0125: Control of cytoplasmic and nuclear PKA activity by β1- and β2-adrenoceptors in cardiac myocytes: role of phosphodiesterases

Abstract

β 1 - and β 2 -adrenoceptors (β-AR) increase cAMP and activate cAMP-dependant protein kinase (PKA) to regulate cardiac contraction and gene expression. Functional differences between β 1 -AR and β 2 -AR stimulations are attributed to spatial confinement of β 2 -AR signalling and differential coupling to cAMP degradation by phosphodiesterases (PDEs). Here, we compared β 1 -AR and β 2 -AR activation of PKA in the cytoplasm and the nucleus of adult rat ventricular myocytes (ARVMs) using adenovirally encoded A-kinase activity reporter (AKAR) based on fluorescence resonance energy transfer (FRET) and targeted to these compartments. Isoprenaline (Iso, 10nM) was used in combination with the β 2 -AR antagonist ICI118551 (ICI, 10nM) or with the β 1 -AR antagonist CGP20712A (100nM) for specific β 1 -AR or β 2 -AR stimulation. β 1 -AR stimulation strongly activated PKA in both compartments, but PKA activation was delayed and reduced in the nucleus (+17±2%, t1/2on=1,7±0,1 min, n=21) compared to the cytoplasm (+35±2%, t1/2on=0,6±0,1 min, n=15). β 2 -AR stimulation induced a small increase in PKA activity only in the cytoplasm (+8,8±1%, n=24, p<0.05). β 1 -AR stimulation with 1 nM Iso + 10nM ICI led to a similar degree of cytoplasmic PKA activation as β 2 -AR stimulation but increased nuclear PKA activity (+3,6±0,5%, n=28, p<0.05). PDE3 inhibition with Cilostamide (Cil, 1μM) potentiated PKA activation by β 1 -AR (1 nM Iso) in the cytoplasm and the nucleus, and by β 2 -AR in the cytoplasm, but failed to reveal β 2 -AR response in the nucleus. PDE4 inhibition with Ro-201724 (Ro, 10μM) potentiated β 1 -AR (1 nM Iso) response in the cytoplasm and the nucleus, increased β 2 -AR responses in the cytoplasm and revealed β 2 -AR dependent nuclear PKA activation. These results show that β 1 -AR and β 2 -AR stimulation differently impact on cytoplasmic and nuclear PKA activity and that PDE4 is important to prevent nuclear PKA activation under β 2 -AR stimulation.