vitamin-k-semiquinone-radical and acivicin

The tripeptide glutathione (GSH) is used by cells to detoxify hydroperoxides, produced during oxidative stress, and is consumed in the process. Previous studies have indicated that cells can be protected against oxidative stress by extracellular GSH through its degradation catalyzed by the exoenzyme gamma-glutamyl transpeptidase (gamma GT) and its de novo synthesis within the cytosol. We hypothesized that gamma GT would be increased as part of the adaptation of cells to oxidative stress. We examined whether oxidative stress could increase gamma GT activity, protein, and mRNA in a lung epithelial cell line (L2). Cultures were subjected to H2O2-mediated toxicity by 15 min of exposure to the redox cycling quinone, menadione. Menadione (50 microM) caused an initial decrease (27 +/- 9% of baseline after 15 min) in intracellular GSH, followed by resynthesis to levels significantly higher than baseline (335 +/- 40% after 24 h, P < 0.001). This elevation was prevented by acivicin, a gamma GT inhibitor. Menadione also caused a dose-dependent increase in gamma GT enzymatic activity (715 +/- 125% of control at 24 h after 15 min of exposure to 100 microM menadione, P < 0.001) that was prevented by actinomycin D. Western blot analysis indicated increased levels of gamma GT protein with increasing menadione. A concentration-dependent increase in gamma GT-mRNA was also observed. Previous investigation has demonstrated that an increase in gamma GT activity enhances the capacity of cells to utilize extracellular GSH. The findings presented here are consistent with a role for gamma GT in cellular adaptation to oxidative stress.

Topics: Animals; Clone Cells; Dactinomycin; Epithelial Cells; gamma-Glutamyltransferase; Glutathione; Isoxazoles; Mice; Molecular Weight; Oxidative Stress; Protein Conformation; Pulmonary Alveoli; Rats; RNA, Messenger; Transcription, Genetic; Vitamin K

The renal processing of the glutathione conjugate of menadione, 2-methyl-3-S-glutathionyl-1,4-naphthoquinone (thiodione) was studied in the isolated perfused rat kidney. Thiodione at an initial concentration of 600 microM was eliminated rapidly from the perfusate (clearance = 6.0 ml/min). Renal disposition could be ascribed to metabolism and transport of the glutathione conjugate. Renal metabolism by gamma-glutamyltranspeptidase was inhibited by AT-125 [L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] (0.5 mM) resulting in a reduction of the thiodione clearance to 0.86 ml/min. Further reduction of the renal clearance of thiodione was achieved by a combination of AT-125 (0.5 mM) and probenecid (0.5 mM), resulting in a renal clearance of 0.58 ml/min which equalled glomerular filtration rate. Addition of thiodione to the perfusate caused loss of renal function and cellular damage, as reflected by a decreased glucose reabsorption and an increased urinary secretion of lactate dehydrogenase, respectively. Thiodione-induced nephrotoxicity was ameliorated by AT-125 and prevented completely by a combination of AT-125 and probenecid. Aminooxyacetic acid (0.5 mM), an inhibitor of beta-lyase, did not afford protection against the nephrotoxic action of thiodione. From our results it can be concluded that the thiodione-mediated toxicity in the isolated perfused rat kidney can be linked to cellular uptake by anionic transport systems and metabolism by gamma-glutamyltranspeptidase.

Topics: Animals; Biological Transport; gamma-Glutamyltransferase; Isoxazoles; Kidney; Male; Perfusion; Probenecid; Rats; Rats, Inbred Strains; Vitamin K