Figure 7.

METH-induced activation of PKA and CaMKII differentially regulates astrocyte EAAT-2. Cultured human astrocyte transfected with siCON (clear bars), siPKA (light gray bars), and siCaMKII (dark gray bars) were treated with METH (500 μM, hatched bars). Alternatively, astrocytes were pretreated with cAMP-dependent PKA inhibitor, PKI (light gray bars), or CaMKII inhibitor, KN62 (dark gray bars), for 1 h prior to METH treatment (hatched bars). RNA was collected from transfected and pretreated astrocytes, and EAAT-2 mRNA levels were analyzed 8 h post-METH treatment (A,C). Glutamate clearance was quantified at 10 h post-glutamate addition (B,D). Astrocytes pretreated with PKA and CaMKII inhibitor +/– METH treatment were immunostained for GFAP (red), EAAT-2 (green), or DAPI [blue, (E–J)]. Statistical analyses were performed using GraphPad Prism V6.0 with one-way ANOVA and Tukey's post-test for multiple comparisons. P ≤ 0.05 were considered statistically significant, and data represent means ± SEM. This figure depicts representative donors chosen from multiple astrocyte donors that were tested; each was analyzed in a minimum of triplicate determinations. Data are shown as cumulative fold changes (*p < 0.05, **p < 0.01, ***p < 0.001, ^###p < 0.001).