Transfected rat cMOAT is functionally expressed on the apical membrane in Madin-Darby canine kidney (MDCK) cells.
OBJECTIVE
The purpose of the present study is to investigate the expression of canalicular multispecific organic anion transporter (cMOAT) by its cDNA transfection in polarized Madin-Darby canine kidney cells (MDCK).


METHODS
MDCK cells were transfected with an expression vector (pCXN2) containing the rat cMOAT cDNA with lipofectamine to obtain the stable transfectant under G418. Cells from a single colony whose cMOAT expression was the highest were seeded to form a tight epithelial monolayer on microporous membrane filters. Export of glutathione S-bimane (GS-B) from monolayers was determined after preloading its precursor, monochloro bimane (MCB).


RESULTS
A comparable amount of GS-B was excreted to the apical and basal compartments in the vector-transfected cells. In contrast, in cMOAT-transfected cells, the amount apically excreted was approximately twice that excreted into the basal compartment. Cyclosporin A (CsA) (30 microM), an inhibitor of cMOAT at higher concentrations, inhibited the preferential apical export of GS-B from cMOAT-transfected cells.


CONCLUSIONS
Rat cMOAT is functionally expressed on the apical membrane of MDCK cells after transfection.
Kinoshita
S
S
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan.
Suzuki
H
H
Ito
K
K
Kume
K
K
Shimizu
T
T
Sugiyama
Y
Y
D000818:Animals; D027321:Anion Transport Proteins; D000838:Anions / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D015152:Blotting, Northern; D001643:Bridged Bicyclo Compounds / Q000493:pharmacokinetics; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002411:Cation Exchange Resins; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D016764:Cell Polarity / Q000187:drug effects; D016572:Cyclosporine / Q000494:pharmacology; D018076:DNA, Complementary; D004285:Dogs; D004847:Epithelial Cells / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D015870:Gene Expression; D005978:Glutathione / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D007202:Indicators and Reagents; D007668:Kidney / Q000166:cytology; D018374:LLC-PK1 Cells; D008055:Lipids; D008565:Membrane Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D013552:Swine; D014162:Transfection
D027321:Anion Transport Proteins; D000838:Anions; D001643:Bridged Bicyclo Compounds; D002352:Carrier Proteins; D002411:Cation Exchange Resins; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008055:Lipids; C086724:Lipofectamine; D008565:Membrane Proteins; D012333:RNA, Messenger; C080120:glutathione-bimane; D016572:Cyclosporine; D005978:Glutathione