Abstract
Transport of many organic anions across the bile canalicular membrane is mediated by the canalicular multispecific organic anion transporter (cMOAT). Previously, we cloned cDNA that may encode cMOAT from Sprague-Dawley rat liver (Ito, K., Suzuki, H., Hirohashi, T., Kume, K., Shimizu, T., and Sugiyama, Y. (1997) Am. J. Physiol. 272, G16-G22). In the present study, the function of this cloned cDNA was investigated by examining the ATP-dependent uptake of S-(2,4-dinitrophenyl)-glutathione (DNP-SG) into membrane vesicles isolated from an NIH/3T3 cell line transfected with an expression vector containing the cloned cDNA. Although the membrane vesicles from the control NIH/3T3 cells exhibited endogenous activity in transporting DNP-SG and leukotriene C4 in an ATP-dependent manner, the transfection of cMOAT cDNA resulted in a significant increase in the transport activity for these ligands. The uptake of DNP-SG into membrane vesicles was osmotically sensitive and was stimulated to some extent by other nucleotide triphosphates (GTP, UTP, and CTP) but not by AMP or ADP. The K(m) and Vmax values for the uptake of DNP-SG by the membrane vesicles were 0.175 +/- 0.031 microM and 11.0 +/- 0.73 pmol/min/mg protein, respectively, for the transfected rat cMOAT and 0.141 +/- 0.036 microM and 3.51 +/- 0.39 pmol/min/mg protein, respectively, for the endogenous transporter expressed on control NIH/3T3 cells. These results suggest that the product of the previously cloned cDNA has cMOAT activity being able to transport organic anions in an ATP-dependent manner. Alternatively, it is possible that the cDNA product encodes an activator of endogenous transporter since the K(m) value for DNP-SG was comparable between the vector- and cMOAT-transfected cells. The transport activity found in the control NIH/3T3 cells may be ascribed to mouse cMOAT since Northern blot analysis indicated the presence of a transcript that hybridyzed to the carboxyl-terminal ATP-binding cassette sequence of the murine protein.
Highlights
It is well established that the biliary excretion of organic anions is mediated by the canalicular multispecific organic anion transporter1 [1,2,3,4]
The membrane vesicles from the control cells exhibited the ability to transport [3H]DNP-SG and [3H]LTC4 in an ATP-dependent manner, the stimulating effect of ATP was greater in the canalicular multispecific organic anion transporter (cMOAT)-transfected NIH/3T3 cells (Fig. 2, A and B)
We found that the expression of rat cMOAT in the transfected cells was reduced during storage in liquid N2; after thawing, expression of the transcript in the transfected cells fell below the limit of detection
Summary
It is well established that the biliary excretion of organic anions is mediated by the canalicular multispecific organic anion transporter (cMOAT)1 [1,2,3,4]. We established an NIH/3T3 cell line transfected with an expression vector containing cMOAT cDNA and examined the transport activity using membrane vesicles isolated from these transfected cells.
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