Uptake of glutathione by renal cortical mitochondria

Abstract

Transport of GSH into renal cortical mitochondria was studied. Mitochondria were highly enriched with little contamination from other subcellular organelles (as assessed by marker enzymes), they exhibited coupled respiration (respiratory control ratio > 3.0), and they had initial GSH concentrations of 5.71 ± 0.65 nmol/mg protein ( n = 47). Incubation of mitochondria with GSH in a triethanolamine, pH 7.4, buffer containing sucrose, potassium phosphate, MgCl 2, and KCl, produced time- and concentration-dependent increases in intramitochondrial GSH content. Uptake was linear versus time for at least 2 min and exhibited kinetics consistent with one low-affinity, high-capacity process ( K m = 1.3 mM, V max = 5.59 nmol/min per mg protein), although the results cannot exclude the presence of other, less quantitatively significant pathways. The initial rate of uptake of 5 m m GSH was not significantly altered by uncouplers (0.1 m m 2,4-dinitrophenol and 25 μ m carbonyl cyanide m-chlorophenylhydrazone) or by 1 m m ADP. In contrast, incubation with 1 m m ATP, 1 m m KCN, 0.1 m m or 1 m m CaCl 2 inhibited uptake by 41, 39, 43, or 55%, respectively. GSH uptake was markedly inhibited by γ-glutamylglutamate and by a series of S-alkyl GSH derivatives. Strong interactions (i.e., both cis and trans effects) were observed with other dicarboxylates (i.e., succinate, malate, glutamate) but not with monocarboxylates (i.e., lactate, pyruvate). Preincubation of mitochondria with GSH protected against tert-butyl hydroperoxide- or methyl vinyl ketone-induced inhibition of state 3 respiration. These results demonstrate uptake of GSH into renal cortical mitochondria that appears to involve electroneutral countertransport (exchange) with other dicarboxylates. Functionally, GSH uptake into mitochondria can protect these organelles from various forms of injury, such as oxidative stress.