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Marco Conti - One of the best experts on this subject based on the ideXlab platform.
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atropine augments cardiac contractility by inhibiting camp specific phosphodiesterase type 4
Scientific Reports, 2017Co-Authors: Ruwan K Perera, Michael Wagner, Matthias Dewenter, Christiane Vettel, Nadja I Bork, Lars S Maier, Marco Conti, Thomas H. Fischer, Julius WessAbstract:Atropine is a clinically relevant anticholinergic drug, which blocks inhibitory effects of the parasympathetic neurotransmitter acetylcholine on heart rate leading to tachycardia. However, many cardiac effects of atropine cannot be adequately explained solely by its antagonism at muscarinic receptors. In isolated mouse ventricular cardiomyocytes expressing a Forster resonance energy transfer (FRET)-based cAMP biosensor, we confirmed that atropine inhibited acetylcholine-induced decreases in cAMP. Unexpectedly, even in the absence of acetylcholine, after G-protein inactivation with pertussis toxin or in myocytes from M2- or M1/3-muscarinic receptor knockout mice, atropine increased cAMP levels that were pre-elevated with the β-adrenergic agonist isoproterenol. Using the FRET approach and in vitro phosphodiesterase (PDE) activity assays, we show that atropine acts as an allosteric PDE type 4 (PDE4) inhibitor. In human atrial myocardium and in both intact wildtype and M2 or M1/3-receptor knockout mouse Langendorff hearts, atropine led to increased contractility and heart rates, respectively. In vivo, the atropine-dependent prolongation of heart rate increase was blunted in PDE4D but not in wildtype or PDE4B knockout mice. We propose that inhibition of PDE4 by atropine accounts, at least in part, for the induction of tachycardia and the arrhythmogenic potency of this drug.
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pde4d phosphorylation a coincidence detector integrating multiple signaling pathways
Cellular Signalling, 2016Co-Authors: Delphine Mika, Marco ContiAbstract:In Eukaryotes, more than 100 different phosphodiesterase (PDE) proteins serve to fine-tune cyclic nucleotide (cAMP and cGMP) signals and contribute to specificity of signaling. In mammals, PDEs are divided into 11 families, of which PDE4 represents the largest family. Four genes (pde4a, pde4b, PDE4C and pde4d) encode for this class of enzymes in mammals and give rise to more than 20 variants. Within this family of genes, PDE4D was discovered on the basis of its regulatory properties and its induction by hormones and cAMP. PDE4D has often been used as the prototype PDE4 and large body of work has been generated on the biochemical, pharmacological, and physiological properties of this enzyme. This review covers the regulation of PDE4D by phosphorylation, the impact of this regulation in the context of the structure of this protein, and the functional consequences of this complex pattern of posttranslational modifications.
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mice deficient in phosphodiesterase 4a display anxiogenic like behavior
Psychopharmacology, 2014Co-Authors: Rolf T Hansen, Marco Conti, Hanting ZhangAbstract:Rationale Phosphodiesterases (PDEs) are a super family of enzymes responsible for the halting of intracellular cyclic nucleotide signaling and may represent novel therapeutic targets for treatment of cognitive disorders. PDE4 is of considerable interest to cognitive research because it is highly expressed in the brain, particularly in the cognition-related brain regions. Recently, the functional role of PDE4B and PDE4D, two of the four PDE4 subtypes (PDE4A, B, C, and D), in behavior has begun to be identified; however, the role of PDE4A in the regulation of behavior is still unknown.
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pde4d and pde4b function in distinct subcellular compartments in mouse embryonic fibroblasts
Journal of Biological Chemistry, 2011Co-Authors: Brigitte E Blackman, Wito Richter, Kathleen Horner, Julia D Heidmann, Thomas C. Rich, Dan Wang, Marco ContiAbstract:Signaling through cAMP regulates most cellular functions. The spatiotemporal control of cAMP is, therefore, crucial for differential regulation of specific cellular targets. Here we investigated the consequences of PDE4B or PDE4D gene ablation on cAMP signaling at a subcellular level using mouse embryonic fibroblasts. PDE4B ablation had no effect on the global or bulk cytosol accumulation of cAMP but increased both basal and hormone-dependent cAMP in a near-membrane pool. Conversely, PDE4D ablation enhanced agonist-induced cAMP accumulation in the bulk cytosol as well as at the plasma membrane. Both PDE4B and PDE4D ablation significantly modified the time course and the level of isoproterenol-induced phosphorylation of vasodilator-stimulated phosphoprotein, a membrane cytoskeletal component. A second membrane response through Toll-like receptor signaling, however, was only affected by PDE4B ablation. PDE4D but not PDE4B ablation significantly prolonged cAMP-response element-binding protein-mediated transcription. These findings demonstrate that PDE4D and PDE4B have specialized functions in mouse embryonic fibroblasts with PDE4B controlling cAMP in a discrete subdomain near the plasma membrane.
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phosphodiesterase 4d knock out and rna interference mediated knock down enhance memory and increase hippocampal neurogenesis via increased camp signaling
The Journal of Neuroscience, 2011Co-Authors: Yufang Cheng, James M Odonnell, Marco Conti, Ying Huang, Steven P Wilson, Hanting ZhangAbstract:Phosphodiesterase-4 (PDE4) plays an important role in mediating memory via the control of intracellular cAMP signaling; inhibition of PDE4 enhances memory. However, development of PDE4 inhibitors as memory enhancers has been hampered by their major side effect of emesis. PDE4 has four subtypes (PDE4A-D) consisting of 25 splice variants. Mice deficient in PDE4D displayed memory enhancement in radial arm maze, water maze, and object recognition tests. These effects were mimicked by repeated treatment with rolipram in wild-type mice. In addition, similarly as rolipram-treated wild-type mice, PDE4D-deficient mice also displayed increased hippocampal neurogenesis and phosphorylated cAMP response element-binding protein (pCREB). Furthermore, microinfusion of lentiviral vectors that contained microRNAs (miRNAs) targeting long-form PDE4D isoforms into bilateral dentate gyri of the mouse hippocampus downregulated PDE4D4 and PDE4D5, enhanced memory, and increased hippocampal neurogenesis and pCREB. Finally, while rolipram and PDE4D deficiency shortened α2 adrenergic receptor-mediated anesthesia, a surrogate measure of emesis, miRNA-mediated PDE4D knock-down in the hippocampus did not. The present results suggest that PDE4D, in particular long-form PDE4D, plays a critical role in the mediation of memory and hippocampal neurogenesis, which are mediated by cAMP/CREB signaling; reduced expression of PDE4D, or at least PDE4D4 and PDE4D5, in the hippocampus enhances memory but appears not to cause emesis. These novel findings will aid in the development of PDE4 subtype- or variant-selective inhibitors for treatment of disorders involving impaired cognition, including Alzheimer's disease.
Claire Lugnier - One of the best experts on this subject based on the ideXlab platform.
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Disease Progression in MRL/lpr Lupus-Prone Mice Is Reduced by NCS 613, a Specific Cyclic Nucleotide Phosphodiesterase Type 4 (PDE4) Inhibitor
2016Co-Authors: Fanny Monneaux, Lucien Gazi, Jean-jacques Bourguignon, Sylviane Muller, Claire LugnierAbstract:Systemic lupus erythematosus is a polymorphic and multigenic inflammatory autoimmune disease. Cyclic AMP (cAMP) modulates inflammation and the inhibition of cyclic nucleotide phosphodiesterase type 4 (PDE4), which specifically hydrolyzes cAMP, inhibits TNFa secretion. This study was aimed at investigating the evolution of PDE activity and expression levels during the course of the disease in MRL/lpr lupus-prone mice, and to evaluate in these mice the biological and clinical effects of treatments with pentoxifylline, denbufylline and NCS 613 PDE inhibitors. This study reveals that compared to CBA/ J control mice, kidney PDE4 activity of MRL/lpr mice increases with the disease progression. Furthermore, it showed that the most potent and selective PDE4 inhibitor NCS 613 is also the most effective molecule in decreasing proteinuria and increasing survival rate of MRL/lpr mice. NCS 613 is a potent inhibitor, which is more selective for the PDE4C subtyp
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Disease progression in MRL/lpr lupus-prone mice is reduced by NCS 613, a specific cyclic nucleotide phosphodiesterase type 4 (PDE4) inhibitor.
PloS one, 2012Co-Authors: Thérèse Keravis, Issaka Yougbaré, Fanny Monneaux, Lucien Gazi, Jean-jacques Bourguignon, Sylviane Muller, Claire LugnierAbstract:Systemic lupus erythematosus is a polymorphic and multigenic inflammatory autoimmune disease. Cyclic AMP (cAMP) modulates inflammation and the inhibition of cyclic nucleotide phosphodiesterase type 4 (PDE4), which specifically hydrolyzes cAMP, inhibits TNFα secretion. This study was aimed at investigating the evolution of PDE activity and expression levels during the course of the disease in MRL/lpr lupus-prone mice, and to evaluate in these mice the biological and clinical effects of treatments with pentoxifylline, denbufylline and NCS 613 PDE inhibitors. This study reveals that compared to CBA/J control mice, kidney PDE4 activity of MRL/lpr mice increases with the disease progression. Furthermore, it showed that the most potent and selective PDE4 inhibitor NCS 613 is also the most effective molecule in decreasing proteinuria and increasing survival rate of MRL/lpr mice. NCS 613 is a potent inhibitor, which is more selective for the PDE4C subtype (IC50 = 1.4 nM) than the other subtypes (PDE4A, IC50 = 44 nM; PDE4B, IC50 = 48 nM; and PDE4D, IC50 = 14 nM). Interestingly, its affinity for the High Affinity Rolipram Binding Site is relatively low (Ki = 148 nM) in comparison to rolipram (Ki = 3 nM). Finally, as also observed using MRL/lpr peripheral blood lymphocytes (PBLs), NCS 613 inhibits basal and LPS-induced TNFα secretion from PBLs of lupus patients, suggesting a therapeutic potential of NCS 613 in systemic lupus. This study reveals that PDE4 represent a potential therapeutic target in lupus disease.
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decreased expression and activity of camp phosphodiesterases in cardiac hypertrophy and its impact on β adrenergic camp signals
Circulation Research, 2009Co-Authors: Aniella Abigerges, Wito Richter, Marco Conti, Claire Lugnier, Florence Lefebvre, Philippe Mateo, Audrey Varin, Christophe Heymes, Janelise Samuel, Rodolphe FischmeisterAbstract:Rationale: Multiple cyclic nucleotide phosphodiesterases (PDEs) degrade cAMP in cardiomyocytes but the role of PDEs in controlling cAMP signaling during pathological cardiac hypertrophy is poorly defined. Objective: Evaluate the -adrenergic regulation of cardiac contractility and characterize the changes in cardiomyocyte cAMP signals and cAMP-PDE expression and activity following cardiac hypertrophy. Methods and Results: Cardiac hypertrophy was induced in rats by thoracic aortic banding over a time period of 5 weeks and was confirmed by anatomic measurements and echocardiography. Ex vivo myocardial function was evaluated in Langendorff-perfused hearts. Engineered cyclic nucleotide-gated (CNG) channels were expressed in single cardiomyocytes to monitor subsarcolemmal cAMP using whole-cell patch-clamp recordings of the associated CNG current (ICNG). PDE variant activity and protein level were determined in purified cardiomyocytes. Aortic stenosis rats exhibited a 67% increase in heart weight compared to sham-operated animals. The inotropic response to maximal -adrenergic stimulation was reduced by 54% in isolated hypertrophied hearts, along with a 32% decrease in subsarcolemmal cAMP levels in hypertrophied myocytes. Total cAMP hydrolytic activity as well as PDE3 and PDE4 activities were reduced in hypertrophied myocytes, because of a reduction of PDE3A, PDE4A, and PDE4B, whereas PDE4D was unchanged. Regulation of -adrenergic cAMP signals by PDEs was blunted in hypertrophied myocytes, as demonstrated by the diminished effects of IBMX (100 mol/L) and of both the PDE3 inhibitor cilostamide (1 mol/L) and the PDE4 inhibitor Ro 201724 (10 mol/L). Conclusions: -Adrenergic desensitization is accompanied by a reduction in cAMP-PDE and an altered modulation of -adrenergic cAMP signals in cardiac hypertrophy. (Circ Res. 2009;105:784-792.)
M D Houslay - One of the best experts on this subject based on the ideXlab platform.
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isoform selective susceptibility of disc1 phosphodiesterase 4 complexes to dissociation by elevated intracellular camp levels
The Journal of Neuroscience, 2007Co-Authors: Hannah Murdoch, David J. Porteous, Kirsty J Millar, Shaun Mackie, Daniel M Collins, Elaine V Hill, Graeme B Bolger, Enno Klussmann, M D HouslayAbstract:Disrupted-in-schizophrenia 1 (DISC1) is a genetic susceptibility factor for schizophrenia and related severe psychiatric conditions. DISC1 is a multifunctional scaffold protein that is able to interact with several proteins, including the independently identified schizophrenia risk factor phosphodiesterase-4B (PDE4B). Here we report that the 100 kDa full-length DISC1 isoform (fl-DISC1) can bind members of each of the four gene, cAMP-specific PDE4 family. Elevation of intracellular cAMP levels, so as to activate protein kinase A, caused the release of PDE4D3 and PDE4C2 isoforms from fl-DISC1 while not affecting binding of PDE4B1 and PDE4A5 isoforms. Using a peptide array strategy, we show that PDE4D3 binds fl-DISC1 through two regions found in common with PDE4B isoforms, the interaction of which is supplemented because of the presence of additional PDE4B-specific binding sites. We propose that the additional binding sites found in PDE4B1 underpin its resistance to release during cAMP elevation. We identify, for the first time, a functional distinction between the 100 kDa long DISC1 isoform and the short 71 kDa isoform. Thus, changes in the expression pattern of DISC1 and PDE4 isoforms offers a means to reprogram their interaction and to determine whether the PDE4 sequestered by DISC1 is released after cAMP elevation. The PDE4B-specific binding sites encompass point mutations in mouse Disc1 that confer phenotypes related to schizophrenia and depression and that affect binding to PDE4B. Thus, genetic variation in DISC1 and PDE4 that influence either isoform expression or docking site functioning may directly affect psychopathology.
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long pde4 camp specific phosphodiesterases are activated by protein kinase a mediated phosphorylation of a single serine residue in upstream conserved region 1 ucr1
British Journal of Pharmacology, 2002Co-Authors: Simon J Mackenzie, George S Baillie, Ian Mcphee, Carolynn Mackenzie, Rachael Seamons, Theresa Mcsorley, Jenni Millen, Matthew B Beard, Gino Van Heeke, M D HouslayAbstract:Challenge of COS1 cells with the adenylyl cyclase activator forskolin led to the activation of recombinant PDE4A8, PDE4B1, PDE4C2 and PDE4D5 cAMP-specific phosphodiesterase long isoforms. Forskolin challenge did not activate mutant long PDE4 isoforms where the serine target residue (STR) within the protein kinase A (PKA) consensus phosphorylation site in Upstream Conserved Region 1 (UCR1) was mutated to alanine. The PKA inhibitor, H89, ablated forskolin activation of wild-type long PDE4 isoforms. Activated PKA caused the in vitro phosphorylation of recombinant wild-type long PDE4 isoforms, but not those where the STR was mutated to alanine. An antiserum specific for the phosphorylated form of the STR detected a single immunoreactive band for recombinant long PDE4 isoforms expressed in COS1 cells challenged with forskolin. This was not evident in forskolin-challenged cells treated with H89. Neither was it evident in forskolin-challenged cells expressing long isoforms where the STR had been mutated to alanine. In transfected COS cells challenged with forskolin, only the phosphorylated PDE4D3 long form showed a decrease in mobility in Western blotting analysis. This decreased mobility of PDE4D3 was ablated upon mutation of either of the two serine targets for PKA phosphorylation in this isoform, namely Ser54 in UCR1 and Ser13 in the isoform-specific N-terminal region. Activation by forskolin challenge did not markedly alter the sensitivity of PDE4A8, PDE4B1, PDE4C2 and PDE4D5 to inhibition by rolipram. Long PDE4 isoforms from all four sub-families can be phosphorylated by protein kinase A (PKA). This leads to an increase in their activity and may thus contribute to cellular desensitization processes in cells where these isoforms are selectively expressed. Keywords: PDE4 cAMP phosphodiesterase, rolipram, phosphorylation, PKA, protein kinase A Introduction cAMP is a ubiquitous second messenger that is pivotal in controlling a wide variety of cellular functions (Houslay & Milligan, 1997). The only known means of degrading cAMP is through the action of cyclic nucleotide phosphodiesterases. It is now well appreciated that a large multigene family of enzymes exhibits the ability to hydrolyse cAMP (Beavo, 1995; Conti & Jin, 1999; Houslay, 2001; Manganiello et al., 1995a, 1995b; Soderling & Beavo, 2000; Thompson, 1991). Recently, however, the use of selective inhibitors has demonstrated the importance of the PDE4 cAMP-specific phosphodiesterase family in controlling inflammatory responses, depression and cognitive function (Barnette, 1999; Bolger, 1994; Cavalla & Frith, 1995; Giembycz, 2000; He et al., 1998; Houslay, 2001; Houslay et al., 1998; Rogers & Giembycz, 1998; Schudt et al., 1995; Souness & Rao, 1997; Spina et al., 1998; Torphy, 1998). Such discoveries have led to the development of PDE4-selective inhibitors as potential therapeutic agents in a variety of inflammatory disease states. Notwithstanding this, it is only recently that we are beginning to understand the complex regulatory mechanisms that control the functioning of the large number of PDE4 isoforms that have been recognized to date (Conti & Jin, 1999; Houslay, 2001; Houslay et al., 1998). PDE4 enzymes are encoded by four genes (PDE4A, PDE4B, PDE4C and PDE4D), each capable of producing a number of isoforms through alternative mRNA splicing and the use of alternative promoters (Conti & Jin, 1999; Houslay, 2001; Houslay et al., 1998). Each PDE4 isoform within a particular PDE4 sub-family possesses a common core region, which consists of the catalytic unit and the C-terminal portion and is defined by its unique extreme N-terminal region. The various PDE4 isoforms are classified further as being either ‘long' or ‘short' isoforms. This classification relates to the presence or absence of two highly conserved sequences that are unique to the PDE4 enzyme family (Bolger, 1994; Bolger et al., 1993). These are the 55 amino acid Upstream Conserved Region 1 (UCR1) and the 76 amino acid Upstream Conserved Region 2 (UCR2) that are both located immediately N-terminal to the catalytic unit. Thus long isoforms exhibit both UCR1 and UCR2 whilst short isoforms lack UCR1 and ‘super-short' isoforms lack UCR1 and have a truncated UCR2 (Houslay, 2001; Houslay et al., 1998). All four PDE4 gene families are known to encode long-form enzymes. N-terminal truncation analyses have led to the suggestion that UCR1 and UCR2 may influence PDE4 catalytic activity (see e.g. Conti & Jin, 1999 for review). More recent studies on the PDE4D3 long isoform have led to an appreciation that UCR1 and UCR2 interact together to form a regulatory module (Beard et al., 2000; Lim et al., 1999) that integrates the regulatory effect of phosphorylation by both protein kinase A (PKA) and by ERK MAP kinase (MacKenzie et al., 2000). Increased intracellular cAMP levels have been demonstrated to increase cellular PDE4 activity (see Houslay, 2001; Houslay et al., 1998 for reviews). This is believed to perform an adaptive role in desensitising cellular processes to increased levels of cAMP. The long-term elevation of intracellular cAMP levels has been shown to cause an increase in the levels of mRNA and protein expression for various PDE4 isoforms, in particular the PDE4D1 and PDE4D2 short forms (Erdogan & Houslay, 1997; Kovala et al., 1994; Sette et al., 1994b; Seybold et al., 1998; Swinnen et al., 1989; Verghese et al., 1995; Vicini & Conti, 1997) where a cAMP-controlled promoter has been identified (Vicini & Conti, 1997). However, a second cAMP-driven controlling mechanism that has been identified is the rapid activation of the PDE4D3 isoform that is achieved through direct PKA-mediated phosphorylation (Alvarez et al., 1995; Hoffmann et al., 1998; Sette & Conti, 1996; Sette et al., 1994a, 1994b). PKA appears to phosphorylate PDE4D3 at two sites, one being Ser13 in the isoform-specific N-terminal region and the other at Ser54 in UCR1. However, only modification of Ser54 in UCR1 is required to elicit activation (Hoffmann et al., 1998; Sette & Conti, 1996). This activation appears to contribute to short-term desensitization, at least in certain cell types (Oki et al., 2000). Such PKA-mediated phosphorylation of PDE4D3 has also been suggested to lead to an increase in the sensitivity of PDE4D3 to inhibition by the PDE4 selective inhibitor, rolipram (Alvarez et al., 1995). Additionally a decrease in mobility of PDE4D3, on SDS – PAGE, has been observed after its phosphorylation by PKA (Sette & Conti, 1996; Sette et al., 1994a). This has been suggested to be diagnostic for the presence of an activated PKA-phosphorylated enzyme (Oki et al., 2000; Sette & Conti, 1996; Sette et al., 1994a). Whilst many analyses have been done on PDE4D3 as a target for PKA-mediated phosphorylation and activation, it has been pointed out (Sette & Conti, 1996; Souness & Rao, 1997) that an identical PKA consensus sequence, namely Arg-Arg-Glu-Ser-Phe, is found in the UCR1 of long isoforms from all four PDE4 sub-families. However, no information is available as to whether long isoforms of other PDE4 sub-families can be phosphorylated and activated by PKA or if this property is unique to PDE4D3. Certainly UCR1 and UCR2 are linked to each other and to the catalytic unit by regions that have very different primary sequences (Houslay et al., 1998), which may alter accessibility of PKA to the site within UCR1, as well as affect any conformational change consequent upon PKA phosphorylation. In addition to this, there are distinct differences in primary sequence of a number of the helices that form the catalytic unit of each of these sub-families (Xu et al., 2000). These differences undoubtedly underpin sub-family variation in sensitivity to the action of certain inhibitors and catalytic activity, for example (Houslay, 2001; Houslay et al., 1998). Thus, there is no a priori reason to expect that long PDE4 isoforms from other sub-families may either be phosphorylated by PKA or will be activated as a consequence of any such phosphorylation event. This study demonstrates that examples of long isoforms from the three other PDE4 sub-families are capable of acting as PKA substrates both in vitro and in intact cells and that this modification leads to enzyme activation. In all cases studied, PKA phosphorylation occurs at a single serine residue in UCR1. For the first time we have developed phospho-specific antisera against this site which allows for the detection of phosphorylated PDE4 long forms from all four PDE4 sub-families. In addition, we show that decreased mobility on SDS – PAGE electrophoresis reported for the PKA-phosphorylated form of PDE4D3 (Oki et al., 2000; Sette & Conti, 1996) is likely to be unique to this isoform and cannot be taken as a universal indicator of PKA phosphorylation of PDE4 long isoforms.
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action of rolipram on specific pde4 camp phosphodiesterase isoforms and on the phosphorylation of camp response element binding protein creb and p38 mitogen activated protein map kinase in u937 monocytic cells
Biochemical Journal, 2000Co-Authors: Simon J Mackenzie, M D HouslayAbstract:U937 monocytic cells are shown here to express a range of PDE4, cAMP-specific phosphodiesterase (PDE) isoenzymes: the long isoenzymes, PDE4A4, PDE4D5 and PDE4D3, plus the short isoenzyme, PDE4B2. These isoenzymes provide around 76% of the total cAMP PDE activity of U937 cells. The specific activities of the total PDE4A, PDE4B and PDE4D activities were 0.63+/-0.09, 8.8+/-0.2 and 34.4+/-2.9 pmol/min per mg of protein respectively. The PDE4 selective inhibitor, rolipram, inhibited immunopurified PDE4B and PDE4D activities similarly, with IC(50) values of approx. 130 nM and 240 nM respectively. In contrast, rolipram inhibited immunopurified PDE4A activity with a dramatically lower IC(50) value of around 3 nM. Rolipram increased phosphorylation of cAMP-response-element-binding protein (CREB) in U937 cells in a dose-dependent fashion, which implied the presence of both high affinity (IC(50) value approx. 1 nM) and low affinity (IC(50) value approx. 120 nM) components. Rolipram dose-dependently inhibited the interferon-gamma (IFN-gamma)-stimulated phosphorylation of p38 mitogen-activated protein (MAP) kinase in a simple monotonic fashion with an IC(50) value of approx. 290 nM. On this basis, it is suggested that rolipram inhibition of PDE4A4 is involved in regulating CREB phosphorylation but not IFN-gamma-stimulated p38 MAP kinase phosphorylation. PDE4A4 was also selectively activated by challenge of U937 cells with either bacterial lipopolysaccharide (LPS) or IFN-gamma through a process which was attenuated by both wortmannin and rapamycin. It is proposed that the PDE4A4 isoform is involved in compartmentalized cAMP signalling responses in U937 monocytes.
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the rack1 signaling scaffold protein selectively interacts with the camp specific phosphodiesterase pde4d5 isoform
Journal of Biological Chemistry, 1999Co-Authors: Stephen J Yarwood, M D Houslay, Grant Scotland, Michael R Steele, Graeme B BolgerAbstract:Abstract The WD-repeat protein receptor for activated C-kinase (RACK1) was identified by its interaction with the cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4D5 in a yeast two-hybrid screen. The interaction was confirmed by co-immunoprecipitation of native RACK1 and PDE4D5 from COS7, HEK293, 3T3-F442A, and SK-N-SH cell lines. The interaction was unaffected by stimulation of the cells with the phorbol ester phorbol 2-myristate 3-acetate. PDE4D5 did not interact with two other WD-repeat proteins, β’-coatomer protein and Gsβ, in two-hybrid tests. RACK1 did not interact with other PDE4D isoforms or with known PDE4A, PDE4B, and PDE4C isoforms. PDE4D5 and RACK1 interacted with high affinity (K a approximately 7 pm) when they were expressed and purified from Escherichia coli, demonstrating that the interaction does not require intermediate proteins. The binding of the E. coli-expressed proteins did not alter the kinetics of cAMP hydrolysis by PDE4D5 but caused a 3–4-fold change in its sensitivity to inhibition by the PDE4 selective inhibitor rolipram. The subcellular distributions of RACK1 and PDE4D5 were extremely similar, with the major amount of both proteins (70%) in the high speed supernatant (S2) fraction. Analysis of constructs with specific deletions or single amino acid mutations in PDE4D5 demonstrated that a small cluster of amino acids in the unique amino-terminal region of PDE4D5 was necessary for its interaction with RACK1. We suggest that RACK1 may act as a scaffold protein to recruit PDE4D5 and other proteins into a signaling complex.
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association with the src family tyrosyl kinase lyn triggers a conformational change in the catalytic region of human camp specific phosphodiesterase hspde4a4b consequences for rolipram inhibition
Journal of Biological Chemistry, 1999Co-Authors: Ian Mcphee, Grant Scotland, Elaine Huston, Matthew B Beard, Stephen J Yarwood, Annette H Ross, Emma S Houslay, M D HouslayAbstract:Abstract The cAMP-specific phosphodiesterase (PDE) HSPDE 4A4B(pde46) selectively bound SH3 domains of SRC family tyrosyl kinases. Such an interaction profoundly changed the inhibition of PDE4 activity caused by the PDE4-selective inhibitor rolipram and mimicked the enhanced rolipram inhibition seen for particulate, compared with cytosolic pde46 expressed in COS7 cells. Particulate pde46 co-localized with LYN kinase in COS7 cells. The unique N-terminal and LR2 regions of pde46 contained the sites for SH3 binding. Altered rolipram inhibition was triggered by SH3 domain interaction with the LR2 region. Purified LYN SH3 and human PDE4A LR2 could be co-immunoprecipitated, indicating a direct interaction. Protein kinase A-phosphorylated pde46 remained able to bind LYN SH3. pde46 was found to be associated with SRC kinase in the cytosol of COS1 cells, leading to aberrant kinetics of rolipram inhibition. It is suggested that pde46 may be associated with SRC family tyrosyl kinases in intact cells and that the ensuing SH3 domain interaction with the LR2 region of pde46 alters the conformation of the PDE catalytic unit, as detected by altered rolipram inhibition. Interaction between pde46 and SRC family tyrosyl kinases highlights a potentially novel regulatory system and point of signaling system cross-talk.
James M Odonnell - One of the best experts on this subject based on the ideXlab platform.
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comparison of the pharmacological profiles of selective pde4b and pde4d inhibitors in the central nervous system
Scientific Reports, 2017Co-Authors: Chong Zhang, Ying Xu, Mark Gurney, James M OdonnellAbstract:Inhibition of cyclic AMP (cAMP)-specific phosphodiesterase 4 (PDE4) has been proposed as a potential treatment for a series of neuropsychological conditions such as depression, anxiety and memory loss. However, the specific involvement of each of the PDE4 subtypes (PDE4A, 4B and 4C) in different categories of behavior has yet to be elucidated. In the present study, we compared the possible pharmacological effects of PDE4B and PDE4D selective inhibitors, A-33 and D159687, in mediating neurological function in mice. Both compounds were equally potent in stimulating cAMP signaling in the mouse hippocampal cell line HT-22 leading to an increase in CREB phosphorylation. In contrast, A-33 and D159687 displayed distinct neuropharmacological effects in mouse behavioral tests. A-33 has an antidepressant-like profile as indicated by reduced immobility time in the forced swim and tail suspension tasks, as well as reduced latency to feed in the novelty suppressed feeding test. D159687, on the other hand, had a procognitive profile as it improved memory in the novel object recognition test but had no antidepressant or anxiolytic benefit. The present data suggests that inhibitors targeting specific subtypes of PDE4 may exhibit differential pharmacological effects and aid a more efficient pharmacotherapy towards neuropsychological conditions.
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phosphodiesterase 4d knock out and rna interference mediated knock down enhance memory and increase hippocampal neurogenesis via increased camp signaling
The Journal of Neuroscience, 2011Co-Authors: Yufang Cheng, James M Odonnell, Marco Conti, Ying Huang, Steven P Wilson, Hanting ZhangAbstract:Phosphodiesterase-4 (PDE4) plays an important role in mediating memory via the control of intracellular cAMP signaling; inhibition of PDE4 enhances memory. However, development of PDE4 inhibitors as memory enhancers has been hampered by their major side effect of emesis. PDE4 has four subtypes (PDE4A-D) consisting of 25 splice variants. Mice deficient in PDE4D displayed memory enhancement in radial arm maze, water maze, and object recognition tests. These effects were mimicked by repeated treatment with rolipram in wild-type mice. In addition, similarly as rolipram-treated wild-type mice, PDE4D-deficient mice also displayed increased hippocampal neurogenesis and phosphorylated cAMP response element-binding protein (pCREB). Furthermore, microinfusion of lentiviral vectors that contained microRNAs (miRNAs) targeting long-form PDE4D isoforms into bilateral dentate gyri of the mouse hippocampus downregulated PDE4D4 and PDE4D5, enhanced memory, and increased hippocampal neurogenesis and pCREB. Finally, while rolipram and PDE4D deficiency shortened α2 adrenergic receptor-mediated anesthesia, a surrogate measure of emesis, miRNA-mediated PDE4D knock-down in the hippocampus did not. The present results suggest that PDE4D, in particular long-form PDE4D, plays a critical role in the mediation of memory and hippocampal neurogenesis, which are mediated by cAMP/CREB signaling; reduced expression of PDE4D, or at least PDE4D4 and PDE4D5, in the hippocampus enhances memory but appears not to cause emesis. These novel findings will aid in the development of PDE4 subtype- or variant-selective inhibitors for treatment of disorders involving impaired cognition, including Alzheimer's disease.
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regulation of phosphodiesterase 4 pde4 expression in mouse brain by repeated antidepressant treatment comparison with rolipram
Brain Research, 2006Co-Authors: Daniel Dlaboga, Hassan Hajjhussein, James M OdonnellAbstract:Cyclic nucleotide phosphodiesterase-4 (PDE4) is a component of signaling pathways involved in the mediation of antidepressant activity. Of the four PDE4 subtypes, PDE4D appears to be of particular importance, given the finding that PDE4D-deficient mice exhibit an antidepressant-like behavioral phenotype. In mouse hippocampus and cerebral cortex, the effects of repeated treatment with the antidepressants desipramine and fluoxetine or the PDE4 inhibitor rolipram on the expression of PDE4D was compared to that of PDE4A and PDE4B, the other two subtypes expressed in the brain. Expression of PDE4D was increased by all drugs tested, with the exception of desipramine in hippocampus. By contrast, these treatments affected PDE4A and PDE4B expression differentially. In hippocampus, antidepressants increased PDE4A and decreased PDE4B, whereas ROL decreased PDE4A and did not change PDE4B. In cerebral cortex, antidepressants increased PDE4A and did not change PDE4B, whereas ROL did not change PDE4A and increased PDE4B. 3H-Rolipram binding was increased in cytosolic, but not in membrane, fractions of cerebral cortex by all drugs tested; there were no changes observed in hippocampus. Overall, the present results suggest some species-dependence of the regulation of PDE4 subtypes, based on data obtained previously using rats. They also suggest that the PDE4D subtype may be of particular importance as an antidepressant target in that it is regulated by repeated treatment with both norepinephrine and serotonin reuptake inhibitors as well as by the PDE4 inhibitor rolipram, drugs that produce antidepressant effects via different neuropharmacological mechanisms.
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antidepressant effects of inhibitors of camp phosphodiesterase pde4
Trends in Pharmacological Sciences, 2004Co-Authors: James M Odonnell, Hanting ZhangAbstract:Abstract Despite initial promise, the development of type 4 phosphodiesterase (PDE4) inhibitors as antidepressants has not advanced significantly. This is due to an incomplete understanding of the functional importance of PDE4 subtypes and high-affinity and low-affinity inhibitor-binding conformers. However, recent developments have rekindled interest in the therapeutic potential of PDE4 inhibitors. First, PDE4 has been shown to be involved in cAMP signaling pathways that are affected by antidepressants. Second, data obtained using mouse knockout lines indicate that PDE4D and PDE4B mediate antidepressant effects. Third, it appears that the interaction of inhibitors with the high-affinity binding conformer of PDE4 is particularly important for antidepressant efficacy. These developments highlight the difficulties of dissociating the actions of PDE4 inhibitors and provide a guide for future research.
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noradrenergic activity differentially regulates the expression of rolipram sensitive high affinity cyclic amp phosphodiesterase pde4 in rat brain
Journal of Neurochemistry, 2002Co-Authors: Marco Conti, Shakeel M Farooqui, Ming Chen, James M OdonnellAbstract:Ina previousstudy,it wasobservedthat the activityof rolipram-sensitive, low-Km,cyclic AMPphos- phodiesterase (PDE4)wasdecreasedin vivowith dimin- ishednoradrenergic stimulation.Theresultsof the pres- entexperiments indicatedthat thereductionintheactivity maybeassociatedwithdown-regulation of PDE4protein. Immunoblotanalysis using PDE4-specific,subfamily- nonspecificantibody(K116)revealedfour majorbands ofPDE4in ratcerebralcortex;thosewithapparentmolec- ularmassesof 109 and 102kDaare variantsof PDE4A. Diminished noradrenergic activity,producedby intracere- broventricular infusionof 6-hydroxydopamine (6-OHDA) or chronic subcutaneousinfusion of propranolol,de- creasedthe intensitiesof the proteinbandsfor the 109- and 102-kDaPDE4Avariantsin rat cerebralcortex but notof the 98- or 91-kDaPDE4forms. 6-OHDA-induced noradrenergiclesioningalso decreasedthe content of 102-kDaPDE4Ain hippocampusas labeledby PDE4A- specific antibody(C-PDE4A).Enhancednoradrenergic stimulationup-regulatedPDE4in cerebralcortex. This wasindicatedby thefindingthat repeatedtreatmentwith desipramineincreasedthe intensityof the protein band for the 102-kDaPDE4but not for the other variantsof PDE4.Theseresultssuggestthat PDE4subtypesaredif- ferentiallyregulatedat the level of expression,as evi- dencedby an apparentchangein the amountof PDE4 protein, following changes in noradrenergicactivity. Theseobservationsare consistentwith the notion that PDE4s,especiallythe PDE4Avariantswith molecular massesof 109 and 102 kDa,play an importantrole in maintaining the homeostasisof the noradrenergicsignal transductionsystemin the brainand may be involved in the mediationof antidepressantactivity. KeyWords: Phosphodiesterase— Noradrenergicsystem — Immuno- blot analysis—6-Hydroxydopamine—Propranolol—De- sipramine. J. Neurochem.69, 2397—2404 (1997).
Wito Richter - One of the best experts on this subject based on the ideXlab platform.
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pde4d and pde4b function in distinct subcellular compartments in mouse embryonic fibroblasts
Journal of Biological Chemistry, 2011Co-Authors: Brigitte E Blackman, Wito Richter, Kathleen Horner, Julia D Heidmann, Thomas C. Rich, Dan Wang, Marco ContiAbstract:Signaling through cAMP regulates most cellular functions. The spatiotemporal control of cAMP is, therefore, crucial for differential regulation of specific cellular targets. Here we investigated the consequences of PDE4B or PDE4D gene ablation on cAMP signaling at a subcellular level using mouse embryonic fibroblasts. PDE4B ablation had no effect on the global or bulk cytosol accumulation of cAMP but increased both basal and hormone-dependent cAMP in a near-membrane pool. Conversely, PDE4D ablation enhanced agonist-induced cAMP accumulation in the bulk cytosol as well as at the plasma membrane. Both PDE4B and PDE4D ablation significantly modified the time course and the level of isoproterenol-induced phosphorylation of vasodilator-stimulated phosphoprotein, a membrane cytoskeletal component. A second membrane response through Toll-like receptor signaling, however, was only affected by PDE4B ablation. PDE4D but not PDE4B ablation significantly prolonged cAMP-response element-binding protein-mediated transcription. These findings demonstrate that PDE4D and PDE4B have specialized functions in mouse embryonic fibroblasts with PDE4B controlling cAMP in a discrete subdomain near the plasma membrane.
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decreased expression and activity of camp phosphodiesterases in cardiac hypertrophy and its impact on β adrenergic camp signals
Circulation Research, 2009Co-Authors: Aniella Abigerges, Wito Richter, Marco Conti, Claire Lugnier, Florence Lefebvre, Philippe Mateo, Audrey Varin, Christophe Heymes, Janelise Samuel, Rodolphe FischmeisterAbstract:Rationale: Multiple cyclic nucleotide phosphodiesterases (PDEs) degrade cAMP in cardiomyocytes but the role of PDEs in controlling cAMP signaling during pathological cardiac hypertrophy is poorly defined. Objective: Evaluate the -adrenergic regulation of cardiac contractility and characterize the changes in cardiomyocyte cAMP signals and cAMP-PDE expression and activity following cardiac hypertrophy. Methods and Results: Cardiac hypertrophy was induced in rats by thoracic aortic banding over a time period of 5 weeks and was confirmed by anatomic measurements and echocardiography. Ex vivo myocardial function was evaluated in Langendorff-perfused hearts. Engineered cyclic nucleotide-gated (CNG) channels were expressed in single cardiomyocytes to monitor subsarcolemmal cAMP using whole-cell patch-clamp recordings of the associated CNG current (ICNG). PDE variant activity and protein level were determined in purified cardiomyocytes. Aortic stenosis rats exhibited a 67% increase in heart weight compared to sham-operated animals. The inotropic response to maximal -adrenergic stimulation was reduced by 54% in isolated hypertrophied hearts, along with a 32% decrease in subsarcolemmal cAMP levels in hypertrophied myocytes. Total cAMP hydrolytic activity as well as PDE3 and PDE4 activities were reduced in hypertrophied myocytes, because of a reduction of PDE3A, PDE4A, and PDE4B, whereas PDE4D was unchanged. Regulation of -adrenergic cAMP signals by PDEs was blunted in hypertrophied myocytes, as demonstrated by the diminished effects of IBMX (100 mol/L) and of both the PDE3 inhibitor cilostamide (1 mol/L) and the PDE4 inhibitor Ro 201724 (10 mol/L). Conclusions: -Adrenergic desensitization is accompanied by a reduction in cAMP-PDE and an altered modulation of -adrenergic cAMP signals in cardiac hypertrophy. (Circ Res. 2009;105:784-792.)
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critical role of pde4d in β2 adrenoceptor dependent camp signaling in mouse embryonic fibroblasts
Journal of Biological Chemistry, 2008Co-Authors: Matthew D Bruss, Wito Richter, Kathleen Horner, Marco ContiAbstract:One of the defining properties of β2-adrenergic receptor (β2AR) signaling is the transient and rapidly reversed accumulation of cAMP. Here we have investigated the contribution of different PDE4 proteins to the generation of this transient response. To this aim, mouse embryonic fibroblasts deficient in PDE4A, PDE4B, or PDE4D were generated, and the regulation of PDE activity, the accumulation of cAMP, and CREB phosphorylation in response to isoproterenol were monitored. Ablation of PDE4D, but not PDE4A or PDE4B, had a major effect on the β-agonist-induced PDE activation, with only a minimal increase in PDE activity being retained in PDE4D knock-out (KO) cells. Accumulation of cAMP was markedly enhanced, and the kinetics of cAMP accumulation were altered in their properties in PDE4DKO but not PDE4BKO cells. Modest effects were observed in PDE4AKO mouse embryonic fibroblasts. The return to basal levels of both cAMP accumulation and CREB phosphorylation was greatly delayed in the PDE4DKO cells, suggesting that PDE4D is critical for dissipation of the β2AR stimulus. This effect of PDE4D ablation was in large part due to inactivation of a negative feedback mechanism consisting of the PKA-mediated activation of PDE4D in response to elevated cAMP levels, as indicated by experiments using the cAMP-dependent protein kinase inhibitors H89 and PKI. Finally, PDE4D ablation affected the kinetics of β2AR desensitization as well as the interaction of the receptor with Gαi. These findings demonstrate that PDE4D plays a major role in shaping the β2AR signal.
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splice variants of the cyclic nucleotide phosphodiesterase pde4d are differentially expressed and regulated in rat tissue
Biochemical Journal, 2005Co-Authors: Wito Richter, Marco ContiAbstract:Cyclic nucleotide PDE4 (phosphodiesterase 4) inhibitors are being developed as potent anti-inflammatory drugs for use in chronic lung diseases, but the complexity of the PDE4 family has hampered this process. The four genes comprising the PDE4 family, PDE4A, PDE4B, PDE4C and PDE4D, are all expressed as multiple splice variants. The most widely used criterion to identify PDE4 variants expressed endogenously is their migration on SDS/PAGE. However, when a PDE4D3-selective antibody was used for immunoprecipitation, the pattern of expression obtained did not confirm the expression predicted by SDS/PAGE. This observation, together with the recent discovery of additional PDE4D transcripts, prompted us to re-evaluate the pattern of expression of these variants. The nine rat PDE4D splice variants, PDE4D1 to PDE4D9, were cloned, their electrophoretic properties compared, and their in vivo mRNA and protein levels determined. Using this approach, we found that the pattern of distribution of the PDE4D splicing variants is more complex than previously reported. Multiple variants co-migrate in single immunoreactive bands, and variant-selective antibodies were necessary to discriminate between splice variants. Tissues that were thought to express only PDE4D3, express three closely related proteins, with PDE4D8 and PDE4D9 as the predominantly expressed forms. In addition, activation of cAMP signalling produces phosphorylation and activation of variants other than PDE4D3, and expression of PDE4D mRNA does not always correlate with the pattern of protein expression. As PDE4 inhibitors have different affinities for distinct PDE4D splicing variants, our results indicate that a better definition of the pattern of PDE4 expression is required for target validation.