Figure 2.

PKA is activated in hepatocytes by β-adrenergic/cAMP stimulation. A and C, pseudocolored ratiometric images of Hep3B cells expressing the FRET PKA biosensor AKAR4-NES before and 5 min after treatment with 50 μm isoproterenol (Iso; A and A′) or 10 μm forskolin (C and C′). B and D, graphs show quantification of FRET/CFP fluorescence intensity in representative cells upon treatment with isoproterenol (B) or forskolin (D), which revealed a 1.14- and 1.45-fold increase in PKA activity in response to these agonists, respectively. This activation reached its peak within just 2 min of treatment. E, Western blot analysis of cell lysates from primary rat hepatocytes shows a substantial increase in the number and intensity of PKA-phosphorylated substrates following 24 h of stimulation with 50 μm isoproterenol or 10 μm forskolin + 0.5 mm IBMX. CT, control.