Changes in the cyclic nucleotide level and lysosomal enzyme activity in the gastric mucosa of rats with experimental ulcer formation

The experiments on rats have proved that ulcerative lesions in the gastric mucosa influenced by intraperitoneal catecholamines (noradrenaline and adrenaline) develop on the background of pronounced decrease of cAMP level in the gastric mucosa during ulceration and relatively slight fluctuations of cGMP level. As a result, cAMP/cGMP ratio in mucosa was significantly decreased during ulceration. These changes in cAMP level and cAMP/cGMP ratio may play an important role in destabilization of lysosomal membranes followed by a chain of pathological reactions resulting in ulcerative lesions of the gastric mucosa.     

Participation of histamine and cyclic nucleotides in the regulation of gastric secretion in experimental thyrotoxicosis

Experiments on 48 Wistar rats evidence that in early times of hyperthyroidism, the action of thyroxine on parietal cells is mediated by histamine and cAMP, while in prolonged hyperthyroidism, its action on parietal cells refractory to endogenous histamine and cAMP is found to be unmediated and direct.     

Lysosomal enzyme activity of the gastric mucosa in rats during experimental ulcer formation

Lysosomal membrane stability has been studied in the gastric mucosa in response to mechanical damage caused by lysosomal fractionation and release of lysosomal enzymes from mucous cells into the gastric cavity of alive animals during induction of acetic ulcer or erosive damage of the gastric mucosa resulting from intraperitoneal introduction of histamine and serotonin. It has been found that all types of ulcerogenesis in the gastric mucosa led to the decrease in lysosomal membrane stability to mechanical stress in the course of lysosomal fractionation. In addition there was a substantial release of lysosomal enzymes into the gastric cavity in different types of ulcerogenesis. The decrease in lysosomal membrane stability combined with a subsequent development of ulcers and erosions in the gastric mucosa seems indicative of the fact that lysosomal enzymes take part in the initial formation of ulcers in the gastric mucosa.     

Regeneration of gastric mucosa during ulcer healing is triggered by growth factors and signal transduction pathways.

An ulcer is a deep necrotic lesion penetrating through the entire thickness of the gastrointestinal mucosa and muscularis mucosae. Ulcer healing is a complex and tightly regulated process of filling the mucosal defect with proliferating and migrating epithelial and connective tissue cells. This process includes the re-establishment of the continuous surface epithelial layer, glandular epithelial structures, microvessels and connective tissue within the scar. Epithelial cells in the mucosa of the ulcer margin proliferate and migrate onto the granulation tissue to re-epithelialize the ulcer. Growth factors, such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), trefoil peptides (TP), platelet derived growth factor (PDGF) and other cytokines produced locally by regenerating cells, control re-epithelialization and the reconstruction of glandular structures. These growth factors, most notably EGF, trigger epithelial cell proliferation via signal transduction pathways involving EGF-R- MAP (Erk1/Erk2) kinases. Granulation tissue, which develops at the ulcer base, consists of fibroblasts, macrophages and proliferating endothelial cells, which form microvessels under the control of angiogenic growth factors. These growth factors [bFGF, vascular endothelial growth factor (VEGF) and angiopoietins] promote angiogenesis--capillary vessel formation--thereby allowing for the reconstruction of microvasculature in the mucosal scar, which is essential for delivery of oxygen and nutrients to the healing site. The primary trigger to activate expression of angiogenic growth factors and their receptors appears to be hypoxia. During ulcer healing expression of growth factor genes is tightly regulated in a temporally and spatially ordered manner.     

Estrogen-producing steroidogenic pathways in parietal cells of the rat gastric mucosa

Recently we demonstrated the presence of aromatase (P450(arom)), estrogen synthetase, and the active production of estrogen in parietal cells of the rat stomach. We therefore investigated the steroidogenic pathways of estrogen and also other steroid metabolites in the gastric mucosa of male rats, by showing the mRNA expression of steroidogenic enzymes using RT-PCR and in situ hybridization histochemistry, and by measuring the blood concentration of steroids in the artery and the portal vein. RT-PCR analysis showed the strong mRNA expression of 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), 17beta-hydroxysteroid dehydrogenase (HSD) type III and P450(arom), and the weak mRNA expression of 17beta-HSD type II, 5alpha-reductase type I and 3alpha-HSD. The other mRNAs of steroidogenic enzymes examined were not detected. In situ hybridization histochemistry demonstrated the localization of mRNAs for P450(17alpha), 17beta-HSD type III and P450(arom) in the parietal cells. Higher levels of progesterone and testosterone were found in the artery compared with the portal vein. Higher amounts of estrone and 17beta-estradiol, by contrast, were present in the portal vein compared with the artery. These results indicate that parietal cells of rat stomach convert circulating progesterone and/through androstenedione and testosterone to synthesize both estrone and 17beta-estradiol, which then enter the liver via the portal vein.     

The A2B adenosine receptor colocalizes with adenosine deaminase in resting parietal cells from gastric mucosa

The A2B adenosine receptor (A2BR) mediates biological responses to extracellular adenosine in a wide variety of cell types. Adenosine deaminase (ADA) can degrade adenosine and bind extracellularly to adenosine receptors. Adenosine modulates chloride secretion in gastric glands and gastric mucosa parietal cells. A close functional link between surface A2BR and ADA has been found on cells of the immune system, but whether this occurs in the gastrointestinal tract is unknown. The goal of this study was to determine whether A2BR and ADA are coexpressed at the plasma membrane of the acid-secreting gastric mucosa parietal cells. We used isolated gastric parietal cells after purification by centrifugal elutriation. The membrane fraction was obtained by sucrose gradient centrifugation. A2BR mRNA expression was analyzed by RT-PCR. The surface expression of A2BR and ADA proteins was evaluated by Western blotting, flow cytometry and confocal microscopy. Our findings demonstrate that A2BR and ADA are expressed in cell membranes isolated from gastric parietal cells. They show a high degree of colocalization that is particularly evident in the surface of contact between parietal cells. The confocal microscopy data together with flow cytometry analysis suggest a tight association between A2BR and ADA that might be specifically linked to glandular secretory function.     

Ultracytochemical study of oxidoreductases in the parietal cells of the gastric mucosa in gastric cancer

Ultracytochemistry was used to study and compare cytochromooxidase, succinate dehydrogenase and NADH-dehydrogenase activity in gastric mucosa parietal cells in health and in gastric carcinoma associated with decreased acidity of gastric juice. The study demonstrated the reduced activity of the enzymes listed in the mucosal parietal cells in gastric carcinoma. This finding is interpreted as a consequence of disturbed energy supply of hydrochloric acid secretion in gastric carcinoma.     

Immunolocalisation of bilitranslocase in mucosecretory and parietal cells of the rat gastric mucosa

SummaryBilitranslocase is a plasma membrane carrier localised at the vascular pole of the rat liver cell, where it mediates uptake of organic anions from the blood into the liver. This carrier is also present in the epithelium of the rat gastric mucosa, with similar molecular mass and functional properties. An immunohistochemical study reveals that both the mucus-secreting cells of the gastric pit and the H⁺-secreting parietal cells express bilitranslocase. These data point to a possible role of bilitranslocase and of its food-borne substrates (anthocyanins and nicotinic acid) in regulating the function and the permeability of the gastric mucosa.     

Basolateral expression of GRP94 in parietal cells of gastric mucosa

GRP94 is a member of the heat shock protein family normally confined to the endoplasmic reticulum that sometimes escapes the KDEL-mediated retention system. It is overexpressed in some gastric and other gastrointestinal carcinomas, but little is known about the physiological role of GRP94 in gastric mucosa. We investigated the membrane presence of GRP94 in parietal cells, which secrete acid into the gastric lumen, using subcellular fractionation, selective solubilization of membrane proteins, Western blotting, and radio-ligand binding and provided evidence of functional GRP94 expression at the surface of gastric mucosa parietal cells anchored to the basolateral domain. Our results show that GRP94 is not an integral membrane protein since 50 mM Na₂CO₃ treatment dissociates part of it from the membrane. However, 100 mM Na₂CO₃ treatment did not extract all GRP94 from the membrane, which indicates that it is strongly associated with it. The presence of GRP94 in isolated plasma membrane was demonstrated by Western blotting and its functionality by radio-lig- and binding experiments. Both the K _D value obtained in saturation experiments with N-ethylcarboxamido-[³H]adenosine at 4°C, at the nanomolar range, and the inhibition constant of its binding by radicicol, the most specific GRP94 inhibitor, indicate that active receptor regions are exposed at the membrane surface. Western blotting of plasma membrane subfractions showed that GRP94 is mainly expressed in the basolateral membrane of gastric parietal cells, while its presence in the apical domain is negligible, thereby inferring a role for GRP94 in processes operating in this membrane domain.     

应激状态下NO的胃粘膜保护作用及其与壁细胞泌酸的关系

目的:探讨应激状态下一氧化氮(NO)的胃粘膜保护作用及其与壁细胞泌酸的关系.方法:采用水浸-束缚应激(WRS)方法制备应激性溃疡(SU)动物模型,检测胃粘膜溃疡指数(UI)、胃粘膜NO含量和壁细胞H ,K -ATPase活性,观察L-硝基精氨酸甲酯(L-NAME)和L-精氨酸(L-Arg)对应激后大鼠壁细胞H ,K -ATPase活性及胃粘膜损伤的影响.结果:L-NAME(20 mg·kg-1)可使胃粘膜NO含量减少(P＜0.01),壁细胞H ,K -AT-Pase活性增加(P＜0.05),并加重应激所致的胃粘膜损伤;L-Arg(300 mg·kg-1)则使胃粘膜NO含量增加(P＜0.01),壁细胞H ,K -ATPase活性下降(P＜0.05),减轻应激所致胃粘膜损伤.结论:NO对应激状态下大鼠胃粘膜具有保护作用,其机制与抑制壁细胞H ,K -ATPase活性有关.     

Metabolism of dog gastric mucosa. Nucleotide levels in parietal cells.

Adenine and pyridine nucleotide levels as well as those of phosphate, phosphocreatine, lactate, pyruvate, beta-hydroxybutyrate, acetoacetate, glucose, and glycogen were measured in histologically defined parietal and mucous cell sections of biopsies of dog gastric mucosa at rest, and in various secretory states. As a result of stimulation of secretion, there appeared to be no change in adenine nucleotide levels, or phosphocreatine, but there was a rise in inorganic phosphate and a fall in phosphorylation potential. However, there was a marked increase in NADH, but no change in NADPH with onset of acid secretion. The increase in the lactate to pyruvate ratio showed that the increased NADH level occurred in the cytoplasm and these data are discussed with reference to change in cell pH.     

Endocrine cells of the gastric mucosa in patients with duodenal ulcer after vagotomy]

Bioptates of the stomach mucous membrane (SMM) have been investigated in 169 patients suffering from duodenal ulcer (DU). According to the nocturnal gastric secretion test among them there are "hypersecretors" and persons with moderate elevation of acid formation. In conformity with the efficiency of the operative treatment among the patients examined, groups are defined: those with recurrent disease and those recovered after vagotomy. The DU endocrine apparatus undergoes both qualitative and quantitative alterations after vagotomy. When recovery after vagotomy takes place, the number of endocrine cells only slightly exceeds these parameters in the patients with a moderately manifested acid production. These alterations are adaptive. The recurrence of DU in patients with moderately manifested acid production before the operation can be explained by hyperplasia of G-cells. A high degree of hyperplasia of all elements of the endocrine apparatus in the "hypersecretors" can be one of the causes of the DU recurrence. The data about the state of G-, Ec- and EcL-cells before and after vagotomy can be used at prognostication the results of surgical treatment of patients with DU.     

Participation of transmembrane proline 82 in angiotensin II AT1 receptor signal transduction

Most of the classical physiological effects of the octapeptide angiotensin II (AngII) are produced by activating the AT1 receptor which belongs to the G-protein coupled receptor family (GPCR). Peptidic GPCRs may be functionally divided in three regions: (i) extracellular domains involved in ligand binding; (ii) intracellular domains implicated in agonist-induced coupling to G protein and (iii) seven transmembrane domains (TM) involved in signal transduction. The TM regions of such receptors have peculiar characteristics such as the presence of proline residues. In this project we aimed to investigate the participation of two highly conserved proline residues (Pro82 and Pro162), located in TM II and TM IV, respectively, in AT1 receptor signal transduction. Both mutations did not cause major alterations in AngII affinity. Functional assays indicated that the P162A mutant did not influence the signal transduction. On the other hand, a potent deleterious effect of P82A mutation on signal transduction was observed. We believe that the Pro82 residue is crucial to signal transduction, although it is not possible to say yet if this is due to a direct participation or if due to a structural rearrangement of TM II. In this last hypothesis, the removal of proline residue might be correlated to a removal of a kink, which in turn can be involved in the correct positioning of residues involved in signal transduction.     

Drebrin E2 is differentially expressed and phosphorylated in parietal cells in the gastric mucosa

Developmentally regulated brain proteins (drebrins) are highly expressed in brain where they may regulate actin filament formation in dendritic spines. Recently, the drebrin E2 isoform was detected in certain epithelial cell types including the gastric parietal cell. In gastric parietal cells, activation of HCl secretion is correlated with actin filament formation and elongation within intracellular canaliculi, which are the sites of acid secretion. The aim of this study was to define the pattern of drebrin expression in gland units in the intact rabbit oxyntic gastric mucosa and to initiate approaches to define the functions of this protein in parietal cells. Drebrin E2 expression was limited entirely or almost entirely to parietal cells and depended upon the localization of parietal cells along the gland axis. Rabbit drebrin E2 was cloned and found to share 86% identity with human drebrin 1a and to possess a number of cross-species conserved protein-protein interaction and phosphorylation consensus sites. Two-dimensional Western blot and phosphoaffinity column analyses confirmed that drebrin is phosphorylated in parietal cells, and several candidate phosphorylation sites were identified by mass spectrometry. Overexpression of epitope-tagged drebrin E2 led to the formation of microspikes and F-actin-rich ring-like structures in cultured parietal cells and suppressed cAMP-dependent acid secretory responses. In Madin-Darby canine kidney cells, coexpression of epitope-tagged drebrin and the Rho family GTPase Cdc42, which induces filopodial extension, produced an additive increase in the length of microspike projections. Coexpression of dominant negative Cdc42 with drebrin E2 did not prevent drebrin-induced microspike formation. These findings suggest that 1) drebrin can induce the formation of F-actin-rich membrane projections by Cdc42-dependent and -independent mechanisms; and that 2) drebrin plays an active role in directing the secretagogue-dependent formation of F-actin-rich filaments on the parietal cell canalicular membrane. Finally, the differential distribution of drebrin in parietal cells along the gland axis suggests that drebrin E2 may be an important marker of parietal cell differentiation and functionality.     

IGF-I and insulin receptor signal transduction in trout muscle cells

In this study, primary cultures of trout skeletal muscle cells were used to investigate the main signal transduction pathways of insulin and IGF-I receptors in rainbow trout muscle. At different stages of in vitro development (myoblasts on day 1, myocytes on day 4, and fully developed myotubes on day 11), we detected in these cells the presence of immunoreactivity against ERK 1/2 MAPK and Akt/PKB proteins, components of the MAPK and the phosphatidylinositol 3-kinase-Akt pathways, respectively, two of the main intracellular transduction pathways for insulin and IGF-I receptors. Both insulin and IGF-I activated both pathways, although the latter provoked higher immunoreactivity of phosphorylated MAPKs and Akt proteins. At every stage, increases in total MAPK immunoreactivity levels were observed when cells were stimulated with IGF-I or insulin, while total Akt immunoreactivity levels changed little under stimulation of peptides. Total Akt and total MAPK levels increased as skeletal muscle cells differentiated in culture. Moreover, when cells were incubated with IGF-I or insulin, MAPK-P immunoreactivity levels showed greater increases over the basal levels on days 1 and 4, with no effect observed on day 11. Although Akt-P immunoreactivity displayed improved responses on days 1 and 4 as well, a stimulatory effect was still observed on day 11. In addition, the present study demonstrates that purified trout insulin receptors possess higher phosphorylative activity per unit of receptor than IGF-I receptors. In conclusion, these results indicate that trout skeletal muscle culture is a suitable model to study the insulin and IGF-I signal transduction molecules and that there is a different regulation of MAPK and Akt pathways depending on the developmental stage of the muscle cells.