PDE Activity Research Articles

Mitochondrial Complex I Core Protein Regulates cAMP Signaling via Phosphodiesterase Pde2 and NAD Homeostasis in Candida albicans.

The cyclic adenosine 3′,5′-monophosphate (cAMP)/protein kinase A (PKA) pathway of Candida albicans responds to nutrient availability to coordinate a series of cellular processes for its replication and survival. The elevation of cAMP for PKA signaling must be both transitory and tightly regulated. Otherwise, any abnormal cAMP/PKA pathway would disrupt metabolic potential and ergosterol synthesis and promote a stress response. One possible mechanism for controlling cAMP levels is direct induction of the phosphodiesterase PDE2 gene by cAMP itself. Our earlier studies have shown that most single-gene-deletion mutants of the mitochondrial electron transport chain (ETC) complex I (CI) are hypersensitive to fluconazole. To understand the fluconazole hypersensitivity observed in these mutants, we focused upon the cAMP/PKA-mediated ergosterol synthesis in CI mutants. Two groups of the ETC mutants were used in this study. Group I includes CI mutants. Group II is composed of CIII and CIV mutants; group II mutants are known to have greater respiratory loss. All mutants are not identical in cAMP/PKA-mediated ergosterol response. We found that ergosterol levels are decreased by 47.3% in the ndh51Δ (CI core subunit mutant) and by 23.5% in goa1Δ (CI regulator mutant). Both mutants exhibited a greater reduction of cAMP and excessive trehalose production compared with other mutants. Despite the normal cAMP level, ergosterol content decreased by 33.0% in the CIII mutant qce1Δ as well, thereby displaying a cAMP/PKA-independent ergosterol response. While the two CI mutants have some unique cAMP/PKA-mediated ergosterol responses, we found that the degree of cAMP reduction correlates linearly with a decrease in total nicotinamide adenine dinucleotide (NAD) levels in all mutants, particularly in the seven CI mutants. A mechanism study demonstrates that overactive PDE2 and cPDE activity must be the cause of the suppressive cAMP-mediated ergosterol response in the ndh51Δ and goa1Δ. While the purpose of this study is to understand the impact of ETC proteins on pathogenesis-associated cellular events, our results reveal the importance of Ndh51p in the regulation of the cAMP/PKA pathway through Pde2p inhibition in normal physiological environments. As a direct link between Ndh51p and Pde2p remains elusive, we suggest that Ndh51p participates in NAD homeostasis that might regulate Pde2p activity for the optimal cAMP pathway state.

NCS 613, a PDE4 inhibitor, by increasing cAMP level suppresses systemic inflammation and immune complexes deposition in kidney of MRL/lpr lupus- prone mice

Nephritis remains the most common severe manifestation of systemic lupus erythematosus in which auto-antibodies mediate chronic inflammation and kidney damage. cAMP-phosphodiesterases regulate sodium excretion and inflammation in various tissues. How cAMP elevation can reduce systemic inflammation and suppress kidney inflammation and damage remains elusive. PDE4 signaling and cAMP metabolism were investigated along immune complex depositions in target tissues and kidney damage (histology). SLE disease progression is associated with changes in kidney PDE4 activity and expression. Moreover, lupus prone mice exhibit low kidney cAMP level which is associated to induction and relocation of nuclear and cytoskeleton PDE4 isoforms. Auto-antibodies-induced kidney damage was attested by mesangial proliferation and cellular infiltration. Interestingly, we reported that NCS 613 treatment decreases systemic auto-antibody secretion and their corresponding immune complex deposition in target tissues. Furthermore, NCS 613 is able to increase cAMP levels in the kidney; hence this compound rescues kidney PDE4 alterations in treated mice. NCS 613 overcomes disease progression in lupus prone mice by improving wellbeing and decreasing inflammation in treated mice. The PDE4 inhibitor, NCS 613, is a new anti-inflammatory compound that is believed to be a leading drug candidate for the treatment of inflammatory diseases such as lupus nephritis.

Purification of 5′-phosphodiesterase from Adzuki (Vigna angularis L.) bean

Adzuki bean is high in 5′-phosphodiesterase (5′-PDE), an enzyme that can form flavor enhancing 5′-nucleotides. In this work, 5′-PDE from adzuki bean is purified. Acetone precipitation (1 crude extract:1.25 cold acetone) is used as the first purification step as it is commonly used in enzyme purification to reduce unwanted proteins, before advancing into an application of the Fast Protein Liquid Chromatography system (FPLC). Three major techniques of purification were carried out in sequence as follows: desalting (using a Hi Prep 26/10 desalting column), anion exchange chromatography (using a Hi-Trap DEAE Fast Flow column), and gel filtration chromatography (using a Superose 12 10/300 GL column). The purification protocol adopted yielded 13.37% 5′-PDE with a purification fold of 6.7 and specific activity of 35.2 µmol p-nitrophenol/min/mg protein. The molecular weight of adzuki bean 5′-PDE was estimated using gel filtration chromatography by comparing the fraction that contained the highest 5′-PDE activity with the molecular weights of standard proteins (1.35–669 kDa). Native PAGE was conducted to determine the homogeneity of proteins in the gel filtration fraction showed one band. SDS-PAGE analysis indicated that the enzyme was composed of two identical polypeptide chains (subunits), each with a molecular weight of 62 kDa. Therefore, the estimated molecular weight of the native protein using SDS-PAGE was 124 kDa.

Role of phosphodiesterases in the development of takotsubo syndrome

Abstract Background/Purpose Takotsubo syndrome (TTS) is characterized by acute transient left ventricular dysfunction in the absence of obstructive coronary lesions. We identified a higher sensitivity to catecholamine-induced stress toxicity as mechanism associated with the TTS phenotype in our former study, but the pathogenesis of TTS is still not completely understood. In this study our aim was to prove the hypothesis of an altered phosphodiesterase (PDE)-dependent 3',5'-cyclic adenosine monophosphate (cAMP)-signaling in TTS in patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Methods and results We generated functional TTS-iPSC-CMs and treated them with catecholamines to mimic a TTS-phenotype. To directly address the hypothesis that local cAMP dynamics might be altered in TTS, we used Förster resonance energy transfer (FRET) based cAMP sensors, which are specifically located in the cytosol or at the sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA) micro domain. We demonstrated that β-adrenergic receptor (β-AR) stimulations resulted in stronger cytosolic FRET responses in TTS-CMs compared to controls. In contrast, no differences of cAMP level were observed in the SERCA-PLN micro domain between TTS- and control-iPSC-CMs. To analyze the interplay of β-AR signaling and specific PDE contribution to the cAMP signaling in TTS, specific PDE-inhibitors were used. We were able to show in the cytosol that after β-AR stimulation, the strong effects of the PDE4 family of control cells were significantly decreased in diseased TTS CMs, which is in line with previously described reduced PDE4 activity in failing mouse hearts. In contrast, the contribution of PDE3 to cytoplasmic cAMP degradation was increased in TTS (Figure 1 A). This is in line with increased PDE3A and down-regulated PDE4D protein expression in TTS-iPSC-CMs compared to control cells. Analysis of PDE-dependent cAMP level in the SERCA micro domain show also a significantly reduced PDE4 activity. But the dynamic cytosolic PDE contribution of PDE2 and PDE3 after catecholamine treatment in TTS is lost in SERCA micro domain (Figure1B). Conclusion Our data showed for the first time alterations of local cAMP signaling in healthy and diseased TTS-iPSC-CMs. We demonstrated an isozym shift from PDE4 in control to PDE3 and PDE2 in TTS and identified PDE4 as an important player in the β-adrenergic cAMP signaling in TTS. Therefore, PDE4 activators may be a possible new therapeutic target option in the treatment of TTS. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): DZHK

Age-Dependent Maturation of iPSC-CMs Leads to the Enhanced Compartmentation of β2AR-cAMP Signalling.

The ability to differentiate induced-pluripotent stem cells into cardiomyocytes (iPSC-CMs) has opened up novel avenues for potential cardiac therapies. However, iPSC-CMs exhibit a range of somewhat immature functional properties. This study explored the development of the beta-adrenergic receptor (βAR) pathway, which is crucial in regulating contraction and signifying the health and maturity of myocytes. We explored the compartmentation of β2AR-signalling and phosphodiesterases (PDEs) in caveolae, as functional nanodomains supporting the mature phenotype. Förster Resonance Energy Transfer (FRET) microscopy was used to study the cyclic adenosine monophosphate (cAMP) levels in iPSC-CMs at day 30, 60, and 90 following βAR subtype-specific stimulation. Subsequently, the PDE2, PDE3, and PDE4 activity was investigated using specific inhibitors. Cells were treated with methyl-β-cyclodextrin (MβCD) to remove cholesterol as a method of decompartmentalising β2AR. As iPSC-CMs mature with a prolonged culture time, the caveolae density is increased, leading to a reduction in the overall cytoplasmic cAMP signal stimulated through β2AR (but not β1AR). Pan-phosphodiesterase inhibition or caveolae depletion leads to an increase in the β2AR-stimulated cytoplasmic cAMP. Moreover, with time in culture, the increase in the βAR-dependent cytoplasmic cAMP becomes more sensitive to cholesterol removal. The regulation of the β2AR response by PDE2 and 4 is similarly increased with the time in culture. We conclude that both the β2AR and PDEs are restricted to the caveolae nanodomains, and thereby exhibit a tighter spatial restriction over the cAMP signal in late-stage compared to early iPSC-CMs.

PDE4 regulates cardiac pacemaker function

Numerous epidemiological and clinical studies have revealed a positive correlation between heart rate (HR) and cardiovascular morbimortality. The autonomic nervous system is the major extracardiac determinant of HR. During sympathetic stimulation, the activation of β-adrenergic receptors (βAR) induces an increase in cAMP levels, leading to positive chronotropic effect. Among the 5 cardiac cAMP-PDE families, PDE4 is critical for controlling excitation-contraction coupling (ECC) during β-stimulation in atrial and ventricular cells. PDE4 may also be important for automaticity. 3 genes encode for PDE4: pde4a,4b,4d. Their respective contribution to the regulation of pacemaker activity remains unknown. Total PDE activity was determined in mouse sinoatrial node (SAN) tissue as the cAMP-hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor Ro-201724. The in vitro pacemaker activity was assessed by measuring spontaneous Ca2+ transients in Fluo4-loaded-SAN tissue. Images were obtained using confocal microscopy. Ro-20-1724 increased beating rate of intact SAN and increased PKA-phosphorylation of key ECC actors (ryanodine receptor, phospholamban and contractile proteins). PDE4 activity was found to account for 60% of total cAMP-PDE activity in SAN ( n = 3). PDE4A, 4B and 4D isoforms were found to be expressed in mouse SAN ( n = 5 independent experiments). In 4D-, but not in 4BKO mice, Ca2+ homeostasis was altered in control conditions (ctrl) and after β-stimulation with isoprenaline (iso). Indeed, ablation of PDE4D induced decreased beating rate (ctrl: 1.00 ± 0.08s-1 vs. 1.57 ± 0.05s-1; iso: 1.71 ± 0.17s-1 vs. 2.39 ± 0.08s-1, P < 0.0001) and increased Ca2+ spark frequency (ctrl: 15.9 ± 5.2 vs. 1.9 ± 0.4sparks/s/100 μm; iso: 22.9 ± 7.1 vs. 0.6 ± 0.2sparks/s/100 μm, P < 0.0001) ( Fig. 1 ). PDE4 controls pacemaker function in mice and PDE4D ablation strongly perturbs normal SAN activity.

Fibroblast growth factor 23 decreases PDE4 expression in heart increasing the risk of cardiac arrhythmia; Klotho opposes these effects.

The concentration of fibroblast growth factor 23 (FGF23) rises progressively in renal failure (RF). High FGF23 concentrations have been consistently associated with adverse cardiovascular outcomes or death, in chronic kidney disease (CKD), heart failure or liver cirrhosis. We identified the mechanisms whereby high concentrations of FGF23 can increase the risk of death of cardiovascular origin. We studied the effects of FGF23 and Klotho in adult rat ventricular cardiomyocytes (ARVMs) and on the heart of mice with CKD. We show that FGF23 increases the frequency of spontaneous calcium waves (SCWs), a marker of cardiomyocyte arrhythmogenicity, in ARVMs. FGF23 increased sarcoplasmic reticulum Ca2+ leakage, basal phosphorylation of Ca2+-cycling proteins including phospholamban and ryanodine receptor type 2. These effects are secondary to a decrease in phosphodiesterase 4B (PDE4B) in ARVMs and in heart of mice with RF. Soluble Klotho, a circulating form of the FGF23 receptor, prevents FGF23 effects on ARVMs by increasing PDE3A and PDE3B expression. Our results suggest that the combination of high FGF23 and low sKlotho concentrations decreases PDE activity in ARVMs, which favors the occurrence of ventricular arrhythmias and may participate in the high death rate observed in patients with CKD.

Activation of SIK1 by phanginin A inhibits hepatic gluconeogenesis by increasing PDE4 activity and suppressing the cAMP signaling pathway

ObjectiveSalt-induced kinase 1 (SIK1) acts as a key modulator in many physiological processes. However, the effects of SIK1 on gluconeogenesis and the underlying mechanisms have not been fully elucidated. In this study, we found that a natural compound phanginin A could activate SIK1 and further inhibit gluconeogenesis. The mechanisms by which phanginin A activates SIK1 and inhibits gluconeogenesis were explored in primary mouse hepatocytes, and the effects of phanginin A on glucose homeostasis were investigated in ob/ob mice. MethodsThe effects of phanginin A on gluconeogenesis and SIK1 phosphorylation were examined in primary mouse hepatocytes. Pan-SIK inhibitor and siRNA-mediated knockdown were used to elucidate the involvement of SIK1 activation in phanginin A-reduced gluconeogenesis. LKB1 knockdown was used to explore how phanginin A activated SIK1. SIK1 overexpression was used to evaluate its effect on gluconeogenesis, PDE4 activity, and the cAMP pathway. The acute and chronic effects of phanginin A on metabolic abnormalities were observed in ob/ob mice. ResultsPhanginin A significantly increased SIK1 phosphorylation through LKB1 and further suppressed gluconeogenesis by increasing PDE4 activity and inhibiting the cAMP/PKA/CREB pathway in primary mouse hepatocytes, and this effect was blocked by pan-SIK inhibitor HG-9-91-01 or siRNA-mediated knockdown of SIK1. Overexpression of SIK1 in hepatocytes increased PDE4 activity, reduced cAMP accumulation, and thereby inhibited gluconeogenesis. Acute treatment with phanginin A reduced gluconeogenesis in vivo, accompanied by increased SIK1 phosphorylation and PDE4 activity in the liver. Long-term treatment of phanginin A profoundly reduced blood glucose levels and improved glucose tolerance and dyslipidemia in ob/ob mice. ConclusionWe discovered an unrecognized effect of phanginin A in suppressing hepatic gluconeogenesis and revealed a novel mechanism that activation of SIK1 by phanginin A could inhibit gluconeogenesis by increasing PDE4 activity and suppressing the cAMP/PKA/CREB pathway in the liver. We also highlighted the potential value of phanginin A as a lead compound for treating type 2 diabetes.

Development of a Rapid Mass Spectrometric Determination of AMP and Cyclic AMP for PDE3 Activity Study: Application and Computational Analysis for Evaluating the Effect of a Novel 2-oxo-1,2-dihydropyridine-3-carbonitrile Derivative as PDE-3 Inhibitor.

A simple, quick, easy and cheap tandem mass spectrometry (MS/MS) method for the determination of adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP) has been newly developed. This novel MS/MS method was applied for the evaluation of the inhibitory effect of a novel 2-oxo-1,2-dihydropyridine-3-carbonitrile derivative, also named DF492, on PDE3 enzyme activity in comparison to its parent drug milrinone. Molecule DF492, with an IC50 of 409.5 nM, showed an inhibition of PDE3 greater than milrinone (IC50 = 703.1 nM). To explain the inhibitory potential of DF492, molecular docking studies toward the human PDE3A were carried out with the aim of predicting the binding mode of DF492. The presence of different bulkier decorating fragments in DF492 was pursued to shift affinity of this novel molecule toward PDE3A compared to milrinone in accordance with both the theoretical and experimental results. The described mass spectrometric approach could have a wider potential use in kinetic and biomedical studies and could be applied for the determination of other phosphodiesterase inhibitor molecules.

PD24-04 DYSREGULATED NO/PDE5 SIGNALING IN THE SICKLE CELL MOUSE LOWER URINARY TRACT: REVERSAL BY ORAL NITRATE THERAPY

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology II (PD24)1 Apr 2020PD24-04 DYSREGULATED NO/PDE5 SIGNALING IN THE SICKLE CELL MOUSE LOWER URINARY TRACT: REVERSAL BY ORAL NITRATE THERAPY Biljana Musicki, Serkan Karakus*, Uzoma Anele, Jeffrey Campbell, Sruti Shiva, Fabio Silva, and Arthur L. Burnett Biljana MusickiBiljana Musicki More articles by this author , Serkan Karakus*Serkan Karakus* More articles by this author , Uzoma AneleUzoma Anele More articles by this author , Jeffrey CampbellJeffrey Campbell More articles by this author , Sruti ShivaSruti Shiva More articles by this author , Fabio SilvaFabio Silva More articles by this author , and Arthur L. BurnettArthur L. Burnett More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000881.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Nitric oxide (NO) has a critical, but not well understood, influence in the physiology of the lower urinary tract. We evaluated the effect of NO/phosphodiesterase (PDE)5 signaling in voiding dysfunction in the sickle cell disease (SCD) mouse, characterized by low NO bioavailability. METHODS: Adult SCD (Sickle) and wild-type (WT) male mice were treated daily with sodium nitrate (10 mM) or vehicle. After 18 days, blood was obtained for nitrite measurement, urethra was collected for organ bath study, and bladder and urethra were collected for Western blot analysis of PDE5 phosphorylation (Ser-92) (activated form). Non-anesthetized mice underwent evaluation of urine volume by void spot assay. eNOS phosphorylation (Ser-1177) and nNOS phosphorylation (Ser-1412) (positive regulatory sites) were evaluated in the bladder and urethra of untreated mice. RESULTS: Sickle mice exhibited decreased eNOS, nNOS, and PDE5 phosphorylation in the bladder and urethra, decreased plasma nitrite levels, increased relaxation of phenylephrine-contracted urethral tissue to an NO donor sodium nitroprusside, and increased total urine volume, compared with WT mice. Nitrate treatment normalized plasma nitrite levels, relaxation of urethra to sodium nitroprusside, PDE5 phosphorylation in the urethra and bladder, and urine volume in Sickle mice. CONCLUSIONS: Derangement in PDE5 activity associated with basally low NO bioavailability in the bladder and urethra contributes to the molecular basis for voiding abnormalities in Sickle mice. Inorganic nitrate supplementation normalized voiding in Sickle mice through mechanisms likely involving upregulation of PDE5 activity. These findings suggest that interventions targeting dysregulatory NO/PDE5 signaling may ameliorate overactive bladder in SCD. Source of Funding: The study was supported by grant from the National Institutes of Health, USA (NIH/NIDDK grant R56DK114095 to A.L.B.) © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e533-e533 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Biljana Musicki More articles by this author Serkan Karakus* More articles by this author Uzoma Anele More articles by this author Jeffrey Campbell More articles by this author Sruti Shiva More articles by this author Fabio Silva More articles by this author Arthur L. Burnett More articles by this author Expand All Advertisement PDF downloadLoading ...

Phosphodiesterase 2 and 3 Regulate Compartmentalized Beta2-Adrenergic Receptor Camp Signaling

The two main β-adrenergic receptor (β-AR) subtypes present in cardiac myocytes, β1-AR and β2-AR, both stimulate production of the diffusible second messenger 3',5'-cyclic adenosine monophosphate (cAMP). However, the cAMP signals from the different receptor subtypes result in distinct protein kinase A (PKA) dependent functional responses. While activation of both receptors produces an increase in the force and rate of cardiac contraction, only β1-AR stimulation causes an increase in the rate of relaxation. It is hypothesized that β1-AR, but not β2-AR, produce cAMP that accesses the subcellular location where PKA-dependent phosphorylation of phospholamban occurs. Once phosphorylated, phospholamban reduces its inhibition of sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) causing an increase in the rate of cardiac relaxation. Using adult rat ventricular myocytes, this hypothesis was tested by comparing cAMP responses detected by a fluorescence resonance energy transfer (FRET) based biosensor (Epac2-camps) expressed uniformly throughout the cytoplasm of the entire cell and a FRET-based biosensor (Epac2-αKAP) targeted to the SERCA/phospholamban complex. Selective activation of β1-AR or β2-AR produced cAMP responses that could be detected by the Epac2-camps probe. However, only β1-AR selective activation produced a change in cAMP that the Epac2-αKAP probe could detect. Furthermore, selective inhibition of individual phosphodiesterases (PDEs) involved in cAMP metabolism (PDE2, PDE3, or PDE4) increased the basal cAMP detected by both probes. Yet only inhibition of PDE2 or PDE3 allowed a β2-AR cAMP signal to be detected by the Epac2-αKAP probe. These results support the conclusions that cAMP produced by β2-AR stimulation is compartmentalized and that this involves the activity of PDE2 and PDE3.

Inhibition of isoform D of phosphodiesterase type 4 reduces cell proliferation and survival in hepatocarcinoma cell lines

Cyclic AMP phosphodiesterases (PDEs) are enzymes that regulate intracellular levels of cAMP by controlling its rate of degradation, in contrast to adenylyl cyclases that synthesize cAMP from intracellular ATP. PDEs are classified in 11 families (PDE1-PDE11). Some of these control the intracellular levels of both cyclic nucleotides, while others (PDE4, 7, 8) are specific for cAMP degradation. Previous experiments showed that isoform D of cAMP specific PDE4 (PDE4D) was aberrantly up-regulated in HCC cells, in particular in the highly tumorigenic Hep3B and Huh7 cell lines, which also showed increased PDE4 activity.

Agonist-specific desensitization of PGE2-stimulated cAMP signaling due to upregulated phosphodiesterase expression in human lung fibroblasts

Pulmonary fibrosis is characterized by fibroblasts persisting in an activated form, producing excessive fibrous material that destroys alveolar structure. The second messenger molecule cyclic 3′,5′-adenosine monophosphate (cAMP) has antifibrotic properties, and prostaglandin E2 (PGE2) can stimulate cAMP production through prostaglandin E (EP)2 and EP4 receptors. Although EP receptors are attractive therapeutic targets, the effects of long-term exposure to PGE2 have not been characterized. To determine the effects of long-term exposure of lung fibroblasts to PGE2, human fetal lung (HFL)-1 cells were treated for 24 h with 100 nM PGE2 or other cAMP-elevating agents. cAMP levels stimulated by acute exposure to PGE2 were measured using a fluorescent biosensor. Pretreatment for 24 h with PGE2 shifted the concentration-response curve to PGE2 rightward by approximately 22-fold but did not affect responses to the beta-adrenoceptor agonist isoproterenol. Neither isoproterenol nor forskolin pretreatment altered PGE2 responses, implying that other cAMP-elevating agents do not induce desensitization. Use of EP2- and EP4-selective agonists and antagonists suggested that PGE2-stimulated cAMP responses in HFL-1 cells are mediated by EP2 receptors. EP2 receptors are resistant to classical mechanisms of agonist-specific receptor desensitization, so we hypothesized that increased PDE activity mediates the loss of signaling after PGE2 pretreatment. PGE2 treatment upregulated messenger RNA for PDE3A, PDE3B, PDE4B, and PDE4D and increased overall PDE activity. The PDE4 inhibitor rolipram partially reversed PGE2-mediated desensitization and PDE4 activity was increased, but rolipram did not alter responses to isoproterenol. The PDE3 inhibitor cilostazol had minimal effect. These results show that long-term exposure to PGE2 causes agonist-specific desensitization of EP2 receptor-stimulated cAMP signaling through the increased expression of PDE isozymes, most likely of the PDE4 family.

An EPAC1/PDE1C-Signaling Axis Regulates Formation of Leading-Edge Protrusion in Polarized Human Arterial Vascular Smooth Muscle Cells.

Pharmacological activation of protein kinase A (PKA) reduces migration of arterial smooth muscle cells (ASMCs), including those isolated from human arteries (HASMCs). However, when individual migration-associated cellular events, including the polarization of cells in the direction of movement or rearrangements of the actin cytoskeleton, are studied in isolation, these individual events can be either promoted or inhibited in response to PKA activation. While pharmacological inhibition or deficiency of exchange protein activated by cAMP-1 (EPAC1) reduces the overall migration of ASMCs, the impact of EPAC1 inhibition or deficiency, or of its activation, on individual migration-related events has not been investigated. Herein, we report that EPAC1 facilitates the formation of leading-edge protrusions (LEPs) in HASMCs, a critical early event in the cell polarization that underpins their migration. Thus, RNAi-mediated silencing, or the selective pharmacological inhibition, of EPAC1 decreased the formation of LEPs by these cells. Furthermore, we show that the ability of EPAC1 to promote LEP formation by migrating HASMCs is regulated by a phosphodiesterase 1C (PDE1C)-regulated “pool” of intracellular HASMC cAMP but not by those regulated by the more abundant PDE3 or PDE4 activities. Overall, our data are consistent with a role for EPAC1 in regulating the formation of LEPs by polarized HASMCs and show that PDE1C-mediated cAMP hydrolysis controls this localized event.

Natural Variation in a Dendritic Scaffold Protein Remodels Experience-Dependent Plasticity by Altering Neuropeptide Expression

SummaryThe extent to which behavior is shaped by experience varies between individuals. Genetic differences contribute to this variation, but the neural mechanisms are not understood. Here, we dissect natural variation in the behavioral flexibility of two Caenorhabditis elegans wild strains. In one strain, a memory of exposure to 21% O2 suppresses CO2-evoked locomotory arousal; in the other, CO2 evokes arousal regardless of previous O2 experience. We map that variation to a polymorphic dendritic scaffold protein, ARCP-1, expressed in sensory neurons. ARCP-1 binds the Ca2+-dependent phosphodiesterase PDE-1 and co-localizes PDE-1 with molecular sensors for CO2 at dendritic ends. Reducing ARCP-1 or PDE-1 activity promotes CO2 escape by altering neuropeptide expression in the BAG CO2 sensors. Variation in ARCP-1 alters behavioral plasticity in multiple paradigms. Our findings are reminiscent of genetic accommodation, an evolutionary process by which phenotypic flexibility in response to environmental variation is reset by genetic change.

The phosphodiesterase-4 inhibitor roflumilast impacts Schistosoma mansoni ovipositing in vitro but displays only modest antischistosomal activity in vivo

Praziquantel (PZQ) is the sole drug used to treat schistosomiasis, and the probability of developing resistance is growing the longer it is relied upon, justifying the search for alternatives. Phosphodiesterases (PDEs), particularly the PDE4 family, have attracted considerable attention as drug targets, including in Schistosoma mansoni, and especially SmPDE4A. This study investigates the potential antischistosomal activity of human PDE4 and potent SmPDE4A inhibitor roflumilast, either alone or combined with PZQ. In vitro, roflumilast resulted in a significant, concentration-dependent reduction in egg production but not of worm viability. In vitro exposure to roflumilast in combination with a low concentration of PZQ was less effective than PZQ alone, pointing to antagonism. S. mansoni-infected mice treated with roflumilast showed significant reductions in worm burden (27%) as well as hepatic and intestinal egg burdens (~28%) two weeks post treatment. Scanning EM of worms isolated from roflumilast-treated and untreated mice did not reveal noticeable changes to their tegument. S. mansoni-infected mice treated with a fixed dosage of roflumilast and a variable dosage of PZQ resulted in a higher reduction in worm burden, reduced hepatic egg counts, absence of immature eggs and a marked increase in dead eggs, compared to PZQ alone. However, the combination resulted in increased animal mortality, probably attributable to pharmacodynamic interactions between the two drugs. Although this study marks the first report of in vivo antischistosomal potential by a PDE inhibitor, an important proof of concept, we conclude that the antischistosomal effects of roflumilast are insufficient to warrant further development.

Dysregulated NO/PDE5 signaling in the sickle cell mouse lower urinary tract: Reversal by oral nitrate therapy

AimsNitric oxide (NO) has a critical, but not well understood, influence in the physiology of the lower urinary tract. We evaluated the effect of NO/phosphodiesterase (PDE)5 signaling in voiding dysfunction in the sickle cell disease (SCD) mouse, characterized by low NO bioavailability. Main methodsAdult SCD (Sickle) and wild-type (WT) male mice were treated daily with sodium nitrate (10 mM) or vehicle. After 18 days, blood was obtained for nitrite measurement, urethra was collected for organ bath study, and bladder and urethra were collected for Western blot analysis of PDE5 phosphorylation (Ser-92) (activated form). Non-anesthetized mice underwent evaluation of urine volume by void spot assay. eNOS phosphorylation (Ser-1177) and nNOS phosphorylation (Ser-1412) (positive regulatory sites) were evaluated in the bladder and urethra of untreated mice. Key findingsSickle mice exhibited decreased eNOS, nNOS, and PDE5 phosphorylation in the bladder and urethra, decreased plasma nitrite levels, increased relaxation of phenylephrine-contracted urethral tissue to an NO donor sodium nitroprusside, and increased total urine volume, compared with WT mice. Nitrate treatment normalized plasma nitrite levels, relaxation of urethra to sodium nitroprusside, PDE5 phosphorylation in the urethra and bladder, and urine volume in Sickle mice. SignificanceDerangement in PDE5 activity associated with basally low NO bioavailability in the bladder and urethra contributes to the molecular basis for voiding abnormalities in Sickle mice. Inorganic nitrate supplementation normalized voiding in Sickle mice through mechanisms likely involving upregulation of PDE5 activity. These findings suggest that interventions targeting dysregulatory NO/PDE5 signaling may ameliorate overactive bladder in SCD.

P2558PDE4 regulates cardiac pacemaker function

Abstract Background Numerous epidemiological and clinical studies have revealed a positive correlation between heart rate (HR) and cardiovascular morbimortality. The autonomic nervous system is the major extracardiac determinant of HR. During sympathetic stimulation, the activation of β-adrenergic receptors (βAR) induces an increase in cAMP levels, leading to positive chronotropic effect. Among the 5 cardiac cAMP-PDE families, PDE4 is critical for controlling excitation-contraction coupling (ECC) during βAR stimulation in atrial and ventricular cells. PDE4 may also be important for automaticity. 3 genes encode for PDE4s: pde4a, pde4b, pde4d. Their respective contribution to the regulation of pacemaker activity remains ill-defined. Purpose Define the role of PDE4 isoforms in the regulation of cardiac pacemaker activity Methods Total PDE activity was determined in mouse sinoatrial node (SAN) tissue as the cAMP-hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor Ro-20-1724. The in vitro pacemaker activity was assessed by measuring spontaneous Ca2+ transients in Fluo4-loaded-SAN tissue. Images were obtained using confocal microscopy. Results Ro-20-1724 increased beating rate of intact SAN and increased PKA-phosphorylation of key ECC actors (ryanodine receptor, phospholamban and contractile proteins). PDE4 activity was found to account for 60% of the total cAMP-PDE activity in SAN (n=3 independent experiments). PDE4A, PDE4B and PDE4D isoforms were found to be expressed in mouse SAN (n=5 independent experiments). In PDE4D-, but not in PDE4B-deficient mice, Ca2+ homeostasis was altered in control conditions (ctrl) and after βAR stimulation with isoprenaline (iso). Indeed, ablation of PDE4D induced decreased beating rate (ctrl: 1.00±0.08 s–1 vs 1.57±0.05 s–1; iso: 1.71±0.17 s–1 vs 2.39±0.08 s–1, p&lt;0.0001) and increased Ca2+ spark frequency (ctrl: 15.9±5.2 sparks/s/100 μm vs 1.9±0.4 sparks/s/100 μm; iso: 22.9±7.1 sparks/s/100 μm vs 0.6±0.2 sparks/s/100 μm, p&lt;0.0001) (Figure). Calcium Homeostasis in SAN cells Conclusion PDE4 controls pacemaker function in mice and PDE4D ablation strongly perturbs normal SAN activity. Acknowledgement/Funding ANR, Fondation Lefoulon Delalande, CORDDIM

3074Analyzing the regulation of a catecholamine-dependent altered cAMP signaling in a patient-specific induced pluripotent stem cell takotsubo-model

Abstract Background/Purpose Takotsubo syndrome (TTS) is characterized by acute transient left ventricular dysfunction in the absence of obstructive coronary lesions. Although, we identified an enhanced β-adrenergic signaling and higher sensitivity to catecholamine-induced stress toxicity as mechanisms associated with the TTS phenotype in our former study, the pathogenesis of TTS is still not completely understood. Here, we aimed to prove the hypothesis of a phosphodiesterase (PDE)-dependent regulation of 3',5'-cyclic adenosine monophosphate (cAMP) signaling in TTS under catecholamine stress. Methods and results We generated functional TTS induced pluripotent stem cell-derived cardiomyocytes (TTS-iPSC-CMs) from 6 patients and treated the cells with catecholamines to mimic a TTS-phenotype. Using a cytosolic Förster resonance energy transfer (FRET) based cAMP sensor, we could observe that β-adrenergic receptor (β-AR) stimulations led to stronger FRET responses in the cytosol of TTS-CMs as compared to controls. Besides β-ARs, PDEs are main players involved in cAMP signaling in CMs. At basal level TTS-CM show a significantly higher PDE3A and a reduced PDE4D protein expression in the TTS-CMs compared to control. In addition, FRET experiments show that after β-AR stimulation, the strong effects of the PDE4 family in the cytosol of control cells were significantly decreased in TTS-CMs. This is in line with previously described reduced PDE4 activity in failing mouse hearts. By analyzing PDE-dependent cAMP downstream effects as PKA-dependent phosphorylation, we could show an additional increase of PLN phosphorylation (PLN-S16), especially in control, when treating iPSC-CMs with a combination of iso and PDE4 inhibitor. In contrast, in TTS-iPSC-CMs the contribution of the PDE-families PDE2, 3 or 4 to phosphorylation of PLN-S16 was increased over iso alone. This suggests that different PDEs in TTS and control are involved in functional segregation of the SERCA2a microdomain from the cytosol in terms of cAMP downstream effects. To directly address the hypothesis that local cAMP dynamics might be altered in TTS, we used a SERCA micro domain targeted FRET based cAMP sensor. In contrast to the cytosolic cAMP regulation, the PDE4 inhibitor effects in the SERCA2 micro domain were only slightly decreased in TTS. Instead, the contribution of PDE2 to local cAMP degradation was slightly increased. Conclusion Our data show for the first time alterations of local cAMP signaling in healthy and diseased TTS-iPSC-CMs. TTS leads to changes in PDE composition in the cytosol but not significantly in SERCA microdomain. Our results uncover a PDE-dependent altered β-adrenergic signaling as a potential disease cause. This data highlight that TTS-iPSC-CMs can be used to provide a versatile tool for evaluating new treatment options for TTS as therapeutic targets.

Detrimental Effects of an Inhaled Phosphodiesterase-4 Inhibitor on Lung Inflammation in Ventilated Preterm Lambs Exposed to Chorioamnionitis Are Dose Dependent.

Background: Treatment of bronchopulmonary dysplasia in preterm infants is challenging due to its multifactorial origin. In rodent models of neonatal lung injury, selective inhibition of phosphodiesterase 4 (PDE4) has been shown to exert anti-inflammatory properties in the lung. We hypothesized that GSK256066, a highly selective, inhalable PDE4 inhibitor, would have beneficial effects on lung injury and inflammation in a triple hit lamb model of Ureaplasma parvum (UP)-induced chorioamnionitis, prematurity, and mechanical ventilation. Methods: Twenty-one preterm lambs were surgically delivered preterm at 129 days after 7 days intrauterine exposure to UP. Sixteen animals were subsequently ventilated for 24 hours and received endotracheal surfactant and intravenous caffeine citrate. Ten animals were randomized to receive twice a high (10 μg/kg) or low dose (1 μg/kg) of nebulized PDE4 inhibitor. Results: Nebulization of high, but not low, doses of PDE4 inhibitor led to a significant decrease in pulmonary PDE activity, and was associated with lung injury and vasculitis, influx of neutrophils, and increased proinflammatory cytokine messenger RNA levels. Conclusion: Contrary to our hypothesis, we found in our model a dose-dependent proinflammatory effect of an inhaled highly selective PDE4 inhibitor in the lung. Our findings indicate the narrow therapeutic range of inhaled PDE4 inhibitors in the preterm population.