Figure 4: PDE4 and PDE3 have distinct roles in regulating cAMP around AKAP95.

The effect of isoform-specific PDE inhibition was tested in the AKAP95 microdomain. Cells expressing AKAP95-targeted ICUE3 and PM-sAC were co-treated with PDE inhibitor and rapamycin (100 nM) and subsequently with NaHCO₃ (LD, 2.5 mM; HD, 15 mM) to generate cAMP at the plasma membrane via PM-sAC. a) AKAP95-targeted ICUE3 emission ratio (cyan/yellow) responses when cells were treated with the PDE4 inhibitor rolipram (1 μM) (purple, N=29 cells), the PDE3 inhibitor milrinone (10 μM) (blue, N=19 cells), or without inhibitor treatment (black, N=39 cells). Cells were additionally treated with a low dose (LD, 2.5 mM) and subsequently a high dose (HD, 15 mM) of NaHCO₃. b) The initial FRET response after inhibitor addition indicates basal PDE activity in the AKAP95 microdomain. The change in normalized ICUE3 emission ratio (cyan/yellow) is shown. There is a statistically significant response in cells treated with rolipram (p<0.0001) but not in cells treated with milrinone (p=0.1293). c) After low-dose NaHCO₃ stimulation, a statistically significant response is observed in PDE4-inhibited cells (p<0.0001) and PDE3-inhibited cells (p=0.0002) compared to cells lacking inhibitor treatment. d) AKAP95-targeted ICUE3 emission ratio (cyan/yellow) responses in cells expressing dnPDE4D5 (purple, N=24 cells) or cells not expressing dominant negative PDE isoforms (black, N=35 cells). Cells were treated with LD (2.5 mM) followed by HD (15 mM) NaHCO₃ to stimulate PM-sAC. e) LD PM-sAC stimulation induced statistically significant responses in cells expressing the dominant negative PDE4D5 compared to control cells (inset, p<0.0001).