title: Acute glutathione depletion restricts mitochondrial ATP export in cerebellar granule neurons.
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.
authors.affiliation: Buck Institute for Age Research, Novato, California 94945, USA. svesce@buckinstitute.org
authors.forename: Sabino
authors.initials: S
authors.lastname: Vesce
authors.forename: Mika B
authors.initials: MB
authors.lastname: Jekabsons
authors.forename: Linda I
authors.initials: LI
authors.lastname: Johnson-Cadwell
authors.forename: David G
authors.initials: DG
authors.lastname: Nicholls
mesh_terms: D033741:Adenine Nucleotide Translocator 1 / Q000037:antagonists & inhibitors; D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001692:Biological Transport; D001865:Bongkrekic Acid / Q000494:pharmacology; D002021:Buffers; D002118:Calcium / Q000378:metabolism; D016923:Cell Death; D002462:Cell Membrane / Q000378:metabolism; D019069:Cell Respiration; D002531:Cerebellum / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D006019:Glycolysis; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry; D007150:Immunohistochemistry; D007477:Ions; D007608:Kainic Acid / Q000494:pharmacology; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D009840:Oligomycins / Q000494:pharmacology; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D011720:Pyrazoles / Q000737:chemistry / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D020112:Rhodamine 123 / Q000494:pharmacology; D012964:Sodium / Q000737:chemistry; D013367:Submitochondrial Particles / Q000378:metabolism; D013816:Thermodynamics; D013997:Time Factors; D014162:Transfection
chemical_list: D033741:Adenine Nucleotide Translocator 1; D002021:Buffers; D005456:Fluorescent Dyes; D007477:Ions; D009840:Oligomycins; D011720:Pyrazoles; D001865:Bongkrekic Acid; D049452:Green Fluorescent Proteins; D020112:Rhodamine 123; D000244:Adenosine Diphosphate; C059597:monochlorobimane; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D012964:Sodium; D005978:Glutathione; D010100:Oxygen; D007608:Kainic Acid; D002118:Calcium