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.

A tissue-engineered stomach shows presence of proton pump and G-cells in a rat model, resulting in improved anemia following total gastrectomy.

Despite advances in surgical reconstruction, total gastrectomy still is accompanied by various complications, especially chronic ones, such as pernicious anemia, resulting in refractory malnutrition. As an alternative approach, we have proposed a tissue-engineered stomach as a replacement of the native stomach. This study aimed to assess the secretory functions of a tissue-engineered stomach in a rat model and the nutritional status of the recipients over an extended time period. Stomach epithelial organoid units were isolated from neonatal rats and seeded onto biodegradable polymers. These constructs were implanted into the omenta of adult recipient rats. After 3 weeks, cyst-like structures had formed, henceforth referred to as tissue-engineered stomachs. The recipient stomachs were resected and replaced by their tissue-engineered counterparts. At 24 weeks after implantation, the secretory function of the tissue-engineered stomach was evaluated using immunohistochemical staining. The hemoglobin levels and nutritional status of the recipients were compared with a control group that had undergone a simple Roux-en-Y reconstruction following total gastrectomy. Recipient rats tolerated the tissue-engineered stomachs well. X-ray examination using barium as contrast showed no bowel stenosis. Staining for proton pump alpha-subunit and gastrin demonstrated the existence of parietal cells and G-cells in the neogastric mucosa, respectively, suggesting secretory functions. The treatment group showed significantly higher hemoglobin levels than the control group, although no differences in the body weight change, total protein, or cholesterol levels were observed between the two groups. A tissue-engineered stomach has the potential to function as a food reservoir following total gastrectomy. It is conjectured that replacement with a tissue-engineered stomach might restore the proton pump parietal cells and G-cells, and thereby improve anemia after a total gastrectomy in a rat model.

Histamine and histidine decarboxylase are hallmark features of ECL cells but not G cells in rat stomach.

The oxyntic mucosa of the rat stomach is rich in ECL cells which produce and secrete histamine in response to gastrin. Histamine and the histamine-forming enzyme histidine decarboxylase (HDC) have been claimed to occur also in the gastrin-secreting G cells in the antrum. In the present study, we used a panel of five HDC antisera and one histamine antiserum to investigate whether histamine and HDC are exclusive to the ECL cells. By immunocytochemistry, we could show that the ECL cells were stained with the histamine antiserum and all five HDC antisera. The G cells, however, were not stained with the histamine antiserum, but with three of the five HDC antisera. Thus, histamine and HDC coexist in the ECL cells (oxyntic mucosa) but not in G cells (antral mucosa). Western blot analysis revealed a typical pattern of HDC-immunoreactive bands (74, 63 and 54 kDa) in oxyntic mucosa extracts with all five antisera. In antral extracts, immunoreactive bands were detected with three of the five HDC antisera (same as above); the pattern of immunoreactivity differed from that in oxyntic mucosa. Food intake of fasted rats or treatment with the proton pump inhibitor omeprazole raised the HDC activity and the HDC protein content of the oxyntic mucosa but not of the antral mucosa; the HDC activity in the antrum was barely detectable. We suggest that the HDC-like immunoreactivity in the antrum represents a cross-reaction with non-HDC proteins and conclude that histamine and HDC are hallmark features of ECL cells but not of G cells.

Immunocytochemical evidence suggesting that diamine oxidase catalyzes biosynthesis of gamma-aminobutyric acid in antropyloric gastrin cells.

gamma-Aminobutyric acid (GABA) is a neurotransmitter that also occurs in a few non-neuronal cell types, where it may serve as a paracrine modulator. GABA is biosynthesized from glutamate by glutamate decarboxylase (GAD) and from putrescine via diamine oxidase (DAO). GAD is demonstrable in several GABA-positive cell types but is undetectable in the GABA-containing gastrin cells and somatostatin cells of the antropyloric mucosa of the stomach. Using two antisera raised against synthetic peptides corresponding to two different regions of rat DAO, we now demonstrate strong reactivity for DAO in gastrin-positive cells of the rat antropyloric mucosa, whereas somatostatin-positive cells as well as other structures of the antrum are unreactive. Western blotting analysis of antrum and colon demonstrate that both antisera react with a single band of 85 kD, consistent with the predicted molecular weight of DAO. Expression of DAO mRNA in the antrum is demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR). Our results strongly indicate that gastrin cells produce GABA via DAO-catalyzed oxidation of putrescine, and experimental data moreover suggest that the biosynthesis of GABA is regulated by the prandial state. Because GABA modulates release of somatostatin, these results point to a new mechanism of paracrine interaction between gastrin cells and somatostatin cells.