Fig. 2.

E2/TX reverses Mn-induced glutamate uptake inhibition in astrocytes. After treatment with Mn or E2/TX, cells were incubated with 100 nM of unlabeled glutamate containing 0.25 μCi of [³H]glutamate (specific activity: 49.0 Ci/mmol) for 10 min. The uptake was terminated by washing the cells with ice-cold PBS buffer as described in the Methods section. (a) Glutamate uptake was THA-sensitive, Na⁺-dependent and GLAST subtype-sensitive. [³H]glutamate uptake was measured in uptake buffer, Na+-free medium, in the presence of THA (a non-selective inhibitor of glutamate transporters), MSO (an inhibitor of glutamine synthase) or DHK (a selective inhibitor of GLT-1). (MSO, methionine sulfoximine; THA, dl-threo-μ-hydroxyaspartic acid; DHK, dihydrokainic acid). (b) Mn (6 h) inhibited glutamate uptake in a concentration-dependent manner. (c) E2/TX (24 h) increased glutamate uptake. (d) E2/TX (18 h pre-treatment) reversed Mn (6 h)-induced glutamate uptake inhibition. (e) E2/TX-induced enhancement of glutamate uptake was ER-dependent. ^###p < 0.001 vs. control (d); *p < 0.05, **p < 0.01, ***p < 0.001, ^##p < 0.01 vs. control (b,c,e) or Mn treatment (d); Tukey's test following anova. Data are expressed as the mean ± SEM (n = 5–8).