Search

Accumulated evidence suggests that a functional loop composed of gastrin and cholecystokinin B receptor (CCK-BR) may exist in gastric carcinogenesis. However, this suggestion is not completely supported due to a lack of direct evidence, and the underlying mechanism is not completely understood. Here, we evaluated the effects of gastrin/CCK-BR signaling on the cell growth, invasion, and expression of MMP-2 and VEGF, as well as xenograft growth in vivo. Furthermore, we detected gastrin mRNA content in human gastric cancer tissues, metastatic lymph nodes, and adjacent nontumor tissues. We found that the forced gastrin could promote the proliferation, migration, and invasion of gastric cancer cells by upregulating the expression of MMP-2 and VEGF. Blocking gastrin/CCK-BR signal using either Proglumide, a CCK-BR antagonist, or shRNA against GASTRIN significantly inhibited the gastrin-promoting effects. In vivo study revealed that the tumor growth in nude mice inoculated with gastrin-overexpressed cells was significantly faster than control cells. The gastrin mRNA content in metastatic lymph nodes was higher in patients with gastric cancer than in primary gastric cancer and adjacent nontumor tissues. In conclusion, we provided direct evidence and possible mechanism of gastrin/CCK-BR signaling in the initiation and progression of gastric cancer.

Background: Lipid metabolic reprogramming was considered as a new hallmark of malignant tumors. It has been reported to play a crucial biological role in cell proliferation, energy homeostasis and signal-transduction. However, the important value of lipid metabolism-related genes(LMRGs) in prognostic prediction and the tumor immune microenvironment has not been explored by large sample studies in colorectal cancer(CRC). Methods: In this study, the lipid metabolism status of 1086 CRC samples was analyzed using RNA expression profiles and clinical data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, of which the former was determined as training set and the latter as validation set. The risk signature was constructed by the univariate Cox regression and Least Absolute Shrinkage and Selection Operator(LASSO) COX regression. The patients were stratified into high- and low-risk groups according to the median value of the risk score. Immune and mutation landscape between low- and high-risk CRC patients were also explored. Additionally, we established a nomogram integrating the risk signature and clinical factors to improve risk assessment of CRC patients. Results: A four LMRGs signature, including PROCA1, CCKBR, CPT2 and FDFT1, was constructed to predict the prognosis of CRC. The risk signature as an independent prognostic factor for CRC was associated with a variety of parameters. Survival analysis showed that patients with low risk score had a better prognosis. There were different immune landscapes between low and high-risk CRC patients, especially in monocytes, dendritic cells, M0 and M2-like macrophages. Patients in the low-risk group were more likely to have higher tumor mutation burden, stem cell characteristics and level of PD-L1 expression. In addition, it was found that genes that played crucial biological functions in tumorigenesis (including TP53, PI3K and MUC16) had significant differences in mutation frequency between two groups. Conclusion: A lipid metabolism-related risk signature for predicting the prognosis of CRC was identified in this study. Furthermore, this prognostic signature may be a potential biomarker for predicting the efficacy of chemotherapy and anti-PD-L1 therapy in CRC.

Cholecystokinin receptors, CCKAR and CCKBR, are important neurointestinal peptide hormone receptors and play a vital role in food intake and appetite regulation. Here, we report three crystal structures of the human CCKAR in complex with different ligands, including one peptide agonist and two small-molecule antagonists, as well as two cryo-electron microscopy structures of CCKBR-gastrin in complex with Gi2 and Gq, respectively. These structures reveal the recognition pattern of different ligand types and the molecular basis of peptide selectivity in the cholecystokinin receptor family. By comparing receptor structures in different conformational states, a stepwise activation process of cholecystokinin receptors is proposed. Combined with pharmacological data, our results provide atomic details for differential ligand recognition and receptor activation mechanisms. These insights will facilitate the discovery of potential therapeutics targeting cholecystokinin receptors.

Cholecystokinin (CCK), the most abundant brain neuropeptide, is involved in relevant behavioral functions like memory, cognition, and reward through its interactions with the opioid and dopaminergic systems in the limbic system. CCK excites neurons by binding two receptors, CCK1 and CCK2, expressed at low and high levels in the brain, respectively. Historically, CCK2 receptors have been related to the induction of panic attacks in humans. Disturbances in brain CCK expression also underlie the physiopathology of schizophrenia, which is attributed to the modulation by CCK1 receptors of the dopamine flux in the basal striatum. Despite this evidence, neither CCK2 receptor antagonists ameliorate human anxiety nor CCK agonists have consistently shown neuroleptic effects in clinical trials. A neglected aspect of the function of brain CCK is its neuromodulatory role in mental disorders. Interestingly, CCK is expressed in pivotal inhibitory interneurons that sculpt cortical dynamics and the flux of nerve impulses across corticolimbic areas and the excitatory projections to mesolimbic pathways. At the basal striatum, CCK modulates the excitability of glutamate, the release of inhibitory GABA, and the discharge of dopamine. Here we focus on how CCK may reduce rather than trigger anxiety by regulating its cognitive component. Adequate levels of CCK release in the basal striatum may control the interplay between cognition and reward circuitry, which is critical in schizophrenia. Hence, it is proposed that disturbances in the excitatory/ inhibitory interplay modulated by CCK may contribute to the imbalanced interaction between corticolimbic and mesolimbic neural activity found in anxiety and schizophrenia.

The cholecystokinin-2/gastrin receptor (CCK2 R) is considered a suitable target for the development of radiolabelled antagonists, due to its overexpression in various tumours, but no such compounds are available in clinical use. Therefore, we designed novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugated ligands based on CCK2 R antagonist Z360/nastorazepide. As a proof of concept that CCK2 R antagonistic activity can be retained by extending the Z360/nastorazepide structure using suitable linker, we present herein three compounds containing various PEG linkers synthesised on solid phase and in solution. The antagonistic properties were measured in a functional assay in the A431-CCK2 R cell line (in the presence of agonist G17), with IC50 values of 3.31, 4.11 and 10.4 nM for compounds containing PEG4 , PEG6 and PEG12 , respectively. All compounds were successfully radiolabelled with indium-111, lutetium-177 and gallium-68 (incorporation of radiometal >95 %). The gallium-68-labelled compounds were stable for up to 2 h (PBS, 37 °C). log D7.4 values were determined for indium-111- and gallium-68-labelled compounds, showing improved hydrophilicity compared to the reference compound.

Neuroendocrine neoplasms (NENs) comprise a heterogeneous group of tumors, mainly localized in the gastrointestinal system. What characterizes NENs is the expression of hormone receptors on the tumor cell surface, making them accessible for diagnostic and therapeutic approaches (theranostics) using radiolabelled peptides. Somatostatin receptors subtype-two (SST2) play an important role in NENs since they are overexpressed and homogeneously distributed at the surface of the majority of NENs. Accordingly, targeting SST2 for diagnostic and therapeutic purposes has been established. Current research aims at upregulating its expression by epigenetic treatment or improving its targeting via use of alternative radioligands. In addition, recent data suggest a future role of SST antagonists as a diagnostic tool and a potential therapeutic option. Another promising target is the glucagon-like peptide-1 (GLP-1) receptor. Targeting GLP-1R using exendin-4 (GLP-1 analogue) has a high sensitivity for the localization of the often SST2-negative sporadic insulinomas and insulinomas in the context of multiple endocrine neoplasia type-1. Further options for patients with insufficient expression of SST2 involve metaiodobenzylguanidine (MIBG) and the molecular target C-X-C motif chemokine receptor-4 (CXCR4), which have been evaluated for potential theranostic approach in symptomatic NENs or dedifferentiated tumors. Recently, new targets such as the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the fibroblast activation protein (FAP) have been identified in NENs. Finally, minigastrin – a ligand targeting the cholecystokinin-2 (CCK2) receptors in medullary thyroid carcinoma and foregut neuroendocrine tumors – may improve future management of these diseases with currently limited therapeutic options. This review summarises the current approaches and future challenges of diagnostic and therapeutic evaluations in neuroendocrine neoplasms.

Gastrin, secreted by stomach G cells in response to ingested sodium, stimulates the renal cholecystokinin B receptor (CCKBR) to increase renal sodium excretion. It is not known how dietary sodium, independent of food, can increase gastrin secretion in human G cells. However, fenofibrate (FFB), a peroxisome proliferator-activated receptor-α (PPAR-α) agonist, increases gastrin secretion in rodents and several human gastrin-secreting cells, via a gastrin transcriptional promoter. We tested the following hypotheses: (1.) the sodium sensor in G cells plays a critical role in the sodium-mediated increase in gastrin expression/secretion, and (2.) dopamine, via the D1R and PPAR-α, is involved. Intact human stomach antrum and G cells were compared with human gastrin-secreting gastric and ovarian adenocarcinoma cells. When extra- or intracellular sodium was increased in human antrum, human G cells, and adenocarcinoma cells, gastrin mRNA and protein expression/secretion were increased. In human G cells, the PPAR-α agonist FFB increased gastrin protein expression that was blocked by GW6471, a PPAR-α antagonist, and LE300, a D1-like receptor antagonist. LE300 prevented the ability of FFB to increase gastrin protein expression in human G cells via the D1R, because the D5R, the other D1-like receptor, is not expressed in human G cells. Human G cells also express tyrosine hydroxylase and DOPA decarboxylase, enzymes needed to synthesize dopamine. G cells in the stomach may be the sodium sensor that stimulates gastrin secretion, which enables the kidney to eliminate acutely an oral sodium load. Dopamine, via the D1R, by interacting with PPAR-α, is involved in this process.

Cholecystokinin (CCK) and its receptors are expressed in mammalian cardiomyocytes and are involved in cardiovascular system regulation; however, the exact effect and underlying mechanism of CCK in cardiomyocyte apoptosis remain to be elucidated. We examined whether sulfated CCK octapeptide (CCK-8) protects H9c2 cardiomyoblast cells against angiotensin II (Ang II)-induced apoptosis. The H9c2 cardiomyoblasts were subjected to Ang II with or without CCK-8 and the viability and apoptotic rate were detected using a Cell Counting Kit-8 assay, Hoechst 33342 staining, terminal deoxyribonucleotide transferase-mediated nick-end labeling assays, and flow cytometry. In addition, specific antiapoptotic mechanisms of CCK-8 were investigated using specific CCK1 (Devazepide) or CCK2 (L365260) receptor antagonists, or the PI3K inhibitor LY294002. The expression of CCK, CCK1 receptor, CCK2 receptor, Akt, p-Akt, Bad, p-Bad, Bax, Bcl-2, and caspase-3 were detected by Western blot analysis and real-time polymerase chain reaction. We found that CCK and its receptor messenger RNA (mRNA) and protein are expressed in H9c2 cardiomyoblasts. Ang II-induced increased levels of CCK mRNA and protein expression and decreased levels of CCK1 receptor protein and mRNA. Pretreatment of CCK-8 attenuated Ang II-induced cell toxicity and apoptosis. In addition, pretreatment of H9c2 cells with CCK-8 markedly induced expression of p-Akt, p-bad, and Bcl-2 and decreased the expression levels of Bax and caspase-3. The protective effects of CCK-8 were partly abolished by Devazepide or LY294002. Our results suggest that CCK-8 protects H9c2 cardiomyoblasts from Ang II-induced apoptosis partly via activation of the CCK1 receptor and the phosphatidyqinositol-3 kinase/protein kinase B (PI3K/Akt) signaling pathway.

Cholecystokinin (CCK) increases core body temperature via CCK2 receptors when administered intracerebroventricularly (icv). The mechanisms of CCK-induced hyperthermia are unknown, and it is also unknown whether CCK contributes to the fever response to systemic inflammation. We studied the interaction between central CCK signaling and the cyclooxygenase (COX) pathway. Body temperature was measured in adult male Wistar rats pretreated with intraperitoneal infusion of the nonselective COX enzyme inhibitor metamizol (120 mg/kg) or a selective COX-2 inhibitor, meloxicam or etoricoxib (10 mg/kg for both) and, 30 minutes later, treated with icv CCK (1.7 µg/kg). In separate experiments, CCK-induced neuronal activation (with and without COX inhibition) was studied in thermoregulation- and feeding-related nuclei with c-Fos immunohistochemistry. CCK increased body temperature by ~0.4°C from 10 min post-infusion, which was attenuated by metamizol. CCK reduced the number of c-Fos-positive cells in the median preoptic area (by ~70%) but increased it in the dorsal hypothalamic area and in the rostral raphe pallidus (by ~50% in both); all these changes were all completely blocked with metamizol. In contrast, CCK-induced satiety and neuronal activation in the ventromedial hypothalamus were not influenced by metamizol. CCK-induced hyperthermia was also completely blocked with both selective COX-2 inhibitors studied. Finally, the CCK2 receptor antagonist YM022 (10 µg/kg; icv) attenuated the late phases of fever induced by bacterial lipopolysaccharide (10 µg/kg; intravenously). We conclude that centrally administered CCK causes hyperthermia through changes in the activity of "classical" thermoeffector pathways, and that the activation of COX-2 is required for the development of this response.

Sulfakinin (SK)/cholecystokinin (CCK)-type neuropeptides regulate feeding and digestion in protostomes (e.g. insects) and chordates. Here, we characterised SK/CCK-type signalling for the first time in a non-chordate deuterostome – the starfish Asterias rubens (phylum Echinodermata). In this species, two neuropeptides (ArSK/CCK1, ArSK/CCK2) derived from the precursor protein ArSK/CCKP act as ligands for an SK/CCK-type receptor (ArSK/CCKR) and these peptides/proteins are expressed in the nervous system, digestive system, tube feet, and body wall. Furthermore, ArSK/CCK1 and ArSK/CCK2 cause dose-dependent contraction of cardiac stomach, tube foot, and apical muscle preparations in vitro, and injection of these neuropeptides in vivo triggers cardiac stomach retraction and inhibition of the onset of feeding in A. rubens. Thus, an evolutionarily ancient role of SK/CCK-type neuropeptides as inhibitory regulators of feeding-related processes in the Bilateria has been conserved in the unusual and unique context of the extra-oral feeding behaviour and pentaradial body plan of an echinoderm.

Highlights • Ribosome display used for recombinant antibody selection and scFv generation targeting CCK-BR. • Engineered scFv have stronger affinity and increased tissue penetrability due to smaller size. • Single dose scFv reduces hypersensitivity, anxiety- and depression-like behavior in two models. • Reduced neuronal firing frequency in TG primary neuronal cultures treated in vitro. • CCK-BR scFv is ideal for neuropathic pain with both nociceptive and emotional components., The cholecystokinin B receptor and its neuropeptide ligand are upregulated in chronic neuropathic pain models. Single-chain Fragment variable antibodies were generated as preferred non-opioid targeting therapy blocking the cholecystokinin B receptor to inhibit chronic neuropathic pain models in vivo and in vitro. Engineered antibodies of this type feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their smaller size. More importantly, single-chain Fragment variable antibodies have promising biotherapeutic applications for both nervous and immune systems, now recognized as interactive in chronic pain. A mouse single-chain Fragment variable antibody library recognizing a fifteen amino acid extracellular peptide fragment of the cholecystokinin B receptor was generated from immunized spleens. Ribosome display, a powerful cell-free technology, was applied for recombinant antibody selection. Antibodies with higher affinity, stability, solubility, and binding specificity for cholecystokinin B not A receptor were selected and optimized for in vivo and in vitro efficacy. A single dose of the lead candidate reduced mechanical and cold hypersensitivity in two rodent models of neuropathic pain for at least seven weeks. Continuing efficacy was evident with either intraperitoneal or intranasal dosing. Likewise, the lead single-chain Fragment variable antibody totally prevented development of anxiety- and depression-like behaviors and cognitive deficits typical in the models. Reduction of neuronal firing frequency was evident in trigeminal ganglia primary neuronal cultures treated in vitro with the cholecystokinin B receptor antibody. Immunofluorescent staining intensity in the trigeminal neuron primary cultures was significantly reduced incrementally after overnight binding with increasingly higher dilutions of the single-chain Fragment variable antibody. While it is reported that single-chain Fragment variable antibodies are removed systemically within 2–6 h, Western blot evidence indicates the His-tag marker remained after 7 weeks in the trigeminal ganglia and in the dorsolateral medulla, providing evidence of brain and ganglia penetrance known to be compromised in overactivated states. This project showcases the in vivo efficacy of our lead single-chain Fragment variable antibody indicating its potential for development as a non-opioid, non-addictive therapeutic intervention for chronic pain. Importantly, studies by others have indicated treatments with cholecystokinin B receptor antagonists suppress maintenance and reactivation of morphine dependence in place preference tests while lowering tolerance and dose requirements. Our future studies remain to address these potential benefits that may accompany the cholecystokinin B receptor biological therapy. Both chronic sciatic and orofacial pain can be unrelenting and excruciating, reducing quality of life as well as diminishing physical and mental function. An effective non-opiate, non-addictive therapy with potential to significantly reduce chronic neuropathic pain long term is greatly needed.

Cholecystokinin (CCK) is an appetite-suppressing hormone that acts in the dorsomedial hypothalamus (DMH) in adult rats to suppress food intake. It remains unknown, however, whether CCK has the same affect in young animals, despite the rising prevalence of childhood obesity and drastic need for research in this area. At the synaptic level, CCK has been shown to inhibit putative orexigenic DMH neurons in young male rats by increasing GABA release onto these neurons via a CCK2 receptor and nitric oxide-dependent pathway. Whether this pathway leads to appetite suppression in young rats is not known. We therefore investigated whether intra-DMH administration of CCK, with or without inhibitors of the CCK2 receptor and nitric oxide signaling pathways, affects food intake in young, male, fasted Sprague-Dawley rats. We implanted bilateral guide cannulas into the DMH and allowed animals to recover from the surgery. Animals were then fasted for 24 h, following which they received intra-DMH microinjections of vehicle, CCK-8S, or CCK-8S combined with either LY-225910 (CCK2 receptor antagonist), L-NAME (a nitric oxide synthase inhibitor), or ODQ (a soluble guanylate cyclase inhibitor) and were given access to regular chow. Following a two hour refeeding period during which food intake, latency to feed, and body weight were measured, brains were subsequently removed to confirm cannula placement in the DMH. The effect of CCK on these parameters in rats given a high fat diet were also measured. Here we show that intra-DMH administration of CCK suppresses food intake and body weight in young rats. This effect requires activation of CCK2 receptors and nitric oxide signaling. Finally, CCK has no effect on consumption of a high fat diet when administered into the DMH. Overall, these findings demonstrate a potential pathway through which CCK might suppress appetite in young rats.

SummaryCholecystokinin receptors, CCKAR and CCKBR, are important neuro-intestinal peptide hormone receptors and play a vital role in food intake and appetite regulation. Here we report three crystal structures of the human CCKAR in complex with different ligands, including one peptide agonist and two small-molecule antagonists, as well as two cryo-electron microscopy structures of CCKBR–gastrin in complex with Gi2 and Gq, respectively. These structures reveal the recognition pattern of different ligand types and the molecular basis of peptide selectivity in the cholecystokinin receptor family. By comparing receptor structures in different conformational states, a stepwise activation process of cholecystokinin receptors is proposed. Combined with pharmacological data, our results provide atomic details for differential ligand recognition and receptor activation mechanisms. These insights will facilitate the discovery of potential therapeutics targeting cholecystokinin receptors.

In this study, tumor necrosis factor-alpha (TNF-α) was incubated to primary hypothalamic cell culture of rat as an important inflammatory cytokine and the effect of TNF-α to neurotransmitter receptors of the hypothalamus in vitro by real-time fluorescence quantitative PCR with PCR array for researching the effect of TNF-α to neuroendocrine system was analyzed. The result showed that the expression level of Avpr1a, Gabrr1, Grin1, Htr4, and Tspo 5 genes was obviously up and the other Adrb2, Cckbr, Chrm5, Chrna6, Gabra5, Gabra6, Grm8, Htr2c, Htr3a, Sstr1, and Tacr1 11 genes were down. Especially, Grin1 and Tacr1 were affected obviously, increased 3.12 and decreased 2.76, respectively. So, TNF-α can modulate the expression of many receptors of neurotransmitters of the hypothalamus by the special cell signal transduction pathways and alter the responsibility and exciting of these neurons to neurotransmitters corresponding. Furthermore, these changes can affect many hormone secretion of the hypothalamus and affect neuroendocrine function of rat.

Memory is stored in neural networks via changes in synaptic strength mediated in part by NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here we show that a cholecystokinin (CCK)-B receptor (CCKBR) antagonist blocks high-frequency stimulation-induced neocortical LTP, whereas local infusion of CCK induces LTP. CCK(−/−) mice lacked neocortical LTP and showed deficits in a cue–cue associative learning paradigm; and administration of CCK rescued associative learning deficits. High-frequency stimulation-induced neocortical LTP was completely blocked by either the NMDAR antagonist or the CCKBR antagonist, while application of either NMDA or CCK induced LTP after low-frequency stimulation. In the presence of CCK, LTP was still induced even after blockade of NMDARs. Local application of NMDA induced the release of CCK in the neocortex. These findings suggest that NMDARs control the release of CCK, which enables neocortical LTP and the formation of cue–cue associative memory.

Animals that communicate using sound are found throughout the animal kingdom. Interestingly, in contrast to human vocal learning, most animals can produce species-specific patterns of vocalization without learning them from their parents. This phenomenon is called innate vocalization. The underlying molecular basis of both vocal learning in humans and innate vocalization in animals remains unknown. The crowing of a rooster is also innately controlled, and the upstream center is thought to be localized in the nucleus intercollicularis (ICo) of the midbrain. Here, we show that the cholecystokinin B receptor (CCKBR) is a regulatory gene involved in inducing crowing in roosters. Crowing is known to be a testosterone (T)-dependent behavior, and it follows that roosters crow but not hens. Similarly, T-administration induces chicks to crow. By using RNA-sequencing to compare gene expression in the ICo between the two comparison groups that either crow or do not crow, we found that CCKBR expression was upregulated in T-containing groups. The expression of CCKBR and its ligand, cholecystokinin (CCK), a neurotransmitter, was observed in the ICo. We also showed that crowing was induced by intracerebroventricular administration of an agonist specific for CCKBR. Our findings therefore suggest that the CCK system induces innate vocalization in roosters.

Recently, He et al. reported that CCKB receptors located in the neocortex of the brain when bound to their bound natural ligand, CCK peptides, enhance memory, bringing up the possibility that agonists targeting the CCKB receptor may act as therapeutic agents in diseases in which memory loss is marked as observed in dementia and Alzheimer’s. In this report, we describe the synthesis of novel low-molecular weight benzoamine CCKB receptor agonists. The compounds made in this series were determined to be mostly partial agonists, although some antagonists were identified, as well, capable of triggering calcium release in a cell line that overexpresses the CCKB receptor. Compound 35 demonstrated an EC50 of 0.15 µM in the cell-based assay, but more importantly, several of the compounds, including 35, demonstrated a physiological effect, inducing long-term potentiation in rat brains comparable to the CCK-8 peptide albeit at much higher concentrations. Based on these findings, benzoamines may be the basis for a new series of CCKB receptor agonists in drug-discovery efforts that seek to develop therapeutics to prevent memory loss.

Thomas Abraham,1 Christopher O McGovern,2 Samuel S Linton,2 Zachary Wilczynski,3 James H Adair,3,4 Gail L Matters2 1Departments of Neural and Behavioral Sciences and the Microscopy Imaging Core Facility, The Pennsylvania State University College of Medicine, Hershey, PA, 17033, USA; 2Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, PA, 17033, USA; 3Departments of Materials Science, The Pennsylvania State University, University Park, PA, 16802, USA; 4Department of Biomedical Engineering and Pharmacology, The Pennsylvania State University, University Park, PA, 16802, USACorrespondence: Gail L MattersDepartment of Biochemistry and Molecular Biology, H171, The Pennsylvania State University College of Medicine, PO Box 850, Hershey, PA, 17033, USATel +1 717 531-4098Fax +1 717 531-7072Email gmatters@pennstatehealth.psu.eduPurpose: Accurate tumor identification and staging can be difficult. Aptamer-targeted indocyanine green (ICG)-nanoparticles can enhance near-infrared fluorescent imaging of pancreatic and prostate tumors and could improve early cancer detection. This project explored whether calcium-phosphosilicate nanoparticles, also known as NanoJackets (NJs), that were bioconjugated with a tumor-specific targeting DNA aptamer could improve the non-invasive detection of pancreatic and prostate tumors.Methods: Using in vivo near-infrared optical imaging and ex vivo fluorescence analysis, DNA aptamer-targeted ICG-loaded NJs were compared to untargeted NJs for detection of tumors.Results: Nanoparticles were bioconjugated with the DNA aptamer AP1153, which binds to the CCK-B receptor (CCKBR). Aptamer bioconjugated NJs were not significantly increased in size compared with unconjugated nanoparticles. AP1153-ICG-NJ accumulation in orthotopic pancreatic tumors peaked at 18 h post-injection and the ICG signal was cleared by 36 h with no evidence on uptake by non-tumor tissues. Ex vivo tumor imaging confirmed the aptamer-targeted NJs accumulated to higher levels than untargeted NJs, were not taken up by normal pancreas, exited from the tumor vasculature, and were well-dispersed throughout pancreatic and prostate tumors despite extensive fibrosis. Specificity for AP1153-NJ binding to the CCK-B receptor on pancreatic tumor cells was confirmed by pre-treating tumor-bearing mice with the CCK receptor antagonist proglumide. Proglumide pre-treatment reduced the in vivo tumoral accumulation of AP1153-NJs to levels comparable to that of untargeted NJs.Conclusion: Through specific interactions with CCK-B receptors, tumor-targeted nanoparticles containing either ICG or rhodamine WT were well distributed throughout the matrix of both pancreatic and prostate tumors. Tumor-targeted NJs carrying various imaging agents can enhance tumor detection.Keywords: tumor detection, ICG nanoparticles, aptamer targeting, proglumide

Genetic factors are recognized to contribute to peptic ulcer disease (PUD) and other gastrointestinal diseases, such as gastro-oesophageal reflux disease (GORD), irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Here, genome-wide association study (GWAS) analyses based on 456,327 UK Biobank (UKB) individuals identify 8 independent and significant loci for PUD at, or near, genes MUC1, MUC6, FUT2, PSCA, ABO, CDX2, GAST and CCKBR. There are previously established roles in susceptibility to Helicobacter pylori infection, response to counteract infection-related damage, gastric acid secretion or gastrointestinal motility for these genes. Only two associations have been previously reported for duodenal ulcer, here replicated trans-ancestrally. The results highlight the role of host genetic susceptibility to infection. Post-GWAS analyses for PUD, GORD, IBS and IBD add insights into relationships between these gastrointestinal diseases and their relationships with depression, a commonly comorbid disorder., Genetic factors contribute to peptic ulcer disease (PUD). Here, the authors perform a genome-wide association analysis on PUD in the UK Biobank, highlighting shared architecture with other gastrointestinal disorders and possible causal links with depression.

Hypertensive nephropathy (HN) is a common cause of end-stage renal disease with renal fibrosis; chronic kidney disease is associated with elevated serum gastrin. However, the relationship between gastrin and renal fibrosis in HN is still unknown. We, now, report that mice with angiotensin II (Ang II)-induced HN had increased renal cholecystokinin receptor B (CCKBR) expression. Knockout of CCKBR in mice aggravated, while long-term subcutaneous infusion of gastrin ameliorated the renal injury and interstitial fibrosis in HN and unilateral ureteral obstruction (UUO). The protective effects of gastrin on renal fibrosis can be independent of its regulation of blood pressure, because in UUO, gastrin decreased renal fibrosis without affecting blood pressure. Gastrin treatment decreased Ang II-induced renal tubule cell apoptosis, reversed Ang II-mediated inhibition of macrophage efferocytosis, and reduced renal inflammation. A screening of the regulatory factors of efferocytosis showed involvement of peroxisome proliferator-activated receptor α (PPAR-α). Knockdown of PPAR-α by shRNA blocked the anti-fibrotic effect of gastrin in vitro in mouse renal proximal tubule cells and macrophages. Immunofluorescence microscopy, Western blotting, luciferase reporter, and Cut&tag-qPCR analyses showed that CCKBR may be a transcription factor of PPAR-α, because gastrin treatment induced CCKBR translocation from cytosol to nucleus, binding to the PPAR-α promoter region, and increasing PPAR-α gene transcription. In conclusion, gastrin protects against HN by normalizing blood pressure, decreasing renal tubule cell apoptosis, and increasing macrophage efferocytosis. Gastrin-mediated CCKBR nuclear translocation may make it act as a transcription factor of PPAR-α, which is a novel signaling pathway. Gastrin may be a new potential drug for HN therapy.

Background Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disease characterized by chronic abdominal discomfort and pain. The mechanisms of abdominal pain, as a relevant symptom, in IBS are still unclear. We aimed to explore the key genes and neurobiological changes specially involved in abdominal pain in IBS. Methods Gene expression data (GSE36701) was downloaded from Gene Expression Omnibus database. Fifty-three rectal mucosa samples from 27 irritable bowel syndrome with diarrhea (IBS-D) patients and 40 samples from 21 healthy volunteers as controls were included. Differentially expressed genes (DEGs) between two groups were identified using the GEO2R online tool. Functional enrichment analysis of DEGs was performed on the DAVID database. Then a protein–protein interaction network was constructed and visualized using STRING database and Cytoscape. Results The microarray analysis demonstrated a subset of genes (CCKBR, CCL13, ACPP, BDKRB2, GRPR, SLC1A2, NPFF, P2RX4, TRPA1, CCKBR, TLX2, MRGPRX3, PAX2, CXCR1) specially involved in pain transmission. Among these genes, we identified GRPR, NPFF and TRPA1 genes as potential biomarkers for irritating abdominal pain of IBS patients. Conclusions Overexpression of certain pain-related genes (GRPR, NPFF and TRPA1) may contribute to chronic visceral hypersensitivity, therefore be partly responsible for recurrent abdominal pain or discomfort in IBS patients. Several synapses modification and biological process of psychological distress may be risk factors of IBS.

Ischemic/reperfusion (I/R) injury is the primary cause of acute kidney injury (AKI). Gastrin, a gastrointestinal hormone, is involved in the regulation of kidney function of sodium excretion. However, whether gastrin has an effect on kidney I/R injury is unknown. Here we show that cholecystokinin B receptor (CCKBR), the gastrin receptor, was significantly up-regulated in I/R-injured mouse kidneys. While pre-administration of gastrin ameliorated I/R-induced renal pathological damage, as reflected by the levels of serum creatinine and blood urea nitrogen, hematoxylin and eosin staining and periodic acid-Schiff staining. The protective effect could be ascribed to the reduced apoptosis for gastrin reduced tubular cell apoptosis bothin vivoandin vitro.In vitrostudies also showed gastrin preserved the viability of hypoxia/reoxygenation (H/R)-treated human kidney 2 (HK-2) cells and reduced the lactate dehydrogenase release, which were blocked by CI-988, a specific CCKBR antagonist. Mechanistically, the PI3K/Akt/Bad pathway participates in the pathological process, because gastrin treatment increased phosphorylation of PI3K, Akt and Bad. While in the presence of wortmannin (1 μM), a PI3K inhibitor, the gastrin-induced phosphorylation of Akt after H/R treatment was blocked. Additionally, wortmannin and Akt inhibitor VIII blocked the protective effect of gastrin on viability of HK-2 cells subjected to H/R treatment. These studies reveals that gastrin attenuates kidney I/R injury via a PI3K/Akt/Bad-mediated anti-apoptosis signaling. Thus, gastrin can be considered as a promising drug candidate to prevent AKI.

Control of appetite and feed intake in fish larvae are still largely unexplored. Two of the key players in controlling vertebrate's feed intake are cholecystokinin (CCK) and peptide YY (PYY). Here we investigated the mRNA expression of pyy, cck and cck receptors (cckr) in the brain (head) and gut of Atlantic halibut larvae in response to three consecutive meals. We used Artemia nauplii cysts that are commonly ingested by halibut larvae when present as inert feed, and three water-soluble extracts as attractants to stimulate appetite. Cyst intake was not affected by the use of attractants and overall ingestion rate was low. Differences in mRNA expression of cck and pyy were observed between the halibut larvae that had eaten and those that had not despite readily available feed (cysts), supporting that mechanisms for control of feed intake are at least partly functional. All genes analysed were present in the brain and gut, however the different expression profiles between paralogues suggest potential divergent functions. In the gut, cck2 and pyyb mRNA expression was significantly higher in the larvae that ate cysts compared to larvae that decided to not eat, indicating that these genes play a satiety function in the halibut larvae similar to the general vertebrate scheme. However, cck2, cck2r1, and pyy mRNA expression in the brain were lower in the fed-filled larvae group compared to larvae before eating, which contrasts with the presumable anorectic function of these genes. Further research is required to fully evaluate how PYY and CCK affect the feeding biology in halibut larvae, contributing to formulate inert diets that can stimulate appetite and feed intake.

Reports of the stimulated release of extracellular vesicles (EVs) are few, and the mechanisms incompletely understood. To our knowledge, the possibility that the activation of any one of the multitudes of G‐protein‐coupled receptors (GPCRs) expressed by a single cell‐type might increase EV release has not been explored. Recently, we identified the expression of cholecystokinin (CCK), gastrin, gastrin/cholecystokinin types A and/or B receptors (CCKAR and/or –BR), and the bitter taste receptor, TAS2R14 in the human and mouse placenta. specifically, trophoblast. These GPCR(s) were also expressed in four different human trophoblast cell lines. The current objective was to employ two of these cell lines—JAR choriocarcinoma cells and HTR‐8/SVneo cells derived from first‐trimester human villous trophoblast—to investigate whether CCK, TAS2R14 agonists, and other GPCR ligands would each augment EV release. EVs were isolated from the cell‐culture medium by filtration and ultracentrifugation. The preparations were enriched in small EVs (, Activation of multiple G protein‐coupled receptors stimulated extracellular vesicle release from a single cell‐type trophoblast‐derived cells. Other cell signaling pathways may underlie receptor‐stimulated extracellular vesicle release besides, or in addition to, calcium.

Suanzaoren decoction (SZRT), a classic Chinese herbal prescription, has been used as a treatment for insomnia for more than a thousand years. However, recent studies have found no significant effects of SZRT as a treatment for insomnia caused by gastric discomfort. Herein, we studied the effects of modified Suanzaoren decoction (MSZRD) on gastrointestinal disorder-related insomnia. The main constituents of MSZRD were spinosin (2.21 mg/g) and 6-feruloylspinosin (0.78 mg/g). A pentobarbital-induced animal model of insomnia showed that MSZRD shortened sleep latency and prolonged sleep time of the male Institute of Cancer Research (ICR) mice treated for 7 days with oral MSZRD. Sprague-Dawley male rats were treated daily with oral MSZRD or placebo for 11 days and then deprived of sleep for the last 4 days to establish a model of insomnia. Of note, MSZRD-treated animals had significantly improved body weight, organ index scores, and fecal moisture relative to placebo-treated animals, as well as reduced temperature. Sleep-deprived rats exhibited more exploratory behaviors in an open-field anxiety test; however, this effect was significantly reduced in MSZRD-treated animals. We found that MSZRD treatment decreased gastric acid pH, decreased the production of gastrin, pepsin, and Orexin-A, and increased the expression of MTL and CCK-8. Importantly, serum GABA concentration was increased by treatment with MSZRD, as reflected by a decreased Glu/GABA ratio. Treated animals had increased the expression of GAD1, GABARA1, and CCKBR but decreased the expression of Orexin R1. In summary, these results suggest that MSZRD has soporific and gastroprotective effects that may be mediated by differential expression of CCK-8 and Orexin-A.

Optimal glucose control remains elusive for many patients with diabetes despite current therapies, necessitating the development of novel therapeutic modalities. While the central nervous system (CNS) plays a crucial role in glucose homeostasis, specific CNS targets for therapeutic intervention in diabetes have remained elusive. We recently demonstrated that the cholecystokinin b receptor (CCKbR)-expressing subpopulation of ventromedial hypothalamic nucleus (VMN; VMNCCKBR) neurons not only mediate the counter-regulatory response, but also elevate blood glucose by insulin-independently modulating hepatic glucose production under normal physiologic conditions. To test the potential therapeutic utility of these neurons for the treatment of diabetes, we cre-dependently expressed tetanus toxin in the VMN of Cckbrcre mice to silence VMNCCKBR neurons in streptozotocin (STZ)-induced (insulinopenic; Type 1 model) and leptin-deficient (ob/ob; obese Type 2 model) mice. While STZ-treated control mice became hyperglycemic and lost approximately 20% of their body weight over two weeks, VMNCCKBR-silenced mice were protected from these effects of STZ, despite similar hormone levels in STZ-treated control and CCKBRTTmice. Similarly, silencing of VMNCCKBR neurons in ob/ob mice normalized fasting and ad libitum-fed blood glucose despite identical food intake, body weight and body composition in control and VMNCCKBR-silenced mice. While VMNCCKBR-silenced ob/ob mice exhibited no alterations in glucose or insulin tolerance compared to controls, glucose clamps demonstrated decreased hepatic glucose output in the VMNCCKBR-silenced animals. These data suggest that VMNCCKBR neurons represent potential targets to reduce hepatic glucose production and ameliorate hyperglycemia in diabetes. Disclosure A. Affinati: Research Support; Self; Novo Nordisk Inc. J. Flak: None. N. Bozadjieva: None. N. Kodur: None. C.M. Cras-Méneur: None. D.A. Sandoval: Research Support; Self; MedImmune, Novo Nordisk A/S. M.G. Myers: Research Support; Self; AstraZeneca, Novo Nordisk Inc. Funding National Institutes of Health (5T32DK7245-42, 1F32DK122660-01)

The lungs and the immune and nervous systems functionally interact to respond to respiratory environmental exposures and infections. The lungs are innervated by vagal sensory neurons of the jugular and nodose ganglia, fused together in smaller mammals as the jugular-nodose complex (JNC). Whereas the JNC shares properties with the other sensory ganglia, the trigeminal (TG) and dorsal root ganglia (DRG), these sensory structures express differential sets of genes that reflect their unique functionalities. Here, we used RNA sequencing (RNA-seq) in mice to identify the differential transcriptomes of the three sensory ganglia types. Using a fluorescent retrograde tracer and fluorescence-activated cell sorting, we isolated a defined population of airway-innervating JNC neurons and determined their differential transcriptional map after pulmonary exposure to lipopolysaccharide (LPS), a major mediator of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) after infection with gram-negative bacteria or inhalation of organic dust. JNC neurons activated an injury response program, leading to increased expression of gene products such as the G protein-coupled receptor Cckbr, inducing functional changes in neuronal sensitivity to peptides, and Gpr151, also rapidly induced upon neuropathic nerve injury in pain models. Unique JNC-specific transcripts, present at only minimal levels in TG, DRG, and other organs, were identified. These included TMC3, encoding for a putative mechanosensor, and urotensin 2B, a hypertensive peptide. These findings highlight the unique properties of the JNC and reveal that ALI/ARDS rapidly induces a nerve injury-related state, changing vagal excitability.

Gastrin and cholecystokinin (CCK) are hormones released from endocrine cells in the antral stomach (gastrin), the duodenum, and the jejunum (CCK). Recent reports, based on secretion experiments in an enteroendocrine cell line (NCI‐H716) and gastrin receptor expression in proglucagon‐expressing cells from the rat colon, suggested that gastrin could be a regulator of glucagon‐like peptide‐1 (GLP‐1) secretion. To investigate these findings, we studied the acute effects of CCK‐8 (a CCK1/CCK2 (gastrin) receptor agonist) and gastrin‐17 (a CCK2(gastrin) receptor agonist) in robust ex vivo models: the isolated perfused rat small intestine and the isolated perfused rat colon. Small intestines from Wistar rats (n = 6), were perfused intraarterially over 80 min. During the perfusion, CCK (1 nmol/L) and gastrin (1 nmol/L) were infused over 10‐min periods separated by washout/baseline periods. Colons from Wistar rats (n = 6) were perfused intraarterially over 100 min. During the perfusion, CCK (1 nmol/L), vasoactive intestinal peptide (VIP) (10 nmol/L), and glucose‐dependent insulinotropic polypeptide (GIP) (1 nmol/L) were infused over 10‐min periods separated by washout/baseline periods. In the perfused rat small intestines neither CCK nor gastrin stimulated the release of GLP‐1 or neurotensin. In the perfused rat colon, neither CCK or VIP stimulated GLP‐1 or peptide YY (PYY) release, but GIP stimulated both GLP‐1 and PYY release. In both sets of experiments, bombesin, a gastrin‐releasing peptide analog, served as a positive control. Our findings do not support the suggestion that gastrin or CCK participate in the acute regulation of intestinal GLP‐1 secretion, but that GIP may play a role in the regulation of hormone secretion from the colon., Recent reports, based on secretion experiments in an enteroendocrine cell line (NCI‐H716) and gastrin receptor expression in proglucagon‐expressing cells from the rat colon, suggested that gastrin could be a regulator of glucagon‐like peptide‐1 (GLP‐1) secretion. To investigate these findings, we studied the acute effects of CCK‐8 (a CCK1/CCK2 (gastrin) receptor agonist) and gastrin‐17 (a CCK2(gastrin) receptor agonist) in the isolated perfused rat small intestine and the isolated perfused rat colon. Our findings do not support the suggestion that gastrin or CCK participate in the acute regulation of intestinal GLP‐1 secretion, but that GIP, unexpectedly, may play a role in the regulation of hormone secretion from the colon.

Background & Aims In patients with autoimmune atrophic gastritis and achlorhydria, hypergastrinemia is associated with the development of type 1 gastric neuroendocrine tumors (gNETs). Twelve months of treatment with netazepide (YF476), an antagonist of the cholecystokinin B receptor (CCKBR or CCK2R), eradicated some type 1 gNETs in patients. We investigated the mechanisms by which netazepide induced gNET regression using gene expression profiling. Methods We obtained serum samples and gastric corpus biopsy specimens from 8 patients with hypergastrinemia and type 1 gNETs enrolled in a phase 2 trial of netazepide. Control samples were obtained from 10 patients without gastric cancer. We used amplified and biotinylated sense-strand DNA targets from total RNA and Affymetrix (Thermofisher Scientific, UK) Human Gene 2.0 ST microarrays to identify differentially expressed genes in stomach tissues from patients with type 1 gNETs before, during, and after netazepide treatment. Findings were validated in a human AGSGR gastric adenocarcinoma cell line that stably expresses human CCK2R, primary mouse gastroids, transgenic hypergastrinemic INS-GAS mice, and patient samples. Results Levels of pappalysin 2 (PAPPA2) messenger RNA were reduced significantly in gNET tissues from patients receiving netazepide therapy compared with tissues collected before therapy. PAPPA2 is a metalloproteinase that increases the bioavailability of insulin-like growth factor (IGF) by cleaving IGF binding proteins (IGFBPs). PAPPA2 expression was increased in the gastric corpus of patients with type 1 gNETs, and immunohistochemistry showed localization in the same vicinity as CCK2R-expressing enterochromaffin-like cells. Up-regulation of PAPPA2 also was found in the stomachs of INS-GAS mice. Gastrin increased PAPPA2 expression with time and in a dose-dependent manner in gastric AGSGR cells and mouse gastroids by activating CCK2R. Knockdown of PAPPA2 in AGSGR cells with small interfering RNAs significantly decreased their migratory response and tissue remodeling in response to gastrin. Gastrin altered the expression and cleavage of IGFBP3 and IGFBP5. Conclusions In an analysis of human gNETS and mice, we found that gastrin up-regulates the expression of gastric PAPPA2. Increased PAPPA2 alters IGF bioavailability, cell migration, and tissue remodeling, which are involved in type 1 gNET development. These effects are inhibited by netazepide., Graphical abstract

Rationale: A high tumor-to-healthy-tissue uptake ratio of radiolabeled ligands is an essential prerequisite for safe and effective peptide receptor radionuclide therapy (PRRT). In the present study, we searched for novel opportunities to increase tumor-specific uptake of the radiolabeled minigastrin analogue [177Lu]Lu-DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 ([177Lu]Lu-PP-F11N), that targets the cholecystokinin B receptor (CCKBR) in human cancers. Methods: A kinase inhibitor library screen followed by proliferation and internalization assays were employed to identify compounds which can increase uptake of [177Lu]Lu-PP-F11N in CCKBR-transfected human epidermoid carcinoma A431 cells and natural CCKBR-expressing rat pancreatic acinar AR42J cells. Western blot (WB) analysis verified the inhibition of the signaling pathways and the CCKBR level, whereas the cell-based assay analyzed arrestin recruitment. Biodistribution and SPECT imaging of the A431/CCKBR xenograft mouse model as well as histological analysis of the dissected tumors were used for in vivo validation. Results: Our screen identified the inhibitors of mammalian target of rapamycin complex 1 (mTORC1), which increased cell uptake of [177Lu]Lu-PP-F11N. Pharmacological mTORC1 inhibition by RAD001 and metformin increased internalization of [177Lu]Lu-PP-F11N in A431/CCKBR and in AR42J cells. Analysis of protein lysates from RAD001-treated cells revealed increased levels of CCKBR (2.2-fold) and inhibition of S6 phosphorylation. PP-F11N induced recruitment of β-arrestin1/2 and ERK1/2 phosphorylation. In A431/CCKBR-tumor bearing nude mice, 3 or 5 days of RAD001 pretreatment significantly enhanced tumor-specific uptake of [177Lu]Lu-PP-F11N (ratio [RAD001/Control] of 1.56 or 1.79, respectively), whereas metformin treatment did not show a significant difference. Quantification of SPECT/CT images confirmed higher uptake of [177Lu]Lu-PP-F11N in RAD001-treated tumors with ratios [RAD001/Control] of average and maximum concentration reaching 3.11 and 3.17, respectively. HE staining and IHC of RAD001-treated tumors showed a significant increase in necrosis (1.4% control vs.10.6% of necrotic area) and the reduction of proliferative (80% control vs. 61% of Ki67 positive cells) and mitotically active cells (1.08% control vs. 0.75% of mitotic figures). No significant difference in the tumor vascularization was observed after five-day RAD001 or metformin treatment. Conclusions: Our data demonstrates, that increased CCKBR protein level by RAD001 pretreatment has the potential to improve tumor uptake of [177Lu]Lu-PP-F11N and provides proof-of-concept for the development of molecular strategies aimed at enhancing the level of the targeted receptor, to increase the efficacy of PRRT and nuclear imaging., Theranostics, 10 (24), ISSN:1838-7640

Pain is associated with negative emotions such as anxiety, but the underlying neurocircuitry and modulators of the association of pain and anxiety remain unclear. The neuropeptide cholecystokinin (CCK) has both pronociceptive and anxiogenic properties, so we explored the role of CCK in anxiety and nociception in the central amygdala (CeA), a key area in control of emotions and descending pain pathways. Local infusion of CCK into the CeA of control rats increased anxiety, as measured in the light-dark box test, but had no effect on mechanical sensitivity. By contrast, intra-CeA CCK infusion 4 days after Complete Freund's Adjuvant (CFA) injection into the hindpaw resulted in analgesia, but also in loss of its anxiogenic capacity. Inflammatory conditions induced changes in the CeA CCK signaling system with an increase of CCK immunoreactivity and a decrease in CCK1, but not CCK2, receptor mRNA. In CFA rats, patch-clamp experiments revealed that CCK infusion increased CeA neuron excitability. It also partially blocked the discharge of wide dynamic range neurons in the dorsal spinal cord. These effects of CCK on CeA and spinal neurons in CFA rats were mimicked by the specific CCK2 receptor agonist, gastrin. This analgesic effect was likely mediated by identified CeA neurons projecting to the periaqueductal gray matter that express CCK receptors. Together, our data demonstrate that intra-CeA CCK infusion activated a descending CCK2 receptor-dependent pathway that inhibited spinal neuron discharge. Thus, persistent pain induces a functional switch to a newly identified analgesic capacity of CCK in the amygdala, indicating central emotion-related circuit controls pain transmission in spinal cord.

Background Gastrin is an important gastrointestinal hormone produced primarily by G-cells in the antrum of the stomach. It normally regulates gastric acid secretion and is implicated in a number of human disease states, but how its function affects breast cancer (BC) development is not documented. The current study investigated the suppressive effects of gastrin on BC and its underlying mechanisms. Methods Serum levels of gastrin were measured by enzyme-linked immunosorbent assay (ELISA) and correlation between gastrin level and development of BC was analyzed by chi-square test. Inhibitory effects of gastrin on BC were investigated by CCK-8 assay and nude mice models. Expressions of CCKBR/ERK/P65 in BC patients were determined through immunohistochemistry (IHC) and Western blot. Survival analysis was performed using the log-rank test. Results The results indicated that the serum level of gastrin in BC patients was lower compared with normal control. Cellular and molecular experiments indicated that reduction of gastrin is associated with inactivation of cholecystokinin B receptor (CCKBR)/ERK/P65 signaling in BC cells which is corresponding to molecular type of estrogen receptor (ER) positive BC. Furthermore, we found that low expression of gastrin/CCKBR/ERK /P65 was correlated to worse prognosis in BC patients. Gastrin or ERK/P65 activators inhibited ER+ BC through CCKBR-mediated activation of ERK/P65. Moreover, combination treatment with gastrin and tamoxifen more efficiently inhibited ER+ BC than tamoxifen alone. Conclusions We concluded that low serum gastrin is related to increased risk of ER+ BC development. The results also established that CCKBR/ERK/P65 signaling function is generally tumor suppressive in ER+ BC, indicating therapies should focus on restoring, not inhibiting, CCKBR/ERK/P65 pathway activity. Electronic supplementary material The online version of this article (10.1186/s12885-018-4717-7) contains supplementary material, which is available to authorized users.

Aim: Cholecystokinin (CCK) participates in the storage of dietary triglycerides in white adipose tissue (WAT). Our goal was to characterize, both in subcutaneous (Sc-WAT) and visceral WAT (Vis-WAT), the functional expression of the two known CCK receptors, CCK-1 (CCK-1R) and CCK-2 (CCK-2R), as well as of CCK. Main methods: Gene and protein expression was assessed in different cell types of rat and human WAT by means of RT-PCR and western-blot, respectively. The functionality of CCK-Rs was tested by quantifying protein kinase B (Akt) phosphorylation after treatment of pre-adipocytes with the bioactive fragment of CCK, CCK-8. The CCK receptor subtype involved in Akt phosphorylation was investigated by using selective CCK-1R (SR-27,897) and CCK-2R antagonists (L-365,260). Key findings: In rats, CCK-1R (Cckar) and CCK-2R (Cckbr) gene expression was detected in the two types of WAT analyzed as well as in isolated adipocytes, mesenchymal stem cells and pre-adipocytes. CCK-1R and CCK-2R proteins were identified in adipocytes and, to a minor extent, in pre-adipocytes. In addition, CCK-2R were detected in subcutaneous mesenchymal stem cells. Gene expression of the CCK precursor preproCCK as well as CCK immunoreactivity were also found in Sc-WAT and Vis-WAT. In human WAT, CCK gene expression as well as CCK-2Rs and CCK were also identified. CCK-8 evoked Akt phosphorylation in rat pre-adipocytes, and this effect was antagonized by SR-27,897 and L-365,260. Significance: Our data show that both human and rat WAT express a complete CCK system, and suggest that CCK may have an autocrine/paracrine role in regulating adipose tissue biology.

Panic disorder is a widespread socially significant disease, which genetic nature is extremely poorly known. The gene of this neuropeptide (CCK) and its receptors (CCKAR, CCK2R) have being actively studied since the discovery of panicogenic properties of cholecystokinin. The purpose of this work was to estimate the degree of incidence of seven single nucleotide substitutions in the CCK, CCKAR and CCKBR genes in the population of patients diagnosed with panic disorder and a control population consisting of unexamined residents of the Moscow region. A significant increase in the degree of incidence of the T allele of the single nucleotide substitution 109C/T (rs1805000) in the CCKBR gene was identified in the patient population as compared with the controls, prompting suggestions that this substitution is involved in the aetiology of panic disorder. It also demonstrated the association of the combination of alleles -36T CCK, -128T CCKAR (rs11571842 and rs1800908, respectively) with the development of a panic disorder.

Activation of the cholecystokinin type B receptor (CCKBR) by cholecystokinin octapeptide (CCK-8) inhibits opioid analgesia. Chronic opiate treatment leads to an increase in the CCK-8 concentration and thus enhances the antagonism of CCK-8 against opioid analgesia; the underlying molecular mechanisms remain of great interest. In the present study, we validated the colocalization of the μ-opioid receptor (MOR) and CCKBR in pain signal transmission-related spinal cord dorsal horn and dorsal root ganglion neurons of rats. Co-immunoprecipitation (Co-IP) and fluorescence lifetime-imaging-microscopy-based fluorescence resonance energy transfer (FLIM-FRET) assays showed that MOR heteromerized with CCKBR directly in transfected HEK293 cells. Combined with MOR mutant construction, the third transmembrane domain of MOR (TM3MOR) was demonstrated to participate in heteromerization with CCKBR. Receptor ligand binding, ERK phosphorylation and cAMP assays showed that MOR heteromerization with CCKBR weakened the activity of MOR. A cell-penetrating interfering peptide consisting of TM3MOR and TAT (a transactivator of HIV-1) sequences from the N terminal to the C terminal disrupted the MOR–CCKBR interaction and restored the activity of MOR in transfected HEK293 cells. Furthermore, intrathecal application of the TM3MOR-TAT peptide alleviated CCK-8-injection-induced antagonism to morphine analgesia in rats. These results suggest a new molecular mechanism for CCK-8 antagonism to opioid analgesia in terms of G-protein-coupled receptor (GPCR) interaction through direct heteromerization. Our study may provide a potential strategy for pain management with opioid analgesics., Pain medicine: Boosting the potency of opioid analgesics A hormone known to weaken the pain-relieving effects of opioid drugs does so because of interaction between the hormone receptor and the opioid receptor. A team from Peking University in Beijing, China, led by Li Su and You Wan showed that an opioid receptor and a receptor that is activated by a peptide hormone involved in digestion are both found in rat neurons of the spinal cord and spinal ganglion. They demonstrated in human cells that the two receptors could form a single structural complex, and that this process weakened the ability of the opioid receptor to respond to drugs such as morphine. A decoy peptide that disrupted the interaction between the receptors boosted the analgesic effects of morphine in rats. Similar strategies could help with pain management.

Allele frequencies of three substitutions in the CCK (rs42891945 and rs42891946) and CCKBR (rs42670352) genes were identified in three Russian cattle breeds: Holstein (dairy), Bestuzhev (dual-purpose), and Kalmyk (beef). The most promising for further associative studies is the rs42670352 substitution in the CCKBR gene.

In some chronic primary pain conditions such as temporomandibular disorder (TMD) and fibromyalgia syndrome (FMS), mild or chronic stress enhances pain. TMD and FMS often occur together, but the underlying mechanisms are unclear. The purpose of this study was to investigate the role of cholecystokinin (CCK) in the spinal cord in somatic hyperalgesia induced by orofacial inflammation combined with stress. Somatic hyperalgesia was detected by the thermal withdrawal latency and mechanical withdrawal threshold. The expression of CCK(1) receptors, CCK(2) receptors, ERK1/2 and p-ERK1/2 in the spinal cord was examined by Western blot. After the stimulation of orofacial inflammation combined with 3 day forced swim, the expression of CCK(2) receptors and p-ERK1/2 protein in the L4-L5 spinal dorsal horn increased significantly, while the expression of CCK(1) receptors and ERK1/2 protein remained unchanged. Intrathecal injection of the CCK(2) receptor antagonist YM-022 or mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor PD98059 blocked somatic hyperalgesia induced by orofacial inflammation combined with stress. Intrathecal administration of the MEK inhibitor blocked somatic sensitization caused by the CCK receptor agonist CCK8. The CCK(2) receptor antagonist YM-022 significantly reduced the expression of p-ERK1/2. These data indicate that upregulation of CCK(2) receptors through the MAPK pathway contributes to somatic hyperalgesia in this comorbid pain model. Thus, CCK(2) receptors and MAPK pathway may be potential targets for the treatment of TMD comorbid with FMS.

Alcohol use disorder or alcoholism is characterized by uncontrollable alcohol use and intoxication, as well as a heightened state of anxiety after alcohol withdrawal. Ethanol-associated stimuli also drive the urge to drink by means of classical conditioning. Alcoholism has been considered a dopamine (DA) dysregulation syndrome that involves the activity of the central amygdala circuitry of anxiety. Cholecystokinin (CCK) is the most abundant neuropeptide in the mammal brain, where it activates two receptors, CCK1 and CCK2. Genetic evidence relates CCK1 receptors to alcoholism in humans. CCK2 activity has been associated with the onset of human anxiety. CCK modulates DA release in the nucleus accumbens (NAc) and it is expressed in the γ-aminobutyric acid (GABA)-expressing basket interneurons in the cerebral cortex. CCK interacts with serotonin (5-HT) neurotransmission through 5-HT3 receptors to regulate mesocorticolimbic pathways and with GABA to attenuate anxiety in the amygdala. Finally, CCK stimulates the release of orexins and oxytocin in the hypothalamus, two relevant hypothalamic neuropeptides involved in signaling satiety for ethanol and well-being respectively. Given the "dimmer-switch" function of endogenous CCK in the neurotransmission by 5-HT, DA, GABA, and glutamate in normal and pathological behaviors (Ballaz and Bourin, 2020), we hypothesize that CCK adjusts functioning of the reward and anxiety circuitries altered by ethanol. This review gathers data supporting this hypothesis, and suggests mechanisms underlying a role for endogenous CCK in alcoholism.

A neurochemical hypothesis of acupuncture analgesia suggests that the pain relief effect of acupuncture is primarily due to activation of a central endorphin system. It has been shown that the primary afferent sensory fibers, a mesolymbic neural circuit, and a descending inhibitory pathway are critical in acupuncture analgesia. The therapeutic effects of electroacupuncture (EA) and related techniques, such as transcutaneous electroacupoint stimulation (TEAS), are frequency-dependent: different frequencies of EA activate different brain regions and release different neuropeptides. EA and TEAS have been used successfully to treat heroin addiction. Activation of endorphin gene expression and release by TEAS can explain the dramatic attenuation of withdrawal syndrome and prolongation of retention time during and after detoxification treatment in patients who are addicted to heroin. However, repeated EA at high intensity should be avoided because it can induce a gradual loss of the analgesic effect. Opioid-receptor desensitization occurs and is manifested as decreased ligand-binding affinity and second-messenger detachment. Repeated large doses of morphine induce morphine tolerance. Cross-tolerance between morphine and EA suggests similar underlying mechanisms. Recent studies have demonstrated that excessive activation of cholecystokinin (CCK), an antiopioid peptide, appears to be responsible. CCK-receptor subtype B (CCKBR) and opioid μ-receptor are co-expressed in the dorsal-horn neurons. Activation of CCKBR promotes formation of heteromerization of morphine-receptor and CCKBR. Interaction of the third transmembrane domain between the 2 receptors resulted in the reduced binding affinity of the opioid receptor.

Gastric cancer is an important disease due to its high mortality. Despite the decline in frequency, most cases are discovered late in its course, and most of the cancer patients die within a few years of diagnosis. In addition to Helicobacter pylori gastritis, gastrin is considered an important factor in the development of this disease, and thus, cholecystokinin-B receptor (CCKBR) becomes of interest. The aim of our study was to explore whether CCKBR is expressed in stomach cancers. Thirty-seven tumors from 19 men and 18 women diagnosed with either adenocarcinoma or neuroendocrine neoplasm (NENs) were included in this study. The tumors were classified into 29 adenocarcinomas and eight NENs. Immunohistochemistry with antibodies against chromogranin A (CgA), synaptophysin and CCKBR, and in situ hybridization with probes against CgA, CCKBR and histidine decarboxylase were used to further explore these tumors. Thirty-three (89%) of the tumors expressed CCKBR protein, whereas only 20 (54%) of all tumors expressed CCKBR mRNA. Of the 20 tumors expressing CCKBR mRNA, eight were NENs and 12 were adenocarcinoma. The highest amount of CCKBR was expressed in NEN. Interestingly, a high degree of co-expression of CCKBR and CgA was observed when the two markers were examined together with in situ hybridization. In conclusion, we found that all eight NENs expressed CCKBR and neuroendocrine markers in a majority of tumor cells. The same markers were also expressed in a proportion of adenocarcinomas supporting the view that gastrin is important in the development of gastric cancer. © The Author(s) 2017. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).

The Noda epileptic rat (NER) exhibits generalized tonic-clonic seizures (GTCS). A genetic linkage analysis identified two GTCS-associated loci, Ner1 on Chr 1 and Ner3 on Chr 5. The wild-type Ner1 and Ner3 alleles suppressed GTCS when combined in double-locus congenic lines, but not when present in single-locus congenic lines. Global expression analysis revealed that cholecystokinin B receptor (Cckbr) and suppressor of tumorigenicity 5 (St5), which map within Ner1, and PHD finger protein 24 (Phf24), which maps within Ner3, were significantly downregulated in NER. De novo BAC sequencing detected an insertion of an endogenous retrovirus sequence in intron 2 of the Phf24 gene in the NER genome, and PHF24 protein was almost absent in the NER brain. Phf24 encodes a Gαi-interacting protein involved in GABAB receptor signaling pathway. Based on these findings, we conclude that Cckbr, St5, and Phf24 are strong candidate genes for GTCS in NER.

Pancreatic ductal adenocarcinomas (PDACs) constitutively express the G-protein-coupled cholecystokinin B receptor (CCKBR). In this study, we identified DNA aptamers (APs) that bind to the CCKBR and describe their characterization and targeting efficacy. Using dual SELEX selection against "exposed" CCKBR peptides and CCKBR-expressing PDAC cells, a pool of DNA APs was identified. Further downselection was based on predicted structures and properties, and we selected eight APs for initial characterizations. The APs bound specifically to the CCKBR, and we showed not only that they did not stimulate proliferation of PDAC cell lines but rather inhibited their proliferation. We chose one AP, termed AP1153, for further binding and localization studies. We found that AP1153 did not activate CCKBR signaling pathways, and three-dimensional Confocal microscopy showed that AP1153 was internalized by PDAC cells in a receptor-mediated manner. AP1153 showed a binding affinity of 15 pM. Bioconjugation of AP1153 to the surface of fluorescent NPs greatly facilitated delivery of NPs to PDAC tumors in vivo. The selectivity of this AP-targeted NP delivery system holds promise for enhanced early detection of PDAC lesions as well as improved chemotherapeutic treatments for PDAC patients.

Gastrin controls gastric acid secretion and mucosal cell growth, especially of enterochromaffin-like cells, via gastrin/cholecystokinin-2 receptor (CCK2R) binding and downstream signalling. Studies in animal models, healthy subjects and patients with gastric neuroendocrine tumours provide compelling evidence to justify developing a CCK2R antagonist (CCK2RA) for preventing or treating the trophic effects of hypergastrinaemia or conditions expressing CCK2R, and with or without a proton pump inhibitor, for treating gastric acid-related conditions. Many compounds have been studied, but most have had problems with potency, selectivity for CCK2 versus CCK1 receptor, solubility or oral bioavailability. None has yet been marketed. Netazepide and Z-360 are currently undergoing clinical development, for treatment of gastric neuroendocrine tumours and pancreatic cancer, respectively. There are several other potential indications for a CCK2RA and an unmet need.