Figure 5.

Neuronal protection by EPO is mediated by the activation of Akt1, phosphorylation of Bad, and the prevention of mitochondrial membrane depolarization and cytochrome c release. In (A) and (B), equal amounts of neuronal protein extracts (50 μg per lane) were immunoblotted with antiphospho-Akt1 (p-Akt1, active Akt1, Ser 473) antibody. Exposure to EPO (1 U ml⁻¹) or NO significantly increased p-Akt1 expression. Application of the PI-3K inhibitor wortmannin (100 nM) or LY294002 (LY) (10 μM) was sufficient to block the expression of active p-Akt1 in the presence of EPO during NO (NOC-9, 300 μM) exposure. (C) At a concentration that blocks activation of p-Akt1 during NO administration (NOC-9 or SNP, 300 μM), wortmannin (100 nM) or LY294002 (10 μM) applied 1 h prior to NO significantly reduced the protective capacity of EPO (1 U ml⁻¹) during NO exposure (^*P<0.01 vs NO; †P<0.01 vs EPO). (D) Equal amounts of neuronal protein extracts (50 μg per lane) were immunoblotted with antiphosphorylated Bad (p-Bad, Ser 136) antibody. Exposure to EPO (1 U ml⁻¹) or NO significantly increased p-Bad expression. EPO application further increased phosphorylation of Bad during NO exposure. Application o of the PI-3K inhibitor wortmannin (100 nM) was sufficient to block the expression of p-Bad in the presence of EPO during NO (NOC-9, 300 μM) exposure. (E, F) Exposure to NO (NOC-9, 300 μM) produced a significant decrease in the red/green fluorescence intensity ratio using a cationic membrane potential indicator JC-1 within 3 h when compared with untreated control cultures, suggesting that NO results in mitochondrial membrane depolarization. Application of EPO (1 U ml⁻¹) 1 h prior to NO exposure significantly increased the red/green fluorescence intensity of neurons, indicating that mitochondrial permeability transition pore membrane potential was restored to baseline (E,F). (G) A representative Western blot with equal amounts of mitochondrial or cytosol protein extracts (50 μg per lane) were immunoblotted demonstrating that application of EPO (1 U ml⁻¹) significantly prevented cytochrome c release from mitochondria during NO exposure. In (C) and (F), to simplify the figures, the results of the two NO donors were combined. In all cases, control=untreated neurons.