Distribution of Glutamate Transporter GLAST in Membranes of Cultured Astrocytes in the Presence of Glutamate Transport Substrates and ATP

Abstract

Neurotransmitter L-glutamate released at central synapses is taken up and "recycled" by astrocytes using glutamate transporter molecules such as GLAST and GLT. Glutamate transport is essential for prevention of glutamate neurotoxicity, it is a key regulator of neurotransmitter metabolism and may contribute to mechanisms through which neurons and glia communicate with each other. Using immunocytochemistry and image analysis we have found that extracellular D-aspartate (a typical substrate for glutamate transport) can cause redistribution of GLAST from cytoplasm to the cell membrane. The process appears to involve phosphorylation/dephosphorylation and requires intact cytoskeleton. Glutamate transport ligands L-trans-pyrrolidine-2,4-dicarboxylate and DL-threo-3-benzyloxyaspartate but not anti,endo-3,4-methanopyrrolidine dicarboxylate have produced similar redistribution of GLAST. Several representative ligands for glutamate receptors whether of ionotropic or metabotropic type, were found to have no effect. In addition, extracellular ATP induced formation of GLAST clusters in the cell membranes by a process apparently mediated by P2 receptors. The present data suggest that GLAST can rapidly and specifically respond to changes in the cellular environment thus potentially helping to fine-tune the functions of astrocytes.

Expression of multiple glutamate transporter splice variants in the rodent testis

“…20 Our ability to detect uptake of D-aspartate into interstitial cells that express immunoreactivity for C-terminal GLAST/GLAST1a/GLAST1b suggests that these proteins may be functional transporters. The lack of detectable amino-terminal GLAST in the tissue, despite being consistently detected in brain tissues in our hands, 19,24,40,41 suggests that the amino-terminus may be modified or cleaved in the testis. Susarla et al 42 have noted that increased transport activity by GLAST can be associated with a paradoxical loss of immunoreactivity for N-and C-terminal epitopes of GLAST, a phenomenon previously noted as indicating the modification or cleavage of these regions.…”

Rottlerin Inhibits (Na+, K+)-ATPase Activity in Brain Tissue and Alters d-Aspartate Dependent Redistribution of Glutamate Transporter GLAST in Cultured Astrocytes

“…Cultured astrocytes were prepared from neocortices of Sprague-Dawley rat pups (0-3 days post natum) as described in detail in a related study [8]. Briefly, the tissue was dissociated with trypsin (0.25% in Hanks balanced salt solution) and the cells were seeded, using Dulbecco modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS), into 25 cm [2] flasks (usually 2-3 neonatal brains per flask) and grown until confluent (10-14 days).…”

“…The procedure has been described in detail elsewhere [8]. The coverslips with astrocytes were washed in serum-free DMEM (sfDMEM) and incubated in the presence of D-aspartate and/or rottlerin dissolved in 500 ll of sfD-MEM.…”

“…Deconvolution microscope (Axioplan 2, Zeiss) was used for image acquisition [8]. AF 488 and AF 594 were excited at 499 nm (emission at 520 nm) and 590 (emission at 618 nm), respectively.…”

“…Moreover, GLAST transporter appears to be activity regulated, i.e. in the presence of ligands for the glutamate recognition site, especially when these are readily transportable substrates such as D-aspartate, GLAST molecules tend to change their cellular distribution by moving into plasma membrane thereby increasing the transport capacity [8] (for reviews see [5,9,10]). The necessary ionic gradients have to be maintained by the (Na ?…”