Altered expression of glutamate transporters under hypoxic conditions in vitro.
Regulation of extracellular excitotoxins by glial and neuronal glutamate transporters is critical to maintain synaptic terminal integrity. Factors interfering with the normal functioning of these transporters might be involved in neurodegeneration. Among them, recent studies have shown that hypoxia alters glutamate transporter function; however, it is unclear if hypoxia has an effect on the expression of glutamate transporters and which intracellular signaling pathways are involved. The C6 rat glial and GT1--7 mouse neuronal cell lines were exposed to hypoxic conditions (5% CO(2), 95% N(2)) and levels of glutamate transporter mRNA were determined by ribonuclease protection assay. After 21 hr, there was a 100% increase in levels of rat excitatory amino acid transporter 3 (EAAT3) mRNA in C6 cells and a 600% increase in levels of murine EAAT2 mRNA in GT1--7 cells. There was a similar increase in mRNA levels after hypoxia in C6 cells transfected with human EAAT2, whereas reoxygenation normalized the expression levels of glutamate transporters. Although the expression of EAATs was associated with increased immunoreactivity by Western blot, functioning of the transporters was decreased as evidenced by D-aspartate uptake. Finally, although the protein kinase C stimulator phorbol-12-myristate-13-acetate enhanced EAAT2 mRNA levels after hypoxia, protein kinase C inhibitor bisindolylmaleimide I had the opposite effect. Taken together, this study suggests that the hypoxia is capable of upregulating levels of EAATs via a protein kinase C-dependent compensatory mechanism. This increased expression is not sufficient to overcome the decreased functioning of the EAATs associated with decreased ATP production and mitochondrial dysfunction.
Hsu
L
L
Department of Neurosciences, University of California, San Diego, La Jolla, 92093-0624, USA.
Rockenstein
E
E
Mallory
M
M
Hashimoto
M
M
Masliah
E
E
D027322:Amino Acid Transport System X-AG; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D016923:Cell Death; D015687:Cell Hypoxia; D027342:Excitatory Amino Acid Transporter 2; D050580:Excitatory Amino Acid Transporter 3; D005786:Gene Expression Regulation / Q000187:drug effects; D005910:Glioma / Q000473:pathology; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000378:metabolism*; D007029:Hypothalamic Neoplasms / Q000473:pathology; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017981:Receptors, Neurotransmitter / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D027981:Symporters; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D027322:Amino Acid Transport System X-AG; D002352:Carrier Proteins; D027342:Excitatory Amino Acid Transporter 2; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D007211:Indoles; D008301:Maleimides; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D017981:Receptors, Neurotransmitter; D011993:Recombinant Fusion Proteins; C493399:Slc1a1 protein, mouse; C493398:Slc1a1 protein, rat; C493396:Slc1a2 protein, mouse; C493395:Slc1a2 protein, rat; D027981:Symporters; D001224:Aspartic Acid; D018698:Glutamic Acid; D011493:Protein Kinase C; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate