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.

Response of germfree rat colonic mucous cells to peroral endotoxin application.

During microbial colonization, mucin-releasing goblet cells of germ-free (GF) rats proliferate and upregulate their mucin synthesis, thus improving the intestinal mucus barrier. The present study determined the significance of bacterial membrane constituents for this development. A single dose of lipopolysaccharide (LPS) (35 micrograms/100 g body weight) and lipid A (3.5 micrograms/100 g body weight, respectively), was perorally administered to GF AS/Ztm rats. One, 3 and 5 days later, sections of the proximal and distal colon served for characterization of mucin-secreting goblet cells, released mucins were isolated in parallel. Maximal goblet cell diameters were evidenced at day 3. LPS generated a maximal goblet cell hyperplasia one day after challenge, lipid A stimulated the goblet cell proliferation continuously up to day 5. Three days after challenge with one of the stimuli, either, intracellular mucins had shifted significantly to neutral constituents. In addition, mucins, adherent to the colon mucosa and submerged to the luminal content, respectively, then were augmented. At day 5, adherent mucins were similar to the controls, while luminal, soluble constituents had further increased. Histometrical and biochemical methods evidenced a transient, inflammatory response of mucin-secreting cells, followed by an upregulated release of immature mucins.

Effects of perorally applied endotoxin on colonic mucins of germfree rats.

BACKGROUND: The intestinal epithelium, with the potential to restrict luminal noxae from the host, secretes a mucous layer with various protective functions. Microbial colonization of germfree (GF) rats stimulates this mucin-secreting tissue. The present study determined the effect of bacterial lipopolysaccharides (LPS) on this process. METHODS: One, 3, and 5 days after peroral application of 35 micrograms LPS/100 g body weight (from Escherichia coli O55:B5), LPS concentrations were monitored in ingesta, intestinal tissue, and liver. Mucin high molecular weight glycoproteins (HMG), released in response to LPS, were isolated and separated into mucins, i) attached to the colonic epithelium (EM) and ii) mixed to the luminal content (LM), respectively. Subsequently, the binding capacity of both mucin fractions for various lectins and for type-1 pili expressing E. coli was determined. RESULTS: Ingesta and tissue had maximal LPS concentrations on days 3 (jejunum) and 5 (colon). Maximal EM secretion was found on day 3, release of LM further increased to day 5. Both mucin fractions had altered glycosylation patterns: augmentation of beta-galactose, alpha-N-acetyl galactosamine, and mannose coincided with a decrease in alpha-fucose. Compared with the controls, attachment of E. coli to EM increased slightly on day 1 only; the binding capacity of LM increased continuously up to day 5. CONCLUSION: Results suggest that mucins, released in response to LPS, in addition to the epithelial protection, support the gut microbial clearance system.

Response of rat colonic mucosa to a high fiber diet.

Secreting lubricating mucins, colonic crypt goblet cells, contribute to the intestinal protection against mechanical challenge. After feeding germ-free (GF) and specific pathogen-free (SPF) AS/Ztm rats for 6 weeks, the proliferative response of colonic goblet to a commercial bulky diet (37.1% fiber) was compared to that of a standard diet. (4.4% fiber). An increased uptake of the high fiber diet by GF rats significantly augmented the capacity for mucin secretion as indicated by the amount and length of crypts, crypt cells and mature goblet cells. The response of SPF rats was limited to a crypt elongation, generated by more crypt cells. In both study groups, the goblet cell replication activity was similar to their controls. The increase in the mucin-secreting capacity, induced by a constant mechanical challenge, highly suggests an improved intestinal protection.

Prostaglandin E2 alters terminal glycosylation of high molecular weight glycoproteins, released by pig gastric mucous cells in vitro.

The gastric mucus layer consists of high molecular weight glycoproteins (HMG). E-Type prostaglandins (PGs) stimulate total HMG release from isolated gastric mucous cells. We determined the effects of PGE2 on HMG glycosylation. Pig gastric mucous cells were cultured for 20 h with 1 mumol/l PGE2. Released HMG were isolated by gel chromatography and periodic acid-Schiff (PAS)-positive sugars and protein-bound [14C]GlcNAc were determined. Monosaccharides terminally linked to HMG oligosaccharide chains were monitored by lectin enzyme linked immunosorbent assay (ELISA): N-acetylglucosamine (GlcNAc) with Datura stramonium agglutinin, N-acetylgalactosamine (GalNAc) with soy bean agglutinin, fucose (Fuc) with Ulex europaeus I agglutinin and sialic acids (Sial) with Sambucus nigra agglutinin. PGE2 stimulated total HMG release, indicated by an increase of PAS-positive sugars to 170% and [14C]GlcNAc to 220% of controls. Terminal GlcNAc increased to 128%, GalNAc to 133%, Fuc to 165% and Sial to 182%. In addition to stimulation of total HMG release, PGE2 caused alterations of HMG glycosylation, which may modulate HMG viscosity and microbiological barrier function.

Mechanical challenge causes alterations of rat colonic mucosa and released mucins. Alterations of mucosa and mucins.

The effect of intraluminal challenge on rat colonic mucin producing cells and the amount and composition of released mucins was investigated. Germfree rats (GF) were maintained on a commercial high fiber (HF) diet (37% of undigestable fiber, Altromin 1640 p), in order to increase volume, dry weight and abrasive effect of the ingesta. GF control rats were fed a standard (ST) laboratory diet with 4.5% fiber (Altromin 1314 f). In the HF diet group, histological sections of the proximal and distal colon revealed a significantly increased number of mucin secreting goblet cells and an elevated goblet cell replication activity, as determined by 5'-bromo-deoxyuridine incorporation. The total amount of colonic mucins, isolated by gel filtration, was increased versus the control group. According to the results of ion exchange chromatography, carbohydrate and amino acid analysis, mucins from rats, given the HF diet, had an elevated content of acidic mucin constituents with alterations in the carbohydrate and amino acid composition. In a parallel study with specified pathogen free rats (SPF), the additional influence of the microflora on mucin secreting cells and isolated mucins was determined. An increased number of mucin secreting cells predominantly was observed in rats given the standard diet. Due to bacterial degradation, significantly less mucin was isolated from both dietary groups. The increase of acidic mucin constituents was less pronounced than in GF rat mucin, coinciding with losses of terminally linked monosaccharides. Alterations of the core protein, accompanying the presence of the microflora, were not detected.

Changes in colonic mucins of germfree rats in response to the introduction of a "normal" rat microbial flora. Rat colonic mucin.

In order to determine the influence of bacterial colonization on amount and composition of colonic mucins, germfree male AS/Ztm rats were colonized with a rat specific intestinal flora for different times (2, 7, 14, 21, 28, 35, 120 days). The amount of colonic mucins was determined by gel filtration on Sepharose CL-4B; the relative amount of acidic mucins was calculated after ion exchange chromatography. In addition, cecal weight and dry matter of feces were monitored. While germfree and SPF rats revealed similar amounts of colonic mucins (7.0 vs. 7.2 mg mucin/300 g body weight), the initial phase of association was characterized by considerably decreasing values. After four weeks of association, the total amount of colonic mucins had almost equalized in the two groups. The amount of acidic mucins, having decreased during the first three weeks of colonization, rendered values comparable to the SPF mucins after four months of adaptation. Cecomegaly in germfree rats disappeared within the first two days, while solidification of the intestinal content occurred within four months. Mucin losses during initial phase of association are attributed 1. to the disappearance of the cecal mucin pool, and 2. to the mucin degrading activity of some bacterial strains known to be present in the intestinal flora. Further development is conducted by a stimulation of mucin secretion, described to follow the colonization. The initially increased secretion of neutral mucins is attributed to a pronounced release of immature mucin glycoproteins, while the shift to more acidic mucins is considered to result from stimulated secretion as well as from a selective bacterial degradation of neutral mucin components.