Opening of the Mitochondrial Permeability Transition Pore Induces Reactive Oxygen Species Production at the Level of the Respiratory Chain Complex I

Cécile Batandier,Xavier Leverve,Eric Fontaine

doi:10.1074/jbc.m310329200

Abstract

We have investigated the consequences of permeability transition pore (PTP) opening on the rate of production of reactive oxygen species in isolated rat liver mitochondria. We found that PTP opening fully inhibited H(2)O(2) production when mitochondria were energized both with complex I or II substrates. Because PTP opening led to mitochondrial pyridine nucleotide depletion, H(2)O(2) production was measured again in the presence of various amounts of NADH. PTP opening-induced H(2)O(2) production began when NADH concentration was higher than 50 microm and reached a maximum at over 300 microm. At such concentrations of NADH, the maximal H(2)O(2) production was 4-fold higher than that observed when mitochondria were permeabilized with the channel-forming antibiotic alamethicin, indicating that the PTP opening-induced H(2)O(2) production was not due to antioxidant depletion. Moreover, PTP opening decreased rotenone-sensitive NADH ubiquinone reductase activity, whereas it did not affect the NADH FeCN reductase activity. We conclude that PTP opening induces a specific conformational change of complex I that (i) dramatically increases H(2)O(2) production so long as electrons are provided to complex I, and (ii) inhibits the physiological pathway of electrons inside complex I. These data allowed the identification of a novel consequence of permeability transition that may partly account for the mechanism by which PTP opening induces cell death.

Highlights

Besides their well established role in energy metabolism, mitochondria are recognized to play a key role in the commitment to cell death [1,2,3,4]
permeability transition pore (PTP) opening led to NADH oxidation, which interfered with the homovanillic acid (HVA) signal
We have shown that PTP opening directly affected electron-transfer through complex I and induced ROS production in the presence of NADH

Summary

Introduction

Besides their well established role in energy metabolism, mitochondria are recognized to play a key role in the commitment to cell death [1,2,3,4]. PTP opening in vitro leads to the collapse of the proton motive force, ATP hydrolysis, the disruption of ionic homeostasis, and mitochondrial swelling In this situation, mitochondrial swelling is due to the presence of matrix proteins that cannot diffuse through the open pore, creating an oncotic pressure gradient [5]. The role of PTP in the commitment to cell death is supported by (i) the fact that mitochondrial swelling leads to outer membrane rupture and release of pro-apoptotic intermembrane space proteins (6 – 8), (ii) the demonstration that PTP opening occurs in intact cells (9 –13), and (iii) the finding that different PTP inhibitors have a protective effect in several models of cell death (9, 14 –22). The mechanisms by which PTP opening leads to the release of pro-apoptotic proteins in vivo needs to be clarified, especially whether PTP opening induces mitochondrial swelling in intact cells (or not) when mitochondria are surrounded by cytosolic proteins. Experiments were started by the addition of 1 mg of mitochondria (data not shown)

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Results

Conclusion