Translocation of intracellular glutathione to membrane-bound γ-glutamyl transpeptidase as a discrete step in the γ-glutamyl cycle: Glutathionuria after inhibition of transpeptidase

Abstract

Several inhibitors of 'y-glutamyl transpeptidase in vitro [L-serine plus borate, 6-diazo-5-oxo-L-norleucine, and L-and Dy-glutamyl-(o-carboxy)phenylhydrazidel are active in vivo, as indicated by their effect in decreasing the conversion of administered D"-glutamyl-L-a-amino[14Cbutyrate to respiratory 14CO2 in mice. The hydrazides (both L and D isomers) are the most ptent inhibitors in vitro and in vivo. Inhibition of y-glutamyl transpeptidase in vivo by the hydrazides is accompanied by extensive glutathionuria. The evidence suggests that a substantial fraction of the urinary glutathione arises from the kidney. The findings support the view that renal intracellular glutathione is normally tra-slocated to the membrane-bound 'y-glutamyl transpeptidase as a separate step in the -utamyl cycle. Studies on in vivo inhibition of glutathione synthesis and of 'y-glutamyl transpeptidase provide direct evidence that glutathione is normally translocated from tissues to the blood plasma and that the turnover of plasma glutathione is relativelyMigh. The data suggest that the low but significant steady-state level of glutathione in the plasma reflects synthesis of glutathione (predominantly in the liver) and its utilization by -y-glutamyl transpeptidase (predominantly in the kidney). Thus, glutathione synthesized in cells that have transpeptidase may be translocated to and used by the membrane-bound enzyme, whereas glutathione synthesized in cells that lack the transtidase may be transported via the plasma to transpeptidase ocated on the membranes of other cells. Previous studies in this laboratory provided evidence that the 'y-glutamyl cycle functions in the kidney by using intracellular glutathione and that the steady-state level of glutathione in the kidney reflects a balance between glutathione synthesis and glutathione utilization (1). Thus, administration of prothionine sulfoximine, an inhibitor of y-glutamyl-cysteine synthetase (and therefore of glutathione synthesis), was followed by a rapid decline in the glutathione level (2). Administration of large doses of amino acids also decreased kidney levels of glutathione, an effect ascribed to increased utilization of glutathione by 'yglutamyl transpeptidase. Inhibition of 'y-glutamyl transpeptidase by administration of L-serine plus borate decreased the rate of glutathione disappearance that occurs after inhibition of glutathione synthesis and also decreased the extent of glutathione disappearance that follows administration of amino acids. [A similar result was observed after inhibition of -y-glutamyl transpeptidase in vivo by administration of 6-diazo-5-oxo-L-norleucine (data cited in ref. 1).]Glutathione occurs predominantly intracellularly, whereas most of the y-glutamyl transpeptidase activity present in tissues is bound to cell membranes, and there is evidence that a substantial fraction of the enzyme is bound to the outer surface of the membrane (3-8). Previous studies indicated that intracellular glutathione must be accessible to the transpeptidase to acc...

Oxidative stress and regulation of glutathione in lung inflammation

“…Thus c-GT acts as a salvage enzyme for cellular GSH synthesis. The lung epithelium has been shown to contain high levels of c-GT activity and utilizes extracellular GSH from the alveolar lining fluid [114,115]. Hence most plasma GSH is catabolized by the enzyme c-GT in lungs [114,115].…”

“…As a result, c-GT may be important in determining the levels of GSH in lung ELF. Endothelial cells, alveolar macrophages and fibroblasts have lower c-GT levels, and, therefore, less easily use extracellular GSH for intracellular GSH synthesis [113,115,116].…”

Metabolism of trichloroethylene.

“…Efflux of GSH and GSH S-conjugates into the lumen by transport across the brushborder membrane is viewed as an essential step in the turnover of these compounds because the active site of GGT is extracellular (135). In fact, inhibition of GGT in vivo produces marked glutathionuria in mice (135). Presumably, a marked increase in urinary excretion of GSH conjugates would also occur under similar conditions.…”

“…For DCVG that is generated by renal GSTs, further processing occurs by efflux of intracellular DCVG by transport across the brush-border membrane into the lumen, where it can then be metabolized by GGT and dipeptidases to generate DCVC. Efflux of GSH and GSH S-conjugates into the lumen by transport across the brushborder membrane is viewed as an essential step in the turnover of these compounds because the active site of GGT is extracellular (135). In fact, inhibition of GGT in vivo produces marked glutathionuria in mice (135).…”

Renal Membrane Transport of Glutathione in Toxicology and Disease

“…26,34,83 Rather than uptake occurring, transport of GSH across the renal BBM is primarily if not entirely efflux from the cell into the tubular lumen. Pathways for GSH transport across the renal BBM are illustrated in Fig.…”