Abstract 16262: Regulation of Mitochondria Redox State by GSH Repletion Triggers Cell Death
Intracellular redox balance is critical for the proper functioning of numerous proteins and biological processes. To control the redox environment of subcellular compartments, cells utilize two regulatory pathways: GSH system and thioredoxin (Trx) redox couples. How the GSH and Trx systems exert their complemenary roles in redox regulaion remains poorly defined. To examine how GSH modulates redox status, we monitored the redox state of specific subcompartments using cytosolic and mitochondrial redox-senstive GFP (ro-GFP) by fluorescence, and performed redox western blots for ro-GFP, Trx1 and mitochondrial Trx2 in H9c2 cells. We depleted GSH using L-Buthinine sulphoximine (BSO) or 1-Chloro-2,4-dinitrobenzene (DNCB), and repleted GSH using N-Acetyl-L-cysteine (NAC) or overexpression of glutamate cysteine ligase catalytic subunit (GCLC). Inhibition of glutamate cysteine ligase by BSO treatment progressively decreased GSH by 2.3 fold but the GSH/GSSG ratio increased significantly. BSO treatment had no effects on the redox state of ro-GFP, Trx1 and Trx2, ROS production and cell viability. In contrast, treatment with DNCB, an inhibitor of thioredoxin reductase, dramatically decreased GSH by 5.1 fold and GSH/GSSG ratio by 6.4 fold, and induced rapid oxidation of the redox potential accompanied increased ROS production and cell death. Oxidized redox states were confirmed using ro-GFP, Trx 1 and Trx2 after DNCB. These data suggest that dose-dependent GSH depletion has differential effects on oxidative stress and cell death. NAC treatment in H9c2 cells, or GCLC over-expression in HEK293T cells increased glutathione biosynthesis by 3.6 fold and 3 fold respectively, without redox changes in cytosolic roGFP and Trx1. Surprisingly, increased GSH profoundly increased the oxidized forms of mitochondrial ro-GFP and Trx2, decreased GSH/GSSG ratio and cell viability, without alterations of ROS levels. Our data indicate that GSH augmentation does not promote a reduced state, but instead induces the oxidation of the mitochondrial environmental. Together, our findings point to a hitherto unrecognized role for GSH biosynthesis, GSH/GSSG ratio and ROS levels on redox regulation at the subcellular level.
- © 2011 by American Heart Association, Inc.