Dynamic protein kinase a activities induced by β-adrenoceptors dictate signaling propagation for substrate phosphorylation and myocyte contraction

CAMP/protein kinase (PK)A activation represents a key signaling mechanism for neurohormonal stimulation of diversified physiological processes. Using real-time, fluorescence resonance energy transfer-based imaging of PKA activity in neonatal cardiac myocytes, we report that sustained activation of PKA induced by β-adrenoceptor (βAR) dictates signaling propagation for substrate phosphorylation and myocyte contraction. Activation of βARs in wild-type myocytes induces strong and sustained PKA activities, which are rapidly attenuated on washing away agonist or adding antagonist to the cells. The sustained PKA activities promote signaling propagation to the sarcoplasmic reticulum for phosphorylation of phospholamban and increases in myocyte contraction. Addition of antagonist after βAR stimulation significantly attenuates PKA phosphorylation of phospholamban and rapidly reduces contraction rate increases. Moreover, stimulation of β₁AR subtype induces PKA activities similar to those in wild-type cells. In contrast, stimulation of β₂AR subtype induces strong initial activation of PKA similar to those induced by β₁AR; however, the activities are rapidly decreased to baseline levels. The transient PKA activities are sufficient for phosphorylation of the overexpressed β₂ARs under agonist stimulation, but not phospholamban. Further analysis reveals that phosphodiesterase 4 is the major family that shapes PKA activities under βAR stimulation. Inhibition of phosphodiesterase 4 extends β₂AR-induced PKA activities, promotes PKA phosphorylation of phospholamban, and ultimately enhances myocyte contraction responses. Together, our data have revealed insights into kinetics of PKA activities in signaling propagation under neurohormonal stimulation.