Human eosinophils can express the cytokines tumor necrosis factor-alpha and macrophage inflammatory protein-1 alpha.

By in situ hybridization, 44-100% of the blood eosinophils from five patients with hypereosinophilia and four normal subjects exhibited intense hybridization signals for TNF-alpha mRNA. TNF-alpha protein was detectable by immunohistochemistry in blood eosinophils of hypereosinophilic subjects, and purified blood eosinophils from three atopic donors exhibited cycloheximide-inhibitable spontaneous release of TNF-alpha in vitro. Many blood eosinophils (39-91%) from hypereosinophilic donors exhibited intense labeling for macrophage inflammatory protein-1 alpha (MIP-1 alpha) mRNA, whereas eosinophils of normal donors demonstrated only weak or undetectable hybridization signals for MIP-1 alpha mRNA. Most tissue eosinophils infiltrating nasal polyps were strongly positive for both TNF-alpha and MIP-1 alpha mRNA. By Northern blot analysis, highly enriched blood eosinophils from a patient with the idiopathic hypereosinophilic syndrome exhibited differential expression of TNF-alpha and MIP-1 alpha mRNA. These findings indicate that human eosinophils represent a potential source of TNF-alpha and MIP-1 alpha, that levels of expression of mRNA for both cytokines are high in the blood eosinophils of hypereosinophilic donors and in eosinophils infiltrating nasal polyps, that the eosinophils of normal subjects express higher levels of TNF-alpha than MIP-1 alpha mRNA, and that eosinophils purified from the blood of atopic donors can release TNF-alpha in vitro.

Effects of extracellular Mg2+ concentration on intracellular signalling and acid secretion in rat gastric parietal cells.

In the present study, the effects of extracellular magnesium concentration ([Mg2+]ex) on stimulus-secretion coupling processes were investigated in rat gastric parietal cells in vitro. Extracellular magnesium reduction resulted in (1) an increase of basal intracellular free calcium concentration ([Ca2+]in), (2) an enhancement of both carbachol and thapsigargin-induced calcium responses, (3) an improved filling state of intracellular calcium stores, (4) an increase of both basal and carbachol-induced acid secretion, whereas intracellular adenosine 3',5'-cyclic monophosphate (cyclicAMP) levels and histamine stimulated acid secretion were not affected. The effects of high [Mg2+]ex were opposite to the described results, except that high [Mg2+]ex was able to decrease significantly histamine-stimulated cyclicAMP levels and acid secretion. These findings indicate a modulatory role of [Mg2+]ex on the intracellular signalling processes and acid secretory properties in rat parietal cells. These effects seemed to be mediated by regulating (1) calcium loading capacity of intracellular stores, (2) the permeability of the calcium influx pathway, and (3) the formation of cyclicAMP.

A case of malignant mastocytosis with circulating mast cell precursors: biologic and phenotypic characterization of the malignant clone.

The phenotypic and biologic properties of malignant cells in a case of aggressive mastocytosis with multi-organ involvement, circulating mast cell precursors and absence of skin infiltrates were analyzed. Circulating mast cell precursors were detected by immunostaining using antibodies against mast cell tryptase as well as by electron microscopy. These progenitors were tryptase+/chymase- (MCT) and accounted for 10 to 20% of nucleated mononuclear blood cells (MNC). A subset of them contained metachromatic granules. As assessed by combined toluidine blue/immunofluorescence staining, the granulated mast cell precursors were found to express CD9 (P24), CD33 (gp67) and CD44 (Pgp-1), but not basophil-related markers (CD11b (C3biR), CDw17 (lactosylceramide), CD123 (il-3R alpha))or monocyte-related antigens (CD14, CD15). Expression of the mast cell growth factor (MGF) receptor, c-kit(CD117), was also demonstrable, whereas the skin mast cell marker C5aR (CD88) could not be detected on mast cell precursors. The ligand of c-kit, recombinant human (rh) stem cell factor (SCF = MGF), induced histamine release from circulating mast cell progenitors, whereas rhC5a, a potent skin mast cell-/basophil-agonist, was ineffective over the dose-range (10(-9) to 10(-7(M)) tested. Analysis of mast cell antigens in malignant mastocytosis or mast cell leukemias may be helpful to establish a diagnosis and to determine the phenotype of the clone.

Differences in the onset of the inflammatory response to cutaneous leishmaniasis in resistant and susceptible mice.

Sites of cutaneous infection with Leishmania major in genetically susceptible (BALB/c) and resistant (C57B1/6) mice were investigated for the early inflammatory response (6 h to 12 days) by electron microscopy combined with enzyme-histochemical methods. Susceptible BALB/c mice spontaneously recruited only polymorphonuclear leukocytes (PMNs) at the site of infection. Infiltrating mononuclear phagocytes (and eosinophils) were first observed at day 1 in a ratio equal to the influx of PMNs (about 40%). This pattern persisted during the following 11 days of infection. In the resistant C57/B16 mice, the first cellular infiltrate at the infected site contained mononuclear phagocytes (25%) and eosinophils (15%) besides PMNs (60%). Within 3 days after infection, mononuclear phagocytes became the dominant population of cells in cutaneous lesions (up to 80%). It was found in situ that L. major accumulated and replicated in immature macrophages, that is, intermediate stages between monocytes and resident macrophages, which were found in lesions of both strains. The burden of parasites was, however, degraded more rapidly by the infiltrating cells of the resistant mice than by those of the susceptible ones. Within the first 4 days of infection, the parasites were found in PMNs, mononuclear phagocytes, and extracellular spaces in both strains. In susceptible mice this distribution pattern persisted up to 12 days after infection; in resistant C57B1/6 mice parasites accumulated inside mononuclear phagocytes within this period. It is concluded that the features of acute inflammation during leishmaniasis in BALB/c mice are sustained over a prolonged period that is ineffective in the elimination of L. major.

Ultrastructural immunogold localization of subcellular sites of TNF-alpha in colonic Crohn's disease.

Tumor necrosis factor-alpha, a proinflammatory cytokine, might have an important role(s) in initiating, modifying, and/or sustaining chronic inflammatory processes such as those that characterize Crohn's disease, an inflammatory bowel disease of unknown etiology. We used an immunogold ultrastructural morphometric approach to localize tumor necrosis factor-alpha in colonic Crohn's disease biopsies. Tumor necrosis factor-alpha was present in seven cell types (fibroblasts, eosinophils, mast cells, macrophages, colonic epithelial absorptive cells, Paneth cells, neutrophils). Tumor necrosis factor-alpha-containing subcellular organelles included lipid bodies (fibroblasts, eosinophils, macrophages, mast cells, colonic epithelial cells, neutrophils), secretory granules (eosinophils, Paneth cells), phagolysosomes (macrophages, colonic epithelial cells), and Golgi structures and vesicle membranes (neutrophils). A gradient of extracellular tumor necrosis factor-alpha immunoreactivity surrounded eosinophils, mast cells, and macrophages. P values of gold counts/microns2 were significant for all cells, organelles, and extracellular spaces measured, and all positive structures significantly exceeded the background labeling density/microns2. Specificity controls (normal rabbit serum, tumor necrosis factor-alpha-absorbed primary antibody) either failed to label these sites or gave markedly reduced specific tumor necrosis factor-alpha labeling, respectively. These findings represent the first ultrastructural localization of the subcellular sites of TNF-alpha in vivo in seven cell lineages in human colonic tissues.

Multiple in vivo passages enhance the ability of a clinical Helicobacter pylori isolate to colonize the stomach of Mongolian gerbils and to induce gastritis.

The Mongolian gerbil is an excellent animal model for Helicobacter pylori-induced gastritis in humans. In this study, initially low colonization rates of the H. pylori strains ATCC 43504, SS1, or HP87 inoculated into gerbils caused difficulties in establishing this model. In order to increase the colonization ability and pathogenicity, the clinical HP87 isolate was selected for adaptation to the gerbil stomach by multiple in vivo passages through gerbils. Development of gastritis was examined histologically at 4-52 weeks after infection. The proportion of gerbils which tested positive for H. pylori by culture at four weeks after inoculation gradually increased from 11.1% of gerbils inoculated with HP87 without prior in vivo passage (P0) to 100% of gerbils inoculated with HP87 with seven in vivo passages (P7). In addition, adaptation of HP87 resulted in more severe histopathological changes. Gerbils infected with adapted HP87 (P7) exhibited severe infiltration by monomorphonuclear and polymorphonuclear leukocytes in the mucosa, submucosa, and subserosa of the gastric antrum, as well as epithelial changes consisting of hyperplasia, erosion, and ulceration. Histopathological changes increased in severity from four to 52 weeks after infection. Adaptation of HP87 during its passages through gerbils could be due to genetic changes in bacterial colonization factors. Identification of these changes might be useful to understand the underlying mechanism of gastric adaptation and pathogenesis of H. pylori.

Phenotypic and functional characterization of mast cells derived from renal tumor tissues.

Mast cells (MCs) originate from multipotent hematopoietic progenitor cells. However, MCs in various organs are heterogenous in terms of mediator or receptor expression and response to diverse stimuli. We characterized the phenotype and functional properties of human renal mast cells (HRMCs). Tissue was obtained from 17 patients suffering from renal tumors (transitional cell carcinoma, n = 4; renal cell carcinoma, n = 13). HRMCs were isolated by collagenase digestion. Double staining with toluidine blue and immunofluorescence using monoclonal antibodies (mAbs) revealed expression of stem cell factor (SCF)-receptor (c-kit/CD117), CD9, CD29, CD33, CD43, CD44, CD54, and CD63 on HRMCs. In contrast, HRMCs were not recognized by mAbs to CD2, CD3, CD4, CD11b, CD14, CD15, CD16, CDw17, CD19, or CD23. HRMCs were also negative for CD116 (granulocyte-macrophage colony-stimulating factor [GM-CSF] receptor alpha), CD123 (interleukin [IL]-3Ralpha), CD121a (IL-1R type I), CD122 (IL-2Rbeta), and CD127 (IL-7R) and were also found to lack C5aR (CD88). Ligand-induced activation of HRMCs through immunoglobulin (Ig)E-R or SCF-R (c-kit) resulted in histamine secretion (control: <10%; alphaIgE, 1 microg/mL: 50.12 +/-5.18%; rhSCF, 100 ng/mL: 29.24 +/- 22.39), whereas recombinant C5a, erythropoietin (EPO), IL-1 through 10, and GM-CSF exerted no effects. As determined by in situ staining, HRMCs contained tryptase, but only low or undetectable amounts of chymase. Electron microscopy confirmed the presence of MCs in renal tissues and revealed a scroll-rich granule population in HRMCs. Together, HRMCs are tryptase+, C5aR- mast cells exhibiting phenotypic and functional properties similar to those of lung MCs.

Variation of human mucin gene expression in gastric cancer cell lines and gastric mucous cell primary cultures.

Human gastric mucous cells - gastric cancer cell lines mucin gene expression - TNFalpha - RT-PCR immunocytochemistry Little is known on the expression pattern of mucin genes in human gastric cancer cell lines in relation to mucin expression in normal gastric epithelial cells. Thus, the aim of this study was to compare gastric cancer cell lines and non-transformed epithelial cells in their expression of the different mucin genes, in order to use these cells as models for physiological MUC expression in human stomach. Human gastric mucous cell primary cultures which were obtained from surgical specimen by collagenase/pronase treatment and a panel of six human gastric cancer cells were screened for mRNA expression of the mucin genes MUC1, MUC2, MUC5AC, MUC5B, and MUC6. Mucin gene expression was analyzed by semi-quantitative RT-PCR, and by Western blotting and immunocytochemistry. Primary cultured human gastric mucous cells retained the stomach-specific pattern of mRNA expression found in gastric mucosal biopsies (MUC1, MUC5AC, MUC6), whereas any gastric cancer cell line exhibited an aberrant mucin gene expression. Mucin gene expression showed large variations in levels and patterns from cell line to cell line, but MUC2 was aberrantly expressed in all cancer cells. Immunocytochemistry confirmed aberrant MUC2 protein expression in cancer cells. The expression of the secretory mucin genes MUC2 and MUC5AC varied in relation to the length of cultivation of the cancer cell lines. Treatment of the gastric cancer cells with TNFalpha resulted in an enhanced mRNA expression of MUC1, MUC2, and MUC5AC (2-fold increase within 3 hours; p <0.05). In contrast, immunocytochemistry disclosed a decrease in MUC2 and MUC5AC staining intensity. Our results indicate that primary cultured human gastric mucous cells provide a physiological in vitro system for investigations of gastric mucin gene regulation. In gastric cancer cells marked changes in the mucin gene expression pattern are found with coexpression of non-gastric type mucins. Gastric mucin gene expression may be regulated by proinflammatory cytokines which could have implications in gastritis.

Ultrastructural immunogold localization of tumor necrosis factor-alpha to the matrix compartment of eosinophil secondary granules in patients with idiopathic hypereosinophilic syndrome.

Peripheral blood eosinophils from two normal donors and two patients with the hypereosinophilic syndrome (HES) were analyzed with a post-embedding immunogold method to detect the substructural location of tumor necrosis factor-alpha (TNF-alpha). In eosinophils of HES patients, TNF-alpha was localized to the matrix compartment of 64% of the specific secondary granules. Other structures in the HES eosinophils were unlabeled. No TNF-alpha was detected in eosinophils of normal donors. These studies document the first ultrastructural subcellular localization of any cytokine within the major population of secretory granules in human eosinophils and support other lines of evidence indicating that the expression of TNF-alpha may be greater in the eosinophils of HES patients than in those of normal donors.

Inhibition of partially purified K+/H+-ATPase from guinea-pig isolated and enriched parietal cells by substituted benzimidazoles.

The cellular and subcellular distributions of adenosinetriphosphatases (ATPases) were examined in guinea-pig gastric mucosal cells. All cell types displayed Mg2+-ATPase and bicarbonate (HCO3-)-stimulated ATPase activity. K+-ATPase was located only in fractions derived from parietal cells. Differential and density-gradient centrifugation of material prepared from parietal cells revealed that K+-ATPase activity was located in a tubulo-vesicular membrane fraction. Enzyme activity was ten fold greater in this fraction than in a crude parietal cell homogenate. The substituted benzimidazoles, omeprazole and picoprazole, inhibited K+-ATPase (IC50 1.8 +/- 0.5 mumol l-1 and 3.1 +/- 0.4 mumol l-1, respectively). Detailed kinetic analysis indicated that these compounds were non-competitive and reversible inhibitors of the enzyme. In contrast cimetidine and verapamil were without effect on the enzyme. The relevance of the inhibition of K+-ATPase to the antisecretory activity of the benzimidazoles, in experimental animals and man, is discussed.

Mechanism of gastric antisecretory effect of SCH 28080.

The mechanism of the gastric antisecretory action of SCH 28080 has been studied utilizing two different in vitro test systems, isolated and enriched parietal cells from the guinea-pig and guinea-pig gastric membranes purified and enriched with K+/H+-ATPase. In guinea-pig isolated and enriched parietal cells SCH 28080 inhibited the acid response to histamine and high K+ concentrations with IC50 values not significantly different from each other. SCH 28080 inhibited the purified K+/H+-ATPase measured in the presence of 5 mM KCl with an IC50 value of 1.3 microM. Kinetic studies indicated a competitive inhibition of ATPase by SCH 28080 with respect to K+. Studies on Na+/K+-ATPase showed that this enzyme was only slightly depressed by SCH 28080. It is concluded that SCH 28080 acts with high selectivity on the parietal cell K%/H+-ATPase, establishing its antisecretory effect by a competitive interaction with the high affinity K+-site of the gastric ATPase.

The sulphoxide moiety of substituted benzimidazoles is essential for inhibition of parietal cell K+/H+-ATPase.

The antisecretory action of the benzimidazole sulphoxide derivative B 823-10, 2[(4-methoxy-3-methyl-2-pyridylmethyl)-sulphinyl]- 5-trifluoromethyl(1H)-benzimidazole, was compared with the effect of the corresponding sulphide B 823-08 in several in vivo and in vitro and in vitro test systems. The sulphide B 823-08 and the sulphoxide B 823-10 were found to be equipotent in the Shay rat. The sulphide was found to inhibit H+ secretion in intact rabbit gastric glands and enriched guinea-pig parietal cells with lower potency than the corresponding sulphoxide. The relative potency in antisecretory activity (sulphide/sulphoxide) decreased in the following rank order: Shay rat: gastric glands: parietal cells. Purified K+/H+-ATPase was not blocked by the sulphide, whereas the sulphoxide inhibited the overall as well as the partial reactions of this enzyme. In all in vitro systems tested, inhibition of H+ secretion and enzyme activity by the sulphoxide, but not by the sulphide, was antagonized by SH-compounds such as dithiothreitol. It is concluded that in vivo sulphoxidation of the sulphide plays an important role in acid inhibition. In vitro an additional inhibitory mechanism of the sulphide has to be considered.

Comparative pharmacology of histamine H2-receptor antagonists.

There was no significant difference between the concentration-dependent inhibitory effects produced by roxatidine acetate, roxatidine and ranitidine on adenylate cyclase derived from isolated and enriched guinea-pig parietal cells. All the compounds shifted the concentration-response curve of histamine to the right and transformation of this data to Schild-plots produced straight lines with slopes greater than 1 but not significantly different from each other. The pA2 values characterising the potencies were roxatidine acetate 6.85 +/- 0.86, roxatidine 7.14 +/- 0.04, and ranitidine 6.92 +/- 0.01. Histamine-stimulated acid production from isolated guinea-pig parietal cells, measured by the 14C-aminopyrine accumulation technique, was similarly affected by the 3 compounds. Schild-plot slopes of roxatidine acetate and ranitidine were not significantly different from unity and pA2 values were similar to those of the adenylate cyclase inhibition, roxatidine acetate 7.15 +/- 0.09, roxatidine 7.03 +/- 0.02, and ranitidine 6.83 +/- 0.10. In conclusion, roxatidine acetate and its major metabolite roxatidine behave like competitive antagonists with potencies similar to ranitidine on H2-receptors on the guinea-pig parietal cell.

Interaction of the anti-inflammatory seleno-organic compound ebselen with acid secretion in isolated parietal cells and gastric H+/K(+)-ATPase.

The effects of the anti-inflammatory seleno-organic compound ebselen on gastric H+/K(+)-ATPase, H+/K(+)-ATPase-mediated proton transport and on parietal cell HCl production was studied. Ebselen inhibited K(+)-stimulated ATPase activity in leaky gastric membranes (IC50:0.15 microM) and H+/K(+)-ATPase-mediated proton transport in intact gastric membrane vesicles (IC50:0.7 microM). Histamine- and dibutyryl-cAMP-stimulated HCl production in isolated and enriched guinea-pig parietal cells was inhibited with an IC50 value of 12 microM. The mercaptan dithioerythritol and the nucleotide ATP prevents the H+/K(+)-ATPase against inactivation and dithioerythritol was found to restore already inhibited enzyme activity and ATPase mediated H+ transport. Furthermore, dithioerythritol could prevent ebselen-induced inhibition of HCl production in the parietal cell preparation. It is concluded that ebselen inhibits acid secretion in the parietal cell by interference with SH groups of the gastric proton pump, the H+/K(+)-ATPase. Therefore ebselen can be regarded as an anti-inflammatory drug for which in vitro anti-secretory properties can be demonstrated.

Studies on the mechanism of action of the omeprazole-derived cyclic sulphenamide.

The inhibitory effects of omeprazole and omeprazole-derived metabolites were studied on Escherichia coli glutaminase activity at pH 2.5 which might represent the conditions present at the target enzyme (K+/H+-ATPase) in the secretory membrane of the intact parietal cell. Omeprazole and the omeprazole-derived cyclic sulphenamide inhibited glutaminase at pH 2.5 with identical potency (IC50 36 microM). The substrate, glutamine as well as the mercaptane, dithiothreitol, protect the enzyme. Furthermore, dithioerythritol was found to reverse inhibition. This indicates that an SH-group localized in the substrate binding center of glutaminase is most likely involved in the reaction leading to enzyme inhibition. Glutaminase inhibition by both compounds was less pronounced at pH 5.0. Omeprazole radical, the metabolite generated from the cyclic sulphenamide at more neutral pH values, failed to affect the enzyme. These findings were in contrast with the properties of the omeprazole-derived cyclic sulphenamide and radical at the K+/H+-ATPase preparation. This enzyme was inhibited by both compounds at pH 7.5 with a high potency, and reversal experiments with dithiothreitol demonstrate that these agents interfere with SH-groups of the K+/H+-ATPase. From these data it is suggested that the cyclic sulphenamide and the radical interfere by different reaction pathways with enzymatic SH-groups.

Omeprazole, SCH 28080 and doxepin differ in their characteristics to inhibit H+/K+-ATPase driven proton accumulation by parietal cell membrane vesicles.

The effects of omeprazole, SCH 28080 and doxepin were studied on H+/K+-ATPase mediated H+ accumulation in parietal cell membrane vesicles. Omeprazole had no effect on the initial rate of H+ accumulation and the initial steady state concentration of H+; an inhibition was found after the vesicles were acidified. This inhibition was counteracted by the SH reducing agent dithioerythritol. SCH 28080 inhibited the initial rate of H+ accumulation and the steady state H+ concentration. The inhibitory effect of SCH 28080 was counteracted by KCl. Doxepin (3-100 microM) reduced the initial steady state H+ concentration. Doxepin concentrations lower than 0.5 microM had no such effect but dissipated the proton gradient after the vesicles were fully acidified. This doxepin effect was partially counteracted by KCl and was also obtained in vesicles in which the pump reaction was stopped by EDTA. These data show that (i) omeprazole is an acid-activated compound which interferes with SH groups of the H+/K+-ATPase localized inside the vesicles; (ii) SCH 28080 interferes with the K+ site of the H+/K+-ATPase; and (iii) doxepin interacts by a K+ antagonistic activity at the H+/K+-ATPase site and in addition by intravesicular neutralization and/or a protonophoric mechanism with the process of H+ formation.

The gastric proton pump, a target for neuroleptics and antidepressant drugs?

The antisecretory action of the antidepressant drugs trimipramine, doxepin and nortriptyline was studied in two different in-vitro test systems; the isolated and enriched guinea-pig parietal cell and the purified H+/K(+)-ATPase preparation. The effect of the antidepressants was compared with that of the neuroleptic agents chlorpromazine, triflupromazine, trifluperazine, haloperidol, fluspirilene and with that of the tricyclic anticholinergic agent pirenzepine. All neuroleptics and antidepressants inhibited acid formation in intact parietal cells with IC50 values in the nanomolar range. The inhibitory potency for each compound was identical regardless of whether histamine or db-cAMP was used as stimulant. Isolated H+/K(+)-ATPase, measured in the presence of 5 mmol litre-1 KCl, was inhibited by all psychotropic drugs with IC50 values in the micromolar range. EGTA did not affect the inhibitory potency at the H+/K(+)-ATPase, indicating that the action of the drugs does not depend on their calmodulin blocking activity. Pirenzepine was ineffective in both test systems. Kinetic studies done with nortriptyline, chlorpromazine and haloperidol showed a competitive type of inhibition with respect to K+ at low inhibitor concentrations. This competitive type was changed to a mixed type of inhibition with increasing inhibitor concentrations, demonstrating cooperative effects between drug binding and K+ activation of the enzyme. From these data it is suggested that antidepressants and neuroleptics act by an allosteric mechanism of action, and that the lipid solubility is a significant factor to establish enzyme inhibition.

Mast cell granule composition and tissue location--a close correlation.

This review provides a survey on mast cell heterogeneity, with aspects differing in humans and rodents or which are subject of conflicting evidence being discussed in greater detail. Mast cell subsets have been first defined in rats by their fixation and dye-binding properties, and detailed studies in humans and pigs reveal very similar observations. The dye-binding properties of rat mast cell subsets are causally related to the absence or presence of heparin in their granules. In humans, this relation has not been shown. Rodent mast cell subsets store different chymase-isoforms. In contrast, just a single chymase has been defined in humans, and mast cells are classified by the presence or relative absence of this chymase. Different investigators find quite different proportions of chymase-positive to chymase-negative mast cells. Tryptase(s) are found in most or every human mast cell, but in rodents, they have hitherto been essentially localised to mast cells in connective tissues. Human mast cell subsets may also be defined by their expression of receptors such as C5aR and possibly the beta-chemokine receptor CCR3; the CCR3 expression seems to be related to the human mast cell chymase expression. Ultrastructural studies are helpful to distinguish human mast cell subsets, and allow to distinguish between chronic and acute activation. The phenotypical characteristics may change in association with inflammation or other disease processes. Studies in humans and pigs show changed dye-binding and fixation properties of the granules. Experimental rodent infection models reveal similar changes of chymase isoform expression. Human lung mast cells have been reported to strongly upregulate their chymase content in pulmonary vascular disease. This line of evidence can explain some inconsistent information on mast cell heterogeneity and may help to understand the physiological role of mast cells.

Helicobacter pylori fatty acid cis 9,10-methyleneoctadecanoic acid increases [Ca2+]i, activates protein kinase C and stimulates acid secretion in parietal cells.

The effect of the Helicobacter pylori (H. pylori) fatty acid cis 9,10-methyleneoctadecanoic acid (MOA) on gastric acid secretion was studied in isolated guinea-pig parietal cells. MOA (1 and 3 micromol/l) stimulated basal and enhanced histamine- and dibutyryl cyclic AMP-stimulated acid secretion in parietal cells. MOA increased intracellular free [Ca2+]i concentration in a concentration-dependent manner. The source of [Ca2+]i was extracellular as demonstrated by depletion of [Ca2+]i with EGTA. Furthermore, MOA caused activation of parietal cell protein kinase C (PKC). The effect of MOA upon PKC activation was [Ca2+]i-dependent but did not require phosphatidylserine as phospholipid co-factor. Similarly to the effect of diolein, MOA increased the stimulatory effect of phosphatidylserine at low [Ca2+]i concentrations. Treatment of parietal cells with MOA caused translocation of PKC from the cytosol to the membrane-associated cell fraction. We propose that MOA stimulates parietal cell acid secretion presumably by an increase of cytosolic free [Ca2+]i concentrations and PKC activation.

Effects of PGE2 on amount and composition of high molecular weight glycoproteins released by human gastric mucous cells in primary culture.

The aim of this study was to determine effects of prostaglandin E2 (PGE2) on amount and composition of high molecular weight glycoproteins (HMG), released by human gastric mucous cells in primary culture. PGE2 stimulated the release of HMG, as evidenced by measurement of total carbohydrate and protein content, in a concentration-dependent manner. At the maximally tested concentration of 10(-5) mol/l, the increase amounted to 53% and 85%, over controls, for carbohydrate and protein, respectively. The stimulated release was accompanied by alterations of HMG glycosylation. As detected by lectin-ELISA, there was a relative decrease in N-acetyl glucosamine and an increase in mannose and galactose content. The sialic acid content increased in parallel to the total carbohydrate content. These results suggest that PGE2 plays a regulatory role in the synthesis and secretion of HMG by human gastric mucous cells.