Compartmentation Of cAMP Research Articles

Phosphodiesterases and Compartmentation of cAMP and cGMP Signaling in Regulation of Cardiac Contractility in Normal and Failing Hearts.

Cardiac contractility is regulated by several neural, hormonal, paracrine, and autocrine factors. Amongst these, signaling through β-adrenergic and serotonin receptors generates the second messenger cyclic AMP (cAMP), whereas activation of natriuretic peptide receptors and soluble guanylyl cyclases generates cyclic GMP (cGMP). Both cyclic nucleotides regulate cardiac contractility through several mechanisms. Phosphodiesterases (PDEs) are enzymes that degrade cAMP and cGMP and therefore determine the dynamics of their downstream effects. In addition, the intracellular localization of the different PDEs may contribute to regulation of compartmented signaling of cAMP and cGMP. In this review, we will focus on the role of PDEs in regulating contractility and evaluate changes in heart failure.

Mechanisms of cAMP compartmentation in cardiac myocytes: experimental and computational approaches to understanding.

The small diffusible second messenger 3',5'-cyclic adenosine monophosphate (cAMP) is found in virtually every cell in our bodies, where it mediates responses to a variety of different G protein coupled receptors (GPCRs). In the heart, cAMP plays a critical role in regulating many different aspects of cardiac myocyte function, including gene transcription, cell metabolism, and excitation-contraction coupling. Yet, not all GPCRs that stimulate cAMP production elicit the same responses. Subcellular compartmentation of cAMP is essential to explain how different receptors can utilize the same diffusible second messenger to elicit unique functional responses. However, the mechanisms contributing to this behaviour and its significance in producing physiological and pathological responses are incompletely understood. Mathematical modelling has played an essential role in gaining insight into these questions. This review discusses what we currently know about cAMP compartmentation in cardiac myocytes and questions that are yet to be answered.

Cardiac Hypertrophy Changes Compartmentation of cAMP in Non-Raft Membrane Microdomains.

3′,5′-Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger which plays critical roles in cardiac function and disease. In adult mouse ventricular myocytes (AMVMs), several distinct functionally relevant microdomains with tightly compartmentalized cAMP signaling have been described. At least two types of microdomains reside in AMVM plasma membrane which are associated with caveolin-rich raft and non-raft sarcolemma, each with distinct cAMP dynamics and their differential regulation by receptors and cAMP degrading enzymes phosphodiesterases (PDEs). However, it is still unclear how cardiac disease such as hypertrophy leading to heart failure affects cAMP signals specifically in the non-raft membrane microdomains. To answer this question, we generated a novel transgenic mouse line expressing a highly sensitive Förster resonance energy transfer (FRET)-based biosensor E1-CAAX targeted to non-lipid raft membrane microdomains of AMVMs and subjected these mice to pressure overload induced cardiac hypertrophy. We could detect specific changes in PDE3-dependent compartmentation of β-adrenergic receptor induced cAMP in non-raft membrane microdomains which were clearly different from those occurring in caveolin-rich sarcolemma. This indicates differential regulation and distinct responses of these membrane microdomains to cardiac remodeling.

Computational modeling approaches to cAMP/PKA signaling in cardiomyocytes

The cAMP/PKA pathway is a fundamental regulator of excitation-contraction coupling in cardiomyocytes. Activation of cAMP has a variety of downstream effects on cardiac function including enhanced contraction, accelerated relaxation, adaptive stress response, mitochondrial regulation, and gene transcription. Experimental advances have shed light on the compartmentation of cAMP and PKA, which allow for control over the varied targets of these second messengers and is disrupted in heart failure conditions. Computational modeling is an important tool for understanding the spatial and temporal complexities of this system. In this review article, we outline the advances in computational modeling that have allowed for deeper understanding of cAMP/PKA dynamics in the cardiomyocyte in health and disease, and explore new modeling frameworks that may bring us closer to a more complete understanding of this system. We outline various compartmental and spatial signaling models that have been used to understand how β-adrenergic signaling pathways function in a variety of simulation conditions. We also discuss newer subcellular models of cardiovascular function that may be used as templates for the next phase of computational study of cAMP and PKA in the heart, and outline open challenges which are important to consider in future models.

Studying signal compartmentation in adult cardiomyocytes.

Multiple intra-cellular signalling pathways rely on calcium and 3′–5′ cyclic adenosine monophosphate (cAMP) to act as secondary messengers. This is especially true in cardiomyocytes which act as the force-producing units of the cardiac muscle and are required to react rapidly to environmental stimuli. The specificity of functional responses within cardiomyocytes and other cell types is produced by the organellar compartmentation of both calcium and cAMP. In this review, we assess the role of molecular localisation and relative contribution of active and passive processes in producing compartmentation. Active processes comprise the creation and destruction of signals, whereas passive processes comprise the release or sequestration of signals. Cardiomyocytes display a highly articulated membrane structure which displays significant cell-to-cell variability. Special attention is paid to the way in which cell membrane caveolae and the transverse-axial tubule system allow molecular localisation. We explore the effects of cell maturation, pathology and regional differences in the organisation of these processes. The subject of signal compartmentation has had a significant amount of attention within the cardiovascular field and has undergone a revolution over the past two decades. Advances in the area have been driven by molecular imaging using fluorescent dyes and genetically encoded constructs based upon fluorescent proteins. We also explore the use of scanning probe microscopy in the area. These techniques allow the analysis of molecular compartmentation within specific organellar compartments which gives researchers an entirely new perspective.

Intracellular compartmentation of cAMP promotes neuroprotection and regeneration of CNS neurons.

Intracellular compartmentation of cAMP promotes neuroprotection and regeneration of CNS neurons.

A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation.

Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal distribution of cAMP at the subcellular level could be important for developing new strategies for the prevention or treatment of unfavorable responses associated with different disease states.

A Computational Investigation Predicts Mechanisms of Subcellular cAMP Compartmentation

For more than 30 years, subcellular compartmentation of the ubiquitous second messenger cAMP has been proposed as a mechanism to explain the distinct cAMP-mediated functional responses that occur after activation of various cell surface receptors. To date, the mechanism of compartmentation is still unknown. One possible mechanism of cAMP compartmentalization is specific localization of phosphodiesterases (PDEs), which degrade cAMP and create a functional barrier to the diffusion of cAMP. In order to assess the plausibility of this mechanism, we developed computational models to represent the subcellular sarcomeric spaces of a cardiac myocyte at varying resolutions. The models include a 2-dimensional continuum model of a subcellular compartment spanning the space between T-tubules of a cardiac sarcomere, a 3-dimensional stochastic model of cAMP diffusion in a microdomain using MCell (mcell.org), and a reduced 1-dimensional continuum model that can be solved analytically. We used these models to predict the contributions of various mechanisms to subcellular cAMP microdomains. We tested the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Predictions from all the models concurred: Under realistic conditions phosphodiesterases were insufficient to generate cAMP gradients. This prediction persisted even with the slowest measured cAMP diffusion rate. Our model simulations suggest that additional mechanisms contribute to cAMP gradients occurring in submicroscopic cellular domains. Additional experimental studies will be needed to understand the contribution of factors required to coordinate spatio-temporal distribution of cAMP at the subcellular level.

Mitochondrial Buffering of cAMP in Adult Cardiac Myocytes

Compartmentation of the diffusible second messenger cAMP plays a key role in producing unique responses to different G‐protein coupled receptors. It has been proposed that this can only happen if the movement of cAMP within the cell occurs at rates well below those of free diffusion. To directly test this hypothesis we used Raster Image Correlation Spectroscopy (RICS) to measure the diffusion coefficient of fluorescently labeled cAMP, 8‐[Pharos‐450]‐cAMP (P450‐cAMP) in live cells. Confocal imaging showed that incubation with the membrane permeable P450‐cAMP led to a distinct longitudinal pattern of fluorescence accumulation in adult rat ventricular myocytes. This was in contrast to the uniform distribution of cAMP‐free dye (P450), suggesting that P450‐cAMP binds to immobilized target. Interestingly, expression of PKA type II regulatory subunit labeled with CFP (RII‐CFP) exhibited a pattern similar to P450‐cAMP, which corresponded with the extra‐myofibrillar space populated by mitochondria, as verified by labeling with MitoTracker Red. RICS analysis found the diffusion coefficient (μm2/s) of P450‐cAMP (9.7±1.6, n=37) to be significantly slower than that of P450 (17.6 ± 1.73, n=14), but similar to that of RII‐CFP (12.1±1.85, n=11). Furthermore, Ht31, a peptide that disrupts interactions between RII and A kinase anchoring proteins (AKAPs), caused the distribution pattern of both P450‐cAMP and RII‐CFP to become more diffuse. Correspondingly, the diffusion coefficients for P450‐cAMP (34.4±4.37, n=15) and RII‐CFP (32.0±4.53, n=8) increased significantly, while that of P450 did not. These data suggest that the movement of cAMP in adult cardiac myocytes is limited by interactions with PKA RII subunits associated specifically with mitochondria.

Role of membrane microdomains in compartmentation of cAMP signaling.

Spatially restricting cAMP production to discrete subcellular locations permits selective regulation of specific functional responses. But exactly where and how cAMP signaling is confined is not fully understood. Different receptors and adenylyl cyclase isoforms responsible for cAMP production are not uniformly distributed between lipid raft and non-lipid raft domains of the plasma membrane. We sought to determine the role that these membrane domains play in organizing cAMP responses in HEK293 cells. The freely diffusible FRET-based biosensor Epac2-camps was used to measure global cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. Disruption of lipid rafts by cholesterol depletion selectively altered cAMP responses produced by raft-associated receptors. The results indicate that receptors associated with lipid raft as well as non-lipid raft domains can contribute to global cAMP responses. In addition, basal cAMP activity was found to be significantly higher in non-raft domains. This was supported by the fact that pharmacologic inhibition of adenylyl cyclase activity reduced basal cAMP activity detected by Epac2-CAAX but not Epac2-MyrPalm or Epac2-camps. Responses detected by Epac2-CAAX were also more sensitive to direct stimulation of adenylyl cyclase activity, but less sensitive to inhibition of phosphodiesterase activity. Quantitative modeling was used to demonstrate that differences in adenylyl cyclase and phosphodiesterase activities are necessary but not sufficient to explain compartmentation of cAMP associated with different microdomains of the plasma membrane.

How uniform is cAMP signaling? Focus on “Systems analysis of GLP-1 receptor signaling in pancreatic β-cells”

the goal of many cell physiologists is to ascribe cellular behavior to specific molecular signaling mechanisms. However, the reductionist experimental approach commonly used to understand molecular function often makes this task harder rather than easier to accomplish because of the complexity of

Mechanisms underlying cAMP compartmentation

There is mounting evidence suggesting that specificity within cAMP signaling pathways is caused by compartmentation. Mechanisms underlying compartmentation are poorly understood. The spatial spread of cAMP is affected by phosphodiesterase (PDE) localization, buffering, cell geometry, and restrictions on diffusion. We developed mathematical models to investigate potential contributions of these mechanisms in the compartmentation of cAMP. Models assumed realistic enzyme concentrations and homogenous rates of diffusion. Initial simulations used an effective diffusion coefficient (D) of 300 μm2/s ‐ D for cAMP in aqueous solution. Simulations of cAMP spread within a spherical cell indicated that PDE activity and buffering by PKA cannot solely account for the segregation of cAMP signals, regardless of their subcellular location. When D was lowered 10,000‐fold, significant cAMP gradients were observed. We then incorporated realistic geometries of endothelial cells into the mathematical model. In simulations of this model with D = 300 μm2/s and PDE activity concentrated at the nuclear membrane, 3‐fold cAMP gradients were observed between cellular processes and the nucleus. Lowering D 100‐fold increased the magnitude of cAMP gradients. These simulations are consistent with a conceptual model in which cell geometry and structural elements contribute to cAMP compartmentation.

Cell Shape and Negative Links in Regulatory Motifs Together Control Spatial Information Flow in Signaling Networks

The role of cell size and shape in controlling local intracellular signaling reactions, and how this spatial information originates and is propagated, is not well understood. We have used partial differential equations to model the flow of spatial information from the beta-adrenergic receptor to MAPK1,2 through the cAMP/PKA/B-Raf/MAPK1,2 network in neurons using real geometries. The numerical simulations indicated that cell shape controls the dynamics of local biochemical activity of signal-modulated negative regulators, such as phosphodiesterases and protein phosphatases within regulatory loops to determine the size of microdomains of activated signaling components. The model prediction that negative regulators control the flow of spatial information to downstream components was verified experimentally in rat hippocampal slices. These results suggest a mechanism by which cellular geometry, the presence of regulatory loops with negative regulators, and key reaction rates all together control spatial information transfer and microdomain characteristics within cells.

Spatiotemporal Dynamics of β-Adrenergic cAMP Signals and L-Type Ca 2+ Channel Regulation in Adult Rat Ventricular Myocytes

Steady-state activation of cardiac beta-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(2+) 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 approximately 5 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 micromol/L Ro 20-1724) increased the amplitude and dramatically slowed down the onset and recovery of cAMP signals, whereas PDE3 blockade (with 1 micromol/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 micromol/L), cAMP signals and I(Ca,L) declined with a time constant >10 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 approximately 50%) dose-dependent increase in total PDE activity, which was mainly attributable to activation of PDE4. These results reveal temporally distinct beta-adrenergic receptor cAMP compartments in ARVMs and shed new light on the intricate roles of PDE3 and PDE4.

Diminished Phosphodiesterase-8B Potentiates Biphasic Insulin Response to Glucose

cAMP activates multiple signal pathways, crucial for the pancreatic beta-cells function and survival and is a major potentiator of insulin release. A family of phosphodiesterases (PDEs) terminate the cAMP signals. We examined the expression of PDEs in rat beta-cells and their role in the regulation of insulin response. Using RT-PCR and Western blot analyses, we identified PDE3A, PDE3B, PDE4B, PDE4D, and PDE8B in rat islets and in INS-1E cells and several possible splice variants of these PDEs. Specific depletion of PDE3A with small interfering (si) RNA (siPDE3A) led to a small (67%) increase in the insulin response to glucose in INS-1E cells but not rat islets. siPDE3A had no effect on the glucagon-like peptide-1 (10 nmol/liter) potentiated insulin response in rat islets. Depletion in PDE8B levels in rat islets using similar technology (siPDE8B) increased insulin response to glucose by 70%, the potentiation being of similar magnitude during the first and second phase insulin release. The siPDE8B-potentiated insulin response was further increased by 23% when glucagon-like peptide-1 was included during the glucose stimulus. In conclusion, PDE8B is expressed in a small number of tissues unrelated to glucose or fat metabolism. We propose that PDE8B, an 3-isobutyl-1-methylxanthine-insensitive cAMP-specific phosphodiesterase, could prove a novel target for enhanced insulin response, affecting a specific pool of cAMP involved in the control of insulin granule trafficking and exocytosis. Finally, we discuss evidence for functional compartmentation of cAMP in pancreatic beta-cells.

Compartmentation of cAMP Signaling in Cardiac Myocytes: A Computational Study

Receptor-mediated changes in cAMP production play an essential role in sympathetic and parasympathetic regulation of the electrical, mechanical, and metabolic activity of cardiac myocytes. However, responses to receptor activation cannot be easily ascribed to a uniform increase or decrease in cAMP activity throughout the entire cell. In this study, we used a computational approach to test the hypothesis that in cardiac ventricular myocytes the effects of β 1-adrenergic receptor ( β 1AR) and M 2 muscarinic receptor (M 2R) activation involve compartmentation of cAMP. A model consisting of two submembrane (caveolar and extracaveolar) microdomains and one bulk cytosolic domain was created using published information on the location of β 1ARs and M 2Rs, as well as the location of stimulatory (G s) and inhibitory (G i) G-proteins, adenylyl cyclase isoforms inhibited (AC5/6) and stimulated (AC4/7) by G i, and multiple phosphodiesterase isoforms (PDE2, PDE3, and PDE4). Results obtained with the model indicate that: 1), bulk basal cAMP can be high (∼1 μM) and only modestly stimulated by β 1AR activation (∼2 μM), but caveolar cAMP varies in a range more appropriate for regulation of protein kinase A (∼100 nM to ∼2 μM); 2), M 2R activation strongly reduces the β 1AR-induced increases in caveolar cAMP, with less effect on bulk cAMP; and 3), during weak β 1AR stimulation, M 2R activation not only reduces caveolar cAMP, but also produces a rebound increase in caveolar cAMP following termination of M 2R activity. We conclude that compartmentation of cAMP can provide a quantitative explanation for several aspects of cardiac signaling.

Cyclic AMP Imaging in Adult Cardiac Myocytes Reveals Far-Reaching β 1 -Adrenergic but Locally Confined β 2 -Adrenergic Receptor–Mediated Signaling

Beta(1)- and beta(2)-adrenergic receptors (betaARs) are known to differentially regulate cardiomyocyte contraction and growth. We tested the hypothesis that these differences are attributable to spatial compartmentation of the second messenger cAMP. Using a fluorescent resonance energy transfer (FRET)-based approach, we directly monitored the spatial and temporal distribution of cAMP in adult cardiomyocytes. We developed a new cAMP-FRET sensor (termed HCN2-camps) based on a single cAMP binding domain of the hyperpolarization activated cyclic nucleotide-gated potassium channel 2 (HCN2). Its cytosolic distribution, high dynamic range, and sensitivity make HCN2-camps particularly well suited to monitor subcellular localization of cardiomyocyte cAMP. We generated HCN2-camps transgenic mice and performed single-cell FRET imaging on freshly isolated cardiomyocytes. Whole-cell superfusion with isoproterenol showed a moderate elevation of cAMP. Application of various phosphodiesterase (PDE) inhibitors revealed stringent control of cAMP through PDE4>PDE2>PDE3. The beta(1)AR-mediated cAMP signals were entirely dependent on PDE4 activity, whereas beta(2)AR-mediated cAMP was under control of multiple PDE isoforms. beta(1)AR subtype-specific stimulation yielded approximately 2-fold greater cAMP responses compared with selective beta(2)-subtype stimulation, even on treatment with the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) (DeltaFRET, 17.3+/-1.3% [beta(1)AR] versus 8.8+/-0.4% [beta(2)AR]). Treatment with pertussis toxin to inactivate G(i) did not affect cAMP production. Localized beta(1)AR stimulation generated a cAMP gradient propagating throughout the cell, whereas local beta(2)AR stimulation did not elicit marked cAMP diffusion. Our data reveal that in adult cardiac myocytes, beta(1)ARs induce far-reaching cAMP signals, whereas beta(2)AR-induced cAMP remains locally confined.

Systems analysis of PKA-mediated phosphorylation gradients in live cardiac myocytes

Compartmentation and dynamics of cAMP and PKA signaling are important determinants of specificity among cAMP's myriad cellular roles. Both cardiac inotropy and the progression of heart disease are affected by spatiotemporal variations in cAMP/PKA signaling, yet the dynamic patterns of PKA-mediated phosphorylation that influence differential responses to agonists have not been characterized. We performed live-cell imaging and systems modeling of PKA-mediated phosphorylation in neonatal cardiac myocytes in response to G-protein coupled receptor stimuli and UV photolysis of "caged" cAMP. cAMP accumulation was rate-limiting in PKA-mediated phosphorylation downstream of the beta-adrenergic receptor. Prostaglandin E1 stimulated higher PKA activity in the cytosol than at the sarcolemma, whereas isoproterenol triggered faster sarcolemmal responses than cytosolic, likely due to restricted cAMP diffusion from submembrane compartments. Localized UV photolysis of caged cAMP triggered gradients of PKA-mediated phosphorylation, enhanced by phosphodiesterase activity and PKA-mediated buffering of cAMP. These findings indicate that combining live-cell FRET imaging and mechanistic computational models can provide quantitative understanding of spatiotemporal signaling.

A Specific Pattern of Phosphodiesterases Controls the cAMP Signals Generated by Different G s -Coupled Receptors in Adult Rat Ventricular Myocytes

Compartmentation of cAMP is thought to generate the specificity of Gs-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 Ca2+ current (I(Ca,L)) on stimulation with beta1-adrenergic receptors (beta1-ARs), beta2-ARs, glucagon receptors (Glu-Rs) and prostaglandin E1 receptors (PGE1-Rs). All receptors but PGE1-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 beta1-AR and Glu-R increased cAMP, whereas beta2-AR and PGE1-R had no detectable effect. Selective inhibition of PDE3 by cilostamide and PDE4 by Ro 20-1724 potentiated beta1-AR cAMP signals, whereas Glu-R cAMP was augmented only by PD4 inhibition. PGE1-R and beta2-AR generated substantial cAMP increases only when PDE3 and PDE4 were blocked. For all receptors except PGE1-R, the measurements of I(Ca,L) closely matched the ones obtained with CNG channels. Indeed, PDE3 and PDE4 controlled beta1-AR and beta2-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. PGE1 had no effect on I(Ca,L) even after blockade of PDE3 or PDE4, suggesting that other mechanisms prevent cAMP produced by PGE1 to diffuse to L-type Ca2+ channels. These results identify specific functional coupling of individual PDE families to Gs-coupled receptors as a major mechanism enabling cardiac cells to generate heterogeneous cAMP signals in response to different hormones.

PDE4: arrested at the border.

Compartmentation plays a critical role in determining the specificity and efficacy of signaling by adenosine 3',5'-monophosphate (cAMP) and other second messengers. It has become apparent that all of the protein components of the cAMP signaling pathway are subject to regulation and may be localized to particular subcellular compartments. This Perspective discusses recent research concerning the importance of cAMP phosphodiesterase localization in cAMP signaling, functional implications of the organization of this pathway's protein components into macromolecular complexes, and common themes in the compartmentation of cAMP- and Ca2+-mediated signaling pathways.