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Circulation Research. 2007;100:950-966
doi: 10.1161/01.RES.0000261934.56938.38
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(Circulation Research. 2007;100:950.)
© 2007 American Heart Association, Inc.


Reviews

cAMP-Specific Phosphodiesterase-4 Enzymes in the Cardiovascular System

A Molecular Toolbox for Generating Compartmentalized cAMP Signaling

Miles D. Houslay, George S. Baillie, Donald H. Maurice

From the Molecular Pharmacology Group (M.D.H., G.S.B.), Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland, United Kingdom; and Department of Pharmacology and Toxicology (D.H.M.), Queen’s University, Kingston, Ontario, Canada.

Correspondence to Prof Miles Houslay, Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK. E-mail M.Houslay{at}bio.gla.ac.uk



This Review is part of a thematic series on Phosphodiesterases, which includes the following articles:

Compartmentation of Cyclic Nucleotide Signaling in the Heart: The Role of Cyclic Nucleotide Phosphodiesterases

Overview of PDEs and Their Regulation

Regulation of Phosphodiesterase 3 and Inducible cAMP Early Repressor in the Heart

cAMP-Specific Phosphodiesterase-4 Enzymes in the Cardiovascular System: A Molecular Toolbox for Generating

Compartmentalized cAMP Signaling

cAMP and cGMP Signaling Cross-Talk: Role of Phosphodiesterases and Implications for Cardiac Pathophysiology

PDE5 and Regulation of Vessel and Heart Function 
David A. Kass Editor


*    Abstract
up arrowTop
*Abstract
down arrowIntroduction
down arrow"PDE4-ology"
down arrowThe Long, Short, and...
down arrowThe Motor in the...
down arrowFinding the Perfect Partner...
down arrowCellular Function and Phenotype...
down arrowMolecular Determinants Mediating...
down arrowCardiomyocyte PDE4s
down arrowPDE4D-RyR2 Interaction
down arrowPDE4D3-mAKAP
down arrowPhenotypic Modulation of VSMCs
down arrowVSMC Phenotypic Modulation and...
down arrowDistinct PDE4D Variants...
down arrowSignificance of a Distinct...
down arrowThe Future
down arrowReferences
 
Cyclic AMP regulates a vast number of distinct events in all cells. Early studies established that its hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) controlled both the magnitude and the duration of its influence. Recent evidence shows that PDEs also act as coincident detectors linking cyclic-nucleotide– and non–cyclic-nucleotide–based cellular signaling processes and are tethered with great selectively to defined intracellular structures, thereby integrating and spatially restricting their cellular effects in time and space. Although 11 distinct families of PDEs have been defined, and cells invariably express numerous individual PDE enzymes, a large measure of our increased appreciation of the roles of these enzymes in regulating cyclic nucleotide signaling has come from studies on the PDE4 family. Four PDE4 genes encode more than 20 isoforms. Alternative mRNA splicing and the use of different promoters allows cells the possibility of expressing numerous PDE4 enzymes, each with unique amino-terminal-targeting and/or regulatory sequences. Dominant negative and small interfering RNA–mediated knockdown strategies have proven that particular isoforms can uniquely control specific cellular functions. Thus the protein kinase A phosphorylation status of the ß2 adrenoceptor and, thereby, its ability to switch its signaling to extracellular signal-regulated kinase activation, is uniquely regulated by PDE4D5 in cardiomyocytes. We describe how cardiomyocytes and vascular smooth muscle cells selectively vary both the expression and the catalytic activities of PDE4 isoforms to regulate their various functions and how altered regulation of these processes can influence the development, or resolution, of cardiovascular pathologies, such as heart failure, as well as various vasculopathies.


Key Words: phosphodiesterase-4 • cAMP • cardiomyocytes • vascular smooth muscle cells • compartmentation • ß2 adrenoceptor • ß-arrestin


*    Introduction
up arrowTop
up arrowAbstract
*Introduction
down arrow"PDE4-ology"
down arrowThe Long, Short, and...
down arrowThe Motor in the...
down arrowFinding the Perfect Partner...
down arrowCellular Function and Phenotype...
down arrowMolecular Determinants Mediating...
down arrowCardiomyocyte PDE4s
down arrowPDE4D-RyR2 Interaction
down arrowPDE4D3-mAKAP
down arrowPhenotypic Modulation of VSMCs
down arrowVSMC Phenotypic Modulation and...
down arrowDistinct PDE4D Variants...
down arrowSignificance of a Distinct...
down arrowThe Future
down arrowReferences
 
Cyclic AMP is used in cells as a second messenger to regulate a large number of key processes.1–5 It can influence cell growth, differentiation and movement, for example, and regulates specialized actions unique to specific cell types. In the case of cardiac myocytes, cAMP influences inotropic and chronotropic actions, in addition to influencing apoptosis and hypertrophy.6,7 In the vasculature, cAMP influences contraction/relaxation of blood vessel smooth muscle cells (VSMCs), as well as their movement, proliferation, shape, and response to vascular trauma and hypoxia.8–11 Furthermore, various actions of cAMP can be altered in cardiovascular diseases such as heart failure and vascular stenosis.7,11

How does cAMP exert such a myriad of sophisticated actions on particular cell types? Individual cells are complex entities whose components are intricately organized in 3D space. This extends to the machinery involved in the control of cAMP levels and the generation of specific responses at discrete intracellular loci.1,2,12–14

cAMP is generated by adenylyl cyclase isoforms, the majority of which are embedded in the cell surface plasma membrane.15 These are activated by transmembrane receptors, such as the ß-adrenergic receptor16 that, on agonist occupancy, couple to the stimulatory G protein Gs, thereby activating adenylyl cyclase. This confers cAMP generation to the cytosol surface of the plasma membrane, from which emanates a cloud of cAMP. However, because cells are polar, then different Gs-coupled receptors and adenylyl cyclase isoenzymes may be restricted to plasma membrane subdomains,15,17 so as to provide distinct "point sources" of cAMP generation at the plasma membrane. This offers potential for compartmentation (compartmentalization) of cAMP signaling in cells, a notion that was first mooted by Buxton and Brunton in milestone studies on cardiac myocytes18 and substantiated by others in such cells.19–26 However, as the free diffusion of cAMP is rapid (130 to 700 µm2 sec–1), the cell interior will quickly be equally distributed with cAMP. Furthermore, without any means of degrading cAMP then, after adenylyl cyclase activation, the cell interior would rapidly be saturated with cAMP. The ability to generate and shape cAMP gradients within the cell depends on the degradation of cAMP to 5'-AMP, which is achieved by cAMP phosphodiesterases (PDEs).27 Cytosol PDE activity would allow the formation of gradients of cAMP that depended on the source of cAMP generation by adenylyl cyclases located within subdomains of the plasma membrane. This adenylyl cyclase–PDE-dependent formation limits the potential for shaping gradients of cAMP in cells, thereby channeling cAMP signaling along specific conduits. To achieve this process, a further level of sophistication is engineered into the system, namely the ability to spatially restrict cAMP gradients at specific intracellular sites by targeting PDEs to specific intracellular sites and signaling complexes within cells.12,14,28–30

Tethering of PDEs allows these enzymes to form and shape localized cAMP gradients that can be visualized with genetically encoded sensors.20,24,31,32 Up until recently the general notion was that tethered PDEs provided a barrier to free diffusion of cAMP from restricted microenvironments surrounding the site of generation at the plasma membrane. However, it is clear that such a model imposes severe limitations on the degree of control that can be exerted, not the least of which, that it confines spatial control to two compartments, namely those defined as being either inside or outside the PDE "barrier." Recent experimental evidence, using siRNA knockdown of cAMP-specific PDE4, has suggested an additional scenario.31 This scenario envisages spatially confined populations of tethered PDEs generating localized "sinks" or "black holes," down which cAMP "disappears" as it is converted into 5'-AMP (Figure 1). Spatially constrained PDE subpopulations coupled with free diffusion of cAMP will allow a myriad of localized gradients of cAMP to be generated and shaped in cells. Such a sophisticated system provides a means of generating a multitude of microenvironments in the cell interior that are under precise control of specific, tethered PDE subpopulations. Each of these PDE subpopulations can be envisaged as regulating distinct cAMP-controlled processes through either protein kinase A (PKA) and its associated substrates or EPAC (exchange protein activated by cAMP) and its associated Rap1/2 effectors, with specific phenotypes therefore being associated with displacing specific PDEs from individual locales. Thus targeted PDEs are fundamental to the generation and control of compartmentalized cAMP signaling processes. These findings clearly underpin the complex nature of cAMP actions in the heart, where there are a plethora of distinct cAMP-regulated systems requiring distinct regulation of cAMP inputs that appear to be regulated by distinct PDEs. Such a system offers potential for sophisticated regulation by manipulating PDE tethering. Indeed, the cell type–specific expression of components of the proteins that form the cAMP signaling toolbox allows specific tailoring of the compartmentation of cAMP signaling. Additionally, it can be envisaged that alterations in either PDE isoform profile or tethering mode could contribute to certain pathologies.


Figure 1
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Figure 1. Compartmentalization of cAMP: barriers, sinks, holes. a, Schematic showing cAMP generation by plasma membrane–located adenylyl cyclase coupled to ß2AR-Gs plus PDE4 tethered to an AKAP scaffold, which generates a localized sink (white/grey) associated cAMP gradient. b, AKAP-tethered PDE4 activity gates the activation of AKAP-associated PKA and its action on tethered substrates, which include PDE4 long isoforms. c, Sink size depends on the tethered [PDE4] and activity regulation by inputs form other signaling pathways.

There are 11 different PDE families, of which 8 encode a plethora of isoenzymes able to degrade cAMP.27 The use of selective inhibitors,27 small interfering RNA (siRNA)-mediated gene knockdown,33 dominant negative constructs,33–35 and targeted gene knockouts36–41 has identified nonredundant, functional roles for an increasing number of PDEs.

Here we focus on the insight that investigation of the PDE4 family has provided into cAMP signal compartmentation, with special emphasis on cells of the cardiovascular system. Indeed, analysis of PDE4 enzymes has provided the paradigm for intracellular targeting of cAMP degradation30 and highlights the fundamental role that individual PDE4 isoforms are poised to play in tailoring compartmentalized cAMP signaling. This, undoubtedly, is a key reason why the complex 4 gene PDE4 family has been highly conserved through evolution.


*    "PDE4-ology"
up arrowTop
up arrowAbstract
up arrowIntroduction
*"PDE4-ology"
down arrowThe Long, Short, and...
down arrowThe Motor in the...
down arrowFinding the Perfect Partner...
down arrowCellular Function and Phenotype...
down arrowMolecular Determinants Mediating...
down arrowCardiomyocyte PDE4s
down arrowPDE4D-RyR2 Interaction
down arrowPDE4D3-mAKAP
down arrowPhenotypic Modulation of VSMCs
down arrowVSMC Phenotypic Modulation and...
down arrowDistinct PDE4D Variants...
down arrowSignificance of a Distinct...
down arrowThe Future
down arrowReferences
 
Four genes (A/B/C/D) encode more than 20 different PDE4 isoforms through alternative mRNA splicing coupled to the use of different promoters.28,42,43

PDE4A is located at Chr19p13.2, PDE4B at Chr1p31, PDE4C at Chr19p13.1, and PDE4D at Chr5q12. These genes span approximately 50 kb and comprise approximately 20 exons, the core catalytic unit of which is encoded by 7 exons.44–46 Additional exons encode regulatory regions and the N-terminal regions that uniquely identify individual isoforms. Various studies have linked the PDE4D gene to stroke47 and to changes in bone mineral density48; other studies have linked the PDE4B gene to schizophrenia.49

Unique to the PDE4 family are the highly conserved regulatory regions upstream conserved region 1 (UCR1) and upstream conserved region 2 (UCR2).50 Each is encoded by 3 separate exons, with UCR1 being formed from some 55 amino acids and UCR2 being formed by some 76 amino acids. PDE4 isoforms are subcategorized into 4 groups based on their UCR1/UCR2 complement. Thus "long" isoforms have UCR1 and UCR2, "short" isoforms lack UCR1, and "super-short" isoforms have just a truncated UCR2, whereas "dead-short" isoforms lack UCR1 and UCR2 and have an inactive catalytic unit that is both N- and C-terminally truncated (Figure 2).51,52 All PDE4 genes encode long isoforms, although only certain PDE4 genes encode types of short forms (Figure 2).


Figure 2
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Figure 2. PDE4 isoform diversity: long, short, super-short and dead-short. Each isoform is defined by its unique N-terminal targeting region. Long isoforms have UCR1 and UCR2 domains; short isoforms lack UCR1; super-short isoforms have an N-terminally truncated UCR2; dead-short isoforms lack UCR1 and UCR2 and have an inactive catalytic unit that is both N- and C-terminally truncated. Current isoforms among the four PDE4 genes are shown.

UCR1 and UCR2 likely interact in PDE4 long isoforms through ionic interactions53 to form a regulatory module that directs the functional outcome of phosphorylation by PKA and extracellular signal-regulated kinase (ERK).54–61

UCR1 is joined to UCR2 by LR1, a region that is encoded by 2 exons, is approximately 22/24 amino acids in length, and shows profound heterogeneity between subfamilies. LR2, which joins UCR2 to the catalytic unit, is encoded by a single exon and, as with LR1, shows no similarity between PDE4 subfamilies and varies in length between 10 and 28 aa. Their functional significance remains to be ascertained.

The final exon encodes part of the core catalytic unit as well as the C-terminal tail unique to each PDE4 subfamily. Indeed, the difference in primary sequence of this region has been exploited by us to make antisera specific to each PDE4 subfamily.

The most 5' isoform for each PDE4 gene, seemingly, has dual 5' exons encoding its unique N-terminal region, whereas other isoforms have a single 5' exon encoding their unique N-terminal region. Specific promoters found immediately 5' to the N-terminal coding exon control the expression of individual isoforms.62–65 Such minimal PDE4 promoters appear to lack a canonical TATA box but contain CpG-rich islands and a series of perfect stimulating protein 1 (Sp1) consensus binding sites that drive basal promoter activity. Undoubtedly, regions 5' to this confer cell type–specific expression and further regulation.

It has also been shown that an alteration in the degree of histone acetylation of the PDE4D1/2 intronic promoter regulates the extent to which these variants are expressed in VSMCs.66 Histone acetylation is among the numerous epigenetic factors that control expression of many genes,67 and it will be interesting to determine whether other PDE4 isoforms are similarly regulated. Additional control of PDE4 isoform expression can occur through regulation of mRNA stability by cross-talk with the ERK pathway,68 the action of which has been implicated in cardiac hypertrophy.69

At a genomic level, the sequence of coding exons is highly conserved among species, indicating that the complexity of organization and plethora of PDE4 isoforms must provide a functional advantage to have survived evolutionary pressures in such an intact state. If changes in the PDE4 isoform and tethering-protein profiles change in pathological states, then this is likely to have a profound effect of compartmentation of cAMP signaling.


*    The Long, Short, and Super-Short of PDE4 Isoforms
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrow"PDE4-ology"
*The Long, Short, and...
down arrowThe Motor in the...
down arrowFinding the Perfect Partner...
down arrowCellular Function and Phenotype...
down arrowMolecular Determinants Mediating...
down arrowCardiomyocyte PDE4s
down arrowPDE4D-RyR2 Interaction
down arrowPDE4D3-mAKAP
down arrowPhenotypic Modulation of VSMCs
down arrowVSMC Phenotypic Modulation and...
down arrowDistinct PDE4D Variants...
down arrowSignificance of a Distinct...
down arrowThe Future
down arrowReferences
 
UCR1 and UCR2 have a major functional role in regulating the activity of the PDE4 catalytic unit, particularly in integrating the effect of phosphorylation. This was first demonstrated for PKA,59,60 which phosphorylates the target serine within the conserved RRESF motif and causes activation of long isoforms from all 4 subfamilies.58 Such activation contributes to the cellular desensitization system for cAMP70 in cells in which long isoforms are expressed, such as cardiomyocytes20 and VSMCs.66

The mitogen-activated protein kinase ERK regulates numerous aspects of cardiomyocyte and VSMC functions, both in health and disease, including their hypertrophic responses.69,71–73 The catalytic unit of all PDE4 subfamilies, save for PDE4A, contains a serine within a consensus site (PQSP) that allows phosphorylation by ERK in vivo, altering activity54,55,57,61 and expression.68 As with all authentic ERK substrates, the catalytic unit of PDE4 enzymes contains both a KIM docking site (VxxKKxxxxxxLL), located on an exposed ß-hairpin loop some 122 amino acids N-terminal to the target serine, and an ERK specificity motif (FQF), located on an exposed {alpha}-helix some 18 amino acids C-terminal to the target serine.57 It is the presence of absence of UCR1/UCR2 that determines the functional outcome of ERK phosphorylation of PDE4, with long isoforms being inhibited, short isoforms being activated, and super-short isoforms being weakly inhibited. Thus cAMP signaling can be either positively or negatively coupled to ERK activation in specific intracellular locales dependent on the complement of short and long isoforms expressed. Such cross-talk can be reprogrammed by changes in the PDE4 isoform expression profile as seen in monocyte to macrophage differentiation,74 and it will be of interest to see whether changes in cross-talk occurs in VSMC differentiation, where the PDE4 long/short profile changes.66

Interestingly, ERK inhibition of PDE4 long isoforms can be negated by PKA phosphorylation.55 This can lead to a situation where ERK-induced PDE4 inhibition can raise cAMP levels, causing PKA to become activated and phosphorylate the long PDE4, thereby ablating the inhibitory effect of ERK phosphorylation. Thus, as a consequence of ERK activation, long PDE4 isoforms may cycle through inhibition followed by activation, thereby causing either a transient, programmed rise in cAMP levels in their immediate locale or even oscillations.

More recently it has been demonstrated61 that the N-terminal portion of the PDE catalytic unit (Ser239 in PDE4D3) can be phosphorylated by an unknown kinase that acts downstream of phosphatidylinositol 3-kinase and is activated by oxidative stress. Phosphorylation at this site alone in PDE4D3 has no effect on catalytic activity. However, oxidative stress also activates ERK and it is when PDE4D3 is phosphorylated both by ERK (Ser579) and the unknown kinase (Ser239) that the function of this kinase is uncovered as reprogramming the effect of inhibitory ERK phosphorylation to now cause activation. Indeed, this now mimics the "loss of UCR1," seen in short isoforms, where ERK phosphorylation of the PDE4 catalytic unit confers activation.57

This unknown kinase is activated by reactive oxygen (ROS), and it may be linked to stress-induced reprofiling evident in cardiovascular disease. Clearly it will be important to identify it.


*    The Motor in the Middle
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrow"PDE4-ology"
up arrowThe Long, Short, and...
*The Motor in the...
down arrowFinding the Perfect Partner...
down arrowCellular Function and Phenotype...
down arrowMolecular Determinants Mediating...
down arrowCardiomyocyte PDE4s
down arrowPDE4D-RyR2 Interaction
down arrowPDE4D3-mAKAP
down arrowPhenotypic Modulation of VSMCs
down arrowVSMC Phenotypic Modulation and...
down arrowDistinct PDE4D Variants...
down arrowSignificance of a Distinct...
down arrowThe Future
down arrowReferences
 
The structure of the core PDE4 catalytic unit has been resolved.28,43,75 It is a compact structure of 17 {alpha}-helices folded into 3 subdomains. These subdomains come together to form a deep pocket containing the cAMP binding active site, which contains tightly bound Zn2+ and loosely bound Mg2+, essential for catalytic activity. This pocket has a volume of 440 Å3, which contains the 232 Å3 cAMP molecule. PDE4 activation by PKA phosphorylation is influenced by [Mg2+] and the dominant "connections" that hold Mg2+ and are links to amino acids on helices 10/11. These connections, together with their connecting loop, fold over the surface of the catalytic center so as to create a "tweezer-like" motif that grips the Mg2+. It is possible that UCR/UCR2 may direct actions to Mg2+ at the catalytic center via helices 10/11 or others that either interact directly with them or indirectly cause conformational changes in them.

Analyses of the PDE4 catalytic unit structure, proposed catalytic mechanism, binding of selective inhibitors and "inside-out" signaling where inhibitor binding might transmit changes to the molecule surface are discussed elsewhere.28,43,76


*    Finding the Perfect Partner and Identifying Targeting "Zip Codes"
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrow"PDE4-ology"
up arrowThe Long, Short, and...
up arrowThe Motor in the...
*Finding the Perfect Partner...
down arrowCellular Function and Phenotype...
down arrowMolecular Determinants Mediating...
down arrowCardiomyocyte PDE4s
down arrowPDE4D-RyR2 Interaction
down arrowPDE4D3-mAKAP
down arrowPhenotypic Modulation of VSMCs
down arrowVSMC Phenotypic Modulation and...
down arrowDistinct PDE4D Variants...
down arrowSignificance of a Distinct...
down arrowThe Future
down arrowReferences
 
PDE isoforms play a pivotal role in creating and underpinning compartmentalized cAMP responses by generating gradients that are subsequently read and acted on by tethered PKA and EPAC subpopulations. This process, undoubtedly, explains the need for diversity among PDE isoforms and explains the importance as evidenced by the maintenance of such diversity against evolutionary pressures. Pivotal to this finding was the insight that the N-terminal regions unique to individual isoforms contain information that confers intracellular targeting.28,43,77 This targeting can take the form of targeting to specific subcellular membranes or to specific signaling complexes. In this way, unique gradients are generated that can be controlled by cross-talk with the ERK signaling pathway and by PKA activation of long isoforms.

The paradigm for this notion came from studies on the PDE4A1 super-short isoform.77 Uniquely, PDE4A1 is exclusively membrane-associated and requires detergents to effect its release. PDE4A1 is uniquely characterized by its 25-aa N-terminal region, the removal of which generates a soluble, fully active species.77–82 Thus all of the information essential for membrane targeting is held within its unique N-terminal region. Consistent with this, chimeric species, made with various soluble, cytosolic proteins transformed them to membrane-bound species that localized within cells as did PDE4A1.

A key feature of individual PDE4 isoforms is their ability to be targeted to specific sites/signaling complexes within cells, leading to the notion of a "PDE4 interactome" (Figure 3).


Figure 3
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Figure 3. PDE4 interactome. Proteins currently known to interact with PDE4 isoforms to form signaling-specific signaling scaffold complexes that in certain instances are targeted to specific cellular regions. Such complexes underpin sink formation and the compartmentation of cAMP signaling.

The N-terminal region of the long PDE4D5 isoform contains binding sites for the signaling scaffold proteins ß-arrestin33,34,83,84 and RACK1,85–87 which we discuss in detail below.

The N-terminal regions of the long PDE4A4/5 and PDE4D4 isoforms contain proline-rich sequences that confer interaction with SH3-domains of certain proteins, such as the tyrosyl kinases Lyn, Fyn, and Src.88–91 Differences in specificity of interaction are seen between PDE4A4/5 and PDE4D4 because of different sequences surrounding their distinct proline- and arginine-rich sequences.

PDE4A4/5 can bind to the immunophilin AIP/XAP2/ARA9, which is known to interact with the aryl hydrocarbon receptor (AHR), a transcription factor required for normal cardiac development92; AHR is upregulated in cardiomyopathy93; and the genetic deletion of AHR leads to cardiac hypertrophy, hypertension, and fibrosis.94 Aryl hydrocarbon receptor interacting protein (AIP) interacts not only with the PDE4A4/5 N-terminal region to give isoform specificity but also interacts with UCR2 to elicit an inhibitory effect on PDE4A4/5 activity.95 This paradigm is a clear indication of how protein–protein interaction may regulate PDE4 catalytic activity. However, many PDE4-interacting proteins, such as RACK1, ß-arrestin, and SH3 domain–containing proteins do not exert any profound effect on catalytic activity.

Interestingly, other proteins have been shown to interact with UCR2, namely the scaffold proteins, myomegalin,96 myeloid translocation gene protein,97 and DISC1.49 However, it remains to be seen whether they interact with additional sites on PDE4 and whether they alter PDE4 activity.

The long PDE4D3 isoform interacts with the PKA anchor protein mAKAP,98–100 which is induced in cardiac hypertrophy101 and serves to relocate PDE4D3 to the perinuclear region of hypertrophic cardiac myocytes.99 PKA phosphorylation of Ser13, within the unique N-terminal region of PDE4D3, increases interaction with mAKAP.98 The activities of mAKAP-associated PDE4D3 and PKA are intertwined with PDE4D3 being phosphorylated at 2 sites by PKA,56,58–60 namely Ser54 in UCR1, causing activation, and Ser13, causing increased binding to mAKAP.

Mapping sites of interaction between proteins has, traditionally, been an arduous process involving truncation and mutation approaches. However, we have recently pioneered peptide array technology as a rapid means of ascertaining interaction sites.85 Here a recombinant interactor protein is used to probe a library of overlapping, immobilized 25-mer peptides that scan the entire sequence of a particular protein. This allows rapid identification of regions that may contribute to the protein–protein binding. An interacting peptide was used as a template to generate a library of progeny where individual amino acids in the 25-mer parent are replaced by alanine, for example, in a corollary to scanning mutagenesis. Thus amino acids of putative importance to binding can be identified and used to direct mutagenesis approaches using intact proteins in 2-hybrid, pull-down, coimmunoprecipitation, and colocalization approaches. Where structural information is also available, this can additionally be used to identify surface residues that likely form a binding site, thereby further facilitating mutagenesis strategies.


*    Cellular Function and Phenotype Conferred by PDE4D5 Association With ß-Arrestin
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrow"PDE4-ology"
up arrowThe Long, Short, and...
up arrowThe Motor in the...
up arrowFinding the Perfect Partner...
*Cellular Function and Phenotype...
down arrowMolecular Determinants Mediating...
down arrowCardiomyocyte PDE4s
down arrowPDE4D-RyR2 Interaction
down arrowPDE4D3-mAKAP
down arrowPhenotypic Modulation of VSMCs
down arrowVSMC Phenotypic Modulation and...
down arrowDistinct PDE4D Variants...
down arrowSignificance of a Distinct...
down arrowThe Future
down arrowReferences
 
Although great strides have been made in determining the location and mode of targeting of various PDE4 isoforms, the exact cellular function attributable to the targeting of individual PDE4 isoforms is now just beginning to be appreciated with the deployment of novel technologies. Paramount to this progress has been our development of the "dominant negative" approach.33–35,85 This took advantage of knowledge of the 3D structure of the PDE4 catalytic unit75 to formulate a catalytic mechanism28 and identify key amino acids in the putative cAMP binding pocket that would be essential for allowing hydrolysis of the phosphodiester bond of cAMP. The mutation of a key aspartate residue sufficed to render PDE4 isoforms from all 4 subfamilies catalytically inactive.35 Thus overexpression of catalytically inactive isoforms in cells would not be expected to generate an overt phenotype unless they acted to replace/displace their cognate, native functional isoform from the site its tethered to in a cell, thereby removing the sink mechanism and allowing for an increase in localized cAMP levels (Figure 4). Such an approach does not require any understanding of the nature of the anchor for the PDE4 isoform. Of course, if the anchor and mode of anchoring were known, then the catalytically inactive PDE4 isoform could be suitably mutated, so that it were unable to bind the anchor, which would lead to a loss of phenotype and its ability to exert a the dominant negative action.85 The paradigm for this approach came from studies on PDE4D5,102 leading to the first demonstration that a single PDE4 isoform can express a unique functional role in cells.33 Thus PDE4D5 bound to the multifunction adapter protein ß-arrestin allows for the dynamic movement of active PDE4D5 from the cytosol to the ß2 adrenoreceptor (ß2AR) after agonist challenge (Figure 5). This generates a localized sink for cAMP adjacent to the ß2AR that controls the activity of a PKA subpopulation tethered to this receptor by AKAP79.33 Both of these actions were unequivocally shown to be ß-arrestin dependent as they were not apparent in ß-arrestin-1/2 double knockout mouse embryo fibroblasts (MEFs) but could be reconstituted in such cells on ectopic reintroduction of ß-arrestin-1 using an adenoviral vector.84


Figure 4
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Figure 4. Ectopic expression of catalytically inactive PDE4 isoforms to uncover dominant negative functionality. a, Schematic of 2 PDE populations, 1 that is distributed randomly in the cytosol and 1 that is sequestered to a specific site. b, The sequestered PDE subpopulation will generate a sink/localized gradient. c, Overexpression of a catalytically inactive PDE that is cognate to the endogenously expressed, tethered species. This causes displacement of the endogenous active species such that it is randomly distributed in the cytosol. d, Loss of the active tethered species destroys the sink and localized gradient, conferring a dominant negative action on the catalytically inactive PDE. This will now allow PKA/EPAC subpopulations in the environment of the tethered inactive PDE to be activated.


Figure 5
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Figure 5. Dual desensitization of ß2AR signaling through recruitment of ß-arrestin/PDE4D5 complex. Isoprenaline (ISO) stimulation of the ß2AR causes activation of adenylyl cyclase and increases cAMP levels. This activates PKA-RII tethered to the receptor by AKAP79, causing PKA phosphorylation of the ß2AR, which can now couple to Gi to activate ERK. Gs activation allows G protein coupled receptor kinase (GRK) recruitment to, and phosphorylation, of the ß2AR. This signals recruitment of a ß-arrestin–bound PDE4D5 complex to the ß2AR, which confers dual desensitization; (1) ß-arrestin attenuates ß2AR coupling to Gs; and (2) PDE4D5 forms a localized cAMP sink, causing deactivation of PKA-RII tethered to the ß2AR by AKAP79 with loss of PKA-phosphorylated ß2AR and ERK activation.

In various cell types including cardiomyocytes PKA phosphorylation of the ß2AR partially attenuates coupling to Gs and switches on coupling of the phosphorylated ß2AR to the guanine nucleotide regulatory protein, Gi (Figure 5).12,103 Through this process, the PKA phosphorylated ß2AR elicits the SRC-dependent activation of ERK.

Recruitment of the ß-arrestin–PDE4 complex serves to orchestrate a dual desensitization event. Thus the recruited ß-arrestin physically interdicts signaling between receptor and Gs, initiating a reduction in adenylyl cyclase activation and a subsequent decrease in cAMP production. However, simultaneously, ß-arrestin–bound PDE4D5 provides a localized sink for cAMP degradation, which acts to downregulate ß2AR associated PKA activity and, thereby, dampen signaling to ERK through Gi. Such a regulatory function has been demonstrated in neonatal cardiomyocytes as well as model cell lines.12,34,83,103,104

It has also been demonstrated that cAMP pools generated by stimulation of different Gs coupled receptors in rat ventricular myocytes are shaped by the differential coupling of each receptor type to different PDE families.19 However, the extent to which G protein–coupled receptor–specific cAMP "pools" are influenced by recruited PDE4D5 in complex with ß-arrestin remains to be ascertained.

In cardiac myocytes, PDE4D5 is preferentially associated with ß-arrestin and selectively recruited to the ß2AR on agonist challenge, despite the fact that PDE4D5 expression was some 5 times lower than that of PDE4D3.34,83 As discussed below, PDE4D5 interacts preferentially with ß-arrestin because of an additional binding site unique to this isoform.83,85

In cardiac myocytes, chemical ablation of PDE4 activity by the specific inhibitor, rolipram enhances both PKA phosphorylation of the ß2AR and the switching of its signaling to ERK activation.34,83 However, rolipram inhibits all PDE4 isoforms similarly and so cannot identify control by any one PDE4 isoform. That selective silencing of all PDE4D isoforms by siRNA-mediated knockdown mimicked such actions of rolipram33 identifies the importance of this subfamily in modulating ß2AR signaling but gives no insight into which particular isoform and whether targeting is required. Isoform-specific, siRNA-mediated knockdown subsequently identified PDE4D5 as the relevant species.33 However, although this technological approach indicates the role of PDE4D5, it gives no insight into whether the entire cellular PDE4D5 pool is of importance or whether a subpopulation is important, namely one that is tethered specifically to ß-arrestin. Such an analysis demanded a new technological approach, and for this we used overexpression of a catalytically inactive PDE4D5 (Asp556Ala), which was introduced into cardiac myocytes by adenoviral-mediated gene delivery.34 This catalytically inactive PDE4D5, when overexpressed, serves to displace endogenous PDE4D5 from ß-arrestin and prevent agonist-mediated delivery of active PDE4D5 to the ß2AR, providing a dominant negative action.33,34,85 Dominant negative PDE4D5 amplifies PKA activity at the plasma membrane but not in the cytoplasm. This mimics the phenotype engendered by treatment with either rolipram or PDE4D5 knockdown. Final verification that the PDE4D5 phenotype resulted from its preferential association with ß-arrestin resulted from the demonstration that a discrete mutation in the N-terminus of catalytically inactive PDE4D5 construct, made so as to compromise its ability to bind ß-arrestin; prevented such a species from displacing endogenous active PDE4D5 from ß-arrestin; and failed to elicit a dominant negative effect.85 This dominant negative approach, undertaken on cardiac myocytes, provided the first indication that a cellular phenotype could be assigned to an individual PDE isoform.

Clearly, a dominant negative strategy provides a means of dissecting out functional roles for anchored subpopulations of PDE4 isoforms that cannot be determined using either active site-directed inhibitors or siRNA knockdown. The identification of small molecules that disrupt targeting of specifically anchored PDE4 isoforms may provide for novel therapeutic agents that are not plagued by the various side effects seen with active site-directed PDE4 inhibitors. Such targeting disruptors can be expected to allow diminution of PDE4 activity at a highly specific spatial locale.


*    Molecular Determinants Mediating PDE4D5–ß-Arrestin Interaction
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*Molecular Determinants Mediating...
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Pull-down and 2-hybrid analyses showed that members from all 4 PDE4 families could bind ß-arrestin.84 Further investigations showed that this was attributable to the presence of a common site within the highly conserved catalytic unit of the enzyme.83 Pull-down studies using truncated PDE4D5 constructs coupled with 2-hybrid analyses went on to demonstrate that the PDE4D5 isoform interacted preferentially with ß-arrestin because, in addition to the binding site for ß-arrestin in its catalytic region, PDE4D5 has an additional binding site in its unique N-terminal region.83 These two sites on PDE4D5 interact with distinct sites on ß-arrestin. Thus the common interaction site in the PDE4 catalytic unit binds to the N-domain of ß-arrestin, whereas the site unique to PDE4D5 binds to the C-domain of ß-arrestin. Such 2-point interaction allows PDE4D5 to straddle ß-arrestin. This underpins the preferential association of PDE4D5 with ß-arrestin, which is pivotal in conferring its precise functional action in regulating the PKA phosphorylation status of the ß2AR.

PDE4D5 can also form a complex with the WD repeat scaffold protein RACK1. This interaction is unique to PDE4D5 because, as with ß-arrestin, it involves a binding site located in the PDE4D5-unique N-terminal region.86,87 Indeed, overlapping binding sites for both ß-arrestin and RACK1 in the unique N-terminal region of PDE4D5 coupled to distinct second sites of interaction on the catalytic unit confers their mutually exclusive binding to PDE4D5.85 Thus ß-arrestin and RACK1 independently sequester PDE4D5, ensuring fidelity of signaling through these distinct scaffold proteins.

Insight into the location and nature of the binding sites for ß-arrestin and RACK1 on PDE4D5 was garnered using peptide array technology (Figure 6).85 This allowed us to demonstrate that both RACK1 and ß-arrestin to PDE4D5 could bind to amino acids between residues 22 and 45 within the N-terminal portion of PDE4D5. Using alanine substitution arrays to scan this region, specific amino acids were identified as involved in determining the binding of either ß-arrestin (E27, D28, L29) or RACK1 (N22, P23, W24, V30, K31), exclusively, or were found in common as important for the binding of each of these signaling scaffold proteins (L33, R34). Analysis of the 3D structure of this portion of the PDE4D5 N-terminal region showed these residues to be surface exposed and that the concomitant binding of both ß-arrestin and RACK1 to PDE4D5 was not possibly attributable to the proximal and overlapping nature of their respective binding sites. Thus in any one cell, there are likely to be specific, spatially distinct subpopulations of PDE4D5 because of the association of PDE4D5 with scaffolding proteins such as ß-arrestin and RACK1.


Figure 6
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Figure 6. Scanning peptide array technology identifies binding sites on PDE4D5 for ß-arrestin. a, Schematic of PDE4D5, the entire sequence of which was used to provide an immobilized overlapping 25-mer peptide array. b, Sections of the PDE4D5 library that interact with ß-arrestin. c, Peptide 131, with a sequence that is within the PDE4D5 catalytic unit, was used as a parent peptide to generate a scanning peptide library where sequential amino acids in the native sequence were substituted with alanine. When probed to identify amino acids important in binding ß-arrestin, namely those failing to interact when changed to alanine, the FxFxLxL motif was identified. d, Surface projection of the modeled PDE4D catalytic unit with the FxFxLxL motif identified as surface exposed on helix-17. Some of the information used in this figure has been adapted from Bolger et al.85

Peptide array analysis also facilitated resolution of the amino acids that contribute to the common ß-arrestin binding region within the conserved catalytic unit of all PDE4s.85 The amino acids identified (F670, F672, L674, and L676) are all surface exposed and located on helix-17, which appears to be attached to the compared core catalytic unit by a mobile hinge region (Figure 6).85 Interestingly, 2 of the amino acids implicated (F670, F672) in ß-arrestin binding also form part of the ERK specificity/docking binding motif on PDE4 enzymes,57 which would preclude PDE4 isoforms from binding directly to both ß-arrestin and ERK. As ERK can phosphorylate and deactivate PDE4 long forms, it is tempting to speculate that by preventing ERK docking to PDE4D5, ß-arrestin–bound PDE4D5 ensures only activated enzyme is found in this complex and so recruited to the ß2AR on agonist activation.

This work highlights the power of peptide array technology for the rapid and informative definition of protein–protein interactions.


*    Cardiomyocyte PDE4s
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up arrow"PDE4-ology"
up arrowThe Long, Short, and...
up arrowThe Motor in the...
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*Cardiomyocyte PDE4s
down arrowPDE4D-RyR2 Interaction
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PDE4B and PDE4D variants are expressed in rodent, murine, and human cardiomyocytes,105 and selective PDE4 inhibitors have been suggested to have small effects on Ca2+ currents and contractility.62,106–108 In particular, it has been suggested that PDE4 inhibitors, although promoting isoprenaline-induced inotropic responses, have little effect alone.106,109 Intriguingly, much analysis of this effect has been performed using diazepam, which has been suggested to function through PDE4 inhibition,109 although any possible action on the "novel" cAMP-hydrolyzing PDE7/8/10/11 forms has yet to be assessed. Indeed, PDE4 inhibitors, which have potential therapeutic use in treating sepsis, improve cardiac contractility in endotoxemia,110 suggesting that they may have a role for treating critically ill patients.

Although several studies have described altered cardiomyocyte PDE3A expression in certain types of heart disease,111,112 no studies have systematically addressed whether the PDE4 expression profile is altered in heart failure. Given the distinct spatial and temporal regulation of cAMP levels afforded by distinct PDE4 subtypes, their altered expression in heart disease would be predicted to, potentially, markedly alter cardiac function. In contrast, significant advances have been made relative to the manner by which these enzymes allow compartmentalized cAMP signaling in cardiomyocytes. Recent elegant molecular studies have determined that individual PDE4 variants can be tethered to distinct sarcoplasmic reticulum (SR) proteins, allowing them to either directly or indirectly control SR function.12,19,20,22,25,31,34,38,108,113 Indeed, tethering of PDE4 variants to specific signaling complexes likely represents the molecular basis for much of the selective actions seen with PDE4 inhibitors, compared with PDE2 and PDE3-selective inhibitors in cardiomyocytes. Moreover, it is possible that loss of tethering for certain PDE4D variants may lead to cardiac failure, as seen in PDE4D-null mice and, perhaps, in humans (see below).38

PDE4B and PDE4D localize to sarcomeric M- and Z-line structures, respectively, in neonatal rat ventricular cardiomyocytes. Consistent with this, in fully differentiated adult cardiomyocytes, PDE4 activity is high at the transverse (T) tubule/SR junctional space of cardiomyocytes, the area regulating excitation–contraction coupling.25,114 Notwithstanding that cardiomyocytes express several long PDE4D isoforms (PDE4D3, PDE4D5, PDE4D8, PDE4D9), to date most studies have limited their analysis to how PDE4D3, and PDE4D5 tethering contributes to compartmented cAMP signaling in cardiomyocytes. Thus PDE4D3 associates with the ryanodine receptor 2 (RyR2)38 and A-kinase anchoring proteins (AKAPs)99,115,116 in cardiomyocytes, so as to spatially and temporally regulate cAMP. PDE4D5 is the key isoform interacting with ß-arrestin,33,34,83,85 which allows it to preferentially regulate ß2AR signaling12,34 but can also interact with the signaling scaffold protein, RACK1.85–87,117,118


*    PDE4D–RyR2 Interaction
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*PDE4D-RyR2 Interaction
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PDE4D3 can integrate into an SR-associated RyR–based complex (RyR2, cardiac, RyR2). RyR2 is a tetrameric SR Ca2+ channel that represents the dominant Ca2+-release channel in cardiomyocytes.119,120 Kinetics of RyR2 channel opening is complex, with the open probability state being stabilized by several factors including cytosolic Ca2+, cyclic ADP-ribose (cADPR), caffeine, and phosphorylation by several protein kinases including PKA and Ca2+/calmodulin-dependent protein kinase II (CaMKII).121,122 The closed state of RyR2 is stabilized by binding of 4 subunits of the {approx}12-kDa FK506-binding protein FKBP12.6. In addition to FKBP12.6, several other proteins interact with the RyR2 within a large macromolecular signaling complex in cardiomyocytes, which may include PKA, CaMKII, PP1, PP2A, mAKAP, spinophilin, PR130, sorcin, triadin, junctin, calsequestrin, and Homer.123

Until very recently, analyses of PDE4D- and PDE4B-null mice had revealed no significant pathological role for these enzymes. Indeed, most of the attention had focused on either PDE3B within a phosphatidylinositol 3-kinase {gamma} complex or on PDE3A, the more abundant isoform expressed in cardiomyocytes.124,125 However, recently, PDE4D-null mice were reported38 to display a very late, age-dependent, cardiac phenotype composed of a progressive cardiomyopathy and an increased incidence of exercise-induced arrhythmias. This phenotype is similar to that reported when a RyR2 defect in patients produces heart failure and sudden cardiac death.126 At a functional level, the PDE4D-null mouse phenotype was associated with RyR2 hyperphosphorylation and a reduced capacity of hyperphosphorylated RyR2 to gate Ca2+. It was suggested that hyperphosphorylation of RyR2 was attributable to the absence of PDE4D within the RyR2-based macromolecular complex of PDE4D-null animals.38,127 The authors suggested that, because the phenotype was suppressed in mice engineered to lack one of the potential PKA phosphorylation sites within RyR2 (S2808) and because less PDE4D3 was associated with RyR2 in human myocardium from heart failure patients, their findings were consistent with the hypothesis that PDE4D deficiency may contribute to heart failure and arrhythmias by promoting defective regulation of the RyR2 channel in humans. In addition, the authors also speculated that prolonged PDE4 inhibitor use might predispose patients to unexpected cardiac events. In this context, because no fewer than 70 individual mutations that alter the biophysical properties of the RyR2 have been described, because phosphorylation of the RyR2 is catalyzed by numerous kinases in addition to PKA, and because PDE4D activity and targeting are also each dynamically regulated by multiple factors, it is likely that further work in human cardiomyocytes will be required to fully assess the significance of the effect in humans. Additional work is needed to explore this proposal in studies using animals with different genetic backgrounds and, importantly, with conditional knockouts, so as to exclude any phenotypic input resulting from loss of PDE4D during development. Also, because the PDE4D knockout was generated by deletion of a catalytic exon such an approach might lead to the generation of truncated proteins that could interact with signaling complexes to exert dominant negative actions independent of loss of PDE4 activity. In this context, it should be noted that no significant cardiac side effects have been reported in clinical trials of PDE4 inhibitors designed for use in treating chronic obstructive pulmonary disease or asthma in humans, and no cardiac toxicology has been reported in animal studies performed using various PDE4-selective inhibitors in development.43,128–130 Thus it is important to extend studies of PDE4 action in the heart to understand fully its role in health and disease.


*    PDE4D3–mAKAP
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up arrow"PDE4-ology"
up arrowThe Long, Short, and...
up arrowThe Motor in the...
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up arrowCellular Function and Phenotype...
up arrowMolecular Determinants Mediating...
up arrowCardiomyocyte PDE4s
up arrowPDE4D-RyR2 Interaction
*PDE4D3-mAKAP
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In addition to regulating the acute contractile functions of the heart, PDE4 enzymes have also been shown to have potential in regulating the trophic responses of cAMP in cardiomyocytes. For example, binding of PDE4D3 to mAKAP in neonatal hypertrophic ventricular cardiomyocytes targets this PDE4 variant to the perinuclear region and allows it to regulate cAMP levels within this locale.99 Thus, PDE4D3 association with mAKAP, a striated muscle-specific AKAP scaffold tethered to nuclear membranes, was shown to promote more efficient control of PKA-mediated phosphorylation of several proteins, including PDE4D3 itself, in cardiomyocytes.99 In this context, further studies will be required to determine whether other PDE4 isoforms can interact with either mAKAP or other cardiomyocyte AKAPs and contribute to compartmentalized signaling in these cells.

Although little is known concerning the number of PDE4 variants that interact with cardiomyocyte AKAPs, recent studies have identified some of the proteins that also populate the mAKAP signaling complex. Indeed, the EPAC, a cAMP-activated Rap-GEF,4 as well as ERK5, may be found together in certain complexes.100 Interestingly, the presence of both PKA and EPAC within such a complex may represent a situation in which local cAMP concentrations can differentially control the activity of 2 cAMP effectors with distinct sensitivities to activation by cAMP. Indeed, whereas cAMP activation of mAKAP-associated PKA served to phosphorylate and thereby activate PDE4D3, reducing local cAMP concentrations, maximal activation of mAKAP-associated ERK5 served to suppress PDE4D3 activity, thus allowing for activation of EPAC. The mechanism by which ERK-mediated phosphorylation inhibits PDE4D3 involves phosphorylation of this long isoform at Ser579.54,55,57

Although it has been suggested that PDE4D3 represents the adaptor protein that recruits EPAC1 to the mAKAP complex,100 this has yet to be established. Indeed, PDE4D3 interacts with mAKAP via its unique 18-aa N-terminal domain.99 Thus, unless EPAC also interacts with this small PDE4D3-specific domain, which seems unlikely as it might then be expected to compete with mAKAP for binding to PDE4D3, we would expect that EPAC binds to a different site. Indeed, this appears to be the case as we (M. Houslay, H. Bos, M. Lynch, G. Baillie, unpublished results, 2006) can show that there is a common binding site on PDE4 isoforms for EPAC. Thus various other PDE4 isoforms may also be able to recruit EPAC to their site of anchorage within the cell. If multiple PDE4 isoforms were able to recruit EPAC to such signaling complexes, this would further increase the need to determine whether the PDE4 isoform expression profile is impacted in heart failure.


*    Phenotypic Modulation of VSMCs
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VSMCs can exist in 2 distinct phenotypes, contractile/quiescent (herein contractile) or synthetic/activated (synthetic).131,132 In healthy blood vessels in vivo, contractile VSMCs have low proliferative and migratory indexes, express contractile proteins, and contract or dilate in response to numerous hormonal or biophysical demands. In contrast, in culture, or in situ following vascular insult, synthetic VSMCs have a higher proliferative and migratory index, express fewer contractile proteins, and release extracellular matrix (ECM) proteins. Synthetic VSMCs do not directly maintain vascular tone, but rather, during developmental vasculogenesis and angiogenesis or in response to vascular injury or insult in the adult, they maintain vascular structural integrity.131–133 Substantial evidence indicates that both acute local effects, such as changes in VSMC–vascular endothelium communications, as well as longer-term genetic and epigenetic effects contribute to such phenotypic switches. These concepts and how they impact the maintenance of blood vessel structural and functional homeostatic integration have been reviewed elsewhere.67


*    VSMC Phenotypic Modulation and PDE3/PDE4 Activity Ratios
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Contractile VSMCs isolated from several distinct rat or human arteries express one variant each of PDE4A and PDE4B, as well as numerous PDE4D gene-derived variants including PDE4D3, PDE4D5, PDE4D7, PDE4D8, and PDE4D9.105 Selective immunoprecipitation of PDE4A, PDE4B, or PDE4D enzymes from these cells identifies PDE4D as representing the dominant catalytic activity.68,134 Although both contractile and synthetic VSMCs use PDE3 and PDE4 to hydrolyze cAMP, the individual gene family variants expressed in these cells, as well as their relative proportions, can differ in VSMCs isolated from different blood vessels within a given species, or in the same blood vessel between species.105 Indeed, whereas cAMP hydrolysis by PDE3 is dominant in rat and human aortic contractile VSMCs, PDE4 activity surpasses PDE3 activity in the synthetic VSMC phenotype (Figure 7).66,135–137 Although a phenotypic modulation-based regulation of the PDE3/PDE4 activity ratios has been reported in all rat and human VSMCs thus far studied, further work will be needed to establish the generality of this event to VSMCs from all vascular beds. Although the molecular basis for the reduced level of PDE3 activity in rat or human synthetic VSMCs is dependent on a marked reduction in PDE3A, the underlying mechanisms, and the factors that limit changes in PDE3B, or PDE4 expressed in these cells, during the phenotypic switch is currently unclear. However, it is apparent that the reduction in PDE3A expression in synthetic VSMCs represents another example in which synthetic VSMCs effectively reduces sensitivity to regulation through cGMP-dependent mechanisms. Indeed, it has been established that several cGMP-sensitive enzymes, including protein kinase G (PKG) and NO-sensitive guanylyl cyclases (sGCs), are downregulated in synthetic VSMCs compared with their contractile counterpart.138 In this context, it may be that PDE3B levels do not become reduced in synthetic VSMCs because PDE3B is significantly less sensitive to cGMP-mediated inhibition than PDE3A.139 PDE4 would be excluded from this regulatory scheme, being insensitive to physiological [cGMP]. Consistent with the proposition that synthetic VSMCs are somewhat more "cAMP-centric" than their contractile equivalent is the observation that the cAMP-hydrolyzing PDE1C is upregulated in human synthetic VSMCs and inhibition of its induction impacts VSMC proliferation.140,141


Figure 7
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Figure 7. Differential expression of PDE4 and PDE3 in contractile and synthetic VSMCs. Contractile VSMCs populate the medial layer of arteries, whereas synthetic VSMCs accumulate in the intimal layer in response to vascular insult. Although PDE3 and PDE4 activities are roughly equivalent in contractile VSMCs, a dramatic downregulation of PDE3A expression in synthetic VSMCs markedly increases the dependence of these cells on PDE4 for cAMP hydrolysis. In human VSMCs, PDE1C may also contribute to cAMP hydrolysis. Prolonged increases in cAMP in contractile VSMCs increases the level of expression of PDE4D1/2 long-form variants in these cells, whereas a similar treatment markedly upregulates expression of the PDE4D1 and PDE4D2 short forms in synthetic VSMCs.

With respect to the mechanism(s) that regulate these changes and render the synthetic VSMCs more dependent on PDE4-mediated regulation of cAMP signaling, it may be relevant that inducible cAMP early repressor (ICER), a transcriptional repressor, was recently shown111,112 to reduce PDE3A, but not PDE3B nor PDE4, expression in cardiomyocytes in response to certain rodent models of heart failure. Although a role for ICER in regulating CREB-mediated expression of some gene products has been reported in rodent synthetic VSMCs,142,143 further studies will be required to assess the involvement of this mechanism in the PDE3/PDE4 switch observed during the VSMC phenotype switch. In addition, although currently untested, it may be possible that changes in histone acetylation of the PDE3A promoter is reduced in synthetic VSMCs,66 an effect known to reduce transcription of several genes during the phenotypic switch.67

Rather than simply providing yet another example of differences between contractile and synthetic VSMCs, we believe that the altered PDE3/PDE4 activity ratio between these cells may be physiologically and therapeutically important. Thus, although it is generally accepted that PDE3 inhibitors are more effective at relaxing contractile VSMCs than PDE4 inhibitors, the increased dependence of synthetic VSMCs on PDE4 may afford selectivity in the regulation of cAMP-mediated events in synthetic VSMCs using PDE4-selective inhibitors.105 Indeed, although a PDE3 inhibitor (cilostamide) reduces accumulation of intimal (synthetic) VSMCs in a rat model of restenotic injury,144 given the potential proarrhythmic potential of these compounds, it is unlikely that they could be used safely to reduce postangioplasty restenosis in humans.145 In contrast, because PDE4 inhibitors have generally been found to have only very modest effects on the heart, selective PDE4 inhibitors could prove useful in this therapeutic arena. Also, because synthetic human VSMCs may also express the dual cAMP/cGMP-hydrolyzing PDE1C, inhibitors of this enzyme in combination with PDE4 inhibitors could also be a very powerful combination in these situations.


*    Distinct PDE4D Variants Coordinate Desensitization to Prolonged cAMP Signaling in Contractile and Synthetic VSMCs
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The transition of rat and human VSMCs from a contractile to a synthetic phenotype is accompanied by a major shift in the ratio between total PDE3 and PDE4 activities (Figure 7). Synthetic and contractile VSMCs both express approximately equal PDE4 activity. In addition, short-term (<30 minutes) treatment of these cells with cAMP-elevating agents causes the PKA-mediated phosphorylation and activation of the expressed PDE4D long isoforms.68,134 In contrast, the mechanisms by which these cells use PDE4 to desensitize the effects of prolonged challenges with cAMP-elevating agents (>1 hour) are markedly different regarding which PDE4D isoforms are upregulated (Figure 8).66 Indeed, a greater increase in PDE4 expression is seen in synthetic VSMCs. Thus, prolonged cAMP elevation in vivo initiates a program involving cAMP-PKA-CREB/CRE, but not EPAC, that stimulates increased expression of the dominant PDE4D variants present in these cells before challenge. Consistent with a role for increased transcription and translation, pharmacological inhibition of such processes abolished the increases. Further work is needed to analyze fully changes in PDE4 isoform profile in these cells by such treatments.


Figure 8
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Figure 8. Differential regulation of basal PDE4D expression and of cAMP-induced upregulated expression of PDE4D in contractile and synthetic VSMCs. A dynamic balance between histone acetylation (Ac) by histone acetyl transferases (HAT) and histone deacetylation by histone deacetylases (HDAC) during the phenotypic switch of VSMCs from contractile to a synthetic phenotypes allows induction of different PDE4D variants in these cells in response to prolonged increases in cAMP.

Interestingly, and of potential therapeutic relevance, when synthetic VSMCs were similarly treated with cAMP-elevating agents, there was no evidence that the levels of the dominant PDE4D variants expressed in these cells were increased. In contrast, prolonged incubations of synthetic VSMCs caused a marked PKA/CREB/CRE signaling cascade-mediated induction of 2 PDE4D short-form variants (PDE4D1 and PDE4D2).66 Again, there was no obvious role for EPAC-Rap signaling in this effect. PDE4D1/2 short-form enzymes were not expressed in either contractile or synthetic VSMCs before treatment and were not induced in contractile VSMCs following similar treatments. As was the case in contractile VSMCs, a role for increased transcription and translation of these short-form PDE4D variants was evident in synthetic VSMCs.

Reversible posttranslational modifications of histones, including ADP ribosylation, methylation, glycosylation, phosphorylation, and acetylation regulates whether individual promoter sequences can bind transcription factors and be "turned on."67 In general, increased levels of histone acetylation within promoter sequences correlate with increased gene expression. Marked advances in our understanding of the role of histone acetylation in regulating gene expression have recently been made, and this process serves as an important factor regulating the differential expression of certain genes during the process of VSMC phenotypic modulation. Interestingly, hyperacetylation of the intronic promoter regulating PDE4D1/2 expression in synthetic VSMCs is consistent with the idea that histone acetylation is involved in determining which PDE4D isoforms are upregulated in response to cAMP elevation (Figure 8).


*    Significance of a Distinct PDE4D Expression Pattern in Contractile and Synthetic VSMCs
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*Significance of a Distinct...
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down arrowReferences
 
Several physiological and therapeutically important consequences may flow from the differences by which contractile and synthetic VSMCs react to prolonged increases in cAMP. Thus, differences in the magnitude of the cAMP-induced increase in PDE4 activity in contractile and synthetic VSMCs may indicate that PDE4D upregulation plays a less significant role in desensitizing contractile VSMCs to prolonged actions of cAMP than in synthetic VSMCs. Also, these data are consistent with the idea that synthetic VSMCs might be much more dependent on increased PDE4D to desensitize the effects of prolonged cAMP elevation, than the contractile cells. Although neither of these hypotheses has yet been formally tested, they are internally consistent with the idea that PDE4 inhibitors might more markedly influence cAMP-mediated signaling in synthetic than contractile VSMCs. Of course, should this be the case, one would further suggest that PDE4 selective, and perhaps even PDE4D-selective inhibitors, would have more marked effects in neointimal VSMCs in stenotic lesions, which are by definition synthetic, than in the contractile VSMCs resident within the medial muscular layers of the artery. Because most attempts to use cAMP-elevating agents to reduce intimal hyperplasia have been hampered by effects of these agents on systemic blood pressure, PDE4-selective agents may prove useful in obviating this limitation.

Several reports54,55,57,68,134 have shown that the differential impact of ERK-mediated phosphorylation of distinct PDE4D variants provides a powerful mechanism through which activation of a mitogen-activated protein kinase–signaling cascade can integrate with cAMP-mediated effects in cells, including cardiomyocytes and VSMCs. Indeed, because ERK-mediated phosphorylation54,55,57 of PDE4D long-form variants can either activate or inhibit these enzymes, whereas the PDE4D short forms are always activated by this event, it is reasonable to propose that the interactions between cAMP-elevating agents and growth factors that activate ERK will be different in these cells. For example, we suggest that because ERK-mediated phosphorylation of PDE4D1 activates this enzyme, physiologically relevant VSMC trophic factors, such as PDGF or angiotensin II, which activate ERK, might reduce the antiproliferative actions of cAMP-elevating agents in synthetic VSMCs. Clearly, further work is required to assess the functional impact of these events. Because activation of ERK signaling by PDGF or angiotensin II can also regulate PDE4D expression through effects dependent on messenger RNA stabilization,68 it is clear that the levels of cross-talk are complex.


*    The Future
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrow"PDE4-ology"
up arrowThe Long, Short, and...
up arrowThe Motor in the...
up arrowFinding the Perfect Partner...
up arrowCellular Function and Phenotype...
up arrowMolecular Determinants Mediating...
up arrowCardiomyocyte PDE4s
up arrowPDE4D-RyR2 Interaction
up arrowPDE4D3-mAKAP
up arrowPhenotypic Modulation of VSMCs
up arrowVSMC Phenotypic Modulation and...
up arrowDistinct PDE4D Variants...
up arrowSignificance of a Distinct...
*The Future
down arrowReferences
 
Exploiting Tethered PDE4
The plethora of PDE4 isoforms confers specific functional attributes that relate to (1) targeting to specific intracellular complexes and membranes so as to exert control by shaping local cAMP gradients and (2) regulation by phosphorylation. These indicate that specific PDE4 isoforms will have particular functional roles. We have provided the paradigm for this in the interaction of PDE4D5 with ß-arrestin. This delineates how recruitment and redistribution of a particular PDE4 isoform confers a specific functional role.

Active site-directed inhibitors cannot discriminate between PDE4 isoforms with a subfamily as they have identical catalytic sites. Indeed, even between subfamilies the similarity of active sites militates against effective selective inhibitors being developed. To demonstrate that specific isoforms can have particular functional roles novel approaches and technologies are required. We have pioneered this in generating catalytically inactive isoforms whose overexpression in cells acts to displace the cognate endogenous, active isoform from its functionally relevant scaffold(s).12,33,34,110 This so-called dominant negative approach can be used to determine the function attributable to tethering of specific isoforms. Additionally, the use of siRNA targeted to specific isoforms allows us to uncover functional attribute(s). However, unlike the dominant negative approach, this does not inform directly on functions attributable to tethering. Such approaches have identified specific nonredundant functional roles for specific PDE4 isoforms, which is consistent with observations from PDE4 subfamily knockout analyses.36,37

Cardiomyocytes
The PDE4D5/ß-arrestin, PDE4D3/mAKAP, PDE4D3/EPAC, and PDE4D3/RyR complexes have been identified in cardiomyocytes. However, as cardiomyocytes express a range of other PDE4 isoforms, it is likely that these represent a fraction of PDE4 complexes in these cells. It is, as yet, unknown whether the PDE4 isoform profile is altered in heart disease. However, should this be the case, it may become possible to identify individual molecular complexes that more strongly influence cardiomyocyte dysfunctions and for which therapeutic strategies may be developed.

Vascular Smooth Muscle Cells
Virtually all cAMP-dependent pharmacological agents used to treat heart conditions have effects on the ability of contractile VSMCs to control blood pressure. Indeed, several cAMP-dependent agents, that might be useful to inhibit VSMC proliferation and migration in the context of restenosis, alter blood pressure by influencing the functions of contractile VSMCs. Clearly, advances in our understanding of how cardiomyocytes and contractile or synthetic VSMCs regulate cAMP-mediated effects will be required to achieve cell-type-selective therapeutic effects. We suggest that recent advances in our understanding of PDE4-mediated regulation of cAMP-signaling in these distinct cell types may be important. Thus, the marked increase in the PDE4/PDE3 activity ratio that occurs during the phenotypic switch of VSMCs may allow PDE4 inhibitors to selectively affect synthetic VSMC functions compared with those in contractile VSMCs. In addition, because synthetic VSMCs markedly induce PDE4D to desensitize the impact of prolonged cAMP-signaling, selective PDE4 inhibitors may prolong the effects of cAMP-elevating agents in these cells compared with those seen in contractile VSMCs. Similarly, the observation that PDE4D3 is anchored via an AKAP in cardiomyocytes, but not in contractile nor synthetic VSMCs, may afford some measure of selectivity if, as proposed above, noncatalytic domain-based strategies of PDE4 inhibition become feasible.


*    Acknowledgments
 
Sources of Funding

This work was supported by Medical Research Council (United Kingdom) grant G8604010 (to M.D.H. and G.S.B.), by The Leducq Foundation (Paris) (to M.D.H. and G.S.B.), by Heart and Stroke Foundation of Ontario grant T5426 (to D.H.M.), and by Canadian Institute of Health Research grant MOP-57699 (to D.H.M.). D.H.M. is a Heart and Stroke Foundation of Ontario Career Investigator.

Disclosures

None.


*    Footnotes
 
Original received November 29, 2006; revision received January 11, 2007; accepted February 6, 2007.


*    References
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrow"PDE4-ology"
up arrowThe Long, Short, and...
up arrowThe Motor in the...
up arrowFinding the Perfect Partner...
up arrowCellular Function and Phenotype...
up arrowMolecular Determinants Mediating...
up arrowCardiomyocyte PDE4s
up arrowPDE4D-RyR2 Interaction
up arrowPDE4D3-mAKAP
up arrowPhenotypic Modulation of VSMCs
up arrowVSMC Phenotypic Modulation and...
up arrowDistinct PDE4D Variants...
up arrowSignificance of a Distinct...
up arrowThe Future
*References
 
1. Smith FD, Langeberg LK, Scott JD. The where’s and when’s of kinase anchoring. Trends Biochem Sci. 2006; 31: 316–323.[CrossRef][Medline] [Order article via Infotrieve]

2. Tasken K, Aandahl EM. Localized effects of cAMP mediated by distinct routes of protein kinase A. Physiol Rev. 2004; 84: 137–167.[Abstract/Free Full Text]

3. Beavo JA, Brunton LL. Cyclic nucleotide research—still expanding after half a century. Nat Rev Mol Cell Biol. 2002; 3: 710–718.[CrossRef][Medline] [Order article via Infotrieve]

4. Bos JL. Epac: a new cAMP target and new avenues in cAMP research. Nat Rev Mol Cell Biol. 2003; 4: 733–738.[CrossRef][Medline] [Order article via Infotrieve]

5. Taylor SS, Yang J, Wu J, Haste NM, Radzio-Andzelm E, Anand G. PKA: a portrait of protein kinase dynamics. Biochim Biophys Acta. 2004; 1697: 259–269.[Medline] [Order article via Infotrieve]

6. Petrashevskaya NN, Koch SE, Bodi I, Schwartz A. Calcium cycling, historic overview and perspectives. Role for autonomic nervous system regulation. J Mol Cell Cardiol. 2002; 34: 885–896.[CrossRef][Medline] [Order article via Infotrieve]

7. Tomita H, Nazmy M, Kajimoto K, Yehia G, Molina CA, Sadoshima J. Inducible cAMP early repressor (ICER) is a negative-feedback regulator of cardiac hypertrophy and an important mediator of cardiac myocyte apoptosis in response to beta-adrenergic receptor stimulation. Circ Res. 2003; 93: 12–22.[Abstract/Free Full Text]

8. Wu YJ, Bond M, Sala-Newby GB, Newby AC. Altered S-phase kinase-associated protein-2 levels are a major mediator of cyclic nucleotide-induced inhibition of vascular smooth muscle cell proliferation. Circ Res. 2006; 98: 1141–1150.[Abstract/Free Full Text]

9. Johnson R, Webb JG, Newman WH, Wang Z. Regulation of human vascular smooth muscle cell migration by beta-adrenergic receptors. Am Surg. 2006; 72: 51–54.[Medline] [Order article via Infotrieve]

10. Fetalvero KM, Shyu M, Nomikos AP, Chiu YF, Wagner RJ, Powell RJ, Hwa J, Martin KA. The prostacyclin receptor induces human vascular smooth muscle cell differentiation via the protein kinase A pathway. Am J Physiol Heart Circ Physiol. 2006; 290: H1337–H1346.[Abstract/Free Full Text]

11. Li RC, Cindrova-Davies T, Skepper JN, Sellers LA. Prostacyclin induces apoptosis of vascular smooth muscle cells by a cAMP-mediated inhibition of extracellular signal-regulated kinase activity and can counteract the mitogenic activity of endothelin-1 or basic fibroblast growth factor. Circ Res. 2004; 94: 759–767.[Abstract/Free Full Text]

12. Baillie GS, Houslay MD. Arrestin times for compartmentalised cAMP signalling and phosphodiesterase-4 enzymes. Curr Opin Cell Biol. 2005; 17: 129–134.[CrossRef][Medline] [Order article via Infotrieve]

13. Baillie GS, Scott JD, Houslay MD. Compartmentalisation of phosphodiesterases and protein kinase A: opposites attract. FEBS Lett. 2005; 579: 3264–3270.[CrossRef][Medline] [Order article via Infotrieve]

14. Fischmeister R. Is cAMP good or bad? Depends on where it’s made. Circ Res. 2006; 98: 582–584.[Free Full Text]

15. Cooper DM. Regulation and organization of adenylyl cyclases and cAMP. Biochem J. 2003; 375: 517–529.[CrossRef][Medline] [Order article via Infotrieve]

16. Lefkowitz RJ. Historical review: a brief history and personal retrospective of seven-transmembrane receptors. Trends Pharmacol Sci. 2004; 25: 413–422.[CrossRef][Medline] [Order article via Infotrieve]

17. Houslay MD, Milligan G. Tailoring cAMP-signalling responses through isoform multiplicity. Trends Biochem Sci. 1997; 22: 217–224.[CrossRef][Medline] [Order article via Infotrieve]

18. Buxton IL, Brunton LL. Compartments of cyclic AMP and protein kinase in mammalian cardiomyocytes. J Biol Chem. 1983; 258: 10233–10239.[Abstract/Free Full Text]

19. Rochais F, Abi-Gerges A, Horner K, Lefebvre F, Cooper DM, Conti M, Fischmeister R, Vandecasteele G. A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gs-coupled receptors in adult rat ventricular myocytes. Circ Res. 2006; 98: 1081–1088.[Abstract/Free Full Text]

20. Mongillo M, McSorley T, Evellin S, Sood A, Lissandron V, Terrin A, Huston E, Hannawacker A, Lohse MJ, Pozzan T, Houslay MD, Zaccolo M. Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases. Circ Res. 2004; 95: 67–75.[Abstract/Free Full Text]

21. Zaccolo M, Pozzan T. Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science. 2002; 295: 1711–1715.[Abstract/Free Full Text]

22. Georget M, Mateo P, Vandecasteele G, Lipskaia L, Defer N, Hanoune J, Hoerter J, Lugnier C, Fischmeister R. Cyclic AMP compartmentation due to increased cAMP-phosphodiesterase activity in transgenic mice with a cardiac-directed expression of the human adenylyl cyclase type 8 (AC8). FASEB J. 2003; 17: 1380–1391.[Abstract/Free Full Text]

23. Lissandron V, Zaccolo M. Compartmentalized cAMP/PKA signalling regulates cardiac excitation-contraction coupling. J Muscle Res Cell Motil. 2006; 27: 399–403.[CrossRef][Medline] [Order article via Infotrieve]

24. Mongillo M, Tocchetti CG, Terrin A, Lissandron V, Cheung YF, Dostmann WR, Pozzan T, Kass DA, Paolocci N, Houslay MD, Zaccolo M. Compartmentalized phosphodiesterase-2 activity blunts beta-adrenergic cardiac inotropy via an NO/cGMP-dependent pathway. Circ Res. 2006; 98: 226–234.[Abstract/Free Full Text]

25. Nikolaev VO, Bunemann M, Schmitteckert E, Lohse MJ, Engelhardt S. Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching beta1-adrenergic but locally confined beta2-adrenergic receptor-mediated signaling. Circ Res. 2006; 99: 1084–1091.[Abstract/Free Full Text]

26. Jurevicius J, Fischmeister R. cAMP compartmentation is responsible for a local activation of cardiac Ca2+ channels by beta-adrenergic agonists. Proc Natl Acad Sci U S A. 1996; 93: 295–299.[Abstract/Free Full Text]

27. Lugnier C. Cyclic nucleotide phosphodiesterase (PDE) superfamily: a new target for the development of specific therapeutic agents. Pharmacol Ther. 2006; 109: 366–398.[CrossRef][Medline] [Order article via Infotrieve]

28. Houslay MD, Adams DR. PDE4 cAMP phosphodiesterases: modular enzymes that orchestrate signalling cross-talk, desensitization and compartmentalization. Biochem J. 2003; 370: 1–18.[CrossRef][Medline] [Order article via Infotrieve]

29. Houslay MD. A RSK(y) relationship with promiscuous PKA. Sci STKE. 2006; 2006: pe32.[Abstract/Free Full Text]

30. Huston E, Houslay TM, Baillie GS, Houslay MD. cAMP phosphodiesterase-4A1 (PDE4A1) has provided the paradigm for the intracellular targeting of phosphodiesterases, a process that underpins compartmentalized cAMP signalling. Biochem Soc Trans. 2006; 34: 504–509.[CrossRef][Medline] [Order article via Infotrieve]

31. Terrin A, Di Benedetto G, Pertegato V, Cheung YF, Baillie G, Lynch MJ, Elvassore N, Prinz A, Herberg FW, Houslay MD, Zaccolo M. PGE1 stimulation of HEK293 cells generates multiple contiguous domains with different [cAMP]: role of compartmentalized phosphodiesterases. J Cell Biol. 2006; 175: 441–451.[Abstract/Free Full Text]

32. Willoughby D, Wong W, Schaack J, Scott JD, Cooper DM. An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics. EMBO J. 2006; 25: 2051–2061.[CrossRef][Medline] [Order article via Infotrieve]

33. Lynch MJ, Baillie GS, Mohamed A, Li X, Maisonneuve C, Klussmann E, van Heeke G, Houslay MD. RNA silencing identifies PDE4D5 as the functionally relevant cAMP phosphodiesterase interacting with beta arrestin to control the protein kinase A/AKAP79-mediated switching of the beta2-adrenergic receptor to activation of ERK in HEK293B2 cells. J Biol Chem. 2005; 280: 33178–33189.[Abstract/Free Full Text]

34. Baillie GS, Sood A, McPhee I, Gall I, Perry SJ, Lefkowitz RJ, Houslay MD. beta-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates beta-adrenoceptor switching from Gs to Gi. Proc Natl Acad Sci U S A. 2003; 100: 940–945.[Abstract/Free Full Text]

35. McCahill A, McSorley T, Huston E, Hill EV, Lynch MJ, Gall I, Keryer G, Lygren B, Tasken K, van Heeke G, Houslay MD. In resting COS1 cells a dominant negative approach shows that specific, anchored PDE4 cAMP phosphodiesterase isoforms gate the activation, by basal cyclic AMP production, of AKAP-tethered protein kinase A type II located in the centrosomal region. Cell Signal. 2005.

36. Ariga M, Neitzert B, Nakae S, Mottin G, Bertrand C, Pruniaux MP, Jin SL, Conti M. Nonredundant function of phosphodiesterases 4D and 4B in neutrophil recruitment to the site of inflammation. J Immunol. 2004; 173: 7531–7538.[Abstract/Free Full Text]

37. Jin SL, Lan L, Zoudilova M, Conti M. Specific role of phosphodiesterase 4B in lipopolysaccharide-induced signaling in mouse macrophages. J Immunol. 2005; 175: 1523–1531.[Abstract/Free Full Text]

38. Lehnart SE, Wehrens XH, Reiken S, Warrier S, Belevych AE, Harvey RD, Richter W, Jin SL, Conti M, Marks AR. Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias. Cell. 2005; 123: 25–35.[CrossRef][Medline] [Order article via Infotrieve]

39. Mehats C, Jin SL, Wahlstrom J, Law E, Umetsu DT, Conti M. PDE4D plays a critical role in the control of airway smooth muscle contraction. FASEB J. 2003; 17: 1831–1841.[Abstract/Free Full Text]

40. Zhang HT, Huang Y, Jin SL, Frith SA, Suvarna N, Conti M, O’Donnell JM. Antidepressant-like profile and reduced sensitivity to rolipram in mice deficient in the PDE4D phosphodiesterase enzyme. Neuropsychopharmacology. 2002; 27: 587–595.[Medline] [Order article via Infotrieve]

41. Yang G, McIntyre KW, Townsend RM, Shen HH, Pitts WJ, Dodd JH, Nadler SG, McKinnon M, Watson AJ. Phosphodiesterase 7A-deficient mice have functional T cells. J Immunol. 2003; 171: 6414–6420.[Abstract/Free Full Text]

42. Conti M, Richter W, Mehats C, Livera G, Park JY, Jin C. Cyclic AMP-specific PDE4 phosphodiesterases as critical components of cyclic AMP signaling. J Biol Chem. 2003; 278: 5493–5496.[Free Full Text]

43. Houslay MD, Schafer P, Zhang KY. Keynote review: phosphodiesterase-4 as a therapeutic target. Drug Discov Today. 2005; 10: 1503–1519.[CrossRef][Medline] [Order article via Infotrieve]

44. Sullivan M, Olsen AS, Houslay MD. Genomic organisation of the human cyclic AMP-specific phosphodiesterase PDE4C gene and its chromosomal localisation to 19p13.1, between RAB3A and JUND. Cell Signal. 1999; 11: 735–742.[CrossRef][Medline] [Order article via Infotrieve]

45. Sullivan M, Rena G, Begg F, Gordon L, Olsen AS, Houslay MD. Identification and characterization of the human homologue of the short PDE4A cAMP-specific phosphodiesterase RD1 (PDE4A1) by analysis of the human HSPDE4A gene locus located at chromosome 19p13.2. Biochem J. 1998; 333 (pt 3): 693–703.[Medline] [Order article via Infotrieve]

46. Vicini E, Conti M. Characterization of an intronic promoter of a cyclic adenosine 3',5'-monophosphate (cAMP)-specific phosphodiesterase gene that confers hormone and cAMP inducibility. Mol Endocrinol. 1997; 11: 839–850.[Abstract/Free Full Text]

47. Houslay MD. The long and short of vascular smooth muscle phosphodiesterase-4 as a putative therapeutic target. Mol Pharmacol. 2005; 68: 563–567.[Abstract/Free Full Text]

48. Reneland RH, Mah S, Kammerer S, Hoyal CR, Marnellos G, Wilson SG, Sambrook PN, Spector TD, Nelson MR, Braun A. Association between a variation in the phosphodiesterase 4D gene and bone mineral density. BMC Med Genet. 2005; 6: 9.[CrossRef][Medline] [Order article via Infotrieve]

49. Millar JK, Pickard BS, Mackie S, James R, Christie S, Buchanan SR, Malloy MP, Chubb JE, Huston E, Baillie GS, Thomson PA, Hill EV, Brandon NJ, Rain JC, Camargo LM, Whiting PJ, Houslay MD, Blackwood DH, Muir WJ, Porteous DJ. DISC1 and PDE4B are interacting genetic factors in schizophrenia that regulate cAMP signaling. Science. 2005; 310: 1187–1191.[Abstract/Free Full Text]

50. Bolger GB. Molecular biology of the cyclic AMP-specific cyclic nucleotide phosphodiesterases: a diverse family of regulatory enzymes. Cell Signal. 1994; 6: 851–859.[CrossRef][Medline] [Order article via Infotrieve]

51. Johnston LA, Erdogan S, Cheung YF, Sullivan M, Barber R, Lynch MJ, Baillie GS, Van Heeke G, Adams DR, Huston E, Houslay MD. Expression, intracellular distribution and basis for lack of catalytic activity of the PDE4A7 isoform encoded by the human PDE4A cAMP-specific phosphodiesterase gene. Biochem J. 2004; 380: 371–384.[CrossRef][Medline] [Order article via Infotrieve]

52. Houslay MD. PDE4 cAMP-specific phosphodiesterases. Prog Nucleic Acid Res Mol Biol. 2001; 69: 249–315.[Medline] [Order article via Infotrieve]

53. Beard MB, Olsen AE, Jones RE, Erdogan S, Houslay MD, Bolger GB. UCR1 and UCR2 domains unique to the cAMP-specific phosphodiesterase family form a discrete module via electrostatic interactions. J Biol Chem. 2000; 275: 10349–10358.[Abstract/Free Full Text]

54. Baillie GS, MacKenzie SJ, McPhee I, Houslay MD. Sub-family selective actions in the ability of Erk2 MAP kinase to phosphorylate and regulate the activity of PDE4 cyclic AMP-specific phosphodiesterases. Br J Pharmacol. 2000; 131: 811–819.[CrossRef][Medline] [Order article via Infotrieve]

55. Hoffmann R, Baillie GS, MacKenzie SJ, Yarwood SJ, Houslay MD. The MAP kinase ERK2 inhibits the cyclic AMP-specific phosphodiesterase HSPDE4D3 by phosphorylating it at Ser579. EMBO J. 1999; 18: 893–903.[CrossRef][Medline] [Order article via Infotrieve]

56. Hoffmann R, Wilkinson IR, McCallum JF, Engels P, Houslay MD. cAMP-specific phosphodiesterase HSPDE4D3 mutants which mimic activation and changes in rolipram inhibition triggered by protein kinase A phosphorylation of Ser-54: Generation of a molecular model. Biochem J. 1998; 333: 139–149.[Medline] [Order article via Infotrieve]

57. MacKenzie SJ, Baillie GS, McPhee I, Bolger GB, Houslay MD. ERK2 MAP kinase binding, phosphorylation and regulation of PDE4D cAMP specific phosphodiesterases: the involvement of C-terminal docking sites and N-terminal UCR regions. J Biol Chem. 2000; 275: 16609–16617.[Abstract/Free Full Text]

58. MacKenzie SJ, Baillie GS, McPhee I, MacKenzie C, Seamons R, McSorley T, Millen J, Beard MB, van Heeke G, Houslay MD. Long PDE4 cAMP specific phosphodiesterases are activated by protein kinase A-mediated phosphorylation of a single serine residue in upstream conserved region 1 (UCR1). Br J Pharmacol. 2002; 136: 421–433.[CrossRef][Medline] [Order article via Infotrieve]

59. Sette C, Conti M. Phosphorylation and activation of a cAMP-specific phosphodiesterase by the cAMP-dependent protein kinase. Involvement of serine 54 in the enzyme activation. J Biol Chem. 1996; 271: 16526–16534.[Abstract/Free Full Text]

60. Sette C, Iona S, Conti M. The short-term activation of a rolipram-sensitive, cAMP-specific phosphodiesterase by thyroid-stimulating hormone in thyroid FRTL-5 cells is mediated by a cAMP-dependent phosphorylation. J Biol Chem. 1994; 269: 9245–9252.[Abstract/Free Full Text]

61. Hill EV, Sheppard CL, Cheung YF, Gall I, Krause E, Houslay MD. Oxidative stress employs phosphatidyl inositol 3-kinase and ERK signalling pathways to activate cAMP phosphodiesterase-4D3 (PDE4D3) through multi-site phosphorylation at Ser239 and Ser579. Cell Signal. 2006; 18: 2056–2069.[CrossRef][Medline] [Order article via Infotrieve]

62. Verde I, Vandecasteele G, Lezoualc’h F, Fischmeister R. Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca2+ current in rat ventricular myocytes. Br J Pharmacol. 1999; 127: 65–74.[CrossRef][Medline] [Order article via Infotrieve]

63. Le Jeune IR, Shepherd M, Van Heeke G, Houslay MD, Hall IP. Cyclic AMP-dependent transcriptional up-regulation of phosphodiesterase 4D5 in human airway smooth muscle cells. Identification and characterization of a novel PDE4D5 promoter. J Biol Chem. 2002; 277: 35980–35989.[Abstract/Free Full Text]

64. Rena G, Begg F, Ross A, MacKenzie C, McPhee I, Campbell L, Huston E, Sullivan M, Houslay MD. Molecular cloning, genomic positioning, promoter identification, and characterization of the novel cyclic amp-specific phosphodiesterase PDE4A10. Mol Pharmacol. 2001; 59: 996–1011.[Abstract/Free Full Text]

65. Wallace DA, Johnston LA, Huston E, MacMaster D, Houslay TM, Cheung YF, Campbell L, Millen JE, Smith RA, Gall I, Knowles RG, Sullivan M, Houslay MD. Identification and characterization of PDE4A11, a novel, widely expressed long isoform encoded by the human PDE4A cAMP phosphodiesterase gene. Mol Pharmacol. 2005; 67: 1920–1934.[Abstract/Free Full Text]

66. Tilley DG, Maurice DH. Vascular smooth muscle cell phenotype-dependent phosphodiesterase 4D short form expression: role of differential histone acetylation on cAMP-regulated function. Mol Pharmacol. 2005; 68: 596–605.[Abstract/Free Full Text]

67. Clayton AL, Hazzalin CA, Mahadevan LC. Enhanced histone acetylation and transcription: a dynamic perspective. Mol Cell. 2006; 23: 289–296.[CrossRef][Medline] [Order article via Infotrieve]

68. Liu H, Palmer D, Jimmo SL, Tilley DG, Dunkerley HA, Pang SC, Maurice DH. Expression of phosphodiesterase 4D (PDE4D) is regulated by both the cyclic AMP-dependent protein kinase and mitogen-activated protein kinase signaling pathways. A potential mechanism allowing for the coordinated regulation of PDE4D activity and expression in cells. J Biol Chem. 2000; 275: 26615–26624.[Abstract/Free Full Text]

69. Sugden PH. Signalling pathways in cardiac myocyte hypertrophy. Ann Med. 2001; 33: 611–622.[Medline] [Order article via Infotrieve]

70. Oki N, Takahashi SI, Hidaka H, Conti M. Short term feedback regulation of cAMP in FRTL-5 thyroid cells. Role Of pde4d3 phosphodiesterase activation. J Biol Chem. 2000; 275: 10831–10837.[Abstract/Free Full Text]

71. Clerk A, Kemp TJ, Harrison JG, Pham FH, Sugden PH. Integration of protein kinase signaling pathways in cardiac myocytes: signaling to and from the extracellular signal-regulated kinases. Adv Enzyme Regul. 2004; 44: 233–248.[Medline] [Order article via Infotrieve]

72. Heineke J, Molkentin JD. Regulation of cardiac hypertrophy by intracellular signalling pathways. Nat Rev Mol Cell Biol. 2006; 7: 589–600.[CrossRef][Medline] [Order article via Infotrieve]

73. Blanc A, Pandey NR, Srivastava AK. Synchronous activation of ERK 1/2, p38mapk and PKB/Akt signaling by H2O2 in vascular smooth muscle cells: potential involvement in vascular disease (review). Int J Mol Med. 2003; 11: 229–234.[Medline] [Order article via Infotrieve]

74. Shepherd MC, Baillie GS, Stirling DI, Houslay MD. Remodelling of the PDE4 cAMP phosphodiesterase isoform profile upon monocyte-macrophage differentiation of human U937 cells. Br J Pharmacol. 2004; 142: 339–351.[CrossRef][Medline] [Order article via Infotrieve]

75. Xu RX, Hassell AM, Vanderwall D, Lambert MH, Holmes WD, Luther MA, Rocque WJ, Milburn MV, Zhao Y, Ke H, Nolte RT. Atomic structure of PDE4: insights into phosphodiesterase mechanism and specificity. Science. 2000; 288: 1822–1825.[Abstract/Free Full Text]

76. Terry R, Cheung YF, Praestegaard M, Baillie GS, Huston E, Gall I, Adams DR, Houslay MD. Occupancy of the catalytic site of the PDE4A4 cyclic AMP phosphodiesterase by rolipram triggers the dynamic redistribution of this specific isoform in living cells through a cyclic AMP independent process. Cell Signal. 2003; 15: 955–971.[CrossRef][Medline] [Order article via Infotrieve]

77. Huston E, Gall I, Houslay TM, Houslay MD. Helix-1 of the cAMP-specific phosphodiesterase PDE4A1 regulates its phospholipase-D-dependent redistribution in response to release of Ca2+. J Cell Sci. 2006; 119: 3799–3810.[Abstract/Free Full Text]

78. Shakur Y, Pryde JG, Houslay MD. Engineered deletion of the unique N-terminal domain of the cyclic AMP-specific phosphodiesterase RD1 prevents plasma membrane association and the attainment of enhanced thermostability without altering its sensitivity to inhibition by rolipram. Biochem J. 1993; 292 (pt 3): 677–686.[Medline] [Order article via Infotrieve]

79. Shakur Y, Wilson M, Pooley L, Lobban M, Griffiths SL, Campbell AM, Beattie J, Daly C, Houslay MD. Identification and characterization of the type-IVA cyclic AMP-specific phosphodiesterase RD1 as a membrane-bound protein expressed in cerebellum. Biochem J. 1995; 306 (pt 3): 801–809.[Medline] [Order article via Infotrieve]

80. Baillie GS, Huston E, Scotland G, Hodgkin M, Gall I, Peden AH, MacKenzie C, Houslay ES, Currie R, Pettitt TR, Walmsley AR, Wakelam MJ, Warwicker J, Houslay MD. TAPAS-1, a novel microdomain within the unique N-terminal region of the PDE4A1 cAMP-specific phosphodiesterase that allows rapid, Ca2+-triggered membrane association with selectivity for interaction with phosphatidic acid. J Biol Chem. 2002; 277: 28298–28309.[Abstract/Free Full Text]

81. Scotland G, Houslay MD. Chimeric constructs show that the unique N-terminal domain of the cyclic AMP phosphodiesterase RD1 (RNPDE4A1A; rPDE-IVA1) can confer membrane association upon the normally cytosolic protein chloramphenicol acetyltransferase. Biochem J. 1995; 308 (pt 2): 673–681.[Medline] [Order article via Infotrieve]

82. Smith KJ, Scotland G, Beattie J, Trayer IP, Houslay MD. Determination of the structure of the N-terminal splice region of the cyclic AMP-specific phosphodiesterase RD1 (RNPDE4A1) by 1H NMR and identification of the membrane association domain using chimeric constructs. J Biol Chem. 1996; 271: 16703–16711.[Abstract/Free Full Text]

83. Bolger GB, McCahill A, Huston E, Cheung YF, McSorley T, Baillie GS, Houslay MD. The unique amino-terminal region of the PDE4D5 cAMP phosphodiesterase isoform confers preferential interaction with beta-arrestins. J Biol Chem. 2003; 278: 49230–49238.[Abstract/Free Full Text]

84. Perry SJ, Baillie GS, Kohout TA, McPhee I, Magiera MM, Ang KL, Miller WE, McLean AJ, Conti M, Houslay MD, Lefkowitz RJ. Targeting of cyclic AMP degradation to ß2-adrenergic receptors by ß-arrestins. Science. 2002; 298: 834–836.[Abstract/Free Full Text]

85. Bolger GB, Baillie GS, Li X, Lynch MJ, Herzyk P, Mohamed A, Mitchell LH, McCahill A, Hundsrucker C, Klussmann E, Adams DR, Houslay MD. Scanning peptide array analyses identify overlapping binding sites for the signalling scaffold proteins, beta-arrestin and RACK1, in cAMP-specific phosphodiesterase PDE4D5. Biochem J. 2006; 398: 23–36.[CrossRef][Medline] [Order article via Infotrieve]

86. Bolger GB, McCahill A, Yarwood SJ, Steele MR, Warwicker J, Houslay MD. Delineation of RAID1, the RACK1 interaction domain located within the unique N-terminal region of the cAMP-specific phosphodiesterase, PDE4D5. BMC Biochem. 2002; 3: 24.[CrossRef][Medline] [Order article via Infotrieve]

87. Yarwood SJ, Steele MR, Scotland G, Houslay MD, Bolger GB. The RACK1 signaling scaffold protein selectively interacts with the cAMP-specific phosphodiesterase PDE4D5 isoform. J Biol Chem. 1999; 274: 14909–14917.[Abstract/Free Full Text]

88. Beard MB, O’Connell JC, Bolger GB, Houslay MD. The unique N-terminal domain of the cAMP phosphodiesterase PDE4D4 allows for interaction with specific SH3 domains. FEBS Lett. 1999; 460: 173–177.[CrossRef][Medline] [Order article via Infotrieve]

89. McPhee I, Yarwood SJ, Scotland G, Huston E, Beard MB, Ross AH, Houslay ES, Houslay MD. Association with the SRC family tyrosyl kinase LYN triggers a conformational change in the catalytic region of human cAMP-specific phosphodiesterase HSPDE4A4B. Consequences for rolipram inhibition. J Biol Chem. 1999; 274: 11796–11810.[Abstract/Free Full Text]

90. O’Connell JC, McCallum JF, McPhee I, Wakefield J, Houslay ES, Wishart W, Bolger G, Frame M, Houslay MD. The SH3 domain of Src tyrosyl protein kinase interacts with the N-terminal splice region of the PDE4A cAMP-specific phosphodiesterase RPDE-6 (RNPDE4A5). Biochem J. 1996; 318 (pt 1): 255–261.[Medline] [Order article via Infotrieve]

91. Beard MB, Huston E, Campbell L, Gall I, McPhee I, Yarwood S, Scotland G, Houslay MD. In addition to the SH3 binding region, multiple regions within the N-terminal noncatalytic portion of the cAMP-specific phosphodiesterase, PDE4A5, contribute to its intracellular targeting. Cell Signal. 2002; 14: 453–465.[CrossRef][Medline] [Order article via Infotrieve]

92. Carney SA, Chen J, Burns CG, Xiong KM, Peterson RE, Heideman W. Aryl hydrocarbon receptor activation produces heart-specific transcriptional and toxic responses in developing zebrafish. Mol Pharmacol. 2006; 70: 549–561.[Abstract/Free Full Text]

93. Mehrabi MR, Steiner GE, Dellinger C, Kofler A, Schaufler K, Tamaddon F, Plesch K, Ekmekcioglu C, Maurer G, Glogar HD, Thalhammer T. The arylhydrocarbon receptor (AhR), but not the AhR-nuclear translocator (ARNT), is increased in hearts of patients with cardiomyopathy. Virchows Arch. 2002; 441: 481–489.[CrossRef][Medline] [Order article via Infotrieve]

94. Lund AK, Goens MB, Nunez BA, Walker MK. Characterizing the role of endothelin-1 in the progression of cardiac hypertrophy in aryl hydrocarbon receptor (AhR) null mice. Toxicol Appl Pharmacol. 2006; 212: 127–135.[CrossRef][Medline] [Order article via Infotrieve]

95. Bolger GB, Peden AH, Steele MR, MacKenzie C, McEwan DG, Wallace DA, Huston E, Baillie GS, Houslay MD. Attenuation of the activity of the cAMP-specific phosphodiesterase PDE4A5 by interaction with the immunophilin XAP2. J Biol Chem. 2003; 278: 33351–33363.[Abstract/Free Full Text]

96. Verde I, Pahlke G, Salanova M, Zhang G, Wang S, Coletti D, Onuffer J, Jin SL, Conti M. Myomegalin is a novel protein of the golgi/centrosome that interacts with a cyclic nucleotide phosphodiesterase. J Biol Chem. 2001; 276: 11189–11198.[Abstract/Free Full Text]

97. Asirvatham AL, Galligan SG, Schillace RV, Davey MP, Vasta V, Beavo JA, Carr DW. A-kinase anchoring proteins interact with phosphodiesterases in T lymphocyte cell lines. J Immunol. 2004; 173: 4806–4814.[Abstract/Free Full Text]

98. Carlisle Michel JJ, Dodge KL, Wong W, Mayer NC, Langeberg LK, Scott JD. PKA-phosphorylation of PDE4D3 facilitates recruitment of the mAKAP signalling complex. Biochem J. 2004; 381: 587–592.[CrossRef][Medline] [Order article via Infotrieve]

99. Dodge KL, Khouangsathiene S, Kapiloff MS, Mouton R, Hill EV, Houslay MD, Langeberg LK, Scott JD. mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module. EMBO J. 2001; 20: 1921–1930.[CrossRef][Medline] [Order article via Infotrieve]

100. Dodge–Kafka KL, Soughayer J, Pare GC, Carlisle Michel JJ, Langeberg LK, Kapiloff MS, Scott JD. The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways. Nature. 2005; 437: 574–578.[CrossRef][Medline] [Order article via Infotrieve]

101. Kapiloff MS, Schillace RV, Westphal AM, Scott JD. mAKAP: an A-kinase anchoring protein targeted to the nuclear membrane of differentiated myocytes. J Cell Sci. 1999; 112 (pt 16): 2725–2736.[Abstract]

102. Bolger GB, Erdogan S, Jones RE, Loughney K, Scotland G, Hoffmann R, Wilkinson I, Farrell C, Houslay MD. Characterization of five different proteins produced by alternatively spliced mRNAs from the human cAMP-specific phosphodiesterase PDE4D gene. Biochem J. 1997; 328 (pt 2): 539–548.[Medline] [Order article via Infotrieve]

103. Lefkowitz RJ, Pierce KL, Luttrell LM. Dancing with different partners: protein kinase a phosphorylation of seven membrane-spanning receptors regulates their G protein-coupling specificity. Mol Pharmacol. 2002; 62: 971–974.[Free Full Text]

104. Martin NP, Whalen EJ, Zamah MA, Pierce KL, Lefkowitz RJ. PKA-mediated phosphorylation of the beta1-adrenergic receptor promotes Gs/Gi switching. Cell Signal. 2004; 16: 1397–1403.[CrossRef][Medline] [Order article via Infotrieve]

105. Maurice DH, Palmer D, Tilley DG, Dunkerley HA, Netherton SJ, Raymond DR, Elbatarny HS, Jimmo SL. Cyclic nucleotide phosphodiesterase activity, expression, and targeting in cells of the cardiovascular system. Mol Pharmacol. 2003; 64: 533–546.[Abstract/Free Full Text]

106. Katano Y, Endoh M. Effects of a cardiotonic quinolinone derivative Y-20487 on the isoproterenol-induced positive inotropic action and cyclic AMP accumulation in rat ventricular myocardium: comparison with rolipram, Ro 20–1724, milrinone, and isobutylmethylxanthine. J Cardiovasc Pharmacol. 1992; 20: 715–722.[Medline] [Order article via Infotrieve]

107. Jurevicius J, Skeberdis VA, Fischmeister R. Role of cyclic nucleotide phosphodiesterase isoforms in cAMP compartmentation following beta2-adrenergic stimulation of ICa,L in frog ventricular myocytes. J Physiol. 2003; 551: 239–252.[Abstract/Free Full Text]

108. Rochais F, Vandecasteele G, Lefebvre F, Lugnier C, Lum H, Mazet JL, Cooper DM, Fischmeister R. Negative feedback exerted by cAMP-dependent protein kinase and cAMP phosphodiesterase on subsarcolemmal cAMP signals in intact cardiac myocytes: an in vivo study using adenovirus-mediated expression of CNG channels. J Biol Chem. 2004; 279: 52095–52105.[Abstract/Free Full Text]

109. Juan-Fita MJ, Vargas ML, Hernandez J. Comparative actions of diazepam and other phosphodiesterase inhibitors on the effects of noradrenaline in rat myocardium. Pharmacol Toxicol. 2003; 93: 23–28.[CrossRef][Medline] [Order article via Infotrieve]

110. Thomas NJ, Carcillo JA, Herzer WA, Mi Z, Tofovic SP, Jackson EK. Type IV phosphodiesterase inhibition improves cardiac contractility in endotoxemic rats. Eur J Pharmacol. 2003; 465: 133–139.[CrossRef][Medline] [Order article via Infotrieve]

111. Ding B, Abe J, Wei H, Huang Q, Walsh RA, Molina CA, Zhao A, Sadoshima J, Blaxall BC, Berk BC, Yan C. Functional role of phosphodiesterase 3 in cardiomyocyte apoptosis: implication in heart failure. Circulation. 2005; 111: 2469–2476.[Abstract/Free Full Text]

112. Ding B, Abe J, Wei H, Xu H, Che W, Aizawa T, Liu W, Molina CA, Sadoshima J, Blaxall BC, Berk BC, Yan C. A positive feedback loop of phosphodiesterase 3 (PDE3) and inducible cAMP early repressor (ICER) leads to cardiomyocyte apoptosis. Proc Natl Acad Sci U S A. 2005; 102: 14771–14776.[Abstract/Free Full Text]

113. Xiang Y, Naro F, Zoudilova M, Jin SL, Conti M, Kobilka B. Phosphodiesterase 4D is required for beta2 adrenoceptor subtype-specific signaling in cardiac myocytes. Proc Natl Acad Sci U S A. 2005; 102: 909–914.[Abstract/Free Full Text]

114. Mongillo M, Zaccolo M. A complex phosphodiesterase system controls beta-adrenoceptor signalling in cardiomyocytes. Biochem Soc Trans. 2006; 34: 510–511.[CrossRef][Medline] [Order article via Infotrieve]

115. Dodge-Kafka KL, Langeberg L, Scott JD. Compartmentation of cyclic nucleotide signaling in the heart: the role of A-kinase anchoring proteins. Circ Res. 2006; 98: 993–1001.[Abstract/Free Full Text]

116. Zakhary DR, Moravec CS, Bond M. Regulation of PKA binding to AKAPs in the heart: alterations in human heart failure. Circulation. 2000; 101: 1459–1464.[Abstract/Free Full Text]

117. McCahill A, Warwicker J, Bolger GB, Houslay MD, Yarwood SJ. The RACK1 scaffold protein: a dynamic cog in cell response mechanisms. Mol Pharmacol. 2002; 62: 1261–1273.[Free Full Text]

118. Steele MR, McCahill A, Thompson DS, MacKenzie C, Isaacs NW, Houslay MD, Bolger GB. Identification of a surface on the beta-propeller protein RACK1 that interacts with the cAMP-specific phosphodiesterase PDE4D5. Cell Signal. 2001; 13: 507–513.[CrossRef][Medline] [Order article via Infotrieve]

119. Song LS, Guatimosim S, Gomez-Viquez L, Sobie EA, Ziman A, Hartmann H, Lederer WJ. Calcium biology of the transverse tubules in heart. Ann N Y Acad Sci. 2005; 1047: 99–111.[CrossRef][Medline] [Order article via Infotrieve]

120. Marks AR. Novel therapy for heart failure and exercise-induced ventricular tachycardia based on ‘fixing’ the leak in ryanodine receptors. Novartis Found Symp. 2006; 274: 132–147.[Medline] [Order article via Infotrieve]

121. Kockskamper J, Pieske B. Phosphorylation of the cardiac ryanodine receptor by Ca2+/calmodulin-dependent protein kinase II: the dominating twin of protein kinase A. Circ Res. 2006; 99: 333–335.[Free Full Text]

122. Guo T, Zhang T, Mestril R, Bers DM. Ca2+/calmodulin-dependent protein kinase II phosphorylation of ryanodine receptor does affect calcium sparks in mouse ventricular myocytes. Circ Res. 2006; 99: 398–406.[Abstract/Free Full Text]

123. Bers DM. Macromolecular complexes regulating cardiac ryanodine receptor function. J Mol Cell Cardiol. 2004; 37: 417–429.[CrossRef][Medline] [Order article via Infotrieve]

124. Marcantoni A, Levi RC, Gallo MP, Hirsch E, Alloatti G. Phosphoinositide 3-kinasegamma (PI3Kgamma) controls L-type calcium current (ICa,L) through its positive modulation of type-3 phosphodiesterase (PDE3). J Cell Physiol. 2006; 206: 329–336.[CrossRef][Medline] [Order article via Infotrieve]

125. Movsesian MA, Bristow MR. Alterations in cAMP-mediated signaling and their role in the pathophysiology of dilated cardiomyopathy. Curr Top Dev Biol. 2005; 68: 25–48.[Medline] [Order article via Infotrieve]

126. Wehrens XH, Lehnart SE, Marks AR. Intracellular calcium release and cardiac disease. Annu Rev Physiol. 2005; 67: 69–98.[CrossRef][Medline] [Order article via Infotrieve]

127. Lehnart SE, Marks AR. Phosphodiesterase 4D and heart failure: a cautionary tale. Expert Opin Ther Targets. 2006; 10: 677–688.[CrossRef][Medline] [Order article via Infotrieve]

128. Lipworth BJ. Phosphodiesterase-4 inhibitors for asthma and chronic obstructive pulmonary disease. Lancet. 2005; 365: 167–175.[CrossRef][Medline] [Order article via Infotrieve]

129. Boswell-Smith V, Spina D, Page CP. Phosphodiesterase inhibitors. Br J Pharmacol. 2006; 147: S252–S257.[CrossRef][Medline] [Order article via Infotrieve]

130. Fan Chung K. Phosphodiesterase inhibitors in airways disease. Eur J Pharmacol. 2006; 533: 110–117.[CrossRef][Medline] [Order article via Infotrieve]

131. Yoshida T, Owens GK. Molecular determinants of vascular smooth muscle cell diversity. Circ Res. 2005; 96: 280–291.[Abstract/Free Full Text]

132. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 2004; 84: 767–801.[Abstract/Free Full Text]

133. Sarkar K, Sharma SK, Sachdeva R, Romeo F, Garza L, Mehta JL. Coronary artery restenosis: vascular biology and emerging therapeutic strategies. Expert Rev Cardiovasc Ther. 2006; 4: 543–556.[CrossRef][Medline] [Order article via Infotrieve]

134. Liu H, Maurice DH. Phosphorylation-mediated activation and translocation of the cyclic AMP-specific phosphodiesterase PDE4D3 by cyclic AMP-dependent protein kinase and mitogen-activated protein kinases. A potential mechanism allowing for the coordinated regulation of PDE4D activity and targeting. J Biol Chem. 1999; 274: 10557–10565.[Abstract/Free Full Text]

135. Tilley DG, Maurice DH. Vascular smooth muscle cell phosphodiesterase (PDE) 3 and PDE4 activities and levels are regulated by cyclic AMP in vivo. Mol Pharmacol. 2002; 62: 497–506.[Abstract/Free Full Text]

136. Dunkerley HA, Tilley DG, Palmer D, Liu H, Jimmo SL, Maurice DH. Reduced phosphodiesterase 3 activity and phosphodiesterase 3A level in synthetic vascular smooth muscle cells: implications for use of phosphodiesterase 3 inhibitors in cardiovascular tissues. Mol Pharmacol. 2002; 61: 1033–1040.[Abstract/Free Full Text]

137. Palmer D, Maurice DH. Dual expression and differential regulation of phosphodiesterase 3A and phosphodiesterase 3B in human vascular smooth muscle: implications for phosphodiesterase 3 inhibition in human cardiovascular tissues. Mol Pharmacol. 2000; 58: 247–252.[Abstract/Free Full Text]

138. Lincoln TM, Wu X, Sellak H, Dey N, Choi CS. Regulation of vascular smooth muscle cell phenotype by cyclic GMP and cyclic GMP-dependent protein kinase. Front Biosci. 2006; 11: 356–367.[Medline] [Order article via Infotrieve]

139. Degerman E, Belfrage P, Manganiello VC. Structure, localization, and regulation of cGMP-inhibited phosphodiesterase (PDE3). J Biol Chem. 1997; 272: 6823–6826.[Free Full Text]

140. Rybalkin SD, Bornfeldt KE, Sonnenburg WK, Rybalkina IG, Kwak KS, Hanson K, Krebs EG, Beavo JA. Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1C) is induced in human arterial smooth muscle cells of the synthetic, proliferative phenotype. J Clin Invest. 1997; 100: 2611–2621.[Medline] [Order article via Infotrieve]

141. Rybalkin SD, Rybalkina I, Beavo JA, Bornfeldt KE. Cyclic nucleotide phosphodiesterase 1C promotes human arterial smooth muscle cell proliferation. Circ Res. 2002; 90: 151–157.[Abstract/Free Full Text]

142. Debey S, Meyer-Kirchrath J, Schror K. Regulation of cyclooxygenase-2 expression by iloprost in human vascular smooth muscle cells. Role of transcription factors CREB and ICER. Biochem Pharmacol. 2003; 65: 979–988.[CrossRef][Medline] [Order article via Infotrieve]

143. Wang X, Murphy TJ. The inducible cAMP early repressor ICERIIgamma inhibits CREB and AP-1 transcription but not AT1 receptor gene expression in vascular smooth muscle cells. Mol Cell Biochem. 2000; 212: 111–119.[CrossRef][Medline] [Order article via Infotrieve]

144. Inoue Y, Toga K, Sudo T, Tachibana K, Tochizawa S, Kimura Y, Yoshida Y, Hidaka H. Suppression of arterial intimal hyperplasia by cilostamide, a cyclic nucleotide phosphodiesterase 3 inhibitor, in a rat balloon double-injury model. Br J Pharmacol. 2000; 130: 231–241.[CrossRef][Medline] [Order article via Infotrieve]

145. Packer M. Effect of phosphodiesterase inhibitors on survival of patients with chronic congestive heart failure. Am J Cardiol. 1989; 63: 41A–45A.[CrossRef][Medline] [Order article via Infotrieve]




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