Gigliozzi A, Fraioli F, Sundaram P, Lee J, Mennone A, Alvaro D, Boyer J L
Department of Internal Medicine and Liver Center, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Gastroenterology. 2000 Oct;119(4):1113-22. doi: 10.1053/gast.2000.18156.
BACKGROUND & AIMS: The multidrug resistance P-glycoprotein 170 gene products (mdr1a and 1b) are glycosylated plasma membrane proteins that function as adenosine triphosphate-dependent transmembrane export pumps for lipophilic xenobiotics of widely different structure. We assessed whether these P-glycoproteins are functionally expressed in cholangiocytes.
A reverse-transcription polymerase chain reaction was performed on RNA from a normal rat cholangiocyte cell line using mdr1-specific primers. Northern and Western blot analyses were performed on cholangiocytes immunoisolated from 2-week bile duct-ligated rats and cholangiocytes and isolated cholangiocyte membrane subfractions, respectively. Functional assays were performed in isolated bile duct units from bile duct-ligated rats and incubated with rhodamine 123, a P-glycoprotein substrate, with or without the P-glycoprotein inhibitors verapamil or GF120918.
A 400-base pair fragment with 99% homology to the cytosolic domain of rat intestinal mdr1a (5' 1953-2350 3') was identified that hybridized to a 5.2-kilobase RNA transcript in a normal rat cholangiocyte cell line, isolated rat cholangiocytes, and ileum. Western analysis localized mdr1 to the apical membrane of cholangiocytes. Confocal microscopy showed active secretion of rhodamine 123 into the lumen of isolated bile duct units that was abolished by vanadate and P-glycoprotein competitive antagonists, verapamil and GF120918, in a dose-dependent manner.
These findings provide the first molecular and functional evidence for the expression of mdr1a on the luminal membrane of cholangiocytes, where it may have a protective role.
多药耐药P-糖蛋白170基因产物（mdr1a和1b）是糖基化的质膜蛋白，作为依赖三磷酸腺苷的跨膜输出泵，作用于结构差异很大的亲脂性外源性物质。我们评估了这些P-糖蛋白在胆管细胞中是否有功能表达。
使用mdr1特异性引物对来自正常大鼠胆管细胞系的RNA进行逆转录聚合酶链反应。分别对从2周胆管结扎大鼠免疫分离的胆管细胞以及胆管细胞和分离的胆管细胞膜亚组分进行Northern和Western印迹分析。在胆管结扎大鼠的分离胆管单位中进行功能测定，并与罗丹明123（一种P-糖蛋白底物）一起孵育，同时加入或不加入P-糖蛋白抑制剂维拉帕米或GF120918。
鉴定出一个与大鼠肠道mdr1a胞质结构域（5' 1953 - 2350 3'）有99%同源性的400碱基对片段，该片段与正常大鼠胆管细胞系、分离的大鼠胆管细胞和回肠中的5.2千碱基RNA转录本杂交。Western分析将mdr1定位到胆管细胞的顶端膜。共聚焦显微镜显示罗丹明123以剂量依赖方式被钒酸盐以及P-糖蛋白竞争性拮抗剂维拉帕米和GF120918抑制后，向分离胆管单位管腔的活性分泌被消除。
这些发现为mdr1a在胆管细胞腔膜上的表达提供了首个分子和功能证据，其在该处可能具有保护作用。
