Molecular identification and functional characterization of Mdr1a in rat cholangiocytes.
OBJECTIVE
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.


METHODS
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.


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. 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.


CONCLUSIONS
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.
Gigliozzi
A
A
Department of Internal Medicine and Liver Center, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Fraioli
F
F
Sundaram
P
P
Lee
J
J
Mennone
A
A
Alvaro
D
D
Boyer
J L
JL
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000032:analysis / Q000235:genetics*; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000032:analysis / Q000235:genetics*; D000166:Acridines / Q000494:pharmacology; D000818:Animals; D001652:Bile Ducts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D002455:Cell Division; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D003600:Cytosol / Q000502:physiology; D018432:Drug Resistance, Multiple; D066298:In Vitro Techniques; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D018613:Microscopy, Confocal; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020112:Rhodamine 123; D044005:Tetrahydroisoquinolines; D014158:Transcription, Genetic / Q000187:drug effects; D014638:Vanadates / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D000166:Acridines; D007546:Isoquinolines; D012333:RNA, Messenger; D044005:Tetrahydroisoquinolines; D020112:Rhodamine 123; D014638:Vanadates; C095122:multidrug resistance protein 3; D014700:Verapamil; C083501:Elacridar