Delineation of the chemomechanosensory regulation of gastrin secretion using pure rodent G cells.

BACKGROUND & AIMS: Gastrin is a key regulator of gastric acid secretion. We aimed to isolate pure G cells to identify the mechanistic basis of luminal- and strain-mediated regulation. METHODS: Using gradient centrifugation and fluorescence-activated cell sorting, rat G cells were prepared and luminal, neural, hormonal, and mechanical activation of secretion and signaling pathways studied. RESULTS: Pure G-cell preparations (>97%) were isolated. Reverse-transcription polymerase chain reaction identified neural, hormonal, bacterial, and luminal G protein-coupled receptors, and immunostaining visualized specific sweet/bitter receptors and the tastant-associated G protein alpha-gustducin. Gastrin release was stimulated by forskolin (adenosine 3',5'-cyclic monophosphate [cAMP] inducer, 10 micromol/L; >3-fold), potentiated by 3-isobutyl-1-methylxanthine (IBMX; phosphodiesterase type 5 inhibitor and adenosine antagonist, 10 micromol/L) and phorbol myristate acetate (phorbol ester, 10 micromol/L), and inhibited by H-89 (protein kinase A inhibitor, 10 micromol/L), PD98059 (MEK1 inhibitor, 0.1 micromol/L), and wortmannin (phosphatidylinositol 3-kinase inhibitor, 1 nmol/L). Gastrin release was stimulated by neuronal G protein-coupled receptor ligands, pituitary adenylate cyclase-activating protein (20 pmol/L, >8-fold) and bombesin (0.1 micromol/L, 8-fold) through cAMP signaling. The tastants sucralose, glucose, caffeine, denatonium, and the vanilloid receptor activator capsaicin all stimulated secretion (>3-fold), as did bacterial lipopolysaccharides Salmonella enteritidis (0.24 nmol/L, 5-fold) greater than Helicobacter pylori (0.57 micromol/L, 3-fold). Secretion was associated with elevated cAMP levels (approximately 2-fold) and could be inhibited by H-89 and PD98059 and potentiated by IBMX and cholera toxin (250 microg/mL). Bacterially mediated secretion also involved activation of nuclear factor kappaB and the c-Jun-N-terminal kinase pathway. Mechanical strain stimulated (2-fold to 8-fold) gastrin release, and decreasing pH from 7.4 to 5.5 inhibited release. The adenosine receptor 2B antagonist MRS1754 inhibited mechanically induced gastrin release. CONCLUSIONS: G cells are luminal sampling chemomechanosensory cells whose secretion is regulated by neural, hormonal, luminal, and mechanical factors through protein kinase A activation, cAMP signaling, and mitogen-activated protein kinase phosphorylation.

G cells and gastrin in chronic alcohol-treated rats.

Numerous reports have described gastric mucosal injury in rats treated with high ethanol concentrations. However, to the best of our knowledge, ultrastructural characteristics of G cells and antral gastrin levels have not been previously reported, either in rats that chronically consumed alcohol or in human alcoholics. The goal of this study was to examine the effect of ethanol consumption (8.5 g/kg) over a 4-month period, under controlled nutritional conditions, on antral and plasma levels of gastrin, ultrastructure of G cells, morphometric characteristics of G cells by stereological methods, and analysis of endocrine cells in the gastric mucosa by immunohistochemistry. The chronic alcohol consumption resulted in a nonsignificant decrease in gastrin plasma levels and unchanged antral gastrin concentrations. A slightly damaged glandular portion of the gastric mucosa and dilatation of small blood vessels detected by histological analysis, suggests that ethanol has a toxic effect on the mucosal surface. Chronic alcohol treatment significantly decreased the number of antral G cells per unit area, and increased their cellular, nuclear, and cytoplasmatic profile areas. In addition, the volume density and diameter of G-cell granules, predominantly the pale and lucent types, were increased, indicating inhibition of gastrin release. Ethanol treatment also decreased the number of gastric somatostatin-, serotonin-, and histamine-immunoreactive cells, except the somatostatin cells in the pyloric mucosa, as well as both G: D: enterochromaffin cells (EC) cell ratios in the antrum and D: ECL cell ratios in the fundus. These results indicate that the change of morphometric parameters in G cells may be related to cellular dysfunction. Our findings also suggest that regulation of G-cell secretion was not mediated by locally produced somatostatin in ethanol-consuming rats, but may involve gastric luminal content and/or neurotransmitters of gastric nerve fibers.

Antral G-cell in gastrin and gastrin-cholecystokinin knockout animals.

The antral hormone gastrin is the key regulator of gastric acid secretion, mucosal growth and differentiation. Gastrin is synthesized in the endocrine G-cells in the antroduodenal mucosa. We have now examined the way in which the loss of gastrin alone or gastrin plus cholecystokinin (CCK) affects the antral G-cell. Immunohistochemistry, radioimmunoassay and quantitative real-time polymerase chain reaction techniques were employed to examine the expression of genes belonging to the G-cell secretory pathway in gastrin and gastrin-CCK knockout mice. Transmission electron microscopy was used to examine the ultrastructure of the G-cells. The number of G-cells increased but the secretory granules were few and abnormally small in the G-cells of both mouse models compared with wildtypes. Thus, gastrin is not necessary for the formation of G-cells as such but the lack of gastrin reduces the number and size of their secretory granules suggesting that gastrin is vital for the formation and/or maintenance of secretory granules in G-cells.

[Exocrinocytes of the gastric mucosa epithelium of Siberian chipmunk under different seasonal conditions].

The objective of this study was to analyze the morpho-functional remodeling of the epitheliocytes of stomach fundic glands in Siberian chipmunk under different seasonal conditions. The methods of light and electron microscopy were used. It was shown that during summer period, the structure of gastric fundic mucosa of Siberian chipmunk was similar to that one in other mammals. In contrast, in winter, during the periods of hypothermia, the cells of the fundic glands in the gastric corpus demonstrated the changes indicative of the suppression of their secretory activity. During the spontaneous arousals, all the elements of the gastric glands were activated, while the animals consumed the stored food. The present results were compared with the materials obtained earlier in the study of ground squirrels which also have a period of winter hibernation. Gastric cell remodeling in chipmunks during hypothermia was comparable in its trend with the changes taking place in ground squirrels, however, these changes were more pronounced in the latter. Food digestion in the stomach of chipmunk took place not only during the periods of normothermia, but also in the course of slow decline of the body temperature, when animals fell into a state of torpidity, and it recommenced at the early stages of animal rewarming during frequent episodes of its spontaneous arousal. Differences in the changes found in chipmunks and ground squirrels are associated with ecological peculiarities of these species and in the characteristics of the adaptation during the winter period.

G and D cells in rat antral mucosa: an immunoelectron microscopic study.

AIM: To investigate the gastrin secreting cells (G cells) and the somatostatin secreting cells (D cells) of antral mucosa in rats at the ultrastructural level. METHODS: Revised immunoelectron microscopic technique was used to detect the G cells and D cells in rat antral mucosa through gastrin and somatostatin antibodies labeled by colloidal gold. Also the relevant quantitative analysis regarding the granular number of colloidal gold in G cells and in D cells was conducted. RESULTS: Immunological granules of colloidal gold were distributed in G cells and D cells. Gastrin labeled golden granules or somatostatin labeled ones presented mainly as lobation-like or island-like congeries. Most of the golden congeries were observed dissociated in cytoplasms of G cells or D cells, near the basement membrane. A few golden congeries were located in nuclei. The number of golden granules in one G cell was around 107.04 +/- 19.68 and was 83.36 +/- 17.58 in one D cell. CONCLUSION: Gastrin secreting granules are located in cytoplasms and nuclei of G cells, and somatostatin secreting granules both in cytoplasms and in nuclei of D cells. The number of golden granules can be quantitatively analyzed to determine the relative amount of gastrin secreting granules or somatostatin secreting granules.

Ultrastructural immunohistochemical localization of gastrin, somatostatin and serotonin in endocrine cells of human antral gastric mucosa.

Five types of endocrine cells are found in the human antral gastric mucosa: gastrin (G) cells, somatostatin (D) cells, enterochromaffin (EC) cells and cells with an unknown secretory product (D1 cells and P cells). The content of secretory granules, gastrin, somatostatin and serotonin, was evaluated using electron microscopic immunohistochemistry and was compared with the granular content in G cells, D cells and EC cells as determined by routine electron microscopy. Semi-quantitative scoring of the granular content was performed on a scale 1-4 (empty-full). The content of gastrin (2.5 +/- 0.2) and somatostatin (3.3 +/- 0.2) in the granules was not different from the granular content in G cells (2.5 +/- 0.3; p > 0.05) and D cells (3.5 +/- 0.2; p > 0.05). Gastrin was also found in G cells in a nongranular form. The content of serotonin in granules (2.8 +/- 0.3) was smaller than the granular content in EC cells (3.7 +/- 0.2; p < 0.05). In intermediate-full and intermediate-empty granules, serotonin was localized in the periphery of granules whereas the granular content in EC cells was localized in an eccentric or central pattern. The granular content of D1 cells and P cells was 3.8 +/- 0.2, and 3.4 +/- 0.2, respectively. It is concluded that gastrin and somatostatin immunostaining in granules of G cells and D cells reflects the granular content in G cells and D cells, respectively, whereas serotonin immunostaining does not agree with the granular content of EC cells.

Antral G cells in rats during dosing with a PPAR alpha agonist: a morphometric and immunocytochemical study.

Gastrin-producing G cells constitute one of the major populations of neuroendocrine cells in the antral mucosa of the stomach. The peroxisome proliferator-activated receptor (PPAR) alpha-agonist ciprofibrate is used as a lipid-lowering drug. Recently, ciprofibrate has been shown to induce hypergastrinemia in rats without reducing gastric acidity, which indicates a direct stimulatory effect on the G cell. Gastrin probably plays an important role in gastric tumorgenesis, and long-term dosing with ciprofibrate results in enterochromaffin-like (ECL) cell carcinoids in the oxyntic mucosa of rats. In this study, we aimed to examine changes of neuroendocrine granules in G cells following ciprofibrate dosing and relate them to changes induced by the proton pump inhibitor pantoprazole. Furthermore, we wanted to study peroxisomes in G cells. Rats received ciprofibrate 80 mg/kg/day or pantoprazole 200 mg/kg/day in 4 weeks. Antral mucosal specimens were processed for conventional staining procedures and immunocytochemistry for both the light and electron micro-scope. Specimens were immunolabeled for gastrin and peroxisome-specific proteins. Electron micrographs were analyzed planimetrically. This study shows that hypergastrinemia induced by ciprofibrate is accompanied by a decrease in granule number per cell and a relative increase in electron-dense granules. These changes were quite similar to those induced by pantoprazole, indicating signs of G-cell activation in general. However, distinctions concerning granule size and composition and both hypertrophy and hyperplasia of G cells are presented. Finally, demonstration of peroxisomes in G cells was only achieved by using the highly sensitive tyramide signal amplification technique in immunostaining for the peroxisome-specific protein PMP-70. Therefore, neither morphological nor quantitative changes of peroxisomes in G cells were detected.

Light and electron microscopic immunohistochemical investigation on G and D cells in antral mucosa in Helicobacter pylori-related gastritis.

Recently, it has been recognized that Helicobacter pylori (H. pylori) infection is associated with an exaggeration of basal and meal gastrin secretion. We investigate whether there is a relationship between H. pylori-related chronic gastritis and G-cell and D-cell number and granule density index of G and D cells. - The number of antral G cells and D cells and granule density index of D and G cells are compared between thirty two patients with H. pylori-related chronic gastritis and twelve patients without H. pylori and inflammation. Antral mucosal biopsy specimens are examined using light and electron immunohistochemical techniques. - The number of G cells is the same in either infected or uninfected patients (98.40 +/- 11.39, 109.25 +/- 12.76 vs 101.17 +/- 7.72 for infected patients with non atrophic and with mild atrophic chronic gastritis and uninfected controls, respectively) except for the cases with moderate gastric mucosal atrophy, where G cells (58.22 +/- 5.63) decrease in number. The number of D cells is decreased in all patients with H. pylori-related gastritis. G cell granule density index is significantly (p < 0.05) increased in patients with H. pylori-related chronic gastritis than in controls (3.15 +/- 0.43 vs 2.528 +/- 0.01). D cell granule density index is similar between patients with H. pylori chronic gastritis and controls (3.18 +/- 0.05 vs 3.166 +/- 0.12). It is concluded that decreased D cells number in patients with H. pylori-related chronic gastritis might be one of the reasons for the existing hypergastrinaemia.

Gastrin producing G-cells after chronic ethanol and low protein nutrition.

Male Wistar rats, (2 months old), randomly divided according to the diet offered to four groups (C-control; A- alcoholized, PD-protein-deprived, A-PD- alcoholized protein-deprived). In group A and A-PD rats, the number of gastrin producing G-cells was significantly lower. The volume density of G-cells was significantly decreased in alcoholic rats. Fasting serum gastrin level (FSGL) significantly raised due to combined effect of alcohol consumption and protein malnutrition. In group A rats, the profile area of G-cells and their nuclei increased. In PD rats, the profile area of G cells also increased. There were no differences in nucleus/cell ratio due to alcohol ingestion alone, but it decreased significantly in PD and A-PD rats. Pale and lucent types of granules were predominantly seen in G-cells of animals of group A and A-PD. Mean diameter of granules increased in A, PD and A-PD rats. Other endocrine cells (ECL, D, EC) also decreased in number in A rats. Somatostatin producing D-cells decreased significantly in A-PD rats, both in fundic and pyloric mucosa.

Ultrastructural study of antral G cells in patients with duodenal ulcer: effect of Helicobacter pylori eradication.

BACKGROUND: It is established now that Helicobacter pylori infection is associated with exaggerated gastrin release to meals and other stimuli and that the abnormal secretion of gastrin is reversed after successful treatment of the infection. By comparing morphology of G cells from the same patients before and after treatment, we were able to investigate the ultrastructural effect of cure of H. pylori on G cells. METHODS: Gastric mucosal biopsy specimens were obtained from 10 patients with duodenal ulcer before and 3, 6, and 9 months after cure of H. pylori infection. Negative controls consisted of four healthy volunteers without H. pylori infection. G cells were evaluated by immunohistochemical and electron microscopy. Treatment with H2-antagonists was continued for 6 months after cure of the H. pylori infection. RESULTS: Ultrastructural studies of secretory granules of antral G cells in controls displayed a broad range of electron density ranging from dark with a full appearance to totally electron-lucent with a "vacuolating" appearance. In duodenal ulcer patients before treatment, electron-lucent vacuolating granules predominated. After elimination of H. pylori, G-cell granules showed a marked increase in electron density close to that of controls. CONCLUSIONS: Our study showed that the density of granules of G cells is decreased in H. pylori-infected duodenal ulcer patients compared to that in H. pylori-negative controls and is consistent with enhanced gastrin release. Cure of H. pylori infection was associated with return to normal density of G-cell granules.