Fig. 4: Misoprostol stimulates EP4 receptors to activate PKA and promote the modulation of a novel PKA phosphorylation site on Bnip3 at Thr-181 in cells and the PND10 neonatal heart.
A Mitochondrial calcium retention capacity (CRC) in isolated mitochondria exposed to 10 μM misoprostol (Miso) or PBS control. B Mitochondrial swelling assay in isolated mitochondria treated as in (A). C H9c2 cells exposed to 1% O2 (HPX) or 21% O2 (control) and treated with 10 μM misoprostol (Miso) or PBS control for 24 h. 10 μM L161,982 was also included in half of the conditions. Cells were stained with TMRM (red) and hoechst (blue) and imaged by standard fluorescence microscopy. D Quantification of cells in (C), where red fluorescent signal was normalized to cell area and quantified in 30 random fields, across 3 independent experiments. E H9c2 cells treated, stained and imaged as in (C). 1 μM L798106 was also included in half of the conditions. F Quantification of cells € (E), as in (D). G Quantification of H9c2 cells treated as in (C) with the addition of 10 μM H-89 for 24 h. Cells were imaged as in (C) and quantified as in (D). H SIM scan of the wild-type peptide spanning the PKA site of Bnip3. The unphosphorylated peptide has a 837 m/z (z = 2+) (Left), putative phosphorylation showing an increased m/z of 40 that corresponds to PO3 (M = 80.00 Da) (Right). I MS2 spectra following collision induced dissociation (CID) of the mass shifted ion from (H) yielding a product-ion consistent with a neutral loss of H3PO4. J MS2 spectra following electron transfer dissociation (ETD) of a triply charged mass-shifted ion following kinase reaction (not shown). Analysis of this fragmentation spectra confirmed that threonine-181 is the preferred phosphorylation residue. K Alignment of evolutionarily conserved T181 phosphorylation site (bold and underlined), and 14-3-3 binding motifs (Red) for Bnip3 in human, mouse and rat. L Immunoblot of H9c2 cells transfected with Myc-Bnip3 and/or PKA for 16 h. M Representative immunoblot of heart protein extracts from post-natal day (PND10) mice exposed to hypoxia (10% O2) ± 10 μg/kg misoprostol from PND3-10. Extracts were immunoblotted as indicated. N Phospho-Bnip3 densitometry for extracts in (M), representing an N of 3 male PND10 mouse hearts. All data are represented as mean ± S.E.M. *P < 0.05 compared with control, while **P < 0.05 compared with hypoxia treatment, determined by 1-way ANOVA or 2-way ANOVA where appropriate.
