Stimulation of β1- and β2-ARs induce differential activation of cytoplasmic and nuclear PKA activity in ARVMs. A–D Representative time course of cytoplasmic and nuclear PKA activities reported by the normalized yellow fluorescent protein (YFP)/cyan fluorescent protein (CFP) ratio in ARVMs transduced with Ad.AKAR3-NES (A and C) or Ad.AKAR3-NLS (B and D) for 24 h at a multiplicity of infection (MOI) of 1000 active viral particles/cell. β1-AR stimulation was achieved using a combination of 10 nM Iso and 10 nM ICI 118, 551 (ICI) (A and B); β2-AR stimulation using 100 nM Iso in combination with 100 nM CGP 20712A (CGP) (C and D). Pseudo-colour images of the YFP/CFP ratio were recorded at the times indicated by the letters on the graphs. Scale bars represent 20 µm. (E and F) Mean variation (±S.E.M.) of the YFP/CFP ratio in ARVMs expressing either AKAR3-NES or AKAR3-NLS upon β1-AR stimulation using 1, 3 and 10 nM Iso in combination with 10 nM ICI (E) or β2-AR stimulation using 10, 30, and 100 nM Iso in combination with 100 nM CGP (F). Number of cells/animals is indicated in brackets. Statistical significance is indicated as ***P < 0.001 vs. ICI+Iso 1 nM or CGP+Iso 10 nM in the cytoplasm, $$P < 0.01 vs. ICI+Iso 1 nM in the nucleus, ###P < 0.001 by nested ANOVA with Tukey’s post hoc test. (G) Nuclear PKA activation (% increase in YFP/CFP ratio in ARVMs expressing AKAR3-NLS) is plotted as a function of cytoplasmic PKA activation (% increase in YFP/CFP ratio in ARVMs expressing AKAR3-NES) for β1- and β2-AR stimulations. Values (±S.E.M.) of (E and F) were used for this graph.
