Na+ dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) in primary astrocyte cultures: effect of oxidative stress.
The Na+ -dependent L-glutamate transporters EAAT1(GLAST), EAAT2 (GLT-1) and EAAT3 (EAAC1) are expressed in primary astrocyte cultures, showing that the EAAT3 transporter is not neuron-specific. The presence of these three transporters was evaluated by RT-PCR, immunoblotting, immunocytochemical techniques, and transport activity. When primary astrocyte cultures were incubated with L-buthionine-(S,R)-sulfoximine (BSO), a selective inhibitor of gamma-glutamylcysteine synthetase, the GSH concentration was significantly lower than in control cultures, but the expression and amount of protein of EAAT1, EAAT2 and EAAT3 and transport of L-glutamate was unchanged. Oxidative stress was created by adding H(2)O(2) or tert.-butyl hydroperoxide (t-bOOH) to the primary astrocyte cultures and cell damage was evaluated by measuring activity of lactate dehydrogenase. Under oxidative stress, GSH levels were significantly lower than in control astrocytes; but the expression and the amount of protein of the three transporters remained unchanged. However, L-glutamate uptake was significantly lower in astrocytes under oxidative conditions when compared to controls. L-Glutamate uptake was not changed in the presence of ascorbate, but was partially recovered in the presence of DTT and GSH ethyl ester. This report emphasizes that oxidative stress and not GSH depletion alters transporter activity without changing transporter expression.
Miralles
V J
VJ
Departamento de Bioquímica y Biología Molecular, Facultades de Medicina y Farmacia, Universidad de Valencia, Valencia, Spain. mirallev@uv.es
Martínez-López
I
I
Zaragozá
R
R
Borrás
E
E
García
C
C
Pallardó
F V
FV
Viña
J R
JR
D027322:Amino Acid Transport System X-AG; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001923:Brain Chemistry / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D003634:DDT / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D027341:Excitatory Amino Acid Transporter 1 / Q000378:metabolism*; D027342:Excitatory Amino Acid Transporter 2 / Q000378:metabolism*; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism / Q000494:pharmacology; D015151:Immunoblotting; D007150:Immunohistochemistry; D007306:Insecticides / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008856:Microscopy, Fluorescence; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000502:physiology*; D027981:Symporters
D027322:Amino Acid Transport System X-AG; D002352:Carrier Proteins; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D007306:Insecticides; D012333:RNA, Messenger; C493398:Slc1a1 protein, rat; C493395:Slc1a2 protein, rat; C493392:Slc1a3 protein, rat; D027981:Symporters; D018698:Glutamic Acid; D012964:Sodium; D003634:DDT; D007770:L-Lactate Dehydrogenase; D005978:Glutathione