Gastrin Response Research Articles

GASTRIN IN SERUM AND MORPHOLOGICAL STATE OF GASTRIN-SECRETING CELLS IN PATIENTS WITH GASTRIC POLYPOSIS

Objective. Numerous studies regarding gastric hormones and their regulation have been performed until now. However, the effect of the hormones on the formation and malignisation of gastric polyps still remains not clear. Our aim was to identify the relation between the level of gastrin in the blood, gastric mucosa, polyp tissue, gastric juice and pathogenesis of gastric polyposis. Materials and methods. A thorough investigation of gastrointestinal hormones in serum and gastric juice, in polyp’s tissue and mucosa, gastrin-secreting cells and proteolytic activity of gastric juice was carried out in 40 patients with gastric polyps. These patients were divided into groups, depending on the location, number, and malignancy of the polyps. As a control group, 10 healthy individuals were used to determine the normal values of the studied indicators. Results: A significant increase (more than two times) in the gastrinemia level before the surgery was noted in patients with polyp recurrence, and gastrin level increased to more significant digits of 227.0+37.4 pg/ml (p<0.05) in one year after polypectomy. Conclusion. Gastrin is apparently involved in the process of polyp formation since polyp’s growth is accompanied by elevation of serum gastrin. This is confirmed by a response of gastrin in the blood to a test meal in individuals with different duration of the disease: a marked increase in gastrinemia appears in patients suffering from gastric polyposis for more than three years. Therefore, evaluation of gastrin level in the patients’ blood can be used to predict a recurrence potential of polyps. This is evidenced by more pronounced hypergastrinemia before polypectomy in patients who had a further recurrence of the disease within one year after the surgery

S2162 Hemorrhagic Shock Following a Secretin Stimulating Test

INTRODUCTION: Zollinger-Ellison Syndrome (ZES) is a rare disorder resulting from inappropriate gastrin release, which causes acid hypersecretion and the development of peptic ulcer disease (PUD), severe gastroesophageal reflux, and diarrhea. Diagnosing ZES is challenging in the age of PPIs. Furthermore, discontinuing PPI therapy in patients with suspected ZES can be dangerous and even life-threatening. CASE DESCRIPTION/METHODS: A 44 y.o. male with a history of PUD presented for evaluation for ZES. Due to concern for ZES, a secretin-stimulating test was ordered. One week prior to his test he was instructed to stop his PPI and start Ranitidine 450 mg every 6 hours until 24 hours prior to the test. Immediately following the secretin-stimulating test he presented to the emergency room with nausea, vomiting, and profuse watery diarrhea. A pantoprazole infusion at 8 mg/ hr was started. Overnight he became tachycardic and developed about 400cc of hematemesis. His Hbg dropped from 10.3 g/dL to 7.7 g/dL. He subsequently developed hemorrhagic shock requiring intubation and transfer to the ICU. On EGD, he had a large clot in the second portion of the duodenum. He had hematin in the stomach and over 10 ulcers in the duodenum (Image 1). The pantoprazole infusion was increased to 12 mg/hr with no further recurrence of hemorrhage. The secretin stim test returned, confirming the diagnosis of ZES. This was followed up with an Octreotide scan, which revealed increased radiotracer near the second portion of the duodenum, confirming the location of the gastrinoma (Image 2). DISCUSSION: This case illustrates a rare complication of a patient with suspected ZES undergoing a secretin stimulating test. In patient’s with ZES, a secretin stimulating test causes a paradoxical gastrin response to secretin, likely from secretin receptors on tumor cells (Image 3). Furthermore, PPI withdrawal can cause rebound acid secretion, which can lead to exaggerated acid hypersecretion in patient's with ZES. Our patient had an exaggerated response to secretin leading to acid hypersecretion causing PUD, diarrhea, and hemorrhagic shock. Although complications from a secretin stimulating test are rare, in patients with prior UGIB and high suspicion for ZES, a secretin-stimulating test may need to be performed in a controlled hospitalized setting to minimize complication. This case further illustrates how the diagnosis of ZES has become difficult and whether we need to propose new diagnostic criteria to avoid complications in establishing a diagnosis.Figure 1.: Image 1. Large Clot and Multiple Ulcers (Yellow Arrows) in the Duodenum.Figure 2.: Image 2. Markedly increased radiotracer uptake in a soft tissue nodule medial to the second portion of the duodenum, suggestive of a neuroendocrine tumor (confirmed to be gastrinoma by surgical resection).Figure 3.: Image 3. A. In the antrum, exogenous secretin stimulates gastrin secretion directly and concomitantly inhibits gastrin secretion by stimulating somatostatin (SST) secretion, resulting in little or no gastrin release. B. In patients with ZES, because the gastrinoma does not contain functionally coupled SST cells, the effect of secretin is solely to stimulate gastrin secretion from the tumor.

Increased oral sodium chloride intake in humans amplifies selectively postprandial GLP-1 but not GIP, CCK, and gastrin in plasma.

Human studies have demonstrated that physiologically relevant changes in circulating glucagon‐like peptide‐1 (GLP‐1) elicit a rapid increase in renal sodium excretion when combined with expansion of the extracellular fluid volume. Other studies support the involvement of various gastrointestinal hormones, e.g., gastrin and cholecystokinin (CCK) in a gut‐kidney axis, responsible for a rapid‐acting feed‐forward natriuretic mechanism. This study was designed to investigate the hypothesis that the postprandial GLP‐1 plasma concentration is sensitive to the sodium content in the meal. Under fixed sodium intake for 4 days prior to each experimental day, 10 lean healthy male participants were examined twice in random order after a 12‐hr fasting period. Arterial blood samples were collected at 10–20‐min intervals for 140 min after 75 grams of oral glucose + 6 grams of oral sodium chloride (NaCl) load versus 75 grams of glucose alone. Twenty‐four‐hour baseline urinary sodium excretions were similar between study days. Arterial GLP‐1 levels increased during both oral glucose loads and were significantly higher at the 40–80 min period during glucose + NaCl compared to glucose alone. The postprandial arterial responses of CCK, gastrin, and glucose‐dependent insulinotropic polypeptide as well as glucose, insulin, and C‐peptide did not differ between the two study days. Arterial renin, aldosterone, and natriuretic peptides levels did not change within subjects or between study days. Angiotensin II levels were significantly lower at the time GLP‐1 was higher (60–80 min) during glucose + NaCl. Sodium intake in addition to a glucose load selectively amplifies the postprandial GLP‐1 plasma concentration. Thus, GLP‐1 may be part of an acute feed‐forward mechanism for natriuresis.

Diagnostic accuracy of selective arterial calcium injection test for localization of gastrinoma.

The appropriate localization of gastrinoma is still difficult. We aimed to evaluate the diagnostic accuracy of selective arterial calcium injection (SACI) for localization of gastrinomas including multiple lesions. This retrospective study included ten patients with surgically proven gastrinomas (gastrinoma group) and six patients without any findings suggesting Zollinger-Ellison syndrome (non-gastrinoma group). For SACI, calcium gluconate was injected into the arteries supplying pancreas, duodenum, and liver. Blood samples from the hepatic vein were obtained before and 30, 60, and 120 seconds after each injection. The results were considered positive when the increase in serum immunoreactive gastrin (IRG) levels within 60 seconds of calcium gluconate injection were more than 80 pg/mL and more than 20% from baseline. We evaluated the efficacy of SACI by comparing the SACI responses with definitive locations diagnosed by clinical and histopathological findings. In the gastrinoma group, false-positive responses were confirmed in seven of the ten patients. False-negative response was observed in one of the feeding arteries of one patient with gastrinomas in multiple locations. Conversely, the greatest increase in serum gastrin levels from baseline at 30 seconds indicated the true-positive responses in all patients with gastrinomas. In the non-gastrinoma group, calcium gluconate injection into gastroduodenal artery evoked positive responses in five of the six patients. In conclusion, our data suggest the strongest gastrin response evoked by SACI indicates the definitive location in patients with gastrinomas. In contrast, SACI could not accurately locate multiple gastrin-secreting lesions due to poor specificity.

1075 - Increased Gastrin Responsiveness after Injury in Esophageal Submucosal Glands

1075 - Increased Gastrin Responsiveness after Injury in Esophageal Submucosal Glands

The impact of EndoBarrier gastrointestinal liner in obese patients with normal glucose tolerance and in patients with type 2 diabetes.

The duodenal-jejunal bypass sleeve ((DJBS) or EndoBarrier Gastrointestinal Liner) induces weight loss in obese subjects and may improve glucose homeostasis in patients with type 2 diabetes (T2D). To explore the underlying mechanisms, we evaluated postprandial physiology including glucose metabolism, gut hormone secretion, gallbladder emptying, appetite and food intake in patients undergoing DJBS treatment. A total of 10 normal glucose-tolerant (NGT) obese subjects and 9 age-, body weight- and body mass index-matched metformin-treated T2D patients underwent a liquid mixed meal test and a subsequent ad libitum meal test before implantation with DJBS and 1 week (1w) and 26 weeks (26w) after implantation. At 26w, both groups had achieved a weight loss of 6 to 7 kg. Postprandial glucagon-like peptide-1 (GLP-1) and peptide YY responses increased at 1w and 26w, but only in T2D subjects. In contrast, glucose-dependent insulinotropic polypeptide responses were reduced only by DJBS in the NGT group. Postprandial glucose, insulin, C-peptide, glucagon, cholecystokinin and gastrin responses were unaffected by DJBS in both groups. Satiety and fullness sensations were stronger and food intake was reduced at 1w in NGT subjects; no changes in appetite measures or food intake were observed in the T2D group. No effect of DJBS on postprandial gallbladder emptying was observed, and gastric emptying was not delayed. DJBS-induced weight loss was associated with only marginal changes in postprandial physiology, which may explain the absence of effect on postprandial glucose metabolism.

Gastrin Secretion After Bariatric Surgery-Response to a Protein-Rich Mixed Meal Following Roux-En-Y Gastric Bypass and Sleeve Gastrectomy: a Pilot Study in Normoglycemic Women.

Recent investigations have linked elevated gastrin levels to the improvement of type 2 diabetes mellitus (T2DM). Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG) are effective treatments for T2DM, but it is not known if this is related to postoperative alterations of gastrin secretion. Twenty women previously operated with RYGB or SG and 13 female controls were enrolled and evaluated for body mass index, lipids, C-peptide, HbA1c, and anti-H. pylori IgG. Glucose, gastrin, insulin, and glucagon-like peptide 1 (GLP-1) concentrations were measured before and 30, 60, 90, and 120min after ingestion of a protein-rich mixed meal. Six participants primarily selected were excluded due to usage of proton pump inhibitors, positive H.pylori IgG, or history of T2DM, yielding the following groups: RYGB (n = 9), SG (n = 8), and controls (n = 10). There were no differences in age, body mass index, HbA1c, or C-peptide levels between groups. RYGB had significantly lower area under the curve (AUC) for glucose during the test compared to controls (p = 0.013). RYGB showed lower serum gastrin levels compared to SG and controls (p < 0.05 for all). There was a non-significant increased gastrin release in SG compared to controls (p = 0.091). For SG and controls, there was a negative correlation between glucose and gastrin response (p = 0.0043). Gastrin secretion is diminished after RYGB. Hypergastrinemia was not present after SG, but a tendency of enhanced gastrin secretion was observed. These findings require further investigation in prospective studies.

Abstract 087: Human Stomach Cell Gastrin Inhibits Renal NHE3 and NaKATPase in Concert With the Renal D1R

The digestive track secretes gastrin in response to sodium ingestion stimulating increased renal sodium excretion. We previously showed that gastrin secreted by human colon cancer cells (SW626) can bind to cell surface cholecystokinin receptors on renal proximal tubule cells (RPTC), modulating the natriuretic dopaminergic system. We tested the hypothesis that a similar gastro/renal axis exists in humans. Novel human stomach antrum G-cell lines from 6 separate individuals were isolated and each shown to express gastrin mRNA and protein, and bind Phaseolus vulgaris Leucoagglutinin (PHA-l) by fluorescence lectin affinity (marker for gastrin secreting cells). It was determined that the D1 receptor (D1R) is also found on G-Cells. In human stomach tissue, gastrin expression was increased by Fenofibrate treatment, (PPARα agonist, p= 0.07, in 3 live ex-vivo cultured human stomachs). Moreover, we tested the effect of gastrin on CCKB2 receptors in human RPTC. Gastrin (100 nM 15 min.) increased RPTC phospholipace-C (PLC) activity by 1.07± 0.01 fold, N=35, p&lt;0.0001, using a PLC-FRET biosensor, CYPHR, but did not increase cAMP levels using the specific cAMP-FRET biosensor, ICUE-YR. Both gastrin and SKF83822 (100 nM, a cAMP specific D1R agonist, 15 min) alone reduced sodium influx into RPTC via NHE3 (from VEH 100± 4.7% to 73.5± 6.2% or 74.1± 4.3% respectively N=6, p&lt;0.05), but gastrin along with SKF83822 decreased sodium influx more than either alone (57.2 ± 5.8% N=6, p&lt;0.05). Sodium efflux via NaKATPase was reduced by SKF83822 (from VEH 100± 3.9% to 84.2± 3.3%, N=6, p&lt;0.05), but not gastrin alone, however SKF83822 along with gastrin reduced sodium efflux more than SKF83822 alone (72.3±5.1% vs N=6, p&lt;0.05). Additionally we found that Angiotensin II (AngII, 10 nM, 15 min.) increased NHE3 activity (12.3± 3.6% N=6, p&lt;0.01) and this increase was completely blocked by gastrin (N=6, p&lt;0.01). The PLC inhibitor U73122 reversed the inhibitory effect of gastrin on NHE3 and NaKATPase. Gastrin was also found to decrease the amount of fluorescent AngII binding to RPTCs (27.4± 6.1% N=6, p&lt;0.01) and this decrease was completely blocked by U73122. Thus, both stomach gastrin and the renal D1R inhibit NHE3 and NaKATPase, to increase sodium elimination from the body after salt is ingested.

Abstract 516: Intestinal Cell Gastrin Secretion is Regulated by a Sodium-Induced Increase in PPAR-A that is Mediated by Dopaminergic Stimulation

Recently a novel gastro-renal axis has been discovered for the regulation of renal sodium excretion at the moment of sodium ingestion. Cells in the digestive track secrete gastrin in response to sodium ingestion which stimulates an increase in renal sodium excretion. Dopamine is largely responsible for the regulation of sodium excretion in the kidney, and has also been shown to decrease sodium transport in intestines. We hypothesized that dopamine might also be involved with the regulation of gastrin secretion in a human intestinal gastrin secreting cell line (SW626, ATCC, Manassas VA) that also contains dopaminergic receptors. We used the ionophore monensin (MON) to increase intracellular sodium in SW626 cells in order to simulate an oral sodium load. We also stimulated the cells with the dopaminergic agonist SKF38393 (SKF, D 1 -like receptor agonist). These two manipulations (compared to vehicle VEH) caused increased relative gene expression of gastrin (ΔΔCt gastrin to actin, VEH 0.00638±0.00283; MON 0.3913372±0.0985926; SKF 0.2139286±0.0253672; P&lt;0.01 VEH vs MON or SKF, N=6 per group). Peroxisome proliferator-activated receptor-alpha (PPAR-α) associates with the gastrin transcriptional promoter by CHIP-SEQ (ENCODE database) and PPAR-α activation by fenofibrate (FENO) has been shown to reduce blood pressure and increase sodium excretion. We hypothesized that PPAR alpha would increase gastrin expression. FENO increased the immunoreactive gastrin and this effect was blocked by a PPAR alpha antagonist GW6471 (VEH 16,440±2,438 RFU; FENO 42,639±4,891; FENO+GW 19,367±1,865; P&lt;0.001 VEH vs FENO and FENO vs FENO+GW). Stimulation with MON or SKF also significantly stimulated the production of immunoreactive gastrin (VEH 16,440±2,438 RFU; MON 22,345±935; SKF 24,386±1,124; GW alone 10,235±1,214; P&lt;0.05 VEH vs MON, SKF, or GW, N=6). SW626 appear to secrete gastrin, therefore we tested the ability of secreted gastrin obtained from the media bathing SW626 cells to bind to i22 human renal proximal tubule cells (RPTC). After incubation of i22 RPTC in SW626 media for 24 hours, we detected surface gastrin staining on the i22 RPTC. We conclude that gastrin is produced by SW626 cells, is secreted into the growth media, and can bind to cell surface receptors on RPTC.

Obituary-Dr. Zahedi

A highly respected, renowned academician of his generation, Dr. Saleh Zahedi-Asl passed away on the 3rd November 2013, leaving behind a lasting legacy. His sudden, totally unexpected demise left us all shocked and bewildered, but forced to come to terms with the tragic loss. Dr. Zahedi was the Editor-in-Chief of both the International Journal of Endocrinology and Metabolism and the Iranian Journal of Endocrinology and Metabolism, and the Deputy Director of the Research Institute for Endocrine Sciences (RIES), Shahid Beheshti University of Medical Sciences, Tehran. In the past he had been the editor in-chief of the Scientific Medical Journal of Ahvaz University of Medical Sciences (AUMS). A member of several editorial boards, both international and national, research councils and national examination boards and societies, Dr. Zahedi was never intimidated by controversy, did not always concur with current trends and was always available to and willing to corroborate with and counsel authors within and outside Iran. Born in Ardebil, Iran, on August 29th 1951, Dr. Zahedi graduated with an honors bachelor’s degree in basic sciences from the University of Tabriz (1973) and went on to obtain his master’s in physiology from the university of Jundishapur, Ahvaz (1978). Dr. Zahedi then proceeded to the University Of Newcastle Upon Tyne, UK for his Ph.D. (1981), during which he developed radioimmunoassay for insulin, two kinds of glucagon (pancreatic & gut) and GIP (glucose-dependent insulinotropic pepetide). He was granted a MRC postdoctoral fellowship to work with Dr. John Brown on projects involving raising monoclonal antibodies to hormones, extraction, purification and sequencing of new biologically active peptides. He had published over 108 papers in Persian and 81 in English and edited one book and translated four books from English to Farsi. Dr. Zahedi’s publications include many papers on both animal and human research, as well as some valuable papers in the field of endocrinology and gastro-intestinal physiology. He had recently worked on the developmental effects of thyroid hormones and demonstrated for the first time that maternal hypothyroidism could affect carbohydrate metabolism and insulin secretion in adult offspring in rats. He also had some other publications on laboratory methods including the development of modified RIA and ELISA methods. He used the assays mentioned to study the plasma pancreatic and gut glucagon, GIP, insulin and gastrin responses to feeding in controls and patients with duodenal ulcer (DU) disease, who were significantly hypoglycaemic before and after breakfast compared with controls, and had significantly higher insulin, GIP and gut glucagon responses after feeding, demonstrating that the relative hypoglycaemia was enough to activate the vagus nerves and to cause peptic ulcer; he further showed that secretin provocation tests, usually used to diagnose pancreatic gastrinoma, produce marked insulin release in patients with primary hyperparathyroidism but not in controls or in patients with gastrinoma or those with DU disease. In another study, he reported that the gut hormone cholecystokinin (CCK) has a similar distribution in human brain compared with results documented for guinea pig and porcine brains. Using the gastrin assay and column chromatography to identity CCK molecules of different sizes, he also showed that in the cerebral cortex most of the CCK is in the grey matter and that in patients with Alzheimer disease there is significantly more glucagon in grey matter than in controls. He was very involved in teaching different aspects of human physiology, especially the GI tract, the endocrine system and circulation to both medical and post-graduate students, and had conducted well over fifty theses. To date he had conducted over 100 research projects, established a research laboratory and was currently working mainly on the behavior of isolated islets and isolated aorta from diabetic animals. He received many awards, including one for outstanding professor in 2011. A workaholic, Dr. Zahedi was a member of the Iranian Academy of Medical Sciences. Apart from this he held almost thirty-five other important key positions; he was a member and chairman of the physiology and pharmacology boards of the country, and was a founding member of continuing education in Iran. So many distinctive compliments come to mind when trying to eulogize Dr. Zahedi for what he was renowned and respected for, but space and words to describe him are lacking. Devout, self- effacing, affable, always the gracious gentleman, Dr. Zahedi is and will remain with us in our thoughts and prayers for a long long time.

Sa1738 Crosstalk Between PKD1 and β-Catenin Signaling Pathways in Intestinal Epithelial Cells

The hormone gastrin helps maintain gastric mucosal integrity by regulating expression of genes such as plasminogen activator inhibitor 2 (PAI-2) and Regenerating protein 1 (Reg1) , but the mechanisms involved are incompletely understood. We hypothesized that the proteasome beta subunit PSMB1, recently implicated in the gastrin responsiveness of vesicular monoamine transporter 2, may be required for gastrin-mediated upregulation of PAI-2 and Reg1. PSMB1 was knocked down by siRNA in the gastric cancer cell line AGS-GR, which expresses the cholecystokinin 2 receptor, and the gastrin response to PAI-2 (1.6kb) and Reg1 (2.1kb) promoter-luciferase reporter constructs determined. In control cells (scrambled RNA) gastrin (2nM G17 for 6h) increased PAI-2 transcription by 9.2±1.5 fold (n=6), but in cells with PSMB1 knocked down (69.5±5.5% protein reduction), this was significantly reduced to 2.1±0.2 fold (p,0.001, ANOVA). In control cells, gastrin increased Reg1 transcription by 2.4±0.9 fold (n=9), but with PSMB1 knocked down, the response was abolished (1.2±0.1 fold, p,0.03). To investigate PSMB1 binding to the promoters, ChIP assays were performed using an anti-PSMB1 antibody for immunoprecipitation, or IgG control. In anti-PSMB1 precipitated chromatin from cells treated with gastrin (2nM G17 for 2h), there was a 7.9±2.5 (n=3) fold enrichment of DNA compared to control, determined by real time PCR using primers that generated a 250bp amplicon incorporating the region of the PAI-2 promoter known to respond to gastrin. Using primers generating a 218bp amplicon including the gastrin responsive region of the Reg1 promoter we observed 10.0±4.6 fold enrichment. Immunocytochemistry of unstimulated AGS-GR cells revealed distribution of PSMB1 throughout the cytoplasm and nucleus. After 2h stimulation with 2nM G17, cytoplasmic PSMB1 was barely detectable and nuclear staining was intensified; after 6h stimulation, PSMB1 was again visible in cytoplasm. Western blotting of nuclear and cytoplasmic extracts showed that the nuclear:cytoplasmic ratio of PSMB1 changed from 1.7±0.4 in untreated cells to 10.6±2.6 (n=7, p,0.01) after 2h stimulation with G17. This subcellular redistribution was not seen with alpha (PSMA5) or regulatory (PSMC1) proteasome subunits. PSMB1 distribution was unaffected by activation of other receptors expressed by AGS-GR cells, including histamine, epidermal growth factor, PGE2, or IL-8. The response to G17 was however mimicked by phorbol 12-myristate 13-acetate (0.1μM), and prevented by the protein kinase C (PKC) antagonist Ro-32-0432 (1μM). We conclude that proteasome beta subunits bind to the PAI-2 and Reg1 promoters in response to gastrin stimulation of AGSGR cells and are required for transcription. Gastrin induces a PKC-dependent subcellular redistribution of beta proteasome subunits that may be linked to their transcriptional functions.

Sa1737 Intracellular CA2+ Oscillations Produced by the CA2+-Sensing Receptor Are Mediated by Negative Feedback by Protein Kinase C At Thr888

The hormone gastrin helps maintain gastric mucosal integrity by regulating expression of genes such as plasminogen activator inhibitor 2 (PAI-2) and Regenerating protein 1 (Reg1) , but the mechanisms involved are incompletely understood. We hypothesized that the proteasome beta subunit PSMB1, recently implicated in the gastrin responsiveness of vesicular monoamine transporter 2, may be required for gastrin-mediated upregulation of PAI-2 and Reg1. PSMB1 was knocked down by siRNA in the gastric cancer cell line AGS-GR, which expresses the cholecystokinin 2 receptor, and the gastrin response to PAI-2 (1.6kb) and Reg1 (2.1kb) promoter-luciferase reporter constructs determined. In control cells (scrambled RNA) gastrin (2nM G17 for 6h) increased PAI-2 transcription by 9.2±1.5 fold (n=6), but in cells with PSMB1 knocked down (69.5±5.5% protein reduction), this was significantly reduced to 2.1±0.2 fold (p,0.001, ANOVA). In control cells, gastrin increased Reg1 transcription by 2.4±0.9 fold (n=9), but with PSMB1 knocked down, the response was abolished (1.2±0.1 fold, p,0.03). To investigate PSMB1 binding to the promoters, ChIP assays were performed using an anti-PSMB1 antibody for immunoprecipitation, or IgG control. In anti-PSMB1 precipitated chromatin from cells treated with gastrin (2nM G17 for 2h), there was a 7.9±2.5 (n=3) fold enrichment of DNA compared to control, determined by real time PCR using primers that generated a 250bp amplicon incorporating the region of the PAI-2 promoter known to respond to gastrin. Using primers generating a 218bp amplicon including the gastrin responsive region of the Reg1 promoter we observed 10.0±4.6 fold enrichment. Immunocytochemistry of unstimulated AGS-GR cells revealed distribution of PSMB1 throughout the cytoplasm and nucleus. After 2h stimulation with 2nM G17, cytoplasmic PSMB1 was barely detectable and nuclear staining was intensified; after 6h stimulation, PSMB1 was again visible in cytoplasm. Western blotting of nuclear and cytoplasmic extracts showed that the nuclear:cytoplasmic ratio of PSMB1 changed from 1.7±0.4 in untreated cells to 10.6±2.6 (n=7, p,0.01) after 2h stimulation with G17. This subcellular redistribution was not seen with alpha (PSMA5) or regulatory (PSMC1) proteasome subunits. PSMB1 distribution was unaffected by activation of other receptors expressed by AGS-GR cells, including histamine, epidermal growth factor, PGE2, or IL-8. The response to G17 was however mimicked by phorbol 12-myristate 13-acetate (0.1μM), and prevented by the protein kinase C (PKC) antagonist Ro-32-0432 (1μM). We conclude that proteasome beta subunits bind to the PAI-2 and Reg1 promoters in response to gastrin stimulation of AGSGR cells and are required for transcription. Gastrin induces a PKC-dependent subcellular redistribution of beta proteasome subunits that may be linked to their transcriptional functions.

Assessment of Gastric Ulceration and Gastrin Response in Horses with History of Crib-Biting

It was hypothesized that horses exhibiting crib-biting (CB) have a greater degree of gastric mucosal damage and higher serum gastrin response to concentrate feeding than non-crib-biting (NCB) horses. Eighteen mature horses, 9 CB and 9 NCB, were used to determine prevalence and severity of gastric mucosal damage and effect of concentrate feeding on circulating gastrin. Horses were maintained on pasture with free access to hay and fed a pelleted concentrate diet twice daily. Number of crib-bites and duration of cribbing bouts were recorded in a 24-hour period. Endoscopic examinations (EE) of the squamous mucosa were performed and gastric fluid sampled after 24 to 28 hour feed removal. Following EE, horses were returned to pasture for 72 hours. Blood was collected following 12-hour feed removal (0 minutes), and at 60 and 120 minutes after consuming 1 kg of concentrate. Mean number of crib bites in 24 hours was 1,558 ± 303 with CB peaking prior to and during the afternoon feeding (3:30 PM, P < .05). There were no differences in the number or severity of ulcers, prevalence of hyperkeratosis, or baseline gastric pH between CB and NCB. Serum gastrin concentration at 60 and 120 minutes was greater (P < .05) and tended to be greater (P < .06), respectively, in CB than in NCB horses following feeding of concentrate. Crib-biting behavior in horses maintained on pasture was not associated with gastric mucosal damage; however, consumption of concentrate feed resulted in greater serum gastrin concentration in CB horses.

Ghrelin and gastrin in advanced gastric cancer before and after gastrectomy

To investigate plasma ghrelin, gastrin and growth hormone secretagogue receptor (GHS-R) expression in advanced gastric cancer (GC) before and after resection. Seventy subjects in whom endoscopy of the upper gastrointestinal tract was performed in the Department of General Surgery at Cracow University during the past decade: (1) 25 patients with GC associated with Helicobacter pylori (H. pylori) infection; (2) 10 patients with GC 4-5 years after (total or subtotal) gastrectomy; (3) 25 healthy H. pylori-negative controls, matched by age and BMI to the above two groups; and (4) 10 GC patients 4-5 years after total gastrectomy. Ghrelin and gastrin plasma concentrations were measured by specific radioimmunoassay under fasting conditions and postprandially at 60 and 90 min after ingestion of a mixed meal. GHS-R expression was examined in biopsy samples from intact healthy mucosa and GC tissue using semi-quantitative reverse transcription-polymerase chain reaction. In healthy controls, fasting plasma ghrelin levels were significantly elevated and declined markedly at 60 and 90 min after a mixed meal. The concomitant enhanced ghrelin, GHS-R and gastrin expression in GC tissue over that recorded in intact mucosa, and the marked rise in plasma gastrin in these subjects under fasting conditions indicate the role of these hormonal factors in GC formation. Fasting plasma levels and postprandial response of ghrelin and gastrin appear to be inversely correlated in healthy subjects. Feeding in the controls resulted in a significant fall in plasma ghrelin with a subsequent rise in plasma gastrin, but in H. pylori-positive GC patients submitted to total or distal gastrectomy, feeding failed to affect significantly the fall in plasma ghrelin that was recorded in these patients before surgery. Fasting ghrelin concentrations were significantly lower in patients 4-5 years after total gastrectomy compared to those in healthy controls and to these in GC patients before surgery. Elevated plasma gastrin and suppression of fasting ghrelin in patients with GC suggest the existence of a close relationship between these two hormones in gastric carcinogenesis.

Gastrin response to candidate messengers in intact conscious rats monitored by antrum microdialysis

We monitored gastrin release in response to locally applied candidate messengers in intact conscious rats. Earlier studies have been performed on anaesthetized animals, isolated pieces of antrum, or purified preparations of gastrin cells. In this study we created an experimental situation to resemble physiological conditions, using reverse microdialysis to administer regulatory peptides and amines that might affect gastrin secretion. Microdialysis probes were implanted in the submucosa of the antrum of the rat stomach. Three days later, putative messenger compounds were administered via the probe. Their effects on basal (24 h fast) and omeprazole-stimulated (400 μmol/kg/day, 4 days peroral administration) gastrin release were monitored by continuous measurement (3 h) of gastrin in the perfusate (radioimmunoassay). Fasted rats (low microdialysate gastrin, 2.1 ± 0.1 pmol l − 1 ) were used to study stimulation of gastrin release. Omeprazole-treated rats (high microdialysate gastrin, 95.8 ± 6.7 pmol l − 1 ) were used to study suppression of gastrin release. The following agents raised the concentration of microdialysate gastrin (peak response): gastrin-releasing peptide (GRP) (11-fold increase at a near-maximal dose), carbachol (5-fold increase), serotonin (2-fold increase) and isoprenaline (20-fold increase). Adrenaline and noradrenaline induced transient but powerful elevation (40- and 20-fold increase). Somatostatin, galanin and bradykinin (at near-maximal doses) suppressed omeprazole-stimulated gastrin release (50% decrease). Calcitonin gene-related peptide, ghrelin, gastric inhibitory peptide, motilin, neurotensin, neuromedin U-25, peptide YY and vasoactive intestinal peptide were without effect on gastrin release, as were aspartate, γ-aminobutyric acid, glutamate, glycine, dopamine and histamine. The results support the view that G cells operate under neurocrine/paracrine control. They were stimulated by agents present in enteric neurons (GRP, galanin, choline ester and catechol amines) and in gastric endocrine cells (serotonin). They were inhibited by somatostatin (D cell peptide), galanin (neuropeptide) and by the inflammatory agent bradykinin.

Gastrin release: Antrum microdialysis reveals a complex neural control

We used microdialysis to monitor local gastrin release in response to food, acid blockade and acute vagal excitation. For the first time, gastrin release has been monitored continuously in intact conscious rats in a physiologically relevant experimental setting in a fashion that minimizes confounding systemic effects. Microdialysis probes were placed in the submucosa on either side of the antrum, 3 days before the experiments. The concentration of gastrin in the antral submucosal compartment was about 20 times higher than in the microdialysate and estimated to be 5–10 times higher than in serum regardless of the prandial state. The rats were conscious during microdialysis except when subjected to electrical vagal stimulation. Acid blockade (omeprazole treatment of freely fed rats for 4 days), or bilateral sectioning of the abdominal vagal trunks (fasted, 3 days post-op.), raised the gastrin concentration in blood as well as microdialysate. The high gastrin concentration following omeprazole treatment was not affected by vagotomy. Vagal excitation stimulated the G cells: electrical vagal stimulation and pylorus ligation (fasted rats) raised the gastrin concentration transiently in both serum and microdialysate. Food intake induced a 2- to 3-fold increase in serum gastrin, while gastrin in antral microdialysate increased 10- to 15-fold. In unilaterally vagotomized rats (fasted, 3 days post-op.), food evoked a prompt peak gastrin release followed by a gradual decline on the intact side. On the vagotomized side of the antrum, the peak response seemed to be reduced while the microdialysate gastrin concentration remained elevated. Thus, unilateral vagotomy surprisingly raised the integrated gastrin response to food on the denervated side compared to the intact side, indicating that vagotomy suppresses an inhibitory as well as a stimulating effect on the G cells. While local infusion of atropine was without effect, infusion of the neuronal blocker tetrodotoxin (TTX) (which had no effect on basal gastrin) virtually abolished the food-evoked gastrin response and lowered the high microdialysate gastrin concentration in omeprazole-treated rats by 65%. We conclude that activated gastrin release, unlike basal gastrin release, is highly dependent on a neural input: 1) Vagal excitation has a transient stimulating effect on the G cells. The transient nature of the response suggests that the vagus has not only a prompt stimulatory but also a slow inhibitory effect on gastrin release. 2) Although vagal denervation did not affect the gastrin response to anacidity, the TTX experiments revealed that both food-evoked and anacidity-evoked gastrin release depends on neural input.

Evidence of extragastric gastrin release in postoperative ulcer patients.

The principal sites of gastrin production in man are localized at the level of the gastric antrum; both the oral glucose load and the protein meal stimulate the gastrin secretion. The aim of this study was to verify the gastrin response in patients with gastric resections presented with various stimuli. In the operated patients, the behavior of serum gastrin after a protein meal was different with respect to that observed in control subjects. After glucose, on the contrary, a very similar result was seen when compared to controls. The increase in serum gastrin of patients with Billroth II provides a further confirmation of an extragastric origin of gastrin.

Pharmacokinetic– pharmacodynamic analysis of the role of CYP2C19 genotypes in short‐term rabeprazole‐based triple therapy against Helicobacter pylori

The aim was to explore the role of CYP2C19 polymorphism in short-term rabeprazole-based triple therapy against Helicobacter pylori infection. Patients with H. pylori infection were tested for CYP2C19 genotype as poor metabolizers (PMs) or extensive metabolizers (EMs, homozygous EM or heterozygous EM) and given rabeprazole for 7 days. Antibiotics (clarithromycin and amoxicillin) were given on days 1-4, days 4-7, or days 1-7. A direct link model with an effect compartment was used in the population pharmacokinetic-pharmacodynamic analysis. The status of H. pylori infection was evaluated. Rabeprazole clearance was lower in CYP2C19 PMs than in EMs (with average values of 10.7 vs. 16.8 l h(-1) in PMs and EMs, respectively), resulting in higher plasma levels in the former group. The values of EC(50) and k(eo) of gastrin response increased with multiple doses of rabeprazole. The k(eo) values were lower in CYP2C19 PMs than in EMs on day 1 (0.012 vs. 0.017 x 10(-4) l min(-1)), and higher than in EMs on day 4 (0.804 vs. 0.169 x 10(-4) l min(-1)) of rabeprazole treatment. The predicted gastrin-time profile showed a higher response in CYP2C19 PMs than in EMs on days 4 and 7. Helicobacter pylori was eradicated in all CYP2C19 PMs except in one patient infected by a resistant strain. In contrast, in CYP2C19 EMs the eradication rates ranged from 58 to 85%. CYP2C19 genotypes play a role in H. pylori eradication therapy. Rabeprazole-based short-term triple therapy may be applicable in CYP2C19 PMs for H. pylori eradication.

Identification of a Gastrin Response Element in the Vesicular Monoamine Transporter Type 2 Promoter and Requirement of 20 S Proteasome Subunits for Transcriptional Activity

Vesicular monoamine transporter type 2 (VMAT2) is crucial for accumulation of monoamine neurotranmitters into neuronal secretory vesicles and histamine into secretory granules of the enterochromaffin-like cell in the acid-secreting gastric mucosa. Gastric VMAT2 expression is regulated by the antral hormone gastrin acting at the CCK(2) receptor. We demonstrate a gastrin response element (-56)ccgccccctc(-47) in the proximal VMAT2 promoter that binds in a gastrin-sensitive manner to nuclear proteins from gastric epithelial cell lines. Mutations within this sequence prevented nuclear protein binding and significantly reduced gastrin-stimulated expression of VMAT2 promoter-reporter constructs in gastric epithelial cells. In a yeast one-hybrid screen of an AR42J cell cDNA library, using the gastrin response element as bait, we identified a beta subunit of the 20 S proteasome, PSMB1, as a potential binding partner. In supershift assays, antibodies to PSMB1 and other proteasome beta subunits disrupted gastrin sensitive nuclear protein binding to the VMAT2 promoter. Moreover, RNA interference of PSMB1 significantly inhibited gastrin-mediated VMAT2 transcription. These data suggest that elements of the 20 S proteasome interact with the VMAT2 promoter to enhance G-protein-coupled receptor-mediated transcription.

Inducible cAMP early repressor suppresses gastrin-mediated activation of cyclin D1 and c-fosgene expression

The gastric hormone gastrin and its precursors promote proliferation in several gastrointestinal cell types. Here we show that gastrin induces transcription of cell cycle gene cyclin D1 and protooncogene c-fos in the neuroendocrine pancreatic cell line AR42J and that this gastrin response is inhibited by endogenous inducible cAMP early repressor (ICER). The transcriptional repressor ICER is known to downregulate both its own expression and the expression of other genes containing cAMP-responsive elements (CREs). Using siRNA, we also show that CRE promoter elements are the targets of endogenous ICER in AR42J cells as well as in the neuroendocrine cell line RIN5F. Our results suggest that ICER plays an important role in molecular mechanisms governing gastrin-mediated growth by modulating gastrin's transcriptional activation of growth-related genes. Our finding that ICER modulates pituitary adenylate cyclase-activating polypeptide-activated gene expression also indicates a regulatory effect of ICER in the responses of neuroendocrine cells to peptides other than gastrin.