Glutathionylation of Adenine Nucleotide Translocase Induced by Carbon Monoxide Prevents Mitochondrial Membrane Permeabilization and Apoptosis

Cláudia S.F Queiroga,Ana S Almeida,Cécile Martel,Catherine Brenner,Paula M Alves,Helena L.A Vieira

doi:10.1074/jbc.m109.065052

Abstract

The present work demonstrates the ability of CO to prevent apoptosis in a primary culture of astrocytes. For the first time, the antiapoptotic behavior can be clearly attributed to the inhibition of mitochondrial membrane permeabilization (MMP), a key event in the intrinsic apoptotic pathway. In isolated non-synaptic mitochondria, CO partially inhibits (i) loss of potential, (ii) the opening of a nonspecific pore through the inner membrane, (iii) swelling, and (iv) cytochrome c release, which are induced by calcium, diamide, or atractyloside (a ligand of ANT). CO directly modulates ANT function by enhancing ADP/ATP exchange and prevents its pore-forming activity. Additionally, CO induces reactive oxygen species (ROS) generation, and its prevention by beta-carotene decreases CO cytoprotection in intact cells as well as in isolated mitochondria, revealing the key role of ROS. On the other hand, CO induces a slight increase in mitochondrial oxidized glutathione, which is essential for apoptosis modulation by (i) delaying astrocytic apoptosis, (ii) decreasing MMP, and (iii) enhancing ADP/ATP translocation activity of ANT. Moreover, CO and GSSG trigger ANT glutathionylation, a post-translational process regulating protein function in response to redox cellular changes. In conclusion, CO protects astrocytes from apoptosis by preventing MMP, acting on ANT (glutathionylation and inhibition of its pore activity) via a preconditioning-like process mediated by ROS and GSSG.

Highlights

Tolerance, which is defined as a condition of transiently increased resistance to injury
Primary cultures of cortical astrocytes were treated with Carbon monoxide (CO)-saturated phosphate-buffered saline (PBS) with a final concentration of 50 ␮M 3 h prior to apoptosis induction
CO partially prevents the dissipation of ⌬⌿m (quantified by DiOC6(3); Fig. 1, A and C), followed by plasma membrane permeabilization, a marker for loss of viability

Summary

Introduction

Tolerance, which is defined as a condition of transiently increased resistance to injury. CO protects astrocytes from apoptosis by preventing MMP, acting on ANT (glutathionylation and inhibition of its pore activity) via a preconditioning-like process mediated by ROS and GSSG. Non-synaptic mitochondria isolated from brain cortex [27] were treated with 10 ␮M CO during 15 min prior to the addition of diamide, atractyloside (a ligand of ANT that prevents ADP/ATP translocation and induces its pore forming function), or calcium to induce MMP.

Results

Conclusion