CELLULAR MECHANISMS OF ALCOHOL INJURY OF GASTRIC MUCOSA

酒精损伤胃粘膜的细胞机制

• 批准号: 3111627
• 负责人: ANDRZEJ S TARNAWSKI
• 金额: $ 16.35万
• 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/3111627
• 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.

摄入的酒精经常会导致胃粘膜损伤和 流血。 酒精引起的胃的细胞机制 粘膜损伤仍然未知。 因此预防和治疗 ODE没有明确的科学基础和成功的结果。 我们 将研究细胞机制和细胞亚靶标 特殊重点的胃粘膜细胞的酒精损伤 关于胃粘膜细胞的钙和细胞骨架的作用， 2）钙传输和钙的维护机制 特定胃粘膜细胞中的稳态，3）酒精的影响 在细胞外钙流入，释放和隔离时 细胞内钙和细胞挤出钙的能力，4） 靶标亚细胞的酒精损伤部位着重于 细胞骨架元素，5）超微结构和功能特征 以及钙和细胞骨架在可逆和 胃粘膜细胞不可逆的酒精损伤，6） 酒精引起的细胞损伤中的钙调蛋白和白三烯，以及 7）胃粘膜适应慢性酒精的机制 行政。 研究将在大鼠的体内进行 以单剂量的胃内接受酒精或喂酒精 2-10周。 胃粘膜损伤将由A评估） 总外观（平面法）b）定量组织学，c） 通过扫描和传输EM进行超微结构，d）功能 和e）生化。 酒精的影响将在 孤立的胃腺（大鼠，兔子和人），并在孤立 在营养培养基中孵育的胃细胞（粘液，首席，顶叶） 含0-15％（v/v）酒精。 将评估细胞损伤：a） 快速绿色排除（可行性），b）通过细胞质泄漏 酶，c）通过扫描和传输EM，d）功能 （对分泌刺激和粘液，胃蛋白酶原和DNA的反应 合成）。 确定钙在酒精诱导的 受伤我们将在培养基中孵育胃腺或分离细胞 没有钙和钙（0.05-4毫米）以及与酒精一起 没有钙离子载体和/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 离子通道的性能，特别是依赖电压的钙 使用斑块夹在损伤过程中孤立的胃细胞中的通道 酒精对细胞外钙涌入的技术和影响 和钙外排，在摄取和钙释放时通过分离 线粒体，微粒体和这些过程的依赖性 钠，镁，ATP和代谢能。 细胞内钙 将评估：a）使用Fura-2（荧光钙指标）， b）通过45CA的吸收和c）超结构与拟甲酸 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 胃粘膜细胞的损伤，20种钙转运机制 和胃粘膜细胞中的稳态，3）钙和钙的作用 酒精损伤中的细胞骨架，4） 胃粘膜适应慢性酒精给药。