Decreased endothelial cell glutathione and increased sensitivity to oxidative stress in an in vitro blood-brain barrier model system.
Using a cell culture model of the blood-brain barrier (BBB) we have evaluated the role of endothelial cell glutathione in protecting barrier integrity against nitric oxide (NO)-induced oxidative stress. The co-culture of human umbilical vein endothelial cells (ECV304) with rat (C6) glioma cells, or incubation with glioma cell or primary astrocytic conditioned medium, resulted in a decline in endothelial cell glutathione. Exposure to a single addition of NO gas induced a rapid breakdown in model barrier integrity in endothelial/glioma co-cultures. Addition of NO gas or tumour necrosis factor-alpha (TNF-alpha) also resulted in a loss of membrane integrity, as measured by an enhanced release of lactate dehydrogenase, only from endothelial cells treated with glioma conditioned medium. Furthermore, assessment of viability in endothelial cells grown alone or treated with glioma conditioned medium, by propidium iodide labelled flow cytometry. demonstrated no difference in the number of positively stained cells after NO exposure. These results indicate that when enhanced endothelial monolayer barrier formation occurs via astrocytic-endothelial interactions, cellular glutathione levels are decreased. This renders the barrier cells, under these conditions, more susceptible to oxidative stress but does no necessarily lead to greater cell death.
Hurst
R D
RD
Department of Neurochemistry, University College London, UK. rhurst@ion.ucl.ac.uk
Heales
S J
SJ
Dobbie
M S
MS
Barker
J E
JE
Clark
J B
JB
D000818:Animals; D001812:Blood-Brain Barrier / Q000502:physiology*; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D016923:Cell Death / Q000502:physiology; D002478:Cells, Cultured; D004351:Drug Resistance; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology; D018384:Oxidative Stress / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar
D004791:Enzyme Inhibitors; D009569:Nitric Oxide; D019328:Buthionine Sulfoximine; D007770:L-Lactate Dehydrogenase; D005978:Glutathione