Elevated Serum Gastrin Is Associated with Melanoma Progression: Putative Role in Increased Migration and Invasion of Melanoma Cells.

Micro-environmental factors, including stromal and immune cells, cytokines, and circulating hormones are well recognized to determine cancer progression. Melanoma cell growth was recently shown to be suppressed by cholecystokinin/gastrin (CCK) receptor antagonists, and our preliminary data suggested that melanoma patients with Helicobacter gastritis (which is associated with elevated serum gastrin) might have an increased risk of cancer progression. Therefore, in the present study, we examined how gastrin may act on melanoma cells. In 89 melanoma patients, we found a statistically significant association between circulating gastrin concentrations and melanoma thickness and metastasis, which are known risk factors of melanoma progression and prognosis. Immunocytochemistry using a validated antibody confirmed weak to moderate CCK2R expression in both primary malignant melanoma cells and the melanoma cell lines SK-MEL-2 and G361. Furthermore, among the 219 tumors in the Skin Cutaneous Melanoma TCGA Pan-Cancer dataset showing gastrin receptor (CCKBR) expression, significantly higher CCKBR mRNA levels were linked to stage III-IV than stage I-II melanomas. In both cell lines, gastrin increased intracellular calcium levels and stimulated cell migration and invasion through mechanisms inhibited by a CCK2 receptor antagonist. Proteomic studies identified increased MMP-2 and reduced TIMP-3 levels in response to gastrin that were likely to contribute to the increased migration of both cell lines. However, the effects of gastrin on tumor cell invasion were relatively weak in the presence of the extracellular matrix. Nevertheless, dermal fibroblasts/myofibroblasts, known also to express CCK2R, increased gastrin-induced cancer cell invasion. Our data suggest that in a subset of melanoma patients, an elevated serum gastrin concentration is a risk factor for melanoma tumor progression, and that gastrin may act on both melanoma and adjacent stromal cells through CCK2 receptors to promote mechanisms of tumor migration and invasion.

Stable Isotope Dynamic Labeling of Secretomes (SIDLS) Identifies Authentic Secretory Proteins Released by Cancer and Stromal Cells.

Analysis of secretomes critically underpins the capacity to understand the mechanisms determining interactions between cells and between cells and their environment. In the context of cancer cell micro-environments, the relevant interactions are recognized to be an important determinant of tumor progression. Global proteomic analyses of secretomes are often performed at a single time point and frequently identify both classical secreted proteins (possessing an N-terminal signal sequence), as well as many intracellular proteins, the release of which is of uncertain biological significance. Here, we describe a mass spectrometry-based method for stable isotope dynamic labeling of secretomes (SIDLS) that, by dynamic SILAC, discriminates the secretion kinetics of classical secretory proteins and intracellular proteins released from cancer and stromal cells in culture. SIDLS is a robust classifier of the different cellular origins of proteins within the secretome and should be broadly applicable to nonproliferating cells and cells grown in short term culture.

Functional Differentiation of Cholecystokinin-Containing Interneurons Destined for the Cerebral Cortex.

Although extensively studied postnatally, the functional differentiation of cholecystokinin (CCK)-containing interneurons en route towards the cerebral cortex during fetal development is incompletely understood. Here, we used CCKBAC/DsRed mice encoding a CCK promoter-driven red fluorescent protein to analyze the temporal dynamics of DsRed expression, neuronal identity, and positioning through high-resolution developmental neuroanatomy. Additionally, we developed a dual reporter mouse line (CCKBAC/DsRed::GAD67gfp/+) to differentiate CCK-containing interneurons from DsRed+ principal cells during prenatal development. We show that DsRed is upregulated in interneurons once they exit their proliferative niche in the ganglionic eminence and remains stably expressed throughout their long-distance migration towards the cerebrum, particularly in the hippocampus. DsRed+ interneurons, including a cohort coexpressing calretinin, accumulated at the palliosubpallial boundary by embryonic day 12.5. Pioneer DsRed+ interneurons already reached deep hippocampal layers by embryonic day 14.5 and were morphologically differentiated by birth. Furthermore, we probed migrating interneurons entering and traversing the cortical plate, as well as stationary cells in the hippocampus by patch-clamp electrophysiology to show the first signs of Na+ and K+ channel activity by embryonic day 12.5 and reliable adult-like excitability by embryonic day 18.5. Cumulatively, this study defines key positional, molecular, and biophysical properties of CCK+ interneurons in the prenatal brain.

The role of chemerin and ChemR23 in stimulating the invasion of squamous oesophageal cancer cells.

BACKGROUND: Stromal cells, including cancer-associated myofibroblasts (CAMs), are recognised to be determinants of cancer progression, but the mechanisms remain uncertain. The chemokine-like protein, chemerin, is upregulated in oesophageal squamous cancer (OSC) CAMs compared with adjacent tissue myofibroblasts (ATMs). In this study, we hypothesised that chemerin stimulates OSC cell invasion. METHODS: Expression of the chemerin receptor, ChemR23, in OSC was examined by immunohistochemistry. The invasion of OSC cells was studied using Boyden chambers and organotypic assays, and the role of chemerin was explored using siRNA, immunoneutralisation and a ChemR23 receptor antagonist. Matrix metalloproteinases (MMPs) were detected by western blot, enzyme assays or immunohistochemistry. RESULTS: Immunohistochemistry indicated expression of the putative chemerin receptor ChemR23 in OSC. It was also expressed in the OSC cell line, OE21. Chemerin stimulated OE21 cell migration and invasion in Boyden chambers. Conditioned medium (CM) from OSC CAMs also stimulated OE21 cell invasion and this was inhibited by chemerin immunoneutralisation, the ChemR23 antagonist CCX832, and by pretreatment of CAMs with chemerin siRNA. In organotypic cultures of OE21 cells on Matrigel seeded with either CAMs or ATMs, there was increased OE21 cell invasion by CAMs that was again inhibited by CCX832. Chemerin increased MMP-1, MMP-2 and MMP-3 abundance, and activity in OE21 cell media, and this was decreased by inhibiting protein kinase C and p44/42 MAPK kinase but not PI-3 kinase. CONCLUSIONS: The data indicate that OSC myofibroblasts release chemerin that stimulates OSC cell invasion. Treatments directed at inhibiting chemerin-ChemR23 interactions might be therapeutically useful in delaying progression in OSC.

Distinct miRNA profiles in normal and gastric cancer myofibroblasts and significance in Wnt signaling.

Stromal cells influence epithelial function in both health and disease. Myofibroblasts are abundant stromal cells that influence the cellular microenvironment by release of extracellular matrix (ECM) proteins, growth factors, proteases, cytokines, and chemokines. Cancer-associated myofibroblasts (CAMs) differ from adjacent tissue (ATMs) and normal tissue myofibroblasts (NTMs), but the basis of this is incompletely understood. We report now the differential expression of miRNAs in gastric cancer CAMs. MicroRNA arrays identified differences in the miRNA profile in gastric and esophageal NTMs and in CAMs from stomach compared with NTMs. miR-181d was upregulated in gastric CAMs. Analysis of differentially regulated miRNAs indicated an involvement in Wnt signaling. Examination of a microarray data set then identified Wnt5a as the only consistently upregulated Wnt ligand in gastric CAMs. Wnt5a stimulated miR-181d expression, and knockdown of miR-181d inhibited Wnt5a stimulation of CAM proliferation and migration. Analysis of miR-181d targets suggested a role in chemotaxis. Conditioned medium from CAMs stimulated gastric cancer cell (AGS) migration more than that from ATMs, and miR-181d knockdown reduced the effect of CAM-CM on AGS cell migration but had no effect on AGS cell responses to ATM conditioned media. The data suggest that dysregulation of miRNA expression in gastric CAMs, secondary to Wnt5a signaling, accounts at least in part for the effect of CAMs in promoting cancer cell migration.

Gastrin stimulates MMP-1 expression in gastric epithelial cells: putative role in gastric epithelial cell migration.

The pyloric antral hormone gastrin plays a role in remodeling of the gastric epithelium, but the specific targets of gastrin that mediate these effects are poorly understood. Glandular epithelial cells of the gastric corpus express matrix metalloproteinase (MMP)-1, which is a potential determinant of tissue remodeling; some of these cells express the CCK-2 receptor at which gastrin acts. We have now examined the hypothesis that gastrin stimulates expression of MMP-1 in the stomach. We determined MMP-1 transcript abundance in gastric mucosal biopsies from Helicobacter pylori negative human subjects with normal gastric mucosal histology, who had a range of serum gastrin concentrations due in part to treatment with proton pump inhibitors (PPI). The effects of gastrin were studied on gastric epithelial AGS-GR cells using Western blot and migration assays. In human subjects with increased serum gastrin due to PPI usage, MMP-1 transcript abundance was increased 2-fold; there was also increased MMP-7 transcript abundance but not MMP-3. In Western blots, gastrin increased proMMP-1 abundance, as well that of a minor band corresponding to active MMP-1, in the media of AGS-GR cells, and the response was mediated by protein kinase C and p42/44 MAP kinase. There was also increased MMP-1 enzyme activity. Gastrin-stimulated AGS-GR cell migration in both scratch wound and Boyden chamber assays was inhibited by MMP-1 immunoneutralization. We conclude that MMP-1 expression is a target of gastrin implicated in mucosal remodeling.

The neuroendocrine phenotype of gastric myofibroblasts and its loss with cancer progression.

Stromal cells influence cancer progression. Myofibroblasts are an important stromal cell type, which influence the tumour microenvironment by release of extracellular matrix (ECM) proteins, proteases, cytokines and chemokines. The mechanisms of secretion are poorly understood. Here, we describe the secretion of marker proteins in gastric cancer and control myofibroblasts in response to insulin-like growth factor (IGF) stimulation and, using functional genomic approaches, we identify proteins influencing the secretory response. IGF rapidly increased myofibroblast secretion of an ECM protein, TGFßig-h3. The secretory response was not blocked by inhibition of protein synthesis and was partially mediated by increased intracellular calcium (Ca(2+)). The capacity for evoked secretion was associated with the presence of dense-core secretory vesicles and was lost in cells from patients with advanced gastric cancer. In cells responding to IGF-II, the expression of neuroendocrine marker proteins, including secretogranin-II and proenkephalin, was identified by gene array and LC-MS/MS respectively, and verified experimentally. The expression of proenkephalin was decreased in cancers from patients with advanced disease. Inhibition of secretogranin-II expression decreased the secretory response to IGF, and its over-expression recovered the secretory response consistent with a role in secretory vesicle biogenesis. We conclude that normal and some gastric cancer myofibroblasts have a neuroendocrine-like phenotype characterized by Ca(2+)-dependent regulated secretion, dense-core secretory vesicles and expression of neuroendocrine marker proteins; loss of the phenotype is associated with advanced cancer. A failure to regulate myofibroblast protein secretion may contribute to cancer progression.

Gastrointestinal hormones and the dialogue between gut and brain.

The landmark discovery by Bayliss and Starling in 1902 of the first hormone, secretin, emerged from earlier observations that a response (pancreatic secretion) following a stimulus (intestinal acidification) occurred after section of the relevant afferent nerve pathway. Nearly 80 years elapsed before it became clear that visceral afferent neurons could themselves also be targets for gut and other hormones. The action of gut hormones on vagal afferent neurons is now recognised to be an early step in controlling nutrient delivery to the intestine by regulating food intake and gastric emptying. Interest in these mechanisms has grown rapidly in view of the alarming global increase in obesity. Several of the gut hormones (cholecystokinin (CCK); peptide YY3-36 (PYY3-36); glucagon-like peptide-1 (GLP-1)) excite vagal afferent neurons to activate an ascending pathway leading to inhibition of food intake. Conversely others, e.g. ghrelin, that are released in the inter-digestive period, inhibit vagal afferent neurons leading to increased food intake. Nutrient status determines the neurochemical phenotype of vagal afferent neurons by regulating a switch between states that promote orexigenic or anorexigenic signalling through mechanisms mediated, at least partly, by CCK. Gut-brain signalling is also influenced by leptin, by gut inflammation and by shifts in the gut microbiota including those that occur in obesity. Moreover, there is emerging evidence that diet-induced obesity locks the phenotype of vagal afferent neurons in a state similar to that normally occurring during fasting. Vagal afferent neurons are therefore early integrators of peripheral signals underling homeostatic mechanisms controlling nutrient intake. They may also provide new targets in developing treatments for obesity and feeding disorders.

Mapping proteolytic processing in the secretome of gastric cancer-associated myofibroblasts reveals activation of MMP-1, MMP-2, and MMP-3.

Cancer progression involves changes in extracellular proteolysis, but the contribution of stromal cell secretomes to the cancer degradome remains uncertain. We have now defined the secretome of a specific stromal cell type, the myofibroblast, in gastric cancer and its modification by proteolysis. SILAC labeling and COFRADIC isolation of methionine containing peptides allowed us to quantify differences in gastric cancer-derived myofibroblasts compared with myofibroblasts from adjacent tissue, revealing increased abundance of several proteases in cancer myofibroblasts including matrix metalloproteinases (MMP)-1 and -3. Moreover, N-terminal COFRADIC analysis identified cancer-restricted proteolytic cleavages, including liberation of the active forms of MMP-1, -2, and -3 from their inactive precursors. In vivo imaging confirmed increased MMP activity when gastric cancer cells were xenografted in mice together with gastric cancer myofibroblasts. Western blot and enzyme activity assays confirmed increased MMP-1, -2, and -3 activity in cancer myofibroblasts, and cancer cell migration assays indicated stimulation by MMP-1, -2, and -3 in cancer-associated myofibroblast media. Thus, cancer-derived myofibroblasts differ from their normal counterparts by increased production and activation of MMP-1, -2, and -3, and this may contribute to the remodelling of the cancer cell microenvironment.

The role of plasminogen activator inhibitor-1 in gastric mucosal protection.

Gastric mucosal health is maintained in response to potentially damaging luminal factors. Aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) disrupt protective mechanisms leading to bleeding and ulceration. The plasminogen activator system has been implicated in fibrinolysis following gastric ulceration, and an inhibitor of this system, plasminogen activator inhibitor (PAI)-1, is expressed in gastric epithelial cells. In Helicobacter pylori-negative patients with normal gastric histology taking aspirin or NSAIDs, we found elevated gastric PAI-1 mRNA abundance compared with controls; the increase in patients on aspirin was independent of whether they were also taking proton pump inhibitors. In the same patients, aspirin tended to lower urokinase plasminogen activator mRNA. Immunohistochemistry indicated PAI-1 localization to epithelial cells. In a model system using MKN45 or AGS-GR cells transfected with a PAI-1 promoter-luciferase reporter construct, we found no evidence for upregulation of PAI-1 expression by indomethacin, and, in fact, cyclooxygenase products such as PGE2 and PGI2 weakly stimulated expression. Increased gastric PAI-1 mRNA was also found in mice following gavage with ethanol or indomethacin, but plasma PAI-1 was unaffected. In PAI-1(-/-) mice, gastric hemorrhagic lesions in response to ethanol or indomethacin were increased compared with C57BL/6 mice. In contrast, in PAI-1-H/Kß mice in which PAI-1 is overexpressed in parietal cells, there were decreased lesions in response to ethanol and indomethacin. Thus, PAI-1 expression is increased in gastric epithelial cells in response to mucosal irritants such as aspirin and NSAIDs probably via an indirect mechanism, and PAI-1 acts as a local autoregulator to minimize mucosal damage.

Functional neuroimaging demonstrates that ghrelin inhibits the central nervous system response to ingested lipid.

OBJECTIVE: Gut-derived humoural factors activate central nervous system (CNS) mechanisms controlling energy intake and expenditure, and autonomic outflow. Ghrelin is secreted from the stomach and stimulates food intake and gastric emptying, but the relevant mechanisms are poorly understood. Nutrient-activated CNS systems can be studied in humans by physiological/pharmacological MRI (phMRI). This method has been used to examine the CNS responses to exogenous ghrelin. DESIGN: phMRI was used to study the CNS responses in healthy people to a ghrelin bolus (0.3 nmol/kg, intravenous) in the post-prandial state, and an intravenous infusion of ghrelin (1.25 pmol/kg/min) alone and after intragastric lipid (dodecanoate, C12) in people who have fasted. RESULTS: A ghrelin bolus decreased the blood oxygenation level dependent (BOLD) signal detected by phMRI in feeding-activated areas of the CNS in the post-prandial state. Infusion of ghrelin reversed the effect of C12 in delaying gastric emptying but had no effect on hunger. Intragastric C12 caused strong bilateral activation of a matrix of CNS areas, including the brain stem, hypothalamus and limbic areas which was attenuated by exogenous ghrelin. Ghrelin infusion alone had a small but significant stimulatory effect on CNS BOLD signals. CONCLUSION: Ghrelin inhibits activation of the hypothalamus and brain stem induced by ingested nutrients, suggesting a role in suppression of gut-derived satiety signals in humans.

Release of TGFßig-h3 by gastric myofibroblasts slows tumor growth and is decreased with cancer progression.

Tumor progression has been linked to changes in the stromal environment. Myofibroblasts are stromal cells that are often increased in tumors but their contribution to cancer progression is not well understood. Here, we show that the secretomes of myofibroblasts derived from gastric cancers [cancer-associated myofibroblasts (CAMs)] differ in a functionally significant manner from those derived from adjacent tissue [adjacent tissue myofibroblasts (ATMs)]. CAMs showed increased rates of migration and proliferation compared with ATMs or normal tissue myofibroblasts (NTMs). Moreover, conditioned medium (CM) from CAMs significantly stimulated migration, invasion and proliferation of gastric cancer cells compared with CM from ATMs or NTMs. Proteomic analysis of myofibroblast secretomes revealed decreased abundance of the extracellular matrix (ECM) adaptor protein like transforming growth factor-ß-induced gene-h3 (TGFßig-h3) in CAMs, which was correlated with lymph node involvement and shorter survival. TGFßig-h3 inhibited IGF-II-stimulated migration and proliferation of both cancer cells and myofibroblasts, and suppressed IGF-II activation of p42/44 MAPkinase; TGFßig-h3 knockdown increased IGF-II- and CM-stimulated migration. Furthermore, administration of TGFßig-h3 inhibited myofibroblast-stimulated growth of gastric cancer xenografts. We conclude that stromal cells exert inhibitory as well as stimulatory effects on tumor cells; TGFßig-h3 is a stromal inhibitory factor that is decreased with progression of gastric cancers.

Gastrin receptor pharmacology.

C-terminally amidated gastrins act at cholecystokinin-2 receptors (CCK2R), which are normally expressed by gastric parietal and enterochromaffin-like (ECL) cells and smooth muscle; there is also extensive expression in the CNS where the main endogenous ligand is cholecystokinin. A variety of neoplasms express CCK2R, or splice variants, including neuroendocrine, pancreatic, medullary thyroid and lung cancers. Other products of the gastrin gene (progastrin, the Gly-gastrins) may stimulate cell proliferation but are not CCK2R ligands. Depending on the cell type, stimulation of CCK2R evokes secretion, increases proliferation and cell migration, inhibits apoptosis, and controls the expression of various genes. These effects are mediated by increased intracellular calcium and activation of protein kinase C, MAPkinase and other protein kinase cascades. There has been recent progress in developing CCK2R ligands that can be used for imaging tumours expressing the receptor. New antagonists have also been developed, and there is scope for using these for suppression of gastric acid and for treatment of neuroendocrine and other CCK2R-expressing tumours.

Gastrin stimulates expression of plasminogen activator inhibitor-1 in gastric epithelial cells.

Plasminogen activator inhibitor (PAI)-1 is associated with cancer progression, fibrosis and thrombosis. It is expressed in the stomach but the mechanisms controlling its expression there, and its biological role, are uncertain. We sought to define the role of gastrin in regulating PAI-1 expression and to determine the relevance for gastrin-stimulated cell migration and invasion. In gastric biopsies from subjects with elevated plasma gastrin, the abundances of PAI-1, urokinase plasminogen activator (uPA), and uPA receptor (uPAR) mRNAs measured by quantitative PCR were increased compared with subjects with plasma concentrations in the reference range. In patients with hypergastrinemia due to autoimmune chronic atrophic gastritis, there was increased abundance of PAI-1, uPA, and uPAR mRNAs that was reduced by octreotide or antrectomy. Immunohistochemistry revealed localization of PAI-1 to parietal cells and enterochromaffin-like cells in micronodular neuroendocrine tumors in hypergastrinemic subjects. Transcriptional mechanisms were studied by using a PAI-1-luciferase promoter-reporter construct transfected into AGS-G(R) cells. There was time- and concentration-dependent increase of PAI-1-luciferase expression in response to gastrin that was reversed by inhibitors of the PKC and MAPK pathways. In Boyden chamber assays, recombinant PAI-1 inhibited gastrin-stimulated AGS-G(R) cell migration and invasion, and small interfering RNA treatment increased responses to gastrin. We conclude that elevated plasma gastrin concentrations are associated with increased expression of gastric PAI-1, which may act to restrain gastrin-stimulated cell migration and invasion.

Cocaine- and amphetamine-regulated transcript mediates the actions of cholecystokinin on rat vagal afferent neurons.

BACKGROUND & AIMS: Cholecystokinin (CCK) acts on vagal afferent neurons to inhibit food intake and gastric emptying; it also increases expression of the neuropeptide cocaine- and amphetamine-regulated transcript (CART), but the significance of this is unknown. We investigated the role of CARTp in vagal afferent neurons. METHODS: Release of CART peptide (CARTp) from cultured vagal afferent neurons was determined by enzyme-linked immunosorbent assay. Expression of receptors and neuropeptides in rat vagal afferent neurons in response to CARTp was studied using immunohistochemistry and luciferase promoter reporter constructs. Effects of CARTp and CCK were studied on food intake. RESULTS: CCK stimulated CARTp release from cultured nodose neurons. CARTp replicated the effect of CCK in stimulating expression of Y2R and of CART itself in these neurons in vivo and in vitro, but not in inhibiting cannabinoid-1, melanin-concentrating hormone, and melanin-concentrating hormone-1 receptor expression. Effects of CCK on Y2R and CART expression were reduced by CART small interfering RNA or brefeldin A. Exposure of rats to CARTp increased the inhibitory action of CCK on food intake after short-, but not long-duration, fasting. CONCLUSIONS: The actions of CCK in stimulating CART and Y2R expression in vagal afferent neurons and in inhibiting food intake are augmented by CARTp; CARTp is released by CCK from these neurons, indicating that it acts as an autocrine excitatory mediator.

Defining the role of cholecystokinin in the lipid-induced human brain activation matrix.

BACKGROUND & AIMS: In human beings, as in most vertebrates, the release of the intestinal peptide cholecystokinin (CCK) by ingested food plays a major role both in digestion and the regulation of further food intake, but the changes in brain function and their underlying activation mechanisms remain unknown. Our aim was to explore, using a novel physiologic magnetic resonance imaging approach, the temporospatial brain activation matrix, in response to ingestion of a lipid meal and, by use of a CCK-1 receptor antagonist, to define the role of CCK in this activation. METHODS: We studied, in 19 healthy subjects, the brain activation responses to ingested lipid (dodecanoic acid) or saline (control) with magnetic resonance imaging. Gallbladder volume, plasma CCK levels, and subjective hunger and fullness scores were also recorded. The experiment was then repeated, with and without prior administration of the CCK-1 receptor antagonist dexloxiglumide (600 mg orally) with a controlled, randomized order, latin-square design. RESULTS: Ingested lipid activated bilaterally a matrix of brain areas, particularly the brain stem, pons, hypothalamus, and also cerebellum and motor cortical areas. These activations were abolished by dexloxiglumide, indicating a CCK-mediated pathway, independent of any nutrient-associated awareness cues. CONCLUSION: The identification of these activations now defines the lipid-activated brain matrix and provides a means by which the gut-derived homeostatic mechanisms of food regulation can be distinguished from secondary sensory and hedonic cues, thereby providing a new approach to exploring aberrant human gastrointestinal responses and eating behavior.

Effects of Proton Pump Inhibitor Therapy, H. pylori Infection and Gastric Preneoplastic Pathology on Fasting Serum Gastrin Concentrations.

Background: Hypergastrinaemia occasionally indicates the presence of a gastrinoma. However it is much more commonly associated with various benign causes including proton pump inhibitor (PPI) use, Helicobacter pylori infection and/or atrophic gastritis. The extent to which these factors interact to influence fasting serum gastrin concentrations remains incompletely understood. Materials and Methods: Fasting serum gastrin concentrations were measured by radioimmunoassay in 1,400 patients attending for diagnostic oesophagogastro-duodenoscopy. After exclusions, 982 patients were divided into four groups and their results analysed. We compared gastrin concentrations in normal patients (no H. pylori infection, no PPI use and no histological evidence of gastric preneoplasia (n=233)), with those in patients who were taking regular PPIs (H. pylori negative with no gastric preneoplasia (n=301)), patients who had active H. pylori infection but no gastric preneoplasia (n=164) and patients with histologically confirmed gastric preneoplasia (n=284). Results: Median fasting gastrin concentration in the normal group was 20pM and was significantly increased in PPI users (46pM, p<0.0001), patients with active H. pylori infection (27pM, p<0.0001), and patients with antral (25pM, p<0.01) or corpus (48pM, p<0.0001) gastric preneoplasia. PPI use resulted in further significant increases in fasting serum gastrin concentrations in patients who were infected with H. pylori (50pM, n=56) or who had antral gastric preneoplasia (53pM, n=87), but did not significantly alter serum gastrin concentrations in patients with corpus preneoplasia (90pM, n=66). Conclusions: PPI use, H. pylori infection and atrophic gastritis all caused significant elevations of median fasting gastrin concentrations. However, several patients who had potential risk factors for hypergastrinaemia still demonstrated fasting serum gastrin concentrations within the normal range.

Expression of cannabinoid CB1 receptors by vagal afferent neurons: kinetics and role in influencing neurochemical phenotype.

The intestinal hormone cholecystokinin (CCK) inhibits food intake via stimulation of vagal afferent neurons (VAN). Recent studies suggest that CCK also regulates the expression of some G protein-coupled receptors and neuropeptide transmitters in these neurons. The aim of the present study was to characterize the expression of cannabinoid (CB)1 receptors in VAN and to determine whether stimulation of these receptors plays a role in regulating neurochemical phenotype. Expression of CB1 in rat VAN was detectable by in situ hybridization or immunohistochemistry after 6 h of fasting and increased to a maximum after 24 h when approximately 50% of neurons in the mid and caudal regions expressed the receptor. Melanin-concentrating hormone (MCH)1 receptors also increased with fasting, but the changes were delayed compared with CB1; in contrast Y2 receptors (Y2R) exhibited reciprocal changes in expression to CB1. Administration of CCK8s (10 nmol ip) to fasted rats decreased expression of CB1 with a t(1/2) of approximately 1 h compared with 3 h for MCH1. The action of CCK8s was inhibited by ghrelin and orexin-A. The CB1 agonist anandamide (intraperitoneally) reversed the effect of CCK8s on CB1, MCH1, and Y2 receptor expression. In contrast, in rats fasted for 18 h, administration of a CB1 antagonist/inverse agonist (AM281 ip) downregulated CB1 expression and increased Y2 receptor expression. Activation of vagal CB1 receptors therefore influences the neurochemical phenotype of these neurons, indicating a new and hitherto unrecognized role for endocannabinoids in gut-brain signaling.

Cholecystokinin regulates expression of Y2 receptors in vagal afferent neurons serving the stomach.

The intestinal hormones CCK and PYY3-36 inhibit gastric emptying and food intake via vagal afferent neurons. Here we report that CCK regulates the expression of Y2R, at which PYY3-36 acts. In nodose ganglia from rats fasted up to 48 h, there was a fivefold decrease of Y2R mRNA compared with rats fed ad libitum; Y2R mRNA in fasted rats was increased by administration of CCK, and by refeeding through a mechanism sensitive to the CCK1R antagonist lorglumide. Antibodies to Y2R revealed expression in both neurons and satellite cells; most of the former (89 +/- 4%) also expressed CCK1R. With fasting there was loss of Y2R immunoreactivity in CCK1R-expressing neurons many of which projected to the stomach, but not in satellite cells or neurons projecting to the ileum or proximal colon. Expression of a Y2R promoter-luciferase reporter (Y2R-luc) in cultured vagal afferent neurons was increased in response to CCK by 12.3 +/- 0.1-fold and by phorbol ester (16.2 +/- 0.4-fold); the response to both was abolished by the protein kinase C inhibitor Ro-32,0432. PYY3-36 stimulated CREB phosphorylation in rat nodose neurons after priming with CCK; in wild-type mice PYY3-36 increased Fos labeling in brainstem neurons but in mice null for CCK1R this response was abolished. Thus Y2R is expressed by functionally distinct subsets of nodose ganglion neurons projecting to the stomach and ileum/colon; in the former expression is dependent on stimulation by CCK, and there is evidence that PYY3-36 effects on vagal afferent neurons are CCK dependent.

Recharacterization data for a geriatric gastrin polycolonal antibody.

Radioimmunoassay data on the recharacterization of a gastrin polycolonal antibody first generated in 1973 is presented. The data include specificity, effect of matrix, and the establishment of a reference range for circulating fasting gastrin concentrations in normal subjects. For a discussion of the interpretation of the data, please see doi, 10.1016/j.peptides.2019.02.001 [1].