Effect of anisotonic cell‐volume modulation on glutathione‐S‐conjugate release, t‐butylhydroperoxide metabolism and the pentose‐phosphate shunt in perfused rat liver

Abstract

1. Addition of 1‐chloro‐2,4‐dinitrobenzene to isolated perfused rat liver results in the rapid formation of its glutathione‐S‐conjugate [S‐(2,4‐dinitrophenyl)glutathione], which is released into both, bile and effluent perfusate. Ahisotonic perfusion did not affect total S‐conjugate formation, but release of the S‐conjugate into the perfusate was increased (decreased) following hypertonic (hypotonic) exposure at the expense of excretion into bile. Stimulation of S‐conjugate release into the perfusate following hypertonic exposure paralleled the time course of volume‐regulatory net K⁺ uptake. 2. Basal steady‐state release of oxidized glutathione (GSSG) into bile was 1.30 ± 0.12 nmol · g⁻¹· min⁻¹ (n= 18) during normotonic (305 mOsmol/I) perfusion and was 3.8±0.3 nmol · g⁻¹· min⁻¹ in the presence of t‐butylhydroperoxide (50 μmol/I). Hypotonic exposure (225 mOsmol/1) lowered both, basal and t‐butylhydroperoxide(50 μmol/I)‐stimulated GSSG release into bile by 35% and 20%, respectively, whereas hypertonic exposure (385 mOsmol/I) increased. Anisotonic exposure was without effect on t‐butylhydroperoxide removal by the liver. GSSG release into bile also decreased by 33% upon liver‐cell swelling due to addition of glutamine plus glycine (2 mmol/I, each). 3. Hypotonic exposure led to a persistent stimulation ¹⁴CO₂ production from [1‐¹⁴C]glucose by about 80%, whereas ¹⁴CO₂ production from [6‐¹⁴C]glucose increased by only 10%. Conversely, hypertonic exposure inhibited ¹⁴CO₂ production from [1‐¹⁴C]glucose by about 40%, whereas ¹⁴CO₂ production from [6‐¹⁴C]glucose was unaffected. The effect of anisotonicity on ¹⁴CO₂ production from [1‐¹⁴C]glucose was also observed in presence of t‐butylhydroperoxide (50 μmol/1), which increased ¹⁴CO₂ production from [1‐¹⁴C]glucose by about 40%. 4. t‐Butylhydroperoxide (50 μmol/1) was without significant effect on volume‐regulatory K⁺ fluxes following exposure to hypotonic (225 mOsmol/1) or hypertonic (385 mOsmol/1) perfusate. Lactate dehydrogenase release from perfused rat liver under the influence of t‐butylhydroperoxide was increased by hypertonic exposure compared to hypotonic perfusions. 5. The data suggest that hypotonic cell swelling stimulates flux through the pentose‐phosphate pathway and diminishes loss of GSSG under conditions of mild oxidative stress. Hypotonically swollen cells are less prone to hydroperoxide‐induced lactate dehydrogenase release than hypertonically shrunken cells. Hypertonic cell shrinkage stimulates the excretion of glutathione‐S‐conjugates into the sinusoidal circulation at the expense of biliary secretion.