Molecular and functional characterisation of glutamate transporters in rat cortical astrocytes exposed to a defined combination of growth factors during in vitro differentiation.
In vitro culture of astroglial progenitors can be obtained from early post-natal brain tissues and several methods have been reported for promoting their maturation into differentiated astrocytes. Hence, a combination of several nutriments/growth factors -- the G5 supplement (insulin, transferrin, selenite, biotin, hydrocortisone, fibroblast growth factor and epidermal growth factor) -- is widely used as a culture additive favouring the growth, differentiation and maturation of primary cultured astrocytes. Considering the key role played by glial cells in the clearance of glutamate in the synapses, cultured astrocytes are frequently used as a model for the study of glutamate transporters. Indeed, it has been shown that when tested separately, growth factors influence the expression and activity of the GLAST and GLT-1. The present study aimed at characterising the functional expression of these transporters during the time course of differentiation of cultured cortical astrocytes exposed to the supplement G5. After a few days, the vast majority of cells exposed to this supplement adopted a typical stellate morphology (fibrous or type II astrocytes) and showed intense expression of the glial fibrillary acidic protein. Both RT-PCR and immunoblotting studies revealed that the expression of both GLAST and GLT-1 rapidly increased in these cells. While this was correlated with a significant increase in specific uptake of radiolabelled aspartate, fluorescence monitoring of the Na+ influx associated with glutamate transporters activity revealed that the exposure to the G5 supplement considerably increased the percentage of cells participating in the uptake. Biochemical and pharmacological studies revealed that this activity did not involve GLT-1 but most likely reflected an increase in GLAST-mediated uptake. Together, these data indicate that the addition of this classical combination of growth factors and nutriments drives the rapid differentiation toward a homogenous culture of fibrous astrocytes expressing functional glutamate transporters.
Vermeiren
Céline
C
Laboratoire de Pharmacologie Expérimentale, Université catholique de Louvain, Av. Hippocrate 54, 1200 Brussels, Belgium.
Najimi
Mustapha
M
Maloteaux
Jean-Marie
JM
Hermans
Emmanuel
E
D027322:Amino Acid Transport System X-AG / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000378:metabolism*; D001693:Biological Transport, Active; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D017931:DNA Primers; D027342:Excitatory Amino Acid Transporter 2 / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006133:Growth Substances / Q000494:pharmacology*; D007150:Immunohistochemistry; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000378:metabolism / Q000502:physiology
D027322:Amino Acid Transport System X-AG; D017931:DNA Primers; D027342:Excitatory Amino Acid Transporter 2; D005904:Glial Fibrillary Acidic Protein; D006133:Growth Substances; D012964:Sodium