Abstract
Decreases in GSH pools detected during ischemia sensitize neurons to excitotoxic damage. Thermodynamic analysis predicts that partial GSH depletion will cause an oxidative shift in the thiol redox potential. To investigate the acute bioenergetic consequences, neurons were exposed to monochlorobimane (mBCl), which depletes GSH by forming a fluorescent conjugate. Neurons transfected with redox-sensitive green fluorescent protein showed a positive shift in thiol redox potential synchronous with the formation of the conjugate. Mitochondria within neurons treated with mBCl for 1 h failed to hyperpolarize upon addition of oligomycin to inhibit their ATP synthesis. A decreased ATP turnover was confirmed by monitoring neuronal oxygen consumption in parallel with mitochondrial membrane potential (Deltapsi(m)) and GSH-mBCl formation. mBCl progressively decreased cell respiration, with no effect on mitochondrial proton leak or maximal respiratory capacity, suggesting adequate glycolysis and a functional electron transport chain. This approach to "state 4" could be mimicked by the adenine nucleotide translocator inhibitor bongkrekic acid, which did not further decrease respiration when administered after mBCl. The cellular ATP/ADP ratio was decreased by mBCl, and consistent with mitochondrial ATP export failure, respiration could not respond to an increased cytoplasmic ATP demand by plasma membrane Na(+) cycling; instead, mitochondria depolarized. More prolonged mBCl exposure induced mitochondrial failure, with Deltapsi(m) collapse followed by cytoplasmic Ca(2+) deregulation. The initial bioenergetic consequence of neuronal GSH depletion in this model is thus an inhibition of ATP export, which precedes other forms of mitochondrial dysfunction.
Highlights
The tripeptide glutathione is a key antioxidant that maintains protein thiols in a reduced state and scavenges H2O2 in a reaction catalyzed by glutathione peroxidase [6, 7]
The intracellular thiol redox potential was detected with a redoxsensitive green fluorescent protein variant [22], whereas population cell respiratory rates were monitored in parallel with cytoplasmic free Ca2ϩ concentration ([Ca2ϩ]c) and mitochondrial membrane potential (⌬m) [23], allowing glutathione depletion to be correlated with a rapid inhibition of ATP turnover that became evident within the first hour of treatment
The data suggest that a restriction of mitochondrial ATP export to the cytoplasm, consistent with adenine nucleotide translocator (ANT) inhibition, is the first event occurring in this model of GSH depletion
Summary
The tripeptide glutathione is a key antioxidant that maintains protein thiols in a reduced state and scavenges H2O2 in a reaction catalyzed by glutathione peroxidase [6, 7]. Thermodynamic and kinetic factors predict that the steady-state thiol redox potential will be very sensitive to changes in total glutathione pool size [7, 13]. This may lead to the oxidative damage of key mitochondrial proteins. An intriguing target for thiol oxidation is represented by the adenine nucleotide translocator (ANT), which functions as a dimer and can be progressively inhibited as the intermolecular oxidation of thiol groups increases [17] This loss of ADP/ATP exchange activity precedes the formation of intramolecular cross-linking, which seems to require stronger oxidative conditions and may be related to mitochondrial PTP opening [18, 19]. The data suggest that a restriction of mitochondrial ATP export to the cytoplasm, consistent with ANT inhibition, is the first event occurring in this model of GSH depletion
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