Glutathione transport across hepatocyte plasma membranes. Analysis using isolated rat-liver sinusoidal-membrane vesicles

Abstract

Transport of reduced glutathione (GSH) and its derivatives was studied in rat-liver sinusoidal plasma-membrane vesicles by a rapid filtration technique. The membrane vesicles exhibited transport of GSH into an osmotically active intravesicular space. Equilibrium uptake of vesicle-associated GSH was similar to that of free ligands which can be trapped by the intravesicular space of sinusoidal membrane samples. Kinetic analysis of the transport process revealed that the sinusoidal membrane vesicles have high-affinity and low-affinity GSH transport systems; the former has an apparent Km of 0.34 mM for GSH and V of 1.4 nmol X mg protein-1 X 20 s-1, and the latter has an apparent Km of 3.3 mM and V of 3.9 nmol X mg protein-1 X 20 s-1. Both Km values are lower than intrahepatic GSH levels, indicating that GSH transport across the sinusoidal membrane occurs via carrier-mediated mechanism and argues simple diffusion mechanism. The presence of oxidized glutathione (GSSG) or S-benzylglutathione inhibited GSH transport by the vesicles; the low-affinity transport system was inhibited more markedly than the high-affinity transport system. This suggests that these glutathione derivatives interacted preferentially with the low-affinity transport system for GSH. S-Dinitrophenylglutathione was also transported by the vesicles by a process which was inhibited by GSH and GSSG. The vesicles also transported GSSG, and this transport was markedly inhibited by S-benzylglutathione or GSH. The transport systems in sinusoidal plasma membranes may function in vivo in translocating GSH and its derivatives from hepatocytes into plasma and play an important role in inter-organ metabolism of these compounds.