Quantitative imaging of glutathione in hippocampal neurons and glia in culture using monochlorobimane.
Glutathione (GSH) is a major antioxidant system in the mammalian central nervous system (CNS). Abnormalities of GSH metabolism have been associated with many disorders of the CNS, including Parkinson's, Alzheimer's, and Huntingdon's diseases and ischaemic/reperfusion injury. Investigation of GSH levels in the CNS generally relies on biochemical assays from cultures enriched for different cell types. Because glia influence neuronal metabolism, we have studied cultures in which neurons and glia are cocultured. This approach demands fluorescence imaging to differentiate between the different cell types in the culture, permitted by the use of monochlorobimane (MCB), which reacts with GSH to produce a fluorescent product. We have defined the conditions required to ensure steady-state MCB loading and show the specificity of MCB for GSH through a reaction catalysed by glutathione-S-transferase (GST). [GSH] was consistently higher in glia than in neurons, and [GSH] in both cell types decreased with time in culture. Inhibition of GSH synthesis by buthionine sulfoximine (BSO) caused a greater proportional depletion of GSH in glia than in neurons. The depletion of GSH induced by BSO was significantly greater in cells cultured for >10 days. Furthermore, release of GSH from glia and its breakdown by the ectoenzyme gamma-glutamyltranspeptidase (gammaGT) maintains [GSH] in neurons. In older cultures, inhibition of gammaGT by acivicin caused significant depletion of neuronal GSH. After inhibition of GSH synthesis by BSO, inhibition of the glia-neuron trafficking pathway by acivicin caused widespread neuronal death. Such neurotoxicity was independent of the endogenous glutamate and nitric oxide synthase, suggesting that it is not due to secondary excitotoxicity.
Keelan
J
J
Department of Physiology, University College London, Gower Street, London WC1N 6BT, United Kingdom.
Allen
N J
NJ
Antcliffe
D
D
Pal
S
S
Duchen
M R
MR
D000818:Animals; D000831:Animals, Newborn; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002478:Cells, Cultured; D004151:Dipeptides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004976:Ethacrynic Acid / Q000494:pharmacology; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005721:Glutamate-Cysteine Ligase / Q000037:antagonists & inhibitors / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D018715:Microscopy, Video; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D018384:Oxidative Stress / Q000502:physiology*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011720:Pyrazoles; D051381:Rats
D004151:Dipeptides; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D011720:Pyrazoles; D019328:Buthionine Sulfoximine; C059597:monochlorobimane; D005982:Glutathione Transferase; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D004976:Ethacrynic Acid; C007740:fluorexon