Graham J. Dockray

Synergistic interaction between hypergastrinemia and Helicobacter infection in a mouse model of gastric cancer.

BACKGROUND & AIMS Hypergastrinemia occurs frequently in association with acid suppression and Helicobacter infection, but its role in the progression to gastric atrophy and gastric cancer has not been well defined. METHODS The effects of hypergastrinemia, and possible synergy with Helicobacter felis infection, were investigated in insulin-gastrin (INS-GAS) transgenic mice. RESULTS INS-GAS mice initially showed mild hypergastrinemia, increased maximal gastric acid secretion, and increased parietal cell number but later progressed to decreased parietal cell number and hypochlorhydria. Development of gastric atrophy was associated with increased expression of growth factors, heparin-binding epidermal growth factor and transforming growth factor alpha. At 20 months of age, INS-GAS mice showed no evidence of increased enterochromaffin-like cell number, but instead exhibited gastric metaplasia, dysplasia, carcinoma in situ, and gastric cancer with vascular invasion. Invasive gastric carcinoma was observed in 6 of 8 INS-GAS mice that were >20 months old. Helicobacter felis infection of INS-GAS mice led to accelerated (< or = 8 mo) development of intramucosal carcinoma (85%), with submucosal invasion (54%) and intravascular invasion (46%; P < or = 0.05). CONCLUSIONS These findings support the unexpected conclusion that chronic hypergastrinemia in mice can synergize with Helicobacter infection and contribute to eventual parietal cell loss and progression to gastric cancer.

Processing and proliferative effects of human progastrin in transgenic mice.

Incompletely processed gastrins have been postulated to play a role in growth of the gastrointestinal tract, but few studies have examined the effects of progastrin on mucosal proliferation in vivo. Human gastrin gene expression and progastrin processing were therefore studied in transgenic mice containing a human gastrin (hGAS) minigene, and compared to processing in mice bearing an insulin gastrin (INS-GAS) transgene that overexpresses amidated gastrin. Progastrin processing was studied using region-specific antisera and radioimmunoassays, biosynthetic labeling, immunoprecipitation, and HPLC. Proliferative effects due to overexpression of processed and unprocessed gastrin in INS-GAS and hGAS mice, respectively, were determined using routine histology and BrdU incorporation. The pancreatic islets of INS-GAS mice were able to produce carboxyamidated G-17, resulting in a twofold elevation of serum amidated gastrin, marked thickening of the oxyntic mucosa, and an increased BrdU labeling index (LI) of the gastric body. In contrast, livers of adult hGAS mice expressed abundant human gastrin mRNA and human progastrin but were unable to process this peptide to the mature amidated form, resulting in markedly elevated serum progastrin levels and normal amidated gastrin levels. Nevertheless, there was a marked increase in the BrdU labeling index of the colon in hGAS mice (LI 7.46+/-1.90%), as well as in INS-GAS mice (LI 6.16+/-1.17%), compared to age-matched, wild type control mice (LI 4.01+/-0.98%, P < 0.05). These studies suggest that incompletely processed gastrin precursors may contribute to colonic mucosal proliferation in vivo.

Gut peptides : biochemistry and physiology

Written by international experts, this reference presents the latest research on the structure, functions, and actions of the gut peptides. The first section of the book explains the general principles of the regulatory peptide systems of the gut. The second section provides a detailed account of the properties and functions of each of the major peptides. The final section focuses on the role of gut peptides in gut physiology and pathophysiology and integrates new information on topics such as gastric secretion, peptic ulcer disease, peristalisis, intestinal salt and water transport, growth regulation, gastrointestinal malignancies and the mucosal immune and inflammatory responses.

The neuronal origin of bombesin-like immunoreactivity in the rat gastrointestinal tract.

Abstract Extracts of muscle and mucosal layers of the rat stomach contained material cross-reacting in radioimmunoassays for the amphibian skin peptide bombesin. In the intestine, immunoreactive bom-besin was confined to the muscle layers. Two molecular forms of immunoreactive bombesin were identified; one of these components was eluted in a similar position to tetradecapeptide bombesin on gel filtration and accounted for about 90% of the immunoreactivity in intestinal extracts, compared with about 40% in stomach. The two components were distinguishable from synthetic bombesin, and from the structurally related peptide substance P, on the basis of their pattern of immunochemical properties with three different antisera. Immunohistochemical studies using the same antisera revealed a rich distribution of nerve fibres in the mucosa of the rat stomach, but few fibres were seen in the intestinal mucosa. Abundant fibres with bombesin-like immunoreactivity were found surrounding nerve cell bodies in the myenteric plexus throughout the gut. Immunoreactivc nerve cell bodies were not identified, neither was convincing evidence obtained to indicate the presence of bombesin in mucosal endocrine cells. The results support the possibilities that bombesin-like peptides are neurotransmitters in the gut and that they could play a role in the modulation of gastrointestinal motility and in the release of gastrin.

Characterization of the peptidergic afferent innervation of the stomach in the rat, mouse and guinea-pig

Retrograde tracing of the fluorescent marker, True Blue, has been used together with immunohistochemistry employing antibodies to substance P, calcitonin gene-related peptide, somatostatin, vasoactive intestinal polypeptide and morphine-modulating peptide to study the afferent innervation of the stomach in rat, mouse and guinea-pig. Up to 85% of spinal afferents to the stomach in all three species contained immunoreactive calcitonin gene-related peptide, and up to 50% contained substance P. In all three species less than 10% of vagal afferents to the stomach reacted with antibodies to calcitonin gene-related peptide, or substance P. Cacitonin gene-related peptide-immunoreactive fibres were found in the myenteric plexus, circular muscle and around submucosal blood vessels in the stomach. In the rat, removal of the coeliac ganglion, splanchnic nerve section, or capsaicin treatment virtually abolished calcitonin gene-related peptide immunoreactivity in the stomach. Capsaicin and splanchnic section also abolished the staining of immunoreactive calcitonin gene-related peptide fibres in the coeliac ganglion. The same treatments abolished substance P staining of fibres around submucosal blood vessels, but in the myenteric plexus and circular smooth muscle there were still abundant immunoreactive fibres, presumably arising from intrinsic cell bodies. No somatostatin-containing visceral afferents could be found, although somatostatin was localized to cell bodies in rat dorsal root ganglia. Immunoreactive vasoactive intestinal polypeptide-containing dorsal root ganglia neurons were not found; although antibodies to morphine-modulatory peptide revealed immunoreactive nerve cell bodies, we were unable to exclude the possibility that this result is attributable to cross reactivity with calcitonin gene-related peptide. These results provide direct evidence that calcitonin gene-related peptide is a marker for a major subset of visceral primary afferent neurons and suggest that this population of spinal afferents makes a major contribution to the total gastric content of calcitonin gene-related peptide.

Expression of cannabinoid CB1 receptors by vagal afferent neurons is inhibited by cholecystokinin

Both inhibitory (satiety) and stimulatory (orexigenic) factors from the gastrointestinal tract regulate food intake. In the case of the satiety hormone cholecystokinin (CCK), these effects are mediated via vagal afferent neurons. We now report that vagal afferent neurons expressing the CCK-1 receptor also express cannabinoid CB1 receptors. Retrograde tracing established that these neurons project to the stomach and duodenum. The expression of CB1 receptors determined by RT-PCR, immunohistochemistry and in situ hybridization in rat nodose ganglia was increased by withdrawal of food for ≥12 hr. After refeeding of fasted rats there was a rapid loss of CB1 receptor expression identified by immunohistochemistry and in situ hybridization. These effects were blocked by administration of the CCK-1 receptor antagonist lorglumide and mimicked by administration of CCK to fasted rats. Because CCK is a satiety factor that acts via the vagus nerve and CB1 agonists stimulate food intake, the data suggest a new mechanism modulating the effect on food intake of satiety signals from the gastrointestinal tract.

Small-intestinal dysfunction accompanies the complex endocrinopathy of human proprotein convertase 1 deficiency

We have previously described the only reported case of human proprotein convertase 1 (PC1) deficiency, in a female (Subject A) with obesity, hypogonadism, hypoadrenalism, and reactive hypoglycemia. We now report the second case of human PC1 deficiency (Subject B), also due to compound heterozygosity for novel missense and nonsense mutations. While both subjects shared the phenotypes of obesity, hypoadrenalism, reactive hypoglycemia, and elevated circulating levels of certain prohormones, the clinical presentation of Subject B was dominated by severe refractory neonatal diarrhea, malabsorptive in type. Subsequent investigation of Subject A revealed marked small-intestinal absorptive dysfunction, which was not previously clinically suspected. We postulate that PC1, presumably in the enteroendocrine cells, is essential for the normal absorptive function of the human small intestine. The differences in the nature and severity of presentation between the two cases cannot readily be explained on the basis of allelic heterogeneity, as the nonsense and missense mutations from both subjects had comparably severe effects on the catalytic activity of PC1. Despite Subject As negligible PC1 activity, some mature ACTH and glucagon-like peptide 17-36(amide) were detectable in her plasma, suggesting that the production of these hormones, at least in humans, does not have an absolute dependence on PC1. The presence of severe obesity and the absence of growth retardation in both subjects contrast markedly with the phenotype of mice lacking PC1 and suggest that the precise physiological repertoire of this enzyme may vary between mammalian species.

The hypothalamic arcuate nucleus-median eminence complex: Immunohistochemistry of transmitters, peptides and DARPP-32 with special reference to coexistence in dopamine neurons

In this paper, we describe the results of a series of experiments which have examined the distribution within the arcuate nucleus of the hypothalamus of neurons containing the following immunoreactivities: TH-LI, GAD-LI, NT-LI, GAL-LI, GRF-LI, Met-ENK-LI, Leu-ENK-LI, Met-ENK-7-LI, Met-ENK-8-LI, metorphamide-LI, DYN-LI, NPY-LI, SOM-LI, FMRFamide-LI, and CLIP-LI and ependymal tanycytes containing DARPP-32-LI. Using elution-restaining and double antibody staining techniques we have established numerous patterns of coexistence of these various neurotransmitters and neuropeptides. Thus, neurons containing TH-LI were, in some instances, also found to contain GAD-LI, NT-LI, GAL-LI, GRF-LI, Met-ENK-8-LI, Leu-ENK-LI, or DYN-LI or combinations of these compounds. For example, some TH-IR neurons also contained GAL-LI and GRF-LI, while other TH-IR. neurons were also seen to contain GRF- and NT-LI. These neurons may, in fact, contain even more compounds. NPY-IR neurons and those containing SOM-LI and CLIP-LI were distinct and separate from those containing TH-LI. The distribution of these different neurochemical types of neurons and their patterns of coexistence are summarized in Fig. 34, while the relative distribution patterns of immunoreactive fibres in the median eminence are summarized in Fig. 35.

Fatty acid chain length determines cholecystokinin secretion and effect on human gastric motility

BACKGROUND & AIMS Fatty acids induce cholecystokinin (CCK) secretion and modify gastric motility, but the chain length requirements for these effects are not known. Nor is it clear whether the effects of fatty acids on gastric motility in humans are CCK mediated or directly exerted. The aim of this study was to determine the role of fatty acyl chain length in CCK secretion and in influencing gastric motility. METHODS Fatty acids were infused into the upper gut in healthy volunteers; plasma CCK was determined by radioimmunoassay. Effects of fatty acids on antral contractility were determined by percutaneous ultrasonography; effects on proximal gastric tone were studied during fundal distention. RESULTS Plasma CCK concentration was consistently and similarly elevated by fatty acids with a chain of 12 carbon atoms or longer, whereas those of 11 or fewer carbon atoms failed to increase plasma CCK. A 12-carbon but not a 10-carbon-long chain fatty acid reduced antral contractile amplitude, an effect that was abolished by loxiglumide (a specific CCK-A receptor antagonist). The 12-carbon fatty acid also reduced proximal gastric tone more than the 10-carbon fatty acid. CONCLUSIONS A highly specific, chain length-sensitive fatty acid recognition system exists in the proximal gut mediating CCK secretion and gastric motility. An additional, probably CCK-independent, effect of fatty acid also regulates proximal gastric tone.

Cholecystokinins in rat cerebral cortex: Identification, purification and characterization by immunochemical methods

Cholecystokinins in rat cerebral cortex were studied by radioimmunoassay using an antiserum specific for the COOH-terminus of CCK8. Total concentrations of immunoreactive CCK in cortex were 3-4 fold higher than in jejunum. Rat cerebral CCK was purified by immuno-affinity adsorption to the IgG fraction of CCK antisera conjugated to Sepharose beads, and by gel filtration and ion exchange chromatography. Over 90% of immunoreactive CCK in cortex was accounted for by a factor with the properties of synthetic CCK8, and by a closely related slightly less acidic peptide. In contrast, intestinal CCK consisted of about equal amounts of CCK8-like activity and a larger less acidic immunoreactive component. In both cortex and intestine CCK8-like activity was obtained in substantially higher yield after extracting at neutral pH in boiling water than after extracting in either 0.2 M HCl or 0.5 M acetic acid. However, the larger molecular weight forms of CCK in the intestine were recovered in similar yield by acid and neutral extraction. The principal large form of rat CCK was distinguishable from porcine CCK33 on the basis of both optimum extraction conditions and chromatographic properties. It is suggested that the different distribution of immunoreactive CCK in rat brain and gut can be explained in terms of different biosynthetic processing pathways.