Reappraisal of potassium permanganate oxidation applied to Lowicryl K4M embedded tissues processed by high pressure freezing/freeze substitution, with special reference to differential staining of the zymogen granules of rat gastric chief cells.
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The high pressure freezing/freeze substitution technique is known to yield a deep vitreous freezing of tissues. Combination of this technique with Lowicryl K4M embedding allows us histochemical studies of dynamic cellular processes with improved structural preservation. The disadvantage of Lowicryl K4M embedding is its poor electron density in electron microscopy. To address this problem, we examined the effects of KMnO4 oxidation applied to Lowicryl K4M embedded rat gastric glands processed by high pressure freezing. The KMnO4 oxidation-uranyl acetate-lead citrate sequence succeeded not only in contrast enhancement of cellular components, but also in differential staining of the zymogen granules of rat gastric chief cells. This technique could be applied to semi-thin sections of Lowicryl K4M embedded rat gastric glands. The KMnO4 oxidation-toluidine blue staining provided sufficient contrast with regard to the zymogen granules. Various experiments used in this study verified that the KMnO4 oxidation plays an essential role in the differential staining of the zymogen granules. Combined use of the KMnO4 oxidation with phospholipase A2-immunostaining demonstrated that gold labeling was localized to the zymogen granules without the loss of immunolabeling. Energy dispersive X-ray microanalysis revealed some manganese depositions on the zymogen granules. It is highly anticipated that the KMnO4 oxidation will become a useful tool for histochemical investigations combined with cryofixation/freeze substitution and low temperature embedding techniques. (+info)
Acute parietal and chief cell changes induced by a lethal dose of lipopolysaccharide in mouse stomach before thrombus formation.
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The common lipopolysaccharide (LPS)-induced gastric lesions, such as erosions or ulcers, have been investigated in depth. Little is known, however, about the acute gastric lesions following a high dose of LPS. In a time-course study, ICR female mice were given a high subcutaneous dose of LPS (50 mg/kg). Mice were sacrificed at 4, 6, 12, and 24 hours after dosing and were assessed histopathologically for acute gastric lesions. The major gastric changes were seen in the fundic region and included vacuolar degeneration of parietal cells and apoptosis of chief cells. The vacuole in parietal cells was apparent as early as 4 hours postinjection (PI), and apoptosis of chief cells was apparent at 12 hours PI. Thrombus formation, in contrast, was not seen until 24 hours PI. No erosion, ulcer, or hemorrhage was seen in any gastric region in any of the treated animals at 24 hours PI. These results indicate that a subcutaneous high dose of LPS in mice causes vacuolar degeneration of parietal cells and apoptosis of chief cells before thrombus formation or subsequent ulcerative lesions. (+info)
The LIM and SH3 domain-containing protein, lasp-1, may link the cAMP signaling pathway with dynamic membrane restructuring activities in ion transporting epithelia.
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Lasp-1 is a unique LIM and src homology 3 (SH3) domain-containing protein that was initially identified as a 40 kDa cAMP-dependent phosphoprotein in the HCl-secreting gastric parietal cell. Because cAMP is a potent stimulator of parietal cell acid secretion, we have hypothesized that changes in lasp-1 phosphorylation might be involved in the regulation of ion transport-related activities, perhaps by modulating interactions among cytoskeletal and/or vesicle-associated proteins. In this study, we demonstrate that the cAMP-dependent acid secretory agonist, histamine, induces a rapid, sustained rise in parietal cell lasp-1 phosphorylation and this increase in phosphorylation is closely correlated with the acid secretory response. In addition, elevation of intracellular cAMP concentrations appear to induce a partial redistribution of lasp-1 from the cell cortex, where it predominates along with the gamma-isoform of actin in unstimulated cells, to the beta-actin enriched, apically-directed intracellular canalicular region, which is the site of active proton transport in the parietal cell. Additional studies demonstrate that although lasp-1 mRNA and protein are expressed in a wide range of tissues, the expression is specific for certain actin-rich cell types present within these tissues. For example, gastric chief cells, which contain relatively little F-actin and secrete the enzyme, pepsinogen, by regulated exocytosis, do not appear to express lasp-1. Similarly, lasp-1 was not detected in pancreatic acinar cells, which secrete enzymes by similar mechanisms and also contain relatively low levels of F-actin. Lasp-1 also was not detectable in proximal tubules in the kidney, in gastrointestinal smooth muscle, heart or skeletal muscle. In contrast, expression was prominent in the cortical regions of ion-transporting duct cells in the pancreas and in the salivary parotid gland as well as in certain F-actin-rich cells in the distal tubule/collecting duct. Interestingly, moderate levels of expression were also detected in podocytes present in renal glomeruli and in vascular endothelium. In primary cultures of gastric fibroblasts, lasp-1 was present mainly within the tips of lamellipodia and at the leading edges of membrane ruffles. Taken together these results support the hypothesis that the lasp-1 plays an important role in the regulation of dynamic actin-based, cytoskeletal activities. Agonist-dependent changes in lasp-1 phosphorylation may also serve to regulate actin-associated ion transport activities, not only in the parietal cell but also in certain other F-actin-rich secretory epithelial cell types. (+info)
Mist1 expression is a common link among serous exocrine cells exhibiting regulated exocytosis.
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Mist1 is a basic helix-loop-helix transcription factor that represses E-box-mediated transcription. Previous studies have suggested that the Mist1 gene is expressed in a wide range of tissues, although a complete characterization of Mist1 protein accumulation in the adult organism has not been described. In an effort to identify specific cell types that contain the Mist1 protein, antibodies specific for Mist1 were generated and used in Western blot and immunohistochemical assays. Our studies show that the Mist1 protein is present in many different tissues but that it is restricted to cell types that are exclusively secretory in nature. Pancreatic acinar cells, serous or seromucous cells of the salivary glands, chief cells of the stomach, and secretory cells of the prostate and seminal vesicle show high levels of Mist1 protein, whereas nonserous exocrine cells, including the mucus-producing cells of the salivary glands, remain Mist1 negative. These results identify Mist1 as the first transcription factor that exhibits this unique serous-specific expression pattern and suggest that Mist1 may have a key role in establishing and maintaining a pathway responsible for the exocytosis of serous secretions. (+info)
Leptin secretion and leptin receptor in the human stomach.
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BACKGROUND AND AIM: The circulating peptide leptin produced by fat cells acts on central receptors to control food intake and body weight homeostasis. Contrary to initial reports, leptin expression has also been detected in the human placenta, muscles, and recently, in rat gastric chief cells. Here we investigate the possible presence of leptin and leptin receptor in the human stomach. METHODS: Leptin and leptin receptor expression were assessed by immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR), and western blot analysis on biopsy samples from 24 normal individuals. Fourteen (10 healthy volunteers and four patients with non-ulcer dyspepsia and normal gastric mucosa histology) were analysed for gastric secretions. Plasma and fundic mucosa leptin content was determined by radioimmunoassay. RESULTS: In fundic biopsies from normal individuals, immunoreactive leptin cells were found in the lower half of the fundic glands. mRNA encoding ob protein was detected in the corpus of the human stomach. The amount of fundic leptin was 10.4 (3.7) ng leptin/g mucosa, as determined by radioimmunoassay. Intravenous infusions of pentagastrin or secretin caused an increase in circulating leptin levels and leptin release into the gastric juice. The leptin receptor was present in the basolateral membranes of fundic and antral gastric cells. mRNA encoding Ob-RL was detected in both the corpus and antrum, consistent with a protein of approximately 120 kDa detected by immunoblotting. CONCLUSION: These data provide the first evidence of the presence of leptin and leptin receptor proteins in the human stomach and suggest that gastric epithelial cells may be direct targets for leptin. Therefore, we conclude that leptin may have a physiological role in the human stomach, although much work is required to establish this. (+info)
Association of protein kinase A with AKAP150 facilitates pepsinogen secretion from gastric chief cells.
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Cross talk between signal transduction pathways augments pepsinogen secretion from gastric chief cells. A-kinase anchoring proteins (AKAPs) associate with regulatory subunits of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2B (PP2B) and localize this protein complex to specific cell compartments. We determined whether an AKAP-signaling protein complex exists in chief cells and whether this modulates secretion. In Western blots, we identified AKAP150, a rodent homologue of human AKAP79 that coimmunoprecipitates with PKA, PKC, and actin. The association of PKA and PP2B was demonstrated by affinity chromatography. Confocal microscopy revealed colocalized staining at the cell periphery for AKAP150 and PKC. Ht31, a peptide that competitively displaces PKA from the AKAP complex, but not Ht31P, a control peptide, inhibited 8-Br-cAMP-induced pepsinogen secretion. Ht31 did not inhibit secretion that was stimulated by agents whose actions are mediated by PKC and/or calcium. However, Ht31, but not Ht31P, inhibited carbachol- and A23187-stimulated augmentation of secretion from cells preincubated with cholera toxin. These data suggest the existence in chief cells of a protein complex that includes AKAP150, PKA, PKC, and PP2B. Disruption of the AKAP-PKA linkage impairs cAMP-mediated pepsinogen secretion and cross talk between signaling pathways. (+info)
Laminins and TGF-beta maintain cell polarity and functionality of human gastric glandular epithelium.
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The human gastric glandular epithelium produces a gastric lipase enzyme (HGL) that plays an important role in digestion of dietary triglycerides. To assess the involvement of extracellular matrix components and transforming growth factor-beta1 (TGF-beta1) in the regulation of this enzymic function, normal gastric epithelial cells were cultured on collagen type I, Matrigel, and laminins (LN)-1 and -2 with or without TGF-beta1. Epithelial morphology and HGL expression were evaluated using microscopy techniques, enzymic assays, Western blot, Northern hybridization, and RT-PCR. A correlation was observed between the cell polarity status and the level of HGL expression. TGF-beta1 alone or individual matrix components stimulated cell spreading and caused a downfall of HGL activity and mRNA. By contrast, Matrigel preserved the morphological features of differentiated epithelial cells and maintained HGL expression. The combination of LNs with TGF-beta1 (two constituents of Matrigel) exerted similar beneficial effects on epithelial cell polarity and evoked a 10-fold increase of HGL levels that was blunted by a neutralizing antibody against the alpha(2)-integrin subunit and by mitogen-activated protein kinase (MAPK) inhibitors PD-98059 (p42/p44) or SB-203580 (p38). This investigation demonstrates for the first time that a powerful synergism between a growth factor and basement membrane LNs positively influences cell polarity and functionality of the human gastric glandular epithelium through an activation of the alpha(2)beta(1)-integrin and effectors of two MAPK pathways. (+info)
Defining epithelial cell progenitors in the human oxyntic mucosa.
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In the human stomach, the oxyntic epithelium includes numerous tubular invaginations consisting of short pits opening into long glands. The pit is lined by pit cells, whereas the gland is composed of three regions: the base, containing zymogenic cells; the neck, containing neck cells; and the isthmus, composed of little known immature cells and of parietal cells, which are also scattered through the neck and base. The aim of this study was to examine the ultrastructure of the immature cells and to determine their relation to mature cells. To do so, normal oxyntic mucosal biopsies from subjects ranging from 20-43 years old were fixed in aldehydes and postfixed in reduced osmium for electron microscopy and morphometric analysis. The immature cells were sorted out into four classes, whose roles were clarified by comparison with the thoroughly investigated mouse oxyntic epithelium. The first class was composed of the least differentiated immature cells, which were rare and characterized by minute, dense, or cored secretory granules and were accordingly named mini-granule cells. Their function was not clarified. The second class consisted of pre-pit cells, which were characterized by few dense mucous granules and give rise to pit cells that ascend the pit wall and, after reaching the luminal surface, die or are extruded. Both pre-pit and pit cells underwent continuous renewal and, therefore, together constituted a renewal system referred to as pit cell lineage. The third class, or pre-neck cells, characterized by cored secretory granules, give rise to neck cells that descend toward the base region and differentiate further into pre-zymogenic cells, which finally become zymogenic cells. The latter eventually degenerate and die. Thus pre-neck cells and their progeny constitute a renewing system, designated zymogenic cell lineage. The fourth class, or pre-parietal cells, characterized by long microvilli and few tubulovesicles, differentiate into parietal cells that descend along the neck and base regions and eventually degenerate and die. Pre-parietal and parietal cells represent a renewing system referred to as parietal cell lineage. While the origin of the last three classes of progenitor cells has not been elucidated, it is likely that they arise either from an unidentified multipotential stem cell, possibly the mini-granule cell itself, or from the mitotic activity of pre-pit and pre-neck cells. In conclusion, the human oxyntic epithelium is composed of continually renewing cells organized in distinct cell lineages. (+info)