KINETIC-ANALYSIS OF THE PRIMARY ACTIVE-TRANSPORT OF CONJUGATED METABOLITES ACROSS THE BILE CANALICULAR MEMBRANE - COMPARATIVE-STUDY OF S-(2,4-DINITROPHENYL)-GLUTATHIONE AND HYL-2-METHYLAMINO-4-(3-PYRIDYLMETHYL)BENZOTHIAZOLE GLUCURONIDE

Eisai hyperbilirubinemic rat (EHBR) is a mutant strain with a heredita ry defect in canalicular multispecific organic anion transporter (cMOA T). We examined the uptake and mutual inhibition of S-(2,4-dinitrophen yl)-glutathione (DNP-SG), which is a typical substrate for cMOAT, and oxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E304 0) glucuronide (E-glu) with canalicular membrane vesicles (CMV) prepar ed from Sprague-Dawley (SD) and EHBR rats to investigate the multiplic ity of the organic anion transporter. The ATP-dependent uptake by cMV from SD rats had an apparent K-m of 17.6 mu M for DNP-SG and 5.7 mu M for E-glu, whereas the corresponding uptake by CMV from EHBR had an ap parent K-m of 44.6 mu M for E-glu. The effects of E-glu, 4-methylumbel liferone glucuronide (4 MUG), E3040 sulfate (E-sul) and 4-methylumbell iferone sulfate (4 MUS) on the uptake of [H-3]DNP-SG were also examine d. The uptake of [H-3]DNP-SG was inhibited by glucuronides (E-glu and 4 MUG) in a concentration-department manner, although it was enhanced by the sulfate conjugates (E-sul and 4 MUS). This enhancement was show n to be caused by an increased DNP-SG affinity for the transporter. In CMV from SD rats, although ATP-dependent uptake of [(3)]DNP-SG was al most completely inhibited by E-glu, that of [C-14]E-glu was only reduc ed to about 30% of controls by DNP-SG, On the other hand, in CMV from EHBR, the ATP-dependent uptake of [C-14]E-glu was not inhibited at all by DNP-SG. Kinetic analysis indicated that E-glu inhibited DNP-SG upt ake competitively. In conclusion: 1) cMOAT recognizes both DNP-SG and E-glu, and another transporter present in SD rats is also involved in E-glu transport along with cMOAT; 2) the latter transporter is kinetic ally similar to the E-glu transporter present in EHBR; 3) E-sul enhanc es the uptake of DNP-SG by increasing the affinity of glucuronide for the transporter.