Abstract-A current challenge in cellular signaling is to decipher the complex intracellular spatiotemporal organization that any given cell type has developed to discriminate among different external stimuli acting via a common signaling pathway. This obviously applies to cAMP and cGMP signaling in the heart, where these cyclic nucleotides determine the regulation of cardiac function by many hormones and neuromediators. Key Words: cAMP Ⅲ cGMP Ⅲ heart Ⅲ G protein-coupled receptor Ⅲ phosphodiesterase T he cyclic nucleotides cyclic adenosine 3Ј,5Ј-monophosphate (cAMP) and cyclic guanosine 3Ј,5Ј-monophosphate (cGMP) were identified more than 4 decades ago. 1,2 Since then, many studies have appeared on how these 2 second messengers are synthesized or degraded, what makes their level go up or down, what they do to target effectors by either covalent (phosphorylation) or noncovalent (direct binding to proteins, such as ion channels or guanine-nucleotide-exchange factors) mechanisms, and how they affect a countless number of cellular functions. [3][4][5][6] In certain tissues and organs, the cyclic nucleotide pathways have been so fully explored over the years that one can wonder what else is there to be found. This is the case for cAMP in the heart, where it plays a key role in the sympathetic nerve/␤-adrenergic receptor (␤-AR)/adenylyl cyclase (AC)/protein kinase A (PKA) axis that serves to stimulate cardiac rhythm (chronotropy) as well as contractile force (inotropy) and relaxation (lusitropy). 7 Yet, there are a number of questions that have always made us wonder but have only lately begun to receive the attention they deserve: how so many different receptors coupled to cAMP or cGMP signaling pathway manage to achieve specific cellular responses? What is the purpose of the different adenylyl and guanylyl cyclases present in the same cell? Why do Original

Abstract-Steady-state activation of cardiac ␤-adrenergic receptors leads to an intracellular compartmentation of cAMP resulting from localized cyclic nucleotide phosphodiesterase (PDE) activity. To evaluate the time course of the cAMP changes in the different compartments, brief (15 seconds) pulses of isoprenaline (100 nmol/L) were applied to adult rat ventricular myocytes (ARVMs) while monitoring cAMP changes beneath the membrane using engineered cyclic nucleotide-gated channels and within the cytosol with the fluorescence resonance energy transfer-based sensor, Epac2-camps. cAMP kinetics in the two compartments were compared to the time course of the L-type Ca 2ϩ channel current (I Ca,L ) amplitude. The onset and recovery of cAMP transients were, respectively, 30% and 50% faster at the plasma membrane than in the cytosol, in agreement with a rapid production and degradation of the second messenger at the plasma membrane and a restricted diffusion of cAMP to the cytosol. I Ca,L amplitude increased twice slower than cAMP at the membrane, and the current remained elevated for Ϸ5 minutes after cAMP had already returned to basal level, indicating that cAMP changes are not rate-limiting in channel phosphorylation/dephosphorylation. Inhibition of PDE4 (with 10 mol/L Ro 20-1724) increased the amplitude and dramatically slowed down the onset and recovery of cAMP signals, whereas PDE3 blockade (with 1 mol/L cilostamide) had a minor effect only on subsarcolemmal cAMP. However, when both PDE3 and PDE4 were inhibited, or when all PDEs were blocked using 3-isobutyl-l-methylxanthine (300 mol/L), cAMP signals and I Ca,L declined with a time constant Ͼ10 minutes. cAMP-dependent protein kinase inhibition with protein kinase inhibitor produced a similar effect as a partial inhibition of PDE4 on the cytosolic cAMP transient. Consistently, cAMP-PDE assay on ARVMs briefly (15 seconds) exposed to isoprenaline showed a pronounced (up to Ϸ50%) dose-dependent increase in total PDE activity, which was mainly attributable to activation of PDE4. These results reveal temporally distinct ␤-adrenergic receptor cAMP compartments in ARVMs and shed new light on the intricate roles of PDE3 and PDE4. (Circ Res. 2008;102:1091-1100.) Key Words: cAMP Ⅲ L-type calcium current Ⅲ 5Ј-3Ј cyclic nucleotide phosphodiesterases Ⅲ ␤-adrenergic receptors Ⅲ compartmentation C AMP is an ubiquitous second messenger regulating a myriad of cellular functions. In the heart, cAMP mediates the positive inotropic and lusitropic effects of ␤-adrenergic receptor (␤-AR) stimulation by activating the cAMP-dependent protein kinase (PKA), thereby promoting the phosphorylation and activation of key components of the excitation-contraction coupling process. These include the sarcolemmal L-type Ca 2ϩ channels (Ca V 1.2), which are responsible for the initial Ca 2ϩ influx; the ryanodine receptors, which allow Ca 2ϩ release from the sarcoplasmic reticulum (SR); troponin I, which controls myofilament sensitivity to Ca 2ϩ ; and phospholamban, which regulates Ca 2ϩ withdrawal from t...

Abstract-Compartmentation of cAMP is thought to generate the specificity of G s -coupled receptor action in cardiac myocytes, with phosphodiesterases (PDEs) playing a major role in this process by preventing cAMP diffusion. We tested this hypothesis in adult rat ventricular myocytes by characterizing PDEs involved in the regulation of cAMP signals and L-type Ca 2ϩ current (I Ca,L ) on stimulation with ␤ 1 -adrenergic receptors (␤ 1 -ARs), ␤ 2 -ARs, glucagon receptors (Glu-Rs) and prostaglandin E 1 receptors (PGE 1 -Rs). All receptors but PGE 1 -R increased total cAMP, and inhibition of PDEs with 3-isobutyl-1-methylxanthine strongly potentiated these responses. When monitored in single cells by high-affinity cyclic nucleotide-gated (CNG) channels, stimulation of ␤ 1 -AR and Glu-R increased cAMP, whereas ␤ 2 -AR and PGE 1 -R had no detectable effect. Selective inhibition of PDE3 by cilostamide and PDE4 by Ro 20-1724 potentiated ␤ 1 -AR cAMP signals, whereas Glu-R cAMP was augmented only by PD4 inhibition. PGE 1 -R and ␤ 2 -AR generated substantial cAMP increases only when PDE3 and PDE4 were blocked. For all receptors except PGE 1 -R, the measurements of I Ca,L closely matched the ones obtained with CNG channels. Indeed, PDE3 and PDE4 controlled ␤ 1 -AR and ␤ 2 -AR regulation of I Ca,L , whereas only PDE4 controlled Glu-R regulation of I Ca,L thus demonstrating that receptor-PDE coupling has functional implications downstream of cAMP. PGE 1 had no effect on I Ca,L even after blockade of PDE3 or PDE4, suggesting that other mechanisms prevent cAMP produced by PGE 1 to diffuse to L-type Ca 2ϩ channels. These results identify specific functional coupling of individual PDE families to G s -coupled receptors as a major mechanism enabling cardiac cells to generate heterogeneous cAMP signals in response to different hormones. Key Words: cAMP Ⅲ heart Ⅲ G-protein-coupled receptor Ⅲ phosphodiesterase C ardiac myocytes express a number of G s -coupled receptors (G s PCRs) that raise intracellular cAMP levels and activate cAMP-dependent protein kinase (PKA) but exert different downstream effects. For instance, ␤ 1 -adrenergic receptor (␤ 1 -AR) stimulation produces a major and sustained increase in force of contraction, accelerates relaxation, and stimulates glycogen phosphorylase. 1 ␤ 2 -AR stimulation also increases contractile force but does not activate glycogen phosphorylase 2 and does not accelerate relaxation 1,3 (see also ref. 2); glucagon receptor (Glu-R) stimulation activates phosphorylase and exerts positive inotropic and lusitropic effects, but the contractile effects fade with time. 4 Finally, prostaglandin E 1 (PGE 1 ) has no effect on contractile activity or glycogen metabolism. 5,6 Such observations led to the proposal that activation of different G s PCRs results in the accumulation of cAMP and phosphorylation of hormone target proteins in distinct compartments. 7 The discovery of A-kinase anchoring proteins, responsible for the subcellular distribution of particulate PKA, 8 and the development of new method...