Shigehisa Kitahara

The Effects Of γ-glutamylcysteine Ethyl Ester, A Prodrug Of Glutathione, On Ischemia-reperfusion-induced Liver Injury In Rats

This study was designed to clarify the effects of changes in liver tissue glutathione (GSH) concentration on postischemic liver injury together with the effects of γ-glutamylcysteine ethyl ester (GCE), a prodrug of GSH, and GSH. Rats were pretreated with GSH (50 mg/kg, i.v.), or GCE (50 mg/kg, i.v.), or untreated. In each rat, liver was isolated, and liver mitochondria were prepared after 2 h of ischemia or 1 h of reperfusion following 2 h of ischemia. Mitochondrial function was measured polarographically. Liver adenine nucleotide concentrations were also determined using high-performance liquid chromatography. Liver tissue GSH, an oxidized form of glutathione (GSSG) concentrations, and activities of GSH peroxidase and GSSG reductase were determined enzymatically. Liver hypoxanthine and xanthine concentrations were determined by HPLC. Liver tissue concentration of lipid peroxide was measured. Leakages of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and adenine nucleotides into the hepatic vein after reperfusion were also measured. Administration of GCE improved the recovery of mitochondrial function and maintained tissue GSH concentration concomitantly. Increases in liver lipid peroxide concentration after reperfusion, and leakage of liver cell enzymes and adenine nucleotides were mitigated by administration of GCE. Administration of GSH itself failed to maintain tissue GSH concentration and had no protective effects. From these results, it is concluded that in the postischemic process, free radical formation might be enhanced, and the radical scavenging system deteriorated. To enhance the radical scavenging system is a possible maneuver to prevent radical-related cell damage associated with reperfusion, because pharmacological reduction of breakdown of ATP to hypoxanthine and xanthine seems to be difficult. GCE maintained liver GSH concentrations and mitigated postischemic liver injury, concomitantly. Clinical use of GCE might be recommended.

Fc portion of intravenous immunoglobulin suppresses the induction of experimental allergic neuritis

To clarify how intravenous immunoglobulin (IVIg) acts on Guillain-Barré syndrome, we investigated the effects of intact-type IVIg treatment on experimental allergic neuritis (EAN) induced by immunizing with synthetic peptide from bovine P2 protein. Treatment with intact-type IVIg (400 mg/kg/day) on days 0, 7, 14, 15 and 16 after immunization prevented the paralysis, whereas treatment with F(ab)2 failed to alter the clinical course. Intact-type IVIg treatment given on days 0 and 1 showed almost the same efficacy. These results suggest that intact-type IVIg is superior to F(ab)2 in ameliorating the clinical course of EAN and that the Fc portion might affect the immune system.

Conversion of γ-glutamylcysteinylethyl ester to glutathione in rat hepatocytes

Abstract The conversion of γ-glutamylcysteinylethyl ester (γ-GCE) to glutathione in a reduced form (GSH) was examined using isolated rat hepatocytes pretreated with diethylmaleate, a GSH-depletor. Incubation of hepatocytes with 0.1 and 5.0 mM γ-GCE (γ-GCE-hepatocytes) over a 30-min period resulted in time-dependent increases in intracellular GSH and nonprotein-SH (NP-SH) concentrations. Hepatocytes incubated with 5.0 mM but not 0.1 mM GSH over a period of 30 min showed a time-dependent increase in intracellular GSH concentration. In the γ-GCE-hepatocytes pretreated with bis-(p-nitrophenyl)phosphate (BNPP), a non-specific esterase inhibitor, an enhancement of intracellular GSH concentration was markedly reduced. γ-GCE concentration in the γ-GCE-hepatocytes with BNPP pretreatment was significantly higher than that in the cells without BNPP pretreatment, although there was no difference in the total amount of intracellular NP-SH, i.e., γ-GCE, GSH, γ-glutamylcysteine, cysteine ethyl ester, and cysteine between both γ-GCE-hepato-cytes. The present results indicate that γ-GCE is transported into liver cells more easily than GSH itself, resulting in its conversion to GSH via esterase and glutathione synthetase within the cells.

The cardioprotective effect of γ-glutamylcysteine ethyl ester during coronary reperfusion in canine hearts

1 The cardioprotective effect of γ‐glutamylcysteine ethyl ester was investigated on ischaemia‐reperfusion‐induced myocardial damage in anaesthetized dogs. 2 Open chest anaesthetized dogs were divided into four groups: 2 h occlusion of the left anterior descending coronary artery (LAD); 2 h LAD occlusion followed by 1 h reperfusion; 2 h LAD occlusion followed by 1 h reperfusion with administration of γ‐glutamylcysteine ethyl ester (10 mg kg−1 just before reperfusion); 2 h LAD occlusion followed by 1 h reperfusion with administration of GSH (the reduced form of glutathione, 10 mg kg−1 just before reperfusion). 3 After occlusion or reperfusion, heart mitochondria were prepared from the normal area and the occluded or the reperfused area, and mitochondrial function (rate of oxygen consumption in State III, and respiratory control index) was measured polarographically. 4 Mitochondrial GSH and GSSG (the oxidized form of glutathione) concentrations, and activities of glutathione peroxidase and glutathione reductase were measured. 5 Two h of LAD occlusion induced mitochondrial dysfunction with depletion of mitochondrial GSH concentration. One h of reperfusion after 2 h LAD occlusion induced significant mitochondrial dysfunction associated with a marked depletion of mitochondrial GSH concentration. 6 γ‐Glutamylcysteine ethyl ester reduced mitochondrial dysfunction and depletion of mitochondrial GSH concentration after 2 h LAD occlusion and 1 h reperfusion. In contrast, GSH did not prevent depletion of mitochondrial GSH concentration and mitochondrial dysfunction after 2 h LAD occlusion followed by 1 h reperfusion. 7 The activities of glutathione peroxidase and glutathione reductase did not change significantly in each group. 8 One h of reperfusion after 2 h occlusion of LAD induced ventricular arrhythmias. γ‐Glutamylcysteine ethyl ester markedly reduced the development of reperfusion arrhythmias, whilst GSH showed no protective effect. 9 γ‐Glutamylcysteine ethyl ester maintained mitochondrial GSH concentration, prevented reperfusion myocardial damage, and reduced reperfusion arrhythmias.