Equilibrium between adenylyl cyclase and phosphodiesterase patterns adrenergic agonist dose-dependent spatiotemporal cAMP/protein kinase A activities in cardiomyocytes.

Beta-adrenergic receptor induces cAMP/Protein kinase A (PKA) activation to regulate cardiac contraction. Using real-time fluorescence resonance energy transfer imaging for highly sensitive detection of cAMP and PKA activities, we show two distinct phases in isoproterenol dose-dependent responses in cardiomyocytes: a transient and dose-dependent increase in cAMP and PKA activities at lower concentrations from 10(-12) to 10(-8) M; and a saturated initial increases at higher concentrations from 10(-8) to 10(-5) M followed by a rapid decrease to different levels that were later sustained in a dose-dependent manner. The dose-dependent temporal responses are patterned by equilibrium between receptor-activated adenylyl cyclase (AC) and phosphodiesterase (PDE). At lower concentrations, cAMP is produced in an agonist dose-dependent manner with AC as a rate-limiting factor. However, the cAMP activities are confined within local domains for phosphorylation of PDE isoforms in the receptor complex but not for phosphorylation of phospholamban and troponin I. At higher concentrations, isoproterenol promotes a dose-dependent selective dissociation of PDE4D but not ACVI from the receptor complex, which shifts the equilibrium between AC and PDE. This shifted balance leads to sustained cAMP accumulation and diffusion for PKA phosphorylation of phospholamban and troponin I, and for myocyte contraction. Pharmacological inhibition or overexpression of either ACVI or PDE4D8 disrupts the balance and shapes the temporal responses in cAMP accumulation. Together, our data reveal a new paradigm for adrenergic agonist dose-dependent cAMP/PKA activities for substrate-specific phosphorylation dictated by dual regulation of AC and PDE in cardiomyocytes.