Abstract
Cellular and molecular mechanisms of methamphetamine (METH)-induced neurotoxicity may involve alterations of cellular redox status and induction of inflammatory genes in endothelial cells. To study these hypotheses, molecular signaling pathways of METH-induced inflammatory responses via activation of redox-sensitive transcription factors were investigated in human brain microvascular endothelial cells (HBMEC). A dose-dependent depletion of total glutathione levels was detected in HBMEC exposed to METH. In addition, electrophoretic mobility shift assay (EMSA) showed significant increases in DNA binding activities of redox-responsive transcription factors, AP-1 and NF-kappaB, in HBMEC treated with METH. METH-mediated AP-1 or NF-kappaB activation was accompanied by induction of transactivation of AP-1 or NF-kappaB, as measured by dual luciferase assay using specific reporter plasmids. Because NF-kappaB and AP-1 are known to regulate expression of inflammatory genes, expression of the gene encoding for tumor necrosis factor-alpha (TNF-alpha) was also studied in METH-treated HBMEC. A dose-dependent overexpression of the TNF-alpha gene was observed in HBMEC treated with METH. The importance of AP-1 and NF-kappaB in METH-induced TNF-alpha gene was confirmed in functional promoter studies using constructs of the TNF-alpha promoter with mutated AP-1 or NF-kappaB sites. These results indicate that METH-induced disturbances in cellular redox status and activation of AP-1 and NF-kappaB can play critical roles in the signaling pathways leading to upregulation of inflammatory genes in human brain endothelial cells.
Copyright 2001 Wiley-Liss, Inc.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amphetamine-Related Disorders / genetics
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Amphetamine-Related Disorders / metabolism
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Amphetamine-Related Disorders / physiopathology
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Binding Sites / drug effects
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Binding Sites / physiology
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Blood-Brain Barrier / drug effects
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Blood-Brain Barrier / physiology
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Brain / drug effects*
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Brain / metabolism
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Brain / physiopathology
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Cells, Cultured / drug effects
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Cells, Cultured / metabolism
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Encephalitis / chemically induced
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Encephalitis / genetics*
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Encephalitis / metabolism
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Endothelium, Vascular / drug effects*
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Endothelium, Vascular / metabolism
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Endothelium, Vascular / physiopathology
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Gene Expression Regulation / drug effects
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Gene Expression Regulation / physiology
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Genes, Reporter / drug effects
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Genes, Reporter / physiology
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Glutathione / drug effects
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Glutathione / metabolism
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Humans
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Methamphetamine / toxicity*
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Microcirculation / drug effects
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Microcirculation / metabolism
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Microcirculation / physiopathology
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NF-kappa B / drug effects*
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NF-kappa B / genetics
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NF-kappa B / metabolism
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Oxidation-Reduction / drug effects
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Oxidative Stress / drug effects*
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Oxidative Stress / genetics
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RNA, Messenger / drug effects
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RNA, Messenger / metabolism
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Signal Transduction / drug effects
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Signal Transduction / genetics
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Transcription Factor AP-1 / drug effects*
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Transcription Factor AP-1 / genetics
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Transcription Factor AP-1 / metabolism
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Transcription, Genetic / drug effects
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Transcription, Genetic / physiology
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Transfection
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Tumor Necrosis Factor-alpha / drug effects
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Tumor Necrosis Factor-alpha / genetics
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Tumor Necrosis Factor-alpha / metabolism
Substances
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NF-kappa B
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RNA, Messenger
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Transcription Factor AP-1
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Tumor Necrosis Factor-alpha
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Methamphetamine
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Glutathione