The role of cysteine in the regulation of blood glutathione-protein mixed disulfides in rats treated with diamide

The kinetics of GSH, GSSG, and thiol-protein mixed disulfides (RS-SP) of GSH (GS-SP) and cysteine (CYS-SP) were studied in rat blood and liver in the time range 0-120 min after treatment with 100 and 200 mg/kg ip of diamide. Total consumption (10 min) and regeneration (120 min) of blood GSH, matched by parallel increases and decreases in RS-SP, were observed. GSSG did not change appreciably. No dose-effect relationship was obtained with either treatment. On the contrary, in vitro treatment of blood with 0.75 mM diamide provoked the same trends of GSH and RS-SP as in vivo (e.g., reversible modifications), whereas treatment with 1.5 mM caused drops and rises in GSH and RS-SP, respectively, without any subsequent return to control values. The presence of a hematic factor responsible for RS-SP regulation is hypothesized in the in vivo experiment. Successive experiments involving in vitro pretreatment with 2 mM diamide and treatment with 0.5 mM of various thiols indicated that cysteine (CYS), but not GSH or N-acetylcysteine, rapidly restored erthyrocyte GSH and RS-SP to their basal levels. No evident sign of hemolysis was observed in these experiments. These results indicate that CYS is a diffusible thiol important for RS-SP regulation. Analysis of whole blood of rats treated with 100 mg/kg ip diamide and the presence of two reversible peaks (about 10 times the corresponding control level) of CYS-SP and free CYS confirmed the plausible role of CYS in maintaining the reversibility of the process. Preliminary results in liver of rats treated with 100 mg/kg diamide indicated that CYS may act by metabolic cooperation between organs. We suggest that CYS may have a role in the regulation of the intracellular redox state of rat erythrocytes during oxidative stress.