Figure 4

Inhibition of RAGE-mediated G-protein receptor activity by the formyl peptide receptor antagonist WRW4 in glial cells. For analysis of ERK1/2 phosphorylation, astrocytes (A) and microglia (B) were each treated with 1 μM Aβ1–42, 1 μM fMLF, 2.4 μM S100B or 0.75 μM AGE-BSA (AGE) with or without 10 μM WRW4 and with WRW4 alone for 5 min at 37°C. Cells were lysed, equal amounts of protein (5 μg) were dissolved in SDS sample buffer, and the levels of total ERK2 and phosphorylated phosphorylated ERK1/2 were determined via immunoblotting. The positions of phospho-ERK1/2 (pERK1/2) and total ERK2 (ERK2) along with those of the molecular mass markers (in kDa) are indicated on the left side. The values representing mean ± standard error of the mean (SEM) of phosphorylation levels derived from densitometric quantification of three independent experiments are indicated in (C) and (D). An asterisk indicates a significant difference (* - p < 0.05) compared to control as determined by one-way ANOVA and Bonferroni post-hoc test. In order to analyse the inhibition of forskolin-stimulated adenylate cyclase activity, astrocytes (E) and microglia (F) were subjected to either 10 μM (E) or 25 μM (F) forskolin as well as well as to 1 μM Aβ1–42, 1 μM fMLF, 2.4 μM S100B or 0.75 μM AGE-BSA (AGE) with or without 10 lM WRW4 and to WRW4 alone for 15 min at 37°C. cAMP levels were determined as described above (see Methods). The values given represent mean ± SEM from four independent experiments. Asterisks indicate significant differences (*, p < 0.05) between forskolin plus agonists and forskolin alone as determined by one-way ANOVA and Bonferroni post-hoc tests.