Critical Role of Mitochondrial Glutathione in the Survival of Hepatocytes during Hypoxia

Montserrat Mari,Carmen Garcia-Ruiz,Albert Morales,Carmen Blasco,Josep M. Lluis,José C. Fernandez-Checa,Anna Colell

doi:10.1074/jbc.m408244200

Abstract

Hypoxia is known to stimulate reactive oxygen species (ROS) generation. Because reduced glutathione (GSH) is compartmentalized in cytosol and mitochondria, we examined the specific role of mitochondrial GSH (mGSH) in the survival of hepatocytes during hypoxia (5% O2). 5% O2 stimulated ROS in HepG2 cells and cultured rat hepatocytes. Mitochondrial complex I and II inhibitors prevented this effect, whereas inhibition of nitric oxide synthesis with Nomega-nitro-L-arginine methyl ester hydrochloride or the peroxynitrite scavenger uric acid did not. Depletion of GSH stores in both cytosol and mitochondria enhanced the susceptibility of HepG2 cells or primary rat hepatocytes to 5% O2 exposure. However, this sensitization was abrogated by preventing mitochondrial ROS generation by complex I and II inhibition. Moreover, selective mGSH depletion by (R,S)-3-hydroxy-4-pentenoate that spared cytosol GSH levels sensitized rat hepatocytes to hypoxia because of enhanced ROS generation. GSH restoration by GSH ethyl ester or by blocking mitochondrial electron flow at complex I and II rescued (R,S)-3-hydroxy-4-pentenoate-treated hepatocytes to hypoxia-induced cell death. Thus, mGSH controls the survival of hepatocytes during hypoxia through the regulation of mitochondrial generation of oxidative stress.

Highlights

In conditions of limited O2 supply cells adapt and survive because of the existence of specific sensors that allow them to acclimate to the deprivation of oxygen and to recover from ischemic conditions
Hypoxia stimulated a reactive oxygen species (ROS) generation in both HepG2 cells and primary rat hepatocytes with respect to normoxic incubation in a time-dependent fashion that increased with the severity of oxygen deprivation (Fig. 1, A and B)
Primary rat hepatocytes were more sensitive to hypoxia than HepG2 cells as reflected by the ROS stimulation and loss of survival observed at 2% O2 exposure (Fig. 1, C and D)

Summary

Introduction

In conditions of limited O2 supply cells adapt and survive because of the existence of specific sensors that allow them to acclimate to the deprivation of oxygen and to recover from ischemic conditions. At low O2 tension, prolyl hydroxylase activity is inhibited, resulting in HIF1␣ protein accumulation Another well recognized response to hypoxia is the generation of reactive oxygen species (ROS). The regulation of intracellular GSH levels by hypoxia is known [13,14,15,16,17,18], the specific role that mitochondrial GSH (mGSH) plays in the survival of cells during hypoxia has not been reported to the best of our knowledge. The abrogation of mitochondrial ROS production rescued HepG2 cells or primary rat hepatocytes from hypoxia-induced cell death despite GSH depletion in both cytosol and mitochondria, using (R,S)-3-hydroxy-4-pentenoate (HP), which is transformed in the mitochondria of hepatocytes into a Michael acceptor resulting in selective mGSH depletion with the sparing of cytosol. We provide evidence for a critical role of mGSH in determining the susceptibility of hepatocytes to hypoxia through control of mitochondrial oxidative stress

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