Human Gastric Mucous Cells Research Articles

EP4 agonist alleviates indomethacin-induced gastric lesionsand promotes chronic gastric ulcer healing

To investigate EP4-selective agonist effect on indomethacin-induced gastric lesions and on the spontaneous healing of chronic gastric ulcers. In a mouse model of gastric bleeding with high dose of indomethacin (20 mg/kg), an EP4-selective agonist was administered orally. Stomach lesions and gastric mucous regeneration were monitored. In a mouse model of chronic gastric ulcer induced by acetic acid, EP4 agonist effect on the healing of chronic gastric ulcer was evaluated in the presence or absence of low dose indomethacin (3 mg/kg). In cultured human gastric mucous cells, EP4 agonist effect on indomethacin-induced apoptosis was assessed by flow cytometry. The EP4-selective agonist reduced high dose indomethacin-induced acute hemorrhagic damage and promoted mucous epithelial regeneration. Low-dose indomethacin aggravated ulcer bleeding and inflammation, and delayed the healing of the established chronic gastric ulcer. The EP4 agonist, when applied locally, not only offset indomethacin-induced gastric bleeding and inflammation, but also accelerated ulcer healing. In the absence of indomethacin, the EP4 agonist even accelerated chronic gastric ulcer healing and suppressed inflammatory cell infiltration in the granulation tissue. In vitro, the EP4 agonist protected human gastric mucous cells from indomethacin-induced apoptosis. EP4-selective agonist may prevent indomethacin-induced gastric lesions and promote healing of existing and indomethacin-aggravated gastric ulcers, via promoting proliferation and survival of mucous epithelial cells.

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

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 TNFα 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.

Expression of leptin and its receptor in the human stomach

Dear Sir: We read with interest the well-written review of L. M. Kaplan entitled “Leptin, Obesity, and Liver Disease” published recently in GASTROENTEROLOGY.[1]Kaplan LM. Leptin, obesity, and liver disease.Gastroenterology. 1998; 115: 997-1001Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar He points out the current knowledge about leptin and its physiological role for gastroenterologists. Since the identification of leptin as a product of the obese gene by Zhang et al. in 1994,[2]Zhang Y Procnca R Maffei M. Positional cloning of the mouse obese gene and its human homologue.Nature. 1994; 372: 425-432Crossref PubMed Scopus (11755) Google Scholar the number of publications on leptin and its effects has increased exponentially. Up to now, there have been only few articles about leptin expression and function in the gastrointestinal tract, especially in humans. The initial perception that leptin is exclusively expressed and secreted by adipocytes had to be revised by the detection of leptin expression and secretion in the placenta, mammary epithelial cells, and hepatic stellate cells. Recently, Bado et al.[3]Bado A Levasseur S Attoub S. The stomach is a source of leptin.Nature. 1998; 394: 790-793Crossref PubMed Scopus (1038) Google Scholar showed leptin expression in the rat gastric mucosa. This led us to investigate leptin expression and secretion in the human gastric mucosa, which, to our knowledge, has not been reported. We screened (1) fundic and antral gastric biopsy specimens, obtained from healthy subjects during endoscopy; (2) human gastric mucous cells, isolated from surgical specimens by collagenase/pronase treatment; and (3) human gastric cancer cell lines (Kato III, 23132/87 cells) for messenger RNA expression of leptin and leptin receptor by reverse-transcription polymerase chain reaction (PCR). Specific oligonucleotide primers, deduced from the cloned human leptin gene and the human leptin receptor gene, were used.[4]Tartaglia LA Dembski M Wenig X. Identification and expression cloning of a leptin receptor, OB-R.Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3231) Google Scholar Human adipose and human placental tissue were used as positive controls. All tested specimens showed the expected PCR amplification product. Furthermore, bound leptin and soluble leptin receptor levels in cultured cell medium supernatant were detected by radioimmunoassay,5Ockenga J. Widjaja A. Holtmannspotter M. Bound leptin is regulated by tumor necrosis factor–alpha in HIV-infected patients: a potential mediator of wasting?.AIDS. 1998; 12: 2233-2235PubMed Google Scholar indicating leptin expression and secretion by human gastric epithelial cells. Finally, immunohistochemistry of gastric mucosal biopsy specimens was reactive for human leptin and human leptin receptor. The presence of the leptin receptor on gastric mucous cells suggests a paracrine effect of leptin on mucous cell function and gastric mucosa protection. In this study we did not differentiate between the different leptin receptor isoforms that have been described previously. Additional in vitro studies are needed to explore the unknown physiological function of leptin and its receptors in the human stomach.

Morphological and molecular characterization of human gastric mucous cells in long-term primary culture.

A primary cell culture of human gastric mucous cells was developed using enzymatic treatment of surgically obtained gastric mucosal specimens. Preferential attachment of gastric mucous cells during a preincubation step resulted in the enrichment of mucous cells [over 90% stained with periodic acid-Schiff (PAS) and mucin-type lectins] in the primary cell culture. Gastric mucous cells could be maintained in culture for 10 days. DNA synthesis peaked during the first 2 days in culture (8+/-1% bromodeoxyuridine-positive cells). During the entire culture period gastric mucous cells released high-molecular-weight glycoproteins into the medium, as determined by gel chromatography on a Sepharose CL-4B column and by metabolic labelling with [14C]-N-acetylglucosamine. Gastric mucin was verified by gas chromatographic analysis of the carbohydrate composition and fractionation of the void-volume fraction by density gradient centrifugation. Determination of the terminal glycosylation of the secreted glycoproteins by a lectin-ELISA revealed that there was a high quantity of alpha-l-fucose. Prostaglandin E2 significantly stimulated glycoprotein secretion during the entire cultivation period by 29-60%. Analysis of mucin-encoding MUC mRNA expression by reverse transcriptase polymerase chain reaction revealed that gastric mucous cells predominantly express MUC1 and MUC5AC, and to a lesser extent MUC6, which reflects the expression pattern obtained following analysis of biopsied samples of gastric mucosa. This primary cell culture model enables the regulation of mucin secretion and mucin gene expression in man to be investigated.

Effects of PGE 2 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 E 2 (PGE 2) on amount and composition of high molecular weight glycoproteins (HMG), released by human gastric mucous cells in primary culture. PGE 2 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 PGE 2 plays a regulatory role in the synthesis and secretion of HMG by human gastric mucous cells.

A method for the isolation of human gastric mucous epithelial cells for primary cell culture: A comparison of biopsy vs surgical tissue

We have developed a method for the isolation and growth of normal human gastric mucous epithelial cells using biopsies or surgically resected tissues as the source of the cells. The attachment and growth of cells were dependent upon: (1) cell planting density, ∼50,000 cells/cm2; (2) extracellular matrix (fibronectin); and (3) and the use of a porous filter. In all experiments we found better cells attachment and growth of human gastric mucous cells isolated from surgical specimens compared with those gastric mucous cells isolated from gastric biopsies. The initial cell viability (as measured by Trypan-blue) was the same in both populations of gastric mucous epithelial cells isolated from either gastric biopsies or surgical specimens. After 4–5 days in culture one could detect various amounts of mucin in all the cells using either periodic acid Schiff (PAS) staining or a specific anti-mucin antibody. A similar pattern of much straining was also found in primary cultures of guinea pig gastric mucous epithelial cells. Immunohistochemical staining for chief cells (anti-pepsinogen) or parietal cells (anti-H+/K+ ATPasc) in the gastric mucous cuboidal-like epithelial cells with tight junctions, desmosomes,short microvilli, a filamentous terminal web, mucous granules, and basal lamina-like structure. We could not detect the presence of fibroblasts during the 7–9 days that the primary cells were in culture. This cell culture method will prove useful in the isolation of normal human gastric mucous epithelial cells for in vitro studies of gastric mucosal injury and repair.

Extracellular Nucleotides Increase Cytosolic Free Calcium by Activating P2u-Receptors in Single Human Gastric Mucous Cells

In single human gastric mucous cells extracellular ATP, ATP-γ-S, ADP and UTP at micromolar concentrations caused a biphasic increase in [Ca2+]i. Consistent with a P2u-receptor the rank-order of potency was ATP greater than or equal to UTP > ATP-γ-S > ADP, and cross-desensitization of the Ca2+ responses occurred between ATP and UTP. The initial transient peak in [Ca2+]i was resistent to extracellular Ca2+ depletion which demonstrates mobilization of internal Ca2+, By contrast, the sustained plateau phase required influx of external Ca2+. Ca2+ influx occurs most likely through a capacitative Ca2+ entry mechanism, which was shown to exist in these cells by experiments performed with thapsigargin. Thus, extracellular ATP and UTP activate a common P2u-receptor most likely coupled to the Ca2+-phosphatidylinositol signalling cascade. Extracellular nucleotides from various sources might be an important factor in the regulation of human gastric mucous cells.

Regulation and time course of mucus and prostaglandin E 2 secretion in human gastric mucous cells in long-term primary culture : [formula omitted], S. Schumann, M.L. Enns, W Beil, U.E.H. Mai, M. Manns. Dept. of Gastroenterology and Hepatology, Medizinische Hochschule Hannover, D-30623 Hannover, Germany

Regulation and time course of mucus and prostaglandin E 2 secretion in human gastric mucous cells in long-term primary culture : [formula omitted], S. Schumann, M.L. Enns, W Beil, U.E.H. Mai, M. Manns. Dept. of Gastroenterology and Hepatology, Medizinische Hochschule Hannover, D-30623 Hannover, Germany