Identification of murine phosphodiesterase 5A isoforms and their functional characterization in HL-1 cardiac cell line

Phosphodiesterase 5A (PDE5A) specifically degrades the ubiquitous second messenger cGMP and experimental and clinical data highlight its important role in cardiac diseases. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2 and mPde5a3). The predicted amino acidic sequences of the three murine isoforms are different in the N-terminal regulatory domain. mPDE5A isoforms were transfected in HEK293T cells and they showed high affinity for cGMP and similar sensitivity to sildenafil inhibition. RT-PCR analysis showed that mPde5a1, mPde5a2 and mPde5a3 had differential tissue distribution. In the adult heart, mPde5a1 and mPde5a2 were expressed at different levels whereas mPde5a3 was undetectable. Overexpression of mPDE5As induced an increase of HL-1 number cells which progress into cell cycle. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL-1 areas and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms. Moreover, mPDE5A isoforms had differential subcellular localization: mPDE5A1 was mainly localized in the cytoplasm, mPDE5A2 and mPDE5A3 were also nuclear localized. These results demonstrate for the first time the existence of three PDE5A isoforms in mouse and highlight their potential role in the induction of hypertrophy. This article is protected by copyright. All rights reserved

Targeting protein–protein interactions within the cyclic AMP signaling system as a therapeutic strategy for cardiovascular disease

The cAMP signaling system can trigger precise physiological cellular responses that depend on the fidelity of many protein–protein interactions, which act to bring together signaling intermediates at defined locations within cells. In the heart, cAMP participates in the fine control of excitation–contraction coupling, hence, any disregulation of this signaling cascade can lead to cardiac disease. Due to the ubiquitous nature of the cAMP pathway, general inhibitors of cAMP signaling proteins such as PKA, EPAC and PDEs would act non-specifically and universally, increasing the likelihood of serious ‘off target’ effects. Recent advances in the discovery of peptides and small molecules that disrupt the protein–protein interactions that underpin cellular targeting of cAMP signaling proteins are described and discussed

Differential Regulation of PDE5 Expression in Left and Right Ventricles of Feline Hypertrophy Models

Though long known to affect smooth muscle biology, recent studies indicate that phosphodiesterase 5 (PDE5) is also expressed in myocardium. Recognizing that the regulation of PDE5 in hypertrophy is not well understood, we assessed the response of PDE5 expression and the level of cGMP-dependent kinase I (cGKI) in the left and right ventricles of feline hypertrophy models.Using a cDNA library of feline aortic smooth muscle cells, we identified and cloned PDE5 cDNA for the first time in this species. The sequence shares 98% identity with its human orthologue at the amino acid level. E. coli expression of the cloned allele allowed selection of antibodies with appropriate specificity, facilitating the analysis of PDE5 expression in feline models created by selective proximal aortic (Ao) or pulmonary artery (PA) banding that resulted in hypertrophy of the left ventricle (LV) and right ventricle (RV), respectively. We demonstrated that PDE5 expression responded differentially with a decreased expression in the LV and an increased expression in the RV in the Ao-banded model. Similarly, in the PA-banded model, LV showed reduced expression while the RV expression was unaltered. In addition, the expression of cGKI was significantly decreased in the RV of Ao-banded group, correlating inversely with the increase in PDE5 expression.The differential regulation of PDE5 and cGKI expression suggests that the mechanisms involved in hypertrophy could be different in RV vs. LV. Reciprocal PDE5 and cGKI expression in the RV of Ao-banded model suggests functional significance for PDE5 up-regulation

Multiple Isozymes of Cyclic Nucleotide Phosphodiesterases in the Rat

Cyclic nucleotide phosphodiesterases catalyse the hydrolysis of 3'5'-cyclic nucleotides, such as cyclic AMP and cyclic GMP, to produce 5'-derivatives. Multiple isozymes of this enzyme exist that differ in their substrate specificities, means of regulation and subcellular localisation. The soluble phosphodiesterases present in homogenates of both rat liver and rat hepatocytes have beed identified and characterised. Using a high resolution anion-exchange system (Mono Q) to rapidly separate soluble phosphodiesterases, it has been shown that five separate activities are present in both of these soluble fractions. On this basis, it was concluded that the multiplicity of phosphodiesterase species in liver is not due to the presence of multiple cell types in this organ. These soluble activities were termed PDE MQ-I to PDE MQ-V and were distinguishable from one another by a number of criteria. These included substrate specificity, kinetic properties, sensitivity to Ca2+/Calmodulin or cyclic GMP, dependency on magnesium, sensitivity to a number of reference phosphodiesterase inhibitors, molecular weights and chromatographic properties

Therapeutic targeting of 3’,5’-cyclic nucleotide phosphodiesterases: inhibition and beyond

Phosphodiesterases (PDEs), enzymes that degrade 3′,5′-cyclic nucleotides, are being pursued as therapeutic targets for several diseases, including those affecting the nervous system, the cardiovascular system, fertility, immunity, cancer and metabolism. Clinical development programmes have focused exclusively on catalytic inhibition, which continues to be a strong focus of ongoing drug discovery efforts. However, emerging evidence supports novel strategies to therapeutically target PDE function, including enhancing catalytic activity, normalizing altered compartmentalization and modulating post-translational modifications, as well as the potential use of PDEs as disease biomarkers. Importantly, a more refined appreciation of the intramolecular mechanisms regulating PDE function and trafficking is emerging, making these pioneering drug discovery efforts tractable

Cyclic Nucleotide Signaling and the Cardiovascular System

The cyclic nucleotides 3',5'-adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) play important roles in the control of cardiovascular function under physiological and pathological conditions. In this book, which is a reprint of a Special Issue of the Journal of Cardiovascular Development and Disease entitled "Cyclic Nucleotide Signaling and the Cardiovascular System", internationally recognized experts give an overview of this vibrant scientific field. The first series of articles deal with the localization and function of membrane-bound and soluble adenylate cyclases, followed by articles on the roles of phosphodiesterase isoforms in the heart. Cyclic nucleotide signaling takes place in nanodomains and the A-kinase anchor proteins (AKAPS) are essential for the compartmentalized assembly of signaling proteins into functional complexes. Reviews on the role of AKAP proteins in the physiology and pathophysiology of the heart are also included in this book. Cyclic nucleotides act through effector proteins and articles on EPAC and POPDC proteins inform the reader of recent developments on these topics. A major advancement in our understanding of cyclic nucleotide signaling came through the use of genetically encoded cAMP sensor molecules, and a series of articles review the current insight that these reporter molecules have provided. The final set of articles in this book deals with the association of the cyclic nucleotide pathway and cardiovascular disease as well as the development of novel therapeutic approaches. Thomas Brand and Enno Klussmann Special Issue Editor

Protein-protein interactions of PDE4 family members - Functions, interactions and therapeutic value

The second messenger cyclic adenosine monophosphate (cAMP) is ubiquitous and directs a plethora of functions in all cells. Although theoretically freely diffusible through the cell from the site of its synthesis it is not evenly distributed. It rather is shaped into gradients and these gradients are established by phospodiesterases (PDEs), the only enzymes that hydrolyse cAMP and thereby terminate cAMP signalling upstream of cAMP's effector systems. Miles D. Houslay has devoted most of his scientific life highly successfully to a particular family of PDEs, the PDE4 family. The family is encoded by four genes and gives rise to around 20 enzymes, all with different functions. M. Houslay has discovered many of these functions and realised early on that PDE4 family enzymes are attractive drug targets in a variety of human diseases, but not their catalytic activity as that is encoded in conserved domains in all family members. He postulated that targeting the intracellular location would provide the specificity that modern innovative drugs require to improve disease conditions with fewer side effects than conventional drugs. Due to the wealth of M. Houslay's work, this article can only summarize some of his discoveries and, therefore, focuses on protein-protein interactions of PDE4. The aim is to discuss functions of selected protein-protein interactions and peptide spot technology, which M. Houslay introduced into the PDE4 field for identifying interacting domains. The therapeutic potential of PDE4 interactions will also be discussed

Role of cyclic nucleotides and their downstream signaling cascades in memory function:Being at the right time at the right spot

A plethora of studies indicate the important role of cAMP and cGMP cascades in neuronal plasticity and memory function. As a result, altered cyclic nucleotide signaling has been implicated in the pathophysiology of mnemonic dysfunction encountered in several diseases. In the present review we provide a wide overview of studies regarding the involvement of cyclic nucleotides, as well as their upstream and downstream molecules, in physiological and pathological mnemonic processes. Next, we discuss the regulation of the intracellular concentration of cyclic nucleotides via phosphodiesterases, the enzymes that degrade cAMP and/or cGMP, and via A-kinase-anchoring proteins that refine signal compartmentalization of cAMP signaling. We also provide an overview of the available data pointing to the existence of specific time windows in cyclic nucleotide signaling during neuroplasticity and memory formation and the significance to target these specific time phases for improving memory formation. Finally, we highlight the importance of emerging imaging tools like Förster resonance energy transfer imaging and optogenetics in detecting, measuring and manipulating the action of cyclic nucleotide signaling cascades