Figure 7

Uptake of 8-[ϕ-575]-cAMP in intact cells. Kinetics of 8-[ϕ-575]-cAMP (a-d) and of Sp-5,6-DCI-cBIMPS (e-h) uptake in living cells. HEK293 cells were transfected for transient expression of the H30 indicator of cAMP and treated with 100 μM IBMX to inhibit phosphodiesterases. The ratio between the background subtracted emission intensities at 480 nm and 545 nm is plotted as a function of time. The vertical bars indicate the administrations of either 500 μM 8-[ϕ-575]-cAMP (a-d) or Sp-5,6-DCl-cBIMPS (e-h) and of 25 μM Forskolin, which activates adenylyl cyclase and saturates the FRET-based probe. The insets show the yellow fluorescent protein fluorescence at the beginning of the experiments and the regions of interest where FRET ratios are calculated.