Prostaglandin E2 output by isolated rat gastric parietal cells and non-parietal epithelial cells

Prostaglandins have acid antisecretory and cytoprotective effects in gastric mucosa when given exogenously. This study's purpose was to isolate preparations of parietal and non-parietal cells from rat stomachs and to compare prostaglandin output by these cells. Gastric epithelial cells were isolated from rat stomachs using pronase. Cells from different incubation times were collected separately and enriched by discontinuous Percoll gradient. Cell types were identified by hematoxylin and eosin stain, succinic dehydrogenase activity (parietal cells), periodic acid Schiff staining (mucous cells), Bowie staining (chief cells) and electron microscopy. Prostaglandin E2 activity was measured by radio-immunoassay. Parietal cells were purified to over 90% while the non-parietal preparation contained 67% chief cells and over 31% mucous cells. By electron-microscopy, cell integrity was seen to be maintained. The parietal cell enriched fraction contained two and one-half times the amount of prostaglandin E2 that the non-parietal chief cell enriched fraction did, p less than 0.01. These results raise the question as to whether output of PGE2 by parietal cells could play a role in modulating gastric acid secretion directly by parietal cells as well as in protecting the deeper layers of gastric mucosa against damaging agents in-vivo.     

溃疡病胃窦G和D细胞的体视学分析

应用真彩色医学院图像分析仪,对１５例ＤＵ、ＧＵ和ＣＯＮ组患者,非病变区胃窦粘膜,双重免疫组织化学显色的３００个Ｇ和Ｄ细胞进行形态学分析。每个细胞检测十二个参数。三组中,Ｇ细胞的周长、面积、长径、短径、比表面、平均截距、平均体积、平均面积、圆球度和平均直径数,在ＤＵ和ＧＵ组都发生改变,它们的显著性差异率分别为１００％;Ｄ细胞的长径、短径、形状因子和平均轴比数的改变,三组中的显著性差异率均在６０％以上,这些结果,进一步增加了溃疡病患者胃窦粘膜非病变区幽门腺Ｇ和Ｄ细胞的体视学形态计量学的参数资料,并提示它们参与了溃疡病的内分泌调控活动     

The intramucosal distribution of gastric alcohol dehydrogenase and aldehyde dehydrogenase activity in rats.

Using qualitative and microquantitative histo-chemical techniques, alcohol dehydrogenase and aldehyde dehydrogenase activity was studied in the gastric mucosa of male and female rats. Alcohol dehydrogenase was demonstrated by staining reactions with maximum activity in surface and neck cells and with clearly weaker activity also in parietal cells. Aldehyde dehydrogenase could be detected in surface and neck cells, and also to a comparable degree in the parietal cells. Quantitative analyses of microdissected samples yielded high values for alcohol dehydrogenase activity exclusively in the superficial part of the gastric mucosa, whereas low-Km aldehyde dehydrogenase activity showed a decreasing gradient from the surface to the deeper parts of the mucosa. Sex differences could not be confirmed.     

Epithelial response of the rat gastric mucosa to chronic superficial injury.

Chronic injury to the healthy gastric mucosa with noxious agents such as aspirin or alcohol induces a progressive strengthening of the stomach wall against these insults. The present study examined the histologic response of the rat gastric mucosa to chronic destruction of the superficial mucosa for one month with hypertonic saline. The number, position and morphology of proliferating, parietal, G and D cells were followed during mucosal injury and one month of recovery. The results showed that chronic injury reduced parietal cell numbers by about 30 percent, particularly in the middle of the mucosal thickness where a clear zone was formed by hypertrophy of mucous neck-like cells. G cells were also reduced by about 50 percent, but there were no changes in D cells. Chronic injury induced a marked increase in the number of antral (+112 percent) and fundic (+250 percent) proliferating cells. CONCLUSION: The rat gastric mucosa responds to chronic superficial injury by down-regulation of acid secretory cells and gastrin secreting cells and an up-regulation of proliferating cells. The appearance of a prominent layer of mucous neck-like cells may indicate a new secretory function for these cells.     

Alternative ultrastructural localization of Dolichos biflorus lectin binding sites in proton secreting parietal cells of mice

High amount of N-acetyl-D-galactosamine specific lectin binding sites were detected on the canalicular membranes of human parietal cells. Our present model investigations on mice showed that the intracellular distribution of the terminal N-acetyl-D-galactosamine containing glycoprotein highly depends on the actual functional state of the parietal cells. In the normal gastric mucosa 40%-60% of parietal cells react positively after staining with horseradish peroxidase or biotin labelled Dolichos biflorus lectin. Ultrastructurally lectin binding sites occur mainly on the basolateral membrane infoldings in fed animals, while they are present exclusively on the canalicular membranes of fasting mice, suggesting that the alternative appearance of lectin binding sites on the opposite membrane areas of parietal cells is tightly coupled to their main function, to H+ secretion.     

Neuronal nitric oxide synthase: expression in rat parietal cells

Nitric oxide synthases (NOS) are enzymes that catalyze the generation of nitric oxide (NO) from L-arginine and require nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. At least three isoforms of NOS have been identified: neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), and endothelial NOS (eNOS or NOS II). Recent studies implicate NO in the regulation of gastric acid secretion. The aim of the present study was to localize the cellular distribution and characterize the isoform of NOS present in oxyntic mucosa. Oxyntic mucosal segments from rat stomach were stained by the NADPH-diaphorase reaction and with isoform-specific NOS antibodies. The expression of NOS in isolated, highly enriched (>98%) rat parietal cells was examined by immunohistochemistry, Western blot analysis, and RT-PCR. In oxyntic mucosa, histochemical staining revealed NADPH-diaphorase and nNOS immunoreactivity in cells in the midportion of the glands, which were identified as parietal cells in hematoxylin and eosin-stained step sections. In isolated parietal cells, decisive evidence for nNOS expression was obtained by specific immunohistochemistry, Western blotting, and RT-PCR. Cloning and sequence analysis of the PCR product confirmed it to be nNOS (100% identity). Expression of nNOS in parietal cells suggests that endogenous NO, acting as an intracellular signaling molecule, may participate in the regulation of gastric acid secretion.     

Histochemical study of carbonic anhydrase and HCO3(-)-stimulated adenosine triphosphatase in the rat stomach during hydrochloric acid secretion]

By means of histochemical methods it was established that carboanhydrase of the parietal cells of fundal glands and HCO-3 stimulated ATP-ase of the rat's gastric mucosa capillaries disposed next to the parietal cells were activated by food and histamine. The obtained data confirm the idea of the multicellular functional essembly sustaining HCL secretion (R. I. Salganik, 1974). Should gastrin induce the formation of histamine in endocrinous cells and the histamine activate carboanhydrase in parietal cells, our data confirm the supposition that HCO-3-ions stimulate ATP-ase sustaining the exchange between HCO-3-cells and C1- of blood to form HCl in the endothelial cells of blood capillaries adjacent to the parietal cells.     

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.     

The intramucosal distribution of gastric alcohol dehydrogenase and aldehyde dehydrogenase activity in rats

Using qualitative and microquantitative histochemical techniques, alcohol dehydrogenase and aldehyde dehydrogenase activity was studied in the gastric mucosa of male and female rats. Alcohol dehydrogenase was demonstrated by staining reactions with maximum activity in surface and neck cells and with clearly weaker activity also in parietal cells. Aldehyde dehydrogenase could be detected in surface and neck cells, and also to a comparable degree in the parietal cells. Quantitative analyses of microdissected samples yielded high values for alcohol dehydrogenase activity exclusively in the superficial part of the gastric mucosa, whereas low-K _m aldehyde dehydrogenase activity showed a decreasing gradient from the surface to the deeper parts of the mucosa. Sex differences could not be confirmed.Dedicated to Professor Dr. K.S. Ludwig on the occasion of his 70th birthday     

Differential immunocytochemical staining patterns of uroplakin observed on neoplastic and nonneoplastic tissue fragments obtained from upper urinary tract brush specimens

OBJECTIVE: To discern any differences in the distribution of uroplakin expression on neoplastic and nonneoplastic upper urinary tract lesions. STUDY DESIGN: Thirty-seven representative 95% ethanol-fixed direct smears of brush specimens, which were subsequently diagnosed histologically as 10 reactive and 27 transitional cell carcinomas (TCCs), were stained with polyclonal uroplakin antibodies utilizing the avidin-biotin-peroxidase method. In order to ascertain any differences in diagnostic accuracy between conventional cytomorphology and uroplakin immunocytochemical staining, the results were compared to the original final cytologic diagnoses for all 37 cases. RESULTS: The linear staining pattern on the luminal surface of umbrella cells was the dominant pattern expressed on tissue fragments from all 10 reactive lesions. Tissue fragments from low grade TCC demonstrated a weaker and less continuous superficial membrane staining pattern along with a variably intense, diffuse, membranous staining pattern throughout the tumor cell groups. This staining pattern was seen in all 17 (sensitivity = 100%) histologically confirmed low grade TCCs, of which only 13 of the 17 (sensitivity = 76.5%) were diagnosed as TCC on the original final cytology report. Tissue fragments from 10 high grade TCCs lacked the superficial linear staining pattern seen in reactive cell groups. Instead, all 10 high grade TCCs displayed a strong diffuse membrane staining pattern in all the cells in the fragment and also demonstrated microluminal structures within the tumor cell groups. CONCLUSION: The distinctive patterns of uroplakin antigen expression observed in nonneoplastic and neoplastic upper urinary tract lesions in the present study can greatly enhance the accuracy of diagnostic interpretation of upper urinary tract lesions in conventional cytologic specimens.     

Differential distribution of transforming growth factor-alpha immunohistochemistry within whole gastric mucosa in rats

Transforming growth factor-alpha (TGF-alpha) plays an important role in both proliferation and differentiation of mucosal cells at the gastrointestinal level, including stomach, where it is constitutively produced. This study evaluated the immunohistochemical distribution of TGF-alpha within whole gastric mucosa in rats, through the examination of seriate sections. Each stomach was opened along the greater curvature, pinned upon a cork plate, fixed in formalin and cut in 2-mm parallel strips which were sequentially superimposed on a glass slide. Sections were immunostained for TGF-alpha and pictures were taken from three areas: greater and lesser curvature; mucosa lying between the two curvatures. The sections were graded on the basis of the intensity of TGF-alpha staining, which was scored as follows: 0) no staining; 1) weakly positive; 2) intensely positive. The percent number of immunopositive cells and a mean intensity were calculated. Gastric mucosa showed a marked immunopositivity to TGF-alpha, mainly in parietal cells whose cytoplasm displayed moderate to intense staining. Positive cells (and the mean intensity) of total mucosa were 15.7+/-6.1% (1.13+/-0.42). However, they were not uniformly distributed, being 26.3+/-1.9% (1.67+/-0.24) in the mucosa lying between the two curvatures, 12.4+/-2.5% (1.52+/-0.22) along the lesser curvature and 8.3+/-2.1% (0.31+/-0.17) along the greater curvature. These results show that parietal cells of rat gastric mucosa exhibit immunoreactivity to TGF-alpha. Considering the gastroprotective effects of this factor, its non-homogeneous distribution within different areas may be of importance in understanding the lesion pattern of gastric damage after the administration of noxious agents.     

人正常腺上皮组织中MUC6基因的表达

应用免疫组织化学和原位杂交方法检测人正常腺上皮中MUC6基因的表达,揭示MUC6基因在正常人腺上皮组织中的分布异质性及其特点.结果显示MUC6基因编码的核心蛋白及其mRNA主要分布于正常胃粘膜胃腺的基底部,上皮细胞无MUC6基因表达,呈细颗粒状,位于细胞核周,胃底、胃窦的表达无区别;十二指肠绒毛上皮内的表达呈弥漫性,均质状,杯状和柱状细胞的表达类似,杯状细胞的粘液滴内未测得MUC6基因产物;空肠、结肠组织中无MUC6基因的表达;胆囊上皮组织内有强阳性MUC6核心蛋白的表达,而宫颈上皮中表达较弱.实验提示MUC6基因的表达存在异质性及器官特异性.     

Demonstration of a pH gradient in the gastric gland of the acid-secreting guinea pig mucosa

The gastric mucosa is covered by a continuous layer of mucus. Although important for understanding the mechanism of this protective function, only scarce information exists about the pH inside the gastric gland and its outlet. pH in the lumen of the gastric glands, in the outlet of gastric crypts, and in the adjacent cells was measured in the isolated acid-secreting mucosa of the guinea pig. Ultrafine double-barreled pH microelectrodes were advanced at high acceleration rates through the gastric mucus and the tissue to ensure precise intracellular and gland lumen pH measurements. A pH gradient was found to exist along the gastric gland, where the pH is 3.0 at parietal cells, i.e., in the deepest regions, and increases to 4.6 at the crypt outlet. Intracellular pH (pH(i)) of epithelial cells bordering a crypt outlet, and of neck cells bordering a gland, was acidic, averaging 6.0 and 6.5, respectively. pH(i) of deep cells bordering a gland was nearly neutral, averaging 7.1, and the secreting parietal cells were characterized by a slightly alkaline pH(i) of 7.5. This gland pH gradient is in general agreement with a model that we recently proposed for proton transport in the gastric mucus, in which protons secreted by the parietal cells are buffered to and transported with the simultaneously secreted mucus toward the gastric lumen, where they are liberated from the degraded mucus.     

Lectin histochemistry of the gastric mucosa in normal and Helicobacter pylori infected guinea-pigs

Helicobacter pylori attaches via lectins, carbohydrate binding proteins, to the carbohydrate residues of gastric mucins. Guinea-pigs are a suitable model for a H. pylori infection and thus the carbohydrate composition of normal and H. pylori infected gastric mucosa was investigated by lectin histochemistry. The stomach of all infected animals showed signs of an active chronic gastritis in their mucosa, whereas no inflammation was present in the control animals. The corpus–fundus regions of the controls showed heterogeneous WGA, SNA-I, UEA-I and HPA binding in almost all parts of the gastric glands. While these lectins labelled the superficial mucous cells and chief cells heterogeneously, the staining of the parietal cells was limited to WGA and PHA-L. Mucous neck cells reacted heterogeneously with UEA-I, HPA, WGA and PHA-L. In the antrum, the superficial mucous cells and glands were stained by WGA, UEA-I, HPA, SNA-I or PHA-L. WGA, UEA-I, SNA-I and HPA labelled the surface lining cells strongly. The mucoid glands reacted heterogeneously with WGA, UEA-I, HPA, SNA-I and PHA-L. In both regions, the H. pylori infected animals showed similar lectin binding pattern as the controls. No significant differences in the lectin binding pattern and thus in the carbohydrate composition between normal and H. pylori infected mucosa could be detected, hence H. pylori does not induce any changes in the glycosylation of the mucosa of the guinea-pig. This unaltered glycosylation is of particular relevance for the sialic acid binding lectin SNA-I as H. pylori uses sialic acid binding adhesin for its attachment to the mucosa. As sialic acid binding sites are already expressed in the normal mucosa H. pylori can immediately attach via its sialic acid binding adhesin to the mucosa making the guinea-pig particularly useful as a model organism.This work is dedicated to Professor B. Tillmann on the occasion of his 65th birthday     

The biochemical and pharmacological properties of a newly synthesized H+-K+ ATPase inhibitor, 2-dimethylamino-4,5-dihydrothiazolo[4,5:3,4]pyridol-[1,2-a]benzimidazol e

This study was designed to determine the effect of a newly synthesized proton pump inhibitor, 2-dimethylamino-4,5-dihydrothiazolo[4,5:3,4]pyridol[1,2-a]be nzimidazole (YJA20379-2), on gastric H(+)-K(+) ATPase activity, acid secretion, and experimental gastroduodenal lesions or ulcers in rats. YJA20379-2 inhibited in a concentration-dependent manner the proton pump (H(+)-K(+) ATPase) activity in isolated hog gastric mucosal microsomes, therefore, confirming its classification as a proton pump inhibitor. The inhibitory efficacy of YJA20379-2 on the proton pump was about eight times higher than that of omeprazole at pH 7.4. The activity of the inhibited enzyme was not restored by dilution and washout method, so this implied that the inhibition of YJA20379-2 is not reversible. YJA20379-2, given intraduodenally or orally, potently suppressed acid secretion in pylorus-ligated rats, with ED50 values of 3.6 and 7.7 mg.kg(-1), respectively. Pretreatment with YJA20379-2 dose dependently protected the gastric mucosa from damage induced by absolute ethanol, water-immersion stress, indomethacin, and the duodenal mucosa from damage induced by mepirizole in rats, with ED50 values of 11.0, 21.0, 0.5, and 18.7 mg.kg(-1), respectively. Repeated administration of YJA20379-2 also dose dependently accelerated spontaneous healing of acetic acid induced gastric ulcers. These results suggest that YJA20379-2 has potent antisecretory and antiulcer effects, which are exerted by suppression of H(+)-K(+) ATPase activity in gastric parietal cells, such that YJA20379-2 may be useful for the clinical treatment of peptic ulcer diseases.     

Myosin II is present in gastric parietal cells and required for lamellipodial dynamics associated with cell activation

Nonmuscle myosin II has been shown to participate in organizing the actin cytoskeleton in polarized epithelial cells. Vectorial acid secretion in cultured parietal cells involves translocation of proton pumps from cytoplasmic vesicular membranes to the apical plasma membrane vacuole with coordinated lamellipodial dynamics at the basolateral membrane. Here we identify nonmuscle myosin II in rabbit gastric parietal cells. Western blots with isoform-specific antibodies indicate that myosin IIA is present in both cytosolic and particulate membrane fractions whereas the IIB isoform is associated only with particulate fractions. Immunofluorescent staining demonstrates that myosin IIA is diffusely located throughout the cytoplasm of resting parietal cells. However, after stimulation, myosin IIA is rapidly redistributed to lamellipodial extensions at the cell periphery; virtually all the cytoplasmic myosin IIA joins the newly formed basolateral membrane extensions. 2,3-Butanedione monoximine (BDM), a myosin-ATPase inhibitor, greatly diminishes the lamellipodial dynamics elicited by stimulation and retains the pattern of myosin IIA cytoplasmic staining. However, BDM had no apparent effect on the stimulation associated redistribution of H,K-ATPase from a cytoplasmic membrane compartment to apical membrane vacuoles. The myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7) also did not alter the stimulation-associated recruitment of H,K-ATPase to apical membrane vacuoles, but unlike BDM it had relatively minor inhibitory effects on lamellipodial dynamics. We conclude that specific disruption of the basolateral actomyosin cytoskeleton has no demonstrable effect on recruitment of H,K-ATPase-rich vesicles into the apical secretory membrane. However, myosin II plays an important role in regulating lamellipodial dynamics and cortical actomyosin associated with parietal cell activation. acid secretion; cytoskeleton; ion channels and pumps     

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.     

Alternative ultrastructural localization of Dolichos biflorus lectin binding sites in proton secreting parietal cells of mice

Summary High amount of N-acetyl-d-galactosamine specific lectin binding sites were detected on the canalicular membranes of human parietal cells. Our present model investigations on mice showed that the intracellular distribution of the terminal N-acetyl-d-galactosamine containing glycoprotein highly depends on the actual functional state of the parietal cells. In the normal gastric mucosa 40%–60% of parictal cells react positively after staining with horseradish peroxidase or biotin labelled Dolichos biflorus lectin. Ultrastructurally lectin binding sites occur mainly on the basolateral membrane infoldings in fed animals, while they are present exclusively on the canalicular membranes of fasting mice, suggesting that the alternative appearance of lectin binding sites on the opposite membrane areas of parietal cells is tightly coupled to their main function, to H⁺ secretion.     

Acid secretion and proton conductance in human airway epithelium

Acid secretion and proton conductive pathways across primary human airway surface epithelial cultures were investigated with the pH stat method in Ussing chambers and by single cell patch clamping. Cultures showed a basal proton secretion of 0.17 +/- 0.04 micromol.h(-1).cm(-2), and mucosal pH equilibrated at 6.85 +/- 0.26. Addition of histamine or ATP to the mucosal medium increased proton secretion by 0.27 +/- 0.09 and 0.24 +/- 0.09 micromol.h(-1).cm(-2), respectively. Addition of mast cells to the mucosal medium of airway cultures similarly activated proton secretion. Stimulated proton secretion was similar in cultures bathed mucosally with either NaCl Ringer or ion-free mannitol solutions. Proton secretion was potently blocked by mucosal ZnCl(2) and was unaffected by mucosal bafilomycin A(1), Sch-28080, or ouabain. Mucosal amiloride blocked proton secretion in tissues that showed large amiloride-sensitive potentials. Proton secretion was sensitive to the application of transepithelial current and showed outward rectification. In whole cell patch-clamp recordings a strongly outward-rectifying, zinc-sensitive, depolarization-activated proton conductance was identified with an average chord conductance of 9.2 +/- 3.8 pS/pF (at 0 mV and a pH 5.3-to-pH 7.3 gradient). We suggest that inflammatory processes activate proton secretion by the airway epithelium and acidify the airway surface liquid.