cAMP Hydrolysis Research Articles

Identification and characterization of a novel phosphodiesterase from the metagenome of an Indian coalbed.

Phosphoesterases are involved in the degradation of organophosphorus compounds. Although phosphomonoesterases and phosphotriesterases have been studied in detail, studies on phosphodiesterases are rather limited. In our search to find novel phosphodiesterases using metagenomic approach, we cloned a gene encoding a putative phosphodiesterase (PdeM) from the metagenome of the formation water collected from an Indian coal bed. Bioinformatic analysis showed that PdeM sequence possessed the characteristic signature motifs of the class III phosphodiesterases and phylogenetic study of PdeM enabled us to identify three distinct subclasses (A, B, and C) within class III phosphodiesterases, PdeM clustering in new subclass IIIB. Bioinformatic, biochemical and biophysical characterization of PdeM further revealed some of the characteristic features of the phosphodiesterases belonging to newly described subclass IIIB. PdeM is a monomer of 29.3 kDa, which exhibits optimum activity at 25°C and pH 8.5, but low affinity for bis(pNPP) as well as pNPPP. The recombinant PdeM possessed phosphodiesterase, phosphonate-ester hydrolase and nuclease activity. It lacked phosphomonoesterase, phosphotriesterase, and RNAse activities. Overexpression of PdeM in E.coli neither affected catabolite respression nor did the recombinant protein hydrolyzed cAMP in vitro, indicating its inability to hydrolyze cAMP. Although Mn2+ was required for the activity of PdeM, but addition of metals (Mn2+ or Fe3+) did not induce oligomerization. Further increase in concentration of Mn2+ upto 3 mM, increased α-helical content as well as the phosphodiesterase activity. Structural comparison of PdeM with its homologs showed that it lacked critical residues required for dimerization, cAMP hydrolysis, and for the high affinity binding of bis(pNPP). PdeM, thus, is a novel representative of new subclass of class III phosphodiesterases.

Oscillation of cAMP and Ca2+ in cardiac myocytes: a systems biology approach

Cyclic adenosine monophosphate (cAMP) and Ca(2+) levels may oscillate in harmony within excitable cells; a mathematical oscillation loop model, the Cooper model, of these oscillations was developed two decades ago. However, in that model all adenylyl cyclase (AC) isoforms were assumed to be inhibited by Ca(2+), and it is now known that the heart expresses multiple AC isoforms, among which the type 5/6 isoforms are Ca(2+)-inhibitable whereas the other five (AC2, 3, 4, 7, and 9) are not. We used a computational systems biology approach with CellDesigner simulation software to develop a comprehensive graphical map and oscillation loop model for cAMP and Ca(2+). This model indicated that Ca(2+)-mediated inhibition of AC is essential to create oscillations of Ca(2+) and cAMP, and the oscillations were not altered by incorporation of phosphodiesterase-mediated cAMP hydrolysis or PKA-mediated inhibition of AC into the model. More importantly, they were created but faded out immediately in the co-presence of Ca(2+)-noninhibitable AC isoforms. Because the subcellular locations of AC isoforms are different, spontaneous cAMP and Ca(2+) oscillations may occur within microdomains containing only Ca(2+)-inhibitable isoforms in cardiac myocytes, which might be necessary for fine tuning of excitation-contraction coupling.

Regulation of the Cardiac β-Adrenergic Pathway via Camp-Cgmp Competition

In the cardiac myocyte, the second messenger cAMP is synthesized by the β-adrenergic signaling pathway upon sympathetic activation. It activates Protein Kinase A (PKA) mediated phosphorylation of downstream targets that are critical to the control of cardiac contractility. The dynamics of cAMP are also controlled indirectly by cGMP-mediated regulation of phosphodiesterase (PDE) isoenzymes. It is not yet clear how the cGMP signal is transduced by the PDEs to regulate cAMP. To better understand this, we have developed mechanistically detailed models of PDEs 1 - 4, the primary cAMP hydrolyzing PDEs in cardiac myocytes, and integrated them into an existing model of the β-adrenergic signaling pathway. Our PDE models are based upon experimental studies performed on purified PDE enzymes which show that cyclic nucleotides (cNs) bind competitively to the domains of PDEs 1, 2, and 3. PDE4 is regulated by PKA but does not interact appreciably with cGMP. Our individual PDE models reproduce cAMP hydrolysis rates as regulated by various cGMP concentrations, and the fully integrated model also replicates experimentally observed cAMP dose-response relationships and temporal dynamics. Our model shows that PDE2 is critical to the regulation of cAMP signals, especially during increased stimulation of the β-adrenergic pathway. In addition, it reveals that high levels of cGMP out-compete cAMP for catalytic sites of PDEs 1, 2, and 3, and suppress their cAMP hydrolysis rates. This leads to a net accumulation of cAMP despite the negative feedback of the cAMP-driven increases in rate of PDE4-mediated cAMP hydrolysis. These results provide insights into how PDEs serve as an integration point for cN signals and how cN interactions regulate β-adrenergic response.

Roles of Phosphodiesterases in Cyclic Nucleotide Cross-Talk in Cardiac Myocytes

The cyclic nucleotides (cNs) cAMP and cGMP regulate the response of cardiac myocytes to both external and internal stimuli. Distinctly-regulated phosphodiesterases (PDEs) control the degradation of these cNs. As a result of their regulation by cNs, PDEs also facilitate communication between the β-adrenergic and Nitric Oxide (NO)/cGMP/Protein Kinase G (PKG) signaling pathways, which regulate the synthesis of cAMP and cGMP, respectively. Activation of the β-adrenergic pathway potentiates cardiac contractility, whereas activation of the NO/cGMP/PKG pathway reduces it. As a result, the balance between cNs plays a critical role in regulating contraction. The phenomena where cAMP influences the dynamics of the cGMP pathway, and vice versa, are commonly referred to as cN cross-talk. However, the cross-talk response and the individual role of each PDE isoenzyme in shaping this response remain to be fully characterized. We have developed a computational model of the cN cross-talk network that mechanistically integrates the β-adrenergic and NO/cGMP/PKG pathways via regulation of PDEs by both cNs. The individual model components and the entire network model replicate experimentally observed activation-response relationships and temporal dynamics. The model predicts that under sub-maximal β-adrenergic stimulation, an increase of PDE2 and a decrease of PDE3 cAMP hydrolysis rates under concomitant NO stimulation results in a net cAMP accumulation, leading to the observed NO-mediated potentiation of Protein Kinase A (PKA) activation. In addition, under concomitant β-adrenergic stimulation, due to cGMP accumulation from increased PDE5 and decreased PDE3 cGMP hydrolysis rates, PKG can be further activated beyond the level achieved under NO alone. By defining cN cross-talk reactions based on the binding affinity of cNs to specific PDE domains and the associated hydrolysis rates, we pin-pointed the principal mechanisms driving the cross-talk response within this non-linear, tightly-coupled reaction system.

Active Site Coupling in PDE:PKA Complexes Promotes Resetting of Mammalian cAMP Signaling

Cyclic 3′5′ adenosine monophosphate (cAMP)-dependent-protein kinase (PKA) signaling is a fundamental regulatory pathway for mediating cellular responses to hormonal stimuli. The pathway is activated by high-affinity association of cAMP with the regulatory subunit of PKA and signal termination is achieved upon cAMP dissociation from PKA. Although steps in the activation phase are well understood, little is known on how signal termination/resetting occurs. Due to the high affinity of cAMP to PKA (KD ∼ low nM), bound cAMP does not readily dissociate from PKA, thus begging the question of how tightly bound cAMP is released from PKA to reset its signaling state to respond to subsequent stimuli. It has been recently shown that phosphodiesterases (PDEs) can catalyze dissociation of bound cAMP and thereby play an active role in cAMP signal desensitization/termination. This is achieved through direct interactions with the regulatory subunit of PKA, thereby facilitating cAMP dissociation and hydrolysis. In this study, we have mapped direct interactions between a specific cyclic nucleotide phosphodiesterase (PDE8A) and a PKA regulatory subunit (RIα isoform) in mammalian cAMP signaling, by a combination of amide hydrogen/deuterium exchange mass spectrometry, peptide array, and computational docking. The interaction interface of the PDE8A:RIα complex, probed by peptide array and hydrogen/deuterium exchange mass spectrometry, brings together regions spanning the phosphodiesterase active site and cAMP-binding sites of RIα. Computational docking combined with amide hydrogen/deuterium exchange mass spectrometry provided a model for parallel dissociation of bound cAMP from the two tandem cAMP-binding domains of RIα. Active site coupling suggests a role for substrate channeling in the PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. This is the first instance, to our knowledge, of PDEs directly interacting with a cAMP-receptor protein in a mammalian system, and highlights an entirely new class of binding partners for RIα. This study also highlights applications of structural mass spectrometry combined with computational docking for mapping dynamics in transient signaling protein complexes. Together, these results present a novel and critical role for phosphodiesterases in moderating local concentrations of cAMP in microdomains and signal resetting.

Abstract 054: Mutations In Pde3a Explain Mendelian Hypertension With Brachydactyly

We identified Mendelian type E brachydactyly (BDE) with hypertension in six different families from Turkey, America, France, Canada and South Africa. The two phenotypes, hypertension and BDE, invariably coincide. The blood pressure in affected individuals increases with increasing age, the mean arterial blood pressure at 50 years exceeds 150 mm Hg, resulting in stroke. Earlier we suggested that a chromosomal rearrangement on 12p could be responsible. We now used whole-genome sequencing and discovered six different so far unknown missense mutations in the gene encoding PDE3A. The mutations are not identical, but adjacent to each other. Each is responsible for an amino acid substitution in a conserved portion of the protein. The mutations were not present in any non-affected family members, in more than 200 additional controls, and not found in the “1000 genome” project. We performed in vitro cell transfections and found that mutated PDE3A showed gain-of-function with increased cAMP hydrolysis. Mesenchymal stromal cell-derived vascular smooth muscle cells and chondrocytes gave insight into the molecular pathogenesis; PDE3A and vasodilator-stimulated phosphoprotein (VASP) were differently phosphorylated and parathyroid hormone-related peptide (PTHrP) was dysregulated. Our preliminary results reveal that the mutated PDE3A enzymes show gain-of-function with increased cAMP hydrolysis and sensitivity to cGMP inhibition. We suggest that the mutations are responsible for both phenotypes. This Mendelian hypertension is the first that is not attributable to increased sodium reabsorption in the distal nephron.

Dual specificity and novel structural folding of yeast phosphodiesterase-1 for hydrolysis of second messengers cyclic adenosine and guanosine 3',5'-monophosphate.

Cyclic nucleotide phosphodiesterases (PDEs) decompose second messengers cAMP and cGMP that play critical roles in many physiological processes. PDE1 of Saccharomyces cerevisiae has been subcloned and expressed in Escherichia coli. Recombinant yPDE1 has a KM of 110 μM and a kcat of 16.9 s–1 for cAMP and a KM of 105 μM and a kcat of 11.8 s–1 for cGMP. Thus, the specificity constant (kcat/KMcAMP)/(kcat/KMcGMP) of 1.4 indicates a dual specificity of yPDE1 for hydrolysis of both cAMP and cGMP. The crystal structures of unliganded yPDE1 and its complex with GMP at 1.31 Å resolution reveal a new structural folding that is different from those of human PDEs but is partially similar to that of some other metalloenzymes such as metallo-β-lactamase. In spite of their different structures and divalent metals, yPDE1 and human PDEs may share a common mechanism for hydrolysis of cAMP and cGMP.

A Long Lasting β1 Adrenergic Receptor Stimulation of cAMP/Protein Kinase A (PKA) Signal in Cardiac Myocytes

Small-molecule, ligand-activated G protein-coupled receptors are generally thought to be rapidly desensitized within a period of minutes through receptor phosphorylation and internalization after repeated or prolonged stimulation. This transient G protein-coupled receptor activation remains at odds with many observed long-lasting cellular and physiological responses. Here, using live cell imaging of cAMP with a FRET-based biosensor and myocyte contraction assay, we show that the catecholamine-activated β1 adrenergic receptor (β1AR) continuously stimulates second messenger cAMP synthesis in primary cardiac myocytes and neurons, which lasts for more than 8 h (a decay t½ of 3.9 h) in cardiac myocytes. However, the β1AR-induced cAMP signal is counterbalanced and masked by the receptor-bound phosphodiesterase (PDE) 4D8-dependent cAMP hydrolysis. Inhibition of PDE4 activity recovers the receptor-induced cAMP signal and promotes contractile response in mouse hearts during extended periods of agonist stimulation. β1AR associates with PDE4D8 through the receptor C-terminal PDZ motif-dependent binding to synaptic-associated protein 97 (SAP97). Knockdown of SAP97 or mutation of the β1AR PDZ motif disrupts the complex and promotes sustained agonist-induced cAMP activity, PKA phosphorylation, and cardiac myocyte contraction response. Together, these findings unveil a long lasting adrenergic signal in neurons and myocytes under prolonged stimulation and an underappreciated role of PDE that is essential in classic receptor signaling desensitization and in maintaining a long lasting cAMP equilibrium for ligand-induced physiological response.

0059: Cyclic AMP response to β-adrenergic stimulation is strongly dependent on cell confluence in vascular smooth muscle cells

The influence of cell confluence on β-adrenergic (β-AR) cAMP signal and its control by phosphodiesterases (PDE) was investigated in vascular smooth muscle cells (SMC). Cultured rat aortic SMC were plated either at low density (LD: 3.103 cells/cm2) or high density (HD: 3.104 cells/cm2) corresponding, respectively, to non-confluent or confluent cells 2 days later. The cells were infected with an adenovirus encoding the fluorescence resonance energy transfer (FRET)-based cAMP sensor, Epac2-camps and imaging experiments were performed 48 h later. A brief (15 s) application of the β-AR agonist isoproterenol (Iso) induced a typical transient FRET signal, with a rapid increase, reflecting cAMP production, followed by a return to baseline. The amplitude of response to 10 or 100 nM Iso was higher in HD than LD cells, but the kinetics were similar. A β1-AR antagonist (CGP 20712, 100 nM) reduced the Iso (100 nM) response in HD but not LD cells. However, a β2-AR antagonist (ICI 118551, 5 nM) reduced the response in both conditions. To evaluate whether the absence of β1-AR response in LD cells was due to an increased PDE activity, we used the non-selective PDE inhibitor IBMX (100 μM). In LD cells pretreated with IBMX, the Iso response was maintained even after Iso washout, showing that the recovery phase is due to cAMP hydrolysis. However, CGP 20712 still did not alter the Iso response. PDE4 inhibition with Ro-20-1724 (10 μM) strongly prolonged Iso (10 nM) response in LD and HD cells, whereas PDE3 inhibition with cilostamide (1 μM) potentiated Iso response in LD cells but had no effect in HD cells, even at a 100 nM Iso concentration. We conclude that in HD cells, Iso response involves both β1- and β2-AR stimulation and is controlled by PDE4 but not PDE3, whereas in LD cells, Iso response involves only β2-AR stimulation and is controlled by both PDE3 and PDE4. Thus, β-AR-mediated cAMP signal in vascular SMC is strongly dependent on the confluence of the cells.

PDE2-mediated cAMP hydrolysis accelerates cardiac fibroblast to myofibroblast conversion and is antagonized by exogenous activation of cGMP signaling pathways

Recent studies suggest that the signal molecules cAMP and cGMP have antifibrotic effects by negatively regulating pathways associated with fibroblast to myofibroblast (MyoCF) conversion. The phosphodiesterase 2 (PDE2) has the unique property to be stimulated by cGMP, which leads to a remarkable increase in cAMP hydrolysis and thus mediates a negative cross-talk between both pathways. PDE2 has been recently investigated in cardiomyocytes; here we specifically addressed its role in fibroblast conversion and cardiac fibrosis. PDE2 is abundantly expressed in both neonatal rat cardiac fibroblasts (CFs) and cardiomyocytes. The overexpression of PDE2 in CFs strongly reduced basal and isoprenaline-induced cAMP synthesis, and this decrease was sufficient to induce MyoCF conversion even in the absence of exogenous profibrotic stimuli. Functional stress-strain experiments with fibroblast-derived engineered connective tissue (ECT) demonstrated higher stiffness in ECTs overexpressing PDE2. In regard to cGMP, neither basal nor atrial natriuretic peptide-induced cGMP levels were affected by PDE2, whereas the response to nitric oxide donor sodium nitroprusside was slightly but significantly reduced. Interestingly, despite persistently depressed cAMP levels, both cGMP-elevating stimuli were able to completely prevent the PDE2-induced MyoCF phenotype, arguing for a double-tracked mechanism. In conclusion, PDE2 accelerates CF to MyoCF conversion, which leads to greater stiffness in ECTs. Atrial natriuretic peptide- and sodium nitroprusside-mediated cGMP synthesis completely reverses PDE2-induced fibroblast conversion. Thus PDE2 may augment cardiac remodeling, but this effect can also be overcome by enhanced cGMP. The redundant role of cAMP and cGMP as antifibrotic meditators may be viewed as a protective mechanism in heart failure.

How Enzymes Access Caged Substrates? Understanding Targeted Hydrolysis of Cyclic AMP by Phosphodiesterase-Protein Kinase a Interactions

Cyclic adenosine monophosphate (cAMP)-Protein Kinase A (PKA) signaling is a fundamental pathway for mediating cellular responses to hormonal stimuli. cAMP activates PKA by binding to its regulatory (RIα) subunit. While steps in the activation phase are well understood, little is known on how signal desensitization/resetting occurs. We specifically set out to determine (i) how cAMP-bound to the regulatory subunit is hydrolyzed to return RIα to the kinase-bound state and (ii) the role of phosphodiesterases in enabling this important regulatory resetting of the system. We report discovery of a novel signaling complex of a phosphodiesterase (PDE8) and PKA Regulatory subunit (RIα) in mammalian cAMP signaling by a combination of amide hydrogen/deuterium exchange mass spectrometry (HDXMS), peptide arrays and computational docking. A model of the PDE8A-PKA RIα complex reveals the phosphodiesterase active site in close proximity to the cAMP binding site on the PKA target and highlights a role for substrate channeling in the PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. This is the first instance of PDEs directly interacting with a cAMP-receptor protein in a mammalian system and highlights an entirely new class of binding partners for RIα. This has major implications for regulation of intensity and duration of the cAMP signal in all cells. Furthermore, this introduces molecular channeling as a new paradigm for macromolecular assemblies in microdomains and localized stimulus-response cycles in cell signaling. Our model for molecular channeling are consistent with Fluorescence Resonance Energy Transfer- reporter assays for cAMP in cells, providing a new model for understanding spatial and temporal regulation of cAMP signaling.

β-Adrenergic Regulation of Cyclic AMP and Ca Current at the T-Tubules and Surface Membrane in Rat Cardiomyocytes

β-Adrenoceptor (β-AR) signalling is severely impaired in heart failure (HF), and this is accompanied by a loss and disorganization of t-tubules. However, how the latter affects the former is unknown. Here, we examined how acute detubulation affects the β-AR response of L-type Ca2+ channel (LTCC) current (ICa,L) and membrane cAMP ([cAMP]m) in adult rat ventricular myocytes (ARVMs) and the contribution of phosphodiesterases PDE3 and PDE4 in this process. ARVMs were infected with an adenovirus encoding a mutant of the olfactory cyclic nucleotide-gated (CNG) channels α subunit to follow [cAMP]m dynamics by recording the associated cationic current. [cAMP]m was also monitored by fluorescent imaging using a FRET-based sensor encoding a plasma-membrane targeted cAMP probe (Epac2-camps). Osmotic shock treatment with 1.5M formamide during 15 min induced a loss of t-tubule network as verified by confocal imaging with di-8-ANEPPS staining. Detubulation of ARVMs reduced membrane capacitance by 40% and basal ICa,L density ∼3-fold. Short (15s) applications of isoprenaline (Iso 100nM) increased ICa,L ∼2.5-fold similarly in both conditions but had a 30% smaller effect on [cAMP]m in detubulated ARVMs. After Iso washout, ICa,L and [cAMP]m returned to basal with a ∼1.5-fold faster kinetic in detubulated ARVMs than in control, suggesting a faster cAMP degradation by PDEs. The contribution of PDE3 and PDE4 was thus tested using the inhibitors cilostamide (1µM) and Ro20-1724 (10µM), respectively. Both inhibitors had a more pronounced effect on Iso response of ICa,L and [cAMP]m in detubulated ARVMs. Thus, detubulation per se has a profound effect on the β-AR signalling cascade which may contribute to its impairment in HF. This is partly due to a change in the respective contributions of PDE3 and PDE4 to the hydrolysis of cAMP near the membrane and LTCCs.

Phosphodiesterases 3 and 5 express activity in the trigeminal ganglion and co-localize with calcitonin gene-related peptide

Understanding of the neuropathology leading to migraine pain has centered on either a vascular or neuronal origin. Sildenafil, a specific inhibitor of phosphodiesterase 5 (PDE5), induces migraine-like headache in a human headache model without concomitant artery dilation. The presence and activity of PDE3 and PDE5 is known in cerebral arteries. However, the presence in the neuronal part of the trigeminovascular pathway, i.e. the trigeminal ganglion and the possible co-localization with calcitonin gene-related peptide (CGRP), is not known. Rat trigeminal ganglia were isolated and immunohistochemistry and in situ hybridization was applied. Evaluations of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) hydrolysis were performed using scintillation proximity assays. PDE3 and PDE5 were present and active in the trigeminal ganglia. A subset of PDE3- and PDE5-positive neurons contained CGRP. In contrast to cGMP, both sildenafil and cilostazol influenced cAMP hydrolysis. Sildenafil may exert its effect on the neuronal part of the migraine pain pathway. In addition to the effects on cGMP signaling, sildenafil may indirectly affect cAMP signaling in the trigeminal ganglion. This result may suggest a common cAMP-related pathway for sildenafil, cilostazol, and CGRP in eliciting migraine pain.

Clinical and molecular genetics of the phosphodiesterases (PDEs).

Cyclic nucleotide phosphodiesterases (PDEs) are enzymes that have the unique function of terminating cyclic nucleotide signaling by catalyzing the hydrolysis of cAMP and GMP. They are critical regulators of the intracellular concentrations of cAMP and cGMP as well as of their signaling pathways and downstream biological effects. PDEs have been exploited pharmacologically for more than half a century, and some of the most successful drugs worldwide today affect PDE function. Recently, mutations in PDE genes have been identified as causative of certain human genetic diseases; even more recently, functional variants of PDE genes have been suggested to play a potential role in predisposition to tumors and/or cancer, especially in cAMP-sensitive tissues. Mouse models have been developed that point to wide developmental effects of PDEs from heart function to reproduction, to tumors, and beyond. This review brings together knowledge from a variety of disciplines (biochemistry and pharmacology, oncology, endocrinology, and reproductive sciences) with emphasis on recent research on PDEs, how PDEs affect cAMP and cGMP signaling in health and disease, and what pharmacological exploitations of PDEs may be useful in modulating cyclic nucleotide signaling in a way that prevents or treats certain human diseases.

Amrinone, Forerunner of Novel Cardiotonic Agents, Caused Paradigm Shift of Heart Failure Pharmacotherapy

Amrinone has been a great forerunner of noncatechol and nonglycoside novel cardiotonic agents and given an extraordinary strong impact in the development of novel cardiotonic agents. Unexpectedly, amrinone failed to rescue the patients with chronic heart failure, thereby produced a prominent paradigm shift from inotropic to cardioprotective therapy, and contributed to advances in the current pharmacotherapy of chronic heart failure. In this study, Alousi et al showed that amrinone is a potent, long-acting positive inotropic agent when given orally or intravenously to either anesthetized or unanesthetized dogs. Amrinone was orally active with a rapid onset of action and duration of >5 hours and showed a wide separation between the positive inotropic and chronotropic effect. Amrinone increased contractile force and the rate of force development without changes in duration of the contractile cycle or time-to-peak tension, and in action potential or excitability even when it was given in relatively high concentrations. The toxicological studies attested to a low toxicity of amrinone ≤10 mg/kg, with less serious side effects such as lowering in blood pressure and an increase in heart rate compared with cardiac glycosides and catecholamines. The therapeutic index of amrinone was wide in contrast to that of cardiac glycosides that have a therapeutic index of ≈2 to 3, with life-threatening arrhythmias. Of greater interest was that amrinone did not increase cardiac output of the normal (nonfailing), but increased that of the failing heart model. The mechanism of action of amrinone, however, was shown only by exclusion criteria. Amrinone did not seem to act via a catecholamine mechanism because its inotropic effect was not blocked by the β-receptor blocking agent propranolol or by depletion of cardiac norepinephrine with reserpine. It was reported that there were no significant changes in the level of cardiac cAMP or phosphodiesterase (PDE) that could be responsible …

Attenuation of TNF production and experimentally induced inflammation by PDE4 inhibitor rolipram is mediated by MAPK phosphatase‐1

3',5'-Cyclic nucleotide PDE4 is expressed in several inflammatory and immune cells, and PDE4 catalyses the hydrolysis of cAMP to 5'AMP, down-regulating cAMP signalling in cells. MAPK phosphatase-1 (MKP-1) is an endogenous p38 MAPK signalling suppressor and limits inflammatory gene expression and inflammation. In the present study, we investigated the effect of a PDE4 inhibitor rolipram on MKP-1 expression and whether MKP-1 is involved in the anti-inflammatory effects of rolipram. The effect of rolipram on TNF production was investigated in J774 mouse macrophage cell line and in primary mouse peritoneal macrophages (PM) from wild-type (WT) and MKP-1(-/-) mice. We also investigated the effect of rolipram on carrageenan-induced paw inflammation in WT and MKP-1(-/-) mice. MKP-1 expression was enhanced by rolipram, by a non-selective PDE inhibitor IBMX and by a cAMP analogue 8-Br-cAMP in J774 cells and in PM. Enhanced MKP-1 mRNA expression by rolipram was reversed by a PKA inhibitor. Rolipram, IBMX and 8-Br-cAMP also inhibited TNF production in activated macrophages. Accordingly, rolipram inhibited TNF production in PMs from WT mice but, interestingly, not in PMs from MKP-1(-/-) mice. Furthermore, rolipram attenuated carrageenan-induced paw inflammation in WT but not in MKP-1(-/-) mice. PDE4 inhibitor rolipram was found to enhance the expression of MKP-1, and MKP-1 mediated, at least partly, the anti-inflammatory effects of PDE4 inhibition. The results suggest that compounds that enhance MKP-1 expression and/or MKP-1 activity hold potential as novel anti-inflammatory drugs.

Phosphodiesterase-2 Is Up-Regulated in Human Failing Hearts and Blunts β-Adrenergic Responses in Cardiomyocytes

This study investigated whether myocardial phosphodiesterase-2 (PDE2) is altered in heart failure (HF) and determined PDE2-mediated effects on beta-adrenergic receptor (β-AR) signaling in healthy and diseased cardiomyocytes. Diminished cyclic adenosine monophosphate (cAMP) and augmented cyclic guanosine monophosphate (cGMP) signaling is characteristic for failing hearts. Among the PDE superfamily, PDE2 has the unique property of being able to be stimulated by cGMP, thus leading to a remarkable increase in cAMP hydrolysis mediating a negative cross talk between cGMP and cAMP signaling. However, the role of PDE2 in HF is poorly understood. Immunoblotting, radioenzymatic- and fluorescence resonance energy transfer-based assays, video edge detection, epifluorescence microscopy, and L-type Ca2(+) current measurements were performed in myocardial tissues and/or isolated cardiomyocytes from human and/or experimental HF, respectively. Myocardial PDE2 expression and activity were ~2-fold higher in advanced human HF. Chronic β-AR stimulation via catecholamine infusions in rats enhanced PDE2 expression ~2-fold and cAMP hydrolytic activity ~4-fold, which correlated with blunted cardiac β-AR responsiveness. In diseased cardiomyocytes, higher PDE2 activity could be further enhanced by stimulation of cGMP synthesis via nitric oxide donors, whereas specific PDE2 inhibition partially restored β-AR responsiveness. Accordingly, PDE2 overexpression in healthy cardiomyocytes reduced the rise in cAMP levels and L-type Ca2(+) current amplitude, and abolished the inotropic effect following acute β-AR stimulation, without affecting basal contractility. Importantly, PDE2-overexpressing cardiomyocytes showed marked protection from norepinephrine-induced hypertrophic responses. PDE2 is markedly up-regulated in failing hearts and desensitizes against acute β-AR stimulation. This may constitute an important defense mechanism during cardiac stress, for example, by antagonizing excessive β-AR drive. Thus, activating myocardial PDE2 may represent a novel intracellular antiadrenergic therapeutic strategy in HF.

Effect of the Mixed Phosphodiesterase 3/4 Inhibitor RPL554 on Human Isolated Bronchial Smooth Muscle Tone

The phosphodiesterase (PDE) enzyme family hydrolyzes cAMP and cGMP, second messengers that regulate a variety of cellular processes, including airway smooth muscle (ASM) relaxation and the inhibition of inflammatory cells. We investigated the activity of RPL554 [9,10-dimethoxy-2(2,4,6-trimethylphenylimino)-3-(n-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one], a dual PDE3/PDE4 inhibitor that exhibits bifunctional activity for its effects on the tone of human isolated ASM and any potential synergistic interactions with muscarinic receptor antagonists or a β2-agonist. We evaluated the influence of RPL554 on the contractile response induced by electrical field stimulation (EFS), acetylcholine (ACh), or histamine on human isolated bronchi. We also analyzed the potential synergistic effect of RPL554 in combination with atropine, glycopyrollate, or salbutamol by using the Berenbaum Bliss Independence (BI), or the dose equivalence methods. RPL554 inhibited the contraction induced by EFS [maximal effectiveness (Emax) 91.33 ± 3.37%, P < 0.001], relaxed bronchi precontracted with ACh (Emax 94.62 ± 2.63%, pD2 4.84 ± 0.12, P < 0.001), and abolished the contraction induced by histamine. Analysis of interactions indicated a weak synergism between RPL554 and salbutamol (interaction index: 0.25 ± 0.06; BI Δeffect: 0.29 ± 0.11; dose equivalence: no synergism) but significant synergism between RPL554 and atropine (interaction index: 0.09 ± 0.07; BI Δeffect: 0.54 ± 0.09; dose equivalence: synergism for low concentrations) or glycopyrrolate (ACh: BI Δeffect 0.46 ± 0.03, Berenbaum Δeffect 0.42 ± 0.02; histamine: BI Δeffect 0.46 ± 0.03, Berenbaum Δeffect 0.42 ± 0.03). This study demonstrates that RPL554 relaxes human bronchi and that it can interact with a muscarinic receptor antagonist to produce a synergistic inhibition of ASM tone. These results suggest that RPL554 may provide a novel treatment of airway diseases, either alone or in combination with antimuscarinic drugs.

A balanced de novo inv(7)(p14.3q22.3) disrupting PDE1C and ATXN7L1 in a 14-year old developmentally delayed boy

We report a 14 year old male patient ascertained for developmental delay, carrying a de novo pericentric inversion on chr(7)(p14.3q22.3). Sequencing revealed that the breakpoints overlap a LTR sequence on 7q22.3 and a LINE on 7p14.3. A TTTAAA motif was found in proximity of the breakpoints on both arms. In addition the sequencing detected several small micro-rearrangements, deletion, duplication, insertion, at the breakpoints. No significant sequence identity exists between the 7p14.3 and 7q22.3 breakpoints. These features at the breakpoint junctions suggest that the inversion was triggered by the TTTAAA motif, LTR and LINE and healed by a Non Homologous End Joining (NHEJ) mechanism. The genes ATXN7L1 and PDE1C are disrupted by the inversion. PDE1C is responsible for the hydrolysis of the second messenger molecules cAMP and cGMP and is highly expressed in the human heart and certain brain regions. In mice, Pde1c is expressed in migrating neuronal cells within the central nervous system during early embryo development. Although neuronal migration disorder was not seen in our patient, this is the first patient described with haploinsufficiency of PDE1C possibly causing developmental delay.

PDE8A runs interference to limit PKA inhibition of Raf-1

Myriad extracellular and intracellular signals constantly poke or prod cells in order to influence the vast number of functions required of cells to maintain homeostasis. Selective reception of these signals and of their subsequent translation and transduction are tasks coordinated by cellular signal-transduction systems. Two of these systems, the Ras/Raf/MEK (mitogen-activated ERK-kinase)/ERK cascade and the cAMP/PKA signaling system, each regulate numerous cellular functions and their influences on these continue to be the subject of intense study (1). Interestingly, research confirms that rather than functioning in mutually independent “tracks,” the Ras/Raf/MEK/ERK cascade and the cAMP/PKA system often interact and numerous reports document where activation of cAMP/PKA signaling either amplified or antagonized Ras/Raf/MEK/ERK-mediated events, both in physiological settings as well as in pathologies, including cancers (1, 2). Although numerous points of intersection between these systems have been described (1, 2), one critical point of interplay involves an inhibitory PKA-mediated phosphorylation of Raf-1 (V-raf-1 murine leukemia viral oncogene homolog 1, also known as C-Raf) at S259. Raf-1 is one of the three known MEK kinases, along with A-Raf and B-Raf, which can stimulate MEK-catalyzed ERK phosphorylation in this cascade. In PNAS, Brown et al., (3) provide mechanistic insights into the manner by which PKA-mediated phosphorylation and inhibition of Raf-1 is regulated in cells, and they highlight a role for localized hydrolysis of cAMP in this important process.