Molecular identification and functional characterization of Mdr1a in rat cholangiocytes

Background & Aims: The multidrug resistance P-glycoprotein 170 gene product s (mdr1a and 1b) are glycosylated plasma membrane proteins that function as adenosine triphosphate- dependent transmembrane export pumps for lipophili c xenobiotics of widely different structure. We assessed whether these P-gl ycoproteins ave functionally expressed in cholangiocytes. Methods: A revers e-transcription polymerase chain reaction was performed on RNA from a norma l rat cholangiocyte cell line using mdr1-specific primers. Northern and Wes tern blot analyses were performed on cholangiocytes immunoisolated from 2-w eek bile duct-ligated rats and cholangiocytes and isolated cholangiocyte me mbrane subfractions, respectively. Functional assays were performed in isol ated bile duct units from bile duct-ligated rats and incubated with rhodami ne 123, a P-glycoprotein substrate, with or without the P-glycoprotein inhi bitors verapamil or GF120918, Results: 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. Weste rn analysis localized mdr1 to the apical membrane of cholangiocytes. Confoc al microscopy showed active secretion of rhodamine 123 into the lumen of is olated bile duct units that was abolished by vanadate and P-glycoprotein co mpetitive antagonists, verapamil and GF120918, in a dose-dependent manner. Conclusions: These findings provide the first molecular and functional evid ence for the expression of mdr1a on the luminal membrane of cholangiocytes, where it may have a protective role.