Figure 4

The LPS-induced enhancement of BDNF secretion mediated by A _2A R involves the recruitment of the AC-cAMP-PKA transducing pathway, whereas PKC controls the constitutive release of BDNF from N9 microglial cells. In all experiments, the medium was collected after 6 hours for quantitative analysis of extracellular BDNF (ELISA). (A) Cells were incubated with the A_2AR agonist CGS21680 (30 nM) in the absence or in the presence of the A_2AR antagonist SCH58261 (50 nM) or LPS (100 ng/mL). (B) Cells were incubated with the adenylyl cyclase (AC) activator, forskolin (1 μM) or with the cAMP analog, 8-Br-cAMP (5 μM) in the absence and in the presence of LPS (100 ng/mL). (C) Cells were incubated with the PKA inhibitor, H89 (1 μM) or with the PKC inhibitor, chelerythrine (6 μM) in the absence and in the presence of LPS (100 ng/mL). Results are expressed as mean ± SEM of n (as indicated in each bar) independent experiments (*** P < 0.001, ** P < 0.01, * P < 0.05, compared with non-treated cells; ·· P < 0.01, compared with LPS-treated cells, using the Newman–Keuls multiple comparison test) and 100% represents the pro- and mBDNF in cells that were not exposed to LPS. We verified (not shown) that none of the vehicles of the tested drugs (water or dimethyl sulfoxide) modified BDNF levels. A_2AR, A_2A receptor; ADA, adenosine deaminase; BDNF, brain-derived neurotrophic factor; cAMP, cyclic AMP; LPS, lipopolysaccharide; PKA, protein kinase A; PKC, protein kinase C; SEM, standard error of the mean.