CELLULAR MECHANISMS OF ALCOHOL INJURY OF GASTRIC MUCOSA

酒精损伤胃粘膜的细胞机制

• 批准号: 3111626
• 负责人: ANDRZEJ S TARNAWSKI
• 金额: $ 15.66万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3111626
• 关键词: alcoholic beverage consumption; alcoholism /alcohol abuse; calcium channel; calcium channel blockers; calcium flux; calmodulin; cellular pathology; cytoskeleton; cytotoxicity; disease /disorder model; drug administration rate /duration; electron microscopy; fluorescent dye /probe; gastric mucosa; histopathology; human tissue; ion transport; laboratory rabbit; laboratory rat; tissue /cell culture; voltage /patch clamp

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.

摄入酒精经常会造成胃粘膜损伤 流血。 酒精诱发胃病的细胞机制 粘膜损伤尚不清楚。 因此预防和治疗 颂歌没有明确的科学依据和成功的结果。 我们 将研究细胞机制和亚细胞靶标 酒精对胃粘膜细胞损伤的研究 对钙和胃粘膜细胞细胞骨架的作用， 2）钙转运和钙维持机制 特定胃粘膜细胞的稳态，3）酒精的影响 细胞外钙流入、释放和隔离 细胞内钙和细胞排出钙的能力，4) 针对酒精损伤的亚细胞位点，重点是 细胞骨架元件，5) 超微结构和功能特征 以及钙和细胞骨架在可逆和 酒精对胃粘膜细胞的不可逆损伤，6）作用 钙调蛋白和白三烯在酒精诱导的细胞损伤中的作用，以及 7）胃粘膜对慢性酒精的适应机制 行政。 研究将在大鼠体内进行 单剂量胃内饮酒或喂食酒精 持续 2-10 周。 胃粘膜损伤将通过 a) 进行评估 总体外观（面积测量）b) 通过定量组织学，c) 通过扫描和传输 EM 实现超微结构，d) 功能 e) 生物化学。 将研究酒精的影响 分离的胃腺（大鼠、兔和人）和分离的胃腺 在营养培养基中培养的胃细胞（粘液细胞、主细胞、壁细胞） 使用 0-15% (v/v) 酒精。 将评估细胞损伤：a) 快速绿色排除（活力），b) 通过细胞质泄漏 酶，c) 通过扫描和传输 EM，以及 d) 功能性 （对分泌刺激和粘液、胃蛋白酶原和 DNA 的反应 合成）。 确定钙在酒精诱导中的作用 损伤后，我们将在培养基中培养胃腺体或分离的细胞 不含钙和含钙 (0.05-4 mM) 加酒精和 不含钙离子载体和/或钙通道阻滞剂或 钙调蛋白拮抗剂。 此外，我们将研究存在和 离子通道的特性，特别是电压依赖性钙 使用膜片钳在损伤过程中分离胃细胞中的通道 酒精对细胞外钙流入的技术和影响 和钙流出，对分离的钙的吸收和释放 线粒体、微粒体以及这些过程对 钠、镁、ATP 和代谢能。 细胞内钙 将进行评估： a) 使用 Fura-2（荧光钙指示剂）， b) 通过 45Ca 摄取和 c) 通过焦锑酸盐进行超微结构分析 细胞化学染色。 我们的长期目标是解释： 1）酒精诱导的细胞机制和亚细胞靶点 胃粘膜细胞损伤、20种钙转运机制 和胃粘膜细胞的稳态，3）钙和的作用 酒精损伤中的细胞骨架，4）细胞机制 胃粘膜对长期饮酒的适应。