Gastrin secretion from human antral G cells in culture

Receptor-dependent and -independent regulation of gastrin secretion from cultured human antral G cells was investigated. Human antral mucosal cell preparations that were enriched for G cells were obtained by sequential incubations with collagenase and ethylenediaminetetraacetic acid, centrifugal elutriation, and short-term culture. After a 2-day incubation period, gastrin- and somatostatin-containing cells accounted for 15% and 5%, respectively, of the total adhered-cell population. Forskolin, A23187, and beta-phorbol 12 myristate 13-acetate stimulated basal gastrin secretion from cultured human G cells in a concentration-dependent fashion. These results indicate that gastrin release could be mediated by elevations in cytosolic cyclic adenosine monophosphate levels, calcium influx, or activation of protein kinase C. A direct stimulatory role for bombesin- and gastrin-releasing peptide was supported by experiments showing concentration-dependent enhancement of gastrin release by bombesin from 0.01 fmol/L to 10 nmol/L. The putative bombesin antagonist [Leu13-psi-CH2NH-Leu14] bombesin augmented basal gastrin levels by itself and produced weak inhibition of bombesin-induced gastrin secretion from human antral G cells. Somatostatin potently suppressed forskolin- and bombesin-mediated gastrin release but did not significantly alter basal gastrin levels. These results suggest that bombesin and somatostatin directly activate and inhibit G-cell function via specific and sensitive receptors. Furthermore, the adenylate cyclase and phosphatidyl inositide second messenger systems seem to be intracellular mediators of gastrin secretion from human antral G cells.     

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.     

Hyperplasia of antral G cells in uraemic patients

In this study we have performed a quantitative analysis of antral gastrin cells in 9 uraemic patients under dialytic treatment compared to a group of 10 chronic gastritis patients with a similar histological degree of gastritis. Immunocytochemistry was carried out on endoscopic biopsies of both groups. Using a computerised image analysing system we showed a significant increase of G-cell density (number of cells/mm2) in uraemic patients compared to non-uraemics (p less than 0.001). These findings provide a further morphological basis for the elevation of gastrin levels in chronic uraemic patients, suggesting the existence of a specific factor inducing G-cell proliferation in these patients.     

Immunocytochemical localisation of parietal cells and G cells in the developing human stomach.

Previous studies on the distribution of parietal cells and G cells in normal adult stomachs have shown that in about 20% of specimens parietal cells extended to the pylorus. This study aimed to examine the distribution of parietal cells and G cells in the body and antrum of the developing human stomach in relation to anatomical landmarks, using histological and immunocytochemical methods. In all 15 fetal stomachs examined, parietal cells extended to the pylorus and expressed intrinsic factor and hydrogen-potassium-ATPase activity from week 13 of gestation. By contrast, in only one of the five infant stomachs did parietal cells extend to the pylorus: this is identical to the distribution in the adult. G cells developed in the antrum from 18 weeks' gestation and their distribution did not differ between the fetal and infant stomachs. These findings indicate that parietal cells disappear from the antrum of the stomach in the third trimester of pregnancy, but this process fails to occur in approximately 20% of the population.     

Dystroglycan is present in rat thyroid and rat thyroid cells and responds to thyrotropin.

Dystroglycan is a high affinity laminin-binding glycoprotein originally described as a member of the dystrophin-associated glycoprotein complex in muscle. We have demonstrated the presence of dystroglycan in the thyroid using immunocytochemistry, immunoblots, ligand binding assays, and relative quantitative RT-PCR. In intact rat thyroid glands, antibodies against the alpha (extracellular, laminin-binding subunit) and beta (cytoplasmic/membrane bound) portions of the dystroglycan protein reacted at basolateral membranes where they colocalized with laminin. Western-blotted protein from the Fischer rat thyroid cell line FRTL-5 reacted with both the alpha- and beta-dystroglycan antibodies. The alpha-dystroglycan-reactive band colocalized with laminin-binding activity, and the protein and binding activity were decreased by TSH. In contrast, in the culture medium of these cells, alpha-dystroglycan was increased by TSH. The beta-dystroglycan antibody recognized the full-length 43-kDa band and an approximately 30-kDa truncated form. The truncated form was reduced in cells cultured with TSH, whereas the full-length form was not significantly diminished by TSH. Immunofluorescence of FRTL-5 cells in the absence of TSH showed a colocalization of dystroglycan and laminin. This was disrupted by the addition of TSH and was correlated to morphological changes. PCR amplification of complementary DNA with primer pairs from alpha- and beta-dystroglycan produced appropriately sized bands, whose sequence had identical protein-coding sequences and more than 96% nucleotide homology to mouse dystroglycan sequences. Relative quantitative RT-PCR of beta-dystroglycan messenger RNA showed reduced expression in cells cultured with TSH. We conclude that dystroglycan is present in rat thyroid and in FRTL5 rat thyroid cells and that TSH reduces its expression.     

High-molecular weight kininogen is present in cultured human endothelial cells: localization, isolation, and characterization

The presence of high-molecular weight (mol wt) kininogen was demonstrated in cultured human endothelial cells derived from the umbilical cord by immunofluorescence techniques. Cultured human endothelial cells contain 58 +/- 11 ng (n = 16) high-mol wt kininogen/10(6) cells as determined by an enzyme-linked immunosorbent assay (ELISA) specific for high-mol wt kininogen. High-mol wt kininogen was isolated from cultured human endothelial cells by immunoaffinity chromatography. Nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that endothelial cell high-mol wt kininogen consisted of five protein bands with mol wts of 95,000, 85,000, 65,000, 46,000, and 30,000 daltons. Immunoblotting of the endothelial cell high-mol wt kininogen by using specific antisera against the heavy and light chain indicated that the 95,000-, 85,000-, and 65,000-dalton bands consisted of the heavy and light chain whereas the 46,000- and 30,000-dalton bands reacted only with the anti-light chain antiserum. Immunoprecipitation studies performed with lysed, metabolically labeled endothelial cells and monospecific antisera directed against high-mol wt kininogen suggested that high-mol wt kininogen is not synthesized by the endothelial cells. Endothelial cells cultured in high-mol wt kininogen-free medium did not contain high-mol wt kininogen. These studies indicate that endothelial cell high-mol wt kininogen was proteolytically cleaved in the culture medium and subsequently internalized by the endothelial cells. Binding and internalization studies performed with 125I-labeled, proteolytically cleaved, high-mol wt kininogen showed that endothelial cells can indeed bind and internalize proteolytically cleaved high-mol wt kininogen in a specific and saturable way.     

Neuropeptide W influences the excitability of neurons in the rat hypothalamic arcuate nucleus

Neuropeptide W (NPW) is a ligand of the recently deorphaned receptor GPR7. Intracerebroventricular injection of this peptide results in reduced serum growth hormone concentration. Using whole-cell patch clamp recordings from somatostatin (SS) neurons in the hypothalamic arcuate nucleus, identified post-hoc using single-cell RT-PCR, we investigated the effects of NPW on membrane excitability. NPW application in acute slices of the arcuate nucleus resulted in the depolarization of the majority (62.5%) of the SS neurons tested, while smaller proportions of cells showed hyperpolarization or no response. Both the depolarization and hyperpolarization of arcuate SS neurons were preserved during recordings where voltage-gated sodium channels were blocked with tetrodotoxin, suggesting direct effects of NPW on the excitability of SS neurons. The observed depolarization of the majority of the SS neurons tested suggests that the central effects of NPW to inhibit growth hormone release results from activation of arcuate SS neurons, which could result in an inhibition of GHRH-releasing neurons.     

Self-replication of enterochromaffin-like cells in the mouse stomach

The renewal mechanisms for enterochromaffin-like (ECL) cells, the predominating endocrine cell population in the oxyntic mucosa of the stomach, were investigated in the mouse. The ECL cells were selectively demonstrated by immunostaining using histamine antibodies. Under basal conditions, when observed during the night, ECL cells in mitosis could be seen. This observation proved their ability to divide. Autoradiography after a single pulse and after multiple injections of 3H-thymidine made it possible to study some of their cytokinetic characteristics. The observed replication rate of the labeled ECL cells suggested that self-replication is the main mechanism by which the ECL cell population is renewed. The time interval between two successive divisions of labeled ECL cells was estimated to be around 60 days. Since ECL cells proliferate through mitosis, it may be expected that specific mitogenic stimuli might promote the induction of ECL cell hyperplasia and eventually ECL cell tumors (gastric carcinoids).     

Localization of ANP-synthesizing cells in rat stomach

INTRODUCTION Since antrial natriuretic peptide (ANP) was isolated from atrium by de Bold et al in 1981[1-3], brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), dendroaspis (DNP), micrurus natriuretic peptide (MNP), and ventricular natriuretic peptide (VNP) have been found in succession. They distribute not only in the heart but all over the body[4-10]. ANP regulates a variety of physiological functions, including natriuresis, diuresis and vasodilation. Three types…     

Exogenous nitric oxide directly inhibits antral circular muscle motility of rat stomach in vitro

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Cytosolic phospholipase A2 type IVA is present in human red cells

Phospholipase A(2) type IVA (IVAPLA(2)) is a cytosolic enzyme that on activation selectively releases arachidonic acid (AA) from cell membrane phospholipids. Both AA and lysophospholipid, products of the enzymic reaction, can function as signal transducers in cellular interactions. The enzyme is present in most cells, including polymorphs, eosinophils, and platelets. This study used affinity purification to extract IVAPLA(2) from red cell lysate prepared from leukocyte- and platelet-depleted human blood to overcome the masking effect of hemoglobin on Western blot detection. We show that IVAPLA(2) is present in red cells as a 90-kDa protein.     

Morphological localisation of sulfonylurea receptor 1 in endocrine cells of human,mouse and rat pancreas

Aims/hypothesis Sulfonylurea receptor 1 (SUR1) is the regulatory subunit of ATP-sensitive K channels in beta cells. Morphological methods (immunohistochemistry and sulfonylurea binding) were used to establish the cellular and subcellular location of SUR1 in human and rodent islets. Results In the human, mouse and rat pancreas, all endocrine cells of the islets were immunolabelled with an anti-SUR1 antibody, whereas tissues containing SUR2 were consistently negative, as were those from Sur1 (also known as Abcc8)^−/− mice. In beta cells of the three species, the plasma membrane was distinctly stained, but SUR1 was mainly present over the cytoplasm, with an intensity that varied between cells. Electron microscopy showed that SUR1 was immunolocalised in insulin, glucagon and somatostatin granules. In rat beta cells degranulated by in vivo treatment with glibenclamide (known as glyburide in the USA and Canada), the insulin and SUR1 staining intensity was similarly decreased by ∼45%, whereas SUR1 staining was not changed in non-beta cells. In all islet cells, binding of glibenclamide labelled with fluorescent dipyrromethane boron difluoride (BODIPY-FL) was punctate over the cytoplasm, compatible with the labelling of endocrine granules. A faint labelling persisted in Sur1 ^−/− mice, but it was not different from that obtained with BODIPY-FL alone used as negative control. Conclusions/interpretation Our study immunolocalised SUR1 in alpha, beta and delta cells of human, mouse and rat islets, and for the first time visualised it in the plasma membrane. We also show that SUR1 is abundant in endocrine granules, where its function remains to be established. No specific sulfonylurea-binding sites other than SUR1 are identified in islet cells by the glibenclamide–BODIPY-FL technique.     

Peptide-containing nerve fibers in the stomach wall of rat and mouse

Peptide-containing nerve fibers were found to be numerous in the glandular stomach of the rat and mouse. The immunoreactive neuropeptides demonstrated included vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), gastrin-releasing peptide (GRP), substance P (SP), enkephalin, somatostatin, cholecystokinin, and neuropeptide Y (NPY). The density and distribution of the various peptide-containing fibers did not differ overtly between the pyloric and oxyntic gland areas except for the GRP fibers, which were fewer in the pyloric than in the oxyntic mucosa. The entire VIP nerve fiber population was found to also contain PHI. Immunoreactive NPY was found to occur in the VIP/PHI fibers (VIP/PHI/NPY fibers) in the smooth muscle and intramural ganglia of both rat and mouse and in the mucosa of the mouse. Mucosal VIP/PHI fibers in the rat did not contain any NPY-like material. Perivascular NPY fibers in both species and mucosal NPY fibers in the rat did not contain VIP or PHI. The mucosa harbored numerous GRP fibers and VIP/PHI (rat) or VIP/PHI/NPY (mouse) fibers, and a modest number of NPY (rat) and SP fibers. In the submucosa the peptide-containing nerve fibers were found mainly in the ganglia and around blood vessels. Blood vessels received a rich supply of NPY fibers; the number of perivascular VIP/PHI, GRP, and SP fibers was much lower by comparison. The smooth muscle and myenteric ganglia harbored not only VIP/PHI/NPY, GRP, and SP fibers but also enkephalin, somatostatin, and cholecystokinin fibers. Gastrin-releasing peptide, VIP/PHI/NPY, SP, and enkephalin nerve cell bodies occurred in the myenteric ganglia. As studied in the rat, vagal denervation did not affect the density and distribution of the various peptide-containing nerve fibers. After sympathectomy, mucosal and perivascular NPY fibers disappeared. The other types of peptide-containing nerve fibers were not affected.     

Enterochromaffin-like cells, a cellular source of uroguanylin in rat stomach

Uroguanylin is an endogenous peptide ligand for guanylyl cyclase-C, an apical membrane receptor predominantly located in the gastrointestinal epithelium. It regulates intestinal and renal fluid and electrolyte transport through the second messenger, cyclic GMP. Uroguanylin messenger RNA and the peptide are present in rat stomach, but the cellular source has not been identified. We separated gastric mucosal cells by size into seven fractions (F1-F7) and enriched endocrine cells into F1-F3 using counterflow elutriation. Uroguanylin messenger RNA and peptide were found in F1-F3 by Northern blot analysis and an RIA specific for rat uroguanylin. Uroguanylin-producing cells were identified as endocrine cells by immunocytochemical methods using antisera for uroguanylin, prouroguanylin, and chromogranin A, as well as by in situ hybridization cytochemistry. Double-staining showed that uroguanylin and histamine are colocalized in enterochromaffin-like (ECL) cells that release histamine, leading to the stimulation of gastric acid secretion from parietal cells. Uroguanylin is synthesized in ECL cells. These findings should contribute to elucidating the physiological functions of ECL cells and the cyclic GMP-mediated gastric ion transport mechanism.     

The human trefoil peptide, TFF1, is present in different molecular forms that are intimately associated with mucus in normal stomach

BACKGROUND: TFF1 is a 6.5 kDa secreted protein that is expressed predominantly in normal gastric mucosa. It is coexpressed with mucins and it can form dimers via a free carboxy terminal cysteine residue. AIMS: To investigate the molecular forms of TFF1 that are present in normal human stomach and the association of the different molecular forms with mucus. SUBJECTS: All subjects had macroscopically normal stomachs at gastroscopy. None had a significant past medical history. METHODS: TFF1 was detected in normal gastric mucosa and adherent mucus by western transfer analysis after electrophoresis on reducing and non-reducing polyacrylamide gels. In some instances, proteins were fractionated by caesium chloride density gradient centrifugation prior to detection of TFF1. The location of TFF1 in gastric mucosa with an intact adherent mucus layer was assessed by immunohistochemistry. RESULTS: Three different molecular forms of TFF1 were detected: TFF1 monomer, TFF1 dimer, and a TFF1 complex with an apparent molecular mass of about 25 kDa. TFF1 was present at higher concentrations than realised previously. The TFF1 complex was present in the adherent mucus gel layer but while its interaction with mucin was destabilised by caesium chloride, the interaction between mucin and the TFF1 dimer was resistant to caesium chloride. CONCLUSIONS: Most of TFF1 in normal human gastric mucosa is present in a complex that is stabilised by a disulphide bond. TFF1 is intimately associated with mucus. The high concentration, colocalisation, and binding of TFF1 to gastric mucus strongly implicate TFF1 in gastric mucus function.     

Neuropeptide Y is a vasoconstrictor of human coronary arteries

Neuropeptide Y (NPY) is a 36-amino-acid polypeptide which coexists with catecholamines in many adrenergic and noradrenergic neurons. It has been demonstrated to exert pressor effects in the perfused guinea pig heart and to constrict large cerebral and coronary blood vessels in animal studies. To determine if NPY might be a human coronary vasoconstrictor, the authors studied its effect on postmortem human coronary arteries. Proximal epicardial coronary rings were studied in a superfusion apparatus in Krebs-Ringer bicarbonate buffer (37 degrees C, pH 7.4) presaturated with 95% O2-5% CO2. Concentration-response curves were obtained using NPY in 0.1% bovine serum albumin in buffer and the responses were compared to those obtained in the presence of alpha 1, beta, and cyclooxygenase antagonists. A dose-related constrictor effect was obtained with NPY, which was significantly more potent than noradrenaline, constriction often being seen at 10(-12) M concentration. A vasorelaxant effect was seen in nonatherosclerotic vessels at higher concentrations. The vasoconstriction produced by noradrenaline was potentiated by subthreshold concentrations of NPY. The vasoconstrictor effect of NPY was not inhibited by prazosin (10(-6) M), and the vasodilatory effect was not inhibited by propranolol (10(-5) M). Indomethacin (3 X 10(-6) M) did not alter either vasoconstriction or vasorelaxation. The authors conclude that NPY is a potent constrictor of the human coronary artery at concentrations that may be achievable in vivo; it may thus be a contributor to sympathetic enhancement of coronary artery tone.