiroshi

2002-05-01

Increases in neuroendocrine phenotype and secretory products are closely correlated with tumor progression and androgen independence in prostate cancer. In this study, we explored this correlation using serum progastrin-releasing peptide (ProGRP), a carboxy-terminal region common to three subtypes of precursors for gastrin-releasing peptide (GRP), which is released from the neuroendocrine phenotype to act as a growth factor. In 60 patients with benign prostatic hyperplasia (BPH) and 200 with prostate cancer, serum ProGRP levels were determined with an enzyme-linked immunosorbent assay (ELISA) kit and evaluated in relation to clinical stage, hormonal treatment, and prostate-specific antigen (PSA) values. Fourteen randomly selected patients were entered in the follow-up study. Additionally, expression of ProGRP as determined by immunohistochemical analysis was compared to that of chromogranin-A (CgA) in tissue samples from several subjects. We found a positive correlation between PSA and ProGRP in patients with untreated prostate cancer; no correlation was found in the treated groups. The increases in the ProGRP value and in the percentage of patients with higher than normal values were significant (P < 0.0001), especially in the androgen-independent group (P < 0.0001). A longitudinal study showed that, in a subset of patients, the ProGRP values tended to increase transiently when the cancer became androgen independent, but remained unchanged or decreased at the androgen-dependent stage. Positive staining for ProGRP occurred in a different distribution in neuroendocrine tissues than that of staining for CgA. The clinical results demonstrated the existence of a regulatory mechanism for GRP, which to date had only been observed in cell lines. These findings suggest that GRP is a growth factor potentially upregulated by androgen but that does not rely principally on androgen modulation. The large overlap in levels of ProGRP among the groups limits the use of this value

Characterization of Gonadotrope Secretoproteome Identifies Neurosecretory Protein VGF-derived Peptide Suppression of Follicle-stimulating Hormone Gene Expression*

PubMed Central

Wang, Qian; Jia, Jingjing; Chikina, Maria; Pincas, Hanna; Dolios, Georgia; Sasaki, Kazuki; Wang, Rong; Minamino, Naoto; Sealfon, Stuart C.

2016-01-01

Reproductive function is controlled by the pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH), which regulates the expression of the gonadotropins luteinizing hormone and FSH in pituitary gonadotropes. Paradoxically, Fshb gene expression is maximally induced at lower frequency GnRH pulses, which provide a very low average concentration of GnRH stimulation. We studied the role of secreted factors in modulating gonadotropin gene expression. Inhibition of secretion specifically disrupted gonadotropin subunit gene regulation but left early gene induction intact. We characterized the gonadotrope secretoproteome and global mRNA expression at baseline and after Gαs knockdown, which has been found to increase Fshb gene expression (1). We identified 1077 secreted proteins or peptides, 19 of which showed mRNA regulation by GnRH or/and Gαs knockdown. Among several novel secreted factors implicated in Fshb gene regulation, we focused on the neurosecretory protein VGF. Vgf mRNA, whose gene has been implicated in fertility (2), exhibited high induction by GnRH and depended on Gαs. In contrast with Fshb induction, Vgf induction occurred preferentially at high GnRH pulse frequency. We hypothesized that a VGF-derived peptide might regulate Fshb gene induction. siRNA knockdown or extracellular immunoneutralization of VGF augmented Fshb mRNA induction by GnRH. GnRH stimulated the secretion of the VGF-derived peptide NERP1. NERP1 caused a concentration-dependent decrease in Fshb gene induction. These findings implicate a VGF-derived peptide in selective regulation of the Fshb gene. Our results support the concept that signaling specificity from the cell membrane GnRH receptor to the nuclear Fshb gene involves integration of intracellular signaling and exosignaling regulatory motifs. PMID:27466366

Expression of CCK Receptors in Carcinoma Gallbladder and Cholelithiasis: A Pilot Study.

PubMed

Faridi, Mohammad Shazib; Jaiswal, Mahabir Saran Das; Goel, Sudhir K

2015-07-01

Gastrin and cholecystokinin (CCK) receptors are trophic for various gastrointestinal malignancies. Their role in gallbladder cancer has not been widely studied. To identify expression of CCK-A and CCK-B receptors in the tissue and blood of patients suffering from carcinoma (CA) gallbladder and gallstone disease and to compare expression of CCK A and B receptors in the gall bladder tissue and blood of healthy individuals and patients of CA gallbladder, and gallstone diseases. Forty nine subjects of both genders were recruited, comprising of 22 patients of CA gall bladder, 19 cases of cholelithiasis and, 8 normal gallbladders obtained from patients operated for trauma of the biliary system or Whipple's procedure. RNA extraction and cDNA formation for CCK-A and CCK-B receptors were carried out. Real Time PCR was performed on cDNA and threshold cycle (Ct) value of each sample was obtained and ΔCt was calculated. Chi-square test for comparing two groups and ANOVA test for comparing multiple groups were applied and if p<0.05 then Dunnett-C test was performed. Both CCK-A and CCK-B receptors were expressed irrespective of its origin in all tissues and blood samples studied; be it normal, Cholelithiasis or CA gallbladder and there was no difference among them (p>0.05). This preliminary study showed higher expression of CCK-A receptors in patients of cholelithiasis and decreased expression of CCK-A receptors in patients of CA gallbladder as compared to normal gallbladder although it did not rise to statistical significance.

Rationally designed chimeric peptide of met-enkephalin and FMRFa-[D-Ala2,p-Cl-Phe4]YFa induce multiple opioid receptors mediated antinociception and up-regulate their expression.

PubMed

Vats, Ishwar Dutt; Chaudhary, Snehlata; Sharma, Ahuti; Nath, Mahendra; Pasha, Santosh

2010-07-25

The physiological role of NPFF/FMRFa family of peptides appears to be complex and exact mechanism of action of these peptides is not yet completely understood. In same line of scrutiny, another analog of YGGFMKKKFMRFamide (YFa), a chimeric peptide of met-enkephalin and FMRFamide, was rationally designed and synthesized which contain D-alanine and p-Cl-phenylalanine residues at 2nd and 4th positions, respectively i.e., Y-(D-Ala)-G-(p-Cl-Phe)-MKKKFMRFamide ([D-Ala(2), p-Cl-Phe(4)]YFa) in order to achieve improved bioavailability and blood brain barrier penetration. Therefore, present study investigates the possible antinociceptive effect of [D-Ala(2), p-Cl-Phe(4)]YFa on intra-peritoneal (i.p.) administration using tail-flick test in rats followed by its opioid receptor(s) specificity using mu, delta and kappa receptor antagonists. Further, its antinociceptive effect was examined during 6 days of chronic i.p. treatment and assessed effect of this treatment on differential expression of opioid receptors. [D-Ala(2), p-Cl-Phe(4)]YFa in comparison to parent peptide YFa, induce significantly higher dose dependent antinociception in rats which was mediated by all three opioid receptors (mu, delta and kappa). Importantly, it induced comparable antinociception in rats throughout the chronic i.p. treatment and significantly up-regulated the overall expression (mRNA and protein) of mu, delta and kappa opioid receptors. Therefore, pharmacological and molecular behavior of [D-Ala(2), p-Cl-Phe(4)]YFa demonstrate that incorporation of D-alanine and p-Cl-phenylalanine residues at appropriate positions in chimeric peptide leads to altered opioid receptor selectivity and enhanced antinociceptive potency, relative to parent peptide. (c) 2010 Elsevier B.V. All rights reserved.

Receptor activity-modifying protein-dependent effects of mutations in the calcitonin receptor-like receptor: implications for adrenomedullin and calcitonin gene-related peptide pharmacology

PubMed Central

Watkins, H A; Walker, C S; Ly, K N; Bailey, R J; Barwell, J; Poyner, D R; Hay, D L

2014-01-01

Background and Purpose Receptor activity-modifying proteins (RAMPs) define the pharmacology of the calcitonin receptor-like receptor (CLR). The interactions of the different RAMPs with this class B GPCR yield high-affinity calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors. However, the mechanism for this is unclear. Experimental Approach Guided by receptor models, we mutated residues in the N-terminal helix of CLR, RAMP2 and RAMP3 hypothesized to be involved in peptide interactions. These were assayed for cAMP production with AM, AM2 and CGRP together with their cell surface expression. Binding studies were also conducted for selected mutants. Key Results An important domain for peptide interactions on CLR from I32 to I52 was defined. Although I41 was universally important for binding and receptor function, the role of other residues depended on both ligand and RAMP. Peptide binding to CLR/RAMP3 involved a more restricted range of residues than that to CLR/RAMP1 or CLR/RAMP2. E101 of RAMP2 had a major role in AM interactions, and F111/W84 of RAMP2/3 was important with each peptide. Conclusions and Implications RAMP-dependent effects of CLR mutations suggest that the different RAMPs control accessibility of peptides to binding residues situated on the CLR N-terminus. RAMP3 appears to alter the role of specific residues at the CLR-RAMP interface compared with RAMP1 and RAMP2. PMID:24199627

Use of immobilized exopeptidases and volatile buffers for analysis of peptides by fast atom bombardment mass spectrometry.

PubMed

Wagner, R M; Fraser, B A

1987-05-01

beta-Lipotrophin (62-77) or Ac-gastrin releasing peptide was incubated with immobilized carboxypeptidase Y or aminopeptidase M. Subsequent aliquots of each incubation mixture were analysed by fast atom bombardment mass spectrometry using a dithiothreitol/dithioerythritol liquid matrix. The use of immobilized enzymes and volatile buffers for exopeptidase digestions enabled rapid and facile separation of enzyme from digestion products. This approach to mass spectral peptide analysis reduced spectral background arising from a glycerol matrix, buffer salts, or enzyme proteins and contaminants, enabling analysis of as little as 200 picomoles of a suitable peptide.

Water-Soluble Nanoparticle Receptors Supramolecularly Coded for Acidic Peptides.

PubMed

Fa, Shixin; Zhao, Yan

2018-01-02

Sequence-specific recognition of peptides is of enormous importance to many chemical and biological applications, but has been difficult to achieve due to the minute differences in the side chains of amino acids. Acidic peptides are known to play important roles in cell growth and gene expression. In this work, we report molecularly imprinted micelles coded with molecular recognition information for the acidic and hydrophobic side chains of acidic peptides. The imprinted receptors could distinguish acidic amino acids from other polar and nonpolar amino acids, with dissociation constants of tens of nanomolar for biologically active peptides containing up to 18 amino acids. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Designer interface peptide grafts target estrogen receptor alpha dimerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chakraborty, S.; Asare, B.K.; Biswas, P.K., E-mail: pbiswas@tougaloo.edu

The nuclear transcription factor estrogen receptor alpha (ERα), triggered by its cognate ligand estrogen, regulates a variety of cellular signaling events. ERα is expressed in 70% of breast cancers and is a widely validated target for anti-breast cancer drug discovery. Administration of anti-estrogen to block estrogen receptor activation is still a viable anti-breast cancer treatment option but anti-estrogen resistance has been a significant bottle-neck. Dimerization of estrogen receptor is required for ER activation. Blocking ERα dimerization is therefore a complementary and alternative strategy to combat anti-estrogen resistance. Dimer interface peptide “I-box” derived from ER residues 503–518 specifically blocks ER dimerization.more » Recently using a comprehensive molecular simulation we studied the interaction dynamics of ERα LBDs in a homo-dimer. Based on this study, we identified three interface recognition peptide motifs LDKITDT (ERα residues 479–485), LQQQHQRLAQ (residues 497–506), and LSHIRHMSNK (residues 511–520) and reported the suitability of using LQQQHQRLAQ (ER 497–506) as a template to design inhibitors of ERα dimerization. Stability and self-aggregation of peptide based therapeutics poses a significant bottle-neck to proceed further. In this study utilizing peptide grafted to preserve their pharmacophoric recognition motif and assessed their stability and potential to block ERα mediated activity in silico and in vitro. The Grafted peptides blocked ERα mediated cell proliferation and viability of breast cancer cells but did not alter their apoptotic fate. We believe the structural clues identified in this study can be used to identify novel peptidometics and small molecules that specifically target ER dimer interface generating a new breed of anti-cancer agents. - Highlights: • Designer peptide grafts retain core molecular recognition motif during MD simulations. • Designer peptide grafts with Poly-ALA helix form stable

Simultaneous monitoring of presynaptic transmitter release and postsynaptic receptor trafficking reveals an enhancement of presynaptic activity in metabotropic glutamate receptor-mediated long-term depression.

PubMed

Xu, Wei; Tse, Yiu Chung; Dobie, Frederick A; Baudry, Michel; Craig, Ann Marie; Wong, Tak Pan; Wang, Yu Tian

2013-03-27

Although the contribution of postsynaptic mechanisms to long-term synaptic plasticity has been studied extensively, understanding the contribution of presynaptic modifications to this process lags behind, primarily because of a lack of techniques with which to directly and quantifiably measure neurotransmitter release from synaptic terminals. Here, we developed a method to measure presynaptic activity through the biotinylation of vesicular transporters in vesicles fused with presynaptic membranes during neurotransmitter release. This method allowed us for the first time to selectively quantify the spontaneous or evoked release of glutamate or GABA at their respective synapses. Using this method to investigate presynaptic changes during the expression of group I metabotropic glutamate receptor (mGluR1/5)-mediated long-term depression (LTD) in cultured rat hippocampal neurons, we discovered that this form of LTD was associated with increased presynaptic release of glutamate, despite reduced miniature EPSCs measured with whole-cell recording. Moreover, we found that specific blockade of AMPA receptor (AMPAR) endocytosis with a membrane-permeable GluR2-derived peptide not only prevented the expression of LTD but also eliminated LTD-associated increase in presynaptic release. Thus, our work not only demonstrates that mGluR1/5-mediated LTD is associated with increased endocytosis of postsynaptic AMPARs but also reveals an unexpected homeostatic/compensatory increase in presynaptic release. In addition, this study indicates that biotinylation of vesicular transporters in live cultured neurons is a valuable tool for studying presynaptic function.

Neonatal oxytocin treatment modulates oxytocin receptor, atrial natriuretic peptide, nitric oxide synthase and estrogen receptor mRNAs expression in rat heart

PubMed Central

Pournajafi-Nazarloo, Hossein; Perry, Adam; Partoo, Leila; Papademeteriou, Eros; Azizi, Feridoun; Carter, C. Sue; Cushing, Bruce S.

2007-01-01

Oxytocin (OT) has been implicated in reproductive functions, induction of maternal behavior as well as endocrine and neuroendocrine regulation of the cardiovascular system. Here we demonstrate that neonatal manipulation of OT can modulate the mRNAs expression for OT receptor (OTR), atrial natriuretic peptide (ANP), endothelial nitric oxide synthase (eNOS) and estrogen receptor alpha (ERα) in the heart. On the first day of postnatal life, female and male rats were randomly assigned to receive one of following treatments; (a) 50 µl i.p. injection of 7 µg OT, (b) 0.7 µg of OT antagonist (OTA), or (c) isotonic saline (SAL). Hearts were collected either on postnatal day 1 or day 21 (D1 or D21) and the mRNAs expression of OTR, ANP, inducible NOS (iNOS), eNOS, ERα and estrogen receptor beta (ERβ) were compared by age, treatment, and sex utilizing Real Time PCR. OT treatment significantly increased heart OTR, ANP and eNOS mRNAs expression on D1 in both males and females, ERα increased only in females. While there were significant changes in the relative expression of all types of mRNA between D1 and D21 there were no significant treatment effects observed in D21 animals. OTA treatment significantly decreased basal ANP and eNOS mRNAs expression on D1 in both sexes. The results indicate that during the early postnatal period OT can have an immediate effect on the expression OTR, ANP, eNOS, and ERα mRNAs and that these effects are mitigated by D21. Also with the exception of ERα mRNA, the effects are the same in both sexes. PMID:17537544

Radioimmunoassay of gastrin

DOE Office of Scientific and Technical Information (OSTI.GOV)

McGuigan, J.E.

1976-01-26

The use of gastrin radioimmunoassay for differentiating between the Zollinger--Ellison syndrome and common peptic ulcer is discussed. This technique makes it possible to detect the syndrome with greater certainty than measurement of gastric acid secretion. Other clinical disorders in which increased serum gastrin levels occur are pernicious anemia, chronic gastritis, achlorhydria, renal failure, and intestinal resection. (HLW)

Glucagon-like peptide-1 acutely affects renal blood flow and urinary flow rate in spontaneously hypertensive rats despite significantly reduced renal expression of GLP-1 receptors.

PubMed

Ronn, Jonas; Jensen, Elisa P; Wewer Albrechtsen, Nicolai J; Holst, Jens Juul; Sorensen, Charlotte M

2017-12-01

Glucagon-like peptide-1 (GLP-1) is an incretin hormone increasing postprandial insulin release. GLP-1 also induces diuresis and natriuresis in humans and rodents. The GLP-1 receptor is extensively expressed in the renal vascular tree in normotensive rats where acute GLP-1 treatment leads to increased mean arterial pressure (MAP) and increased renal blood flow (RBF). In hypertensive animal models, GLP-1 has been reported both to increase and decrease MAP. The aim of this study was to examine expression of renal GLP-1 receptors in spontaneously hypertensive rats (SHR) and to assess the effect of acute intrarenal infusion of GLP-1. We hypothesized that GLP-1 would increase diuresis and natriuresis and reduce MAP in SHR. Immunohistochemical staining and in situ hybridization for the GLP-1 receptor were used to localize GLP-1 receptors in the kidney. Sevoflurane-anesthetized normotensive Sprague-Dawley rats and SHR received a 20 min intrarenal infusion of GLP-1 and changes in MAP, RBF, heart rate, dieresis, and natriuresis were measured. The vasodilatory effect of GLP-1 was assessed in isolated interlobar arteries from normo- and hypertensive rats. We found no expression of GLP-1 receptors in the kidney from SHR. However, acute intrarenal infusion of GLP-1 increased MAP, RBF, dieresis, and natriuresis without affecting heart rate in both rat strains. These results suggest that the acute renal effects of GLP-1 in SHR are caused either by extrarenal GLP-1 receptors activating other mechanisms (e.g., insulin) to induce the renal changes observed or possibly by an alternative renal GLP-1 receptor. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Expression and GTP sensitivity of peptide histidine isoleucine high-affinity-binding sites in rat.

PubMed

Debaigt, Colin; Meunier, Annie-Claire; Goursaud, Stephanie; Montoni, Alicia; Pineau, Nicolas; Couvineau, Alain; Laburthe, Marc; Muller, Jean-Marc; Janet, Thierry

2006-07-01

High-affinity-binding sites for the vasoactive intestinal peptide (VIP) analogs peptide histidine/isoleucine-amide (PHI)/carboxyterminal methionine instead of isoleucine (PHM) are expressed in numerous tissues in the body but the nature of their receptors remains to be elucidated. The data presented indicate that PHI discriminated a high-affinity guanosine 5'-triphosphate (GTP)-insensitive-binding subtype that represented the totality of the PHI-binding sites in newborn rat tissues but was differentially expressed in adult animals. The GTP-insensitive PHI/PHM-binding sites were also observed in CHO cells over expressing the VPAC2 but not the VPAC1 VIP receptor.

Characterization of bombesin receptors in peripheral contractile organs.

PubMed Central

Rouissi, N.; Rhaleb, N. E.; Nantel, F.; Dion, S.; Drapeau, G.; Regoli, D.

1991-01-01

1 Guinea-pig and rat urinary bladders, rat stomach and the guinea-pig gall bladder, four isolated organs that show high sensitivity to bombesin, were used to characterize bombesin receptors in peripheral organs. 2 The order of potency of agonists was determined with several naturally occurring peptides of the bombesin series, namely bombesin (BBS), litorin (Lit), neuromedin B (NMB), the gastrin-releasing peptide (GRP 18-27), neuromedin C (NMC) and with some bombesin fragments. It was found that bombesin, neuromedin C, litorin and two bombesin fragments, BBS (6-14) and AcBBS (6-14) had similar activities in the four preparations, while neuromedin B and [Phe6]-neuromedin C were more active on the rat urinary bladder than on the other tissues. 3 The order of potency of agonists determined in the rat urinary bladder was as follows: BBS = NMB greater than Lit greater than NMC greater than [Phe6]NMC = GRP and it was found to be different from that observed in the other preparations: BBS greater than GRP = Lit greater than or equal to NMC much greater than NMB greater than [Phe6]NMC, suggesting the existence of two different bombesin receptors, BBS1 and BBS2. 4 This interpretation was convalidated by the finding that bombesin antagonists, namely Ac.GRP(20-26)OCH3 and Ac.GRP(20-26)OC2H5 reduced or blocked the effects of bombesin-related peptides on BBS2 receptor systems while being completely inactive on the rat urinary bladder (BBS1 system). PMID:1652341

Characterization of Gonadotrope Secretoproteome Identifies Neurosecretory Protein VGF-derived Peptide Suppression of Follicle-stimulating Hormone Gene Expression.

PubMed

Choi, Soon Gang; Wang, Qian; Jia, Jingjing; Chikina, Maria; Pincas, Hanna; Dolios, Georgia; Sasaki, Kazuki; Wang, Rong; Minamino, Naoto; Salton, Stephen R J; Sealfon, Stuart C

2016-09-30

Reproductive function is controlled by the pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH), which regulates the expression of the gonadotropins luteinizing hormone and FSH in pituitary gonadotropes. Paradoxically, Fshb gene expression is maximally induced at lower frequency GnRH pulses, which provide a very low average concentration of GnRH stimulation. We studied the role of secreted factors in modulating gonadotropin gene expression. Inhibition of secretion specifically disrupted gonadotropin subunit gene regulation but left early gene induction intact. We characterized the gonadotrope secretoproteome and global mRNA expression at baseline and after Gα s knockdown, which has been found to increase Fshb gene expression (1). We identified 1077 secreted proteins or peptides, 19 of which showed mRNA regulation by GnRH or/and Gα s knockdown. Among several novel secreted factors implicated in Fshb gene regulation, we focused on the neurosecretory protein VGF. Vgf mRNA, whose gene has been implicated in fertility (2), exhibited high induction by GnRH and depended on Gα s In contrast with Fshb induction, Vgf induction occurred preferentially at high GnRH pulse frequency. We hypothesized that a VGF-derived peptide might regulate Fshb gene induction. siRNA knockdown or extracellular immunoneutralization of VGF augmented Fshb mRNA induction by GnRH. GnRH stimulated the secretion of the VGF-derived peptide NERP1. NERP1 caused a concentration-dependent decrease in Fshb gene induction. These findings implicate a VGF-derived peptide in selective regulation of the Fshb gene. Our results support the concept that signaling specificity from the cell membrane GnRH receptor to the nuclear Fshb gene involves integration of intracellular signaling and exosignaling regulatory motifs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The in vivo disposition and in vitro transmembrane transport of two model radiometabolites of DOTA-conjugated receptor-specific peptides labelled with (177) Lu.

PubMed

Volková, Marie; Mandíková, Jana; Bárta, Pavel; Navrátilová, Lucie; Lázníčková, Alice; Trejtnar, František

2015-01-01

In vivo metabolism of the radiolabelled receptor-specific peptides has been described; however, information regarding the pharmacokinetic behaviour of the degradation products within the body is very scarce. The present study was designed to obtain new knowledge on the disposition and elimination of low-molecular radiometabolites of receptor-specific peptides in the organism and to reveal the potential involvement of selected membrane transport mechanisms in the cellular uptake of radiometabolites, especially in the kidney. The study compared pharmacokinetics of two radiometabolites: a final metabolite of somatostatin analogues, (177)Lu-DOTA-DPhe, and a tripeptide metabolite of (177)Lu-DOTA-minigastrin 11, (177)Lu-DOTA-DGlu-Ala-Tyr. Their pharmacokinetics was compared with that of respective parent (177)Lu-radiopeptide. Both radiometabolites exhibited relative rapid clearing from most body tissues in rats in vivo along with predominant renal excretion. The long-term renal retention of the smaller radiometabolite (177)Lu-DOTA-DPhe was lower than that of (177)Lu-DOTA-DGlu-Ala-Tyr. An uptake of (177)Lu-DOTA-DPhe by human renal influx transporter organic cation transporter 2 was found in vitro in a cellular model. The study brings the first experimental data on the in vivo pharmacokinetics of radiometabolites of receptor-specific somatostatin and gastrin analogues. The found results may indicate a negative correlation between the degree of decomposition of the parent peptide chain and the renal retention of the metabolite. Copyright © 2015 John Wiley & Sons, Ltd.

Ghrelin-Related Peptides Exert Protective Effects in the Cerebral Circulation of Male Mice Through a Nonclassical Ghrelin Receptor(s)

PubMed Central

Ku, Jacqueline M.; Andrews, Zane B.; Barsby, Tom; Reichenbach, Alex; Lemus, Moyra B.; Drummond, Grant R.; Sleeman, Mark W.; Spencer, Sarah J.; Sobey, Christopher G.

2015-01-01

The ghrelin-related peptides, acylated ghrelin, des-acylated ghrelin, and obestatin, are novel gastrointestinal hormones. We firstly investigated whether the ghrelin gene, ghrelin O-acyltransferase, and the ghrelin receptor (GH secretagogue receptor 1a [GHSR1a]) are expressed in mouse cerebral arteries. Secondly, we assessed the cerebrovascular actions of ghrelin-related peptides by examining their effects on vasodilator nitric oxide (NO) and superoxide production. Using RT-PCR, we found the ghrelin gene and ghrelin O-acyltransferase to be expressed at negligible levels in cerebral arteries from male wild-type mice. mRNA expression of GHSR1a was also found to be low in cerebral arteries, and GHSR protein was undetectable in GHSR-enhanced green fluorescent protein mice. We next found that exogenous acylated ghrelin had no effect on the tone of perfused cerebral arteries or superoxide production. By contrast, exogenous des-acylated ghrelin or obestatin elicited powerful vasodilator responses (EC50 < 10 pmol/L) that were abolished by the NO synthase inhibitor Nω-nitro-L-arginine methyl ester. Furthermore, exogenous des-acylated ghrelin suppressed superoxide production in cerebral arteries. Consistent with our GHSR expression data, vasodilator effects of des-acylated ghrelin or obestatin were sustained in the presence of YIL-781 (GHSR1a antagonist) and in arteries from Ghsr-deficient mice. Using ghrelin-deficient (Ghrl−/−) mice, we also found that endogenous production of ghrelin-related peptides regulates NO bioactivity and superoxide levels in the cerebral circulation. Specifically, we show that NO bioactivity was markedly reduced in Ghrl−/− vs wild-type mice, and superoxide levels were elevated. These findings reveal protective actions of exogenous and endogenous ghrelin-related peptides in the cerebral circulation and show the existence of a novel ghrelin receptor(s) in the cerebral endothelium. PMID:25322462

Peptide receptor targeting in cancer: the somatostatin paradigm.

PubMed

Barbieri, Federica; Bajetto, Adriana; Pattarozzi, Alessandra; Gatti, Monica; Würth, Roberto; Thellung, Stefano; Corsaro, Alessandro; Villa, Valentina; Nizzari, Mario; Florio, Tullio

2013-01-01

Peptide receptors involved in pathophysiological processes represent promising therapeutic targets. Neuropeptide somatostatin (SST) is produced by specialized cells in a large number of human organs and tissues. SST primarily acts as inhibitor of endocrine and exocrine secretion via the activation of five G-protein-coupled receptors, named sst1-5, while in central nervous system, SST acts as a neurotransmitter/neuromodulator, regulating locomotory and cognitive functions. Critical points of SST/SST receptor biology, such as signaling pathways of individual receptor subtypes, homo- and heterodimerization, trafficking, and cross-talk with growth factor receptors, have been extensively studied, although functions associated with several pathological conditions, including cancer, are still not completely unraveled. Importantly, SST exerts antiproliferative and antiangiogenic effects on cancer cells in vitro, and on experimental tumors in vivo. Moreover, SST agonists are clinically effective as antitumor agents for pituitary adenomas and gastro-pancreatic neuroendocrine tumors. However, SST receptors being expressed by tumor cells of various tumor histotypes, their pharmacological use is potentially extendible to other cancer types, although to date no significant results have been obtained. In this paper the most recent findings on the expression and functional roles of SST and SST receptors in tumor cells are discussed.

Expression of CCK Receptors in Carcinoma Gallbladder and Cholelithiasis: A Pilot Study

PubMed Central

Jaiswal, Mahabir Saran Das; Goel, Sudhir K.

2015-01-01

Background: Gastrin and cholecystokinin (CCK) receptors are trophic for various gastrointestinal malignancies. Their role in gallbladder cancer has not been widely studied. Objectives: To identify expression of CCK-A and CCK-B receptors in the tissue and blood of patients suffering from carcinoma (CA) gallbladder and gallstone disease and to compare expression of CCK A and B receptors in the gall bladder tissue and blood of healthy individuals and patients of CA gallbladder, and gallstone diseases. Materials and Methods: Forty nine subjects of both genders were recruited, comprising of 22 patients of CA gall bladder, 19 cases of cholelithiasis and, 8 normal gallbladders obtained from patients operated for trauma of the biliary system or Whipple’s procedure. RNA extraction and cDNA formation for CCK-A and CCK-B receptors were carried out. Real Time PCR was performed on cDNA and threshold cycle (Ct) value of each sample was obtained and ΔCt was calculated. Chi-square test for comparing two groups and ANOVA test for comparing multiple groups were applied and if p<0.05 then Dunnett-C test was performed. Observation and Results: Both CCK-A and CCK-B receptors were expressed irrespective of its origin in all tissues and blood samples studied; be it normal, Cholelithiasis or CA gallbladder and there was no difference among them (p>0.05). Conclusion: This preliminary study showed higher expression of CCK-A receptors in patients of cholelithiasis and decreased expression of CCK-A receptors in patients of CA gallbladder as compared to normal gallbladder although it did not rise to statistical significance. PMID:26393162

Design of chimeric peptide ligands to galanin receptors and substance P receptors.

PubMed

Langel, U; Land, T; Bartfai, T

1992-06-01

Several chimeric peptides were synthesized and found to be high-affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N-terminal part of galanin and C-terminal part of substance P (SP), galanin-(1-12)-Pro-SP-(5-11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (KD(1) less than 0.1 nM and KD(2) approximately 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (KD approximately 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin-(1-29) (KD approximately 1 nM) or its N-terminal fragment galanin-(1-13) (KD approximately 1 microM), which constitutes the N-terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C-terminal SP fragment-(4-11) amide (KD = 0.4 microM), which constitutes its C-terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp2], in the galanin portion of the chimeric peptide or substitution of ([Phe7] or [Met11]-amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin- and the SP-receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and functional characterization of hemorphins VV-H-7 and LVV-H-7 as low-affinity agonists for the orphan bombesin receptor subtype 3

PubMed Central

Lammerich, Hans-Peter; Busmann, Annette; Kutzleb, Christian; Wendland, Martin; Seiler, Petra; Berger, Claudia; Eickelmann, Peter; Meyer, Markus; Forssmann, Wolf-Georg; Maronde, Erik

2003-01-01

The human orphan G-protein coupled receptor bombesin receptor subtype 3 (hBRS-3) was screened for peptide ligands by a Ca2+ mobilization assay resulting in the purification and identification of two specific ligands, the naturally occurring VV-hemorphin-7 (VV-H-7) and LVV-hemorphin-7 (LVV-H-7), from human placental tissue. These peptides were functionally characterized as full agonists with unique specificity albeit low affinity for hBRS-3 compared to other bombesin receptors. VV-H-7 and LVV-H-7 induced a dose-dependent response in hBRS-3 overexpressing CHO cells, as well as in NCI-N417 cells expressing the hBRS-3 endogenously. The affinity of VV-H-7 was higher in NCI-N417 cells compared to overexpressing CHO cells. In detail, the EC50 values were 45±15 μM for VV-H-7 and 183±60 μM for LVV-H-7 in CHO cells, and 19±6 μM for VV-H-7 and 38±18 μM for LVV-H-7 in NCI-N417 cells. Other hemorphins had no effect. Gastrin-releasing peptide (GRP) and neuromedin B (NMB) showed similar EC50 values of 13–20 μM (GRP) and of 1–2 μM (NMB) on both cell lines. Structure-function analysis revealed that both the N-terminal valine and the C-terminal phenylalanine residues of VV-H-7 are critical for the ligand-receptor interaction. Endogenous hBRS-3 in NCI-N417 activated by VV-H-7 couples to phospholipase C resulting in changes of intracellular calcium, which is initially released from an inositol trisphosphate (IP3)-sensitive store followed by a capacitive calcium entry from extracellular space. VV-H-7-induced hBRS-3 activation led to phosphorylation of p42/p44-MAP kinase in NCI-N417 cells, but did not stimulate cell proliferation. In contrast, phosphorylation of focal adhesion kinase (p125FAK) was not observed. PMID:12721098

Expression and potential role of the peptide orexin-A in prostate cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valiante, Salvatore; Liguori, Giovanna; Tafuri, Simona

The peptides orexin-A and orexin-B and their G protein-coupled OX1 and OX2 receptors are involved in multiple physiological processes in the central nervous system and peripheral organs. Altered expression or signaling dysregulation of orexins and their receptors have been associated with a wide range of human diseases including narcolepsy, obesity, drug addiction, and cancer. Although orexin-A, its precursor molecule prepro-orexin and OX1 receptor have been detected in the human normal and hyperplastic prostate tissues, their expression and function in the prostate cancer (PCa) remains to be addressed. Here, we demonstrate for the first time the immunohistochemical localization of orexin-A inmore » human PCa specimens, and the expression of prepro-orexin and OX1 receptor at both protein and mRNA levels in these tissues. Orexin-A administration to the human androgen-dependent prostate carcinoma cells LNCaP up-regulates OX1 receptor expression resulting in a decrease of cell survival. Noteworthy, nanomolar concentrations of the peptide counteract the testosterone-induced nuclear translocation of the androgen receptor in the cells: the orexin-A action is prevented by the addition of the OX1 receptor antagonist SB-408124 to the test system. These findings indicate that orexin-A/OX1 receptor interaction interferes with the activity of the androgen receptor which regulates PCa onset and progression, thus suggesting that orexin-A and its receptor might represent novel therapeutic targets to challenge this aggressive cancer. - Highlights: • Orexin-A and OX1 receptor are present in human cancer prostate tissues. • Orexin-A up-regulates OX1 receptor expression in LNCaP cells. • Orexin-A inhibits testosterone-induced nuclear translocation of androgen receptor.« less

Milk bioactive peptides and beta-casomorphins induce mucus release in rat jejunum.

PubMed

Trompette, Aurélien; Claustre, Jean; Caillon, Fabienne; Jourdan, Gérard; Chayvialle, Jean Alain; Plaisancié, Pascale

2003-11-01

Intestinal mucus is critically involved in the protection of the mucosa. An enzymatic casein hydrolysate and beta-casomorphin-7, a mu-opioid peptide generated in the intestine during bovine casein digestion, markedly induce mucus discharge. Because shorter mu-opioid peptides have been described, the effects of the opioid peptides in casein, beta-casomorphin-7, -6, -4, -4NH2 and -3, and of opioid neuropeptides met-enkephalin, dynorphin A and (D-Ala2,N-Me-Phe4,glycinol5)enkephalin (DAMGO) on intestinal mucus secretion were investigated. The experiments were conducted with isolated perfused rat jejunum. Mucus secretion under the influence of beta-casomorphins and opioid neuropeptides administered intraluminally or intra-arterially was evaluated using an ELISA for rat intestinal mucus. Luminal administration of beta-casomorphin-7 (1.2 x 10(-4) mol/L) provoked a mucus discharge (500% of controls) that was inhibited by naloxone, a specific opiate receptor antagonist. Luminal beta-casomorphin-6, -4 and -4NH2 did not modify basal mucus secretion, whereas intra-arterial administration of beta-casomorphin-4 (1.2 x 10(-6) mol/L) induced a mucus discharge. In contrast, intra-arterial administration of the nonopioid peptide beta-casomorphin-3 did not release mucus. Among the opioid neuropeptides, intra-arterial infusion of Met-enkephalin or dynorphin-A did not provoke mucus secretion. In contrast, beta-endorphin (1.2 x 10(-8) to 1.2 x 10(-6) mol/L) induced a dose-dependent release of mucus (maximal response at 500% of controls). DAMGO (1.2 x 10(-6) mol/L), a mu-receptor agonist, also evoked a potent mucus discharge. Our findings suggest that mu-opioid neuropeptides, as well as beta-casomorphins after absorption, modulate intestinal mucus discharge. Milk opioid-derived peptides may thus be involved in defense against noxious agents and could have dietary and health applications.

Short-term effects of beta-amyloid25-35 peptide aggregates on transmitter release in neuromuscular synapses.

PubMed

Garcia, Neus; Santafé, Manel M; Tomàs, Marta; Lanuza, Maria A; Tomàs, Josep

2008-03-01

The beta-amyloid (AB) peptide25-35 contains the functional domain of the AB precursor protein that is both required for neurotrophic effects in normal neural tissues and is involved in the neurotoxic effects in Alzheimer disease. We demonstrated the presence of the amyloid precursor protein/AB peptide in intramuscular axons, presynaptic motor nerve terminals, terminal and myelinating Schwann cells, and the postsynaptic and subsarcolemmal region in the Levator auris longus muscle of adult rats by immunocytochemistry. Using intracellular recording, we investigated possible short-term functional effects of the AB fragment (0.1-10 micromol/L) on acetylcholine release in adult and newborn motor end plates. We found no change in evoked, spontaneous transmitter release or resting membrane potential of the muscle cells. A previous block of the presynaptic muscarinic receptor subtypes and a previous block or stimulation of protein kinase C revealed no masked effect of the peptide on the regulation of transmitter release. The aggregated form of AB peptide25-35, however, interfered acutely with acetylcholine release (quantal content reduction) when synaptic activity was maintained by electric stimulation. The possible relevance of this inhibition of neurotransmission by AB peptide25-35 to the pathogenesis of Alzheimer remains to be determined.

The effect of water deprivation on the expression of atrial natriuretic peptide and its receptors in the spinifex hopping mouse, Notomys alexis.

PubMed

Heimeier, Rachel A; Davis, Belinda J; Donald, John A

2002-08-01

This study investigated the mRNA expression of the atrial natriuretic peptide (ANP) system (peptide and receptors) during water deprivation in the spinifex hopping mouse, Notomys alexis, a native of central and western Australia that is well adapted to survive in arid environments. Initially, ANP, NPR-A and NPR-C cDNAs (partial for receptors) were cloned and sequenced, and were shown to have high homology with those of rat and mouse. Using a semi-quantitative multiplex PCR technique, the expression of cardiac ANP mRNA and renal ANP, NPR-A, and NPR-C mRNA was determined in 7- and 14-day water-deprived hopping mice, in parallel with control mice (access to water). The levels of ANP mRNA expression in the heart remained unchanged, but in the kidney ANP mRNA levels were increased in the 7-day water-deprived mice, and were significantly decreased in the 14-day water-deprived mice. NPR-A mRNA levels were significantly higher in 7-day water-deprived mice while no change for NPR-A mRNA expression was observed in 14-day water-deprived mice. No variation in NPR-C mRNA levels was observed. This study shows that water deprivation differentially affects the expression of the ANP system, and that renal ANP expression is more important than cardiac ANP in the physiological adjustment to water deprivation.

Renal C-type natriuretic peptide and natriuretic peptide receptor B mRNA expression are affected by water deprivation in the Spinifex Hopping mouse.

PubMed

Heimeier, Rachel A; Donald, John A

2003-11-01

This study investigated the effect of water deprivation on the expression of C-type natriuretic peptide (CNP) and natriuretic peptide receptor B (NPR-B) mRNA, and the ability of NPR-B to generate cGMP in the Spinifex Hopping mouse, Notomys alexis. This rodent is a native of central and western Australia that is well adapted to survive in arid environments. Initially, CNP and NPR-B cDNAs (partial for NPR-B) were cloned and sequenced, and were shown to have high homology with those of rat and mouse. RT-PCR analysis showed CNP mRNA expression in the kidney, proximal and distal colon and small intestine, whilst NPR-B mRNA expression was found in the kidney, proximal and distal colon and the atria. Using a semi-quantitative multiplex PCR technique, the expression of renal CNP and NPR-B mRNA was determined in 7- and 14-day water-deprived hopping mice, in parallel with control hopping mice (access to water). Water deprivation significantly decreased the relative levels of CNP and NPR-B mRNA expression in both the 7- and 14-day water-deprived hopping mice, when compared to control hopping mice. In contrast, the ability of CNP to stimulate cGMP production was significantly increased after 14 days of water deprivation. This study shows that alterations in the renal CNP/NPR-B system may be an important physiological adjustment when water is scarce.

Enduring, Handling-Evoked Enhancement of Hippocampal Memory Function and Glucocorticoid Receptor Expression Involves Activation of the Corticotropin-Releasing Factor Type 1 Receptor

PubMed Central

Fenoglio, Kristina A.; Brunson, Kristen L.; Avishai-Eliner, Sarit; Stone, Blake A.; Kapadia, Bhumika J.; Baram, Tallie Z.

2011-01-01

Early-life experience, including maternal care, influences hippocampus-dependent learning and memory throughout life. Handling of pups during postnatal d 2–9 (P2–9) stimulates maternal care and leads to improved memory function and stress-coping. The underlying molecular mechanisms may involve early (by P9) and enduring reduction of hypothalamic corticotropin-releasing factor (CRF) expression and subsequent (by P45) increase in hippocampal glucocorticoid receptor (GR) expression. However, whether hypothalamic CRF levels influence changes in hippocampal GR expression (and memory function), via reduced CRF receptor activation and consequent lower plasma glucocorticoid levels, is unclear. In this study we administered selective antagonist for the type 1 CRF receptor, NBI 30775, to nonhandled rats post hoc from P10–17 and examined hippocampus-dependent learning and memory later (on P50–70), using two independent paradigms, compared with naive and vehicle-treated nonhandled, and naive and antagonist-treated handled rats. Hippocampal GR and hypothalamic CRF mRNA levels and stress-induced plasma corticosterone levels were also examined. Transient, partial selective blockade of CRF1 in nonhandled rats improved memory functions on both the Morris watermaze and object recognition tests to levels significantly better than in naive and vehicle-treated controls and were indistinguishable from those in handled (naive, vehicle-treated, and antagonist-treated) rats. GR mRNA expression was increased in hippocampal CA1 and the dentate gyrus of CRF1-antagonist treated nonhandled rats to levels commensurate with those in handled cohorts. Thus, the extent of CRF1 activation, probably involving changes in hypothalamic CRF levels and release, contributes to the changes in hippocampal GR expression and learning and memory functions. PMID:15932935

Synthesis of a 68Ga-Labeled Peptoid−Peptide Hybrid for Imaging of Neurotensin Receptor Expression in Vivo

PubMed Central

2010-01-01

The neurotensin receptor subtype 1 (NTS1) represents an attractive molecular target for imaging various tumors. Positron emission tomography (PET) gained widespread importance due to its sensitivity. We combined the design of a metabolically stable neurotensin analogue with a 68Ga-radiolabeling approach. The 68Ga-labeled peptoid−peptide hybrid [68Ga]3 revealed high stability, specific tumor uptake (0.7%ID/g, 65 min p.i.), and advantageous biokinetics in vivo using HT29 tumor-bearing nude mice. Because of the ability to internalize into NTS1-expressing tumor cells, [68Ga]3 proved to be highly suitable for a reliable and practical visualization of NTS1-expressing tumors in vivo by small animal PET. PMID:24900199

Synthesis of a (68)ga-labeled peptoid-Peptide hybrid for imaging of neurotensin receptor expression in vivo.

PubMed

Maschauer, Simone; Einsiedel, Jürgen; Hocke, Carsten; Hübner, Harald; Kuwert, Torsten; Gmeiner, Peter; Prante, Olaf

2010-08-12

The neurotensin receptor subtype 1 (NTS1) represents an attractive molecular target for imaging various tumors. Positron emission tomography (PET) gained widespread importance due to its sensitivity. We combined the design of a metabolically stable neurotensin analogue with a (68)Ga-radiolabeling approach. The (68)Ga-labeled peptoid-peptide hybrid [(68)Ga]3 revealed high stability, specific tumor uptake (0.7%ID/g, 65 min p.i.), and advantageous biokinetics in vivo using HT29 tumor-bearing nude mice. Because of the ability to internalize into NTS1-expressing tumor cells, [(68)Ga]3 proved to be highly suitable for a reliable and practical visualization of NTS1-expressing tumors in vivo by small animal PET.

The three subtypes of atrial natriuretic peptide (ANP) receptors are expressed in the rat adrenal gland.

PubMed

Grandclément, B; Ronsin, B; Morel, G

1997-03-01

Atrial natriuretic peptide (ANP) actions are mediated by highly selective and specific receptors. Three subtypes have been characterized and cloned: ANP receptor-A (or GC-A), -B (or GC-B) and -C (the so-called clearance receptor). In rat adrenal gland, the mRNA for each subtype was detected using 35S-dUTP or digoxigenin-11-dUTP specific labeled probes, and in situ hybridization at light and electron microscopic levels respectively. The three subtypes were expressed the most abundantly in the zona glomerulosa. The amount of GC-A mRNA expression, quantified using macro-autoradiography and densitometry, was higher than the amounts of GC-B mRNA and ANPR-C mRNA both in zona glomerulosa and medullary of adrenal gland. At electron microscopic level, the three subtypes of ANPR were revealed in glomerulosa cells. A noticeable signal was also present in the medullary area, especially for GC-A mRNA, in adrenaline-containing chromaffin cells. No signal was detected in noradrenaline-containing chromaffin cells. The subcellular localization of the three mRNAs is similar: in the cytoplasmic matrix and in the euchromatin of the nucleus in each cell of glomerulosa, and in the same compartments of the adrenaline-containing chromaffin cells. These data indicate that the adrenal gland is an important target tissue for ANP action both in glomerulosa cells and adrenaline-containing chromaffin cells. The mRNA expression levels were different for each ANPR subtype.

Ghrelin, a novel growth hormone-releasing peptide, in the treatment of cardiopulmonary-associated cachexia.

PubMed

Nagaya, Noritoshi; Kojima, Masakazu; Kangawa, Kenji

2006-01-01

Ghrelin is a novel growth hormone (GH)-releasing peptide, isolated from the stomach, which has been identified as an endogenous ligand for GH secretagogue receptor. The discovery of ghrelin indicates that the release of GH from the pituitary might be regulated not only by hypothalamic GH-releasing hormone, but also by ghrelin derived from the stomach. This peptide also stimulates food intake and induces adiposity through GH-independent mechanisms. In addition, ghrelin acts directly on the central nervous system to decrease sympathetic nerve activity. Thus, ghrelin plays important roles for maintaining GH release and energy homeostasis. Repeated administration of ghrelin improves body composition, muscle wasting, functional capacity, and sympathetic augmentation in cachectic patients with heart failure or chronic obstructive pulmonary disease. These results suggest that ghrelin has anti-cachectic effects through GH-dependent and independent mechanisms. Thus, administration of ghrelin may be a new therapeutic strategy for the treatment of cardiopulmonary-associated cachexia.

Targeting prostate cancer cells with hybrid elastin-like polypeptide/liposome nanoparticles

PubMed Central

Zhang, Wei; Song, Yunmei; Eldi, Preethi; Guo, Xiuli; Hayball, John D; Garg, Sanjay; Albrecht, Hugo

2018-01-01

Prostate cancer cells frequently overexpress the gastrin-releasing peptide receptor, and various strategies have been applied in preclinical settings to target this receptor for the specific delivery of anticancer compounds. Recently, elastin-like polypeptide (ELP)-based self-assembling micelles with tethered GRP on the surface have been suggested to actively target prostate cancer cells. Poorly soluble chemotherapeutics such as docetaxel (DTX) can be loaded into the hydrophobic cores of ELP micelles, but only limited drug retention times have been achieved. Herein, we report the generation of hybrid ELP/liposome nanoparticles which self-assembled rapidly in response to temperature change, encapsulated DTX at high concentrations with slow release, displayed the GRP ligand on the surface, and specifically bound to GRP receptor expressing PC-3 cells as demonstrated by flow cytometry. This novel type of drug nanocarrier was successfully used to reduce cell viability of prostate cancer cells in vitro through the specific delivery of DTX. PMID:29391790

Role of Indigenous Lactobacilli in Gastrin-Mediated Acid Production in the Mouse Stomach ▿

PubMed Central

Takahashi, Hidenori; Nakano, Yasuhiro; Matsuoka, Takashi; Kumaki, Nobue; Asami, Yukio; Koga, Yasuhiro

2011-01-01

It is known that the stomach is colonized by indigenous lactobacilli in mice. The aim of this study was to examine the role of such lactobacilli in the development of the stomach. For a DNA microarray analysis, germ-free BALB/c mice were orally inoculated with 109 CFU lactobacilli, and their stomachs were excised after 10 days to extract RNA. As a result, lactobacillus-associated gnotobiotic mice showed dramatically decreased expression of the gastrin gene in comparison to germ-free mice. The mean of the log2 fold change in the gastrin gene was −4.3. Immunohistochemistry also demonstrated the number of gastrin-positive (gastrin+) cells to be significantly lower in the lactobacillus-associated gnotobiotic mice than in the germ-free mice. However, there was no significant difference in the number of somatostatin+ cells in these groups of mice. Consequently, gastric acid secretion also decreased in the mice colonized by lactobacilli. In addition, an increase in the expression of the genes related to muscle system development, such as nebulin and troponin genes, was observed in lactobacillus-associated mice. Moreover, infection of germ-free mice with Helicobacter pylori also showed the down- and upregulation of gastrin and muscle genes, respectively, in the stomach. These results thus suggested that indigenous lactobacilli in the stomach significantly affect the regulation of gastrin-mediated gastric acid secretion without affecting somatostatin secretion in mice, while H. pylori also exerts such an effect on the stomach. PMID:21803885

The effects of sex and neonatal stress on pituitary adenylate cyclase-activating peptide expression.

PubMed

Mosca, E V; Rousseau, J P; Gulemetova, R; Kinkead, R; Wilson, R J A

2015-02-01

What is the central question of this study? Does sex or neonatal stress affect the expression of pituitary adenylate cyclase-activating peptide or its receptors? What is the main finding and its importance? Neonatal-maternal separation stress has little long-lasting effect on the expression of pituitary adenylate cyclase-activating peptide or its receptors, but sex differences exist in these genes between males and females at baseline. Sex differences in classic stress hormones have been studied in depth, but pituitary adenylate cyclase-activating peptide (PACAP), recently identified as playing a critical role in the stress axes, has not. Here we studied whether baseline levels of PACAP differ between sexes in various stress-related tissues and whether neonatal-maternal separation stress has a sex-dependent effect on PACAP gene expression in stress pathways. Using quantitative RT-PCR, we found sex differences in PACAP and PACAP receptor gene expression in several respiratory and/or stress-related tissues, while neonatal-maternal separation stress did little to affect PACAP signalling in adult animals. We propose that sex differences in PACAP expression are likely to contribute to differences between males and females in responses to stress. © 2015 The Authors. Experimental Physiology © 2015 The Physiological Society.

Application of 2-chlorotrityl resin in solid phase synthesis of (Leu15)-gastrin I and unsulfated cholecystokinin octapeptide. Selective O-deprotection of tyrosine.

PubMed

Barlos, K; Gatos, D; Kapolos, S; Poulos, C; Schäfer, W; Yao, W Q

1991-12-01

The carboxyl terminal dipeptide amide, Fmoc-Asp-Phe-NH2, of gastrin and cholecystokinin (CCK) has been attached in high yield through its free side chain carboxyl group to the acid labile 2-chlorotrityl resin. The obtained peptide resin ester has been applied in the solid phase synthesis of partially protected (Leu15)-gastrin I utilising Fmoc-amino acids. Quantitative cleavage of this peptide from resin, with the t-butyl type side chain protection intact is achieved using mixtures of acetic acid/trifluoroethanol/dichloromethane. Under the same conditions complete detritylation of the tyrosine phenoxy function occurs simultaneously. Thus, the solid-phase synthesis of peptides selectively deprotected at the side chain of tyrosine is rendered possible by the use of 2-chlorotrityl resin and Fmoc-Tyr(Trt)-OH. The efficiency of this approach has been proved by the subsequent high-yield synthesis of three model peptides and the CCK-octapeptide.

Calcitonin and Amylin Receptor Peptide Interaction Mechanisms

PubMed Central

Lee, Sang-Min; Hay, Debbie L.; Pioszak, Augen A.

2016-01-01

Receptor activity-modifying proteins (RAMP1–3) determine the selectivity of the class B G protein-coupled calcitonin receptor (CTR) and the CTR-like receptor (CLR) for calcitonin (CT), amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8–37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity. PMID:26895962

Enhanced AMPA Receptor Trafficking Mediates the Anorexigenic Effect of Endogenous Glucagon-like Peptide-1 in the Paraventricular Hypothalamus.

PubMed

Liu, Ji; Conde, Kristie; Zhang, Peng; Lilascharoen, Varoth; Xu, Zihui; Lim, Byung Kook; Seeley, Randy J; Zhu, J Julius; Scott, Michael M; Pang, Zhiping P

2017-11-15

Glucagon-like Peptide 1 (GLP-1)-expressing neurons in the hindbrain send robust projections to the paraventricular nucleus of the hypothalamus (PVN), which is involved in the regulation of food intake. Here, we describe that stimulation of GLP-1 afferent fibers within the PVN is sufficient to suppress food intake independent of glutamate release. We also show that GLP-1 receptor (GLP-1R) activation augments excitatory synaptic strength in PVN corticotropin-releasing hormone (CRH) neurons, with GLP-1R activation promoting a protein kinase A (PKA)-dependent signaling cascade leading to phosphorylation of serine S845 on GluA1 AMPA receptors and their trafficking to the plasma membrane. Finally, we show that postnatal depletion of GLP-1R in the PVN increases food intake and causes obesity. This study provides a comprehensive multi-level (circuit, synaptic, and molecular) explanation of how food intake behavior and body weight are regulated by endogenous central GLP-1. VIDEO ABSTRACT. Copyright © 2017 Elsevier Inc. All rights reserved.

Enhanced AMPA receptor trafficking mediates the anorexigenic effect of endogenous glucagon like peptide-1 in the paraventricular hypothalamus

PubMed Central

Liu, Ji; Conde, Kristie; Zhang, Peng; Lilascharoen, Varoth; Xu, Zihui; Lim, Byung Kook; Seeley, Randy J.; Zhu, Julius J.; Scott, Michael M.; Pang, Zhiping P.

2017-01-01

SUMMARY Glucagon Like Peptide 1 (GLP-1)-expressing neurons in the hindbrain send robust projections to the paraventricular nucleus of the hypothalamus (PVN), which is involved in the regulation of food intake. Here, we describe that stimulation of GLP-1 afferent fibers within the PVN is sufficient to suppress food intake independent of glutamate release. We also show that GLP-1 receptor (GLP-1R) activation augments excitatory synaptic strength in PVN corticotropin-releasing hormone (CRH) neurons, with GLP-1R activation promoting a protein kinase A (PKA) dependent signaling cascade leading to phosphorylation of serine S845 on GluA1 AMPA receptors and their trafficking to the plasma membrane. Finally, we show that postnatal depletion of GLP-1R in the PVN increases food intake and causes obesity. This study provides a comprehensive multi-level (circuit, synaptic, and molecular) explanation of how food intake behavior and body weight are regulated by endogenous central GLP-1. PMID:29056294

Effects of corticotropin-releasing hormone and its antagonist on the gene expression of gonadotrophin-releasing hormone (GnRH) and GnRH receptor in the hypothalamus and anterior pituitary gland of follicular phase ewes.

PubMed

Ciechanowska, Magdalena; Łapot, Magdalena; Malewski, Tadeusz; Mateusiak, Krystyna; Misztal, Tomasz; Przekop, Franciszek

2011-01-01

There is no information in the literature regarding the effect of corticotropin-releasing hormone (CRH) on genes encoding gonadotrophin-releasing hormone (GnRH) and the GnRH receptor (GnRHR) in the hypothalamus or on GnRHR gene expression in the pituitary gland in vivo. Thus, the aim of the present study was to investigate, in follicular phase ewes, the effects of prolonged, intermittent infusion of small doses of CRH or its antagonist (α-helical CRH 9-41; CRH-A) into the third cerebral ventricle on GnRH mRNA and GnRHR mRNA levels in the hypothalamo-pituitary unit and on LH secretion. Stimulation or inhibition of CRH receptors significantly decreased or increased GnRH gene expression in the hypothalamus, respectively, and led to different responses in GnRHR gene expression in discrete hypothalamic areas. For example, CRH increased GnRHR gene expression in the preoptic area, but decreased it in the hypothalamus/stalk median eminence and in the anterior pituitary gland. In addition, CRH decreased LH secretion. Blockade of CRH receptors had the opposite effect on GnRHR gene expression. The results suggest that activation of CRH receptors in the hypothalamus of follicular phase ewes can modulate the biosynthesis and release of GnRH through complex changes in the expression of GnRH and GnRHR genes in the hypothalamo-anterior pituitary unit. © CSIRO 2011 Open Access

Relaxin, its receptor (RXFP1), and insulin-like peptide 4 expression through gestation and in placenta accreta.

PubMed

Goh, William; Yamamoto, Sandra Y; Thompson, Karen S; Bryant-Greenwood, Gillian D

2013-08-01

This study was designed to show whether placental relaxin (RLN), its receptor (RXFP1), or insulin-like peptide 4 (INSL4) might have altered expression in patients with placenta accreta. The baseline expression of their genes through gestation (n = 34) was quantitated in the placental basal plate (BP) and villous trophoblast (TR), and compared to their expression in placenta accreta (n = 6). The proteins were also immunolocalized and quantitated in the accreta tissues. The messenger RNAs (mRNAs) of matrix metalloproteinase 9, -2, and tissue inhibitors of matrix metalloproteinase (TIMP)-1 were also measured. Results demonstrated that the BP and TR expressed low levels of RLN/RXFP1 and INSL4 through gestation. In accreta, increased RLN gene and protein in BP were associated with antepartum bleeding whereas INSL4 expression decreased throughout the TR. There were no changes in mRNAs for MMPs, but TIMP-1 was increased only in the invasive TR.

Relaxin, Its Receptor (RXFP1), and Insulin-Like Peptide 4 Expression Through Gestation and in Placenta Accreta

PubMed Central

Yamamoto, Sandra Y.; Thompson, Karen S.; Bryant-Greenwood, Gillian D.

2013-01-01

This study was designed to show whether placental relaxin (RLN), its receptor (RXFP1), or insulin-like peptide 4 (INSL4) might have altered expression in patients with placenta accreta. The baseline expression of their genes through gestation (n = 34) was quantitated in the placental basal plate (BP) and villous trophoblast (TR), and compared to their expression in placenta accreta (n = 6). The proteins were also immunolocalized and quantitated in the accreta tissues. The messenger RNAs (mRNAs) of matrix metalloproteinase 9, -2, and tissue inhibitors of matrix metalloproteinase (TIMP)-1 were also measured. Results demonstrated that the BP and TR expressed low levels of RLN/RXFP1 and INSL4 through gestation. In accreta, increased RLN gene and protein in BP were associated with antepartum bleeding whereas INSL4 expression decreased throughout the TR. There were no changes in mRNAs for MMPs, but TIMP-1 was increased only in the invasive TR. PMID:23302396

G cells and gastrin in chronic alcohol-treated rats.

PubMed

Todorović, Vera; Koko, Vesna; Budec, Mirela; Mićić, Mileva; Micev, Marjan; Pavlović, Mirjana; Vignjević, Sanja; Drndarević, Neda; Mitrović, Olivera

2008-02-01

Numerous reports have described gastric mucosal injury in rats treated with high ethanol concentrations. However, to the best of our knowledge, ultrastructural characteristics of G cells and antral gastrin levels have not been previously reported, either in rats that chronically consumed alcohol or in human alcoholics. The goal of this study was to examine the effect of ethanol consumption (8.5 g/kg) over a 4-month period, under controlled nutritional conditions, on antral and plasma levels of gastrin, ultrastructure of G cells, morphometric characteristics of G cells by stereological methods, and analysis of endocrine cells in the gastric mucosa by immunohistochemistry. The chronic alcohol consumption resulted in a nonsignificant decrease in gastrin plasma levels and unchanged antral gastrin concentrations. A slightly damaged glandular portion of the gastric mucosa and dilatation of small blood vessels detected by histological analysis, suggests that ethanol has a toxic effect on the mucosal surface. Chronic alcohol treatment significantly decreased the number of antral G cells per unit area, and increased their cellular, nuclear, and cytoplasmatic profile areas. In addition, the volume density and diameter of G-cell granules, predominantly the pale and lucent types, were increased, indicating inhibition of gastrin release. Ethanol treatment also decreased the number of gastric somatostatin-, serotonin-, and histamine-immunoreactive cells, except the somatostatin cells in the pyloric mucosa, as well as both G: D: enterochromaffin cells (EC) cell ratios in the antrum and D: ECL cell ratios in the fundus. These results indicate that the change of morphometric parameters in G cells may be related to cellular dysfunction. Our findings also suggest that regulation of G-cell secretion was not mediated by locally produced somatostatin in ethanol-consuming rats, but may involve gastric luminal content and/or neurotransmitters of gastric nerve fibers.

Novel fluorescent contrast agents for optical imaging of in vivo tumors based on a receptor-targeted dye-peptide conjugate platform

NASA Astrophysics Data System (ADS)

Bugaj, Joseph E.; Achilefu, Samuel I.; Dorshow, Richard B.; Rajagopalan, Raghavan

2001-04-01

We have designed, synthesized, and evaluated the efficacy of novel dye-peptide conjugates that are receptor specific. Contrary to the traditional approach of conjugating dyes to large proteins and antibodies, we used small peptide-dye conjugates that target over-expressed receptors on tumors. Despite the fact that the peptide and the dye probe have similar molecular mass, our results demonstrate that the affinity of the peptide for its receptor and the dye fluorescence properties are both retained. The use of small peptides has several advantages over large biomolecules, including ease of synthesis of a variety of compounds for potential combinatorial screening of new targets, reproducibility of high purity compounds, diffusiveness to solid tumors, and the ability to incorporate a variety of functional groups that modify the pharmacokinetics of the peptide-dye conjugates. The efficacy of these new fluorescent optical contrast agents was evaluated in vivo in well-characterized rat tumor lines expressing somatostatin (sst2) and bombesin receptors. A simple continuous wave optical imaging system was employed. The resulting optical images clearly show that successful specific tumor targeting was achieved. Thus, we have demonstrated that small peptide- dye conjugates are effective as contrast agents for optical imaging of tumors.

Functional expression of cysteinyl leukotriene receptors on human platelets.

PubMed

Hasegawa, Shunji; Ichiyama, Takashi; Hashimoto, Kunio; Suzuki, Yasuo; Hirano, Reiji; Fukano, Reiji; Furukawa, Susumu

2010-01-01

Normal peripheral blood leukocytes, such as basophils, eosinophils, B lymphocytes and monocytes/macrophages, have a cysteinyl leukotriene 1 (CysLT1) receptor, while the cysteinyl leukotriene 2 (CysLT2) receptor is expressed in cardiac Purkinje cells, endothelium, brain and leukocytes. However, it is unknown whether or not platelets express the CysLT1 or CysLT2 receptor. In this study we identify and characterize the biological function of the CysLT receptor of human platelets. We determined the CysLT1 or CysLT2 receptor mRNA expression in normal human platelets by RT-PCR and determined protein expression by Western blotting and flow cytometry. Moreover, we examined the effect of cysteinyl leukotrienes (CysLTs) in platelets on the induction of RANTES (Regulated on Activation, Normal T Expressed, and presumably Secreted). We also investigated whether the CysLT1 receptor antagonist pranlukast inhibits CysLT-induced RANTES release. In conclusion, we showed the functional expression of CysLT receptors on human platelets and demonstrated that CysLTs induced the release of significant amounts of RANTES, which suggests a novel role for human platelets in CysLT-mediated allergic inflammation.

Serum Gastrin in Chronic Gastritis

PubMed Central

Korman, M. G.; Strickland, R. G.; Hansky, J.

1971-01-01

Fasting gastrin levels in serum were measured in 49 patients with different types of chronic gastritis and in matched controls. In 15 patients with established pernicious anaemia the mean (± S.E. of mean) level of gastrin was greatly raised (699 ± 99 pg/ml). In 17 patients with chronic atrophic gastritis, seropositive for parietal cell antibody but with adequate vitamin-B12 absorption, the level was also raised (476 ± 74 pg/ml). By contrast, in “simple” atrophic gastritis seronegative for parietal cell antibody the gastrin levels were significantly lower for both diffuse atrophic gastritis (129 ± 31 pg/ml) and multifocal gastritis (14 ± 4 pg/ml). These levels were similar to those in the controls (46 ± 7 pg/ml). The mechanism of the raised gastrin levels remains uncertain, but neither achlorhydria nor in vivo action of the parietal cell antibody wholly accounted for the hypergastrinaemia. We conclude that hypergastrinaemia is characteristic of gastritis associated with autoimmune reactions to gastric antigens and pernicious anaemia and that a raised serum gastrin is a useful marker of the type of gastritis that tends to progress to the gastric lesion of pernicious anaemia. The findings suggest that this type of gastritis is an essentially different disease from “simple” atrophic gastritis, and the differences in gastrin levels may be due to sparing of the antral mucosa in the autoimmune type but not in “simple” gastritis. PMID:5550864

A second trigeminal CGRP receptor: function and expression of the AMY1 receptor

PubMed Central

Walker, Christopher S; Eftekhari, Sajedeh; Bower, Rebekah L; Wilderman, Andrea; Insel, Paul A; Edvinsson, Lars; Waldvogel, Henry J; Jamaluddin, Muhammad A; Russo, Andrew F; Hay, Debbie L

2015-01-01

Objective The trigeminovascular system plays a central role in migraine, a condition in need of new treatments. The neuropeptide, calcitonin gene-related peptide (CGRP), is proposed as causative in migraine and is the subject of intensive drug discovery efforts. This study explores the expression and functionality of two CGRP receptor candidates in the sensory trigeminal system. Methods Receptor expression was determined using Taqman G protein-coupled receptor arrays and immunohistochemistry in trigeminal ganglia (TG) and the spinal trigeminal complex of the brainstem in rat and human. Receptor pharmacology was quantified using sensitive signaling assays in primary rat TG neurons. Results mRNA and histological expression analysis in rat and human samples revealed the presence of two CGRP-responsive receptors (AMY1: calcitonin receptor/receptor activity-modifying protein 1 [RAMP1]) and the CGRP receptor (calcitonin receptor-like receptor/RAMP1). In support of this finding, quantification of agonist and antagonist potencies revealed a dual population of functional CGRP-responsive receptors in primary rat TG neurons. Interpretation The unexpected presence of a functional non-canonical CGRP receptor (AMY1) at neural sites important for craniofacial pain has important implications for targeting the CGRP axis in migraine. PMID:26125036

Calcitonin and Amylin Receptor Peptide Interaction Mechanisms: INSIGHTS INTO PEPTIDE-BINDING MODES AND ALLOSTERIC MODULATION OF THE CALCITONIN RECEPTOR BY RECEPTOR ACTIVITY-MODIFYING PROTEINS.

PubMed

Lee, Sang-Min; Hay, Debbie L; Pioszak, Augen A

2016-04-15

Receptor activity-modifying proteins (RAMP1-3) determine the selectivity of the class B G protein-coupled calcitonin receptor (CTR) and the CTR-like receptor (CLR) for calcitonin (CT), amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8-37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Trafficking of presynaptic AMPA receptors mediating neurotransmitter release: neuronal selectivity and relationships with sensitivity to cyclothiazide.

PubMed

Pittaluga, Anna; Feligioni, Marco; Longordo, Fabio; Luccini, Elisa; Raiteri, Maurizio

2006-03-01

Postsynaptic glutamate AMPA receptors (AMPARs) can recycle between plasma membrane and intracellular pools. In contrast, trafficking of presynaptic AMPARs has not been investigated. AMPAR surface expression involves interactions between the GluR2 carboxy tail and various proteins including glutamate receptor-interacting protein (GRIP), AMPA receptor-binding protein (ABP), protein interacting with C kinase 1 (PICK1), N-ethyl-maleimide-sensitive fusion protein (NSF). Here, peptides known to selectively block the above interactions were entrapped into synaptosomes to study the effects on the AMPA-evoked release of [3H]noradrenaline ([3H]NA) and [3H]acetylcholine ([3H]ACh) from rat hippocampal and cortical synaptosomes, respectively. Internalization of pep2-SVKI to prevent GluR2-GRIP/ABP/PICK1 interactions potentiated the AMPA-evoked release of [3H]NA but left unmodified that of [3H]ACh. Similar potentiation was caused by pep2-AVKI, the blocker of GluR2-PICK1 interaction. Conversely, a decrease in the AMPA-evoked release of [3H]NA, but not of [3H]ACh, was caused by pep2m, a selective blocker of the GluR2-NSF interaction. In the presence of pep2-SVKI the presynaptic AMPARs on noradrenergic terminals lost sensitivity to cyclothiazide. AMPARs releasing [3H]ACh, but not those releasing [3H]NA, were sensitive to spermine, suggesting that they are GluR2-lacking AMPARs. To conclude: (i) release-regulating presynaptic AMPARs constitutively cycle in isolated nerve terminals; (ii) the process exhibits neuronal selectivity; (iii) AMPAR trafficking and desensitization may be interrelated.

Monitoring multiple myeloma by idiotype-specific peptide binders of tumor-derived exosomes.

PubMed

Iaccino, Enrico; Mimmi, Selena; Dattilo, Vincenzo; Marino, Fabiola; Candeloro, Patrizio; Di Loria, Antonio; Marimpietri, Danilo; Pisano, Antonio; Albano, Francesco; Vecchio, Eleonora; Ceglia, Simona; Golino, Gaetanina; Lupia, Antonio; Fiume, Giuseppe; Quinto, Ileana; Scala, Giuseppe

2017-10-13

Tumor-derived exosomes (TDEs) play a pivotal role in tumor establishment and progression, and are emerging biomarkers for tumor diagnosis in personalized medicine. To date, there is a lack of efficient technology platforms for exosome isolation and characterization. Multiple myeloma (MM) is an incurable B-cell malignancy due to the rapid development of drug-resistance. MM-released exosomes express the immunoglobulin B-cell receptor (Ig-BCR) of the tumor B-cells, which can be targeted by Idiotype-binding peptides (Id-peptides). In this study, we analyzed the production of MM-released exosomes in the murine 5T33MM multiple myeloma model as biomarkers of tumor growth. To this end, we selected Id-peptides by screening a phage display library using as bait the Ig-BCR expressed by 5T33MM cells. By FACS, the FITC-conjugated Id-peptides detected the MM-released exosomes in the serum of 5T33MM-engrafted mice, levels of which are correlated with tumor progression at an earlier time point compared to serum paraprotein. These results indicate that Id-peptide-based recognition of MM-released exosomes may represent a very sensitive diagnostic approach for clinical evaluation of disease progression.

On the G-Protein-Coupled Receptor Heteromers and Their Allosteric Receptor-Receptor Interactions in the Central Nervous System: Focus on Their Role in Pain Modulation

PubMed Central

Borroto-Escuela, Dasiel O.; Romero-Fernandez, Wilber; Rivera, Alicia; Van Craenenbroeck, Kathleen; Tarakanov, Alexander O.; Agnati, Luigi F.; Fuxe, Kjell

2013-01-01

The modulatory role of allosteric receptor-receptor interactions in the pain pathways of the Central Nervous System and the peripheral nociceptors has become of increasing interest. As integrators of nociceptive and antinociceptive wiring and volume transmission signals, with a major role for the opioid receptor heteromers, they likely have an important role in the pain circuits and may be involved in acupuncture. The delta opioid receptor (DOR) exerts an antagonistic allosteric influence on the mu opioid receptor (MOR) function in a MOR-DOR heteromer. This heteromer contributes to morphine-induced tolerance and dependence, since it becomes abundant and develops a reduced G-protein-coupling with reduced signaling mainly operating via β-arrestin2 upon chronic morphine treatment. A DOR antagonist causes a return of the Gi/o binding and coupling to the heteromer and the biological actions of morphine. The gender- and ovarian steroid-dependent recruitment of spinal cord MOR/kappa opioid receptor (KOR) heterodimers enhances antinociceptive functions and if impaired could contribute to chronic pain states in women. MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, mediating morphine induced itch. Other mechanism for the antinociceptive actions of acupuncture along meridians may be that it enhances the cross-desensitization of the TRPA1 (chemical nociceptor)-TRPV1 (capsaicin receptor) heteromeric channel complexes within the nociceptor terminals located along these meridians. Selective ionotropic cannabinoids may also produce cross-desensitization of the TRPA1-TRPV1 heteromeric nociceptor channels by being negative allosteric modulators of these channels leading to antinociception and antihyperalgesia. PMID:23956775

Elastin receptor (S-gal) occupancy by elastin peptides modulates T-cell response during murine emphysema.

PubMed

Meghraoui-Kheddar, Aïda; Pierre, Alexandre; Sellami, Mehdi; Audonnet, Sandra; Lemaire, Flora; Le Naour, Richard

2017-09-01

Chronic obstructive pulmonary disease and emphysema are associated with increased elastin peptides (EP) production because of excessive breakdown of lung connective tissue. We recently reported that exposure of mice to EP elicited hallmark features of emphysema. EP effects are largely mediated through a receptor complex that includes the elastin-binding protein spliced-galactosidase (S-gal). In previous studies, we established a correlation between cytokine production and S-gal protein expression in EP-treated immune cells. In this study, we investigated the S-gal-dependent EP effects on T-helper (Th) and T-cytotoxic (Tc) responses during murine EP-triggered pulmonary inflammation. C57BL/6J mice were endotracheally instilled with the valine-glycine-valine-alanine-proline-glycine (VGVAPG) elastin peptide, and, 21 days after treatment, local and systemic T-lymphocyte phenotypes were analyzed at cytokine and transcription factor expression levels by multicolor flow cytometry. Exposure of mice to the VGVAPG peptide resulted in a significant increase in the proportion of the CD4 + and CD8 + T cells expressing the cytokines IFN-γ or IL-17a and the transcription factors T-box expressed in T cells or retinoic acid-related orphan receptor-γt (RORγt) without effects on IL-4 and Gata-binding protein 3 to DNA sequence [A/T]GATA[A/G] expression. These effects were maximized when each T-cell subpopulation was challenged ex vivo with EP, and they were inhibited in vivo when an analogous peptide antagonizing the EP/S-gal interactions was instilled together with the VGVAPG peptide. This study demonstrates that, during murine emphysema, EP-S-gal interactions contribute to a Th-1 and Th-17 proinflammatory T-cell response combined with a Tc-1 response. Our study also highlights the S-gal receptor as a putative pharmacological target to modulate such an immune response. Copyright © 2017 the American Physiological Society.

A Secreted Peptide and Its Receptors Shape the Auxin Response Pattern and Leaf Margin Morphogenesis.

PubMed

Tameshige, Toshiaki; Okamoto, Satoshi; Lee, Jin Suk; Aida, Mitsuhiro; Tasaka, Masao; Torii, Keiko U; Uchida, Naoyuki

2016-09-26

Secreted peptides mediate intercellular communication [1, 2]. Several secreted peptides in the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family regulate morphogenesis of tissues, such as stomata and inflorescences in plants [3-15]. The biological functions of other EPFL family members remain unknown. Here, we show that the EPFL2 gene is required for growth of leaf teeth. EPFL2 peptide physically interacts with ERECTA (ER) family receptor-kinases and, accordingly, the attenuation of ER family activities leads to formation of toothless leaves. During the tooth growth process, responses to the phytohormone auxin are maintained at tips of the teeth to promote their growth [16-19]. In the growing tooth tip of epfl2 and multiple er family mutants, the auxin response becomes broader. Conversely, overexpression of EPFL2 diminishes the auxin response, indicating that the EPFL2 signal restricts the auxin response to the tooth tip. Interestingly, the tip-specific auxin response in turn organizes characteristic expression patterns of ER family and EPFL2 by enhancing ER family expression at the tip while eliminating the EPFL2 expression from the tip. Our findings identify the novel ligand-receptor pairs promoting the tooth growth, and further reveal a feedback circuit between the peptide-receptor system and auxin response as a mechanism for maintaining proper auxin maxima during leaf margin morphogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Recognition of peptide-MHC class I complexes by activating killer immunoglobulin-like receptors.

PubMed

Stewart, C Andrew; Laugier-Anfossi, Fanny; Vély, Frédéric; Saulquin, Xavier; Riedmuller, Jenifer; Tisserant, Agnès; Gauthier, Laurent; Romagné, François; Ferracci, Géraldine; Arosa, Fernando A; Moretta, Alessandro; Sun, Peter D; Ugolini, Sophie; Vivier, Eric

2005-09-13

Inhibitory receptors for MHC class I molecules increase the threshold of lymphocyte activation. Natural Killer (NK) cells express a large number of such inhibitory receptors, including the human killer Ig-like receptors (KIR). However, activating members of the KIR family have poorly defined ligands and functions. Here we describe the use of activating KIR tetramer reagents as probes to detect their ligands. Infection of cells with Epstein-Barr virus leads to expression of a detectable ligand for the activating receptor KIR2DS1. In this case, KIR2DS1 interacts with up-regulated peptide-MHC class I complexes on Epstein-Barr virus-infected cells in a transporter associated with antigen processing (TAP)-dependent manner. In tetramer-based cellular assays and direct affinity measurements, this interaction with MHC class I is facilitated by a broad spectrum of peptides. KIR2DS1 and its inhibitory homologue, KIR2DL1, share sensitivity to peptide sequence alterations at positions 7 and 8. These results fit a model in which activating and inhibitory receptors recognize the same sets of self-MHC class I molecules, differing only in their binding affinities. Importantly, KIR2DS1 is not always sufficient to trigger NK effector responses when faced with cognate ligand, consistent with fine control during NK cell activation. We discuss how our results for KIR2DS1 and parallel studies on KIR2DS2 relate to the association between activating KIR genes and susceptibility to autoimmune disorders.

Early modulation by the dopamine D4 receptor of morphine-induced changes in the opioid peptide systems in the rat caudate putamen.

PubMed

Gago, Belén; Fuxe, Kjell; Brené, Stefan; Díaz-Cabiale, Zaida; Reina-Sánchez, María Dolores; Suárez-Boomgaard, Diana; Roales-Buján, Ruth; Valderrama-Carvajal, Alejandra; de la Calle, Adelaida; Rivera, Alicia

2013-12-01

The peptides dynorphin and enkephalin modulate many physiological processes, such as motor activity and the control of mood and motivation. Their expression in the caudate putamen (CPu) is regulated by dopamine and opioid receptors. The current work was designed to explore the early effects of the acute activation of D4 and/or μ opioid receptors by the agonists PD168,077 and morphine, respectively, on the regulation of the expression of these opioid peptides in the rat CPu, on transcription factors linked to them, and on the expression of μ opioid receptors. In situ hybridization experiments showed that acute treatment with morphine (10 mg/kg) decreased both enkephalin and dynorphin mRNA levels in the CPu after 30 min, but PD168,077 (1 mg/kg) did not modify their expression. Coadministration of the two agonists demonstrated that PD168,077 counteracted the morphine-induced changes and even increased enkephalin mRNA levels. The immunohistochemistry studies showed that morphine administration also increased striatal μ opioid receptor immunoreactivity but reduced P-CREB expression, effects that were blocked by the PD168,077-induced activation of D4 receptors. The current results present evidence of functional D4 -μ opioid receptor interactions, with consequences for the opioid peptide mRNA levels in the rat CPu, contributing to the integration of DA and opioid peptide signaling. Copyright © 2013 Wiley Periodicals, Inc.

New Insights into Ligand-Receptor Pairing and Coevolution of Relaxin Family Peptides and Their Receptors in Teleosts

PubMed Central

Good, Sara; Yegorov, Sergey; Martijn, Joran; Franck, Jens; Bogerd, Jan

2012-01-01

Relaxin-like peptides (RLN/INSL) play diverse roles in reproductive and neuroendocrine processes in placental mammals and are functionally associated with two distinct types of receptors (RXFP) for each respective function. The diversification of RLN/INSL and RXFP gene families in vertebrates was predominantly driven by whole genome duplications (2R and 3R). Teleosts preferentially retained duplicates of genes putatively involved in neuroendocrine regulation, harboring a total of 10-11 receptors and 6 ligand genes, while most mammals have equal numbers of ligands and receptors. To date, the ligand-receptor relationships of teleost Rln/Insl peptides and their receptors have largely remained unexplored. Here, we use selection analyses based on sequence data from 5 teleosts and qPCR expression data from zebrafish to explore possible ligand-receptor pairings in teleosts. We find support for the hypothesis that, with the exception of RLN, which has undergone strong positive selection in mammalian lineages, the ligand and receptor genes shared between mammals and teleosts appear to have similar pairings. On the other hand, the teleost-specific receptors show evidence of subfunctionalization. Overall, this study underscores the complexity of RLN/INSL and RXFP ligand-receptor interactions in teleosts and establishes theoretical background for further experimental work in nonmammals. PMID:23008798

An interleukin 13 receptor α 2–specific peptide homes to human Glioblastoma multiforme xenografts

PubMed Central

Pandya, Hetal; Gibo, Denise M.; Garg, Shivank; Kridel, Steven; Debinski, Waldemar

2012-01-01

Interleukin 13 receptor α 2 (IL-13Rα2) is a glioblastoma multiforme (GBM)–associated plasma membrane receptor, a brain tumor of dismal prognosis. Here, we isolated peptide ligands for IL-13Rα2 with use of a cyclic disulphide-constrained heptapeptide phages display library and 2 in vitro biopanning schemes with GBM cells that do (G26-H2 and SnB19-pcDNA cells) or do not (G26-V2 and SnB19-asIL-13Rα2 cells) over-express IL-13Rα2. We identified 3 peptide phages that bind to IL-13Rα2 in cellular and protein assays. One of the 3 peptide phages, termed Pep-1, bound to IL-13Rα2 with the highest specificity, surprisingly, also in a reducing environment. Pep-1 was thus synthesized and further analyzed in both linear and disulphide-constrained forms. The linear peptide bound to IL-13Rα2 more avidly than did the disulphide-constrained form and was efficiently internalized by IL-13Rα2–expressing GBM cells. The native ligand, IL-13, did not compete for the Pep-1 binding to the receptor and vice versa in any of the assays, indicating that the peptide might be binding to a site on the receptor different from the native ligand. Furthermore, we demonstrated by noninvasive near infrared fluorescence imaging in nude mice that Pep-1 binds and homes to both subcutaneous and orthotopic human GBM xenografts expressing IL-13Rα2 when injected by an intravenous route. Thus, we identified a linear heptapeptide specific for the IL-13Rα2 that is capable of crossing the blood-brain tumor barrier and homing to tumors. Pep-1 can be further developed for various applications in cancer and/or inflammatory diseases. PMID:21946118

Tachykinin-Related Peptides Share a G Protein-Coupled Receptor with Ion Transport Peptide-Like in the Silkworm Bombyx mori

PubMed Central

Nagai-Okatani, Chiaki; Nagasawa, Hiromichi

2016-01-01

Recently, we identified an orphan Bombyx mori neuropeptide G protein-coupled receptor (BNGR)-A24 as an ion transport peptide-like (ITPL) receptor. BNGR-A24 belongs to the same clade as BNGR-A32 and -A33, which were recently identified as natalisin receptors. Since these three BNGRs share high similarities with known receptors for tachykinin-related peptides (TRPs), we examined whether these BNGRs can function as physiological receptors for five endogenous B. mori TRPs (TK-1–5). In a heterologous expression system, BNGR-A24 acted as a receptor for all five TRPs. In contrast, BNGR-A32 responded only to TK-5, and BNGR-A33 did not respond to any of the TRPs. These findings are consistent with recent studies on the ligand preferences for B. mori natalisins. Furthermore, we evaluated whether the binding of ITPL and TRPs to BNGR-A24 is competitive by using a Ca2+ imaging assay. Concomitant addition of a TRP receptor antagonist, spantide I, reduced the responses of BNGR-A24 not only to TK-4 but also to ITPL. The results of a binding assay using fluorescent-labeled BNGR-A24 and ligands demonstrated that the binding of ITPL to BNGR-A24 was inhibited by TK-4 as well as by spantide I, and vice versa. In addition, the ITPL-induced increase in cGMP levels of BNGR-A24-expressing BmN cells was suppressed by the addition of excess TK-4 or spantide I. The intracellular levels of cAMP and cGMP, as second messenger candidates of the TRP signaling, were not altered by the five TRPs, suggesting that these peptides act via different signaling pathways from cAMP and cGMP signaling at least in BmN cells. Taken together, the present findings suggest that ITPL and TRPs are endogenous orthosteric ligands of BNGR-A24 that may activate discrete signaling pathways. This receptor, which shares orthosteric ligands, may constitute an important model for studying ligand-biased signaling. PMID:27248837

Peptides released by ameboid microglia regulate astroglial proliferation

PubMed Central

1985-01-01

Peptides that stimulate astroglial proliferation are produced in traumatized adult rat brain by 10 d after injury. These same peptides are released by ameboid microglia activated in vitro. Our findings suggest that astroglial scarring is regulated in part by the release of factors from ameboid microglia near the site of brain injury. PMID:4066764

Corticotropin-releasing factor peptide antagonists: design, characterization and potential clinical relevance.

PubMed

Rivier, Jean E; Rivier, Catherine L

2014-04-01

Elusive for more than half a century, corticotropin-releasing factor (CRF) was finally isolated and characterized in 1981 from ovine hypothalami and shortly thereafter, from rat brains. Thirty years later, much has been learned about the function and localization of CRF and related family members (Urocortins 1, 2 and 3) and their 2 receptors, CRF receptor type 1 (CRFR1) and CRF receptor type 2 (CRFR2). Here, we report the stepwise development of peptide CRF agonists and antagonists, which led to the CRFR1 agonist Stressin1; the long-acting antagonists Astressin2-B which is specific for CRFR2; and Astressin B, which binds to both CRFR1 and CRFR2.This analog has potential for the treatment of CRF-dependent diseases in the periphery, such as irritable bowel syndrome. Copyright © 2013 Elsevier Inc. All rights reserved.

Neurotensin is an antagonist of the human neurotensin NT2 receptor expressed in Chinese hamster ovary cells.

PubMed

Vita, N; Oury-Donat, F; Chalon, P; Guillemot, M; Kaghad, M; Bachy, A; Thurneyssen, O; Garcia, S; Poinot-Chazel, C; Casellas, P; Keane, P; Le Fur, G; Maffrand, J P; Soubrie, P; Caput, D; Ferrara, P

1998-11-06

The human levocabastine-sensitive neurotensin NT2 receptor was cloned from a cortex cDNA library and stably expressed in Chinese hamster ovary (CHO) cells in order to study its binding and signalling characteristics. The receptor binds neurotensin as well as several other ligands already described for neurotensin NT1 receptor. It also binds levocabastine, a histamine H1 receptor antagonist that is not recognised by neurotensin NT1 receptor. Neurotensin binding to recombinant neurotensin NT2 receptor expressed in CHO cells does not elicit a biological response as determined by second messenger measurements. Levocabastine, and the peptides neuromedin N and xenin were also ineffective on neurotensin NT2 receptor activation. Experiments with the neurotensin NT1 receptor antagonists SR48692 and SR142948A, resulted in the unanticipated discovery that both molecules are potent agonists on neurotensin NT2 receptor. Both compounds, following binding to neurotensin NT2 receptor, enhance inositol phosphates (IP) formation with a subsequent [Ca2+]i mobilisation; induce arachidonic acid release; and stimulate mitogen-activated protein kinase (MAPK) activity. Interestingly, these activities are antagonised by neurotensin and levocabastine in a concentration-dependent manner. These activities suggest that the human neurotensin NT2 receptor may be of physiological importance and that a natural agonist for the receptor may exist.

Cholecystokinin (CCK) and CCK receptor expression by human gliomas: Evidence for an autocrine/paracrine stimulatory loop.

PubMed

Oikonomou, Eftychia; Buchfelder, Michael; Adams, Eric F

2008-06-01

Cholecystokinin (CCK) is a gut-brain peptide has been described to be able to induce mitosis according to recent studies. Additionally, conflicting data has been published on whether tumours of the central and peripheral nervous system in general, and gliomas in particular, express CCK receptors. In the present in vitro study we employed reverse transcription followed by the polymerase chain reaction (RT-PCR) to investigate whether mRNA for CCK-A and CCK-B receptors as well as CCK peptide itself is present in primary human gliomas and the U-87 MG GBM cell line. The data show that 14/14 (100%) of the primary gliomas exhibited mRNA expression for the CCK peptide gene and the B receptor including the U-87 MG cells, whereas, only 2/14 (14%) showed presence of the CCK-A receptor. The presence of CCK receptors together with CCK peptide expression itself suggests presence of an autocrine loop controlling glioma cell growth. In support of this conclusion, a neutralizing antibody against the CCK peptide exhibited a dose dependent inhibition of cell growth whereas, antagonists to CCK caused a dose depend inhibition of exogenous stimulated glioma cell growth in vitro, via the CCK-B receptor which is PKC activated. Assessment of apoptosis and proteasome activity were undertaken and we report that treatment with CCK antagonists decreased proteasome and increased caspase-3 activity. These data indicate that CCK peptide and CCK-B are abundant in human gliomas and they act to stimulate cell growth in an autocrine manner, primarily via the high affinity CCK-B receptor, which was blocked by antagonists to CCK, perhaps via apoptosis.

Synthesis and characterization of an 111In-labeled peptide for the in vivo localization of human cancers expressing the urokinase-type plasminogen activator receptor (uPAR)

PubMed Central

Liu, Dijie; Overbey, Douglas; Watkinson, Lisa; Giblin, Michael F.

2009-01-01

This study describes the synthesis and preliminary biologic evaluation of an 111Inlabeled peptide antagonist of the urokinase-type plasminogen activator receptor (uPAR) as a potential probe for assessing metastatic potential of human breast cancer in vivo. The peptide (NAc-dD-CHA-F-dS-dR-Y-L-W-S-βAla)2-K-K(DOTA)-NH2 was synthesized and conjugated with the DOTA chelating moiety via conventional Solid-Phase Peptide Synthesis (SPPS), purified by reversed-phase HPLC, and characterized by MALDI-TOF MS and receptor binding assay. In vitro receptor binding studies demonstrated an IC50 of 240 ± 125 nM for the peptide, compared with IC50’s of 0.44 ± 0.02 and 0.75 ± 0.01 nM for the amino terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA) and full-length uPA, respectively. In vivo biodistribution studies were carried out using SCID mice bearing MDA-MB-231 human breast cancer xenografts. Biodistribution data was collected at 1, 4, and 24 hr post-injection of 111In-DOTA-peptide, and compared with data obtained using a scrambled control peptide, as well as with data obtained using wild-type ATF radiolabeled with I-125. Biodistribution studies showed rapid elimination of the 111In-labeled peptide from the blood pool, with 0.12 ± 0.06% ID/g remaining in blood at 4 hr pi. Elimination was seen primarily via the renal/urinary route, with 83.9 ± 2.2%ID in the urine at the same timepoint. Tumor uptake at this time was 0.53 ± 0.11%ID/g, resulting in tumor: blood and tumor: muscle ratios of 4.2 and 9.4, respectively. Uptake in tumor was significantly higher than that obtained using a scrambled control peptide that showed no specific binding to uPAR (p < 0.05). In vitro and ex vivo results both suggested that the magnitude of tumor-specific binding was reduced in this model by endogenous expression of uPA. The results indicate that radiolabeled peptide uPAR antagonists may find application in the imaging and therapy of uPAR-expressing breast cancers in vivo

Functional expression of the thyrotropin receptor in C cells: new insights into their involvement in the hypothalamic-pituitary-thyroid axis

PubMed Central

Morillo-Bernal, Jesús; Fernández-Santos, José M; Utrilla, José C; de Miguel, Manuel; García-Marín, Rocío; Martín-Lacave, Inés

2009-01-01

Thyroid C cells, or parafollicular cells, are mainly known for producing calcitonin, a hormone involved in calcium homeostasis with hypocalcemic and hypophosphatemic effects. Classically, the main endocrine activity of this cell population has been believed to be restricted to its roles in serum calcium and bone metabolism. Nonetheless, in the last few years evidence has been accumulating in the literature with regard to local regulatory peptides secreted by C cells, such as somatostatin, ghrelin, thyrotropin releasing hormone or the recently described cocaine- and amphetamine-related transcript, which could modify thyroid function. As thyrotropin is the main hormone controlling the hypothalamic-pituitary-thyroid axis and, accordingly, thyroid function, we have examined the functional expression of the thyrotropin receptor in C-cell lines and in thyroid tissues. We have found that rat and human C-cell lines express the thyrotropin receptor at both mRNA and protein levels. Furthermore, incubation of C cells with thyrotropin resulted in a 10-fold inhibition of thyrotropin-receptor expression, and a concomitant decrease of the steady-state mRNA levels for calcitonin and calcitonin gene-related peptide determined by quantitative real-time PCR was found. Finally, thyrotropin receptor expression by C cells was confirmed at protein level in both normal and pathological thyroid tissues by immunohistochemistry and immunofluorescence. These results confirm that C cells, under regulation by thyrotropin, are involved in the hypothalamic-pituitary-thyroid axis and suggest a putative role in local fine-tuning of follicular cell activity. PMID:19493188

Helicobacter pylori potentiates epithelial:mesenchymal transition in gastric cancer: links to soluble HB-EGF, gastrin and matrix metalloproteinase-7

PubMed Central

Yin, Yinfei; Grabowska, Anna M; Clarke, Philip A; Whelband, Elisabeth; Robinson, Karen; Argent, Richard H; Tobias, Amanda; Kumari, Rajendra; Atherton, John C

2010-01-01

Background and aims Helicobacter pylori (H pylori) infection is a major risk factor in the development of distal gastric adenocarcinoma. Development of the invasive phenotype is associated with the phenomenon of epithelial:mesenchymal transition (EMT). Soluble heparin-binding epidermal growth factor (HB-EGF) has been implicated in this process. A study was undertaken to investigate the possibility that matrix metalloproteinase (MMP)-7 is upregulated in H pylori infection as a result of hypergastrinaemia, which may enhance shedding of HB-EGF and contribute towards EMT in gastric adenocarcinoma cell lines. Methods Three gastric epithelial cell lines (AGS, MGLVA1 and ST16) were co-cultured with the pathogenic H pylori strain 60190 and non-pathogenic strain Tx30a in an in vitro infection model. Gene expression was quantified by real-time PCR, HB-EGF shedding by ELISA and protein expression by immunofluorescence or immunohistochemistry. The INS-GAS mouse, a transgenic mouse model of gastric carcinogenesis which overexpresses amidated gastrin, was used to investigate the in vivo relationship between HB-EGF, MMP-7, gastrin and EMT. Results The pathogenic strain of H pylori significantly upregulated EMT-associated genes Snail, Slug and vimentin in all three gastric cell lines to a greater degree than the non-pathogenic strain. Pathogenic H pylori also upregulated HB-EGF shedding, a factor implicated in EMT, which was partially dependent on both gastrin and MMP-7 expression. Gastrin and MMP-7 siRNAs and MMP-7 neutralising antibody significantly reduced upregulation of HB-EGF shedding in H pylori infected gastric cell lines and reduced EMT gene expression. The effect of H pylori on EMT was also reversed by gastrin siRNA. Neutralisation of gastrin in the INS-GAS mouse model reduced expression of MMP-7, HB-EGF and key EMT proteins. Conclusion The upregulation of MMP-7 by pathogenic H pylori is partially dependent on gastrin and may have a role in the development of gastric

Identification and Characterization of Receptors for Ion Transport Peptide (ITP) and ITP-like (ITPL) in the Silkworm Bombyx mori*

PubMed Central

Nagai, Chiaki; Mabashi-Asazuma, Hideaki; Nagasawa, Hiromichi; Nagata, Shinji

2014-01-01

Ion transport peptide (ITP) and its alternatively spliced variant, ITP-like (ITPL), are insect peptides that belong to the crustacean hyperglycemic hormone family. These peptides modulate the homeostatic mechanisms for regulating energy metabolism, molting, and reproduction and are specifically conserved in ecdysozoans. Many of the details of the molecular mechanisms by which crustacean hyperglycemic hormone family peptides exert pleiotropy remain to be elucidated, including characterization of their receptors. Here we identified three Bombyx mori orphan neuropeptide G protein-coupled receptors (BNGRs), BNGR-A2, -A24, and -A34, as receptors for ITP and ITPL (collectively referred to as ITPs). BNGR-A2 and -A34 and BNGR-A24 respond to recombinant ITPs, respectively, with EC50 values of 1.1–2.6 × 10−8 m, when expressed in a heterologous expression system. These three candidate BNGRs are expressed at larval B. mori tissues targeted by ITPs, with cGMP elevation observed after exposure to recombinant ITPs. ITPs also increased the cGMP level in B. mori ovary-derived BmN cells via membrane-bound and soluble guanylyl cyclases. The simultaneous knockdown of bngr-A2 and -A34 significantly decreased the response of BmN cells to ITP, whereas knockdown of bngr-A24 led to decreased responses to ITPL. Conversely, transient expression of bngr-A24 potentiated the response of BmN cells to ITPL. An in vitro binding assay showed direct interaction between ITPs and heterologously expressed BNGRs in a ligand-receptor-specific manner. Taken together, these data demonstrate that BNGR-A2 and -A34 are ITP receptors and that BNGR-A24 is an ITPL receptor in B. mori. PMID:25278025

Expression of proteinase-activated receptor (PAR)-2 in monocytes from allergic patients and potential molecular mechanism.

PubMed

Ge, Shuqing; Li, Tao; Yao, Qijian; Yan, Hongling; Huiyun, Zhang; Zheng, Yanshan; Zhang, Bin; He, Shaoheng

2016-12-01

Serine proteases play an important role in inflammation via PARs. However, little is known of expression levels of PARs on monocytes of allergic patients, and influence of serine proteases and PARs on TNF-α secretion from monocytes. Using quantitative real-time PCR (qPCR) and flowcytometry techniques, we observed that the expression level of PAR-2 in monocytes of patients with allergic rhinitis and asthma was increased by 42.9 and 38.2 %. It was found that trypsin, thrombin, and tryptase induced up to 200, 320, and 310 % increase in TNF-α release from monocytes at 16 h, respectively. PAR-1 agonist peptide, SFLLR-NH 2 , and PAR-2 agonist peptide tc-LIGRLO-NH 2 provoked up to 210 and 240 % increase in release of TNF-α. Since SCH 79797, a PAR-1 antagonist, and PD98059, an inhibitor of ERK inhibited thrombin- and SFLLR-NH 2 -induced TNF-α release, the action of thrombin is most likely through a PAR-1- and ERK-mediated signaling mechanism. Similarly, because FSLLRN-NH 2 , an inhibitor of PAR-2 diminished tryptase- and tc-LIGRLO-NH 2 -induced TNF-α release, the action of tryptase appears PAR-2 dependent. Moreover, in vivo study showed that both recombinant cockroach major allergens Per a 1 and Per a 7 provoked upregulation of PAR-2 and PAR-1 expression on CD14+ cells in OVA-sensitized mouse peritoneum. In conclusion, increased expression of PAR-2 in monocytes of AR and asthma implicates that PAR-2 likely play a role in allergy. PAR-2- and PAR-1-mediated TNF-α release from monocytes suggests that these unique protease receptors are involved in the pathogenesis of inflammation.

Effects of Porphyromonas gingivalis LPS and LR12 peptide on TREM-1 expression by monocytes.

PubMed

Dubar, Marie; Carrasco, Kevin; Gibot, Sebastien; Bisson, Catherine

2018-05-19

Periodontal disease involves the activation of host immune response, acting not only as defender of periodontal tissues against bacterial aggression but also as mediator of tissue destruction. Triggering receptor expressed on myeloid cells 1 (TREM-1) is an immune receptor that synergizes with Toll-like receptors in amplifying the inflammatory response mediated by microbial molecules. To investigate the role of P. gingivalis lipopolysaccharide (LPS) and the effect of LR12, a TREM-1 inhibitory peptide, on the expression of membrane-bound and soluble form of TREM-1 on human primary monocytes, as well as the production of proinflammatory cytokines. Cells were stimulated with 1 μg/ml of LPS with or without LR12. PCR, flow cytometry and ELISA were used to determine TREM-1 expressions and cytokines release by monocytes. P. gingivalis LPS can induce a significant increase in TREM-1 expression (mRNA, membrane-bound and soluble form, p < 0.001) as well as cytokines (IL-1β, TNFα) and chemokines (IL-8) production by monocytes. This monocytes' activation was partly prevented by LR12. TREM-1 inhibitors such as LR12 could be interesting for the modulation of the excessive inflammatory response that occurs during periodontal disease. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The human gastrin precursor. Characterization of phosphorylated forms and fragments.

PubMed Central

Varro, A; Desmond, H; Pauwels, S; Gregory, H; Young, J; Dockray, G J

1988-01-01

There is a potential phosphorylation site in the C-terminal region of the precursor for the acid-stimulating hormone gastrin, which is immediately adjacent to an important cleavage point. In the present study we have sought to identify, separate, quantify and characterize phosphorylated and unphosphorylated forms of human progastrin and its fragments. Identification was made by two radioimmunoassays: (a) a novel assay employing an antibody raised to intact human progastrin; and (b) an assay using antibody reacting with the C-terminal tryptic fragment of human progastrin, as well as progastrin itself. Two forms of human progastrin isolated from a gastrinoma were separated by ion-exchange h.p.l.c., and had similar elution positions on reverse-phase h.p.l.c. and on gel filtration. The more acidic peptide contained close to equimolar amounts of phosphate. On trypsinization, peptides were released that co-eluted on ion-exchange h.p.l.c. with, and had the immunochemical properties of, naturally occurring C-terminal fragments of progastrin. One of the latter was isolated and shown by Edman degradation after derivatization with ethanethiol to have the sequence Ser (P)-Ala-Glu-Asp-Glu-Asn. Similar peptides occur in antral mucosa resected from ulcer patients. The unphosphorylated forms of progastrin predominated, whereas the phosphorylated forms of the C-terminal fragments were predominant. This distribution could be explained by preferential cleavage of phosphorylated progastrin. We conclude that in human progastrin, Ser-96 can occur in the phosphorylated form; this residue immediately follows a pair of basic residues (Arg-Arg) that are cleaved during synthesis of the biologically active product. PMID:3223964

High Contrast PET Imaging of GRPR Expression in Prostate Cancer Using Cobalt-Labeled Bombesin Antagonist RM26

PubMed Central

Thisgaard, Helge; Rosenström, Ulrika; Dam, Johan Hygum; Larhed, Mats

2017-01-01

High gastrin releasing peptide receptor (GRPR) expression is associated with numerous cancers including prostate and breast cancer. The aim of the current study was to develop a 55Co-labeled PET agent based on GRPR antagonist RM26 for visualization of GRPR-expressing tumors. Labeling with 57Co and 55Co, stability, binding specificity, and in vitro and in vivo characteristics of 57Co-NOTA-PEG2-RM26 were studied. NOTA-PEG2-RM26 was successfully radiolabeled with 57Co and 55Co with high yields and demonstrated high stability. The radiopeptide showed retained binding specificity to GRPR in vitro and in vivo. 57Co-NOTA-PEG2-RM26 biodistribution in mice was characterized by rapid clearance of radioactivity from blood and normal non-GRPR-expressing organs and low hepatic uptake. The clearance was predominantly renal with a low degree of radioactivity reabsorption. Tumor-to-blood ratios were approximately 200 (3 h pi) and 1000 (24 h pi). The favorable biodistribution of cobalt-labeled NOTA-PEG2-RM26 translated into high contrast preclinical PET/CT (using 55Co) and SPECT/CT (using 57Co) images of PC-3 xenografts. The initial biological results suggest that 55Co-NOTA-PEG2-RM26 is a promising tracer for PET visualization of GRPR-expressing tumors. PMID:29097932

Postsynaptic Depolarization Enhances GABA Drive to Dorsomedial Hypothalamic Neurons through Somatodendritic Cholecystokinin Release.

PubMed

Crosby, Karen M; Baimoukhametova, Dinara V; Bains, Jaideep S; Pittman, Quentin J

2015-09-23

Somatodendritically released peptides alter synaptic function through a variety of mechanisms, including autocrine actions that liberate retrograde transmitters. Cholecystokinin (CCK) is a neuropeptide expressed in neurons in the dorsomedial hypothalamic nucleus (DMH), a region implicated in satiety and stress. There are clear demonstrations that exogenous CCK modulates food intake and neuropeptide expression in the DMH, but there is no information on how endogenous CCK alters synaptic properties. Here, we provide the first report of somatodendritic release of CCK in the brain in male Sprague Dawley rats. CCK is released from DMH neurons in response to repeated postsynaptic depolarizations, and acts in an autocrine fashion on CCK2 receptors to enhance postsynaptic NMDA receptor function and liberate the retrograde transmitter, nitric oxide (NO). NO subsequently acts presynaptically to enhance GABA release through a soluble guanylate cyclase-mediated pathway. These data provide the first demonstration of synaptic actions of somatodendritically released CCK in the hypothalamus and reveal a new form of retrograde plasticity, depolarization-induced potentiation of inhibition. Significance statement: Somatodendritic signaling using endocannabinoids or nitric oxide to alter the efficacy of afferent transmission is well established. Despite early convincing evidence for somatodendritic release of neurohypophysial peptides in the hypothalamus, there is only limited evidence for this mode of release for other peptides. Here, we provide the first evidence for somatodendritic release of the satiety peptide cholecystokinin (CCK) in the brain. We also reveal a new form of synaptic plasticity in which postsynaptic depolarization results in enhancement of inhibition through the somatodendritic release of CCK. Copyright © 2015 the authors 0270-6474/15/3513160-11$15.00/0.

Introduction of D-phenylalanine enhanced the receptor binding affinities of gonadotropin-releasing hormone peptides.

PubMed

Lu, Jie; Hathaway, Helen J; Royce, Melanie E; Prossnitz, Eric R; Miao, Yubin

2014-02-01

The purpose of this study was to examine whether the introduction of D-Phe could improve the GnRH receptor binding affinities of DOTA-conjugated D-Lys(6)-GnRH peptides. Building upon the construct of DOTA-Ahx-(D-Lys(6)-GnRH1) we previously reported, an aromatic amino acid of D-Phe was inserted either between the DOTA and Ahx or between the Ahx and D-Lys(6) to generate new DOTA-D-Phe-Ahx-(D-Lys(6)-GnRH) or DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) peptides. Compared to DOTA-Ahx-(D-Lys(6)-GnRH1) (36.1 nM), the introduction of D-Phe improved the GnRH receptor binding affinities of DOTA-D-Phe-Ahx-(D-Lys(6)-GnRH) (16.3 nM) and DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) (7.6 nM). The tumor targeting and pharmacokinetic properties of (111)In-DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) was determined in MDA-MB-231 human breast cancer-xenografted nude mice. Compared to (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1), (111)In-DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) exhibited comparable tumor uptake with faster renal and liver clearance. The MDA-MB-231 human breast cancer-xenografted tumors were clearly visualized by single photon emission computed tomography (SPECT) using (111)In-DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) as an imaging probe, providing a new insight into the design of new GnRH peptides in the future. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos

PubMed Central

David, Marion; Lécorché, Pascaline; Masse, Maxime; Faucon, Aude; Abouzid, Karima; Gaudin, Nicolas; Varini, Karine; Gassiot, Fanny; Ferracci, Géraldine; Jacquot, Guillaume; Vlieghe, Patrick

2018-01-01

Insufficient membrane penetration of drugs, in particular biotherapeutics and/or low target specificity remain a major drawback in their efficacy. We propose here the rational characterization and optimization of peptides to be developed as vectors that target cells expressing specific receptors involved in endocytosis or transcytosis. Among receptors involved in receptor-mediated transport is the LDL receptor. Screening complex phage-displayed peptide libraries on the human LDLR (hLDLR) stably expressed in cell lines led to the characterization of a family of cyclic and linear peptides that specifically bind the hLDLR. The VH411 lead cyclic peptide allowed endocytosis of payloads such as the S-Tag peptide or antibodies into cells expressing the hLDLR. Size reduction and chemical optimization of this lead peptide-vector led to improved receptor affinity. The optimized peptide-vectors were successfully conjugated to cargos of different nature and size including small organic molecules, siRNAs, peptides or a protein moiety such as an Fc fragment. We show that in all cases, the peptide-vectors retain their binding affinity to the hLDLR and potential for endocytosis. Following i.v. administration in wild type or ldlr-/- mice, an Fc fragment chemically conjugated or fused in C-terminal to peptide-vectors showed significant biodistribution in LDLR-enriched organs. We have thus developed highly versatile peptide-vectors endowed with good affinity for the LDLR as a target receptor. These peptide-vectors have the potential to be further developed for efficient transport of therapeutic or imaging agents into cells -including pathological cells—or organs that express the LDLR. PMID:29485998

Elabela/Toddler Is an Endogenous Agonist of the Apelin APJ Receptor in the Adult Cardiovascular System, and Exogenous Administration of the Peptide Compensates for the Downregulation of Its Expression in Pulmonary Arterial Hypertension

PubMed Central

Yang, Peiran; Read, Cai; Kuc, Rhoda E.; Buonincontri, Guido; Southwood, Mark; Torella, Rubben; Upton, Paul D.; Crosby, Alexi; Sawiak, Stephen J.; Carpenter, T. Adrian; Glen, Robert C.; Morrell, Nicholas W.; Maguire, Janet J.

2017-01-01

Background: Elabela/toddler (ELA) is a critical cardiac developmental peptide that acts through the G-protein–coupled apelin receptor, despite lack of sequence similarity to the established ligand apelin. Our aim was to investigate the receptor pharmacology, expression pattern, and in vivo function of ELA peptides in the adult cardiovascular system, to seek evidence for alteration in pulmonary arterial hypertension (PAH) in which apelin signaling is downregulated, and to demonstrate attenuation of PAH severity with exogenous administration of ELA in a rat model. Methods: In silico docking analysis, competition binding experiments, and downstream assays were used to characterize ELA receptor binding in human heart and signaling in cells expressing the apelin receptor. ELA expression in human cardiovascular tissues and plasma was determined using real-time quantitative polymerase chain reaction, dual-labeling immunofluorescent staining, and immunoassays. Acute cardiac effects of ELA-32 and [Pyr1]apelin-13 were assessed by MRI and cardiac catheterization in anesthetized rats. Cardiopulmonary human and rat tissues from PAH patients and monocrotaline- and Sugen/hypoxia-exposed rats were used to show changes in ELA expression in PAH. The effect of ELA treatment on cardiopulmonary remodeling in PAH was investigated in the monocrotaline rat model. Results: ELA competed for binding of apelin in human heart with overlap for the 2 peptides indicated by in silico modeling. ELA activated G-protein– and β-arrestin–dependent pathways. We detected ELA expression in human vascular endothelium and plasma. Comparable to apelin, ELA increased cardiac contractility, ejection fraction, and cardiac output and elicited vasodilatation in rat in vivo. ELA expression was reduced in cardiopulmonary tissues from PAH patients and PAH rat models, respectively. ELA treatment significantly attenuated elevation of right ventricular systolic pressure and right ventricular hypertrophy and

Elabela/Toddler Is an Endogenous Agonist of the Apelin APJ Receptor in the Adult Cardiovascular System, and Exogenous Administration of the Peptide Compensates for the Downregulation of Its Expression in Pulmonary Arterial Hypertension.

PubMed

Yang, Peiran; Read, Cai; Kuc, Rhoda E; Buonincontri, Guido; Southwood, Mark; Torella, Rubben; Upton, Paul D; Crosby, Alexi; Sawiak, Stephen J; Carpenter, T Adrian; Glen, Robert C; Morrell, Nicholas W; Maguire, Janet J; Davenport, Anthony P

2017-03-21

Elabela/toddler (ELA) is a critical cardiac developmental peptide that acts through the G-protein-coupled apelin receptor, despite lack of sequence similarity to the established ligand apelin. Our aim was to investigate the receptor pharmacology, expression pattern, and in vivo function of ELA peptides in the adult cardiovascular system, to seek evidence for alteration in pulmonary arterial hypertension (PAH) in which apelin signaling is downregulated, and to demonstrate attenuation of PAH severity with exogenous administration of ELA in a rat model. In silico docking analysis, competition binding experiments, and downstream assays were used to characterize ELA receptor binding in human heart and signaling in cells expressing the apelin receptor. ELA expression in human cardiovascular tissues and plasma was determined using real-time quantitative polymerase chain reaction, dual-labeling immunofluorescent staining, and immunoassays. Acute cardiac effects of ELA-32 and [Pyr 1 ]apelin-13 were assessed by MRI and cardiac catheterization in anesthetized rats. Cardiopulmonary human and rat tissues from PAH patients and monocrotaline- and Sugen/hypoxia-exposed rats were used to show changes in ELA expression in PAH. The effect of ELA treatment on cardiopulmonary remodeling in PAH was investigated in the monocrotaline rat model. ELA competed for binding of apelin in human heart with overlap for the 2 peptides indicated by in silico modeling. ELA activated G-protein- and β-arrestin-dependent pathways. We detected ELA expression in human vascular endothelium and plasma. Comparable to apelin, ELA increased cardiac contractility, ejection fraction, and cardiac output and elicited vasodilatation in rat in vivo. ELA expression was reduced in cardiopulmonary tissues from PAH patients and PAH rat models, respectively. ELA treatment significantly attenuated elevation of right ventricular systolic pressure and right ventricular hypertrophy and pulmonary vascular remodeling in

Serum gastrin concentrations in dogs with liver disorders.

PubMed

Mazaki-Tovi, M; Segev, G; Yas-Natan, E; Lavy, E

2012-07-07

Dogs with liver disorders often display gastrointestinal signs that may be triggered by ulceration. The liver is important for inactivation of some forms of gastrin. Therefore, hypergastrinaemia has been implicated in the pathogenesis of gastrointestinal ulcerations related to liver dysfunction. The aim of this study was to determine serum gastrin concentrations in dogs with liver disease. Fasted blood samples were collected from 15 dogs with newly diagnosed liver disease and 18 healthy dogs. Gastrin concentrations were significantly lower in dogs with congenital portosystemic shunt compared with healthy dogs (P=0.003). No significant difference (P=0.6) in gastrin concentration was revealed between dogs with hepatocellular disease and healthy dogs. Serum gastrin concentrations were not significantly associated with the occurrence of vomiting, anorexia, diarrhoea, or melaena in dogs with liver disorders. These findings did not provide support for the role of hypergastrinaemia in the development of gastrointestinal signs associated with liver disease in dogs. Decreased serum concentrations of gastrin in a dog with liver disease may suggest the presence of portosystemic shunt. Further investigation is warranted to determine the importance of hyopogastrinaemia in congenital postosystemic shunts in dogs and to evaluate potential alterations in serum gastrin concentrations in specific hepatocellular diseases.

Expression and characterization of human CB1 cannabinoid receptor in methylotrophic yeast Pichia pastoris.

PubMed

Kim, Tae-Kang; Zhang, Rundong; Feng, Wenke; Cai, Jian; Pierce, William; Song, Zhao-Hui

2005-03-01

For the purpose of purification and structural characterization, the CB1 cannabinoid receptors are expressed in methylotrophic yeast Pichia pastoris. The expression plasmid was constructed in which the CB1 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase I gene. To facilitate easy detection and purification, a FLAG tag was introduced at the N-terminal, a c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB1. In membrane preparations of CB1 gene transformed yeast cells, Western blot analysis detected the expression of CB1 proteins. Radioligand binding assays demonstrated that the tagged CB1 receptors expressed in P. pastoris have a pharmacological profile similar to that of the untagged CB1 receptors expressed in mammalian systems. Furthermore, the tagged CB1 receptors were purified by anti-FLAG M2 affinity chromatography and the identity of the purified CB1 receptor proteins was confirmed by Western blot analysis. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions of purified CB1 preparations detected 17 peptide fragments derived from the CB1, thus further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope tagged, functional CB1 cannabinoid receptors can be expressed in P. pastoris for purification and mass spectrometry characterization.

Effects of long-term treatment with growth hormone-releasing peptide-2 in the GHRH knockout mouse.

PubMed

Alba, Maria; Fintini, Danilo; Bowers, Cyril Y; Parlow, A F; Salvatori, Roberto

2005-11-01

Growth hormone (GH) secretagogues (GHS) stimulate GH secretion in vivo in humans and in animals. They act on the ghrelin receptor, expressed in both the hypothalamus and the pituitary. It is unknown whether GHSs act predominantly by increasing the release of hypothalamic GH-releasing hormone (GHRH) or by acting directly on the somatotroph cells. We studied whether a potent GHS could stimulate growth in the absence of endogenous GHRH. To this end, we used GHRH knockout (GHRH-KO) mice. These animals have proportionate dwarfism due to severe GH deficiency (GHD) and pituitary hypoplasia due to reduced somatotroph cell mass. We treated male GHRH-KO mice for 6 wk (from week 1 to week 7 of age) with GH-releasing peptide-2 (GHRP-2, 10 microg s.c. twice a day). Chronic treatment with GHRP-2 failed to stimulate somatotroph cell proliferation and GH secretion and to promote longitudinal growth. GHRP-2-treated mice showed an increase in total body weight compared with placebo-treated animals, due to worsening of the body composition alterations typical of GHD animals. These data demonstrate that GHRP-2 failed to reverse the severe GHD caused by lack of GHRH.

Role of GABA Release From Leptin Receptor-Expressing Neurons in Body Weight Regulation

PubMed Central

Xu, Yuanzhong; O'Brien, William G.; Lee, Cheng-Chi; Myers, Martin G.

2012-01-01

It is well established that leptin regulates energy balance largely through isoform B leptin receptor-expressing neurons (LepR neurons) in the brain and that leptin activates one subset of LepR neurons (leptin-excited neurons) while inhibiting the other (leptin-inhibited neurons). However, the neurotransmitters released from LepR neurons that mediate leptin action in the brain are not well understood. Previous results demonstrate that leptin mainly acts on γ-aminobutyric acid (GABA)ergic neurons to reduce body weight, and that leptin activates proopiomelanocortin neuron activity by reducing GABA release onto these neurons, suggesting a body weight-promoting role for GABA released from leptin-inhibited neurons. To directly examine the role of GABA release from LepR neurons in body weight regulation, mice with disruption of GABA release specifically from LepR neurons were generated by deletion of vesicular GABA transporter in LepR neurons. Interestingly, these mice developed mild obesity on chow diet and were sensitive to diet-induced obesity, which were associated with higher food intake and lower energy expenditure. Moreover, these mice showed blunted responses in both food intake and body weight to acute leptin administration. These results demonstrate that GABA plays an important role in mediating leptin action. In combination with the previous studies that leptin reduces GABA release onto proopiomelanocortin neurons through leptin-inhibited neurons and that disruption of GABA release from agouti gene-related protein neurons, one subset of LepR-inhibited neurons, leads to a lean phenotype, our results suggest that, under our experimental conditions, GABA release from leptin-excited neuron dominates over leptin-inhibited ones. PMID:22334723

Receptor binding properties and antinociceptive effects of chimeric peptides consisting of a micro-opioid receptor agonist and an ORL1 receptor antagonist.

PubMed

Kawano, Susumu; Ito, Risa; Nishiyama, Miharu; Kubo, Mai; Matsushima, Tomoko; Minamisawa, Motoko; Ambo, Akihiro; Sasaki, Yusuke

2007-07-01

Receptor binding properties and antinociceptive activities of chimeric peptides linked by spacers were investigated. The peptides consisted of the micro-opioid receptor ligand dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) or its analog YRFB (Tyr-D-Arg-Phe-betaAla-NH(2)) linked to the ORL1 receptor ligand Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) (Ac-RYYRIK-NH(2)). All chimeric peptides were found to possess high receptor binding affinities for both micro-opioid and ORL1 receptors in mouse brain membranes although their binding affinities for both receptors in spinal membranes were significantly lower. Among them, chimeric peptide 2, which consists of dermorphin and Ac-RYYRIK-NH(2) connected by a long spacer, had the highest binding affinity towards both receptors. In the tail-flick test following intrathecal (i.t.) administration to mice, all chimeric peptides showed potent and dose-dependent antinociceptive activities with an ED(50) of 1.34-4.51 (pmol/mouse), nearly comparable to dermorphin alone (ED(50); 1.08 pmol/mouse). In contrast to their micro-opioid receptor binding profiles, intracerebroventricular (i.c.v.) administration of the chimeric peptides resulted in much less potent antinociceptive activity (ED(50) 5.55-100< pmol/mouse) than when administered i.t. (ED(50): 1.34-4.51 pmol/mouse). These results suggest the involvement of nociceptin-like agonistic effects of the Ac-RYYRIK pharmacophore in the peptides, and the regulation of mu-opioid receptor-mediated antinociception in brain. The present chimeric peptides may be useful as pharmacological tools for studies on micro-opioid receptor/ORL1 receptor heterodimers.

Substance P receptor binding sites are expressed by glia in vivo after neuronal injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mantyh, P.W.; Johnson, D.J.; Boehmer, C.G.

1989-07-01

In vitro studies have demonstrated that glia can express functional receptors for a variety of neurotransmitters. To determine whether similar neurotransmitter receptors are also expressed by glia in vivo, the authors examined the glial scar in the transected optic nerve of the albino rabbit by quantitative receptor autoradiography. Receptor binding sites for radiolabeled calcitonin gene-related peptide, cholecystokinin, galanin, glutamate, somatostatin, substance P, and vasoactive intestinal peptide were examined. Specific receptor binding sites for each of these neurotransmitters were identified in the rabbit forebrain but were not detected in the normal optic nerve or tract. In the transected optic nerve andmore » tract, only receptor binding sites for substance P were expressed at detectable levels. The density of substance P receptor binding sites observed in this glial scar is among the highest observed in the rabbit forebrain. Ligand displacement and saturation experiments indicate that the substance P receptor binding site expressed by the glial scar has pharmacological characteristics similar to those of substance P receptors in the rabbit striatum, rat brain, and rat and canine gut. The present study demonstrates that glial cells in vivo express high concentrations of substance P receptor binding sites after transection of retinal ganglion cell axons. Because substance P has been shown to regulate inflammatory and immune responses in peripheral tissues, substance P may also, by analogy, be involved in regulating the glial response to injury in the central nervous system.« less

[Mechanism of action and control in the digestion of proteins and peptides in humans].

PubMed

Frenhani, P B; Burini, R C

1999-01-01

This review aims to report the major control mechanisms of protein and peptides digestion of special interest in human patients. Regarding protein assimilation its digestive process begins at the stomach with some not so indispensable actions comparatively to those of duodenal/jejunal lumen. However even the intestine processes are partially under gastric secretion control. Proteolytic enzyme activities are related to protein structure and amino acid constituents, tertiary and quartenary structures need HCl denaturation prior to enzymatic hydrolysis. Thereafter the exopeptidases are guided by either NH2 (aminopeptidases) or COOH (carboxypeptidases) terminals of the molecule while endopeptidases are oriented by the specific amino acids constituents of the peptide. Both dietary and luminal secreted proteins and polypeptides undergo to either limited or complete proteolysis resulting basic or neutral free-amino acids (40%) or dioctapeptides. The brush border peptidases continue to degrade oligopeptide to di-tripeptides and neutral free-amino acids. Some peptides are uptaked by the enterocytes whose cytosolic peptidases complete the hydrolysis. Hence the digestive products flowing in the portal vein are mainly free-amino acids from either luminal or cytosolic hydrolysis and some di-tripeptides intactly absorbed. Both mechanical and chemical processes of digestion are under neural (vagal), neuroendocrinal (acetilcholine), endocrinal (gastrin, secretin and cholecystokinin) or paracrinal (histamine) controls. The gastric phase (hydrochloric acid and pepsinogen secretions) is activated by gastrin, histamine and acetilcholine which respond to both dietary-amino acids (tryptophan and phenylalanine) and mechanic distention of stomach. The pancreatic secretion is stimulated by either cephalic or gastric phases and has influence on the intestinal phase of digestion. The intestinal types of cells S and I release secretin and cholecystokinin respectively in response of acid quimo

Spinal N-methyl-D-aspartate receptors and nociception-evoked release of primary afferent substance P.

PubMed

Nazarian, A; Gu, G; Gracias, N G; Wilkinson, K; Hua, X Y; Vasko, M R; Yaksh, T L

2008-03-03

Dorsal horn N-methyl-D-aspartate (NMDA) receptors contribute significantly to spinal nociceptive processing through an effect postsynaptic to non-primary glutamatergic axons, and perhaps presynaptic to the primary afferent terminals. The present study sought to examine the regulatory effects of NMDA receptors on primary afferent release of substance P (SP), as measured by neurokinin 1 receptor (NK1r) internalization in the spinal dorsal horn of rats. The effects of intrathecal NMDA alone or in combination with D-serine (a glycine site agonist) were initially examined on basal levels of NK1r internalization. NMDA alone or when co-administered with D-serine failed to induce NK1r internalization, whereas activation of spinal TRPV1 receptors by capsaicin resulted in a notable NK1r internalization. To determine whether NMDA receptor activation could potentiate NK1r internalization or pain behavior induced by a peripheral noxious stimulus, intrathecal NMDA was given prior to an intraplantar injection of formalin. NMDA did not alter the formalin-induced NK1r internalization nor did it enhance the formalin paw flinching behavior. To further characterize the effects of presynaptic NMDA receptors, the NMDA antagonists DL-2-amino-5-phosphonopentanoic acid (AP-5) and MK-801 were intrathecally administered to assess their regulatory effects on formalin-induced NK1r internalization and pain behavior. AP-5 had no effect on formalin-induced NK1r internalization, whereas MK-801 produced only a modest reduction. Both antagonists, however, reduced the formalin paw flinching behavior. In subsequent in vitro experiments, perfusion of NMDA in spinal cord slice preparations did not evoke basal release of SP or calcitonin gene-related peptide (CGRP). Likewise, perfusion of NMDA did not enhance capsaicin-evoked release of the two peptides. These results suggest that presynaptic NMDA receptors in the spinal cord play little if any role on the primary afferent release of SP.

Bile Acid-regulated Peroxisome Proliferator-activated Receptor-α (PPARα) Activity Underlies Circadian Expression of Intestinal Peptide Absorption Transporter PepT1/Slc15a1*

PubMed Central

Okamura, Ayako; Koyanagi, Satoru; Dilxiat, Adila; Kusunose, Naoki; Chen, Jia Jun; Matsunaga, Naoya; Shibata, Shigenobu; Ohdo, Shigehiro

2014-01-01

Digested proteins are mainly absorbed as small peptides composed of two or three amino acids. The intestinal absorption of small peptides is mediated via only one transport system: the proton-coupled peptide transporter-1 (PepT1) encoded from the soluble carrier protein Slc15a1. In mammals, intestinal expression of PepT1/Slc15a1 oscillates during the daily feeding cycle. Although the oscillation in the intestinal expression of PepT1/Slc15a1 is suggested to be controlled by molecular components of circadian clock, we demonstrated here that bile acids regulated the oscillation of PepT1/Slc15a1 expression through modulating the activity of peroxisome proliferator-activated receptor α (PPARα). Nocturnally active mice mainly consumed their food during the dark phase. PPARα activated the intestinal expression of Slc15a1 mRNA during the light period, and protein levels of PepT1 peaked before the start of the dark phase. After food intake, bile acids accumulated in intestinal epithelial cells. Intestinal accumulated bile acids interfered with recruitment of co-transcriptional activator CREB-binding protein/p300 on the promoter region of Slc15a1 gene, thereby suppressing PPARα-mediated transactivation of Slc15a1. The time-dependent suppression of PPARα-mediated transactivation by bile acids caused an oscillation in the intestinal expression of PepT1/Slc15a1 during the daily feeding cycle that led to circadian changes in the intestinal absorption of small peptides. These findings suggest a molecular clock-independent mechanism by which bile acid-regulated PPARα activity governs the circadian expression of intestinal peptide transporter. PMID:25016014

Comparison of effect of an increased dosage of vonoprazan versus vonoprazan plus lafutidine on gastric acid inhibition and serum gastrin.

PubMed

Suzuki, Takahiro; Kagami, Takuma; Uotani, Takahiro; Yamade, Mihoko; Hamaya, Yasushi; Iwaizumi, Moriya; Osawa, Satoshi; Sugimoto, Ken; Miyajima, Hiroaki; Furuta, Takahisa

2018-01-01

Vonoprazan, a novel potassium-competitive acid blocker, elicits potent acid inhibition and hypergastrinemia at a dose of 20 mg. Its recommended maintenance dose for gastro-esophageal reflux disease is 10 mg, which is sometimes insufficient for preventing nocturnal acid breakthrough (NAB). Concomitant use of a histamine 2 receptor antagonist (H 2 RA) is effective for NAB. However, further acid inhibition by addition of H2RA has concern of hypergastrinemia again. Lafutidine (H2RA) is known to stimulate somatostatin release. The aim of this study is to compare the levels of acid inhibition and serum gastrin attained by addition of lafutidine to vonoprazan 10 mg with levels after a dose increase of vonoprazan from 10 to 20 mg. Thirteen healthy volunteers underwent 24-h intragastric pH monitoring and serum gastrin measurements on day 7 of three different regimens: vonoprazan 10 mg, vonoprazan 10 mg plus lafutidine 10 mg, and vonoprazan 20 mg. Median pH 4 holding time ratios (range) by vonoprazan 10 mg, vonoprazan 10 mg plus lafutidine 10 mg, and vonoprazan 20 mg were 82% (47-88%), 88% (76-93%), and 99% (95-100%) while those at nighttime from 10 p.m. to 8 a.m. were 94% (29-100%), 100% (95-100%), and 100%, respectively. The incidences of NAB with vonoprazan 10 mg, vonoprazan plus lafutidine, and vonoprazan 20 mg were 38, 8, and 0%, respectively. Respective serum gastrin levels were 420 (173-508), 323 (196-521), and 504 (400-812) pg/ml. Addition of lafutidine 10 mg to vonoprazan 10 mg achieved sufficient acid inhibition, especially at nighttime, without further increase of serum gastrin levels.

Role of Endogenous Cholecystokinin on Growth of Human Pancreatic Cancer

PubMed Central

Matters, Gail L.; McGovern, Christopher; Harms, John F.; Markovic, Kevin; Anson, Krystal; Jayakumar, Calpurnia; Martenis, Melissa; Awad, Christina; Smith, Jill P.

2012-01-01

Cholecystokinin (CCK) and gastrin stimulate growth of pancreatic cancer. Although down regulation of gastrin inhibits growth of pancreatic cancer, the contribution of endogenous CCK to tumor growth is unknown. The purpose of this study was to evaluate the role of endogenous CCK on autocrine growth of pancreatic cancer. Pancreatic cancer cell lines were analyzed for CCK mRNA and peptide expression by real time RT-PCR and radioimmunoassay, respectively. The effect of endogenous CCK on growth was evaluated by treating cancer cells with CCK neutralizing antibodies and by down regulating CCK mRNA by RNAi. Wild type pancreatic cancer cells expressed significantly lower CCK mRNA and peptide levels than gastrin. Neither treatment of pancreatic cancer cells with CCK antibodies nor the down regulation of CCK mRNA and peptide by shRNAs altered growth in vitro or in vivo. Conversely, when gastrin mRNA expression was down regulated, the same cells failed to produce tumors in spite of having sustained levels of endogenous CCK. Pancreatic cancer cells produce CCK and gastrin; however, the autocrine production of gastrin is more important for stimulating tumor growth. PMID:21186400

Discovery of an artificial peptide agonist to the fibroblast growth factor receptor 1c/βKlotho complex from random peptide T7 phage display.

PubMed

Sakamoto, Kotaro; Kawata, Yayoi; Masuda, Yasushi; Umemoto, Tadashi; Ito, Takashi; Asami, Taiji; Takekawa, Shiro; Ohtaki, Tetsuya; Inooka, Hiroshi

2016-11-04

Fibroblast growth factor receptor-1c (FGFR1c)/βKlotho (KLB) complex is a receptor of fibroblast growth factor 21 (FGF21). Pharmacologically, FGF21 shows anti-obesity and anti-diabetic effects upon peripheral administration. Here, we report the development of an artificial peptide agonist to the FGFR1c/KLB heterodimer complex. The peptide, F91-8A07 (LPGRTCREYPDLWWVRCY), was discovered from random peptide T7 phage display and selectively bound to the FGFR1c/KLB complex, but not to FGFR1c and KLB individually. After subsequent peptide dimerization using a short polyethyleneglycol (PEG) linker, the dimeric F91-8A07 peptide showed higher potent agonist activity than that of FGF21 in cultured primary human adipocytes. Moreover, the dimeric peptide led to an expression of the early growth response protein-1 (Egr-1) mRNA in vivo, which is a target gene of FGFR1c. To the best of our knowledge, this is the first report of a FGFR1c/KLB complex-selective artificial peptide agonist. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Brain Natriuretic Peptide Stimulates Lipid Metabolism through Its Receptor NPR1 and the Glycerolipid Metabolism Pathway in Chicken Adipocytes.

PubMed

Huang, H Y; Zhao, G P; Liu, R R; Li, Q H; Zheng, M Q; Li, S F; Liang, Z; Zhao, Z H; Wen, J

2015-11-03

Brain natriuretic peptide (BNP) is related to lipid metabolism in mammals, but its effect and the molecular mechanisms underlying it in chickens are incompletely understood. We found that the level of natriuretic peptide precursor B (NPPB, which encodes BNP) mRNA expression in high-abdominal-fat chicken groups was significantly higher than that of low-abdominal-fat groups. Partial correlations indicated that changes in the weight of abdominal fat were positively correlated with NPPB mRNA expression level. In vitro, compared with the control group, preadipocytes with NPPB interference showed reduced levels of proliferation, differentiation, and glycerin in media. Treatments of cells with BNP led to enhanced proliferation and differentiation of cells and glycerin concentration, and mRNA expression of its receptor natriuretic peptide receptor 1 (NPR1) was upregulated significantly. In cells exposed to BNP, 482 differentially expressed genes were identified compared with controls without BNP. Four genes known to be related to lipid metabolism (diacylglycerol kinase; lipase, endothelial; 1-acylglycerol-3-phosphate O-acyltransferase 1; and 1-acylglycerol-3-phosphate O-acyltransferase 2) were enriched in the glycerolipid metabolism pathway and expressed differentially. In conclusion, BNP stimulates the proliferation, differentiation, and lipolysis of preadipocytes through upregulation of the levels of expression of its receptor NPR1 and key genes enriched in the glycerolipid metabolic pathway.

Structural Insight into Recognition of Plant Peptide Hormones by Receptors.

PubMed

Zhang, Heqiao; Han, Zhifu; Song, Wen; Chai, Jijie

2016-11-07

Secreted signaling peptides or peptide hormones play crucial roles in plant growth and development through coordination of cell-cell communication. Perception of peptide hormones in plants generally relies on membrane-localized receptor kinases (RKs). Progress has recently been made in structural elucidation of interactions between posttranslationally modified peptide hormones and RKs. The structural studies suggest conserved receptor binding and activation mechanisms of this type of peptide hormones involving their conserved C-termini. Here, we review these structural data and discuss how the conserved mechanisms can be used to match peptide-RK pairs. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

The effect of melatonin from slow-release implants on basic and TLR-4-mediated gene expression of inflammatory cytokines and their receptors in the choroid plexus in ewes.

PubMed

Kowalewska, M; Herman, A P; Szczepkowska, A; Skipor, J

2017-08-01

The present study concerns the effect of melatonin from slow-release implants on the expression of genes coding interleukin-1β (Il1B), inerleukin-6 (Il6), tumour necrosis factor α (Tnf) and their receptors: IL-1 receptor type I (Il1r1) and type II (Il1r2), IL-6 receptor (Il6r) and signal transducer (Il6st), TNFα receptor type I (Tnfrsf1a) and II (Tnfrsf1b) and retinoid-related orphan receptor α (RorA) and Rev.-erbα in the ovine choroid plexus (CP) under basal and lipopolysaccharide (LPS)-challenged conditions. Studies were performed on four groups: 1) sham-implanted and placebo-treated, 2) melatonin-implanted (Melovine, 18mg) and placebo-treated, 3) sham-implanted and LPS-treated (400ng/kg of body weight) and 4) melatonin-implanted and LPS-treated. Under basal conditions, we observed weak expression of Tnf, low expression of Il1B, Il6 and Il1r2 and intermediate expression of other cytokines receptors. LPS treatment induced (P≤0.05) expression in all cytokines and their receptors, except Il6r 3h after the administration. Melatonin attenuated (P≤0.05) LPS-induced up-regulation of Il6 but had no effect on other cytokines and their receptors and up-regulated (P≤0.05) Rev.-erbα expression under basal conditions. This indicates that melatonin from slow-release implants suppresses TLR4-mediated Il6 expression in the ovine CP via a mechanism likely involving clock genes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effect of nephrectomy on the rate and pattern of the disappearance of exogenous gastrin in dogs

PubMed Central

Clendinnen, B. Guy; Reeder, David D.; Brandt, E. N.; Thompson, James C.

1973-01-01

Studies of gastrin metabolism were performed in four dogs before and after nephrectomy. Synthetic human gastrin I was infused for two hours and serum samples were obtained at various times during and after infusion. Serum concentrations of gastrin were measured by radioimmunoassay. A two-compartment model was employed to calculate half-lives under each of four experimental conditions, low and high infusion rates, used both before and after nephrectomy. The model half-life was greatly prolonged after nephrectomy at both infusion rates (from 2·54 min to 5·15 min at the low rate, and from 2·85 min to 7·88 min at the high rate). The metabolic clearance rate, an expression of the rate of catabolism during infusion, decreased significantly after nephrectomy at both infusion rates. These observations indicate that the kidney is an important organ for the catabolism of exogenous gastrin. PMID:4719213

Frequency Domain Fluorescent Molecular Tomography and Molecular Probes for Small Animal Imaging

NASA Astrophysics Data System (ADS)

Kujala, Naresh Gandhi

Fluorescent molecular tomography (FMT) is a noninvasive biomedical optical imaging that enables 3-dimensional quantitative determination of fluorochromes distributed in biological tissues. There are three methods for imaging large volume tissues based on different light sources: (a) using a light source of constant intensity, through a continuous or constant wave, (b) using a light source that is intensity modulated with a radio frequency (RF), and (c) using ultrafast pulses in the femtosecond range. In this study, we have developed a frequency domain fluorescent molecular tomographic system based on the heterodyne technique, using a single source and detector pair that can be used for small animal imaging. In our system, the intensity of the laser source is modulated with a RF frequency to produce a diffuse photon density wave in the tissue. The phase of the diffuse photon density wave is measured by comparing the reference signal with the signal from the tissue using a phasemeter. The data acquisition was performed by using a Labview program. The results suggest that we can measure the phase change from the heterogeneous inside tissue. Combined with fiber optics and filter sets, the system can be used to sensitively image the targeted fluorescent molecular probes, allowing the detection of cancer at an early stage. We used the system to detect the tumor-targeting molecular probe Alexa Fluor 680 and Alexa Fluor 750 bombesin peptide conjugates in phantoms as well as mouse tissues. We also developed and evaluated fluorescent Bombesin (BBN) probes to target gastrin-releasing peptide (GRP) receptors for optical molecular imaging. GRP receptors are over-expressed in several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. BBN is a 14 amino acid peptide that is an analogue to human gastrin-releasing peptide that binds specifically to GRPr receptors. BBN conjugates are significant in cancer detection and therapy. The

Changes in gonadotropin-releasing hormone and gonadotropin-releasing hormone receptor gene expression after an increase in carbon monoxide concentration in the cavernous sinus of male wild boar and pig crossbread.

PubMed

Romerowicz-Misielak, M; Tabecka-Lonczynska, A; Koziol, K; Gilun, P; Stefanczyk-Krzymowska, S; Och, W; Koziorowski, M

2016-06-01

Previous studies indicate that there are at least a few regulatory systems involved in photoperiodic synchronisation of reproductive activity, which starts with the retina and ends at the gonadotropin-releasing hormone (GnRH) pulse generator. Recently we have shown indicated that the amount of carbon monoxide (CO) released from the eye into the ophthalmic venous blood depends on the intensity of sunlight. The aim of this study was to test whether changes in the concentration of carbon monoxide in the ophthalmic venous blood may modulate reproductive activity, as measured by changes in GnRH and GnRH receptor gene expression. The animal model used was mature male swine crossbred from wild boars and domestic sows (n = 48). We conducted in vivo experiments to determine the effect of increased CO concentrations in the cavernous sinus of the mammalian perihypophyseal vascular complex on gene expression of GnRH and GnRH receptors as well as serum luteinizing hormone (LH) levels. The experiments were performed during long photoperiod days near the summer solstice (second half of June) and short photoperiod days near the winter solstice (second half of December). These crossbred swine demonstrated a seasonally-dependent marked variation in GnRH and GnRH receptor gene expression and systemic LH levels in response to changes in CO concentration in ophthalmic venous blood. These results seem to confirm the hypothesis of humoral phototransduction as a mechanism for some of bright light's effects in animal chronobiology and the effect of CO on GnRH and GnRH receptor gene expression.

Pancreatic Beta Cells Synthesize Neuropeptide Y and Can Rapidly Release Peptide Co-Transmitters

PubMed Central

Whim, Matthew D.

2011-01-01

Background In addition to polypeptide hormones, pancreatic endocrine cells synthesize a variety of bioactive molecules including classical transmitters and neuropeptides. While these co-transmitters are thought to play a role in regulating hormone release little is known about how their secretion is regulated. Here I investigate the synthesis and release of neuropeptide Y from pancreatic beta cells. Methodology/Principal Findings NPY appears to be an authentic co-transmitter in neonatal, but not adult, beta cells because (1) early in mouse post-natal development, many beta cells are NPY-immunoreactive whereas no staining is observed in beta cells from NPY knockout mice; (2) GFP-expressing islet cells from an NPY(GFP) transgenic mouse are insulin-ir; (3) single cell RT-PCR experiments confirm that the NPY(GFP) cells contain insulin mRNA, a marker of beta cells. The NPY-immunoreactivity previously reported in alpha and delta cells is therefore likely to be due to the presence of NPY-related peptides. INS-1 cells, a beta cell line, are also NPY-ir and contain NPY mRNA. Using the FMRFamide tagging technique, NPY secretion was monitored from INS-1 beta cells with high temporal resolution. Peptide release was evoked by brief depolarizations and was potentiated by activators of adenylate cyclase and protein kinase A. Following a transient depolarization, NPY-containing dense core granules fused with the cell membrane and discharged their contents within a few milliseconds. Conclusions These results indicate that after birth, NPY expression in pancreatic islets is restricted to neonatal beta cells. The presence of NPY suggests that peptide co-transmitters could mediate rapid paracrine or autocrine signaling within the endocrine pancreas. The FMRFamide tagging technique may be useful in studying the release of other putative islet co-transmitters in real time. PMID:21559341

Characterization of atrial natriuretic peptide receptors in brain microvessel endothelial cells

NASA Technical Reports Server (NTRS)

Whitson, P. A.; Huls, M. H.; Sams, C. F.

1991-01-01

Atrial natriuretic peptide (ANP) binding and ANP-induced increases in cyclic guanosine monophosphate (cGMP) levels have been observed in brain microvessels (Chabrier et al., 1987; Steardo and Nathanson, 1987), suggesting that this fluid-regulating hormone may play a role in the fluid homeostasis of the brain. This study was initiated to characterize the ANP receptors in primary cultures of brain microvessel endothelial cells (BMECs). The apparent equilibrium dissociation constant, Kd, for ANP increased from 0.25 nM to 2.5 nM, and the number of ANP binding sites as determined by Scatchard analysis increased from 7,100 to 170,000 sites/cell between 2 and 10 days of culture following monolayer formation. Time- and concentration-dependent studies on the stimulation of cGMP levels by ANP indicated that guanylate cyclase-linked ANP receptors were present in BMECs. The relative abilities of ANP, brain natriuretic peptide (BNP), and a truncated analog of ANP containing amino acids 5-27 (ANP 5-27) to modulate the accumulation of cGMP was found to be ANP greater than BNP much greater than ANP 5-27. Affinity cross-linking with disuccinimidyl suberate and radiolabeled ANP followed by gel electrophoresis under reducing conditions demonstrated a single band corresponding to the 60-70 kD receptor, indicating the presence of the nonguanylate cyclase-linked ANP receptor. Radiolabeled ANP binding was examined in the presence of various concentrations of either ANP, BNP, or ANP 5-27 and suggested that a large proportion of the ANP receptors present in blood-brain barrier endothelial cells bind all of these ligands similarly. These data indicate both guanylate cyclase linked and nonguanylate cyclase linked receptors are present on BMECs and that a higher proportion of the nonguanylate cyclase linked receptors is expressed. This in vitro culture system may provide a valuable tool for the examination of ANP receptor expression and function in blood-brain barrier endothelial cells.

Structural insight into the activation of a class B G-protein-coupled receptor by peptide hormones in live human cells

PubMed Central

Seidel, Lisa; Zarzycka, Barbara; Zaidi, Saheem A; Katritch, Vsevolod; Coin, Irene

2017-01-01

The activation mechanism of class B G-protein-coupled receptors (GPCRs) remains largely unknown. To characterize conformational changes induced by peptide hormones, we investigated interactions of the class B corticotropin-releasing factor receptor type 1 (CRF1R) with two peptide agonists and three peptide antagonists obtained by N-truncation of the agonists. Surface mapping with genetically encoded photo-crosslinkers and pair-wise crosslinking revealed distinct footprints of agonists and antagonists on the transmembrane domain (TMD) of CRF1R and identified numerous ligand-receptor contact sites, directly from the intact receptor in live human cells. The data enabled generating atomistic models of CRF- and CRF(12-41)-bound CRF1R, further explored by molecular dynamics simulations. We show that bound agonist and antagonist adopt different folds and stabilize distinct TMD conformations, which involves bending of helices VI and VII around flexible glycine hinges. Conservation of these glycine hinges among all class B GPCRs suggests their general role in activation of these receptors. DOI: http://dx.doi.org/10.7554/eLife.27711.001 PMID:28771403

Expression of messenger molecules and receptors in rat and human sphenopalatine ganglion indicating therapeutic targets.

PubMed

Steinberg, Anna; Frederiksen, Simona D; Blixt, Frank W; Warfvinge, Karin; Edvinsson, Lars

2016-12-01

Migraine and Cluster Headache (CH) are two primary headaches with severe disease burden. The disease expression and the mechanisms involved are poorly known. In some attacks of migraine and in most attacks of CH, there is a release of vasoactive intestinal peptide (VIP) originating from parasympathetic cranial ganglia such as the sphenopalatine ganglion (SPG). Patients suffering from these diseases are often deprived of effective drugs. The aim of the study was to examine the localization of the botulinum toxin receptor element synaptic vesicle glycoprotein 2A (SV-2A) and the vesicular docking protein synaptosomal-associated protein 25 (SNAP25) in human and rat SPG. Additionally the expression of the neurotransmitters pituitary adenylate cyclase activating polypeptide (PACAP-38), nitric oxide synthase (nNOS), VIP and 5-hydroxttryptamine subtype receptors (5-HT1B,1D,1F) were examined. SPG from adult male rats and from humans, the later removed at autopsy, were prepared for immunohistochemistry using specific antibodies against neurotransmitters, 5-HT1B,1D,1F receptors, and botulinum toxin receptor elements. We found that the selected neurotransmitters and 5-HT receptors were expressed in rat and human SPG. In addition, we found SV2-A and SNAP25 expression in both rat and human SPG. We report that all three 5-HT receptors studied occur in neurons and satellite glial cells (SGCs) of the SPG. 5-HT1B receptors were in addition found in the walls of intraganglionic blood vessels. Recent focus on the SPG has emphasized the role of parasympathetic mechanisms in the pathophysiology of mainly CH. The development of next generation's drugs and treatment of cranial parasympathetic symptoms, mediated through the SPG, can be modulated by treatment with BoNT-A and 5-HT receptor agonists.

Expression and Role of Gonadotropin-Releasing Hormone 2 and Its Receptor in Mammals

PubMed Central

Desaulniers, Amy T.; Cederberg, Rebecca A.; Lents, Clay A.; White, Brett R.

2017-01-01

Gonadotropin-releasing hormone 1 (GnRH1) and its receptor (GnRHR1) drive mammalian reproduction via regulation of the gonadotropins. Yet, a second form of GnRH (GnRH2) and its receptor (GnRHR2) also exist in mammals. GnRH2 has been completely conserved throughout 500 million years of evolution, signifying high selection pressure and a critical biological role. However, the GnRH2 gene is absent (e.g., rat) or inactivated (e.g., cow and sheep) in some species but retained in others (e.g., human, horse, and pig). Likewise, many species (e.g., human, chimpanzee, cow, and sheep) retain the GnRHR2 gene but lack the appropriate coding sequence to produce a full-length protein due to gene coding errors; although production of GnRHR2 in humans remains controversial. Certain mammals lack the GnRHR2 gene (e.g., mouse) or most exons entirely (e.g., rat). In contrast, old world monkeys, musk shrews, and pigs maintain the coding sequence required to produce a functional GnRHR2. Like GnRHR1, GnRHR2 is a 7-transmembrane, G protein-coupled receptor that interacts with Gαq/11 to mediate cell signaling. However, GnRHR2 retains a cytoplasmic tail and is only 40% homologous to GnRHR1. A role for GnRH2 and its receptor in mammals has been elusive, likely because common laboratory models lack both the ligand and receptor. Uniquely, both GnRH2 and GnRHR2 are ubiquitously expressed; transcript levels are abundant in peripheral tissues and scarcely found in regions of the brain associated with gonadotropin secretion, suggesting a divergent role from GnRH1/GnRHR1. Indeed, GnRH2 and its receptor are not physiological modulators of gonadotropin secretion in mammals. Instead, GnRH2 and GnRHR2 coordinate the interaction between nutritional status and sexual behavior in the female brain. Within peripheral tissues, GnRH2 and its receptor are novel regulators of reproductive organs. GnRH2 and GnRHR2 directly stimulate steroidogenesis within the porcine testis. In the female, GnRH2 and its receptor

Increased mast cell expression of PAR-2 in skin inflammatory diseases and release of IL-8 upon PAR-2 activation.

PubMed

Carvalho, Ricardo Filipe da Silva; Nilsson, Gunnar; Harvima, Ilkka Tapani

2010-02-01

Mast cells are increasingly present in the lesional skin of chronic skin inflammatory diseases including psoriasis and basal cell carcinoma (BCC). It has previously been shown that proteinase-activated receptor (PAR)-2 is expressed by mast cells, and tryptase is a potent activator of this receptor. In this study, skin biopsies from both healthy-looking and lesional skin of patients with psoriasis and superficial spreading BCC were collected and the expression of PAR-2 immunoreactivity in tryptase-positive mast cells was analysed. PAR-2 expression was confirmed in vitro in different mast cell populations. Cord-blood derived mast cells (CBMC) were stimulated with a PAR-2 activating peptide, 2-furoyl-LIGRLO-NH(2). Consequently, IL-8 and histamine production was analysed in the supernatants. We observed a significant increase in the percentage of mast cells expressing PAR-2 in the lesional skin of psoriasis and BCC patients compared with the healthy-looking skin. HMC-1.2, LAD-2 and CBMC mast cells all expressed PAR-2 both intracellularly and on the cell surface. CBMC activation with the PAR-2 activating peptide resulted in an increased secretion of IL-8, but no histamine release was observed. Furthermore, both PAR-2 and IL-8 were co-localized to the same tryptase-positive mast cells in the lesional BCC skin. These results show that mast cells express increased levels of PAR-2 in chronic skin inflammation. Also, mast cells can be activated by a PAR-2 agonist to secrete IL-8, a chemokine which can contribute to the progress of inflammation.

Exploring peptide hormones in plants: identification of four peptide hormone-receptor pairs and two post-translational modification enzymes

PubMed Central

MATSUBAYASHI, Yoshikatsu

2018-01-01

The identification of hormones and their receptors in multicellular organisms is one of the most exciting research areas and has lead to breakthroughs in understanding how their growth and development are regulated. In particular, peptide hormones offer advantages as cell-to-cell signals in that they can be synthesized rapidly and have the greatest diversity in their structure and function. Peptides often undergo post-translational modifications and proteolytic processing to generate small oligopeptide hormones. In plants, such small post-translationally modified peptides constitute the largest group of peptide hormones. We initially explored this type of peptide hormone using bioassay-guided fractionation and later by in silico gene screening coupled with biochemical peptide detection, which led to the identification of four types of novel peptide hormones in plants. We also identified specific receptors for these peptides and transferases required for their post-translational modification. This review summarizes how we discovered these peptide hormone–receptor pairs and post-translational modification enzymes, and how these molecules function in plant growth, development and environmental adaptation. PMID:29434080

Exploring peptide hormones in plants: identification of four peptide hormone-receptor pairs and two post-translational modification enzymes.

PubMed

Matsubayashi, Yoshikatsu

2018-01-01

The identification of hormones and their receptors in multicellular organisms is one of the most exciting research areas and has lead to breakthroughs in understanding how their growth and development are regulated. In particular, peptide hormones offer advantages as cell-to-cell signals in that they can be synthesized rapidly and have the greatest diversity in their structure and function. Peptides often undergo post-translational modifications and proteolytic processing to generate small oligopeptide hormones. In plants, such small post-translationally modified peptides constitute the largest group of peptide hormones. We initially explored this type of peptide hormone using bioassay-guided fractionation and later by in silico gene screening coupled with biochemical peptide detection, which led to the identification of four types of novel peptide hormones in plants. We also identified specific receptors for these peptides and transferases required for their post-translational modification. This review summarizes how we discovered these peptide hormone-receptor pairs and post-translational modification enzymes, and how these molecules function in plant growth, development and environmental adaptation.

Role of stress peptides during human pregnancy and labour.

PubMed

Hillhouse, Edward W; Grammatopoulos, Dimitris K

2002-09-01

Premature birth is the major source of perinatal death and disability. Furthermore, the intrauterine health of the baby is important for preventing certain adult diseases. However, the molecular mechanisms driving the onset of human labour remain uncertain, although several key players have been identified. It is becoming clear that there are many pathways to parturition in humans. Stress peptides, in particular placental corticotrophin releasing hormone (CRH) and possibly the related peptide urocortin, appear to play important roles throughout pregnancy. Plasma CRH is a predictor of the duration of human gestation. During most of pregnancy, CRH, acting via specific CRH receptor subtypes, plays a 'protective' role by promoting myometrial quiescence via the generation of cAMP and cGMP, and upregulation of nitric oxide synthase expression. At term, myometrial contractility is enhanced by a complex series of molecular switches, involving the upregulation of oxytocin receptor expression and crosstalk between the oxytocin and CRH receptors. This results in protein kinase C-induced phosphorylation of specific CRH receptor subtypes, with subsequent desensitization and a shift in the intracellular microenvironment to enhance contractility. CRH/urocortin, via specific receptor isoforms, is now able to activate Gq and potentially enhance the oxytocin-driven generation of inositol triphosphate. In addition, CRH/urocortin, via specific CRH receptor subtypes, may generate prostaglandins from the fetal membranes and decidua, play a role in placental vasodilatation and participate in fetal adrenal function and organ maturation. These peptides and receptors are phylogenetically ancient and well preserved across species. They may have evolved as a mechanism to protect against the 'stress' of premature birth.

Comparison of mechanisms of action of luteinizing hormone-releasing hormone (LHRH) antagonist cetrorelix and LHRH agonist triptorelin on the gene expression of pituitary LHRH receptors in rats

PubMed Central

Kovacs, Magdolna; Schally, Andrew V.

2001-01-01

The mechanisms through which luteinizing hormone (LH)-releasing hormone (LHRH) antagonists suppress pituitary gonadotroph functions and LHRH-receptor (LHRH-R) expression are incompletely understood. Consequently, we investigated the direct effect of LHRH antagonist cetrorelix in vitro on the expression of the pituitary LHRH-R gene and its ability to counteract the exogenous LHRH and the agonist triptorelin in the regulation of this gene. We also compared the effects of chronic administration of cetrorelix and triptorelin on the LHRH-R mRNA level and gonadotropin secretion in ovariectomized (OVX) and normal female rats. The exposure of pituitary cells in vitro to 3-min pulses of 1 nM LHRH or 0.1 nM triptorelin for 5 h increased the LHRH-R mRNA level by 77–88%. Continuous perfusion of the cells with 50 nM cetrorelix did not cause any significant changes, but prevented the stimulatory effect of LHRH pulses on the receptor mRNA expression. In OVX rats, 10 days after administration of a depot formulation of cetrorelix, releasing 100 μg of peptide daily, the elevated LHRH-R mRNA level was decreased by 73%, whereas daily injection of 100 μg of triptorelin caused a 41% suppression. In normal female rats, cetrorelix treatment suppressed the LHRH-R mRNA level by 33%, but triptorelin increased it by 150%. The highly elevated serum LH levels in OVX rats and the normal LH concentration of cycling rats were rapidly and completely suppressed by cetrorelix. Triptorelin decreased the serum LH in OVX rats to the precastration level, but had no effect on basal LH in normal rats. Our results confirm that LHRH antagonists, such as cetrorelix, inhibit the gene expression of pituitary LHRH-R indirectly, by counteracting the stimulatory effect of LHRH. A rapid suppression of serum LH by LHRH antagonists would be advantageous in the treatment of sex hormone-dependent tumors and other conditions. PMID:11593037

Immunomodulatory effects of endogenous and synthetic peptides activating opioid receptors.

PubMed

Pomorska, Dorota K; Gach, Katarzyna; Janecka, Anna

2014-01-01

The main role of endogenous opioid peptides is the modulation of pain. Opioid peptides exert their analgesic activity by binding to the opioid receptors distributed widely in the central nervous system (CNS). However, opioid receptors are also found on tissues and organs outside the CNS, including the cells of the immune system, indicating that opioids are capable of exerting additional effects in periphery. Morphine, which is a gold standard in the treatment of chronic pain, is well-known for its immunosuppressive effects. Much less is known about the immunomodulatory effects exerted by endogenous (enkephalins, endorphins, dynorphins and endomorphins) and synthetic peptides activating opioid receptors. In this review we tried to summarize opioid peptide-mediated modulation of immune cell functions which can be stimulatory as well as inhibitory.

Bacterial expression of self-assembling peptide hydrogelators

NASA Astrophysics Data System (ADS)

Sonmez, Cem

For tissue regeneration and drug delivery applications, various architectures are explored to serve as biomaterial tools. Via de novo design, functional peptide hydrogel materials have been developed as scaffolds for biomedical applications. The objective of this study is to investigate bacterial expression as an alternative method to chemical synthesis for the recombinant production of self-assembling peptides that can form rigid hydrogels under physiological conditions. The Schneider and Pochan Labs have designed and characterized a 20 amino acid beta-hairpin forming amphiphilic peptide containing a D-residue in its turn region (MAX1). As a result, this peptide must be prepared chemically. Peptide engineering, using the sequence of MAX1 as a template, afforded a small family of peptides for expression (EX peptides) that have different turn sequences consisting of natural amino acids and amenable to bacterial expression. Each sequence was initially chemically synthesized to quickly assess the material properties of its corresponding gel. One model peptide EX1, was chosen to start the bacterial expression studies. DNA constructs facilitating the expression of EX1 were designed in such that the peptide could be expressed with different fusion partners and subsequently cleaved by enzymatic or chemical means to afford the free peptide. Optimization studies were performed to increase the yield of pure peptide that ultimately allowed 50 mg of pure peptide to be harvested from one liter of culture, providing an alternate means to produce this hydrogel-forming peptide. Recombinant production of other self-assembling hairpins with different turn sequences was also successful using this optimized protocol. The studies demonstrate that new beta-hairpin self-assembling peptides that are amenable to bacterial production and form rigid hydrogels at physiological conditions can be designed and produced by fermentation in good yield at significantly reduced cost when compared to

Prohormone convertase and autocrine growth factor mRNAs are coexpressed in small cell lung carcinoma.

PubMed

Rounseville, M P; Davis, T P

2000-08-01

A hallmark of small cell lung carcinoma (SCLC) is the expression of autocrine growth factors such as neurotensin and gastrin-releasing peptide, which bind to cellular receptors and stimulate cell division. The biological activity of autocrine growth factors requires the concurrent expression of prohormone convertases that cleave the growth factors to their active form, suggesting the expression of these genes is linked in SCLCs. RNase protection assays were used to detect the expression of autocrine growth factor and prohormone convertase mRNAs in a panel of lung cancer cell lines. These mRNAs are coexpressed in SCLC and lung carcinoid cell lines, but not in normal lung epithelium or in non-small cell lung cancers. These findings, together with earlier results from our laboratory, suggest the expression of prohormone convertases has an important role in the development and maintenance of the SCLC phenotype and that autocrine growth factor and prohormone convertase genes respond to a common transcriptional activator in SCLC.

Plant proteases for bioactive peptides release: A review.

PubMed

Mazorra-Manzano, M A; Ramírez-Suarez, J C; Yada, R Y

2017-04-10

Proteins are a potential source of health-promoting biomolecules with medical, nutraceutical, and food applications. Nowadays, bioactive peptides production, its isolation, characterization, and strategies for its delivery to target sites are a matter of intensive research. In vitro and in vivo studies regarding the bioactivity of peptides has generated strong evidence of their health benefits. Dairy proteins are considered the richest source of bioactive peptides, however proteins from animal and vegetable origin also have been shown to be important sources. Enzymatic hydrolysis has been the process most commonly used for bioactive peptide production. Most commercial enzymatic preparations frequently used are from animal (e.g., trypsin and pepsin) and microbial (e.g., Alcalase® and Neutrase®) sources. Although the use of plant proteases is still relatively limited to papain and bromelain from papaya and pineapple, respectively, the application of new plant proteases is increasing. This review presents the latest knowledge in the use and diversity of plant proteases for bioactive peptides release from food proteins including both available commercial plant proteases as well as new potential plant sources. Furthermore, the properties of peptides released by plant proteases and health benefits associated in the control of disorders such as hypertension, diabetes, obesity, and cancer are reviewed.

Structure-activity analysis of synthetic alpha-thrombin-receptor-activating peptides.

PubMed

Van Obberghen-Schilling, E; Rasmussen, U B; Vouret-Craviari, V; Lentes, K U; Pavirani, A; Pouysségur, J

1993-06-15

alpha-Thrombin stimulates G-protein-coupled effectors leading to secretion and aggregation in human platelets, and to a mitogenic response in CCL39 hamster fibroblasts. alpha-Thrombin receptors can be activated by synthetic peptides corresponding to the receptor sequence starting with serine-42, at the proposed cleavage site. We have previously determined that the agonist domain of receptor-activating peptides resides within the five N-terminal residues [Vouret-Craviari, Van Obberghen-Schilling, Rasmussen, Pavirani, Lecocq and Pouysségur (1992) Mol. Biol. Cell. 3, 95-102], although the 7-residue peptide (SFFLRNP) corresponding to the hamster alpha-thrombin receptor was 10 times more potent than the 5-residue peptide for activation of human platelets. In the present study we have analysed the role of individual amino acids in receptor activation by using a series of modified hexa- or hepta-peptides derived from the human alpha-thrombin-receptor sequence. Cellular events examined here include phospholipase C activation, adenylyl cyclase inhibition and DNA synthesis stimulation in non-transformed CCL39 fibroblasts and a tumorigenic variant of that line (A71 cells). Modification of the peptide sequence had similar functional consequence for each of the assays described, indicating that either a unique receptor or pharmacologically indistinguishable receptor subtypes activate distinct G-protein signalling pathways. Furthermore, we found that: (1) the N-terminal serine can be replaced by small or intermediately sized amino acids (+/- hydroxyl groups) without loss of activity. However, its replacement by an aromatic side-chain or omission of the N-terminal amino group severely reduces activity. (2) An aromatic side-chain on the penultimate N-terminal residue appears to play a critical role since phenylalanine in this position can be substituted by tyrosine without complete loss of activity whereas an alanine in its place is not tolerated. (3) Deletion of the first

Structure-activity analysis of synthetic alpha-thrombin-receptor-activating peptides.

PubMed Central

Van Obberghen-Schilling, E; Rasmussen, U B; Vouret-Craviari, V; Lentes, K U; Pavirani, A; Pouysségur, J

1993-01-01

alpha-Thrombin stimulates G-protein-coupled effectors leading to secretion and aggregation in human platelets, and to a mitogenic response in CCL39 hamster fibroblasts. alpha-Thrombin receptors can be activated by synthetic peptides corresponding to the receptor sequence starting with serine-42, at the proposed cleavage site. We have previously determined that the agonist domain of receptor-activating peptides resides within the five N-terminal residues [Vouret-Craviari, Van Obberghen-Schilling, Rasmussen, Pavirani, Lecocq and Pouysségur (1992) Mol. Biol. Cell. 3, 95-102], although the 7-residue peptide (SFFLRNP) corresponding to the hamster alpha-thrombin receptor was 10 times more potent than the 5-residue peptide for activation of human platelets. In the present study we have analysed the role of individual amino acids in receptor activation by using a series of modified hexa- or hepta-peptides derived from the human alpha-thrombin-receptor sequence. Cellular events examined here include phospholipase C activation, adenylyl cyclase inhibition and DNA synthesis stimulation in non-transformed CCL39 fibroblasts and a tumorigenic variant of that line (A71 cells). Modification of the peptide sequence had similar functional consequence for each of the assays described, indicating that either a unique receptor or pharmacologically indistinguishable receptor subtypes activate distinct G-protein signalling pathways. Furthermore, we found that: (1) the N-terminal serine can be replaced by small or intermediately sized amino acids (+/- hydroxyl groups) without loss of activity. However, its replacement by an aromatic side-chain or omission of the N-terminal amino group severely reduces activity. (2) An aromatic side-chain on the penultimate N-terminal residue appears to play a critical role since phenylalanine in this position can be substituted by tyrosine without complete loss of activity whereas an alanine in its place is not tolerated. (3) Deletion of the first

Multiple melanocortin receptors are expressed in bone cells

NASA Technical Reports Server (NTRS)

Zhong, Qing; Sridhar, Supriya; Ruan, Ling; Ding, Ke-Hong; Xie, Ding; Insogna, Karl; Kang, Baolin; Xu, Jianrui; Bollag, Roni J.; Isales, Carlos M.

2005-01-01

Melanocortin receptors belong to the seven transmembrane domain, G-protein coupled family of receptors. There are five members of this receptor family labeled MC1R-MC5R. These receptors are activated by fragments derived from a larger molecule, proopiomelanocortin (POMC) and include ACTH, alpha beta and gamma-MSH and beta-endorphin. Because of in vitro and in vivo data suggesting direct effects of these POMC molecules on bone and bone turnover, we examined bone and bone derived cells for the presence of the various members of the melanocortin receptor family. We report that the five known melanocortin receptors are expressed to varying degrees in osteoblast-like and osteoclastic cells. POMC fragments increased proliferation and expression of a variety of genes in osteoblastic cells. Furthermore, POMC mRNA was detected in osteoclastic cells. These data demonstrate that POMC-derived peptide hormones acting through high affinity melanocortin receptors have specific effects on bone cells. Thus, in addition to the indirect effects of POMC-derived hormones on bone turnover through their modulation of steroid hormone secretion, POMC fragments may have direct and specific effects on bone cell subpopulations.

Activation of TRPM3 by a potent synthetic ligand reveals a role in peptide release

PubMed Central

Held, Katharina; Kichko, Tatjana; De Clercq, Katrien; Klaassen, Hugo; Van Bree, Rieta; Vanherck, Jean-Christophe; Marchand, Arnaud; Reeh, Peter W.; Chaltin, Patrick; Voets, Thomas; Vriens, Joris

2015-01-01

Transient receptor potential (TRP) cation channel subfamily M member 3 (TRPM3), a member of the TRP channel superfamily, was recently identified as a nociceptor channel in the somatosensory system, where it is involved in the detection of noxious heat; however, owing to the lack of potent and selective agonists, little is known about other potential physiological consequences of the opening of TRPM3. Here we identify and characterize a synthetic TRPM3 activator, CIM0216, whose potency and apparent affinity greatly exceeds that of the canonical TRPM3 agonist, pregnenolone sulfate (PS). In particular, a single application of CIM0216 causes opening of both the central calcium-conducting pore and the alternative cation permeation pathway in a membrane-delimited manner. CIM0216 evoked robust calcium influx in TRPM3-expressing somatosensory neurons, and intradermal injection of the compound induced a TRPM3-dependent nocifensive behavior. Moreover, CIM0216 elicited the release of the peptides calcitonin gene-related peptide (CGRP) from sensory nerve terminals and insulin from isolated pancreatic islets in a TRPM3-dependent manner. These experiments identify CIM0216 as a powerful tool for use in investigating the physiological roles of TRPM3, and indicate that TRPM3 activation in sensory nerve endings can contribute to neurogenic inflammation. PMID:25733887

Scorpion venom peptide SPVII promotes irradiated cells proliferation and increases the expression of the IL-3 receptor

PubMed Central

2013-01-01

Background The previous investigation demonstrated the radioprotective efficacy of peptides isolated from the venom of Buthus Martti Karsch. In this study, the effect of isolated scorpion venom peptide II (SVPII) on irradiated M-NFS-60 cells and mouse bone marrow mononuclear cells (BM-MNCs) was observed. The AlamarBlue cell viability assay, a colony-forming unit (CFU) assay, flow cytometry (FCM), immunofluorescence, and Western blotting were used to evaluate cell proliferation, cell cycle progression, and the expression of the IL-3 receptor (IL-3R) protein in non-irradiated and irradiated cells. Results Proliferation of irradiated M-NFS-60 cells was significantly accelerated by SPVII, and this effect was further enhanced by co-application of IL-3. Similarly, SPVII increased the number of BM-MNC CFUs and this proliferative effect was greater in the presence of SVPII plus IL-3. In addition, SPVII significantly altered cell cycle progression; SVPII enhanced the fraction of unirradiated M-NFS-60 cells in S phase and the fraction of irradiated M-NFS-60 cells arrested in G2/M. The expression of IL-3R protein by unirradiated M-NFS-60 cells was enhanced significantly by SVPII, and SVPII-induced IL-3R overexpression was 10-fold greater in irradiated M-NFS-60 cells. Conclusions These results indicated the hematopoietic growth factor (HGF)-like effects of SVPII on irradiated cells, possibly mediated by upregulation of IL-3R. PMID:23835458

A novel Toll like receptor with two TIR domains (HcToll-2) is involved in regulation of antimicrobial peptide gene expression of Hyriopsis cumingii.

PubMed

Ren, Qian; Lan, Jiang-Feng; Zhong, Xue; Song, Xiao-Jun; Ma, Fei; Hui, Kai-Min; Wang, Wen; Yu, Xiao-Qiang; Wang, Jin-Xing

2014-07-01

Animal Toll-like receptors (TLRs) are involved in innate immunity. Toll proteins are generally transmembrane proteins. In this study, an atypical Toll-like receptor (HcToll-2) was identified from the triangle-shell pearl mussel Hyriopsis cumingii, which belongs to phylum Mollusca. Unlike the typical Toll like receptors with extracellular leucine-rich repeats (LRRs), transmembrane, and intracellular Toll/interleukin-1 receptor (TIR) domains, HcToll-2 has two homologous TIR domains located at the C-terminal (designated as HcTIR1 and HcTIR2) and lacks a transmembrane domain. Phylogenetic analysis showed that HcTIR1 was clustered with TIR of sea anemone Toll, and HcTIR2 was clustered with TIR of Drosophila Toll. HcToll-2 mRNA could be detected in the hepatopancreas and was upregulated after challenge with Escherichia coli and Staphylococcus aureus. Recombinant HcLRR protein with GST tag could bind to bacteria and also to LPS and PGN. Over-expression of both HcTIR1 and HcTIR2 induced drosomycin genes in Drosophila S2 cells. RNAi analysis showed that HcToll-2 was required for the expression of theromacin, which is a cysteine-rich antimicrobial peptide (AMP) gene. This research is the first report of an atypical Toll-like receptor HcToll-2 involved in antibacterial immunity through induction of AMP expression. Copyright © 2014 Elsevier Ltd. All rights reserved.

Angiotensin AT1A receptors on leptin receptor-expressing cells control resting metabolism.

PubMed

Claflin, Kristin E; Sandgren, Jeremy A; Lambertz, Allyn M; Weidemann, Benjamin J; Littlejohn, Nicole K; Burnett, Colin M L; Pearson, Nicole A; Morgan, Donald A; Gibson-Corley, Katherine N; Rahmouni, Kamal; Grobe, Justin L

2017-04-03

Leptin contributes to the control of resting metabolic rate (RMR) and blood pressure (BP) through its actions in the arcuate nucleus (ARC). The renin-angiotensin system (RAS) and angiotensin AT1 receptors within the brain are also involved in the control of RMR and BP, but whether this regulation overlaps with leptin's actions is unclear. Here, we have demonstrated the selective requirement of the AT1A receptor in leptin-mediated control of RMR. We observed that AT1A receptors colocalized with leptin receptors (LEPRs) in the ARC. Cellular coexpression of AT1A and LEPR was almost exclusive to the ARC and occurred primarily within neurons expressing agouti-related peptide (AgRP). Mice lacking the AT1A receptor specifically in LEPR-expressing cells failed to show an increase in RMR in response to a high-fat diet and deoxycorticosterone acetate-salt (DOCA-salt) treatments, but BP control remained intact. Accordingly, loss of RMR control was recapitulated in mice lacking AT1A in AgRP-expressing cells. We conclude that angiotensin activates divergent mechanisms to control BP and RMR and that the brain RAS functions as a major integrator for RMR control through its actions at leptin-sensitive AgRP cells of the ARC.

Expression and role of gonadotropin-releasing hormone 2 and its receptor in mammals

USDA-ARS?s Scientific Manuscript database

Gonadotropin-releasing hormone (GnRH1) and its receptor (GnRHR1) drive mammalian reproduction via regulation of the gonadotropins. Yet, a second form of GnRH (GnRH2) and its receptor (GnRHR2) also exist in some mammals. GnRH2 has been completely conserved throughout 500 million years of evolution, s...

International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors.

PubMed

Poyner, David R; Sexton, Patrick M; Marshall, Ian; Smith, David M; Quirion, Remi; Born, Walter; Muff, Roman; Fischer, Jan A; Foord, Steven M

2002-06-01

The calcitonin family of peptides comprises calcitonin, amylin, two calcitonin gene-related peptides (CGRPs), and adrenomedullin. The first calcitonin receptor was cloned in 1991. Its pharmacology is complicated by the existence of several splice variants. The receptors for the other members the family are made up of subunits. The calcitonin-like receptor (CL receptor) requires a single transmembrane domain protein, termed receptor activity modifying protein, RAMP1, to function as a CGRP receptor. RAMP2 and -3 enable the same CL receptor to behave as an adrenomedullin receptor. Although the calcitonin receptor does not require RAMP to bind and respond to calcitonin, it can associate with the RAMPs, resulting in a series of receptors that typically have high affinity for amylin and varied affinity for CGRP. This review aims to reconcile what is observed when the receptors are reconstituted in vitro with the properties they show in native cells and tissues. Experimental conditions must be rigorously controlled because different degrees of protein expression may markedly modify pharmacology in such a complex situation. Recommendations, which follow International Union of Pharmacology guidelines, are made for the nomenclature of these multimeric receptors.

Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Booe, Jason M.; Walker, Christopher S.; Barwell, James

Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind relatedmore » GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. Lastly, the structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.« less

Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor

DOE PAGES

Booe, Jason M.; Walker, Christopher S.; Barwell, James; ...

2015-05-14

Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind relatedmore » GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. Lastly, the structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.« less

Metabotropic GABAB receptors mediate GABA inhibition of acetylcholine release in the rat neuromuscular junction.

PubMed

Malomouzh, Artem I; Petrov, Konstantin A; Nurullin, Leniz F; Nikolsky, Evgeny E

2015-12-01

Gamma-aminobutyric acid (GABA) is an amino acid which acts as a neurotransmitter in the central nervous system. Here, we studied the effects of GABA on non-quantal, spontaneous, and evoked quantal acetylcholine (ACh) release from motor nerve endings. We found that while the application of 10 μM of GABA had no effect on spontaneous quantal ACh release, as detected by the frequency of miniature endplate potentials, GABA reduced the non-quantal ACh release by 57%, as determined by the H-effect value. Finally, the evoked quantal ACh release, estimated by calculating the quantal content of full-sized endplate potentials (EPPs), was reduced by 34%. GABA's inhibitory effect remained unchanged after pre-incubation with picrotoxin, an ionotropic GABAA receptor blocker, but was attenuated following application of the GABAB receptor blocker CGP 55845, which itself had no effect on ACh release. An inhibitor of phospholipase C, U73122, completely prevented the GABA-induced decrease in ACh release. Immunofluorescence demonstrated the presence of both subunits of the GABAB receptor (GABAB R1 and GABAB R2) in the neuromuscular junction. These findings suggest that metabotropic GABAB receptors are expressed in the mammalian neuromuscular synapse and their activation results in a phospholipase C-mediated reduction in the intensity of non-quantal and evoked quantal ACh release. We investigated the effect of gamma-aminobutyric acid (GABA) on neuromuscular transmission. GABA reduced the non-quantal and evoked quantal release of acetylcholine. These effects are mediated by GABAB receptors and are implemented via phospholipase C (PLC) activation. Our findings suggest that in the mammalian neuromuscular synapse, metabotropic GABAB receptors are expressed and their activation results in a reduction in the intensity of acetylcholine release. © 2015 International Society for Neurochemistry.

Opioid Receptor Function Is Regulated by Post-endocytic Peptide Processing*

PubMed Central

Gupta, Achla; Gomes, Ivone; Wardman, Jonathan; Devi, Lakshmi A.

2014-01-01

Most neuroendocrine peptides are generated in the secretory compartment by proteolysis of the precursors at classical cleavage sites consisting of basic residues by well studied endopeptidases belonging to the subtilisin superfamily. In contrast, a subset of bioactive peptides is generated by processing at non-classical cleavage sites that do not contain basic residues. Neither the peptidases responsible for non-classical cleavages nor the compartment involved in such processing has been well established. Members of the endothelin-converting enzyme (ECE) family are considered good candidate enzymes because they exhibit functional properties that are consistent with such a role. In this study we have explored a role for ECE2 in endocytic processing of δ opioid peptides and its effect on modulating δ opioid receptor function by using selective inhibitors of ECE2 that we had identified previously by homology modeling and virtual screening of a library of small molecules. We found that agonist treatment led to intracellular co-localization of ECE2 with δ opioid receptors. Furthermore, selective inhibitors of ECE2 and reagents that increase the pH of the acidic compartment impaired receptor recycling by protecting the endocytosed peptide from degradation. This, in turn, led to a substantial decrease in surface receptor signaling. Finally, we showed that treatment of primary neurons with the ECE2 inhibitor during recycling led to increased intracellular co-localization of the receptors and ECE2, which in turn led to decreased receptor recycling and signaling by the surface receptors. Together, these results support a role for differential modulation of opioid receptor signaling by post-endocytic processing of peptide agonists by ECE2. PMID:24847082

The Effect of Salt Intake and Potassium Supplementation on Serum Gastrin Levels in Chinese Adults: A Randomized Trial

PubMed Central

Wang, Yuan-Yuan; He, Wen-Wen; Liu, Yan-Chun; Lin, Yi-Feng; Hong, Lu-Fei

2017-01-01

Excess dietary salt is strongly correlated with cardiovascular disease, morbidity, and mortality. Conversely, potassium likely elicits favorable effects against cardiovascular disorders. Gastrin, which is produced by the G-cells of the stomach and duodenum, can increase renal sodium excretion and regulate blood pressure by acting on the cholecystokinin B receptor. The aim of our study was to assess the effects of altered salt and potassium supplementation on serum gastrin levels in humans. A total of 44 subjects (38–65 years old) were selected from a rural community in northern China. All subjects were sequentially maintained on a relatively low-salt diet for 7 days (3.0 g/day of NaCl), a high-salt diet for 7 days (18.0 g/day of NaCl), and then a high-salt diet supplemented with potassium for another 7 days (18.0 g/day of NaCl + 4.5 g/day of KCl). The high-salt intake significantly increased serum gastrin levels (15.3 ± 0.3 vs. 17.6 ± 0.3 pmol/L). This phenomenon was alleviated through potassium supplementation (17.6 ± 0.3 vs. 16.5 ± 0.4 pmol/L). Further analyses revealed that serum gastrin was positively correlated with 24 h urinary sodium excretion (r = 0.476, p < 0.001). By contrast, gastrin level was negatively correlated with blood pressure in all dietary interventions (r = −0.188, p = 0.031). The present study indicated that variations in dietary salt and potassium supplementation affected the serum gastrin concentrations in the Chinese subjects. PMID:28420122

The Antimicrobial Peptide Human Beta-Defensin-3 Is Induced by Platelet-Released Growth Factors in Primary Keratinocytes

PubMed Central

Lammel, Justus; Tohidnezhad, Mersedeh; Lippross, Sebastian; Behrendt, Peter; Klüter, Tim; Pufe, Thomas; Cremer, Jochen; Jahr, Holger; Rademacher, Franziska; Gläser, Regine; Harder, Jürgen

2017-01-01

Platelet-released growth factors (PRGF) and its related clinically used formulations (e.g., Vivostat Platelet-Rich Fibrin (PRF®)) contain a variety of chemokines, cytokines, and growth factors and are therefore used to support healing of chronic, hard-to-heal, or infected wounds. Human beta-defensin-3 (hBD-3) is an antimicrobial peptide inducibly expressed in human keratinocytes especially upon wounding. The potent antimicrobial activity of hBD-3 together with its wound closure-promoting activities suggests that hBD-3 may play a crucial role in wound healing. Therefore, we analyzed the influence of PRGF on hBD-3 expression in human primary keratinocytes in vitro. In addition, we investigated the influence of Vivostat PRF on hBD-3 expression in artificially generated human skin wounds in vivo. PRGF treatment of primary keratinocytes induced a significant, concentration- and time-dependent increase in hBD-3 gene expression which was partially mediated by the epidermal growth factor receptor (EGFR). In line with these cell culture data, in vivo experiments revealed an enhanced hBD-3 expression in experimentally produced human wounds after the treatment with Vivostat PRF. Thus, the induction of hBD-3 may contribute to the beneficial effects of thrombocyte concentrate lysates in the treatment of chronic or infected wounds. PMID:28811680

Crystal structure of glucagon-like peptide-1 in complex with the extracellular domain of the glucagon-like peptide-1 receptor.

PubMed

Underwood, Christina Rye; Garibay, Patrick; Knudsen, Lotte Bjerre; Hastrup, Sven; Peters, Günther H; Rudolph, Rainer; Reedtz-Runge, Steffen

2010-01-01

GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic beta-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9-39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Aresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous alpha-helix from Thr(13) to Val(33) when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor.

[Expression of A-type atrial natriuretic peptide receptor in the kidneys of renovascular hypertension rats and its implication].

PubMed

Liu, Rong-Tao; Xiao, Jing; Guo, Hui-Ling; Qiu, Dun-Guo; Yin, Hua-Hu; Wang, Zheng-Rong

2005-11-01

To investigate the expression of A-type atrial natriuretic peptide receptor (ANPR-A) in the kidneys of renovascular hypertension rats and evaluate the significance of the expression. The rat model of renovascular hypertension was produced by constricting one lateral renal artery. After the renal artery being constricted for 4 weeks and 8 weeks, the systolic BP of rats was measured with a manometer using the tail-cuff method. Then, the expression of ANPR-A was respectively detected by immunohistochemical technique in the kidneys of the two-kidney, one-clip (2K1C) rats, and the expression level of ANPR-A was semi-quantitatively measured by Mias-2000 computer image analyzer. At 4 weeks after the artery-constricted operation,the expression of ANPR-A increased significantly in 2K1C hypertensive rat glomeruli and decreased significantly in renal tubules, compared with control (P<0.01), but there was no marked change in medullar collecting tubules. At 8 weeks after the artery-constricted operation, the expression of ANPR-A decreased significantly in 2K1C hypertensive rat renal tubules and medullar collecting tubules, compared with control (P<0.01); however, there was weak expression in glomeruli, and no statistically significant difference was seen when compared with control (P>0.05). The expression of ANPR-A decreased significantly in kidney tissues of renovascular

The Studies on the Gastrin Levels in the Patients with Renal Failure

PubMed Central

Kim, Myung Hwan; Kim, Han Su; Rim, Kyu Sung; Bang, Ik Soo; Kim, Myung Jae; Chang, Rin; Min, Young II

1986-01-01

Fasting and postprandial gastrin levels were measured by radioimmunoassay in serum from 15 patients with renal failure and compared with those in 15 healthy controls. Pre- and posthemodialysis gastrin levels were also measured. The fasting serum gastrin levels and serum gastrin response to a standard meal in the patients with renal failure were significantly higher than those in normal controls. Fasting and meal stimulated gastrin levels were not significantly different in renal failure patients with peptic ulcer when compared with those in renal failure patients without peptic ulcer. There were no statistically significant differences in the serum gastrin levels before and after hemodialysis in patients with renal failure. PMID:15759375

Synthetic non-peptide low molecular weight agonists of the relaxin receptor 1.

PubMed

Agoulnik, Alexander I; Agoulnik, Irina U; Hu, Xin; Marugan, Juan

2017-05-01

Relaxin is a small heterodimeric peptide hormone of the insulin/relaxin superfamily produced mainly in female and male reproductive organs. It has potent antifibrotic, vasodilatory and angiogenic effects and regulates the normal function of various physiological systems. Preclinical studies and recent clinical trials have shown the promise of recombinant relaxin as a therapeutic agent in the treatment of cardiovascular and fibrotic diseases. However, there are the universal drawbacks of peptide-based pharmacology that apply to relaxin: a short half-life in vivo requires its continuous delivery, and there are high costs of production, storage and treatment, as well as the possibility of immune responses. All these issues can be resolved by the development of low non-peptide MW agonists of the relaxin receptors which are stable, bioavailable, easily synthesized and specific. In this review, we describe the discovery and characterization of the first series of such compounds. The lead compound, ML290, binds to an allosteric site of the relaxin GPCR, RXFP1. ML290 shows high activity and efficacy, measured by cAMP response, in cells expressing endogenous or transfected RXFP1. Relaxin-like effects of ML290 were shown in various functional cellular assays in vitro. ML290 has excellent absorption, distribution, metabolism and excretion properties and in vivo stability. The identified series of low MW agonists does not activate rodent RXFP1 receptors and thus, the production of a RXFP1 humanized mouse model is needed for preclinical studies. The future analysis and clinical perspectives of relaxin receptor agonists are discussed. This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc. © 2016 The British Pharmacological Society.

Decoding Corticotropin-Releasing Factor Receptor Type 1 Crystal 
Structures

PubMed Central

Doré, Andrew S.; Bortolato, Andrea; Hollenstein, Kaspar; Cheng, Robert K.Y.; Read, Randy J.; Marshall, Fiona H.

2017-01-01

The structural analysis of class B G protein-coupled receptors (GPCR), cell surface proteins responding to peptide hormones, has until recently been restricted to the extracellular domain (ECD). Cor-ticotropin-releasing factor receptor type 1 (CRF1R) is a class B receptor mediating stress response and also considered a drug target for depression and anxiety. Here we report the crystal structure of the trans-membrane domain of human CRF1R in complex with the small-molecule antagonist CP-376395 in a hex-agonal setting with translational non-crystallographic symmetry. Molecular dynamics and metadynamics simulations on this novel structure and the existing TMD structure for CRF1R provides insight as to how the small molecule ligand gains access to the induced-fit allosteric binding site with implications for the observed selectivity against CRF2R. Furthermore, molecular dynamics simulations performed using a full-length receptor model point to key interactions between the ECD and extracellular loop 3 of the TMD providing insight into the full inactive state of multidomain class B GPCRs. PMID:28183242

Photoaffinity-labeling and fluorescence-distribution studies of gonadotropin-releasing hormone receptors in ovarian granulosa cells.