Ingested alcohol frequently produces gastric mucosal injury and bleeding. The cellular mechanisms of alcohol-induced gastric mucosal injury remain unknown. Therefore prevention and treatment odes not have a clear scientific basis and successful outcome. We will investigate cellular mechanisms and the sub-cellular targets of alcohol injury of the gastric mucosal cells with special focus on the role of calcium and cytoskeleton of gastric mucosal cells, 2) mechanisms of calcium transport and maintenance of calcium homeostasis in specific gastric mucosal cells, 3) effect of alcohol on extracellular calcium influx, on release and sequestration of intracellular calcium and on cell ability to extrude calcium, 4) target subcellular sites of alcohol injury with the focus on cytoskeletal elements, 5) ultrastructural and functional features and the role of calcium and cytoskeleton in reversible and irreversible alcohol injury of gastric mucosal cells, 6) the role of calmodulin and leukotrienes in alcohol-induced cell injury, and 7) the mechanisms of gastric mucosal adaptation to chronic alcohol administration. Studies will be performed in vivo in rats receiving alcohol intragastrically as a single dose, or fed alcohol for 2-10 weeks. Gastric mucosal injury will be assessed by a) gross appearance (planimetry) b) by quantitative histology, c) ultrastructurally by scanning and transmission EM, d) functionally and e) biochemically. Effect of alcohol will be studied in isolated gastric glands (rat, rabbit and human) and in isolated gastric cells (mucus, chief, parietal) incubated in nutrient media with 0-15% (v/v) alcohol. Cell damage will be assessed: a) by Fast green exclusion (viability), b) by leakage of cytoplasmic enzymes, c) by scanning and transmission EM, and d) functionally (response to secretory stimulation and mucus, pepsinogen and DNA synthesis). To determine the role of calcium in alcohol-induced injury we will incubate gastric glands or isolated cells in media without calcium and with calcium (0.05-4 mM) plus alcohol with and without calcium ionophore and/or calcium channel blockers or calmodulin antagonists. In addition we will study presence and properties of ion channels especially voltage dependent calcium channels in isolated gastric cells during injury using patch clamp technique and effect of alcohol on influx of extracellular calcium and calcium efflux, on uptake and release of calcium by isolated mitochondria, microsomes and dependence of these processes on sodium, magnesium, ATP and metabolic energy. Intracellular calcium will be assessed: a) with Fura-2, (fluorescent calcium indicator), b) by 45Ca uptake and c) ultrastructurally with pyroantimonate cytochemical staining. Our long term objectives are to explain: 1) cellular mechanisms and subcellular targets of alcohol-induced injury of gastric mucosal cells, 20 mechanisms of calcium transport and homeostasis in gastric mucosal cells, 3) role of calcium and cytoskeleton in alcohol injury, 4) the cellular mechanisms of adaptation of gastric mucosa to chronic alcohol administration.
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