title: Co-culture of neurones with glutathione deficient astrocytes leads to increased neuronal susceptibility to nitric oxide and increased glutamate-cysteine ligase activity.
abstract: The antioxidant glutathione (GSH) plays an important role in protecting the mitochondrial electron transport chain (ETC) from damage by oxidative stress in astrocytes and neurones. Neurones co-cultured with astrocytes have greater GSH levels, compared to neurones cultured alone, leading to the hypothesis that astrocytes play a key role in brain GSH metabolism by supplying essential GSH precursors to neurones. A previous study has postulated that damage to the ETC following exposure to reactive nitrogen species (RNS) is less in co-cultured neurones, compared to neurones cultured alone, because of the greater GSH levels in the former cells. To investigate this further, primary culture rat neurones were co-cultured with either rat astrocytes activated with IFN-gamma and LPS to produce NO, or NO-generating astrocytes that had been depleted of intracellular GSH by 87% following incubation with the GSH synthesis inhibitor L-buthionine-S,R-sulfoximine (L-BSO). Neurones incubated with NO-generating astrocytes depleted of GSH were unable to elevate GSH levels, unlike neurones co-cultured with NO-generating astrocytes. Complexes II + III and IV of the neuronal ETC were significantly inhibited following exposure to NO-generating astrocytes depleted of GSH. No ETC damage was observed in neurones co-cultured with NO-generating astrocytes. Although neurones co-cultured with GSH depleted astrocytes did not increase cellular GSH levels, the activity of glutamate cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis, was increased by 218%, compared to neurones cultured with control astrocytes. This suggests that neuronal GCL activity could be modulated when GSH metabolism is inhibited in neighboring astrocytes.
authors.affiliation: Cellular Therapy, Institute of Ophthalmology, University College London, London EC1V 9EL, UK. m.gegg@ucl.ac.uk
authors.forename: M E
authors.initials: ME
authors.lastname: Gegg
authors.forename: J B
authors.initials: JB
authors.lastname: Clark
authors.forename: S J R
authors.initials: SJ
authors.lastname: Heales
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001921:Brain / Q000378:metabolism / Q000503:physiopathology; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques; D045222:Electron Transport Chain Complex Proteins / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005721:Glutamate-Cysteine Ligase / Q000378:metabolism*; D005978:Glutathione / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018836:Inflammation Mediators / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism* / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
chemical_list: D045222:Electron Transport Chain Complex Proteins; D004791:Enzyme Inhibitors; D018836:Inflammation Mediators; D009569:Nitric Oxide; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione