GSSG Efflux Research Articles

Glutathione efflux from perfused rat liver after phenobarbital treatment, during drug oxidations, and in selenium deficiency.

1 The efflux of reduced (GSH) and oxidized (GSSG) glutathione from perfused rat liver was studied in different metabolic conditions. 2 At 37 °C in non-recirculating hemoglobin-free perfusion, efflux of glutathione per gram of liver was 14 nmol/min, of which GSH was 12 nmol/min and GSSG 1 nmol/min. Similar rates were observed in livers from phenobarbital-pretreated rats. This is interpreted to indicate that the increased capacity for H2O2 production described in the literature for isolated microsomal membranes is not detectably realized in the intact cell, since H2O2 production from other intracellular sources, e.g. benzylamine, leads to an extra GSSG efflux. Similarly, the glutathione release from livers of selenium-deficient rats low in GSH peroxidase (8% of the selenium-supplemented controls, H2O2 as substrate) was not significantly different from the controls. This observation contrasts with that of Burk et al. [J. Biol. Chem. 253, 43–46] who found a 2-fold increase in glutathione release in livers from rats exposed to selenium-deficiency for a more extended time period. Thus, elevated rates of lipid peroxidation may occur in more severe selenium-deficiency where factors other than a low GSH peroxidase activity may contribute to the effect. 3 An increased rate of GSSG efflux was observed in livers from phenobarbital-pretreated rats during drug oxidations dependent on cytochrome P-450, using aminopyrine, ethylmorphine and hexobarbital as substrates. Half-maximal aminopyrine concentration for GSSG efflux, 0.1–0.2 mM, was similar to that of the extra O2 uptake but lower than that for aminopyrine N-demethylation, 0.5–0.6 mM. Aminopyrine-dependent GSSG efflux was present also in livers from selenium-deficient rats whereas H2O2-dependent or benzylamine-dependent GSSG efflux was abolished. These results do not support extra production of free H2O2 during drug oxidation in the intact cell. Future investigations must evaluate whether glutathione oxidation occurs by reactive drug metabolites or by an enhanced rate of lipid peroxidation, or by back-equilibration of glutathione reductase concomitant with the decreased NADPH levels.

Transport and metabolism of glutathione in the lens

Measurements were made of influx and efflux of reduced and oxidized glutathione (GSH and GSSG) in rabbit lens. Three times as much radioactivity was found in the lens when the anterior surface was exposed to [ 35S]-GSH compared with the posterior side. Nonlabeled GSH added to the medium decreased the penetration of labeled GSH, primarily across the anterior surface. The entry of [ 35S]-GSSG into the lens was approximately the same across both surfaces. In cultured lenses, the accumulation of [ 35S]-GSH decreased significantly in the absence of glucose and at reduced temperature, but was unaffected by ouabain. The entry process for GSH was also shown to be a saturable mechanism. Of the three constituent amino acids in the tripeptide only cysteine was found to compete for the transport system of GSH, suggesting that GSH and cysteine may share a common carrier. The rate of entry of 35S-GSSG in cultured lenses was unaffected by the presence of either nonlabeled cysteine or GSH. No significant efflux of GSH or GSSG occurs from intact lenses. The efflux is extremely rapid when both capsule and epithelium are removed from the lens. When capsule alone was removed with collagenase, leaving the epithelum intact, no detectable amount of GSH or GSSG was found to leave the lens. A significant amount of [ 35S]-GSH that entered the lens was degraded, giving rise to labeled cysteine. Lenses cultured in the presence of 14C-labeled cysteine rapidly incorporated the amino acid into GSH, suggesting that GSH is totally degraded and resynthesized in the lens. From the time course of decrease in the specific activity of glutathione introduced into the lens, the coefficient of turnover of GSH was calculated to be 0·014 hr −1, a value found to be in reasonable agreement with the turnover rate of GSH calculated previously from the rates of incorporation of constituent amino acids.