Fig. 3.

PKA modulation of E2-mediated calpain activation in cultured neurons and effects of ERα and ERβ agonists. (A and B) Cultured hippocampal neurons were preloaded with the FRET substrate as described in Materials and Methods and treated with inhibitors of adenylate cyclase or PKA (SQ22536, 10 μM and KT5720, 10 μM, respectively) before application of 10 nM E2. Confocal images were taken at the indicated times. (C) Cortical neurons were preloaded with the FRET probe and pretreated with inhibitors of adenylate cyclase or PKA (SQ22536, 10 μM and KT5720, 10 μM, respectively); they were then treated with E2 (10 nM) for 5 min. FRET was analyzed by spectrofluorometry and fluorescence intensity was normalized by subtraction of values from untreated controls; data represent means ± SEM of eight experiments. *, P < 0.01, as compared to control; †, P < 0.001, as compared to E2 alone (ANOVA followed by Bonferroni test). (D) Cultured cortical neurons were preloaded with the FRET probe and treated for 5 min with agonists of ER-α or of ER-β, PPT (10 nM) and DPN (10 nM), respectively. FRET was analyzed by spectrofluorometry and fluorescence intensity was normalized by subtraction of values from untreated controls; data were then expressed as percentage of values found in untreated control and represent means of ± SEM of eight experiments. *, P < 0.01 as compared to control.