Amino acid transport and glutathione homeostasis: What is the mechanism for cysteine uptake from bile?

Abstract

Transport of L-cysteine and a cysteine S-conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC) was investigated in rat liver canalicular plasma membrane (cLPM) vesicles. Cysteine uptake into an osmotically active intravesicular space was temperature sensitive and further enhanced by an inwardly directed Na+ gradient. Na+ -dependent and -independent L-cysteine uptake exhibited saturation kinetics with apparent Km of 53 ± 0.7 and 1300 ± 300 μM and Vmax of 95 ± 21 and 1600 ± 200 pmol ± mg protein−1; 10 sec−1 for the Na+ dependent components, and an apparent Km of 207 ± 48 μM and a Vmax of 355 ± 71 pmol ± mg protein−1 ± 10 sec−1 for the Na+ -independent component. Na+-dependent uptake was inhibited by L-alanine, glycine, L-phenylalanine and L-leucine, whereas Na+-independent uptake was inhibited by L-phenylalanine, L-leucine and 2-amino-2-norbornanecarboxylic acid. Both Na+ -dependent and -independent L-cysteine transport processes were inhibited by several cysteine S-conjugates, with DCVC having the strongest effect. Inhibition of [35S]-cysteine uptake by DCVC was noncompetitive with a K, of 1.2 ± 0.1 mM. On the other hand, uptake of [35 S IDCVC by the rat cLPM vesicles was not stimulated by a Na+-gradient, but was inhibited by several other amino acids, including L-cysteine. Further investigation of [35SlDCVC uptake in rat cLPM vesicles indicated a saturable Na+ -independent process with an apparent K, of 155 f 42 μM, and a Vmax of 393 f 53 pmol mg protein −1 5 sec −1. Time-dependent nonspecific binding of DCVC to rat cLPM vesicles was diminished by addition of aminooxyacetic acid, an inhibitor of cysteine conjugate β-lyase activity. These findings demonstrate that cysteine and DCVC uptake into cLPM vesicles occurs by both saturable and nonsaturable transport mechanisms. These transport pathways may function in vivo for uptake and recycling of biliary L-cysteine for glutathione turnover and some cysteine S-conjugates for intrahepatic mercapturic acid biosynthesis.