Albalak A, Zeidel M L, Zucker S D, Jackson A A, Donovan J M
Department of Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Biochemistry. 1996 Jun 18;35(24):7936-45. doi: 10.1021/bi960497i.
Bile salts have been hypothesized to mediate cytotoxicity by increasing membrane permeability to aqueous solutes. We examined whether submicellar bile salt concentrations affect model and native membrane permeability to small uncharged molecules such as water, urea, and ammonia. Osmotic water permeability (Pf) and urea permeability were measured in large unilamellar vesicles composed with egg yolk phosphatidylcholine (EYPC) +/- cholesterol (Ch) or rat liver microsomal membranes by monitoring self-quenching of entrapped carboxyfluorescein (CF). Ammonia permeability was determined utilizing the pH dependence of CF fluorescence. Submicellar bile salt concentrations did not significantly alter Pf of EYPC +/- Ch or rat liver microsomal membranes. At taurodeoxycholate (TDC) or tauroursodeoxycholate concentrations approaching those that solubilized membrane lipids, CF leakage occurred from vesicles, but Pf remained unchanged. Higher bile salt concentrations (0.5-2 mM TDC) did not alter Pf of equimolar EYPC/Ch membranes. The activation energy for transmembrane water flux was unchanged (12.1 +/- 1.2 kcal/mol for EYPC) despite the presence of bile salts in one or both membrane hemileaflets, suggesting strongly that bile salts do not form transmembrane pores that facilitate water flux. Furthermore, submicellar bile salt concentrations did not increase membrane permeability to urea or ammonia. We conclude that at submicellar concentrations, bile salts do not form nonselective convective channels that facilitate transmembrane transport of small uncharged molecules. These results suggest that bile salt-mediated transport of specific substrates, rather than nonselective enhancement of membrane permeability, underlies bile salt cytotoxicity for enterocytes and hepatocytes.
胆汁盐被认为可通过增加膜对水溶性溶质的通透性来介导细胞毒性。我们研究了亚胶束浓度的胆汁盐是否会影响模型膜和天然膜对水、尿素和氨等不带电荷小分子的通透性。通过监测包封的羧基荧光素（CF）的自猝灭，在由蛋黄磷脂酰胆碱（EYPC）±胆固醇（Ch）组成的大单层囊泡或大鼠肝微粒体膜中测量渗透水通透性（Pf）和尿素通透性。利用CF荧光的pH依赖性测定氨通透性。亚胶束浓度的胆汁盐不会显著改变EYPC±Ch或大鼠肝微粒体膜的Pf。在接近溶解膜脂质浓度的牛磺脱氧胆酸盐（TDC）或牛磺熊去氧胆酸盐浓度下，囊泡出现CF泄漏，但Pf保持不变。更高的胆汁盐浓度（0.5 - 2 mM TDC）不会改变等摩尔EYPC/Ch膜的Pf。尽管在一个或两个膜半叶中存在胆汁盐，但跨膜水通量的活化能未改变（EYPC为12.1±1.2 kcal/mol），这强烈表明胆汁盐不会形成促进水通量的跨膜孔。此外，亚胶束浓度的胆汁盐不会增加膜对尿素或氨的通透性。我们得出结论，在亚胶束浓度下，胆汁盐不会形成促进不带电荷小分子跨膜转运的非选择性对流通道。这些结果表明，胆汁盐对特定底物的转运，而非对膜通透性的非选择性增强，是胆汁盐对肠细胞和肝细胞产生细胞毒性的基础。
