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...
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Published in | Brain research Vol. 802; no. 1; pp. 232 - 240 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
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London
Elsevier B.V
17.08.1998
Amsterdam Elsevier New York, NY |
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Abstract | 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-α (TNF-α) 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 not necessarily lead to greater cell death. |
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AbstractList | 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-α (TNF-α) 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 not necessarily lead to greater cell death. 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. 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 not necessarily lead to greater cell death. |
Author | Barker, Jane E Dobbie, Michael S Clark, John B Heales, Simon J.R Hurst, Roger D |
Author_xml | – sequence: 1 givenname: Roger D surname: Hurst fullname: Hurst, Roger D organization: Department of Neurochemistry, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK – sequence: 2 givenname: Simon J.R surname: Heales fullname: Heales, Simon J.R organization: Department of Neurochemistry, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK – sequence: 3 givenname: Michael S surname: Dobbie fullname: Dobbie, Michael S organization: Department of Neurochemistry, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK – sequence: 4 givenname: Jane E surname: Barker fullname: Barker, Jane E organization: The Bernard O'Brien Institute of Microsurgery, Fitzroy, Victoria 3065, Australia – sequence: 5 givenname: John B surname: Clark fullname: Clark, John B organization: Department of Neurochemistry, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK |
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Keywords | Oxidative stress GSH, reduced glutathione Endothelial cell TNF-α, tumour necrosis factor-α TEER, transendothelial electrical resistance LDH, lactate dehydrogenase l-BSO, l-buthionine sulfoximine PBS, phosphate buffered saline BBB, blood–brain barrier Blood–brain barrier NO, nitric oxide Nitric oxide Transendothelial resistance Glutathione Sensitivity Central nervous system Models Blood brain barrier In vitro Brain (vertebrata) |
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Snippet | 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... 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... |
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SubjectTerms | Animals Biological and medical sciences Blood-Brain Barrier - physiology Blood–brain barrier Buthionine Sulfoximine - pharmacology Cell Death - physiology Cells, Cultured Cerebral circulation. Blood-brain barrier. Choroid plexus. Cerebrospinal fluid. Circumventricular organ. Meninges Drug Resistance Electric Impedance Endothelial cell Endothelium, Vascular - cytology Endothelium, Vascular - metabolism Endothelium, Vascular - physiology Enzyme Inhibitors - pharmacology Fundamental and applied biological sciences. Psychology Glutathione Glutathione - metabolism Humans L-Lactate Dehydrogenase - metabolism Nitric oxide Nitric Oxide - pharmacology Oxidative stress Oxidative Stress - physiology Rats Rats, Wistar Transendothelial resistance Vertebrates: nervous system and sense organs |
Title | Decreased endothelial cell glutathione and increased sensitivity to oxidative stress in an in vitro blood–brain barrier model system |
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