Transient upregulation of the glial glutamate transporter GLAST in response to fibroblast growth factor, insulin-like growth factor and epidermal growth factor in cultured astrocytes
Keiko Suzuki1,
Yuji Ikegaya1,*,
Sigeru Matsuura1,
Yoshikatsu Kanai2,
Hitoshi Endou2 and
Norio Matsuki1
1 Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
2 Department of Pharmacology and Toxicology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 180-8611, Japan
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Fig. 1. bFGF enhances THA-sensitive glutamate uptake activity in astrocyte cultures. (A) [3H]glutamate uptake activity of confluent astrocyte cultures was measured in [3H]glutamate-free or Na+-free medium or in the presence of THA, a non-selective inhibitor of glutamate transporters, or DHK, a selective inhibitor of GLT-1. (B) [3H]glutamate uptake activity in the absence or presence of these inhibitors was measured in the astrocytes treated with bFGF for 24 hours. The relative activity is expressed as a percentage of that in intact astrocytes (Control). bFGF enhanced THA-sensitive glutamate transport activity in a concentration-dependent manner. (*<0.05; **p<0.01 vs control; #p<0.05; ##p<0.01 vs 3 ng/ml bFGF, Tukey’s test following ANOVA). Data are means±s.e.m. of 4-8 cases.
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Fig. 2. Effects of growth factors on glutamate uptake activity in astrocyte cultures. Astrocytes were treated with IGF-1, EGF, insulin, PDGF-AB or HGF for 24 hours, and the glutamate uptake activity was measured. The relative activity is expressed as a percentage of control levels. Of these growth factors, IGF-1 and EGF induced an increase in the uptake activity in a concentration-dependent manner. (*p<0.05; **p<0.01 vs control, Tukey’s test following ANOVA). Data are means±s.e.m. of 4-6 cultures.
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Fig. 3. Analysis of the glutamate transport activity of growth factor-treated astrocytes. A concentration dependence of glutamate transport activity was assessed in the astrocytes treated with 3 ng/ml bFGF (A), 100 ng/ml IGF-1 (B), and 30 ng/ml EGF (C) for 24 hours. The Vmax and Km values were calculated by a linear regression analysis of an Eadie-Hofstee transformation. All growth factors significantly enhanced the Vmax value for transport activity (P<0.001, Student’s t-test). Data are means±s.e.m. of 4-12 cultures.
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Fig. 4. Transcriptional and translational dependency of the effects of growth factors. In the presence of 1 µM actinomycin D (A) or 1 µM cycloheximide (B), astrocytes were treated with 3 ng/ml bFGF, 100 ng/ml IGF-1, or 30 ng/ml EGF for 24 hours. The facilitatory effects of the growth factors on glutamate transport activity were completely blocked by either the translational inhibitor cycloheximide or the transcriptional inhibitor actinomycin D. (*p<0.05; **p<0.01 vs control; ##p<0.01 vs the corresponding growth factor, Tukey’s test following ANOVA). Data represent means±s.e.m. of 4 cultures.
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Fig. 5. Growth factors increase the expression level of GLAST. (A) Western blot analysis of the glutamate transporter GLAST (a) or GLT-1 (b) was performed for the rat whole brain or the cortical astrocytes cultured with either 3 ng/ml bFGF, 100 ng/ml IGF-1 or 30 ng/ml EGF for 36 hours. Anti-GLAST or GLT-1 antibody recognized a protein with a molecular weight of  60 kDa. GLAST expression was increased by the incubation in the presence of bFGF, IGF-1 or EGF. GLT-1 expression was virtually undetected in the astrocyte cultures. (B) Immunohistochemical localization of GLAST (green) in the astrocytes cultured in the absence (a) or presence (b) of 3 ng/ml bFGF for 36 hours (left panels). Propidium iodide (red) was used for the counterstaining. Right panels show a nonspecific staining of the secondary IgG-FITC in the absence of anti-GLAST antibody. The result confirmed that bFGF enhanced the GLAST protein levels. (C) Northern blots were performed by using mRNA isolated from the rat whole brain, or untreated (Control) and 3 ng/ml bFGF-treated astrocytes. Treatment with bFGF for 24 hours induced an increase of GLAST mRNA levels.
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Fig. 6. Transient upregulation of GLAST expression following bFGF treatment. Time course of changes in glutamate uptake (A) and GLAST mRNA (B) was assessed in cultured astrocytes treated with 3 ng/ml bFGF. (A) The level of glutamate uptake is expressed relative to the uptake rate of untreated astrocytes (pre). The bFGF effect reached a peak after 36 hours, and rapidly returned to baseline by 48 hours. (*p<0.05; **p<0.01 vs pre, Tukey’s test following ANOVA). Data represent means±s.e.m. of 4 cultures. (B) A representative northern blot of GLAST mRNA. Numbers above the lanes indicate the duration of bFGF treatment. An increase in the mRNA levels was observed at 18 to 36 hours.
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Fig. 7. Additive effects of growth factor combinations on glutamate uptake in cultured astrocytes. Astrocytes were treated with each growth factor alone or together with others for 36 hours. A combination of IGF-1 with either bFGF or EGF showed an additive effect on glutamate uptake activity, but a combination of bFGF and EGF did not. (*p<0.05 vs bFGF alone; #p<0.05; ##p<0.01 vs IGF-1 alone; $p<0.05 vs EGF alone; !!p<0.01 vs a combination of bFGF and EGF, Tukey’s test following ANOVA). Data represent means±s.e.m. of 4 cultures.
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© The Company of Biologists Ltd 2001
