Abstract-Cardiac myocytes have provided a key paradigm for the concept of the compartmentalized cAMP generation sensed by AKAP-anchored PKA. Phosphodiesterases (PDEs) provide the sole route for degrading cAMP in cells and are thus poised to regulate intracellular cAMP gradients. PDE3 and PDE4 represent the major cAMP degrading activities in rat ventriculocytes. By performing real-time imaging of cAMP in situ, we establish the hierarchy of these PDEs in controlling cAMP levels in basal conditions and on stimulation with a ␤-adrenergic receptor agonist. PDE4, rather than PDE3, appears to be responsible for modulating the amplitude and duration of the cAMP response to beta-agonists. PDE3 and PDE4 localize to distinct compartments and this may underpin their different functional roles.

Abstract-␤-Adrenergic signaling via cAMP generation and PKA activation mediates the positive inotropic effect of catecholamines on heart cells. Given the large diversity of protein kinase A targets within cardiac cells, a precisely regulated and confined activity of such signaling pathway is essential for specificity of response. Phosphodiesterases (PDEs) are the only route for degrading cAMP and are thus poised to regulate intracellular cAMP gradients. Their spatial confinement to discrete compartments and functional coupling to individual receptors provides an efficient way to control local [cAMP] i in a stimulus-specific manner. By performing real-time imaging of cyclic nucleotides in living ventriculocytes we identify a prominent role of PDE2 in selectively shaping the cAMP response to catecholamines via a pathway involving ␤ 3 -adrenergic receptors, NO generation and cGMP production. In cardiac myocytes, PDE2, being tightly coupled to the pool of adenylyl cyclases activated by ␤-adrenergic receptor stimulation, coordinates cGMP and cAMP signaling in a novel feedback control loop of the ␤-adrenergic pathway. In this, activation of ␤ 3 -adrenergic receptors counteracts cAMP generation obtained via stimulation of ␤ 1 /␤ 2 -adrenoceptors. Our study illustrates the key role of compartmentalized PDE2 in the control of catecholamine-generated cAMP and furthers our understanding of localized cAMP signaling.

Abstract-Protein kinase A (PKA) is a key regulatory enzyme that, on activation by cAMP, modulates a wide variety of cellular functions. PKA isoforms type I and type II possess different structural features and biochemical characteristics, resulting in nonredundant function. However, how different PKA isoforms expressed in the same cell manage to perform distinct functions on activation by the same soluble intracellular messenger, cAMP, remains to be established. Here, we provide a mechanism for the different function of PKA isoforms subsets in cardiac myocytes and demonstrate that PKA-RI and PKA-RII, by binding to AKAPs (A kinase anchoring proteins), are tethered to different subcellular locales, thus defining distinct intracellular signaling compartments. Within such compartments, PKA-RI and PKA-RII respond to distinct, spatially restricted cAMP signals generated in response to specific G protein-coupled receptor agonists and regulated by unique subsets of the cAMP degrading phosphodiesterases. Key Words: cAMP Ⅲ compartmentalization Ⅲ compartmentation Ⅲ adrenergic stimulation Ⅲ prostaglandin Ⅲ protein kinase A P rotein kinase A (PKA) is a key regulatory enzyme in the heart that is involved in the catecholamine-mediated control of excitation-contraction coupling, as well as in a myriad of other functions including activation of transcription factors and control of metabolic enzymes. The second messenger cAMP activates PKA by binding to the regulatory (R) subunits, causing release of the activated catalytic (C) subunits.The fact that, following cAMP-engagement, PKA mediates a plethora of cellular responses has raised the question of how specificity is maintained. In recent years, features of this pathway that contribute to specificity have been uncovered. 1 A key role is played by AKAPs (A kinase anchoring proteins), a family of proteins that act as molecular scaffolds to anchor PKA in the vicinity of specific substrate molecules, 2 thus focusing PKA activity toward relevant substrates.A second mechanism contributing to specificity revolves around the spatial control of the cAMP signal itself. Restriction of intracellular diffusion of cAMP has been shown by using a variety of approaches, [3][4][5] including direct imaging of gradients of cAMP in response to activation of various G protein-coupled receptors (GPCRs). 6 A key role in shaping cAMP intracellular pools is played by phosphodiesterases (PDEs), the enzymes that hydrolyze cAMP. 7 Indeed, individual PDE isoforms have been shown to be functionally coupled to specific GPCRs to degrade cAMP selectively in response to a given stimulus. 8 Cardiac myocytes express all four types of PKA isozymes, PKA-RI␣, PKA-RII␣, PKA-RI␤, and PKA-RII␤. 9 PKA isoforms show different subcellular localization, with PKA-RII being mainly associated with the particulate fraction of cell lysates whereas PKA-RI has been found preferentially in the cytosol. 10,11 PKA isoforms also show different biochemical properties. PKA-RI is more readily dissociated by cAMP than PKA-RII, 12,13 an...

There is a growing appreciation that the cyclic adenosine monophosphate (cAMP)–protein kinase A (PKA) signaling pathway is organized to form transduction units that function to deliver specific messages. Such organization results in the local activation of PKA subsets through the generation of confined intracellular gradients of cAMP, but the mechanisms responsible for limiting the diffusion of cAMP largely remain to be clarified. In this study, by performing real-time imaging of cAMP, we show that prostaglandin 1 stimulation generates multiple contiguous, intracellular domains with different cAMP concentration in human embryonic kidney 293 cells. By using pharmacological and genetic manipulation of phosphodiesterases (PDEs), we demonstrate that compartmentalized PDE4B and PDE4D are responsible for selectively modulating the concentration of cAMP in individual subcellular compartments. We propose a model whereby compartmentalized PDEs, rather than representing an enzymatic barrier to cAMP diffusion, act as a sink to drain the second messenger from discrete locations, resulting in multiple and simultaneous domains with different cAMP concentrations irrespective of their distance from the site of cAMP synthesis.

Rationale 3′-5′-cyclic adenosine monophosphate (cAMP) and 3′-5′-cyclic guanosine monophosphate (cGMP) are intracellular second messengers involved in heart pathophysiology. cGMP can potentially affect cAMP signals via cGMP-regulated phosphodiesterases (PDEs). Objective To study the effect of cGMP signals on the local cAMP response to catecholamines in specific subcellular compartments. Methods and results We used real-time FRET imaging of living rat ventriculocytes expressing targeted cAMP and cGMP biosensors to detect cyclic nucleotides levels in specific locales. We found that the compartmentalized, but not the global, cAMP response to isoproterenol is profoundly affected by cGMP signals. The effect of cGMP is to increase cAMP levels in the compartment where the PKA-RI isoforms reside but to decrease cAMP in the compartment where the PKA-RII isoforms reside. These opposing effects are determined by the cGMP-regulated PDEs, namely PDE2 and PDE3, with the local activity of these PDEs being critically important. The cGMP-mediated modulation of cAMP also affects the phosphorylation of PKA targets and myocyte contractility. Conclusions cGMP signals exert opposing effects on local cAMP levels via different PDEs the activity of which is exerted in spatially distinct subcellular domains. Inhibition of PDE2 selectively abolishes the negative effects of cGMP on cAMP and may have therapeutic potential.

Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity. Specificity of synaptic plasticity requires that cAMP activates PKA in a highly localized manner despite the speed with which cAMP diffuses. Two mechanisms have been proposed to produce localized elevations in cAMP, known as microdomains: impeded diffusion, and high phosphodiesterase (PDE) activity. This paper investigates the mechanism of localized cAMP signaling using a computational model of the biochemical network in the HEK293 cell, which is a subset of pathways involved in PKA-dependent synaptic plasticity. This biochemical network includes cAMP production, PKA activation, and cAMP degradation by PDE activity. The model is implemented in NeuroRD: novel, computationally efficient, stochastic reaction-diffusion software, and is constrained by intracellular cAMP dynamics that were determined experimentally by real-time imaging using an Epac-based FRET sensor (H30). The model reproduces the high concentration cAMP microdomain in the submembrane region, distinct from the lower concentration of cAMP in the cytosol. Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity. The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains. The simulations reported here further demonstrate the utility of the new stochastic reaction-diffusion algorithm for exploring signaling pathways in spatially complex structures such as neurons.

Rationale : Chronic elevation of 3′-5′-cyclic adenosine monophosphate (cAMP) levels has been associated with cardiac remodeling and cardiac hypertrophy. However, enhancement of particular aspects of cAMP/protein kinase A signaling seems to be beneficial for the failing heart. cAMP is a pleiotropic second messenger with the ability to generate multiple functional outcomes in response to different extracellular stimuli with strict fidelity, a feature that relies on the spatial segregation of the cAMP pathway components in signaling microdomains. Objective : How individual cAMP microdomains affect cardiac pathophysiology remains largely to be established. The cAMP-degrading enzymes phosphodiesterases (PDEs) play a key role in shaping local changes in cAMP. Here we investigated the effect of specific inhibition of selected PDEs on cardiac myocyte hypertrophic growth. Methods and Results : Using pharmacological and genetic manipulation of PDE activity, we found that the rise in cAMP resulting from inhibition of PDE3 and PDE4 induces hypertrophy, whereas increasing cAMP levels via PDE2 inhibition is antihypertrophic. By real-time imaging of cAMP levels in intact myocytes and selective displacement of protein kinase A isoforms, we demonstrate that the antihypertrophic effect of PDE2 inhibition involves the generation of a local pool of cAMP and activation of a protein kinase A type II subset, leading to phosphorylation of the nuclear factor of activated T cells. Conclusions : Different cAMP pools have opposing effects on cardiac myocyte cell size. PDE2 emerges as a novel key regulator of cardiac hypertrophy in vitro and in vivo, and its inhibition may have therapeutic applications.

The direct measurement of mitochondrial [Ca 2؉ ] with highly specific probes demonstrated that major swings in organellar [Ca 2؉ ] parallel the changes occurring in the cytosol and regulate processes as diverse as aerobic metabolism and cell death by necrosis and apoptosis. Despite great biological relevance, insight was limited by the complete lack of molecular understanding. The situation has changed, and new perspectives have emerged following the very recent identification of the mitochondrial Ca 2؉ uniporter, the channel allowing rapid Ca 2؉ accumulation across the inner mitochondrial membrane.