Stieger B, Zhang J, O'Neill B, Sjövall J, Meier P J
University Hospital, Department of Medicine, Zürich, Switzerland.
Eur J Biochem. 1997 Feb 15;244(1):39-44. doi: 10.1111/j.1432-1033.1997.00039.x.
People with genetic or acquired defects in the biosynthesis of bile acids may suffer from cholestasis. Patients with a deficiency of 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase from 3 beta, 7 alpha-dihydroxy- and 3 beta, 7 alpha, 12 alpha-trihydroxy-5-cholenoic acids, the sulfated and partially glycine-conjugated forms of which are found in their urine and bile. 3-Oxo-delta 4 bile acids are detected in the urine of patients with a deficiency of 5 beta-reductase. It has been postulated that these unusual bile acids might act as cholestatic agents in these patients. The aim of the present study was to test this hypothesis in an in vitro system, since the abnormal bile acids would be metabolized in in vivo experiments. Basolateral (sinusoidal) and canalicular plasma membrane vesicles were isolated from rat liver. A rapid filtration method was used to determine transport of cholyltaurine in the presence of model bile acids into the isolated vesicles. It was found that 3 beta, 7 alpha-dihydroxy-5-cholenoic acid and 7 alpha-hydroxy-3-oxo-4-cholenoic acid both inhibited the apical, ATP-dependent transport system for cholyltaurine in a competitive manner with K(m) values of 15 microM and 16 microM, respectively. Radioactively labeled 3 beta, 7 alpha-dihydroxy-5-cholenoyltaurine and 7 alpha-hydroxy-3-oxo-4-cholenoyltaurine were not transported by the same transport system. The same types of experiments were performed with basolateral plasma membrane vesicles. It was found that, in contrast to the canalicular ATP-dependent bile acid transport system, only 7 alpha-hydroxy-3-oxo-4-cholenoyltaurine was a competitive inhibitor of the sodium-dependent transport system for cholyltaurine with a K(m) of 16 microM. Studies with radioactively labeled 7 alpha-hydroxy-3-oxo-4-cholenoyltaurine and 3 beta, 7 alpha-dihydroxy-5-cholenoyltaurine revealed that 7 alpha-hydroxy-3-oxo-4-cholenoyltaurine was transported in a sodium-dependent manner into basolateral rat liver plasma membrane vesicles, whereas 3 beta, 7 alpha-dihydroxy-5-cholenoyltaurine was not transported in a sodium-dependent way. These results support the hypothesis that the unusual bile acids found in patients with defects in bile acid biosynthesis might act as cholestatic agents by inhibiting the canalicular ATP-dependent transport system for bile acids which constitutes the rate-limiting step in the overall process of bile acid transport across hepatocytes. Furthermore, the experiments demonstrated that, despite similar substrate specificities, the basolateral sodium-dependent and the apical ATP-dependent transport system for cholyltaurine might have different recognition sites for bile acids.
胆汁酸生物合成存在遗传或后天缺陷的人可能会患胆汁淤积症。患有3β-羟基-δ5-C27-类固醇脱氢酶/异构酶缺乏症的患者，其尿液和胆汁中存在3β,7α-二羟基-和3β,7α,12α-三羟基-5-胆烯酸的硫酸化和部分甘氨酸共轭形式。在5β-还原酶缺乏症患者的尿液中检测到3-氧代-δ4胆汁酸。据推测，这些异常胆汁酸可能在这些患者中充当胆汁淤积剂。本研究的目的是在体外系统中验证这一假设，因为异常胆汁酸会在体内实验中被代谢。从大鼠肝脏中分离出基底外侧（窦状）和胆小管质膜囊泡。采用快速过滤法测定在模型胆汁酸存在下胆酰牛磺酸向分离囊泡中的转运。结果发现，3β,7α-二羟基-5-胆烯酸和7α-羟基-3-氧代-4-胆烯酸均以竞争性方式抑制胆酰牛磺酸的顶端ATP依赖性转运系统，其K(m)值分别为15 microM和16 microM。放射性标记的3β,7α-二羟基-5-胆烯酰牛磺酸和7α-羟基-3-氧代-4-胆烯酰牛磺酸不是通过同一转运系统转运的。对基底外侧质膜囊泡进行了相同类型的实验。结果发现，与胆小管ATP依赖性胆汁酸转运系统不同，只有7α-羟基-3-氧代-4-胆烯酰牛磺酸是胆酰牛磺酸钠依赖性转运系统的竞争性抑制剂，K(m)为16 microM。对放射性标记的7α-羟基-3-氧代-4-胆烯酰牛磺酸和3β,7α-二羟基-5-胆烯酰牛磺酸的研究表明，7α-羟基-3-氧代-4-胆烯酰牛磺酸以钠依赖性方式转运至基底外侧大鼠肝脏质膜囊泡，而3β,7α-二羟基-5-胆烯酰牛磺酸不以钠依赖性方式转运。这些结果支持了以下假设：胆汁酸生物合成缺陷患者中发现的异常胆汁酸可能通过抑制胆汁酸的胆小管ATP依赖性转运系统而充当胆汁淤积剂，该转运系统是胆汁酸跨肝细胞转运整个过程中的限速步骤。此外，实验表明，尽管底物特异性相似，但胆酰牛磺酸的基底外侧钠依赖性和顶端ATP依赖性转运系统对胆汁酸可能具有不同的识别位点。
