E. Piotrowski

Induction of acute gastritis and epithelial apoptosis by Helicobacter pylori lipopolysaccharide.

BACKGROUND The preservation of gastric mucosal homeostasis is a complex biologic process, controlled by a dynamic equilibrium of cell loss by apoptosis with that of cellular proliferation, and its abrogation is a prominent feature of Helicobacter pylori-associated gastritis. In this report, we show that H. pylori lipopolysaccharide induces histologic lesions typical of acute gastritis and that these changes are reflected in the increased epithelial cell apoptosis. METHODS The experiments were conducted with groups of rats subjected to intragastric surface epithelial application of the lipopolysaccharide at 50 and 200 micrograms per animal. The histologic assessment of the mucosal tissue and quantification of apoptotic epithelial cells was performed 2 and 10 days after the lipopolysaccharide treatment. RESULTS Histologic examination showed that H. pylori lipopolysaccharide at both doses within 2 days induced infiltration of lamina propria with lymphocytes and plasma cells, edema, hyperemia, and hemorrhage extending from the lamina propria to the surface of mucosa, and the effect persisted beyond the 10 days. The in situ DNA fragmentation assay showed that lipopolysaccharide caused a marked increase in epithelial cell apoptosis, with the numerous apoptotic cells present not only in the superficial epithelium but also deeper in the glands. The mean apoptotic index in the mucosa was 59% when assessed 2 days after the administration of the 50-microgram lipopolysaccharide dose and 71.9% after the 200-microgram dose, whereas in the sections assessed 10 days after the lipopolysaccharide treatment the apoptotic index averaged 46% for a 50-microgram dose and 76.8% for a 200-microgram dose. Moreover, the apoptotic index showed positive correlation (r = 0.71) with the grade of the induced inflammatory changes. CONCLUSIONS Our findings demonstrate that H. pylori lipopolysaccharide can cause gastric mucosal responses typical of acute gastritis and identify the lipopolysaccharide as a virulence factor responsible for the induction of gastric epithelial cell apoptosis by H. pylori.

Activation of arachidonoyl phospholipase A2 in prostaglandin-mediated action of sucralfate.

1. The mechanism of sucralfate-induced gastric mucosal prostaglandin generation was investigated using mucosal cells labeled with [14C]choline and [3H]arachidonic acid. 2. In comparison to the controls, the cells maintained in the presence of sucralfate showed a concentration dependent increase in lysophosphatidylcholine (LPC) synthesis and PGE2 generation. The maximal effect was attained at 25 microM sucralfate giving a 45.7% increase in LPC and 70% increase in PGE2. 3. Pretreatment with indomethacin prior to sucralfate, while causing inhibition in PGE2 generation, had no effect on LPC production and led to accumulation of free arachidonic acid. In the case of pretreatment with NDGA, the sucralfate caused increased LPC synthesis accompanied by enhanced PGE2 generation without free arachidonic acid accumulation. 4. The stimulatory effect of sucralfate on LPC synthesis and PGE2 generation was inhibited by phospholipase A2 inhibitors, mepacrine and BPB. The inhibitory effect was concentration dependent and attained maximum at 40 microM for BPB and 80 microM for mepacrine. 5. The results for the first time demonstrate that the enhancement in gastric mucosal prostaglandin generation by sucralfate results from the stimulation of mucosal phospholipase A2 for arachidonic acid release.

Activation of apoptotic caspase‐3 and nitric oxide synthase‐2 in buccal mucosa with chronic alcohol ingestion

Apoptosis, the process of programmed cell death, involves activation of caspase proteases cascade that remains under the regulatory control of nitric oxide. In this study, we investigated the activity of a key apoptotic protease, caspase‐3, and the expression of nitric oxide synthase‐2 (NOS‐2) associated with buccal epithelial cells apoptosis induced by chronic ethanol diet. The assays revealed that a 7.9‐fold enhancement in buccal epithelial cells apoptosis, observed in the alcohol diet group, was accompanied by a 37.6‐fold increase in caspase‐3 activity and a 10.1‐fold increase in NOS‐2. Furthermore, the expression of NOS‐2 showed a positive correlation (r = 0.92) with the extent of changes induced in caspase‐3 activity. These results implicate caspase‐3 in the process of alcohol‐induced epithelial cells apoptosis, and point towards participation of NOS‐2 in the amplification of the cell death signaling cascade.

Adrenergic and Cholinergic Regulation of Gastric Mucus Phospholipid Secretion

The influence of adrenergic and cholinergic agonists on phospholipid secretion in gastric mucosal cells maintained in the presence of [3H]choline was investigated. The secretion of [3H]choline phospholipids over a 30-min period averaged 1.98% of the total cellular labeled phospholipids in the absence of any mediator and was enhanced by the beta-adrenergic agonist isoproterenol to a greater extent than by the cholinergic agonist pilocarpine. A 2-fold increase in phospholipid secretion was achieved with isoproterenol, whereas pilocarpine produced a 1.3-fold increase. The stimulatory effect of isoproterenol was inhibited by alprenolol, and that of pilocarpine by atropine. The phospholipids secreted in response to isoproterenol showed a 30% decrease in lysophosphatidylcholine, whereas a 2.1-fold enrichment in this phospholipid occurred with pilocarpine. The results demonstrate the involvement of neural mediators in the regulation of phospholipid secretion in gastric mucus.

Control of gastric mucus phospholipid content and composition by cholinergic and adrenergic mediators.

1. The secretion of choline-containing phospholipids by gastric mucosal cells in response to neural mediators was investigated using beta-adrenergic and cholinergic agents. 2. A 2.7-fold increase in phospholipid secretion occurred with isoproterenol, while pilocarpine evoked 1.4-fold increase and the effects were inhibited by the respective antagonists. 3. The phospholipid secretory responses were stimulated by dibutyryl-cAMP and phorbol myristate acetate (PMA), but not by 4 alpha-phorbol-12,13-didecanoate which does not activate protein kinase C. The effects of dibutyryl-cAMP and PMA were additive, the the PMA induced phospholipid secretion was inhibited by a protein kinase C inhibitor, tetracaine. 4. The phospholipids secreted in response to isoproterenol showed a 2.1-fold decrease in lysophosphatidylcholine, while those secreted in response to pilocarpine were enriched 2.3-fold in lysophosphatidylcholine, and 1.5-fold in sphingomyelin, and showed 23% lower content of phosphatidylcholine. 5. The results suggest that cholinergic and beta-adrenergic mediators participate in defining the gastric mucus phospholipid content and composition, and hence influence the mucosal protective capability.

Enhancement of gastric mucus phospholipid secretion by an antiulcer agent, ebrotidine.

1. Rat gastric mucosal cells, subjected to phospholipid labeling by incubating the cell suspension in DMEM with [3H]choline, were exposed to different concentrations (0-150 microM) of H2-receptor antagonists, ebrotidine and ranitidine, and the phospholipid secretory responses were evaluated. 2. In the absence of the drugs, the secretion of choline-containing phospholipids over a 1 hr period averaged 3.97% of the total cellular labeled phospholipids. Ebrotidine caused a dose-dependent increase in the rate of phospholipid secretion which was most pronounced at 1 hr and persisted for at least 2 hr. The maximal effect was attained at 120 microM ebrotidine giving a 36% increase in phospholipid secretion. 3. The phospholipid secretory response to ebrotidine was accompanied by an increase in gastric mucosal cell cAMP level which reached a maximum value of 2.1-fold over that of controls at 1 hr. Ranitidine, in contrast, neither evoked increase in cAMP level nor caused any stimulation in phospholipid secretion. 4. The results indicate that the gastroprotective properties of ebrotidine are associated with the ability of the drug to elicit a rapid stimulation in gastric mucus phospholipid secretion, and that ranitidine does not possess such property.