Methylmercury-mediated inhibition of 3H-D-aspartate transport in cultured astrocytes is reversed by the antioxidant catalase.
Astrocytes are essential for removal of glutamate from the extracellular space in the central nervous system. The neurotoxic heavy metal methylmercury potently and specifically inhibits the transport of glutamate in cultured astrocytes by an unknown mechanism. Glutamate transport in astrocytes is also inhibited by reactive oxygen species. A glutamate-induced transporter current is inhibited both by reactive oxygen species and thiol oxidizing agents. These observations suggest that oxidation of the transporter might mediate methylmercury-induced inhibition of glutamate transport. In the present study, we examined the ability of thiol reducing or oxidizing agents to inhibit transport of 3H-D-aspartate, a glutamate analog, in primary cultures of neonatal rat astrocytes. To assess if methylmercury-mediated inhibition of 3H-aspartate transport was due to overproduction of reactive oxygen species, we tested the ability of Trolox, alpha-phenyl-tert-butyl nitrone (PBN), or catalase to attenuate the methylmercury-induced inhibition of aspartate uptake. Neither the thiol reducing agent dithiothreitol (DTT), nor the thiol oxidizing agent 5,5'-dithio-bis(2-nitrobenzoic) acid (DTNB) had any effect on 3H-aspartate transport suggesting that the thiol redox state does not alter transporter function. In contrast, the antioxidant catalase (1000 U/ml) significantly attenuated methylmercury-induced inhibition of 3H-aspartate uptake, suggesting that excess reactive oxygen species, specifically H2O2, inhibit the function of an astrocytic excitatory amino acid transporter (EAAT1). Prolonged exposure (6 h) to inhibitors of glutamate transport significantly decreased EAAT1 mRNA levels suggesting that transporter expression is related to function. This study suggests that methylmercury-induced overproduction of H2O2 is a mechanism for inhibition of glutamate transport and transporter expression in cultured astrocytes.
Allen
J W
JW
Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Medical Center Boulevard, , Winston-Salem, NC 27157-1083, USA.
Mutkus
L A
LA
Aschner
M
M
D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D027322:Amino Acid Transport System X-AG; D000818:Animals; D000831:Animals, Newborn; D000975:Antioxidants / Q000494:pharmacology*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D002374:Catalase / Q000494:pharmacology*; D002417:Cattle; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D002839:Chromans / Q000494:pharmacology; D003497:Cyclic N-Oxides; D004136:Dinitrobenzenes / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism; D008767:Methylmercury Compounds / Q000494:pharmacology*; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009589:Nitrogen Oxides / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology*; D010084:Oxidation-Reduction; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D019163:Reducing Agents / Q000494:pharmacology*; D013113:Spin Labels
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D000975:Antioxidants; D002839:Chromans; D003497:Cyclic N-Oxides; D004136:Dinitrobenzenes; D008767:Methylmercury Compounds; D009419:Nerve Tissue Proteins; D009589:Nitrogen Oxides; D016877:Oxidants; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D019163:Reducing Agents; D013113:Spin Labels; C011274:3-hydroxyaspartic acid; D001224:Aspartic Acid; C029217:phenyl-N-tert-butylnitrone; D006861:Hydrogen Peroxide; D002374:Catalase; C010936:2,4-dinitrothiocyanatobenzene; C004925:methylmercuric chloride; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; D004229:Dithiothreitol