BILE SALTS, MEMBRANES, AND CYTOTOXICITY

胆盐、膜和细胞毒性

• 批准号: 6238913
• 负责人: DOUGLAS HEUMAN
• 金额: $ 19.41万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6238913
• 关键词: adsorption; cell membrane; chemical structure function; cholestane compound; cholesterol; cytotoxicity; gel filtration chromatography; hamsters; high performance liquid chromatography; human tissue; laboratory rat; light scattering; lipid bilayer membrane; liver cells; mathematical model; membrane lipids; membrane permeability; model design /development; neoplastic cell culture for noncancer research; protein kinase C; radionuclides; thin layer chromatography; ultrafiltration; ursodeoxycholate; vesicle /vacuole

Bile salts adsorb to membranes, at high concentrations causing membrane disruption. Adsorption of bile salts to intracellular membranes may determine many of their physiological effects, and bile salt induced membrane injury may be important in pathogenesis of cholestatic liver disease and gallstones. We have studied the adsorption of bile salts to lecithin-cholesterol vesicles and have developed and validated a quantitative model which predicts the distribution of bile salt taurine conjugates in mixed bile salt solutions between lecithin-cholesterol bilayers and the aqueous phase. In the studies proposed, this model will be generalized to a broad array of bile acids and other organic anions, membrane lipids, and solution conditions. Using large unilamellar vesicles of varying lipid composition, we will examine the relationship between membrane binding of bile salts, mixed micellar dissolution of membrane lipids (observed with quasielastic light scattering) and altered membrane permeability (release of trapped soluble markers assessed by ultrafiltration) to determine if the mixed micellar threshold concentration and the permeation threshold at which membrane leakage begins are predictable consequences of the membrane-bound ionized bile salt/lecithin ratio. Pure protein kinase C isoenzymes (alpha, betaII, delta, epsilon) prepared in a baculovirus system will be employed to test the hypothesis that bile salts activate protein kinase C isoenzymes by binding to membranes and serving as a "bridge" between the enzymes and membrane lipids. The model of bile salt-lecithin interactions will be extended beyond the limits of the two phase (monomer-membrane) region into micellar regions of the phase diagram by combining techniques of gel filtration and ultrafiltration, in order to permit modelling of detergent effects of mixed bile salt solutions. Using synthetic vesicles, isolated canalicular plasma membranes, and living cells (erythrocytes, cultured neoplastic gallbladder epithelia) we will test the hypothesis that lecithin in bile normally protects high cholesterol plasma membranes from bile salt injury by depressing the non-lecithin- associated bile salt concentration to non-toxic levels, and that this protective effect declines predictably as the cholesterol content of biliary vesicles increases. Finally the hepatoprotective role of biliary lipids and biliary bile salt-lipid interactions will be studied in two in vivo models of bile salt-induced liver injury: acute infusion of bile salts in the choline deficient bile fistula rat and chronic feeding of bile salts in hamsters fed lithogenic diets. The ultimate goal of these studies is to provide a conceptual framework for understanding the toxic and protective properties of bile salts and the role of bile salt toxicity in human disease.

胆盐吸附在膜上，在高浓度下， 膜破裂 胆盐在细胞内的吸附 膜可以决定它们的许多生理效应， 胆盐引起的膜损伤可能在发病机制中起重要作用 胆汁淤积性肝病和胆结石 我们研究了 胆盐对卵磷脂-胆固醇囊泡的吸附， 开发并验证了一个定量模型，预测 胆汁盐牛磺酸结合物在混合胆汁盐溶液中的分布 在卵磷脂-胆固醇双层和水相之间。 在 研究提出，这个模型将被推广到广泛的阵列， 胆汁酸和其他有机阴离子、膜脂质和溶液 条件 使用不同脂质的大单层囊泡 组成，我们将研究膜之间的关系 胆汁盐的结合，膜脂的混合胶束溶解 （用准弹性光散射观察）和改变的膜 渗透性（通过以下方法评估捕获的可溶性标记物的释放 超滤）以确定混合胶束阈值 浓度和渗透阈值， 泄漏开始是膜结合的可预测的后果， 游离胆汁盐/卵磷脂比率。 纯蛋白激酶C同工酶 在杆状病毒系统中制备的（α，β II，δ，β 3）将 胆盐激活蛋白质的假设 激酶C同功酶通过与细胞膜结合并作为 酶和膜脂之间的“桥梁”。 模型 胆盐-卵磷脂相互作用将超出 两相（单体-膜）区进入胶束区， 结合凝胶过滤技术和 超滤，以允许模拟洗涤剂的效果， 混合胆汁盐溶液。 使用合成囊泡，分离 小管质膜，和活细胞（红细胞， 培养的肿瘤性胆囊上皮细胞），我们将检验这一假设 胆汁中的卵磷脂通常保护高胆固醇血浆， 膜从胆盐损伤通过抑制非卵磷脂- 相关胆盐浓度至无毒水平， 保护作用下降，可预见的胆固醇含量， 胆汁囊泡增多。 最后，胆汁的保肝作用 脂质和胆汁胆汁盐-脂质相互作用将在两个研究中进行研究， 胆盐肝损伤动物模型的建立：急性输注胆汁 胆碱缺乏性胆瘘大鼠和慢性喂养 饲喂致石饲料的仓鼠中的胆汁盐。 这些项目的最终目标 研究的目的是提供一个概念框架， 胆盐的毒性和保护特性以及胆盐的作用 人类疾病中的毒性。