title: The glutamate transporter GLAST-1 (EAAT-1) is expressed in the plasma membrane of osteocytes and is responsive to extracellular glutamate concentration.
abstract: The glutamate/aspartate transporter GLAST-1 is expressed in bone in vivo and also exists as a splice variant (GLAST-1a) in which exon 3 is excluded. Since GLAST-1 expression is regulated in bone in response to osteogenic mechanical stimuli in vivo and binding of glutamate to receptors on osteoblasts increases osteoblast number and activity in vitro, control of extracellular glutamate concentrations may be critical for balanced bone remodelling. To determine whether GLAST isoforms may act to regulate extracellular glutamate concentration in bone we investigated whether their pattern or level of expression is responsive to glutamate concentration in bone cells. GLAST-1a mRNA is expressed at lower levels than GLAST-1 mRNA in all cells examined. The GLAST-1a/GLAST-1 mRNA ratio is greater in MLO-Y4 osteocytes than in SaOS-2 osteoblast-like cells, although this does vary in SaOS-2 cells in response to extracellular glutamate concentration. Transfection of MLO-Y4 cells with green fluorescent protein (GFP)-tagged GLAST isoforms revealed a plasma membrane localization of GLAST-1, consistent with its transporter function, whereas GLAST-1a appeared to be expressed within internal vesicles. Interestingly, low extracellular glutamate concentrations redistributed GLAST-1-GFP into a similar internal expression pattern. Regulation of the expression and distribution of GLAST-1 by extracellular glutamate in bone cells indicates that it may regulate glutamate signalling in bone, consistent with its operation in the central nervous system.
authors.affiliation: Connective Tissue Research Laboratories, School of Biosciences, Cardiff University, Museum Avenue, Wales, UK.
authors.forename: J F
authors.initials: JF
authors.lastname: Huggett
authors.forename: A
authors.initials: A
authors.lastname: Mustafa
authors.forename: L
authors.initials: L
authors.lastname: O'neal
authors.forename: D J
authors.initials: DJ
authors.lastname: Mason
mesh_terms: D000818:Animals; D015139:Blotting, Southern; D002462:Cell Membrane / Q000378:metabolism*; D027341:Excitatory Amino Acid Transporter 1 / Q000096:biosynthesis*; D018698:Glutamic Acid / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D010011:Osteocytes / Q000378:metabolism*; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013977:Tibia / Q000166:cytology; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D027341:Excitatory Amino Acid Transporter 1; D008164:Luminescent Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid