Direct evidence for the role of the membrane potential in glutathione transport by renal brush-border membranes

Abstract

Transport of GSH was studied in isolated rat kidney cortical brush-border membrane vesicles in which gamma-glutamyltransferase had been inactivated by a specific affinity labeling reagent, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125). Transport of intact 2-3H-glycine-labeled GSH occurred into an osmotically active intravesicular space of AT-125-treated membranes. The initial rate of transport followed saturation kinetics with respect to GSH concentrations; an apparent Km of 0.21 mM and Vmax of 0.23 nmol/mg protein X 20 were calculated at 25 degrees C with a 0.1 M NaCl gradient (vesicle inside less than vesicle outside). Sodium chloride in the transport medium could be replaced with KCl without affecting transport activity. The rate of GSH uptake was enhanced by replacing KCl in the transport medium with K2SO4, providing a less permeant anion, and was reduced by replacing KCl with KSCN, providing a more permeant anion. The rate of GSH transport markedly decreased in the absence of a K+ gradient across the vesicular membranes and was enhanced by a valinomycin-induced K+ diffusion potential (vesicle-inside-positive). These results indicate that GSH transport is dependent on membrane potential and involves the transfer of negative charge. The rate of GSH transport was inhibited by S-benzyl glutathione but not by glycine, glutamic acid, and gamma-glutamyl-p-nitroanilide. When incubated with [2-3H]glycine-labeled GSH, intact untreated vesicles also accumulated radioactivity; the rate of uptake was significantly higher in a Na+ gradient than in a K+ gradient. Sodium-dependent transport, but not sodium-independent uptake, was almost completely inhibited by a high concentration of unlabeled glycine. At equilibrium, most of the radioactivity which accumulated in the intravesicular space was accounted for by free glycine. These results suggest that GSH which is secreted into the tubular lumen by a specific translocase in the lumenal membranes or filtered by the glomerulus may be degraded in situ by membranous gamma-glutamyltransferase and peptidase activities which hydrolyze peptide bonds of cysteinylglycine and its derivatives. The resulting free amino acids can be reabsorbed into tubule cells by sodium-dependent transport systems in renal cortical brush-border membranes.