Insights into Effects/Risks of Chronic Hypergastrinemia and Lifelong PPI Treatment in Man Based on Studies of Patients with Zollinger-Ellison Syndrome.

The use of proton pump inhibitors (PPIs) over the last 30 years has rapidly increased both in the United States and worldwide. PPIs are not only very widely used both for approved indications (peptic ulcer disease, gastroesophageal reflux disease (GERD), Helicobacter pylori eradication regimens, stress ulcer prevention), but are also one of the most frequently off-label used drugs (25-70% of total). An increasing number of patients with moderate to advanced gastroesophageal reflux disease are remaining on PPI indefinitely. Whereas numerous studies show PPIs remain effective and safe, most of these studies are <5 years of duration and little data exist for >10 years of treatment. Recently, based primarily on observational/epidemiological studies, there have been an increasing number of reports raising issues about safety and side-effects with very long-term chronic treatment. Some of these safety issues are related to the possible long-term effects of chronic hypergastrinemia, which occurs in all patients taking chronic PPIs, others are related to the hypo-/achlorhydria that frequently occurs with chronic PPI treatment, and in others the mechanisms are unclear. These issues have raised considerable controversy in large part because of lack of long-term PPI treatment data (>10-20 years). Zollinger-Ellison syndrome (ZES) is caused by ectopic secretion of gastrin from a neuroendocrine tumor resulting in severe acid hypersecretion requiring life-long antisecretory treatment with PPIs, which are the drugs of choice. Because in <30% of patients with ZES, a long-term cure is not possible, these patients have life-long hypergastrinemia and require life-long treatment with PPIs. Therefore, ZES patients have been proposed as a good model of the long-term effects of hypergastrinemia in man as well as the effects/side-effects of very long-term PPI treatment. In this article, the insights from studies on ZES into these controversial issues with pertinence to chronic PPI use in non-ZES patients is reviewed, primarily concentrating on data from the prospective long-term studies of ZES patients at NIH.

Pb2+ induces gastrin gene expression by extracellular signal-regulated kinases 1/2 and transcription factor activator protein 1 in human gastric carcinoma cells.

Divalent lead ions (Pb(2+) ) are toxic environmental pollutants known to cause serious health problems in humans and animals. Absorption of Pb(2+) from air, water, and food takes place in the respiratory and digestive tracts. The ways in which absorbed Pb(2+) affects cell physiology are just beginning to be understood at the molecular level. Here, we used reverse transcription PCR and Western blotting to analyze cultures of human gastric carcinoma cells exposed to 10 µM lead nitrate. We found that Pb(2+) induces gastrin hormone gene transcription and translation in a time-dependent manner. Promoter deletion analysis revealed that activator protein 1 (AP1) was necessary for gastrin gene transcription in cells exposed to Pb(2+) . MitogIen-activated protein kinase (MAPK)/ERK kinase inhibitor PD98059 suppressed the Pb(2+) -induced increase in messenger RNA. Epidermal growth factor receptor (EGFR) inhibitors AG1478 and PD153035 reduced both transcription and phosphorylation by extracellular signal-regulated kinase (ERK1/2). Cells exposed to Pb(2+) also increased production of c-Jun protein, a component of AP1, and over-expression of c-Jun enhanced activation of the gastrin promoter. In sum, the findings suggest the EGFR-ERK1/2-AP1 pathway mediates the effects of Pb(2+) on gastrin gene activity in cell culture.

Toxicoproteomic analysis of a mouse model of nonsteroidal anti-inflammatory drug-induced gastric ulcers.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are valuable agents; however, their use has been limited by their association with mucosal damage in the upper gastrointestinal tract. NSAIDs inhibit cyclooxygenase and consequently block the synthesis of prostaglandins, which have cytoprotective effects in gastric mucosa; these effects on prostaglandins have been thought to be major cause of NSAID-induced ulceration. However, studies indicate that additional NSAID-related mechanisms are involved in formation of gastric lesions. Here, we used a toxicoproteomic approach to understand cellular processes that are affected by NSAIDs in mouse stomach tissue during ulcer formation. We used fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS)-which consists of fluorogenic derivatization, separation and fluorescence detection by LC, and identification by LC-tandem mass spectrometry-in this proteomic analysis of pyrolic stomach from control and diclofenac (Dic)-treated mice. FD-LC-MS/MS results were highly sensitive; 10 differentially expressed proteins were identified, and all 10 were more highly expressed in Dic-treated mice than in control mice. Specifically, expression levels of 78 kDa glucose-regulated protein (GRP78), heat shock protein beta-1 (HSP27), and gastrin were more than 3-fold higher in Dic-treated mice than in control mice. This study represents a first step to ascertain the precise actors of early NSAID-induced ulceration.

The protective effects of calcitonin gene-related peptide on gastric mucosa injury of gastric ischemia reperfusion in rats.

Gastric mucosa is one of the most vulnerable tissues in human and animal. However, little is known about the effects of calcitonin gene-related peptide (CGRP) on gastric mucosa injuries induced by gastric ischemia reperfusion. The purpose of the present study was to investigate the protective effects and mechanism of CGRP on gastric mucosa injury after gastric ischemia reperfusion in rats. Thirty-six healthy Wistar rats were randomly divided into CGRP-treated, sham-operated, and control groups. Twelve rats were involved in each group. These groups were further divided into 24-h and 48-h subgroups. Gastric ischemia reperfusion injury (GI-RI) rat model was established by a 30-min celiac artery occlusion by an artery clamp, followed by 24 h or 48 h of reperfusion. CGRP (1 µg/ml) at the dose of 3 µg/kg was given intraperiloneally (IP) at the beginning of reperfusion for rats in CGRP-treated group. Saline as vehicle (3 ml/kg body weight), IP, was administered at the beginning of reperfusion for rats in control group. Sham-operated animals were subjected to an operation without GI-RI. Twenty-four hours or 48 h after operation, the samples were taken out and processed for calculating stomach mucous membrane damage index according to Guth method, detecting pathological changes of gastric mucosa tissue by light microscopy and observing the expression of gastrin (Gas) and somatostatin (SST) by immunohistochemical staining. The results showed the following: (i) gastric mucosa with diffuse edema, splinter hemorrhage and erosion, numerous endothelial cells necrosis, mucosa dissociation, and infiltration of inflammatory cells were observed in both control and CGRP-treated animals, especially in the earlier period (24 h) and then gradually healing. CGRP administration could reduce the damage of gastric mucosa. The injury index of gastric mucosa was lower in CGRP-treated group as compared with that in control group (P < 0.01). (ii) Gas expression in gastric antrum mucosa was lower in CGRP-treated group than that in control group (P < 0.01). SST expression in gastric antrum mucosa was higher in CGRP-treated group than that in control group (P < 0.01). It is concluded that CGRP regulated the secretion of Gas and SST and thus alleviated the damage of gastric mucosa induced by ischemia and reperfusion. CGRP might be a potential candidate for clinical therapy on modulating gastric mucosal protection and maintaining gastric mucosal integrity after ischemia and reperfusion of the stomach.

Long-term proton pump induced hypergastrinaemia does induce lineage-specific restitution but not clonal expansion in benign Barrett's oesophagus in vivo.

BACKGROUND: Barrett's oesophagus is a common premalignant lesion caused partly by acid reflux. Although the requisite therapy, proton pump inhibitors (PPIs), have been implicated in the progression of Barrett's oesophagus in animal models, harmful effects of prolonged PPI therapy in Barrett's oesophagus is both inconclusive and controversial. We therefore aimed to test the role of PPI-induced hypergastrinaemia in vitro and see whether any biological parameters were useful surrogates of long-term therapy in man. METHODS: We undertook detailed serological and tissue assessment of gastrin and CCK(2) receptors in 90 patients randomised to different doses of PPI therapy during a detailed 2-year follow-up. We also undertook a comprehensive study of cell models to study the consequential biological effects of gastrin on the mucosa. RESULTS: Gastrin and its cognate receptor CCK(2)R were expressed highest in the stomach, then less in Barrett's oesophagus and least in squamous oesophagus (SqE) (n=20 paired t-test, p<0.01). Analysis of the change in Barrett's oesophagus segment length change in 70 patients who were randomised to high or low PPI dose showed no difference over 2 years (n=70 t-test, p=0.8). Prolonged PPI use did, however, increase the serum gastrin, (36 pg/ml+/-57 pg/ml to 103 pg/ml+/-94 pg/ml (paired t test, p<0.05)). In vitro gastrin also induced changes in OE33(E)(cckr) Barrett's oesophagus cells, but not OE21(E)(cckr) squamous cells, transfected with CCK(2)R; migration was induced by 1 ng/ml of gastrin but proliferation only increased with 100 ng/ml (paired t-test, p<0.01) and both were abolished by antagonists. CONCLUSION: While the short-term effects of gastrin enhance epithelial restitution in Barrett's oesophagus (but not squamous mucosa) there is no clinical evidence that Barrett's oesophagus length expands over time. This study, which is the largest and longest term randomised controlled trial of gastrin biology in Barrett's oesophagus, is further proof of the clinical safety of PPI therapy.

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

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

Helicobacter pylori induction of the gastrin promoter through GC-rich DNA elements.

BACKGROUND: Helicobacter pylori (H. pylori) infection has been linked to the development of chronic gastritis, duodenal ulcer disease, and gastric cancer. Helicobacter pylori- infected patients and animal models develop hypergastrinemia, chronic gastritis, and gastric atrophy. Since gastrin is an important regulator of gastric acid secretion and cell growth, H. pylori regulation of this hormone has been implicated in its pathogenesis. OBJECTIVES: To investigate the effect of H. pylori on gastrin gene expression in mice and of human bacterial isolates on gastrin mRNA expressed in a human cell line. METHODS: Gastrin mRNA was measured by qRT-PCR in H. pylori-infected mice. H. pylori were co-cultured with AGS cells to study regulation of human gastrin gene expression. Various MAP kinases were implicated in signal transduction from the bacteria using specific inhibitors. Gastrin reporter constructs and gel shift assays were used to map DNA responsive elements. RESULTS: In addition to an increase in gastrin mRNA in H. pylori-infected mice, H. pylori induced the endogenous human gastrin gene through MAP kinase-dependent signaling but not NFκB-dependent signaling. Activation of gastrin through MAPK signaling did not require CagA or VacA virulence factors. Transfection studies demonstrated that a GC-rich motif mediated H. pylori-induction of the gastrin promoter and that the motif inducibly binds Sp1 and Sp3 transcription factors. CONCLUSIONS: Direct contact of live H. pylori bacteria with human cells is sufficient to induce gastrin gene expression.

Unusual multiple gastric carcinoids with hypergastrinemia: report of a case.

We report the case of a patient demonstrating multiple gastric carcinoids with hypergastrinemia. A 50-year-old Japanese woman was admitted to our hospital for the further examination of multiple carcinoids of the stomach with hypergastrinemia, although she was asymptomatic. However, based on our clinical examination, this case seemed to be neither type I nor II carcinoid. We performed a total gastrectomy with D1 lymph node dissection. A pathological examination showed numerous endocrine micronests, hyperplasia of the parietal cells extending to the foveolar neck region, and numerous dilated oxyntic glands filled with eosinophilic secretions. Many parietal cells exhibited vacuolated cytoplasms and apical snouts. Furthermore, the dilated glands at the base of the mucosa had hyperchromatic nuclei and ciliated surfaces. The postoperative serum gastrin level was soon normalized to 47 pg/ml. This is only the third reported case of multiple gastric carcinoids with hypergastrinemia due to an intrinsic abnormality in the acid secretion of the parietal cells.

[Helicobacter pylori cytotoxin associated protein CagA regulates gastrin gene promoter activity].

OBJECTIVE: To study the regulatory effect of Helicobacter pylori CagA protein on gastrin promoter and the related signaling pathways as to further elucidate the mechanism of the development and progression of human gastric carcinoma. METHODS: After pcDNA3.1ZEO(-)/CagAand PGL/GP were identified by double restriction enzyme digestion, PCR and sequencing, the gastric cancer cell lines AGS and SGC-7901 cells were co-transfected with pcDNA3.1ZEO(-)/CagA and PGL/GP for 48 h. Alternatively, AGS and SGC-7901 cells were transfected by PGL/GP for 36 h later, and infected with Helicobacter pylori for additional 12 h. Meanwhile, the transfected and infected cells were treated using the JAK2 signaling pathway inhibitor AG490 and the ERK signaling pathway inhibitor U0126. The untreated cells and empty-vector-transfected cells were used as the control. Finally, luciferase activity was detected using the luciferase reporter assay system in transfected and infected cells. The levels of gastrin mRNA was determined by TaqMan® real-time quantitative PCR. RESULTS: After co-transfection with pcDNA3.1ZEO(-)/CagA and PGL/GP, the activities of luciferase were increased by 251.3, 106.1 and 2.4 times in AGS cells and 35.8, 22.7 and 13.4 times in SGC-7901 cells, respectively, as compared with that of the control, pcDNA3.1 ZEO(-)/CagA + PGL3/Basic and pcDNA3.1 ZEO(-) + PGL/GP groups. The activities of luciferase in PGL/GP transfection and HP infection group were also increased by 1673.2, 33.5, 1.4 times in AGS cells and 1180.2, 72.2 and 1.5 times in SGC-7901 cells, respectively, as compared with that of the control, PGL3/Basic + HP and PGL/GP groups. There were statistically significant differences between them (P < 0.05), which suggested that the transcription activity of gastrin promoter increased significantly. But after adding the inhibitor AG490 and U0126, respectively, the activities of luciferase were significantly decreased by 95.7% (U0126) and 33.0% (AG490) in co-transfected AGS cells and 94.8% (U0126) and 86.2% (AG490) in co-transfected SGC-7901 cells with pcDNA3.1ZEO(-)/CagA and PGL/GP (P < 0.05). In the PGL/GP transfection and HP infection group, the activities of luciferase were significantly decreased by 24.6% (U0126) and 25.8% (AG490) in AGS cells and 57.3% (U0126) and 14.1% (AG490) after adding the inhibitor AG490 and U0126, respectively (P < 0.05). The results showed that the gastrin promoter activities were significantly inhibited. The gastrin mRNA levels were 3.0 and 4.5 times higher in HP-infected AGS and SGC-7901 cells, respectively, than that in the control groups. In the cells transfected with pcDNA3.1ZEO(-)/CagA, the gastrin mRNA levels were raised 10.8 and 2.3 times (AGS cells) and 10.9 and 16.2 times (SGC-7901 cells), respectively, as compared with that of control and pcDNA3.1ZEO(-) groups. All of the differences were statistically significant (P < 0.05). CONCLUSION: These results suggest that CagA may activate the gastrin promoter and up-regulate the expression of gastrin gene, and CagA is one of the important proteins in regulating gastrin gene expression. The ERK/MAPK and JAK/STAT signaling pathways may be involved in the controlling of gastrin gene expression by CagA.

Immunohistochemical relationships of huntingtin-associated protein 1 with enteroendocrine cells in the pyloric mucosa of the rat stomach.

Huntingtin-associated protein 1 (HAP1) is a neuronal cytoplasmic protein that is predominantly expressed in the brain and spinal cord. In addition to the central nervous system, HAP1 is also expressed in the peripheral organs including endocrine system. Different types of enteroendocrine cells (EEC) are present in the digestive organs. To date, the characterization of HAP1-immunoreactive (ir) cells remains unreported there. In the present study, the expression of HAP1 in pyloric stomach in adult male rats and its relationships with different chemical markers for EEC [gastrin, marker of gastrin (G) cells; somatostatin, marker of delta (D) cells; 5-HT, marker of enterochromaffin (EC) cells; histamine, marker of enterochromaffin-like (ECL) cells] were examined employing single- or double-labelled immunohistochemistry and with light-, fluorescence- or electron-microscopy. HAP1-ir cells were abundantly expressed in the glandular mucosa but were very few or none in the surface epithelium. Double-labelled immunofluorescence staining for HAP1 and markers for EECs showed that almost all the G-cells expressed HAP1. In contrast, HAP1 was completely lacking in D-cells, EC-cells or ECL-cells. Our current study is the first to clarify that HAP1 is selectively expressed in G-cells in rat pyloric stomach, which probably reflects HAP1's involvement in regulation of the secretion of gastrin.

Gastric-and-intestinal mixed endocrine cell phenotypic expression of carcinoid tumors in the rectum.

We have previously demonstrated that gastric and intestinal endocrine cell (End-cell) marker expression is important for assessment of the histogenesis of endocrine cell tumors. However, the End-cell phenotypes of carcinoid tumors in the rectum remain largely unclear. We therefore examined marker expression of rectal carcinoid tumors. We evaluated 20 rectal carcinoid tumors (as well as 8 from the stomach for comparison) phenotypically, using gastrin, gastric inhibitory polypeptide (GIP) and glucagons-like peptide-1 (GLP-1) as End-cell markers. Rectal carcinoid tumors were divided into 3 endocrine-gastric (e-G), 16 endocrine-gastric-and-intestinal mixed (e-GI), 1 endocrine-intestinal (e-I), and 0 endocrine-null (e-N) types, thus 19 (e-G+ e-GI types, 95%) had gastric phenotypic expression, while 17 (e-GI+ e-I types, 85%) harbored intestinal elements. Stomach carcinoid tumors were classified as 6 e-G and 2 e-N types, respectively. In conclusion, most rectal carcinoid tumors exhibited the e-GI type, suggesting the importance of gastric End-cell marker expression for histogenesis of the rectal carcinoid tumors. Further studies of pathological and biological analyses are needed to clarify the histogenesis of the carcinoid tumors.

Day/night differences in stress-induced gastric lesions in rats with an intact pineal gland or after pinealectomy.

The formation of acute gastric lesions depends upon the balance between the aggressive factors promoting mucosal damage and the natural defense mechanisms. Previous studies have shown that melatonin inhibits gastric acid secretion, enhances the release of gastrin, augments gastric blood flow (GBF), increases the cyclooxygenase-2 (COX-2)-prostaglandin (PG) system and scavenges free radicals, resulting in the prevention of stress-induced gastric lesions. Besides the pineal gland, melatonin is also generated in large amounts in the gastrointestinal tract and due to its antioxidant and anti-inflammatory properties; this indole might serve as local protective endogen preventing the development of acute gastric damage. The results of the present study indicate that stress-induced gastric lesions show circadian variations with an increase in the day time and a decline at night. These changes are inversely related to plasma melatonin levels. Following pinealectomy, stress-induced gastric mucosal lesions were more pronounced both during the day and at night, and were accompanied by markedly reduced plasma melatonin levels with a pronounced reduction in mucosal generation of prostaglandin E(2) (PGE(2)), GBF and increased free radical formation and by small rise in plasma melatonin during the dark phase. We conclude that stress-induced gastric ulcerations exhibit a circadian variation with an increase in the day and attenuation at night and that these fluctuations of gastric stress ulcerogenesis occur also after pinealectomy, depending upon the interaction of COX-PG and free radicals, probably mediated by the changes in local gastric melatonin.

Role of the gastrin-gastrin receptor system in the expansive growth of human gastric neoplasms.

BACKGROUND AND AIM: Gastrin is one of the most important gut hormones. However, the role of the gastrin-gastrin receptor (GR) system in the growth of gastric tumors is still unclear. METHODS: We examined serum gastrin levels in 957 patients with early gastric carcinoma. Next, we raised antibody against the GR and examined GR expression in 5 gastric carcinoma cell lines and 48 human gastric tumor tissues. In 28 cases, Helicobacter pylori eradication therapy was performed and morphological tumor changes were examined. RESULTS: Serum gastrin levels were significantly higher in patients with elevated tumors than in patients with depressed tumors (p = 0.02). All gastric carcinoma cell lines expressed GR. Thirty-one of 48 (65%) gastric tumors expressed GR, and its expression was prominent in elevated-type tumor with an intestinal histologic feature. Of 28 patients who underwent eradication therapy, 9 showed gastric tumors that became flat or depressed. In these 9 cases, GR expression was detected in all tumors, and the decrease in gastrin levels was more prominent than in those without morphological change (p = 0.01). CONCLUSION: The gastrin-GR system plays an important role in the elevated morphology of gastric tumors.

The gastrin gene promoter is regulated by p73 isoforms in tumor cells.

p73, a new p53 family member, is a transcription factor that is increasingly recognized in cancer research as an important player in tumorigenesis as well as in chemotherapeutic drug sensitivity. Despite the substantial structural and functional similarities to p53, accumulating evidence suggests that p53 and p73 may differently regulate their transcriptional targets. In this study, we have investigated the role of p73 in regulation of the gastrin gene promoter. Gastrin is a peptide hormone and an important factor in determining the progression of a number of human malignancies. Our results show that p73 can bind to the gastrin promoter. This leads to transcriptional upregulation of gastrin mRNA. We also found that the levels of gastrin and p73 transcripts correlate in primary gastric tumors. Taken together, our results demonstrate a novel mechanism for regulation of gastrin gene transcription and support a concept that p53 and p73 may have different biological roles in tumors.

Pathways of processing of the gastrin precursor in rat antral mucosa.

The precursor of the acid-stimulating hormone gastrin gives rise to multiple peptides differing markedly in biological activity, but the relevant biosynthetic pathways are poorly understood. We have used antibodies to amidated gastrins, gastrins with COOH-terminal glycine (Gly) gastrins with COOH-terminal hydroxyglycine (GlyOH) and to the COOH terminus of progastrin, to immunoprecipitate peptides labeled with [35S]sulfate or [3H]tyrosine during incubation of rat antral mucosa in vitro. Labeled progastrin was detectable after 30 min of continuous incubation with isotopic precursors, G34 and G34-Gly after 60 min, and G17 and G17-Gly after 120 min. Pulse chase experiments indicated that progastrin is converted to G34-Gly which then follows one of two pathways: (a) hydroxylation of COOH-terminal Gly and conversion to G34 followed by cleavage yielding G17, or (b) cleavage to G17-Gly. The kinetics of G17-Gly and G17 labeling were similar, suggesting that G17-Gly is a product in its own right, and not simply an intermediate in G17 synthesis. Since the two peptides are reported to have distinct biological activities, they appear to be alternative mature products of progastrin processing.

EGF receptor activation stimulates endogenous gastrin gene expression in canine G cells and human gastric cell cultures.

Gastrin release from the antral gastrin-expressing cell (G cell) is regulated by bombesin and luminal factors. Yet, these same extracellular regulators do not stimulate expression of the gene. Since the gastric mucosa expresses large quantities of EGF receptor ligands such as TGFalpha, we examined whether EGF receptor ligands stimulate gastrin gene expression in gastrin-expressing cell cultures. EGF receptor activation of primary cultures stimulated gastrin gene expression about twofold; whereas bombesin treatment of antral G cell cultures stimulated gastrin release but not gene expression. EGF and TGFalpha were weak stimulants of gastrin release. EGF receptor activation of AGS human gastric adenocarcinoma cell line stimulated gastrin gene expression nearly fourfold; and gastrin reporter constructs transfected into AGS cells were stimulated more than fourfold by EGF. EGF induction was conferred by the previously defined GC-rich gastrin EGF response element (gERE) element located at -68 to -53 bp upstream from the cap site since a mutation of the gERE element abolished both basal and EGF induction. Moreover, EGF treatment of AGS cells stimulated binding of the transcription factor Sp1 to this element. Collectively, these results demonstrate that gastrin gene expression and gastrin release are regulated by different signaling pathways: gene expression by EGF receptor activation and gastrin secretion by neuropeptides and luminal factors.

Stimulation of gastrin secretion and synthesis in antral organ culture.

The purpose of the present study was to examine stimulation of gastrin release and the synthesis of gastrin directly by measurement of incorporation of [(3)H]tryptophan into gastrin in rat antral mucosal explants maintained in organ culture. Gastrin synthesis and secretion were assessed simultaneously at intervals over the 24-h duration of explant culture. Antral mucosal explants from fed female Wistar rats (4-5 wk, 100-150 g) were cultured at 37 degrees C (95% O(2)/5% CO(2)) in medium containing 70% Trowell-T8 and 10% NCTC-135 without unlabeled tryptophan, 10% dialyzed fetal calf serum and [(3)H]tryptophan (100 muCi/ml). Antral tissue was harvested at regular intervals during 24-h culture periods. Incorporation of [(3)H]tryptophan into immunoreactive gastrin was determined by techniques utilizing double-antibody immunoprecipitation. Antral tissue protein synthesis was assessed by measurements of incorporation of [(3)H]tryptophan into tissue protein of cultured antral explants. In paired experiments, gastrin synthesis and secretion in the presence of dibutyryl cAMP (DBCAMP) were compared to those observed under control conditions. Gastrin and protein specific activity progressively increased with time. Gastrin specific activity at 30 min increased from 3.3+/-0.5 (SEM) to 55.2+/-10.6 fmol [(3)H]tryptophan/pmol gastrin (or from 1.57+/-0.48 to 26.28+/-5.05 pmol [(3)H]tryptophan/mug gastrin) at 24 h: specific activity of antral tissue protein at 30 min increased from 33.6+/-8.4 to 1,660+/-236 fmol [(3)H]tryptophan/mug protein at 16 h. Culturing of explants for 4 h in the presence of cycloheximide (100 mug/ml) inhibited both gastrin synthesis and protein synthesis by greater than 90 and 95%, respectively. DBCAMP (10 mM) significantly increased both the synthesis and secretion of antral gastrin when compared with control cultured explants. Results of these experiments provide direct demonstration of gastrin synthesis by rat antral mucosal explants in organ culture, indicate that both gastrin and total antral protein synthesis are inhibited by cycloheximide, and demonstrate DBCAMP-induced stimulation of both gastrin synthesis and secretion, suggesting the potentially important role of cyclic AMP in gastrin cell function.

Reciprocal regulation of antral gastrin and somatostatin gene expression by omeprazole-induced achlorhydria.

Gastric acid exerts a feedback inhibition on the secretion of gastrin from antral G cells. This study examines whether gastrin gene expression is also regulated by changes in gastric pH. Achlorhydria was induced in rats by the gastric H+/K+ ATPase inhibitor, omeprazole (100 mumol/kg). This resulted in fourfold increases in both serum gastrin (within 2 h) and gastrin mRNA levels (after 24 h). Antral somatostatin D cells probably act as chemoreceptors for gastric acid to mediate a paracrine inhibition on gastrin secretion from adjacent G cells. Omeprazole-induced achlorhydria reduced D-cell activity as shown by a threefold decrease in antral somatostatin mRNA levels that began after 24 h. Exogenous administration of the somatostatin analogue SMS 201-995 (10 micrograms/kg) prevented both the hypergastrinemia and the increase in gastrin mRNA levels caused by omeprazole-induced achlorhydria. Exogenous somatostatin, however, did not influence the decrease in antral somatostatin mRNA levels seen with achlorhydria. These data, therefore, support the hypothesis that antral D cells act as chemoreceptors for changes in gastric pH, and modulates somatostatin secretion and synthesis to mediate a paracrine inhibition on gastrin gene expression in adjacent G cells.

A GC-rich element confers epidermal growth factor responsiveness to transcription from the gastrin promoter.

Epidermal growth factor (EGF) and transforming growth factor alpha are important determinants of mucosal integrity in the gastrointestinal tract, and they act both directly and indirectly to prevent ulceration in the stomach. Consistent with this physiological role, EGF stimulates transcription of gastrin, a peptide hormone which regulates gastric acid secretion and mucosal growth. EGF stimulation of gastrin transcription is mediated by a GC-rich gastrin EGF response element (gERE) (GGGGCGGGGTGGGGGG) which lies between -54 and -68 in the human gastrin promoter. The gERE sequence also confers weaker responsiveness to phorbol ester stimulation. The gERE sequence differs from previously described EGF response elements. The gERE DNA sequence specifically interacts with a GH4 DNA-binding protein distinct from previously described transcription factors (Egr-1 and AP2) which bind GC-rich sequences and mediate transcriptional activation by growth factors. Furthermore, the gERE element does not bind the Sp1 transcription factor even though the gERE sequence contains a high-affinity Sp1-binding site (GGCGGG).

[Expressions of gastrin and apoptosis-associated proteins involved in mitochondrial pathway in gastric cancer tissues and the clinical significance].

Objective To investigate the expressions and correlations of gastrin and apoptosis-associated proteins involved in mitochondrial apoptotic pathway in gastric cancer tissues, and explore their clinicopathological characteristics. Methods The tissue chip technology and immunohistochemistry (IHC) were used to detect the expressions of gastrin and apoptosis-associated proteins (Bcl2, caspase-9 and caspase-3) in human gastric cancer tissues and their paracancerous tissues. The correlations of these markers and their clinicopathological characteristics were analyzed using Spearman rank correlation analysis and Chi-square test. Results The expressions of gastrin and Bcl2 in gastric cancer tissues were significantly higher than those in the corresponding paracancerous tissues, whereas the expressions of caspase-9 and caspase-3 in gastric cancer tissues was significantly lower than those in the corresponding tissues. There was a significant positive correlation between the expressions of gastrin and Bcl2 in gastric cancer tissues (r=0.237). The expression of gastrin was associated with tumor position, and the expression of Bcl2 was associated with tumor size, TNM stage and lymph node metastasis. Conclusion Gastrin and Bcl2 are highly expressed in gastric cancer tissues, and they are correlated with the clinicopathologic features.