Figure 4

Blockade of adenosine A _2A receptors prevents the interleukin (IL)-1β-induced activation of the mitogen-activated protein kinases (MAPKs) Jun kinase (JNK) and p38 in hippocampal cultured neurons. Rat hippocampal neuronal cultures at 7 days in vitro (DIV) were exposed to 100 ng/ml IL-1β for 15 minutes in the absence or presence of 50 nmol/l SCH58261, an antagonist of adenosine A_2A receptors, which was added 20 minutes before addition of IL-1β. Cells were then lysed on ice in a minimum volume of radio-immunoprecipitation assay (RIPA) buffer for western blotting analysis to quantify the immunoreactivity of the activated (phosphorylated) form of (A) p38 (p-p38) and (B) JNK (p-JNK). In both panels, the bar graphs display the mean ± SEM of five to six experiments, and the blots below illustrate a representative experiment showing that SCH58261 prevented the IL-1β-induced phosphorylation of (A) p38 and (B) JNK, without having any effect itself on either of these. In each experiment, membranes were first used to detect each phosphorylated MAPK and then reprobed for the total amount of MAPK, so that the activation of each MAPK was calculated as a ratio between the phosphorylated and total immunoreactivities, which were expressed as a percentage of control (CTR) values (that is, in the absence of any added drug). *P < 0.05 compared with control; #P < 0.05 and ##P < 0.01 compared with neurons exposed to IL-1β only.