Iodide transport in isolated cells of mouse submaxillary gland

A method has been developed to isolate cells from the submaxillary gland of mouse by treatment with pronase. Three fractions of cells have been isolated having almost equal iodide concentrating activity. The isolated cells show time dependent uphill transport of iodide. The transport is substrate-saturable, having aK _m value of 0.3 μM for iodide. The transport is sensitive to antithyroid drugs, metabolic inhibitors and to some extent to ouabain. Pseudohalide such as thiocyanate competes with the transport of iodide. Thyroid hormones or thyroid stimulating hormone have no significant effect on the iodide transport in these cells.     

新鲜分离小鼠胃ICC样细胞的鉴定及其电生理学特性

将免疫荧光及传统全细胞膜片钳技术应用于新鲜分离的小鼠胃Cajal 间质细胞样细胞上,探讨了小鼠胃Cajal 间质细胞样细胞形态和电生理学特性。经胶原酶消化得到的Cajal间质细胞样细胞胞体呈短梭形,且自胞体发出多个较短的毛刺状突起。免疫细胞化学结果表明,Cajal 间质细胞样细胞胞体和突起酪氨酸激酶受体c-kit表达呈阳性。在传统全细胞记录模式、膜电位钳制在-60 mV 的条件下,可以记录到自发、节律性内向电流,即起搏电流。钙调蛋白抑制剂W-7 (50µmol/L）明显增强了起搏电流幅度并引发明显的内向钳制电流。当电极内液中EGTA 的浓度由0.1 mmol/L增加到10 mmol/L时,也明显增强了起搏电流幅度并引发明显的内向钳制电流。实验结果提示,新鲜分离的小鼠胃Cajal 间质细胞样细胞可以产生自发、自律性内向电流,而这种电流对胞内低钙或钙调蛋白抑制剂敏感。这种具有自发性电活动的Cajal 间质细胞样细胞可能就是胃Cajal 间质细胞。     

Force: velocity relationship in single isolated toad stomach smooth muscle cells

The relationship between force and shortening velocity (F:V) in muscle is believed to reflect both the mechanics of the myosin cross-bridge and the kinetics of its interaction with actin. To date, the F:V for smooth muscle cells has been inferred from F:V data obtained in multicellular tissue preparations. Therefore, to determine F:V in an intact single smooth muscle cell, cells were isolated from the toad (Bufo marinus) stomach muscularis and attached to a force transducer and length displacement device. Cells were electrically stimulated at 20 degrees C and generated 143 mN/mm2 of active force per muscle cross-sectional area. At the peak of contraction, cells were subjected to sudden changes in force (dF = 0.10-0.90 Fmax) and then maintained at the new force level. The force change resulted in a length response in which the cell length (Lcell) rapidly decreased during the force step and then decreased monotonically with a time constant between 75 and 600 ms. The initial length change that coincided with the force step was analyzed and an active cellular compliance of 1.9% cell length was estimated. The maintained force and resultant shortening velocity (V) were fitted to the Hill hyperbola with constants a/Fmax of 0.268 and b of 0.163 Lcell/s. Vmax was also determined by a procedure in which the cell length was slackened and the time of unloaded shortening was recorded (slack test). From the slack test, Vmax was estimated as 0.583 Lcell/s, in agreement with the F:V data. The F:V data were analyzed within the framework of the Huxley model (Huxley. 1957. Progress in Biophysics and Biophysical Chemistry. 7:255-318) for contraction and interpreted to indicate that in smooth muscle, as compared with fast striated muscle, there may exist a greater percentage of attached force-generating cross-bridges.     

Neurohormonal regulation of secretion from isolated rat stomach ECL cells: a critical reappraisal

ECL cells are endocrine/paracrine cells in the oxyntic mucosa. They produce, store and secrete histamine and chromogranin A-derived peptides such as pancreastatin. The regulation of ECL-cell secretion has been studied by several groups using purified ECL cells, isolated from rat stomachs. Reports from different laboratories often disagree. The purpose of the present study was to re-evaluate the discrepancies by studying histamine (or pancreastatin) secretion from standardized preparations of pure, well-functioning ECL cells. Cells from rat oxyntic mucosa were dispersed by pronase digestion, purified by repeated counter-flow elutriation and subjected to density gradient centrifugation. The final preparation consisted of more than 90% ECL cells (verified by histamine and/or histidine decarboxylase immunocytochemistry). They were maintained in primary culture for 48 h before they were exposed to candidate stimulants and inhibitors for 30 min after which the medium was collected for determination of mobilized histamine (or pancreastatin). Gastrin-17 and sulphated cholecystokinin octapeptide (CCK-8s) raised histamine secretion 4-fold, the EC(50) for both peptides being around 100 pM. The neuropeptide pituitary adenylate cyclase activating peptide (PACAP-27) (5-fold increase) and the related neuropeptides vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) (3-fold increase) mobilized histamine with similar potency (EC(50) ranging from 80 to 140 pM). Adrenaline, isoprenaline and terbutaline stimulated secretion by activating a beta2 receptor subtype, while acetylcholine and carbachol were without effect. Secretion experiments were invariably run in parallel with a gastrin standard curve. Somatostatin, prostaglandin E2 (PGE2) and the PGE1 congener misoprostol inhibited PACAP- and gastrin-stimulated secretion by more than 90%, with IC(50) values ranging from 90-720 (somatostatin) to 40-200 (misoprostol) pM. The neuropeptide galanin inhibited secretion by 60-70% with a potency similar to that of somatostatin. Proposed inhibitors such as peptide YY, neuropeptide Y and the cytokines interleukin 1-beta and tumor necrosis factor alpha induced at best a moderate inhibition of gastrin- or PACAP-stimulated secretion at high concentrations, while calcitonin gene-related peptide, pancreatic polypeptide and histamine itself were without effect. Inhibition of gastrin- or PACAP-stimulated secretion was routinely compared to a somatostatin standard curve. In conclusion, gastrin, PACAP, VIP/PHI and adrenaline stimulated secretion. Somatostatin and PGE2 were powerful inhibitors of both gastrin- and PACAP-stimulated secretion; although equally potent, galanin was less effective than somatostatin and PGE2.     

Secretion of intrinsic factor from dispersed mucosal cells isolated from guinea pig and rabbit stomach

In dispersed mucosal cells prepared from rabbit and guinea pig stomach, the secretion of intrinsic factor was constant (0.3–0.4%/min) for at least 30 min incubation at 37°C. Histamine or isobutyl methylxanthine increased cyclic AMP and intrinsic factor secretion in both cell preparations. Isobutyl methylxanthine potentiated and cimetidine competitively inhibited (K_i=5·10^?7 M) both effects of histamine. Dibutyryl cyclic AMP (1.0 mM), also caused a 3-fold increase in intrinsic factor secretion. These results suggest that in rabbit and guinea pig histamine interacts with H₂-receptors to increase cyclic AMP which mediates the rise in the rate of intrinsic factor secretion.     

Effect of alpha-1-antichymotrypsin on activity of DNA primase isolated from human stomach adenocarcinoma cells

This paper describes an investigation into the effect of alpha-1-antichymotrypsin (ACT) on DNA primase. DNA primase was partially purified from human stomach carcinoma cells. It was found that poly(dC)-dependent DNa primase activity was inhibited by ACT and the inhibition was proportional to the concentration of the inhibitor. The inhibitory effect of ACT remained even after ACT lost most of its chymotrypsin-inhibitory activity by heat treatment. Poly(dT)-dependent primase activity was enhanced by the presence of ACT. The enhancement was effective up to a concentration of 1mg/ml.     

Association of glycoproteins and pepsinogen in the secretory granules of fundic epithelial cells isolated from guinea pig stomach

Epithelial cells were isolated from the fundic portion of the guinea pig stomach. Cells were separated by velocity sedimentation at unit gravity in a Ficoll 70 gradient and pooled in three fractions. By morphological and biochemical criteria, each fraction was characterized as a population highly enriched in one of the three main functional types: oxyntic cell; chief cell and mucus-secreting cell. Measure of the pepsinogen content and specific stainings of the secretory granules for light and electron microscopy led to the definition of two types of mucus-secreting cells in nearly equal quantity; mucous cells with smaller secretory granules entirely glycoproteic in nature and muco-peptic cells containing larger heterogeneous secretory granules. These granules were made of a proteic core containing pepsinogen surrounded by a thin membrane and a voluminous cap, both containing carbohydrates. The cap appeared as if built of orderly packed layers of glycoproteins. Secretory granules of chief cells were also surrounded by a membrane containing glycoproteins and occasionally a small glycoproteic cap. Pepsinogen content was estimated to be three times higher in a single chief cell than in a muco-peptic cell.     

Apelin cells in the rat stomach

Apelin is a recently discovered peptide that is the endogenous ligand for the APJ receptor. Apelin is produced in the central nervous system, heart, lung, mammary gland and gastrointestinal (GI) tract. The aim of this study was to identify by immunohistochemistry (IHC) cell types in the rat stomach that produce apelin peptide. IHC revealed abundant apelin-positive cells, primarily in the neck and upper base regions of the gastric glands in the mucosal epithelium. Apelin is not detected in the muscle layer. Apelin-positive cells were identified as mucous neck, parietal cells, and chief cells. Apelin is also identified in gastric epithelial cells that produce chromogranin A (CGA), a marker of enteroendocrine cells. The findings that apelin is expressed in gastric exocrine and endocrine cells agrees with and extends other data showing that apelin peptide is measurable in the gut lumen and in the systemic circulation by immunoassay.     

Regeneration of endocrine cells in the stomach

A mucosal defect was produced by cryosurgery in the antral and the fundic wall of the rat stomach, and regeneration of gastric endocrine cells was studied 50, 100 and 200 days after operation. Fifty days after the operation, the mucosal defect was completely covered with regenerated epithelium. The regenerated mucosa both in the antral and in the fundic region consisted of mucinous glandular structures. The regenerated mucosa in the corpus remained pseudopyloric in type even 200 days after operation. Regardless of the time after operation, regeneration of endocrine cells was always observed. We could identify G cells and EC cells in the regenerated mucosa of the antrum, and EC cells, A cells and AL cells in the regenerated mucosa of the corpus, respectively. By electron microscopy, endocrine-exocrine cells were frequently encountered. These cells had two different types of intra-cytoplasmic granules; one was an endocrine-specific, small electron-dense granule, and the other a large, lucent mucin droplet-like granule. These findings indicate that the endocrine cells of the stomach are formed from endodermal precursor cells.     

Ontogeny of histamine-immunoreactive cells in rat stomach

Summary An antiserum against hemocyanin-conjugated histamine was used to study the cellular stores of histamine in the stomach, especially the oxyntic mucosa, of fetal and early postnatal rats. Tissues were fixed in 4% 1-ethyl-3(3-dimethyl-aminopropyl) carbodiimide (EDC-DI) and standard immunofluorescence technique was used. Histamine was first detected on the 16th embryonic (E16) day when a few histamine-immunoreactive (HA-ir) cells and nerve fibers were observed in the muscular layer of the stomach wall. On day E18, HA-ir cells were visualized for the first time in the oxyntic mucosa of the stomach, and from that day on the number of such cells increased slowly initially and after day E20 more rapidly. At birth many of the HA-ir cells in the oxyntic mucosa possessed processes giving them a paracrine-like appearance typical of enterochromaffin-like cells (ECL cells). Only a very small number of the HA-ir cells represented metachromatically stained mast cells and were located in the submucosa. After birth, the number of HA-ir ECL cells increased steadily, until day 21 when the distribution and number was very similar to that of the adult. The results suggest that histamine-containing neurons and ECL cells appear in the stomach wall before birth, and that there are histamine-containing ECL cells in the mucosa and mast cells in the submucosa of the stomach wall at birth.     

Somatostatin release from isolated perfused rat stomach.

Gastric somatostatin release from the isolated rat stomach was studied using a perfusion technique. Somatostatin released from the isolated perfused rat stomach was found to be identical in molecular size and immunoreactively with synthetic somatostatin. Infusion of glucagon (10^?7 M) caused biphasic increase of gastric somatostatin release. Gastric somatostatin release was also stimulated by infusion of theophylline (10^?3 M) and dibutyryl cyclic AMP (10^?3 M). These results indicate the possible involvement of adenylate cyclase-cyclic AMP system in the regulatory mechanism of gastric somatostatin release.     

Lactobacilli isolated from the stomach of conventional mice.

Twenty strains of lactobacilli isolated from the stomach of conventional mice were tested for their ability to ferment or hydrolyze substrates that may be present in the stomach habitat. The lactobacilli could be placed in four groups (A to D) depending on their ability to ferment N-acetylglucosamine, dextrin, cellobiose, gum arabic, and xylan. The majority of the isolates belonged to groups A and D. Group A strains did not resemble previously described Lactobacillus species, but group D strains were identified as L. leichmannii. A representative group A isolate colonized the surface of the nonsecretory epithelium of the stomach of gnotobiotic mice; a group D isolate did not.     

Histamine in endocrine cells in the stomach

Summary Antibodies to histamine were used to examine the localization of the amine in cells of the stomach and upper small intestine of a great variety of species, including cartilaginous and bony fish, amphibia, reptiles (lizard), birds (chicken) and a large number of mammals. In all species gastric histamine was localized in endocrine cells (invariably found in the epithelium) and mast cells (usually with an extra-epithelial localization). The endocrine cells were identified as such by immunostaining with antibodies to chromogranin A and the mast cells were identified by toluidine blue staining. Histamine-immunoreactive endocrine cells were found almost exclusively in the acid-producing part of the stomach; only rarely were such cells observed in the pyloric gland area. They were fairly numerous in the gastric mucosa of the two subclasses of fish as well as in the amphibia and reptile species studied. Here, the majority of the histamine-immunoreactive endocrine cells seemed to have contact with the gastric lumen (open type cells) and were located in the surface epithelium (certain fish only) or together with mucous neck cells at the bottom of the pits. In the chicken, histamine-immunoreactive endocrine cells were numerous and located peripherally in the deep compound glands. They were without contact with the lumen (closed type) and had long basal extensions (paracrine appearance), running close to the base of the oxyntico-peptic cells. In mammals, the number of histamine-immunoreactive endocrine cells in the stomach varied greatly. They were particularly numerous in the rat and notably few in the dog, monkey and man. In all mammals, the histamine-immunoreactive endocrine cells were of the closed type and located basally in the oxyntic glands. They often had a paracrine appearance with long basal processes. Histamine-storing mast cells, finally, were few in both subclasses of fish as well as in the amphibian species and in the lizard. They were fairly numerous in chicken proventriculus (beneath the surface epithelium), few in the oxyntic mucosa of mouse, rat and hamster, moderate in number in hedge-hog, guinea-pig, rabbit, pig and monkey, and numerous in cat, dog and man. In the oxyntic mucosa of the latter three species mast cells sometimes seemed to have an intraepithelial localization which made their distinction from endocrine cells difficult. In newborn cats (1–3 days old) in human foetuses (17–24 weeks gestational age) mast cells were relatively few in the gastric mucosa and the histamine-containing endocrine cells were easier to demonstrate as a consequence. Patients with achlorhydria (and pernicious anemia) or suffering from hypergastrinemia due to gastrinoma had a greatly increased number of histamine-storing endocrine cells in the oxyntic mucosa compared with normal individuals.