Mazzone Amelia, Tietz Pamela, Jefferson John, Pagano Richard, LaRusso Nicholas F
Center for Basic Research in Digestive Diseases, Mayo Medical School, Clinic and Foundation, Rochester, MN 55905, USA.
Hepatology. 2006 Feb;43(2):287-96. doi: 10.1002/hep.21039.
Canalicular bile is formed by the osmotic filtration of water in response to osmotic gradients generated by active transport at the apical and basolateral plasma membrane domains of hepatocytes. We recently demonstrated that mixed plasma membrane fractions isolated from rat hepatocyte couplets contain lipid microdomains ("rafts") enriched in cholesterol and sphingolipids and AQP8 and 9. We isolated lipid microdomains from hepatocyte apical and basolateral plasma membrane domains using Triton X-100 as detergent, and characterized their lipid and protein composition. A Triton-insoluble band ("raft fraction") at the 5%/30% sucrose interface in both apical and basolateral fractions was enriched for alkaline phosphatase (apical) and Na/K ATPase (basolateral) and was negative for amino peptidase-N. This detergent-insoluble band was also positive for caveolin-1 (a "raft" associated protein) and negative for clathrin (a "raft" negative protein). Lipid analysis showed that, the Triton-insoluble fraction was highly enriched in cholesterol and sphingolipids. Immunofluorescence staining on hepatocyte couplets for both caveolin-1 and cholera toxin B showed a punctate distribution on both the apical and basolateral plasma membranes, consistent with localized membrane microdomains. Dot blot analysis showed that the "raft" associated ganglioside GM1 was enriched in the detergent-insoluble fraction both domains. Furthermore, exposure of isolated hepatocytes to glucagon, a choleretic agonist, significantly increased the expression of AQP8 associated with the apical microdomain fractions but had no effect on AQP9 expression in the basolateral microdomain fractions. In conclusion, "rafts" represent target microdomains for exocytic insertion and retrieval of "flux proteins", including AQPs, involved in canalicular bile secretion.
胆小管胆汁是肝细胞顶端和基底外侧质膜结构域通过主动转运产生渗透梯度后，水经渗透过滤形成的。我们最近证明，从大鼠肝细胞双联体分离的混合质膜组分含有富含胆固醇、鞘脂以及水通道蛋白8和9的脂质微区（“脂筏”）。我们用Triton X-100作为去污剂从肝细胞顶端和基底外侧质膜结构域分离脂质微区，并对其脂质和蛋白质组成进行了表征。顶端和基底外侧组分在5%/30%蔗糖界面处的Triton不溶性条带（“脂筏组分”）富含碱性磷酸酶（顶端）和钠钾ATP酶（基底外侧），而氨肽酶-N呈阴性。这个去污剂不溶性条带对小窝蛋白-1（一种“脂筏”相关蛋白）也呈阳性，对网格蛋白（一种“脂筏”阴性蛋白）呈阴性。脂质分析表明，Triton不溶性组分高度富含胆固醇和鞘脂。对肝细胞双联体进行小窝蛋白-1和霍乱毒素B的免疫荧光染色显示，在顶端和基底外侧质膜上均呈点状分布，这与局部膜微区一致。斑点印迹分析表明，“脂筏”相关神经节苷脂GM1在两个结构域的去污剂不溶性组分中均有富集。此外，将分离的肝细胞暴露于促胆汁分泌激动剂胰高血糖素中，显著增加了与顶端微区组分相关的水通道蛋白8的表达，但对基底外侧微区组分中的水通道蛋白9表达没有影响。总之，“脂筏”代表了参与胆小管胆汁分泌的“通量蛋白”（包括水通道蛋白）胞吐插入和回收的靶微区。
