Abstract
We have characterized the transport of GSH and the mechanism for impaired GSH transport in mutant Eisai hyperbilirubinemic rats (EHBR) using isolated canalicular membrane-enriched vesicles (cLPM). In control animals, the transport of GSH is an electrogenic process and is trans-stimulated by preloading the vesicles with GSH and is not enhanced in the presence of ATP. GSH transport in cLPM is saturable with a single component having a Km of approximately 16 mM and a Vmax of 6.7 nmol/mg/15 s. EHBR is a Sprague-Dawley rat with hyperbilirubinemia due to impaired bile secretion of organic anions by the ATP-dependent organic anion/GSH-conjugate transporter. In cLPM from EHBR we confirmed the defective stimulation by ATP of the transport of LTC4 and GSSG. In the mutant cLPM, the characteristics and kinetics of GSH transport were the same as in the controls. 2,4-(dinitrophenyl)-glutathione (DNP-GSH), which is a substrate for the ATP-dependent canalicular organic anion carrier, in the absence of ATP, cis-inhibited the transport of GSH into cLPM vesicles; however, when the vesicles were preloaded with DNP-GSH, there was a dose-dependent trans-stimulation of GSH transport. In contrast, in the presence of ATP, DNP-GSH enhanced GSH transport in cLPM vesicles; at 0.25 mM DNP-GSH, a concentration which did not cis-inhibit GSH, addition of ATP resulted in accelerated GSH transport; at 1.0 mM DNP-GSH, cis-inhibition was completely reversed by the addition of ATP despite a negligible fall in the medium DNP-GSH. Interestingly, sulfobromophthalein-glutathione (BSP-GSH) neither cis-inhibited nor trans-stimulated GSH transport in cLPM. This contrasts with bLPM where BSP-GSH interacts with the GSH carrier. Therefore, GSH is transported into bile by a multispecific low affinity electrogenic carrier which is distinct from the multispecific high affinity ATP-driven organic anion transporter. Although both carriers have overlapping specificities, BSP-GSH and GSH are uniquely specific for only one of the carriers. The near absence of GSH in the bile of mutant rats can be best explained as a secondary defect due to cis-inhibition from retained substrates for the defective carrier and/or loss of trans-stimulation by these same substrates which normally are concentratively transported into the bile. Other possibilities such as change in GSH carrier activity upon isolation or loss of a negative protein regulator during membrane isolation, although theoretical alternatives are less easily reconciled with the defect in the ATP-driven organic anion transporter.
Highlights
From the $.Division of Gastrointestinal and Liver Diseases, Schoolof Medicine, University of Southern California, Los Angeles, California90033, the TDepartment of Medicine, Teikyo University Schoolof Medicine, Tokyo 173, Japan, and the
A multispecific electrogeniclow affintion which did not cis-inhibit GSH, addition of ATP itytransportsystemshared by GSHand organic anions resulted in accelerateGdSH transport; at1.0 mM DNP- appears to govern the release of GSH and organic anions, GSH, cis-inhibition was completely reversed by the such as GSH conjugates, into sinusoidalplasma
A T P dependence, and Kinetics of GSH in cLPM from Control and EHBR Rats- -To confirm that EHBR rats were defectivein thecanalicular ATP-dependent transportof organic anions, we examined LTCl and GSSG transport
Summary
From the $.Division of Gastrointestinal and Liver Diseases, Schoolof Medicine, University of Southern California, Los Angeles, California90033, the TDepartment of Medicine, Teikyo University Schoolof Medicine, Tokyo 173, Japan, and the. Kinetic Analyses of GSH Transport-The concentration dependlular organelles such as mitochondria, microsomes, and lysoence of GSH transportwas determined by initial rates (15s) of GSH uptake by cLPM with different concentrations of radiolabeled GSH (25 p M to 50 mM) in a medium containing 0.25 M sucrose, 0.2 mM CaCI,, 10 mM MgCL, 10 mM Hepes-Tris, pH 7.4, and 10 mM dithiothreitol in a final volume of 25 pl. The crude LPM fraction obtained after differential and flo- carrier-mediated transport of GSH rather than binding to tation centrifugation [30] consisted of a mixture of bLPM cLPM vesicles. No significant differences were found between normal controls and EHBR
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