Platelet-derived Growth Factor Rapidly Increases Activity and Cell Surface Expression of the EAAC1 Subtype of Glutamate Transporter through Activation of Phosphatidylinositol 3-Kinase

Abstract

Na؉ -dependent glutamate transporters are the primary mechanism for removal of excitatory amino acids (EAAs) from the extracellular space of the central nervous system and influence both physiologic and pathologic effects of these compounds. Recent evidence suggests that the activity and cell surface expression of a neuronal subtype of glutamate transporter, EAAC1, are rapidly increased by direct activation of protein kinase C and are decreased by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K). We hypothesized that this regulation could be analogous to insulin-induced stimulation of the GLUT4 subtype of glucose transporter, which is dependent upon activation of PI3-K. Using C6 glioma, a cell line that endogenously and selectively expresses EAAC1, we report that platelet-derived growth factor (PDGF) increased Na3 H]-glutamate transport activity within 30 min. This effect of PDGF was not due to a change in total cellular EAAC1 immunoreactivity but was instead correlated with an increase cell surface expression of EAAC1, as measured using a membrane impermeant biotinylation reagent combined with Western blotting. A decrease in nonbiotinylated intracellular EAAC1 was also observed. These studies suggest that PDGF causes a redistribution of EAAC1 from an intracellular compartment to the cell surface. These effects of PDGF were accompanied by a 35-fold increase in PI3-K activity and were blocked by the PI3-K inhibitors, wortmannin and LY 294002, but not by an inhibitor of protein kinase C. Other growth factors, including insulin, nerve growth factor, and epidermal growth factor had no effect on glutamate transport nor did they increase PI3-K activity. These studies suggest that, as is observed for insulin-mediated translocation of GLUT4, EAAC1 cell surface expression can be rapidly increased by PDGF through activation of PI3-K. It is possible that this PDGF-mediated increase in EAAC1 activity may contribute to the previously demonstrated neuroprotective effects of PDGF.The rapid clearance of glutamate from the extracellular space of the central nervous system by Na ϩ -dependent high affinity glutamate transporters is critical to the maintenance of effective synaptic transmission and the prevention of excitotoxic injury. Increases in extracellular EAAs 1 after head trauma and ischemic events have been described (1-3) and are presumably related to both a failure of inward transport and increased reverse operation of the carriers (4, 5). A family of glutamate transporters mediates this high affinity uptake and includes five members, the glial transporters GLT-1 (human homologue EAAT2) and GLAST (EAAT1), the neuronal transporters EAAC1 (EAAT3) and EAAT4, and the retinal transporter EAAT5 (6 -10). The EAAC1 subtype of transporter is enriched in the pyramidal cells of hippocampus and cortex (11,12), two areas rich in glutamatergic transmission and exquisitely sensitive to excitotoxic insults (13). Animals treated with antisense oligonucleotides to "knock down" EAAC1 expression develop a seizure phenotype,...

Glutamate transporters in platelets: EAAT1 decrease in aging and in Alzheimer’s disease

“…These results do not exclude EAAT2 involvement in physiological platelet uptake, since it could be a platelet transporter with a slower kinetic rate and it could begin to work at higher glutamate concentrations, when EAAT1 and EAAT3 could be endangered. The EAAT2 subcellular distribution (70% in cytoplasmic fraction) could also justify its minor involvement in physiological glutamate uptake of platelet; EAAT2 could be available in platelet membranes after translocation induced by particular stimuli, as described for EAAT3 in CNS-derived cell lines [48]. It must be also underlined that GFD-8 cell line did not express any EAAT2 immunoreactivity, but clearly showed EAAT2 mRNA.…”

“…As recent studies have also shown that glutamate transporter expression and activity may be regulated by different endogenous or exogenous factors [6,8,10,13,15,27,31,35,44,48,53], we are now studying the possible role of physiological stimuli, such as PKC activation, or oxidative stress on glutamate transporter activation/inactivation or translocation in platelets. The final goal of these studies is the possibility of using platelets as peripheral model to investigate the role of glutamate, its transporters and its effect pathway in neurodegenerative disorders, with the clear advantage of being easy to obtain, suitable for pharmacological and clinical studies and, compared to human brain tissue studies, of being independent from postmortem or end-stage changes.…”

The Transcription Factor Regulatory Factor X1 Increases the Expression of Neuronal Glutamate Transporter Type 3

“…It has been shown that multiple agents, such as volatile anesthetics, cause acute changes of EAAT activity (9,10). In EAAT3, this acute change of activity is mediated by the redistribution of EAAT3 from intracellular compartments to the plasma membrane, the functional site of EAAT3 (9).…”

Fyn-mediated Phosphorylation of NR2B Tyr-1336 Controls Calpain-mediated NR2B Cleavage in Neurons and Heterologous Systems

“…Twenty-four hours after transfection, cell surface proteins were labeled with biotin and isolated (16). Cells were washed twice with cold rinsing solution (phosphate-buffered saline with Ca/Mg (1 mM MgCl 2 and 0.1 mM CaCl 2 , pH 7.35)) (27) and incubated in rinsing solution containing 1 mg/ml EZ-Link Sulfo-NHS-Biotin (Pierce) for 30 min at 4°C. Cells were washed twice with quenching solution (rinsing solution with 100 mM glycine added), incubated in quenching solution for 20 min, and solubilized at 4°C in radioimmune precipitation buffer ((150 mM NaCl, 1 mM EDTA, and 100 mM Tris-HCl, pH 7.4), 1% (v/v) Triton X-100, 1% (w/v) deoxycholate, and 0.1% (w/v) SDS containing protease inhibitors (1 g/ml leupeptin, 250 M phenylmethylsulfonyl fluoride, 1 g/ml aprotinin, 1 mg/ml trypsin inhibitor, and 1 mM iodoacetamide).…”