J. Sarosiek

Campylobacter pyloridis degrades mucin and undermines gastric mucosal integrity

The role of Campylobacter pyloridis, a spiral bacteria associated with gastritis and peptic ulcers in weakening the mucus component of gastric mucosal barrier was investigated. The colonies of bacteria, cultured from antral mucosal biopsies of patients undergoing gastroscopy, were washed with saline, passed through sterilization filter and the filtrate was examined for protease and glycosylhydrolase activities. The obtained results revealed that the filtrate exhibited a strong proteolytic activity not only towards the typical protein substrates such as albumin but also towards gastric mucin. Optimum enzymatic activity for degradation of mucin was attained at pH 7.0 and the protease activity was found in a low m.w. (less than 50K) protein fraction. The filtrate showed little glycosylhydrolase activity and did not cause the hydrolysis of mucin carbohydrates. The data suggest that C pyloridis infection weakens the gastric mucosal defense by causing proteolytic degradation of mucin component of the protective mucus layer.

Effect of lipids and proteins on the viscosity of gastric mucus glycoprotein

The effect of associated lipids and covalently bound fatty acids, and the contribution of serum albumin and secretory IgA to the viscosity of dog gastric mucus glycoprotein was investigated. Using a cone/plate viscometer at shear rates between 1.15 - 230s -1, it was found that extraction of associated lipids from the glycoprotein lead to 80-85% decrease in the viscosity. Further loss (39%) in viscosity of the delipidated glycoprotein occurred following removal of covalently bound fatty acids. Reassociation of the delipidated glycoprotein with its neutral lipids increased the viscosity 3-fold, a 2.5-fold increase was obtained with glycolipids, and 2-fold with phospholipids. Preincubation of purified mucus glycoprotein with albumin or IgA resulted in the increase in viscosity. This increase in viscosity was proportional to albumin concentration up to 10%, and to IgA concentration up to 5%. The results show that interaction of lipids and proteins with mucus glycoprotein contributes significantly to the viscosity of gastric mucus.

Hydrogen ion diffusion in dog gastric mucus glycoprotein: Effect of associated lipids and covalently bound fatty acids

The effect of neutral lipids, glycolipids and phospholipids associated with dog gastric mucus glycoprotein, and that of covalently bound fatty acids on the ability of glycoprotein to retard the diffusion of hydrogen ion was investigated. Purified mucus glycoprotein in its native form, placed between equimolar (0.155M) solutions of HCl and NaCl in a specially designed two-compartment chamber, caused a 90% reduction in permeability to hydrogen ion when compared with a layer of NaCl. Extraction of associated lipids lead to a 68% increase in permeability of the glycoprotein to hydrogen ion, while removal of the covalently bound fatty acids increased further the diffusion rate by 6%. Reassociation of the delipidated glycoprotein with its neutral lipids reduced the permeability to hydrogen ion by 34%, an 11% reduction was obtained with glycolipids, and 23% with phospholipids. Since neutral lipids account for 47% of the glycoprotein lipids, glycolipids 41.1% and phospholipids 11.9%, the quantitative decrease in permeability of the delipidated glycoprotein following its reassociation with phospholipids is 2.7 times greater than that of neutral lipids and 7.3 times greater than that of glycolipids.

Enhancement of the lipid content and physical properties of gastric mucus by geranylgeranylacetone.

The effects of intragastric administration of geranylgeranylacetone (GGA) on the content, composition and physical properties of the mucus component of the gastric mucosal barrier were investigated. One group of rats received twice daily for 3 consecutive days a dose of 100 mg/kg body weight of GGA, while the control group was subjected to daily doses of the vehicle. Sixteen hours following the last dose, the animals were killed, and their stomach was cut open and subjected to measurements of the adherent mucus gel content, analysis of its lipids and molecular forms of elaborated mucin, and evaluation of the viscosity and H+ retardation capacity. The results revealed that GGA elicited a 62% increase in the adherent mucus gel and caused a marked decrease in the proportion of the lower molecular weight mucin. Furthermore, the mucus of the GGA group exhibited a 67% higher content of covalently bound fatty acids and contained 46% more total lipids which were greatly (143%) enriched in phospholipids. The physical measurements demonstrated that mucus elaborated in the presence of GGA also exhibited 2.3 times higher viscosity and had a 32% greater ability to retard the diffusion of H+ than the mucus of the control group. The results suggest that GGA exerts a profound effect on the lipid content and the properties of gastric mucus associated with the maintenance of the mucosal integrity.

The Role of Surface and Intracellular Mucus in Gastric Mucosal Protection against Hydrogen Ion: Compositional Differences

The role of surface and intracellular mucus in gastric mucosal protection against hydrogen ion was investigated. Gastric mucosa, prepared from dog and rat stomachs at various stages of mucus depletion, was mounted in the permeability chamber, and the diffusion of hydrogen ion from the luminal to the serosal side was measured. Removal of the surface mucus caused a 42.9% increase in the permeability of rat gastric mucosa and a 47.4% increase in the permeability of dog gastric mucosa. The permeability to hydrogen ion of gastric mucosa depleted of its surface and intracellular mucus increased 216% in the case of the dog and 280% with the rat. Compositional analysis showed that in both animals the intracellular mucus had a higher content of lipids, covalently bound fatty acids, and carbohydrates, whereas the protein content was higher in the surface mucus. The results suggest that, although both the surface and intracellular mucus participate in the retardation of hydrogen ion diffusion, the contribution of the latter appears to be greater.

Retardation of hydrogen ion diffusion by gastric mucus constituents: Effect of proteolysis

The role of mucus glycoprotein, lipid and protein in the retardation of hydrogen ion diffusion by dog gastric mucus was investigated. The uniform layer of mucus (1.0 +/- 0.2mm thick), formed on the sintered glass support placed between equimolar (0.155M) solutions of HCl and NaCl, caused a 73% reduction in hydrogen ion diffusion when compared with a layer of NaCl. The rate of hydrogen ion diffusion through purified mucus glycoprotein was similar to that of native mucus, whereas a 30% increase in permeability of glycoprotein to hydrogen ion was observed following its delipidation. Preincubation of the purified mucus glycoprotein with serum albumin or IgA led to an increase in the ability of glycoprotein to retard the diffusion of hydrogen ion. This effect of albumin and IgA occurred at a physiological dose. About 70% decrease in hydrogen ion retardation was obtained following digestion of purified mucus glycoprotein with pepsin, and 74% following its digestion with pronase. The results show that the ability of gastric mucus to retard the diffusion of hydrogen ion depends upon the polymeric structure of undegraded mucus glycoprotein and the extent of interaction of this glycoprotein with lipids, serum albumin, and secretory IgA.

Effect of acetylsalicylic acid on gastric mucin viscosity, permeability to hydrogen ion, and susceptibility to pepsin

The effect of acetylsalicylic acid (aspirin) on peptic degradation of gastric mucin, its viscosity and the ability to retard the diffusion of hydrogen ion was investigated. The results of peptic degradation indicated that, in the absence of the drug, the rate of proteolysis was proportional to mucin concentration up to 400 micrograms and remained constant with time for up to 1 hr. Introduction of aspirin led to an enhancement in the rate of proteolysis. The apparent Km value of pepsin toward mucus glycoprotein was 8.7 X 10(-7) M in the absence of the drug and 6.9 X 10(-7) M in its presence. Viscosity measurements showed a drop in mucin viscosity following preincubation with aspirin. This decrease was concentration dependent and at a 4.0 X 10(-5) M concentration of the drug reached a value of 75%. Permeability studies revealed that preincubation with 2.0 X 10(-5) M aspirin increased the permeability of mucin to hydrogen ion by 10%, while an 18% increase was obtained with 4.0 X 10(-5) M aspirin. The results suggest that aspirin weakens the integrity of the gastric mucus layer by promoting its peptic degradation, decreasing viscosity, and reducing the ability to resist hydrogen ion penetration.

Lipids associated with rat small-intestinal mucus glycoprotein

The lipid content and composition of rat small-intestinal mucus, and the purified mucus glycoprotein before and after Pronase digestion were investigated. The mucus, obtained by the instillation of intestine with 2M NaCl, was fractionated on Bio-Gel A-50 in the presence of 6M urea and the mucus glycoprotein free of noncovalently bound protein was isolated. A portion of the purified glycoprotein was subjected to Pronase digestion to yield glycopeptides. The native mucus, and the purified glycoprotein and glycopeptides were extracted with chloroform-methanol, and the lipids contained in the extracts were analyzed. The lipids accounted for 17.6 of the dry weight of mucus, 26.4 of the mucus glycoprotein, and 25.3% of the glycopeptides. In comparison to mucus, the lipids associated with mucus glycoprotein contained 1.9 times more phospholipids and 2.1 times more glycolipids, showed a 26% increase in neutral lipids, and were virtually free of glycosphingolipids. Treatment of the purified glycoprotein with Pronase led to a moderate (22.3%) loss in neutral lipids, 4.3-fold decrease in phospholipids, and 52.3% increase in glyceroglucolipids. The results indicate that while the interaction of mucus glycoprotein with phospholipids involves its Pronase-susceptible region, the interaction with glyceroglucolipids occurs in the glycosylated region of the glycoprotein that is resistant to proteolysis.

Role of carbohydrates in the viscosity and permeability of gastric mucin to hydrogen ion

The effect of carbohydrate removal on the viscosity of gastric mucin and its ability to impede the diffusion of hydrogen ion was investigated. The mucin, purified from dog gastric mucus, was subjected to partial or extensive deglycosylation with specific exoglycosidases and then used in the measurements. The obtained results revealed that removal of peripheral fucose or N-acetylglucosamine caused in each case only about 5% reduction of the glyco-protein viscosity. An 18% drop in the viscosity, however, occurred following removal of sialic acid, while extensive deglycosylation (removal of 86% carbohydrate) reduced the glycoprotein viscosity by 40%. The ability of mucin to retard the diffusion of hydrogen ion increased by 7% following removal of fucose or N-acetylgalactosamine, a 28% increase was obtained following removal of sialic acid, while the permeability to hydrogen ion of the extensively deglycosylated glycoprotein decreased by 42%. The results suggest that carbohydrates contribute significantly to the viscoelastic and permselective properties of gastric mucin.

Prostaglandin effect on the physical properties of gastric mucus glycoprotein and its susceptibility to pepsin

The effect of 16,16-dimethyl prostaglandin E2 (DMPGE2) on gastric mucus glycoprotein viscosity, permeability to hydrogen ion and degradation by pepsin was investigated. Preincubation with DMPGE2 produced a marked enhancement in the glycoprotein viscosity. The increase was concentration dependent and at 2.6 X 10(-5)M DMPGE2 reached a value of 178%. Permeability measurements revealed that 2.6 X 10(-7)M DMPGE2 increased the retardation ability of the glycoprotein to hydrogen ion by 10%, while 22% increase was obtained with 2.6 X 10(-4)M DMPGE2. The results of peptic activity assay showed that DMPGE2 had no inhibitory effect on the rate of glycoprotein proteolysis, and actually a small stimulatory influence was consistently observed. The results suggest that prostaglandins beneficially affect the physical properties of mucus glycoprotein which are considered to be essential for the protective function of gastric mucus.