Compartmentalized cAMP Signaling Research Articles

Nanodomain cAMP signalling in cardiac pathophysiology: potential for developing targeted therapeutic interventions.

3', 5'-cyclic adenosine monophosphate (cAMP) mediates the effects of sympathetic stimulation on the rate and strength of cardiac contraction. Beyond this pivotal role, in cardiac myocytes cAMP also orchestrates a diverse array of reactions to various stimuli. To ensure specificity of response, the cAMP signaling pathway is intricately organized into multiple, spatially confined, subcellular domains, each governing a distinct cellular function. In this review, we describe the molecular components of the cAMP signalling pathway, how they organized are inside the intracellular space and how they achieve exquisite regulation of signalling within nanometer-size domains. We delineate the key experimental findings that lead to the current model of compartmentalised cAMP signaling and we offer an overview of our present understanding of how cAMP nanodomains are structured and regulated within cardiac myocytes. Furthermore, we discuss how compartmentalized cAMP signaling is affected in cardiac disease and consider the potential therapeutic opportunities arising from understanding such organization. By exploiting the nuances of compartmentalized cAMP signaling, novel and more effective therapeutic strategies for managing cardiac conditions may emerge. Finally, we highlight the unresolved questions and hurdles that must be addressed to translate these insights into interventions that may benefit patients.

Mitochondria-associated restricted spaces regulate compartmentalized cAMP signaling in adult ventricular myocytes

Mitochondria-associated restricted spaces regulate compartmentalized cAMP signaling in adult ventricular myocytes

ELUCIDATING LOCAL PHOSPHODIESTERASE 3A SIGNALING AS A BASIS FOR GAINING INSIGHT INTO HYPERTENSION-DRIVING MECHANISMS

Objective: Mutant PDE3A causes hypertension by driving mechanisms that increase peripheral vascular resistance. However, the media to lumen ratio of vessels in mutant PDE3A rat models varied between second-order mesenteric arteries and thoracic aorta. This observation may be related to alterations of the mutant PDE3A in its intracellular location, its hyperactivity and aberrant phosphorylation, which could lead to altered compartmentalized cAMP signaling. PDE3A hydrolyzes cAMP. Therefore, we hypothesized that alterations in compartmentalized cAMP signaling in vascular smooth muscle cells (VSMC) contributes to the hypertension. Design and method: Three different VSMC models were established: VSMCs from rat thoracic aorta, VSMCs from rat secondary mesenteric arteries and VSMCs expressing mutant PDE3A that were differentiated from human induced pluripotent stem cells. The expression levels of PDE3A and candidate genes were detected by immunofluorescence microscopy, Western blotting and qPCR based on results of RNA sequencing. cAMP and Ca2+ signaling pathways are being evaluated by FRET technology and Ca2+ imaging. Results: VSMCs differentiated from human induced pluripotent stem cells could detect abundant expression of vascular smooth muscle cell markers. Differential expression levels of PDE3A and the two screened candidate genes were found in different types of VSMCs. Conclusions: Elucidating differences in cAMP and Ca2+ cycling between different types of VSMCs yielded new insight into the mechanisms of hypertension. The mutant PDE3A signaling may be a starting point for an innovative, PDE3A compartment-specific pharmacological concept for the treatment of hypertension.

Compartmentalised cAMP signalling in the primary cilium.

cAMP is a universal second messenger that relies on precise spatio-temporal regulation to control varied, and often opposing, cellular functions. This is achieved via selective activation of effectors embedded in multiprotein complexes, or signalosomes, that reside at distinct subcellular locations. cAMP is also one of many pathways known to operate within the primary cilium. Dysfunction of ciliary signaling leads to a class of diseases known as ciliopathies. In Autosomal Dominant Polycystic Kidney Disease (ADPKD), a ciliopathy characterized by the formation of fluid-filled kidney cysts, upregulation of cAMP signaling is known to drive cystogenesis. For decades it has been debated whether the primary cilium is an independent cAMP sub-compartment, or whether it shares a diffusible pool of cAMP with the cell body. Recent studies now suggest it is a specific pool of cAMP generated in the cilium that propels cyst formation in ADPKD, supporting the notion that this antenna-like organelle is a compartment within which cAMP signaling occurs independently from cAMP signaling in the bulk cytosol. Here we present examples of cAMP function in the cilium which suggest this mysterious organelle is home to more than one cAMP signalosome. We review evidence that ciliary membrane localization of G-Protein Coupled Receptors (GPCRs) determines their downstream function and discuss how optogenetic tools have contributed to establish that cAMP generated in the primary cilium can drive cystogenesis.

Compartmentalized cAMP signalling and control of cardiac rhythm.

In the last 30 years, the field of cyclic adenosine 3',5'-monophosphate (cAMP) signalling has witnessed a transformative development with the realization that cAMP is compartmentalized and that spatial regulation of cAMP is critical for faithful signal propagation and hormonal specificity. This recognition has changed our understanding of cAMP signalling from the canonical model, where a linear pathway connects a plasma membrane receptor to intracellular effectors and their targets, to a model where signal transduction occurs within a complex network of alternative branches and where an individual receptor leads to activation of a limited fraction of the network, enabled by local regulation of independent signalling units, resulting in a specific functional outcome. The cardiac myocyte has served as the cell model for many of the original findings leading to this paradigm. In this review, we cover some of the evidence supporting this new perspective and discuss how this model is providing novel mechanistic insight into cardiac myocyte physiology. With a focus on the regulation of cardiac rhythm, we consider how this model can provide an original framework for the identification of disease mechanisms. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Using the Proteomics Toolbox to Resolve Topology and Dynamics of Compartmentalized cAMP Signaling.

cAMP is a second messenger that regulates a myriad of cellular functions in response to multiple extracellular stimuli. New developments in the field have provided exciting insights into how cAMP utilizes compartmentalization to ensure specificity when the message conveyed to the cell by an extracellular stimulus is translated into the appropriate functional outcome. cAMP compartmentalization relies on the formation of local signaling domains where the subset of cAMP signaling effectors, regulators and targets involved in a specific cellular response cluster together. These domains are dynamic in nature and underpin the exacting spatiotemporal regulation of cAMP signaling. In this review, we focus on how the proteomics toolbox can be utilized to identify the molecular components of these domains and to define the dynamic cellular cAMP signaling landscape. From a therapeutic perspective, compiling data on compartmentalized cAMP signaling in physiological and pathological conditions will help define the signaling events underlying disease and may reveal domain-specific targets for the development of precision medicine interventions.

Mitochondrial bound PKA contributes to cyclic-AMP compartmentation in human airway smooth muscle cells.

In human airway smooth muscle (hASM) cells, compartmentation of cAMP signaling is necessary to maintain the fidelity of responses by different G protein-coupled receptors sharing this common diffusible second messenger. Using FRET-based biosensors targeted to different subcellular locations, we found that the EP2 prostaglandin receptors (EP2Rs) stimulate cAMP production that is restricted to locations associated with non-lipid raft regions of the plasma membrane. Furthermore, inhibition of phosphodiesterase type 4 (PDE4) allowed EP2R production of cAMP to spread to subcellular locations associated with lipid-raft membrane domains. However, computational modeling has suggested that PDEs may only contribute to compartmentation if cAMP diffusion is slowed by some other means. To test this hypothesis, we used raster image correlation spectroscopy (RICS) to estimate the diffusion coefficient of fluorescently labeled cAMP (φ575-cAMP) in hASM cells and found that it is 3.9 ± 0.49 μm2/s, or nearly two orders of magnitude slower than the predicted rate of free diffusion. Disrupting protein kinase A (PKA) interactions with A kinase anchoring proteins (AKAPs) using the anchoring inhibitor Ht31 nearly doubled the diffusion coefficient of φ575-cAMP to 7.9 ± 1.5 μm2/s, suggesting that cAMP movement was being buffered by PKA anchored by one or more AKAPs. Confocal imaging demonstrated that φ575-cAMP colocalizes with mitochondria, and Western blot analysis found that D-AKAP2 is the primary mitochondrial AKAP found in hASM cells. Knockdown of D‑AKAP2 expression was achieved using adenovirus-mediated delivery of shRNA, and in knockdown cells EP2R activation was able to stimulate cAMP production in subcellular locations associated with lipid-raft domains, similar to effects previously seen with PDE4 inhibition. These results suggest that buffering of cAMP movement by PKA anchored to the outer membrane of mitochondria contributes to compartmentation of EP2R-mediated responses in hASM cells.

Whole-heart multiparametric optical imaging reveals sex-dependent heterogeneity in cAMP signaling and repolarization kinetics.

Cyclic adenosine 3',5'-monophosphate (cAMP) is a key second messenger in cardiomyocytes responsible for transducing autonomic signals into downstream electrophysiological responses. Previous studies have shown intracellular heterogeneity and compartmentalization of cAMP signaling. However, whether cAMP signaling occurs heterogeneously throughout the intact heart and how this drives sex-dependent functional responses are unknown. Here, we developed and validated a novel cardiac-specific fluorescence resonance energy transfer-based cAMP reporter mouse and a combined voltage-cAMP whole-heart imaging system. We showed that in male hearts, cAMP was uniformly activated in response to pharmacological β-adrenergic stimulation. In contrast, female hearts showed that cAMP levels decayed faster in apical versus basal regions, which was associated with nonuniform action potential changes and notable changes in the direction of repolarization. Apical phosphodiesterase (PDE) activity was higher in female versus male hearts, and PDE inhibition prevented repolarization changes in female hearts. Thus, our imaging approach revealed sex-dependent regional breakdown of cAMP and associated electrophysiological differences.

BS-400-08 HETEROGENOUS CAMP SIGNALING IN THE INTACT HEART IS SEX DEPENDENT

cAMP is key for transducing autonomic signals into downstream electrophysiological responses. Previous studies have shown intracellular heterogeneity and compartmentalization of cAMP signaling. Yet, if cAMP signaling occurs heterogeneously throughout the intact heart, and how this translates into functional responses, has not been explored. To determine the spatiotemporal kinetics of cAMP signaling in the intact heart and the underlying mechanisms responsible. Male and female cardiac-specific CAMPER reporter mice that report cAMP binding by changes in FRET were used at 12 weeks. Hearts were excised and Langendorff-perfused for simultaneous cAMP and Vm imaging on a novel integrated whole heart optical imaging system. Following perfusion, tissue samples from base and apex were assessed for phosphodiesterase (PDE) activity. In male hearts, cAMP was uniformly activated in response to β-AR stimulation with bolus norepinephrine (NE, 1.5 μM). Conversely, in female hearts NE led to a greater change in cAMP activity in basal regions vs. the apex (n=7, p<0.05). Moreover, cAMP deactivation was slower in the base vs. apex, in female (n=6, p<0.01) but not male hearts (n=6). Apex-base differences were also evident following PDE inhibition with IBMX (100 μM), with a greater change in cAMP activity in the apex vs. base in both female (n=7, p<0.001) and male hearts (n=5, p<0.05). Likewise, PDE activity assays showed higher total PDE activity in apical regions (n=10, p<0.01), with more apical PDE activity in female vs. male hearts (n=5, p<0.05). In female hearts, faster apical cAMP deactivation following bolus NE was associated with a significant difference in action potential duration (APD80) between apex and base (n=3, p<0.05), but APD80 was not significantly different between regions in male hearts. Using novel whole heart imaging, we have shown female hearts display lower maximal cAMP activity and faster deactivation in the apex, in part, due to elevated PDE activity in this region. This heterogeneity was not observed in male hearts. These findings may have important implications for electrophysiological responses regulated by the cAMP pathway, particularly in heart failure, where PDE activity is altered.

PO-616-02 HETEROGENOUS CAMP SIGNALING IN THE INTACT HEART IS SEX DEPENDENT

cAMP is key for transducing autonomic signals into downstream electrophysiological responses. Previous studies have shown intracellular heterogeneity and compartmentalization of cAMP signaling. Yet, if cAMP signaling occurs heterogeneously throughout the intact heart, and how this translates into functional responses, has not been explored.

Mitochondrial A-kinase anchoring proteins in cardiac ventricular myocytes.

Compartmentation of cAMP signaling is a critical factor for maintaining the integrity of receptor‐specific responses in cardiac myocytes. This phenomenon relies on various factors limiting cAMP diffusion. Our previous work in adult rat ventricular myocytes (ARVMs) indicates that PKA regulatory subunits anchored to the outer membrane of mitochondria play a key role in buffering the movement of cytosolic cAMP. PKA can be targeted to discrete subcellular locations through the interaction of both type I and type II regulatory subunits with A‐kinase anchoring proteins (AKAPs). The purpose of this study is to identify which AKAPs and PKA regulatory subunit isoforms are associated with mitochondria in ARVMs. Quantitative PCR data demonstrate that mRNA for dual specific AKAP1 and 2 (D‐AKAP1 & D‐AKAP2), acyl‐CoA‐binding domain‐containing 3 (ACBD3), optic atrophy 1 (OPA1) are most abundant, while Rab32, WAVE‐1, and sphingosine kinase type 1 interacting protein (SPHKAP) were barely detectable. Biochemical and immunocytochemical analysis suggests that D‐AKAP1, D‐AKAP2, and ACBD3 are the predominant mitochondrial AKAPs exposed to the cytosolic compartment in these cells. Furthermore, we show that both type I and type II regulatory subunits of PKA are associated with mitochondria. Taken together, these data suggest that D‐AKAP1, D‐AKAP2, and ACBD3 may be responsible for tethering both type I and type II PKA regulatory subunits to the outer mitochondrial membrane in ARVMs. In addition to regulating PKA‐dependent mitochondrial function, these AKAPs may play an important role by buffering the movement of cAMP necessary for compartmentation.

Compartmentalized cAMP signaling in arterial myocytes

Cyclic AMP (cAMP) is an important physiological second messenger that plays a central role in arterial myocyte excitability. While it is well-known that cAMP production by different Gs protein coupled receptors (GsPCRs) is confined to discrete subcellular compartments in many excitable cells, it is unclear if and how this spatial organization occurs in arterial myocytes that wrap around blood vessels. We hypothesized that ligand-induced cAMP signals are compartmentalized in arterial myocytes. Here, live-cell imaging with new targeted cAMP FRET-based (CUTie) biosensors was used to visualize cAMP signals in male and female human and mouse arterial myocytes. We found that receptor-mediated cAMP production was compartmentalized within the sarcoplasmic reticulum (SR) region of male, but not female arterial myocytes. PLA imaging suggested SR is closely associated with phosphodiesterase 4 (PDE4) and further analysis revealed that basal PDE4D expression was significantly higher in male arterial myocytes, which may contribute to spatially restricted subcellular cAMP signaling. Intriguingly, PDE4 activity was found to modulate SR Ca2+ load, as well as receptor-mediated vasorelaxation and blood flow (BF) in male arteries. These findings established a key role for PDE4 in regulating unique sexually dimorphic cAMP compartmentalization in arterial myocytes.

Dysregulation of ion transport in the lung epithelium infected with SARS-CoV-2.

Dysregulation of ion transport in the lung epithelium infected with SARS-CoV-2.

Local cyclic adenosine monophosphate signalling cascades-Roles and targets in chronic kidney disease.

The molecular mechanisms underlying chronic kidney disease (CKD) are poorly understood and treatment options are limited, a situation underpinning the need for elucidating the causative molecular mechanisms and for identifying innovative treatment options. It is emerging that cyclic 3',5'-adenosine monophosphate (cAMP) signalling occurs in defined cellular compartments within nanometre dimensions in processes whose dysregulation is associated with CKD. cAMP compartmentalization is tightly controlled by a specific set of proteins, including A-kinase anchoring proteins (AKAPs) and phosphodiesterases (PDEs). AKAPs such as AKAP18, AKAP220, AKAP-Lbc and STUB1, and PDE4 coordinate arginine-vasopressin (AVP)-induced water reabsorption by collecting duct principal cells. However, hyperactivation of the AVP system is associated with kidney damage and CKD. Podocyte injury involves aberrant AKAP signalling. cAMP signalling in immune cells can be local and slow the progression of inflammatory processes typical for CKD. A major risk factor of CKD is hypertension. cAMP directs the release of the blood pressure regulator, renin, from juxtaglomerular cells, and plays a role in Na+ reabsorption through ENaC, NKCC2 and NCC in the kidney. Mutations in the cAMP hydrolysing PDE3A that cause lowering of cAMP lead to hypertension. Another major risk factor of CKD is diabetes mellitus. AKAP18 and AKAP150 and several PDEs are involved in insulin release. Despite the increasing amount of data, an understanding of functions of compartmentalized cAMP signalling with relevance for CKD is fragmentary. Uncovering functions will improve the understanding of physiological processes and identification of disease-relevant aberrations may guide towards new therapeutic concepts for the treatment of CKD.

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.

Subcellular Organization of the cAMP Signaling Pathway.

The field of cAMP signaling is witnessing exciting developments with the recognition that cAMP is compartmentalized and that spatial regulation of cAMP is critical for faithful signal coding. This realization has changed our understanding of cAMP signaling from a model in which cAMP connects a receptor at the plasma membrane to an intracellular effector in a linear pathway to a model in which cAMP signals propagate within a complex network of alternative branches and the specific functional outcome strictly depends on local regulation of cAMP levels and on selective activation of a limited number of branches within the network. In this review, we cover some of the early studies and summarize more recent evidence supporting the model of compartmentalized cAMP signaling, and we discuss how this knowledge is starting to provide original mechanistic insight into cell physiology and a novel framework for the identification of disease mechanisms that potentially opens new avenues for therapeutic interventions.Significance StatementcAMP mediates the intracellular response to multiple hormones and neurotransmitters. Signal fidelity and accurate coordination of a plethora of different cellular functions is achieved via organization of multiprotein signalosomes and cAMP compartmentalization in subcellular nanodomains. Defining the organization and regulation of subcellular cAMP nanocompartments is necessary if we want to understand the complex functional ramifications of pharmacological treatments that target G protein–coupled receptors and for generating a blueprint that can be used to develop precision medicine interventions.

Axelrod Symposium 2019: Phosphoproteomic Analysis of G-Protein-Coupled Pathways.

By limiting unrestricted activation of intracellular effectors, compartmentalized signaling of cyclic nucleotides confers specificity to extracellular stimuli and is critical for the development and health of cells and organisms. Dissecting the molecular mechanisms that allow local control of cyclic nucleotide signaling is essential for our understanding of physiology and pathophysiology, but mapping the dynamics and regulation of compartmentalized signaling is a challenge. In this minireview we summarize advanced imaging and proteomics techniques that have been successfully used to probe compartmentalized cAMP signaling in eukaryotic cells. Subcellularly targeted fluorescence resonance energy transfer sensors can precisely locate and measure compartmentalized cAMP, and this allows us to estimate the range of effector activation. Because cAMP effector proteins often cluster together with their targets and cAMP regulatory proteins to form discrete cAMP signalosomes, proteomics and phosphoproteomics analysis have more recently been used to identify additional players in the cAMP-signaling cascade. We propose that the synergistic use of the techniques discussed could prove fruitful in generating a detailed map of cAMP signalosomes and reveal new details of compartmentalized signaling. Compiling a dynamic map of cAMP nanodomains in defined cell types would establish a blueprint for better understanding the alteration of signaling compartments associated with disease and would provide a molecular basis for targeted therapeutic strategies. SIGNIFICANCE STATEMENT: cAMP signaling is compartmentalized. Some functionally important cellular signaling compartments operate on a nanometer scale, and their integrity is essential to maintain cellular function and appropriate responses to extracellular stimuli. Compartmentalized signaling provides an opportunity for precision medicine interventions. Our detailed understanding of the composition, function, and regulation of cAMP-signaling nanodomains in health and disease is essential and will benefit from harnessing the right combination of advanced biochemical and imaging techniques.

Reshaping cAMP nanodomains through targeted disruption of compartmentalised phosphodiesterase signalosomes.

Spatio-temporal regulation of localised cAMP nanodomains is highly dependent upon the compartmentalised activity of phosphodiesterase (PDE) cyclic nucleotide degrading enzymes. Strategically positioned PDE-protein complexes are pivotal to the homeostatic control of cAMP-effector protein activity that in turn orchestrate a wide range of cellular signalling cascades in a variety of cells and tissue types. Unsurprisingly, dysregulated PDE activity is central to the pathophysiology of many diseases warranting the need for effective therapies that target PDEs selectively. This short review focuses on the importance of activating compartmentalised cAMP signalling by displacing the PDE component of signalling complexes using cell-permeable peptide disrupters.

Specificity of NHERF1 regulation of GPCR signaling and function in human airway smooth muscle.

Na+/H+ exchanger regulatory factor 1 (NHERF1; also known as ezrin-radixin-moesin-binding phosphoprotein 50) is a PSD-95, disc large, zona occludens-1 adapter that acts as a scaffold for signaling complexes and cytoskeletal-plasma membrane interactions. NHERF1 is crucial to β-2-adrenoceptor (β2AR)-mediated activation of cystic fibrosis transmembrane conductance regulator (CFTR) in epithelial cells, and NHERF1 has been proposed to mediate the recycling of internalized β2AR back to the cell membrane. In the current study, we assessed the role of NHERF1 in regulating cAMP-mediated signaling and immunomodulatory functions in airway smooth muscle (ASM). NHERF1 knockdown attenuated the induction of (protein kinase A) phospho-vasodilator-stimulated phosphoprotein (p-VASP) by isoproterenol (ISO), prostaglandin E2 (PGE2), or forskolin (FSK) as well as the induction of p-heat shock protein 20 after 4 h of stimulation with ISO and FSK. NHERF1 knockdown fully abrogated the ISO-, PGE2-, and FSK-induced IL-6 gene expression and cytokine production without affecting cAMP-mediated phosphodiesterase 4D (PDE4D) gene expression, phospho-cAMP response element-binding protein (p-CREB), and cAMP response element (CRE)-Luc, or PDGF-induced cyclin D1 expression. Interestingly, NHERF1 knockdown prevented ISO-induced chromatin-binding of the transcription factor CCAAT-enhancer-binding protein-β (c/EBPβ). c/EBPβ knockdown almost completely abrogated the cAMP-mediated IL-6 but not PDE4D gene expression. The differential regulation of cAMP-induced signaling and gene expression in our study indicates a role for NHERF1 in the compartmentalization of cAMP signaling in ASM.-Pera, T., Tompkins, E., Katz, M., Wang, B., Deshpande, D. A., Weinman, E. J., Penn, R. B. Specificity of NHERF1 regulation of GPCR signaling and function in human airway smooth muscle.

Compartmentalized cAMP Signaling Associated With Lipid Raft and Non-raft Membrane Domains in Adult Ventricular Myocytes.

Aim: Confining cAMP production to discrete subcellular locations makes it possible for this ubiquitous second messenger to elicit unique functional responses. Yet, factors that determine how and where the production of this diffusible signaling molecule occurs are incompletely understood. The fluid mosaic model originally proposed that signal transduction occurs through random interactions between proteins diffusing freely throughout the plasma membrane. However, it is now known that the movement of membrane proteins is restricted, suggesting that the plasma membrane is segregated into distinct microdomains where different signaling proteins can be concentrated. In this study, we examined what role lipid raft and non-raft membrane domains play in compartmentation of cAMP signaling in adult ventricular myocytes.Methods and Results: The freely diffusible fluorescence resonance energy transfer-based biosensor Epac2-camps was used to measure global cytosolic 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. We found that β-adrenergic receptors, which are expressed in lipid raft and non-raft domains, produce cAMP responses near the plasma membrane that are distinctly different from those produced by E-type prostaglandin receptors, which are expressed exclusively in non-raft domains. We also found that there are differences in basal cAMP levels associated with lipid raft and non-raft domains, and that this can be explained by differences in basal adenylyl cyclase activity associated with each of these membrane environments. In addition, we found evidence that phosphodiesterases 2, 3, and 4 work together in regulating cAMP activity associated with both lipid raft and non-raft domains, while phosphodiesterase 3 plays a more prominent role in the bulk cytoplasmic compartment.Conclusion: These results suggest that different membrane domains contribute to the formation of distinct pools of cAMP under basal conditions as well as following receptor stimulation in adult ventricular myocytes.