BACKGROUND AND PURPOSEThe hippocampal cell line HT22 is an excellent model for studying the consequences of endogenous oxidative stress. Extracellular glutamate depletes cellular glutathione by blocking the glutamate/cystine antiporter system xc−. Glutathione depletion induces a well-defined programme of cell death characterized by an increase in reactive oxygen species and mitochondrial dysfunction.
EXPERIMENTAL APPROACHWe compared the mitochondrial shape, the abundance of mitochondrial complexes and the mitochondrial respiration of HT22 cells, selected based on their resistance to glutamate, with those of the glutamate-sensitive parental cell line.
KEY RESULTSGlutamate-resistant mitochondria were less fragmented and displayed seemingly contradictory features: mitochondrial calcium and superoxide were increased while high-resolution respirometry suggested a reduction in mitochondrial respiration. This was interpreted as a reverse activity of the ATP synthase under oxidative stress, leading to hydrolysis of ATP to maintain or even elevate the mitochondrial membrane potential, suggesting these cells endure ineffective energy metabolism to protect their membrane potential. Glutamate-resistant cells were also resistant to oligomycin, an inhibitor of the ATP synthase, but sensitive to deoxyglucose, an inhibitor of hexokinases. Exchanging glucose with galactose rendered resistant cells 1000-fold more sensitive to oligomycin. These results, together with a strong increase in cytosolic hexokinase 1 and 2, a reduced lactate production and an increased activity of glucose-6-phosphate dehydrogenase, suggest that glutamate-resistant HT22 cells shuttle most available glucose towards the hexose monophosphate shunt to increase glutathione recovery.
CONCLUSIONS AND IMPLICATIONSThese results indicate that mitochondrial and metabolic adaptations play an important role in the resistance of cells to oxidative stress.
Cerebral ischemia is a key pathophysiological feature of various brain insults. Inadequate oxygen supply can manifest regionally in stroke or as a result of traumatic brain injury or globally following cardiac arrest, all leading to irreversible brain damage. Mitochondrial function is essential for neuronal survival, since neurons critically depend on ATP synthesis generated by mitochondrial oxidative phosphorylation. Mitochondrial activity depends on Ca(2+) and is fueled either by Ca(2+) from the extracellular space when triggered by neuronal activity or by Ca(2+) released from the endoplasmic reticulum (ER) and taken up through specialized contact sites between the ER and mitochondria known as mitochondrial-associated ER membranes. The coordination of these Ca(2+) pools is required to synchronize mitochondrial respiration rates and ATP synthesis to physiological demands. In this review, we discuss the role of the proteins involved in mitochondrial Ca(2+) homeostasis in models of ischemia. The proteins include those important for the Ca(2+)-dependent motility of mitochondria and for Ca(2+) transfer from the ER to mitochondria, the tethering proteins that bring the two organelles together, inositol 1,4,5-triphosphate receptors that enable Ca(2+) release from the ER, voltage-dependent anion channels that allow Ca(2+) entry through the highly permeable outer mitochondrial membrane and the mitochondrial Ca(2+) uniporter together with its regulatory proteins that permit Ca(2+) entry into the mitochondrial matrix. Finally, we address those proteins important for the extrusion of Ca(2+) from the mitochondria such as the mitochondrial Na(+)/Ca(2+) exchanger or, if the mitochondrial Ca(2+) concentration exceeds a certain threshold, the mitochondrial permeability transition pore.
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