BILE SALTS, MEMBRANES, AND CYTOTOXICITY

胆盐在高浓度时吸附在膜上，导致 膜破裂。胆盐在胞内的吸附 膜可能决定它们的许多生理效应，并且 胆盐致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。我们已经研究了 胆盐在卵磷脂-胆固醇囊泡上的吸附 开发并验证了一个定量模型，该模型预测了 胆盐牛磺酸结合物在混合胆盐溶液中的分布 卵磷脂-胆固醇双层膜和水相之间的相互作用。在 研究表明，该模型将被推广到更广泛的 胆汁酸和其他有机阴离子、膜脂和溶液 条件。使用不同脂类的大单层囊泡 组成，我们将考察膜之间的关系 胆盐的结合、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的释放 超滤)以确定混合胶束阈值 浓度和膜的渗透阈值 开始渗漏是可以预见的膜结合的后果 电离胆盐/卵磷脂比值。纯蛋白激酶C同工酶 在杆状病毒系统中制备的(Alpha，BetaII，Delta，epsilon)将 被用来检验胆盐激活蛋白质的假设 通过与膜结合并作为一种 酶和膜脂之间的“桥梁”。的模型。 胆汁盐与卵磷脂的相互作用将超出 两相(单体-膜)区域转变为胶束区域 凝胶过滤和凝胶过滤技术相结合的相图 超滤，以允许对洗涤剂效果的模拟 混合胆盐溶液。使用合成囊泡，分离 小管质膜和活细胞(红细胞， 培养的肿瘤胆囊上皮)我们将检验这一假设 胆汁中的卵磷脂通常可以保护高胆固醇血浆 胆汁盐膜通过抑制非卵磷脂损伤- 相关的胆盐浓度达到无毒水平，而这 保护作用如可预见的那样下降，因为 胆囊泡增多。胆汁的保肝作用 脂类和胆汁盐-脂类的相互作用将在两个月内进行研究 胆盐致肝损伤的活体模型：急性胆汁输注 胆碱缺乏性胆瘘大鼠的盐分及慢性喂养 喂食致石饲料的仓鼠胆汁盐。这些项目的最终目标是 研究的目的是提供一个概念性的框架来理解 胆盐的毒性和保护性及胆盐的作用 对人类疾病的毒性。

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.