Distinct phosphodiesterase 5A-containing compartments allow selective regulation of cGMP-dependent signalling in human arterial smooth muscle cells.

Cyclic GMP (cGMP) translates and integrates much of the information encoded by nitric oxide (NO·) and several natriuretic peptides, including the atrial natriuretic peptide (ANP). Previously, we reported that integration of a cGMP-specific cyclic nucleotide phosphodiesterase, namely phosphodiesterase 5A (PDE5A), into a protein kinase G (PKG)- and inositol-1,4,5-trisphosphate receptor (IP3R)-containing endoplasmic reticulum (ER) signalosome allows localized control of PDE5A activity and of PKG-dependent inhibition of IP3-mediated release of ER Ca2+ in human platelets. Herein, we report that PDE5A integrates into an analogous signalosome in human arterial smooth muscle cells (HASMC), wherein it regulates muscarinic agonist-dependent Ca2+ release and is activated selectively by PKG-dependent phosphorylation. In addition, we report that PDE5A also regulates HASMC functions via events independent of PKG, but rather through actions coordinated by competitive cGMP-mediated inhibition of cAMP hydrolysis by the so-called cGMP-inhibited cAMP PDE, namely phosphodiesterase 3A (PDE3A). Indeed, we show that ANP increases both cGMP and cAMP levels in HASMC and promotes phosphorylation of vasodilator-stimulated phospho-protein (VASP) at each the PKG and PKA phospho-acceptor sites. Since selective inhibition of PDE5 decreased DNA synthesis and chemotaxis of HASMC, and that PDE3A knockdown obviated these effects, our findings are consistent with a role for a PDE5A-PDE3A-PKA axis in their regulation. Our findings provide insight into the existence of distinct "pools" of PDE5A in HASMC and support the idea that these discrete compartments regulate distinct cGMP-dependent events. As a corollary, we suggest that it may be possible to target these distinct PDE5A-regulated pools and in so-doing differentially impact selected cGMP-regulated functions in these cells.

A systematic review and comparison of functional assessments of community-dwelling elderly patients.

PURPOSE: To provide advanced practice nurses in primary care with information about self-reported functional assessments and physical performance-based functional assessments of geriatric patients living alone within the community at greatest risk of functional decline. DATA SOURCES: Databases searched include CINAHL, Healthsource: Nursing/Academic Edition, MEDLINE, PsycINFO, PsycARTICLES, Cochrane Library, and National Clearinghouse Guidelines. The review was limited to English, research, and the years 2000-2014. Key search words included geriatric, community-dwelling, functional assessment, activities and instrumental activities of daily living, Barthel Index, Katz Index, Lawton Scale, Vulnerable Elders Survey, Timed Up and Go Test, Gait Speed Test, Functional Reach Test, and primary care. CONCLUSIONS: Forty-three million individuals, age 65 and older, are currently living in the United States with numbers expected to double by 2050. Nurse practitioners will be at the forefront of assessing for functional decline and can use tools such as the Barthel Index and Gait Speed Test to improve elderly outcomes. IMPLICATIONS FOR PRACTICE: Self-reported functional questionnaires and physical functional performance tests can quickly be completed in the office to track the risk of functional decline over time. Interventions, such as physical therapy or other community resources, can be initiated when needed to reduce negative outcomes of functional decline.

Emerging biology of PDE10A.

Cyclic AMP and cyclic GMP are essential second messengers that regulate multiple signaling pathways in virtually all cell types. Their accumulation in cells is finely regulated by cyclic nucleotide phosphodiesterases (PDEs), the only enzymes that can degrade these signaling molecules and thus provide exquisite control over intracellular signaling processes. One PDE family, PDE10A, is highly enriched in the brain and its unique expression profile in specific brain regions of interest, in particular to antipsychotic treatment, has made it an attractive therapeutic target for the treatment of schizophrenia. However, after a Phase II trial failure of a selective PDE10A inhibitor for the treatment of schizophrenia, it has encouraged the field to reexamine the role of this enzyme in the brain, and the possible CNS disorders in which PDE10A inhibition could be therapeutic. We will review the localization of PDE10A, both within the brain and the neuron and discuss how its role in regulating cAMP and cGMP accumulation modulates intracellular signaling pathways. Since this cellular signaling has best been documented in the striatum, we will focus our discussion of PDE10A in the context of disorders that affect the basal ganglia, including psychiatric disorders such as bipolar disorder and autism spectrum disorders and the movement disorders, including Parkinson's disease and Huntington's disease.

Cyclic nucleotide phosphodiesterases (PDEs): coincidence detectors acting to spatially and temporally integrate cyclic nucleotide and non-cyclic nucleotide signals.

The cyclic nucleotide second messengers cAMP and cGMP each affect virtually all cellular processes. Although these hydrophilic small molecules readily diffuse throughout cells, it is remarkable that their ability to activate their multiple intracellular effectors is spatially and temporally selective. Studies have identified a critical role for compartmentation of the enzymes which hydrolyse and metabolically inactivate these second messengers, the PDEs (cyclic nucleotide phosphodiesterases), in this specificity. In the present article, we describe several examples from our work in which compartmentation of selected cAMP- or cGMP-hydrolysing PDEs co-ordinate selective activation of cyclic nucleotide effectors, and, as a result, selectively affect cellular functions. It is our belief that therapeutic strategies aimed at targeting PDEs within these compartments will allow greater selectivity than those directed at inhibiting these enzymes throughout the cells.

Phosphodiesterase 4D regulates baseline sarcoplasmic reticulum Ca2+ release and cardiac contractility, independently of L-type Ca2+ current.

RATIONALE: Baseline contractility of mouse hearts is modulated in a phosphatidylinositol 3-kinase-γ-dependent manner by type 4 phosphodiesterases (PDE4), which regulate cAMP levels within microdomains containing the sarcoplasmic reticulum (SR) calcium ATPase type 2a (SERCA2a). OBJECTIVE: The goal of this study was to determine whether PDE4D regulates basal cardiac contractility. METHODS AND RESULTS: At 10 to 12 weeks of age, baseline cardiac contractility in PDE4D-deficient (PDE4D(-/-)) mice was elevated mice in vivo and in Langendorff perfused hearts, whereas isolated PDE4D(-/-) cardiomyocytes showed increased whole-cell Ca2+ transient amplitudes and SR Ca2+content but unchanged L-type calcium current, compared with littermate controls (WT). The protein kinase A inhibitor R(p)-adenosine-3',5' cyclic monophosphorothioate (R(p)-cAMP) lowered whole-cell Ca2+ transient amplitudes and SR Ca2+ content in PDE4D(-/-) cardiomyocytes to WT levels. The PDE4 inhibitor rolipram had no effect on cardiac contractility, whole-cell Ca2+ transients, or SR Ca2+ content in PDE4D(-/-) preparations but increased these parameters in WT myocardium to levels indistinguishable from those in PDE4D(-/-). The functional changes in PDE4D(-/-) myocardium were associated with increased PLN phosphorylation but not cardiac ryanodine receptor phosphorylation. Rolipram increased PLN phosphorylation in WT cardiomyocytes to levels indistinguishable from those in PDE4D(-/-) cardiomyocytes. In murine and failing human hearts, PDE4D coimmunoprecipitated with SERCA2a but not with cardiac ryanodine receptor. CONCLUSIONS: PDE4D regulates basal cAMP levels in SR microdomains containing SERCA2a-PLN, but not L-type Ca2+ channels or ryanodine receptor. Because whole-cell Ca2+ transient amplitudes are reduced in failing human myocardium, these observations may have therapeutic implications for patients with heart failure.

A phosphodiesterase 3B-based signaling complex integrates exchange protein activated by cAMP 1 and phosphatidylinositol 3-kinase signals in human arterial endothelial cells.

Enzymes of the phosphodiesterase 3 (PDE3) and PDE4 families each regulate the activities of both protein kinases A (PKAs) and exchange proteins activated by cAMP (EPACs) in cells of the cardiovascular system. At present, the mechanisms that allow selected PDEs to individually regulate the activities of these two effectors are ill understood. The objective of this study was to determine how a specific PDE3 variant, namely PDE3B, interacts with and regulates EPAC1-based signaling in human arterial endothelial cells (HAECs). Using several biochemical approaches, we show that PDE3B and EPAC1 bind directly through protein-protein interactions. By knocking down PDE3B expression or by antagonizing EPAC1 binding with PDE3B, we show that PDE3B regulates cAMP binding by its tethered EPAC1. Interestingly, we also show that PDE3B binds directly to p84, a PI3Kγ regulatory subunit, and that this interaction allows PI3Kγ recruitment to the PDE3B-EPAC1 complex. Of potential cardiovascular importance, we demonstrate that PDE3B-tethered EPAC1 regulates HAEC PI3Kγ activity and that this allows dynamic cAMP-dependent regulation of HAEC adhesion, spreading, and tubule formation. We identify and molecularly characterize a PDE3B-based "signalosome" that integrates cAMP- and PI3Kγ-encoded signals and show how this signal integration regulates HAEC functions of importance in angiogenesis.

Cyclic AMP phosphodiesterase 4D (PDE4D) Tethers EPAC1 in a vascular endothelial cadherin (VE-Cad)-based signaling complex and controls cAMP-mediated vascular permeability.

Vascular endothelial cell (VEC) permeability is largely dependent on the integrity of vascular endothelial cadherin (VE-cadherin or VE-Cad)-based intercellular adhesions. Activators of protein kinase A (PKA) or of exchange protein activated by cAMP (EPAC) reduce VEC permeability largely by stabilizing VE-Cad-based intercellular adhesions. Currently, little is known concerning the nature and composition of the signaling complexes that allow PKA or EPAC to regulate VE-Cad-based structures and through these actions control permeability. Using pharmacological, biochemical, and cell biological approaches we identified and determined the composition and functionality of a signaling complex that coordinates cAMP-mediated control of VE-Cad-based adhesions and VEC permeability. Thus, we report that PKA, EPAC1, and cyclic nucleotide phosphodiesterase 4D (PDE4D) enzymes integrate into VE-Cad-based signaling complexes in human arterial endothelial cells. Importantly, we show that protein-protein interactions between EPAC1 and PDE4D serve to foster their integration into VE-Cad-based complexes and allow robust local regulation of EPAC1-based stabilization of VE-Cad-based adhesions. Of potential translational importance, we mapped the EPAC1 peptide motif involved in binding PDE4D and show that a cell-permeable variant of this peptide antagonizes EPAC1-PDE4D binding and directly alters VEC permeability. Collectively, our data indicate that PDE4D regulates both the activity and subcellular localization of EPAC1 and identify a novel mechanism for regulated EPAC1 signaling in these cells.

Compartmentation and compartment-specific regulation of PDE5 by protein kinase G allows selective cGMP-mediated regulation of platelet functions.

It is generally accepted that nitric oxide (NO) donors, such as sodium nitroprusside (SNP), or phosphodiesterase 5 (PDE5) inhibitors, including sildenafil, each impact human platelet function. Although a strong correlation exists between the actions of NO donors in platelets and their impact on cGMP, agents such as sildenafil act without increasing global intra-platelet cGMP levels. This study was undertaken to identify how PDE5 inhibitors might act without increasing cGMP. Our data identify PDE5 as an integral component of a protein kinase G1beta (PKG1beta)-containing signaling complex, reported previously to coordinate cGMP-mediated inhibition of inositol-1, 4, 5-trisphosphate receptor type 1 (IP(3)R1)-mediated Ca(2+)-release. PKG1beta and PDE5 did not interact in subcellular fractions devoid of IP(3)R1 and were not recruited to IP(3)R1-enriched membranes in response to cGMP-elevating agents. Activation of platelet PKG promoted phosphorylation and activation of the PDE5 fraction tethered to the IP(3)R1-PKG complex, an effect not observed for the nontethered PDE5. Based on these findings, we elaborate a model in which PKG selectively activates PDE5 within a defined microdomain in platelets and propose that this mechanism allows spatial and temporal regulation of cGMP signaling in these cells. Recent reports indicate that sildenafil might prove useful in limiting in-stent thrombosis and the thrombotic events associated with the acute coronary syndromes (ACS), situations poorly regulated with currently available therapeutics. We submit that our findings may define a molecular mechanism by which PDE5 inhibition can differentially impact selected cellular functions of platelets, and perhaps of other cell types.

Numerous distinct PKA-, or EPAC-based, signalling complexes allow selective phosphodiesterase 3 and phosphodiesterase 4 coordination of cell adhesion.

By activating two distinct classes of effector enzymes, namely Protein Kinases A [PKA] or Exchange Proteins Activated by cAMP [EPAC], the ubiquitous second messenger cAMP selectively coordinates numerous events simultaneously in virtually all cells. Studies focused on dissecting the manner by which cAMP simultaneously regulates multiple cellular events have shown that cAMP activates its effectors non-uniformly in cells and that this localized cAMP-mediated signalling is made possible, at least in part, by anchoring of cAMP effectors to selected subcellular structures. In the work described here, we report that HEK293T cells ["293T"] contain several PKA- and EPAC1-based signalling complexes. Interestingly, our data do not identify signalling complexes in which both PKA and EPAC are each present but rather are consistent with the idea that these two effectors operate in distinct complexes in these cells. Similarly, we report that while individual PKA- or EPAC-containing complexes can contain either phosphodiesterase 3B, [PDE3B] or phosphodiesterase 4D [PDE4D], they do not contain both these phosphodiesterases. Indeed, although PDE4D enzymes were identified in both PKA- and EPAC-based complexes, PDE3B was largely identified in EPAC-based complexes. Using a combination of approaches, we identified that integration of PDE3B into EPAC-based complexes occurred through its amino terminal fragment [PDE3B(AT)]. Consistent with the idea that integration of PDE3B within EPAC-based complexes was dynamic and regulated PDE3 inhibitor-mediated effects on cellular functions, expression of PDE3B(AT) competed with endogenous PDE3B for integration into EPAC-based complexes and antagonized PDE3 inhibitor-based cell adhesion. Our data support the concept that cells can contain several non-overlapping PKA- and EPAC-based signalling complexes and that these complexes may also represent sites within cells were the effects of family-selective PDE inhibitors could be integrated to affect cell functions, including adhesion.

Both protein kinase A and exchange protein activated by cAMP coordinate adhesion of human vascular endothelial cells.

cAMP regulates integrin-dependent adhesions of vascular endothelial cells (VECs) to extracellular matrix proteins, their vascular endothelial cadherin-dependent intercellular adhesions, and their proliferation and migration in response to growth and chemotactic factors. Previously, we reported that cAMP-elevating agents differentially inhibited migration of human VECs isolated from large vascular structures (macro-VECs, human aortic endothelial cells [HAECs]) or small vascular structures (micro-VECs, human microvascular endothelial cells [HMVECs]) and that cAMP hydrolysis by phosphodiesterase (PDE)3 and PDE4 enzymes was important in coordinating this difference. Here we report that 2 cAMP-effector enzymes, namely protein kinase (PK)A and exchange protein activated by cAMP (EPAC), each regulate extracellular matrix protein-based adhesions of both macro- and micro-VECs. Of interest and potential therapeutic importance, we report that although specific pharmacological activation of EPAC markedly stimulated adhesion of micro-VECs to extracellular matrix proteins when PKA was inhibited, this treatment only modestly promoted adhesion of macro-VECs. Consistent with an important role for cAMP PDEs in this difference, PDE3 or PDE4 inhibitors promoted EPAC-dependent adhesions in micro-VECs when PKA was inhibited but not in macro-VECs. At a molecular level, we identify multiple, nonoverlapping, PKA- or EPAC-based signaling protein complexes in both macro- and micro-VECs and demonstrate that each of these complexes contains either PDE3B or PDE4D but not both of these PDEs. Taken together, our data support the concept that adhesion of macro- and micro-VECs is differentially regulated by cAMP and that these differences are coordinated through selective actions of cAMP at multiple nonoverlapping signaling complexes that contain PKA or EPAC and distinct PDE variants.

PI3Kgamma is required for PDE4, not PDE3, activity in subcellular microdomains containing the sarcoplasmic reticular calcium ATPase in cardiomyocytes.

We recently showed that phosphoinositide-3-kinase-gamma-deficient (PI3Kgamma(-/-)) mice have enhanced cardiac contractility attributable to cAMP-dependent increases in sarcoplasmic reticulum (SR) Ca(2+) content and release but not L-type Ca(2+) current (I(Ca,L)), demonstrating PI3Kgamma locally regulates cAMP levels in cardiomyocytes. Because phosphodiesterases (PDEs) can contribute to cAMP compartmentation, we examined whether the PDE activity was altered by PI3Kgamma ablation. Selective inhibition of PDE3 or PDE4 in wild-type (WT) cardiomyocytes elevated Ca(2+) transients, SR Ca(2+) content, and phospholamban phosphorylation (PLN-PO(4)) by similar amounts to levels observed in untreated PI3Kgamma(-/-) myocytes. Combined PDE3 and PDE4 inhibition caused no further increases in SR function. By contrast, only PDE3 inhibition affected Ca(2+) transients, SR Ca(2+) loads, and PLN-PO(4) levels in PI3Kgamma(-/-) myocytes. On the other hand, inhibition of PDE3 or PDE4 alone did not affect I(Ca,L) in either PI3Kgamma(-/-) or WT cardiomyocytes, whereas simultaneous PDE3 and PDE4 inhibition elevated I(Ca,L) in both groups. Ryanodine receptor (RyR(2)) phosphorylation levels were not different in basal conditions between PI3Kgamma(-/-) and WT myocytes and increased in both groups with PDE inhibition. Our results establish that L-type Ca(2+) channels, RyR(2), and SR Ca(2+) pumps are regulated differently in distinct subcellular compartments by PDE3 and PDE4. In addition, the loss of PI3Kgamma selectively abolishes PDE4 activity, not PDE3, in subcellular compartments containing the SR Ca(2+)-ATPase but not RyR(2) or L-type Ca(2+) channels.

Protein kinase A phosphorylation of human phosphodiesterase 3B promotes 14-3-3 protein binding and inhibits phosphatase-catalyzed inactivation.

Recent studies confirm that intracellular cAMP concentrations are nonuniform and that localized subcellular cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is important in maintaining these cAMP compartments. Human phosphodiesterase 3B (HSPDE3B), a member of the PDE3 family of PDEs, represents the dominant particulate cAMP-PDE activity in many cell types, including adipocytes and cells of hematopoietic lineage. Although several previous reports have shown that phosphorylation of HSPDE3B by either protein kinase A (PKA) or protein kinase B (PKB) activates this enzyme, the mechanisms that allow cells to distinguish these two activated forms of HSPDE3B are unknown. Here we report that PKA phosphorylates HSPDE3B at several distinct sites (Ser-73, Ser-296, and Ser-318), and we show that phosphorylation of HSPDE3B at Ser-318 activates this PDE and stimulates its interaction with 14-3-3 proteins. In contrast, although PKB-catalyzed phosphorylation of HSPDE3B activates this enzyme, it does not promote 14-3-3 protein binding. Interestingly, we report that the PKA-phosphorylated, 14-3-3 protein-bound, form of HSPDE3B is protected from phosphatase-dependent dephosphorylation and inactivation. In contrast, PKA-phosphorylated HSPDE3B that is not bound to 14-3-3 proteins is readily dephosphorylated and inactivated. Our data are presented in the context that a selective interaction between PKA-activated HSPDE3B and 14-3-3 proteins represents a mechanism by which cells can protect this enzyme from deactivation. Moreover, we propose that this mechanism may allow cells to distinguish between PKA- and PKB-activated HSPDE3B.