The study of ethanol effects on intracellular transport and membrane biogenesis in rat hepatocytes revealed that, during synthesis of transport vesicles, the cytosolic phosphatidylinositol 3-kinase incorporated into the membrane of Golgi transport vesicles and a portion of the vesicular phosphatidylinositol was phosphorylated to phosphatidylinositol 3-phosphate. Association of the enzyme with Golgi transport vesicles and the transport to the apical portion of the cell membrane was not affected by 0 to 120 mM ethanol, but was dependent on the presence of the p85 subunit of the phosphatidylinositol 3-kinase. In the presence of ATP-enriched cytosol and calcium ions, association of Golgi transport vesicles with the apical membrane was followed by phospholipase A2-specific hydrolysis of phosphatidylinositol 3-phosphate and incorporation of the transport vesicle membrane into the apical membrane. Association of Golgi transport vesicles with apical membranes was not affected by preincubation of the cell membrane or Golgi transport vesicles with 0 to 120 mM ethanol, but was inhibited when the p85 phosphatidylinositol 3-kinase was incorporated into the membrane before incubation with Golgi transport vesicles. The fusion of Golgi transport vesicles with the apical membrane and generation of lysophosphatidylinositol 3-phosphate and arachidonate was inhibited with EGTA or after depletion of ATP from cytosol. Results of these studies provide evidence that phosphatidylinositol 3-kinase and phospholipase A2 activities are crucial for the final step of exocytotic transport. The process consists of two stages. First, the p85 subunit of phosphatidylinositol 3-kinase is involved in the specific association of the vesicle with membrane receptor, and that is followed by phospholipase A2-specific lysophospholipid generation, perturbation of the membranes, and fusion of the transport vesicle membrane with the apical membrane. Addition of ethanol to the in vitro transport system decreased production of Golgi transport vesicles, but had no effect on their association with apical membrane or fusion with the membrane.

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.

In this study, we investigated gastric epithelial cells' apoptosis and tumor necrosis factor-c~ (TNF-cc) expression with ethanol-induced mucosal injury, and the effect of antiulcer agents on this process.Rats received intragastric pretreatment with the agent or vehicle followed lh later by ethanol, and after 30 min the gastric mucosa was assessed for TNF-c~ and apoptosis. In the absence of antiulcer agents, ethanol caused extensive mucosal lesions accompanied by a 9.5-fold enhancement in apoptosis and a 2.5-fold increase in TNF-c~. Pretreatment with omeprazole evoked a 54% reduction in TNF-c~, but had no effect on ethanol-induced mucosal damage or apoptosis, the sucralfate reduced the extent of mucosal damage by 95%, apoptosis by 39% and TNF-c~ by 52%, while ebrotidine not only prevented mucosal injury and rise in TNF-c~, but also caused a 70% reduction in epithelial cells' apoptosis. The results demonstrate that ethanol-induced gastric epithelial cells apoptosis triggered by the enhancement in mucosal TNF-c~ is efficiently counteracted by ebrotidine.

SUMMARYIn this study we investigated buccal mucosal cells' apoptosis and the expression of regulatory cytokJnes, tumor necrosis factor-~ (TNF-~) and inetrleukin-4(IL-4) with chronic ethanol ingestion. The buccal mucosa of rats maintained for 23 days on alcohol-containing and control liquid diet was assessed for IL-4 and TNF-c~ content, and the extent of epithelial cells apoptosis. While the expression of TNF-c~ in alcohol diet group showed a significant increase (1.9-fold) over that of the controls, less apparent differences between the two groups were observed in the content of IL-4 (141.8 ___28.2 vs. 119.8 _+7.3 pg/mg protein). The DNA fragmentation assays revealed that alcohol diet group also exhibited a 3.5-fold enhancement in buccal mucosal cells apoptosis. Moreover, the apoptotic index showed positive correlation (r = 0.53) with the extent of induced changes in TNF-~. These results demonstrate that ethanol-induced buccal mucosal cells apoptosis is triggered by the enhancement in TNF-~ expression.

SUMMARYApoptosis, 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.