Figure 5

Inhibition of Neisseria meningitidis - and Streptococcus pneumoniae- induced ERK1/2 phosphorylation, and changes of cAMP levels by WRW4 and PTX in glial cells. For analysis of ERK1/2 phosphorylation, astrocytes (A) and microglia (B) were each treated with Neisseria meningitidis (NM) or Streptococcus pneumoniae (SP); with NM as well as SP for 5 min at 37°C with 200 ng/ml PTX (16 h preincubation) or 10 μM WRW4 (30 min preincubation); they were also treated with PTX (16 h preincubation) or WRW4 (30 min preincubation) alone as control. Cells were lysed, equal amounts of protein (5 μg) were dissolved in SDS sample buffer, and the levels of total ERK2 and 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 right or left side, respectively. The values representing mean ± standard error of the mean (SEM) of phosphorylation levels derived from densitometric quantification of three independent experiments in astrocytes and microglia are indicated in (C) and (D), respectively. An asterisk indicates a significant difference (*, p < 0.05; **, p < 0,01; ***, p < 0,001) compared to controls (also with DMSO in equivalent amount) as determined by one-way ANOVA and Bonferroni post-hoc tests. 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 to NM, SP or 1 μM fMLF and NM, SP or fMLF with 10 μM WRW4 (30 min preincubation) 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; ***, p < 0,001) between forskolin plus agonists and forskolin alone as determined by one-way ANOVA and Bonferroni post-hoc tests.