Increased glutamate uptake and GLAST expression by cyclic AMP in retinal glial cells.
BACKGROUND
The aim was to investigate the effect of cyclic adenosine monophosphate (cAMP) on glutamate uptake and glial glutamate aspartate transporter (GLAST) expression in cultured rat retinal glial cells.


METHODS
Retinal glial cell cultures were prepared from 1- to 3-day-old Sprague-Dawley rats. Cultures were treated with dibutyryl-cAMP (dBcAMP) for 24 h. Glutamate uptake was measured as 3H-glutamate content of the lysates as determined by scintillation counting. The expression of GLAST in dBcAMP-treated cells was examined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting.


RESULTS
Cells were identified by immunocytochemistry using antibodies against vimentin and carbonic anhydrase C. Treatment of cultured cells with dBcAMP significantly increased glutamate uptake after 60 min compared with untreated cells (p < 0.01). Maintenance of cells in medium supplemented with dBcAMP increased GLAST mRNA and protein levels. GLAST mRNA levels were elevated 2.9-fold 24 h after treatment. GLAST protein levels were elevated 2.5-fold 24 h after treatment.


CONCLUSIONS
These results indicate that treatment with cAMP increases glutamate uptake through upregulated GLAST activity in cultured retinal glial cells. These findings suggest that retinal glial cells may regulate extracellular glutamate concentration and protect retinal neurons against glutamate excitotoxicity.
Sakai
Tsutomu
T
Department of Ophthalmology, Jikei University School of Medicine, 3-25-8 Nishishinbashi, Minato-ku, Tokyo, 105-8461, Japan. tstmski@jikei.ac.jp
Yoshitoshi
Takashi
T
Nagai
Yukiko
Y
Kitahara
Kenji
K
D027322:Amino Acid Transport System X-AG / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D003994:Bucladesine / Q000494:pharmacology; D024402:Carbonic Anhydrase II / Q000378:metabolism; D002478:Cells, Cultured; D003470:Culture Media; D000242:Cyclic AMP / Q000494:pharmacology*; D018698:Glutamic Acid / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D009457:Neuroglia / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000166:cytology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014746:Vimentin / Q000378:metabolism
D027322:Amino Acid Transport System X-AG; D003470:Culture Media; D012333:RNA, Messenger; D014746:Vimentin; D018698:Glutamic Acid; D003994:Bucladesine; D000242:Cyclic AMP; D024402:Carbonic Anhydrase II