Transfected rat cMOAT is functionally expressed on the apical membrane in Madin-Darby canine kidney (MDCK) cells

Abstract

Purpose: 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.