Human dehydroepiandrosterone sulfotransferase: purification and characterization of a recombinant protein

Dehydroepiandrosterone sulfate is the most abundant sulfated steroid transformed in human tissues and serves as a precursor for steroid hormones. Recombinant human dehydroepiandrosterone sulfotransferase (DHEA-ST) expressed in glutathione sulfotransferase fusion form in E. coli was purified using glutathione sepharose 4B affinity adsorption chromatography, a Factor Xa cleavage step, and Q-sepharose fast flow column chromatography. The homogeneous preparation had an activity toward dehydroepiandrosterone (DHEA) of 150+/-40 nmol/min per mg of protein under the assay conditions at an overall yield of 38.4%. The recombinant human DHEA-ST was shown to have a subunit mass of 34 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, while having a molecular mass of 67.2 kDa by Superose-12 gel filtration. Our results indicate that the active recombinant enzyme expressed in E. coli is a homodimer.Biochemical properties for purified DHEA-ST were studied using DHEA as a substrate. The optimum pH ranged from pH 7 to 8, and the optimum temperature 40-45 degrees C. Ninety percent of basal DHEA-ST activity remained even after the enzyme was treated at 45 degrees C for 15 min. The 50% inactivation concentration of NaCl for DHEA-ST activity was determined to be around 500 mM. The K(m) value for DHEA was 1.9+/-0.3 microM and V(max)=190+/-18 nmol/min per mg of protein at 37 degrees C, pH 7.5.     

Sonic hedgehog is associated with H+-K+-ATPase-containing membranes in gastric parietal cells and secreted with histamine stimulation

Sonic hedgehog (Shh) is found within gastric parietal cells and processed from a 45-kDa to a 19-kDa bioactive protein by an acid- and protease-dependent mechanism. To investigate whether Shh is associated with the parietal cell membrane compartment that becomes exposed to both acid and proteolytic enzymes during acid secretion, the cellular location of Shh within resting and stimulated gastric parietal cells was examined. Immunofluorescence microscopy of rabbit stomach sections showed that Shh colocalized predominantly with parietal and pit, not chief/zymogen or neck, cell markers. In resting and histamine-stimulated rabbit gastric glands Shh was expressed only in parietal cells close to H+-K+-ATPase-containing tubulovesicular and secretory membranes with some colocalizing with gamma-actin at the basolateral membrane. Gastric gland microsomal membranes were prepared by differential and sucrose gradient centrifugation and immunoisolation with an anti-H+-K+-ATPase-alpha subunit antibody. The 45- and 19-kDa Shh proteins were detected by immunoblot in immunopurified H+-K+-ATPase-containing membranes from resting and stimulated gastric glands, respectively. Incubating glands with a high KCl concentration removed Shh from the membranes. Histamine stimulated 19-kDa Shh secretion from gastric glands into the medium. In human gastric cancer 23132/87 cells cultured on permeable membranes, histamine increased 19-kDa Shh secretion into both apical and basolateral media. These findings show that Shh is a peripheral protein associated with resting and stimulated H+-K+-ATPase-expressing membranes. In addition, Shh appears to be expressed at or close to the basolateral membrane of parietal cells.     

Contribution of carbon monoxide-producing cells in the gastric mucosa of rat and monkey

 Recent studies have shown that carbon monoxide (CO) may function as a gaseous signaling molecule in a similar way to nitric oxide. In the gastrointestinal tract, immunoreactivity against a CO-producing enzyme, heme oxygenase-2 (HO-2), was reported in epithelial cells and neurons of submucosal and myenteric plexus. However, details of the epithelial cells in the gastric mucosa remain unknown. The aim of this study was to clarify if mRNA for HO-2 is expressed in the rat stomach, if HO-2 protein is present in the mucosa, and to define the cell types of the HO-2-immunoreactive cells. HO-2 mRNA and protein were detected in fundic and pyloric mucosa of rat stomach using an RNA protection assay and western blot analysis. Immunohistochemical study showed that HO-2 was localized in parietal cells of the fundic glands and gastrin cells of the pyloric glands of both rat and monkey. The results suggest that HO-2 enzyme is produced in the gastric mucosa, and that CO is released from parietal cells and gastrin cells. Accepted: 12 November 1997     

Demonstration of a functional apical sodium hydrogen exchanger in isolated rat gastric glands

Previous studies have shown that gastric glands express at least sodium-hydrogen exchanger (NHE) isoforms 1-4. Our aim was to study NHE-3 localization in rat parietal cells and to investigate the functional activity of an apical membrane NHE-3 isoform in parietal cells of rats. Western blot analysis and immunohistochemistry showed expression of NHE-3 in rat stomach colocalizing the protein in parietal cells together with the beta-subunit of the H(+)-K(+)-ATPase. Functional studies in luminally perfused gastric glands demonstrated the presence of an apical NHE isoform sensitive to low concentrations of 5-ethylisopropyl amiloride (EIPA). Intracellular pH measurements in parietal cells conducted in omeprazole-pretreated superfused gastric glands showed an Na+-dependent proton extrusion pathway that was inhibited both by low concentrations of EIPA and by the NHE-3 specific inhibitor S3226. This pathway for proton extrusion had a higher activity in resting glands and was inhibited on stimulation of histamine-induced H(+)-K(+)-ATPase proton extrusion. We conclude that the NHE-3 isoform located on the apical membrane of parietal cells offers an additional pathway for proton secretion under resting conditions. Furthermore, the gastric NHE-3 appears to work under resting conditions and inactivates during periods of H(+)-K(+)-ATPase activity.     

Immunohistochemical localization in human tissues of GPI-80, a novel glycosylphosphatidyl inositol-anchored protein that may regulate neutrophil extravasation

We molecular-cloned a novel 80-kDa human glycosyl-phosphatidyl inositol (GPI)-anchored protein, designated GPI-80, that may regulate neutrophil extravasation. To identify the possible role of GPI-80 in vivo, we examined the immunohistochemical localization of GPI-80 in various human tissues. Our data show that GPI-80 is mainly located in polymorphonuclear leukocytes (PMNs), endothelial cells of the vessels, parietal cells and mucous neck cells of the stomach, goblet cells of the jejunum, and alveolar macrophages of the lung. The pathomechanisms of these positive findings in the gastric glands and the intestinal glands are not well elucidated and further studies will be needed.     

Helicobacter pylori VacA disrupts apical membrane-cytoskeletal interactions in gastric parietal cells

Helicobacter pylori persistently colonize the human stomach and have been linked to atrophic gastritis and gastric carcinoma. Although it is well known that H. pylori infection can result in hypochlorhydria, the molecular mechanisms underlying this phenomenon remain poorly understood. Here we show that VacA permeabilizes the apical membrane of gastric parietal cells and induces hypochlorhydria. The functional consequences of VacA infection on parietal cell physiology were studied using freshly isolated rabbit gastric glands and cultured parietal cells. Secretory activity of parietal cells was judged by an aminopyrine uptake assay and confocal microscopic examination. VacA permeabilization induces an influx of extracellular calcium, followed by activation of calpain and subsequent proteolysis of ezrin at Met(469)-Thr(470), which results in the liberation of ezrin from the apical membrane of the parietal cells. VacA treatment inhibits acid secretion by preventing the recruitment of H,K-ATPase-containing tubulovesicles to the apical membrane of gastric parietal cells. Electron microscopic examination revealed that VacA treatment disrupts the radial arrangement of actin filaments in apical microvilli due to the loss of ezrin integrity in parietal cells. Significantly, expression of calpain-resistant ezrin restored the functional activity of parietal cells in the presence of VacA. Proteolysis of ezrin in VacA-infected parietal cells is a novel mechanism underlying H. pylori-induced inhibition of acid secretion. Our results indicate that VacA disrupts the apical membrane-cytoskeletal interactions in gastric parietal cells and thereby causes hypochlorhydria.     

The calcium-sensing receptor acts as a modulator of gastric acid secretion in freshly isolated human gastric glands

Gastric acid secretion is activated by two distinct pathways: a neuronal pathway via the vagus nerve and release of acetylcholine and an endocrine pathway involving gastrin and histamine. Recently, we demonstrated that activation of H(+)-K(+)-ATPase activity in parietal cells in freshly isolated rat gastric glands is modulated by the calcium-sensing receptor (CaSR). Here, we investigated if the CaSR is functionally expressed in freshly isolated gastric glands from human patients undergoing surgery and if the CaSR is influencing histamine-induced activation of H(+)-K(+)-ATPase activity. In tissue samples obtained from patients, immunohistochemistry demonstrated the expression in parietal cells of both subunits of gastric H(+)-K(+)-ATPase and the CaSR. Functional experiments using the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein and measurement of intracellular pH changes allowed us to estimate the activity of H(+)-K(+)-ATPase in single freshly isolated human gastric glands. Under control conditions, H(+)-K(+)-ATPase activity was stimulated by histamine (100 microM) and inhibited by omeprazole (100 microM). Reduction of the extracellular divalent cation concentration (0 Mg(2+), 100 microM Ca(2+)) inactivated the CaSR and reduced histamine-induced activation of H(+)-K(+)-ATPase activity. In contrast, activation of the CaSR with the trivalent cation Gd(3+) caused activation of omeprazole-sensitive H(+)-K(+)-ATPase activity even in the absence of histamine and under conditions of low extracellular divalent cations. This stimulation was not due to release of histamine from neighbouring enterochromaffin-like cells as the stimulation persisted in the presence of the H(2) receptor antagonist cimetidine (100 microM). Furthermore, intracellular calcium measurements with fura-2 and fluo-4 showed that activation of the CaSR by Gd(3+) led to a sustained increase in intracellular Ca(2+) even under conditions of low extracellular divalent cations. These experiments demonstrate the presence of a functional CaSR in the human stomach and show that this receptor may modulate the activity of acid-secreting H(+)-K(+)-ATPase in parietal cells. Furthermore, our results show the viability of freshly isolated human gastric glands and may allow the use of this preparation for experiments investigating the physiological regulation and properties of human gastric glands in vitro.     

Comparative morphology of the stomach of some laboratory mammals

Histomorphology of the stomach of mouse, rat, hamster, guineapig, gerbil, and rabbit was studied. Although a common structural basis existed in the stomach between these species, the occurrence and distribution of various cells in gastric glands differed considerably between them. In mice, rats, hamsters and gerbils, the lower one-third of the glandular lamina propria was seemingly occupied by a varying proportion of parietal and chief cells. In rabbits, the predominantly occurring chief cells were distributed in the lower three-quarters of the glands intermingling with parietal cells, but in guinea-pigs the chief cells were not discernible. In hamsters, there was, however, a gradual increase of chief cells from the junction between nonglandular-glandular stomach toward the pyloric region. In all these species, parietal cells were the predominant cell type in the upper half to upper one-third of the gastric glands, often extending up to the neck of the glands interspersing between mucus neck cells and occasionally between chief cells.     

Immunoreactive-FSH in human normal gastric mucosa

Using indirect immuno-peroxidase staining technique, localization of immunoreactive follicle-stimulating hormone (IR-FSH) is demonstrated in the cytoplasm of the epithelial cells of normal human stomach. In view of their triangular shape and central nucleus and their predominance in the intermediate glands of the gastric mucosa, these cells are identified as parietal cells. The stromal tissue is devoid of staining reaction.     

The Cardiogastric Gland and Alimentary Tract of Caenolestid Marsupials

The stomach of the South American marsupial family Caenolestidae has a gland on its lesser curvature around the cardia. This cardiogastric gland is bi-lobed, typically 11times5 mm and bears a distinctive, highly folded mucosa which forms sac-like invaginations. These open into the stomach lumen via 40–60 slit-like orifices. The gland mucosa contains unbranched gastric glands which are considerably longer than those of other gastric glands present elsewhere in the stomach. The cells within the cardiogastric gland show intense eosinophilic staining properties, with the parietal cells being larger than those found in other regions of the stomach, as well as being arranged in clusters. Argentaffin cells are not present in the stomach mucosa. The gross morphology of the stomach and intestine is similar to that found in small carnivorous marsupials.     

Role of ADP-ribosylation factor 6 (ARF6) in gastric acid secretion

ADP-ribosylation factor (ARF) proteins are monomeric GTPases that are essential for membrane transport and exocytosis in a number of secretory cells. We investigated ARF6, the activation of which is insensitive to brefeldin A, to determine whether it regulates membrane traffic in gastric parietal cells. ARF6 translocated from cytosol to tubulovesicle in the presence of GTPgammaS, a potential inhibitor of acid secretion in permeabilized cells, whereas under the Mg2+-chelated condition where activity of ARF-GTPase activating protein is inhibited, ARF6 translocated to the apical secretory membrane. Immunohistochemical examination revealed that ARF6 mainly located in parietal cell within the gastric glands, and it translocated from the cytosol to the intracellular canaliculi when the glands were stimulated. These results indicated that the distribution of ARF6 between cytosol and the two different membranes was regulated by its GTPase activity. In cultured gastric glands infected with adenovirus expressing ARF6 Q67L, a mutant lacking GTP hydrolysis activity, gastric acid secretion was inhibited. These results suggest that ARF6 regulates gastric acid secretion in parietal cell and that the GTP hydrolysis cycle of ARF6 is essential for the activation pathway.     

3H]thymidine autoradiographic and alkaline phosphatase histochemical studies of intestinal metaplasia of the human stomach

The relationship between cell proliferation and enzyme activity in intestinal metaplasia of the human stomach was studied using a combined method of [3H]thymidine autoradiography and alkaline phosphatase histochemistry on the same section. Three types of intestinal metaplasia were observed depending on variations in both enzymatic activity and isotope labelling. One type shows alkaline phosphatase-positive cells along the entire length of the glands with [3H]thymidine-labelled cells localized only at the bottom of the glands, resembling the duodenum. In another type of intestinal metaplasia, alkaline phosphatase-positive cells are present on the surface and/or upper half of the glands with mitotically active cells occupying the lower part of the glands. The third variety of intestinal metaplasia is characterized by the absence of alkaline-phosphatase activity and [3H]thymidine-labelled cells present in an extended zone in the lower half of the glands. Differences in labelling patterns of [3H]thymidine and the activity of marker enzyme in various types of intestinal metaplasia seem to reflect variations in cell differentiation during intestinalization of gastric mucosa.     

正常大鼠胃黏膜中肌球蛋白轻链激酶定性和定位观察

目的观察正常大鼠胃组织中肌球蛋白轻链激酶的表达及分布特点。方法取11只SD正常雄性大鼠,在饥饿状态下,处死后取胃组织。通过免疫组化染色,观察正常大鼠胃组织中MLCK的表达及分布。结果MLCK在黏膜肌层、肌层和黏膜下层血管壁平滑肌均有大量表达;在胃底腺中,MLCK主要表达于壁细胞和主细胞胞浆内。结论MLCK不仅存在于平滑肌细胞内,还分布于胃底腺壁细胞和主细胞内,可能参与胃底腺腺细胞的分泌活动。     

Is the ClC-2 chloride channel involved in the Cl- secretory mechanism of gastric parietal cells?

It has been controversial whether the ClC-2 chloride channel is involved in hydrochloric acid secretion of gastric parietal cells. Here, we investigated whether ClC-2 is the apical Cl- channel associated with gastric acid secretion. Two anti-ClC-2 antibodies used in this study reacted with cloned ClC-2 protein expressed in HEK293 cells. In isolated rabbit gastric glands, significant expression of ClC-2 mRNA was observed, but the presence of ClC-2 protein was not clear. Furthermore, no expression of ClC-2 protein was observed in isolated rat and human gastric mucosa. Immunohistochemistry on the rat gastric mucosa showed no significant expression of ClC-2 protein in the parietal cells which showed abundant expression of H+,K+-ATPase. These results indicate that ClC-2 may not be a Cl- -transporting protein for gastric acid secretion in parietal cells.     

Functional-morphologic changes in the gastric and duodenal mucosa during prolonged administration of prednisolone

A prolonged administration (up to 25 days) of prednisolone to rats (0.4 mg per 100 g of the body mass) reveals some functional-morphological changes in the secretory apparatus and mucin production in the stomach and the duodenum: an increased functional activity of parietal, principle, Ecl- and G-cells of the stomach, Ec-cells and goblet cells of the duodenum (except Ec-cells and goblet cells in the outlet part of the stomach), and by the end of the experiment--hyperplasia of the parietal, G-cells and goblet cells. A conclusion is made that the increased acido-peptic activity of the gastric juice and the erosive-ulcerous complications under a prolonged administration of prednisolone result from an increased functional activity of the fundal glands, G-, Ecl-cells and Ec-cells of the duodenum with a subsequent hyperplasia of the G-cells by the 25th day of the experiment, as well as an insufficient mucin formation in the mucous membrane of the stomach, especially in its outlet part as a consequence of a decreased functional activity of the Ec-cells.     

Nitric oxide inhibits gastric acid secretion by increasing intraparietal cell levels of cGMP in isolated human gastric glands

We have previously identified cells containing the enzyme nitric oxide (NO) synthase (NOS) in the human gastric mucosa. Moreover, we have demonstrated that endogenous and exogenous NO has been shown to decrease histamine-stimulated acid secretion in isolated human gastric glands. The present investigation aimed to further determine whether this action of NO was mediated by the activation of guanylyl cyclase (GC) and subsequent production of cGMP. Isolated gastric glands were obtained after enzymatic digestion of biopsies taken from the oxyntic mucosa of healthy volunteers. Acid secretion was assessed by measuring [(14)C]aminopyrine accumulation, and the concentration of cGMP was determined by radioimmunoassay. In addition, immunohistochemistry was used to examine the localization of cGMP in mucosal preparations after stimulation with the NO donor S-nitroso-N-acetylpenicillamine (SNAP). SNAP (0.1 mM) was shown to decrease acid secretion stimulated by histamine (50 microM); this effect was accompanied by an increase in cGMP production, which was histologically localized to parietal cells. The membrane-permeable cGMP analog dibuturyl-cGMP (db-cGMP; 0.1-1 mM) dose dependently inhibited acid secretion. Additionally, the effect of SNAP was prevented by preincubating the glands with the GC inhibitor 4H-8-bromo-1,2,4-oxadiazolo[3,4-d]benz[b][1,4]oxazin-1-one (10 microM). We therefore suggest that NO in the human gastric mucosa is of physiological importance in regulating acid secretion. Furthermore, the results show that NO-induced inhibition of gastric acid secretion is a cGMP-dependent mechanism in the parietal cell involving the activation of GC.     

CORRELATION OF THE FINE STRUCTURE OF THE GASTRIC PARIETAL CELL (DOG) WITH FUNCTIONAL ACTIVITY OF THE STOMACH

The fine structure of the parietal (oxyntic) cell in the gastric glands (corpus of the stomach) of the dog was examined under conditions of active gastric acid secretion and compared with cellular structure in the non-acid-secretory (basal) state. Animals, in both acute and chronic experiments, were equipped with gastric fistulae so that gastric juice could be collected for analysis of total acidity, free acidity, volume, and pH prior to biopsy of the gastric mucosa. The specimens of mucosa were fixed in buffered OsO₄ and embedded in n-butyl methacrylate and the thin sections were stained with lead hydroxide before examination in the electron microscope. A majority of parietal cells showed an alteration of fine structure during stimulation of gastric acid secretion by a number of different techniques (electrical vagal stimulation, histamine administration, or insulin injection). The changes in fine structure affected mainly the smooth surfaced vesicular elements and the intracellular canaliculi in the cytoplasm of the cell. The mitochondria also appeared to be involved to some extent. During acid secretion a greater concentration of smooth surface profiles is found adjacent to the walls of the intracellular canaliculi; other parietal cells exhibited a marked decrease in number of smooth surfaced elements. Intracellular canaliculi, always present in non-acid-secreting oxyntic cells, develop more extensively in cells of acid-secreting gastric glands. The surface area of these canaliculi is greatly increased by the elaboration of a large number of closely approximated and elongated microvilli. Still other parietal cells apparently in a different stage of the secretory cycle exhibit non-patent canaliculi lacking prominence; such cells have very few smooth surfaced vesicular elements. These morphological findings correlated with the acid-secretory state of the stomach provide evidence that the parietal cell participates in the process of acid secretion.     

Detection of ouabain-insensitive H(+)-transporting, K(+)-stimulated p-nitrophenylphosphatase activity in rat gastric glands by cerium-based cytochemistry

We employed a modification of our previously reported cerium-based cytochemical method for ouabain-sensitive, K-dependent p-nitrophenylphosphatase (Na-K ATPase) activity to detect ouabain-insensitive, K-stimulated p-nitrophenylphosphatase (K-pNPPase) activity in rat gastric glands. Biochemically, the enzyme activity of gastric glands incubated in a medium containing 50 mM Tricine buffer (pH 7.5), 50 mM KCl, 10 mM MgCl2, 2 mM CeCl3, 2 mM p-nitrophenylphosphate (pNPP), 2.5 mM levamisole, 10 mM ouabain, and 0.00015% Triton X-100, was optimal at pH 7.5-8.0 and decreased above pH 8.5. The amount of p-nitrophenol after incubation increased linearly in proportion to the amount of tissue in the medium. The enzyme activity was inhibited by omeprazole, sodium flouride (NaF), N-ethylmaleimide (NEM), and dicyclohexylcarbodiimide (DCCD). Heat-treated specimens had no enzyme activity. The enzyme activity increased with addition of K ions up to the concentration of 50 mM, and became constant above 50 mM. Cytochemically, the parietal cells of the gastric glands reacted positively for ouabain-insensitive K-pNPPase activity. Intense reaction was observed at the microvilli of the luminal surface and the intracellular canaliculi. The tubulovesicular system showed weak enzyme activity. The reaction products were found as fine, granular, electron-dense deposits in the cytoplasm just beneath the plasma membrane. The ouabain-insensitive K-pNPPase activity detected in this study appears, therefore, to be associated with that of H-transporting, K-stimulated adenosine triphosphatase (H-K ATPase).     

Changes in the mucosa of the duodenum and stomach as a result of experimental duodenal ulcers and vagotomy

By means of the transmissive and scanning electron microscopy methods and radioautography, structure of mucous membrane of the stomach and duodenum has been studied under experimentally induced duodenal ulcers before and after vagotomy during various time. The vagotomy results in accelerated healing of the ulcer defect. This is connected with an increased proliferative activity in the crypta cells, however, this is accompanied with deceleration of their differentiation. Under the duodenal ulcers the amount of chief and parietal cells increases in the gastric mucous membrane, this depends on gastrostasis produced by stenosis of the pylorus. At vagotomy the amount of the chief and parietal cells in the fundal glands of the mucous membrane decreases; this is accompanied with a lowered secretory activity.