181 results on '"Beavo JA"'
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2. Comparison of adenosine 3':5'-monophosphate-dependent protein kinases from rabbit skeletal and bovine heart muscle.
- Author
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Hofmann, F, primary, Beavo, JA, additional, Bechtel, PJ, additional, and Krebs, EG, additional
- Published
- 1975
- Full Text
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3. Phosphoproteomic Analysis as an Approach for Understanding Molecular Mechanisms of cAMP-Dependent Actions.
- Author
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Beavo JA, Golkowski M, Shimizu-Albergine M, Beltejar MC, Bornfeldt KE, and Ong SE
- Subjects
- Animals, Humans, Proteomics methods, Signal Transduction physiology, Cyclic AMP metabolism, Phosphorylation physiology, Proteome metabolism
- Abstract
In recent years, highly sensitive mass spectrometry-based phosphoproteomic analysis is beginning to be applied to identification of protein kinase substrates altered downstream of increased cAMP. Such studies identify a very large number of phosphorylation sites regulated in response to increased cAMP. Therefore, we now are tasked with the challenge of determining how many of these altered phosphorylation sites are relevant to regulation of function in the cell. This minireview describes the use of phosphoproteomic analysis to monitor the effects of cyclic nucleotide phosphodiesterase (PDE) inhibitors on cAMP-dependent phosphorylation events. More specifically, it describes two examples of this approach carried out in the authors' laboratories using the selective PDE inhibitor approach. After a short discussion of several likely conclusions suggested by these analyses of cAMP function in steroid hormone-producing cells and also in T-cells, it expands into a discussion about some newer and more speculative interpretations of the data. These include the idea that multiple phosphorylation sites and not a single rate-limiting step likely regulate these and, by analogy, many other cAMP-dependent pathways. In addition, the idea that meaningful regulation requires a high stoichiometry of phosphorylation to be important is discussed and suggested to be untrue in many instances. These new interpretations have important implications for drug design, especially for targeting pathway agonists. SIGNIFICANCE STATEMENT: Phosphoproteomic analyses identify thousands of altered phosphorylation sites upon drug treatment, providing many possible regulatory targets but also highlighting questions about which phosphosites are functionally important. These data imply that multistep processes are regulated by phosphorylation at not one but rather many sites. Most previous studies assumed a single step or very few rate-limiting steps were changed by phosphorylation. This concept should be changed. Previous interpretations also assumed substoichiometric phosphorylation was not of regulatory importance. This assumption also should be changed., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)
- Published
- 2021
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4. Analyses of PDE-regulated phosphoproteomes reveal unique and specific cAMP-signaling modules in T cells.
- Author
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Beltejar MG, Lau HT, Golkowski MG, Ong SE, and Beavo JA
- Subjects
- Algorithms, Humans, Jurkat Cells, Metabolic Networks and Pathways, 3',5'-Cyclic-AMP Phosphodiesterases antagonists & inhibitors, Phosphoproteins metabolism, T-Lymphocytes metabolism
- Abstract
Specific functions for different cyclic nucleotide phosphodiesterases (PDEs) have not yet been identified in most cell types. Conventional approaches to study PDE function typically rely on measurements of global cAMP, general increases in cAMP-dependent protein kinase (PKA), or the activity of exchange protein activated by cAMP (EPAC). Although newer approaches using subcellularly targeted FRET reporter sensors have helped define more compartmentalized regulation of cAMP, PKA, and EPAC, they have limited ability to link this regulation to downstream effector molecules and biological functions. To address this problem, we have begun to use an unbiased mass spectrometry-based approach coupled with treatment using PDE isozyme-selective inhibitors to characterize the phosphoproteomes of the functional pools of cAMP/PKA/EPAC that are regulated by specific cAMP-PDEs (the PDE-regulated phosphoproteomes). In Jurkat cells we find multiple, distinct PDE-regulated phosphoproteomes that can be defined by their responses to different PDE inhibitors. We also find that little phosphorylation occurs unless at least two different PDEs are concurrently inhibited in these cells. Moreover, bioinformatics analyses of these phosphoproteomes provide insight into the unique functional roles, mechanisms of action, and synergistic relationships among the different PDEs that coordinate cAMP-signaling cascades in these cells. The data strongly suggest that the phosphorylation of many different substrates contributes to cAMP-dependent regulation of these cells. The findings further suggest that the approach of using selective, inhibitor-dependent phosphoproteome analysis can provide a generalized methodology for understanding the roles of different PDEs in the regulation of cyclic nucleotide signaling., Competing Interests: The authors declare no conflict of interest.
- Published
- 2017
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5. SCAP/SREBP pathway is required for the full steroidogenic response to cyclic AMP.
- Author
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Shimizu-Albergine M, Van Yserloo B, Golkowski MG, Ong SE, Beavo JA, and Bornfeldt KE
- Subjects
- Animals, Carrier Proteins, Cholesterol metabolism, Gene Expression Regulation, Hydroxymethylglutaryl CoA Reductases drug effects, Hydroxymethylglutaryl CoA Reductases metabolism, Intracellular Signaling Peptides and Proteins genetics, Leydig Cells metabolism, Lipoproteins metabolism, Luteinizing Hormone metabolism, Male, Membrane Proteins genetics, Mitochondria genetics, Mitochondria metabolism, Phosphorylation, Steroids chemistry, Sterol Regulatory Element Binding Protein 2 genetics, Cyclic AMP metabolism, Intracellular Signaling Peptides and Proteins metabolism, Membrane Proteins metabolism, Steroids biosynthesis, Sterol Regulatory Element Binding Protein 2 metabolism
- Abstract
Luteinizing hormone (LH) stimulates steroidogenesis largely through a surge in cyclic AMP (cAMP). Steroidogenic rates are also critically dependent on the availability of cholesterol at mitochondrial sites of synthesis. This cholesterol is provided by cellular uptake of lipoproteins, mobilization of intracellular lipid, and de novo synthesis. Whether and how these pathways are coordinated by cAMP are poorly understood. Recent phosphoproteomic analyses of cAMP-dependent phosphorylation sites in MA10 Leydig cells suggested that cAMP regulates multiple steps in these processes, including activation of the SCAP/SREBP pathway. SCAP [sterol-regulatory element-binding protein (SREBP) cleavage-activating protein] acts as a cholesterol sensor responsible for regulating intracellular cholesterol balance. Its role in cAMP-mediated control of steroidogenesis has not been explored. We used two CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associated protein 9) knockout approaches to test the role of SCAP in steroidogenesis. Our results demonstrate that SCAP is required for progesterone production induced by concurrent inhibition of the cAMP phosphodiesterases PDE4 and PDE8. These inhibitors increased SCAP phosphorylation, SREBP2 activation, and subsequent expression of cholesterol biosynthetic genes, whereas SCAP deficiency largely prevented these effects. Reexpression of SCAP in SCAP-deficient cells restored SREBP2 protein expression and partially restored steroidogenic responses, confirming the requirement of SCAP-SREBP2 in steroidogenesis. Inhibitors of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase and isoprenylation attenuated, whereas exogenously provided cholesterol augmented, PDE inhibitor-induced steroidogenesis, suggesting that the cholesterol substrate needed for steroidogenesis is provided by both de novo synthesis and isoprenylation-dependent mechanisms. Overall, these results demonstrate a novel role for LH/cAMP in SCAP/SREBP activation and subsequent regulation of steroidogenesis., Competing Interests: The authors declare no conflict of interest.
- Published
- 2016
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6. Studying mechanisms of cAMP and cyclic nucleotide phosphodiesterase signaling in Leydig cell function with phosphoproteomics.
- Author
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Golkowski M, Shimizu-Albergine M, Suh HW, Beavo JA, and Ong SE
- Subjects
- 3',5'-Cyclic-AMP Phosphodiesterases antagonists & inhibitors, Animals, Cell Cycle Proteins metabolism, Cell Line, Cluster Analysis, Databases as Topic, Endocytosis drug effects, Extracellular Signal-Regulated MAP Kinases metabolism, Gene Ontology, Insulin metabolism, Isotope Labeling, Leydig Cells drug effects, Male, Mice, Phosphodiesterase Inhibitors pharmacology, Phosphorylation drug effects, Proteome metabolism, Transport Vesicles drug effects, Transport Vesicles metabolism, 3',5'-Cyclic-AMP Phosphodiesterases metabolism, Cyclic AMP metabolism, Leydig Cells enzymology, Phosphoproteins metabolism, Proteomics methods, Signal Transduction drug effects
- Abstract
Many cellular processes are modulated by cyclic AMP and nucleotide phosphodiesterases (PDEs) regulate this second messenger by catalyzing its breakdown. The major unique function of testicular Leydig cells is to produce testosterone in response to luteinizing hormone (LH). Treatment of Leydig cells with PDE inhibitors increases cAMP levels and the activity of its downstream effector, cAMP-dependent protein kinase (PKA), leading to a series of kinase-dependent signaling and transcription events that ultimately increase testosterone release. We have recently shown that PDE4B and PDE4C as well as PDE8A and PDE8B are expressed in rodent Leydig cells and that combined inhibition of PDE4 and PDE8 leads to dramatically increased steroid biosynthesis. Here we investigated the effect of PDE4 and PDE8 inhibition on the molecular mechanisms of cAMP actions in a mouse MA10 Leydig cell line model with SILAC mass spectrometry-based phosphoproteomics. We treated MA10 cells either with PDE4 family specific inhibitor (Rolipram) and PDE8 family specific inhibitor (PF-04957325) alone or in combination and quantified the resulting phosphorylation changes at five different time points between 0 and 180min. We identified 28,336 phosphosites from 4837 proteins and observed significant regulation of 749 sites in response to PDE4 and PDE8 inhibitor treatment. Of these, 132 phosphosites were consensus PKA sites. Our data strongly suggest that PDE4 and PDE8 inhibitors synergistically regulate phosphorylation of proteins required for many different cellular processes, including cell cycle progression, lipid and glucose metabolism, transcription, endocytosis and vesicle transport. Our data suggests that cAMP, PDE4 and PDE8 coordinate steroidogenesis by acting on not one rate-limiting step but rather multiple pathways. Moreover, the pools of cAMP controlled by these PDEs also coordinate many other metabolic processes that may be regulated to assure timely and sufficient testosterone secretion in response to LH., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2016
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7. Luteinizing Hormone Causes Phosphorylation and Activation of the cGMP Phosphodiesterase PDE5 in Rat Ovarian Follicles, Contributing, Together with PDE1 Activity, to the Resumption of Meiosis.
- Author
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Egbert JR, Uliasz TF, Shuhaibar LC, Geerts A, Wunder F, Kleiman RJ, Humphrey JM, Lampe PD, Artemyev NO, Rybalkin SD, Beavo JA, Movsesian MA, and Jaffe LA
- Subjects
- Animals, Cells, Cultured, Female, Mice, Mice, Inbred C57BL, Oocytes drug effects, Oocytes metabolism, Ovarian Follicle metabolism, Phosphorylation drug effects, Rats, Rats, Sprague-Dawley, Cyclic GMP metabolism, Cyclic Nucleotide Phosphodiesterases, Type 1 metabolism, Cyclic Nucleotide Phosphodiesterases, Type 5 metabolism, Luteinizing Hormone pharmacology, Meiosis drug effects, Ovarian Follicle drug effects
- Abstract
The meiotic cell cycle of mammalian oocytes in preovulatory follicles is held in prophase arrest by diffusion of cGMP from the surrounding granulosa cells into the oocyte. Luteinizing hormone (LH) then releases meiotic arrest by lowering cGMP in the granulosa cells. The LH-induced reduction of cGMP is caused in part by a decrease in guanylyl cyclase activity, but the observation that the cGMP phosphodiesterase PDE5 is phosphorylated during LH signaling suggests that an increase in PDE5 activity could also contribute. To investigate this idea, we measured cGMP-hydrolytic activity in rat ovarian follicles. Basal activity was due primarily to PDE1A and PDE5, and LH increased PDE5 activity. The increase in PDE5 activity was accompanied by phosphorylation of PDE5 at serine 92, a protein kinase A/G consensus site. Both the phosphorylation and the increase in activity were promoted by elevating cAMP and opposed by inhibiting protein kinase A, supporting the hypothesis that LH activates PDE5 by stimulating its phosphorylation by protein kinase A. Inhibition of PDE5 activity partially suppressed LH-induced meiotic resumption as indicated by nuclear envelope breakdown, but inhibition of both PDE5 and PDE1 activities was needed to completely inhibit this response. These results show that activities of both PDE5 and PDE1 contribute to the LH-induced resumption of meiosis in rat oocytes, and that phosphorylation and activation of PDE5 is a regulatory mechanism., (© 2016 by the Society for the Study of Reproduction, Inc.)
- Published
- 2016
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8. Roles of cGMP-dependent protein kinase I (cGKI) and PDE5 in the regulation of Ang II-induced cardiac hypertrophy and fibrosis.
- Author
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Patrucco E, Domes K, Sbroggió M, Blaich A, Schlossmann J, Desch M, Rybalkin SD, Beavo JA, Lukowski R, and Hofmann F
- Subjects
- Animals, Cardiomegaly chemically induced, Cyclic GMP metabolism, Fibrosis chemically induced, Fibrosis metabolism, Genetic Markers, Hypertension chemically induced, Mice, Muscle, Smooth metabolism, Myocardial Contraction drug effects, Myocardial Contraction physiology, Myocytes, Cardiac drug effects, Myocytes, Cardiac metabolism, Nitric Oxide metabolism, Phosphodiesterase 5 Inhibitors pharmacology, Piperazines pharmacology, Purines pharmacology, Sildenafil Citrate, Sulfones pharmacology, Vasoconstrictor Agents pharmacology, Angiotensin II pharmacology, Cardiomegaly metabolism, Cyclic GMP-Dependent Protein Kinase Type I metabolism, Cyclic Nucleotide Phosphodiesterases, Type 5 metabolism, Hypertension metabolism
- Abstract
Conflicting results have been reported for the roles of cGMP and cGMP-dependent protein kinase I (cGKI) in various pathological conditions leading to cardiac hypertrophy and fibrosis. A cardioprotective effect of cGMP/cGKI has been reported in whole animals and isolated cardiomyocytes, but recent evidence from a mouse model expressing cGKIβ only in smooth muscle (βRM) but not in cardiomyocytes, endothelial cells, or fibroblasts has forced a reevaluation of the requirement for cGKI activity in the cardiomyocyte antihypertrophic effects of cGMP. In particular, βRM mice developed the same hypertrophy as WT controls when subjected to thoracic aortic constriction or isoproterenol infusion. Here, we challenged βRM and WT (Ctr) littermate control mice with angiotensin II (AII) infusion (7 d; 2 mg ⋅ kg(-1) ⋅ d(-1)) to induce hypertrophy. Both genotypes developed cardiac hypertrophy, which was more pronounced in Ctr animals. Cardiomyocyte size and interstitial fibrosis were increased equally in both genotypes. Addition of sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, in the drinking water had a small effect in reducing myocyte hypertrophy in WT mice and no effect in βRM mice. However, sildenafil substantially blocked the increase in collagen I, fibronectin 1, TGFβ, and CTGF mRNA in Ctr but not in βRM hearts. These data indicate that, for the initial phase of AII-induced cardiac hypertrophy, lack of cardiomyocyte cGKI activity does not worsen hypertrophic growth. However, expression of cGKI in one or more cell types other than smooth muscle is necessary to allow the antifibrotic effect of sildenafil.
- Published
- 2014
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9. A yeast-based chemical screen identifies a PDE inhibitor that elevates steroidogenesis in mouse Leydig cells via PDE8 and PDE4 inhibition.
- Author
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Demirbas D, Wyman AR, Shimizu-Albergine M, Cakici O, Beavo JA, and Hoffman CS
- Subjects
- Animals, Cell Line, Tumor, Cyclic Nucleotide Phosphodiesterases, Type 4 chemistry, High-Throughput Screening Assays, Humans, Male, Mice, Molecular Docking Simulation, Phosphodiesterase 4 Inhibitors chemistry, Phosphodiesterase 4 Inhibitors metabolism, Protein Conformation, Small Molecule Libraries chemistry, Small Molecule Libraries metabolism, Small Molecule Libraries pharmacology, Cyclic Nucleotide Phosphodiesterases, Type 4 metabolism, Drug Evaluation, Preclinical methods, Leydig Cells drug effects, Leydig Cells metabolism, Phosphodiesterase 4 Inhibitors pharmacology, Schizosaccharomyces genetics, Steroids biosynthesis
- Abstract
A cell-based high-throughput screen (HTS) was developed to detect phosphodiesterase 8 (PDE8) and PDE4/8 combination inhibitors. By replacing the Schizosaccharomyces pombe PDE gene with the murine PDE8A1 gene in strains lacking adenylyl cyclase, we generated strains whose protein kinase A (PKA)-stimulated growth in 5-fluoro orotic acid (5FOA) medium reflects PDE8 activity. From our previously-identified PDE4 and PDE7 inhibitors, we identified a PDE4/8 inhibitor that allowed us to optimize screening conditions. Of 222,711 compounds screened, ∼0.2% displayed composite Z scores of >20. Additional yeast-based assays using the most effective 367 compounds identified 30 candidates for further characterization. Among these, compound BC8-15 displayed the lowest IC₅₀ value for both PDE4 and PDE8 inhibition in in vitro enzyme assays. This compound also displays significant activity against PDE10A and PDE11A. BC8-15 elevates steroidogenesis in mouse Leydig cells as a single pharmacological agent. Assays using BC8-15 and two structural derivatives support a model in which PDE8 is a primary regulator of testosterone production by Leydig cells, with an additional role for PDE4 in this process. BC8-15, BC8-15A, and BC8-15C, which are commercially available compounds, display distinct patterns of activity against PDE4, PDE8, PDE10A, and PDE11A, representing a chemical toolkit that could be used to examine the biological roles of these enzymes in cell culture systems.
- Published
- 2013
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10. PDE3 and PDE4 isozyme-selective inhibitors are both required for synergistic activation of brown adipose tissue.
- Author
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Kraynik SM, Miyaoka RS, and Beavo JA
- Subjects
- 3',5'-Cyclic-AMP Phosphodiesterases antagonists & inhibitors, 3',5'-Cyclic-AMP Phosphodiesterases genetics, 3',5'-Cyclic-AMP Phosphodiesterases metabolism, Adipose Tissue, Brown cytology, Adipose Tissue, Brown metabolism, Adrenergic beta-Agonists pharmacology, Animals, Cells, Cultured, Cyclic Nucleotide Phosphodiesterases, Type 3 metabolism, Cyclic Nucleotide Phosphodiesterases, Type 4 metabolism, Drug Synergism, Glucose metabolism, Ion Channels genetics, Ion Channels metabolism, Isoenzymes metabolism, Lipolysis, Mice, Mice, Inbred C57BL, Mice, Knockout, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, RNA, Messenger metabolism, Stem Cells drug effects, Stem Cells metabolism, Uncoupling Protein 1, Adipose Tissue, Brown drug effects, Phosphodiesterase 3 Inhibitors pharmacology, Phosphodiesterase 4 Inhibitors pharmacology
- Abstract
Brown adipose tissue (BAT) is a highly thermogenic organ that converts lipids and glucose into heat. Many of the metabolic and gene transcriptional hallmarks of BAT activation, namely increased lipolysis, uncoupling protein-1 (UCP1) mRNA, and glucose uptake, are regulated by the adrenergic second messenger, cAMP. Cyclic nucleotide phosphodiesterases (PDEs) catalyze the breakdown of cAMP, thereby regulating the magnitude and duration of this signaling molecule. In the absence of adrenergic stimulus, we found that it required a combination of a PDE3 and a PDE4 inhibitor to fully induce UCP1 mRNA and lipolysis in brown adipocytes, whereas neither PDE inhibitor alone had any substantial effect under basal conditions. Under submaximal β-adrenoceptor stimulation of brown adipocytes, a PDE3 inhibitor alone could potentiate induction of UCP1 mRNA, whereas a PDE4 inhibitor alone could augment lipolysis, indicating differential roles for each of these two PDEs. Neither induction of UCP1 nor lipolysis was altered by inhibition of PDE1, PDE2, or PDE8A. Finally, when injected into mice, the combination of PDE3 and PDE4 inhibitors stimulated glucose uptake in BAT under thermoneutral and fasted conditions, a response that was further potentiated by the global ablation of PDE8A. Taken together, these data reveal that multiple PDEs work in concert to regulate three of the important pathways leading to BAT activation, a finding that may provide an improved conceptual basis for the development of therapies for obesity-related diseases.
- Published
- 2013
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11. Phosphodiesterase-8A binds to and regulates Raf-1 kinase.
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Brown KM, Day JP, Huston E, Zimmermann B, Hampel K, Christian F, Romano D, Terhzaz S, Lee LC, Willis MJ, Morton DB, Beavo JA, Shimizu-Albergine M, Davies SA, Kolch W, Houslay MD, and Baillie GS
- Subjects
- 3',5'-Cyclic-AMP Phosphodiesterases genetics, Animals, Blotting, Western, DNA Primers genetics, Drosophila melanogaster, Gene Deletion, HEK293 Cells, HeLa Cells, Humans, Immunoprecipitation, MAP Kinase Signaling System genetics, Mass Spectrometry, Mice, Mice, Knockout, Mutagenesis, Site-Directed, Phosphorylation, Surface Plasmon Resonance, 3',5'-Cyclic-AMP Phosphodiesterases metabolism, MAP Kinase Signaling System physiology, Proto-Oncogene Proteins c-raf metabolism
- Abstract
V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) is a key activator of the ERK pathway and is a target for cross-regulation of this pathway by the cAMP signaling system. The cAMP-activated protein kinase, PKA, inhibits Raf-1 by phosphorylation on S259. Here, we show that the cAMP-degrading phosphodiesterase-8A (PDE8A) associates with Raf-1 to protect it from inhibitory phosphorylation by PKA, thereby enhancing Raf-1's ability to stimulate ERK signaling. PDE8A binds to Raf-1 with high (picomolar) affinity. Mapping of the interaction domain on PDE8A using peptide array technology identified amino acids 454-465 as the main binding site, which could be disrupted by mutation. A cell-permeable peptide corresponding to this region disrupted the PDE8A/Raf-1 interaction in cells, thereby reducing ERK activation and the cellular response to EGF. Overexpression of a catalytically inactive PDE8A in cells displayed a dominant negative phenotype on ERK activation. These effects were recapitulated at the organism level in genetically modified (PDE8A(-/-)) mice. Similarly, PDE8 deletion in Drosophila melanogaster reduced basal ERK activation and sensitized flies to stress-induced death. We propose that PDE8A is a physiological regulator of Raf-1 signaling in some cells.
- Published
- 2013
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12. Enzyme assays for cGMP hydrolyzing phosphodiesterases.
- Author
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Rybalkin SD, Hinds TR, and Beavo JA
- Subjects
- Calorimetry methods, Cyclic AMP metabolism, Hydrolysis, Kinetics, Substrate Specificity, Temperature, Cyclic GMP metabolism, Enzyme Assays methods, Phosphoric Diester Hydrolases metabolism
- Abstract
Cyclic nucleotides (cAMP and cGMP) as second messengers regulate a wide variety of biological processes such as cellular growth, secretary signaling, and neuroplasticity. These processes can be regulated by increasing the synthesis of cyclic nucleotides (cyclases), by regulation of cAMP and cGMP effector proteins such as cAMP- and cGMP-dependent protein kinases, or by regulation of cyclic nucleotide degradation via cyclic nucleotide phosphodiestases (PDEs). At present PDEs are classified into 11 gene families, each containing several different isoforms and splice variants. All PDEs share considerable homology in their catalytic domains but substantially differ in their N-terminal regions, that contain different types of regulatory. The different PDEs show complex substrate specificity. PDE5, PDE6, and PDE9 are considered to be cGMP specific, while PDE1, PDE2, PDE3, PDE10, and PDE11 can hydrolyze both cGMP and cAMP. PDE4, PDE7, and PDE8 use mainly cAMP as their substrates at physiological substrate levels. Here we describe two methods designed for measuring cGMP (cAMP) hydrolytic activities. The first one is a traditional method using radioactive substrates and the second one is a recently developed nonradioactive method based on Isothermal Titration Calorimetry.
- Published
- 2013
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13. Inactivation of Pde8b enhances memory, motor performance, and protects against age-induced motor coordination decay.
- Author
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Tsai LC, Chan GC, Nangle SN, Shimizu-Albergine M, Jones GL, Storm DR, Beavo JA, and Zweifel LS
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- 3',5'-Cyclic-AMP Phosphodiesterases metabolism, Age Factors, Animals, Anxiety genetics, Brain enzymology, Brain metabolism, Conditioning, Psychological, Fear, Gene Expression, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Motor Activity physiology, 3',5'-Cyclic-AMP Phosphodiesterases genetics, Memory, Motor Activity genetics
- Abstract
Phosphodiesterases (PDEs) are critical regulatory enzymes in cyclic nucleotide signaling. PDEs have diverse expression patterns within the central nervous system (CNS), show differing affinities for cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), and regulate a vast array of behaviors. Here, we investigated the expression profile of the PDE8 gene family members Pde8a and Pde8b in the mouse brain. We find that Pde8a expression is largely absent in the CNS; by contrast, Pde8b is expressed in select regions of the hippocampus, ventral striatum, and cerebellum. Behavioral analysis of mice with Pde8b gene inactivation (PDE8B KO) demonstrate an enhancement in contextual fear, spatial memory, performance in an appetitive instrumental conditioning task, motor-coordination, and have an attenuation of age-induced motor coordination decline. In addition to improvements observed in select behaviors, we find basal anxiety levels to be increased in PDE8B KO mice. These findings indicate that selective antagonism of PDE8B may be an attractive target for enhancement of cognitive and motor functions; however, possible alterations in affective state will need to be weighed against potential therapeutic value., (© 2012 The Authors. Genes, Brain and Behavior © 2012 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.)
- Published
- 2012
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14. Sildenafil reduces respiratory muscle weakness and fibrosis in the mdx mouse model of Duchenne muscular dystrophy.
- Author
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Percival JM, Whitehead NP, Adams ME, Adamo CM, Beavo JA, and Froehner SC
- Subjects
- Animals, Creatine Kinase blood, Cyclic GMP metabolism, Diaphragm metabolism, Diaphragm pathology, Disease Models, Animal, Evans Blue metabolism, Fibrosis etiology, Male, Mice, Mice, Inbred C57BL, Mice, Inbred mdx, Muscle Contraction drug effects, Muscle Fatigue drug effects, Muscle Fatigue physiology, Muscle Weakness etiology, Muscular Dystrophy, Duchenne complications, Muscular Dystrophy, Duchenne pathology, Nitric Oxide metabolism, Purines pharmacology, Sildenafil Citrate, Diaphragm drug effects, Fibrosis drug therapy, Muscle Weakness drug therapy, Muscular Dystrophy, Duchenne drug therapy, Phosphodiesterase 5 Inhibitors pharmacology, Piperazines pharmacology, Sulfones pharmacology
- Abstract
Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy caused by mutations in the dystrophin gene. Loss of dystrophin initiates a progressive decline in skeletal muscle integrity and contractile capacity which weakens respiratory muscles including the diaphragm, culminating in respiratory failure, the leading cause of morbidity and mortality in DMD patients. At present, corticosteroid treatment is the primary pharmacological intervention in DMD, but has limited efficacy and adverse side effects. Thus, there is an urgent need for new safe, cost-effective, and rapidly implementable treatments that slow disease progression. One promising new approach is the amplification of nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signalling pathways with phosphodiesterase 5 (PDE5) inhibitors. PDE5 inhibitors serve to amplify NO signalling that is attenuated in many neuromuscular diseases including DMD. We report here that a 14-week treatment of the mdx mouse model of DMD with the PDE5 inhibitor sildenafil (Viagra(®), Revatio(®)) significantly reduced mdx diaphragm muscle weakness without impacting fatigue resistance. In addition to enhancing respiratory muscle contractility, sildenafil also promoted normal extracellular matrix organization. PDE5 inhibition slowed the establishment of mdx diaphragm fibrosis and reduced matrix metalloproteinase-13 (MMP-13) expression. Sildenafil also normalized the expression of the pro-fibrotic (and pro-inflammatory) cytokine tumour necrosis factor α (TNFα). Sildenafil-treated mdx diaphragms accumulated significantly less Evans Blue tracer dye than untreated controls, which is also indicative of improved diaphragm muscle health. We conclude that sildenafil-mediated PDE5 inhibition significantly reduces diaphragm respiratory muscle dysfunction and pathology in the mdx mouse model of Duchenne muscular dystrophy. This study provides new insights into the therapeutic utility of targeting defects in NO-cGMP signalling with PDE5 inhibitors in dystrophin-deficient muscle., (Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)
- Published
- 2012
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15. Regulation of adrenal steroidogenesis by the high-affinity phosphodiesterase 8 family.
- Author
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Tsai LC and Beavo JA
- Subjects
- Adrenal Cortex cytology, Animals, Corticosterone urine, Humans, Phosphodiesterase Inhibitors pharmacology, 3',5'-Cyclic-AMP Phosphodiesterases metabolism, Adrenal Cortex metabolism, Multigene Family, Steroids biosynthesis
- Abstract
The main function of cyclic AMP phosphodiesterases (PDEs) is to degrade cAMP, a ubiquitous second messenger. Therefore, PDEs can function as prime regulators of cAMP/PKA-dependent processes such as steroidogenesis. Until recently, the roles of the PDE8 family have been largely unexplored, presumably due to the lack of a selective inhibitor. This review focuses on recent reports about the regulatory roles of the PDE8 family in adrenal steroidogenesis, as well as the inhibitory properties and specificity of a new PDE8-selective inhibitor, PF-04957325. We also describe a method of measuring urinary corticosterone levels in vivo as a minimally invasive way of monitoring the stress level in a mouse., (© Georg Thieme Verlag KG Stuttgart · New York.)
- Published
- 2012
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16. cAMP-specific phosphodiesterases 8A and 8B, essential regulators of Leydig cell steroidogenesis.
- Author
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Shimizu-Albergine M, Tsai LC, Patrucco E, and Beavo JA
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- 3',5'-Cyclic-AMP Phosphodiesterases genetics, Animals, Immunoprecipitation, Isoenzymes genetics, Leydig Cells enzymology, Male, Mice, Mice, Knockout, 3',5'-Cyclic-AMP Phosphodiesterases metabolism, Isoenzymes metabolism, Leydig Cells metabolism, Steroids biosynthesis
- Abstract
Phosphodiesterase (PDE) 8A and PDE8B are high-affinity, cAMP-specific phosphodiesterases that are highly expressed in Leydig cells. PDE8A is largely associated with mitochondria, whereas PDE8B is broadly distributed in the cytosol. We used a new, PDE8-selective inhibitor, PF-04957325, and genetically ablated PDE8A(-/-), PDE8B(-/-) and PDE8A(-/-)/B(-/-) mice to determine roles for these PDEs in the regulation of testosterone production. PF-04957325 treatment of WT Leydig cells or MA10 cells increased steroid production but had no effect in PDE8A (-/-)/B(-/-) double-knockout cells, confirming the selectivity of the drug. Moreover, under basal conditions, cotreatment with PF-04957325 plus rolipram, a PDE4-selective inhibitor, synergistically potentiated steroid production. These results suggest that the pool(s) of cAMP regulating androgen production are controlled by PDE8s working in conjunction with PDE4. Likewise, PDE8A (-/-)/B(-/-) cells had higher testosterone production than cells from either PDE8A(-/-) or PDE8B(-/-) mice, suggesting that both PDE8s work in concert to regulate steroid production. We further demonstrate that combined inhibition of PDE8s and PDE4 greatly increased PKA activity including phosphorylation of cholesterol-ester hydrolase (CEH)/hormone-sensitive lipase (HSL). CEH/HSL phosphorylation also was increased in PDE8A(-/-)/B(-/-) cells compared with WT cells. Finally, combined inhibition of PDE8s and PDE4 increased the expression of steroidogenic acute regulatory (StAR) protein. Together these findings suggest that both PDE8A and PDE8B play essential roles to maintain low cAMP levels, thereby suppressing resting steroidogenesis by keeping CEH/HSL inactive and StAR protein expression low. They also suggest that in order for PDE inhibitor therapy to be an effective stimulator of steroidogenesis, both PDE8 isozymes and PDE4 need to be simultaneously targeted.
- Published
- 2012
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17. The roles of cyclic nucleotide phosphodiesterases (PDEs) in steroidogenesis.
- Author
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Tsai LC and Beavo JA
- Subjects
- 3',5'-Cyclic-AMP Phosphodiesterases genetics, 3',5'-Cyclic-GMP Phosphodiesterases genetics, Adrenal Cortex enzymology, Adrenal Cortex metabolism, Adrenal Hyperplasia, Congenital enzymology, Adrenal Hyperplasia, Congenital genetics, Animals, Atrial Natriuretic Factor metabolism, Cushing Syndrome enzymology, Cushing Syndrome genetics, Cyclic AMP physiology, Cyclic GMP physiology, Female, Humans, Isoenzymes genetics, Isoenzymes metabolism, Leydig Cells enzymology, Leydig Cells metabolism, Male, Mutation, Second Messenger Systems, 3',5'-Cyclic-AMP Phosphodiesterases metabolism, 3',5'-Cyclic-GMP Phosphodiesterases metabolism, Aldosterone metabolism, Corticosterone metabolism, Testosterone metabolism
- Abstract
The second messenger, cAMP, is one of the most important regulatory signals for control of steroidogenesis. This review focuses on current knowledge about regulation of cyclic nucleotides by phosphodiesterases (PDEs) in steroidogenic tissues. The first PDE known to directly regulate steroidogenesis was PDE2, the cGMP-stimulated PDE. PDE2 mediates ANP/cGMP-induced decreases in aldosterone production. Recently, the PDE8 family has been shown to control steroidogenesis in two tissues. Specifically, PDE8A regulates testosterone production by itself and in concert with additional IBMX-sensitive PDEs. PDE8B modulates basal corticosterone synthesis via acute and chronic mechanisms. In addition to cAMP-dependent pathways, cGMP signaling also can promote steroidogenesis, and PDE5 modulates this process. Finally, PDE mutations may lead to several human diseases characterized by abnormal steroid levels., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
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18. Kaempferia parviflora, a plant used in traditional medicine to enhance sexual performance contains large amounts of low affinity PDE5 inhibitors.
- Author
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Temkitthawon P, Hinds TR, Beavo JA, Viyoch J, Suwanborirux K, Pongamornkul W, Sawasdee P, and Ingkaninan K
- Subjects
- Animals, Chickens, Cyclic Nucleotide Phosphodiesterases, Type 6 metabolism, Humans, Lung enzymology, Male, Mice, Molecular Structure, Phosphodiesterase 5 Inhibitors chemistry, Phosphodiesterase 5 Inhibitors isolation & purification, Plant Extracts chemistry, Plant Extracts isolation & purification, Plants, Medicinal, Retina enzymology, Rhizome, Structure-Activity Relationship, Cyclic Nucleotide Phosphodiesterases, Type 5 metabolism, Penile Erection drug effects, Phosphodiesterase 5 Inhibitors pharmacology, Plant Extracts pharmacology, Zingiberaceae chemistry
- Abstract
Aim of the Study: A number of medicinal plants are used in traditional medicine to treat erectile dysfunction. Since cyclic nucleotide PDEs inhibitors underlie several current treatments for this condition, we sought to show whether these plants might contain substantial amounts of PDE5 inhibitors., Materials and Methods: Forty one plant extracts and eight 7-methoxyflavones from Kaempferia parviflora Wall. ex Baker were screened for PDE5 and PDE6 inhibitory activities using the two-step radioactive assay. The PDE5 and PDE6 were prepared from mice lung and chicken retinas, respectively. All plant extracts were tested at 50 μg/ml whereas the pure compounds were tested at 10 μM., Results: From forty one plant extracts tested, four showed the PDE5 inhibitory effect. The chemical constituents isolated from rhizomes of Kaempferia parviflora were further investigated on inhibitory activity against PDE5 and PDE6. The results showed that 7-methoxyflavones from this plant showed inhibition toward both enzymes. The most potent PDE5 inhibitor was 5,7-dimethoxyflavone (IC(50) = 10.64 ± 2.09 μM, selectivity on PDE5 over PDE6 = 3.71). Structure activity relationship showed that the methoxyl group at C-5 position of 7-methoxyflavones was necessary for PDE5 inhibition., Conclusions: Kaempferia parviflora rhizome extract and its 7-methoxyflavone constituents had moderate inhibitory activity against PDE5. This finding provides an explanation for enhancing sexual performance in the traditional use of Kaempferia parviflora. Moreover, 5,7-dimethoxyflavones should make a useful lead compound to further develop clinically efficacious PDE5 inhibitors., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2011
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19. The high-affinity cAMP-specific phosphodiesterase 8B controls steroidogenesis in the mouse adrenal gland.
- Author
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Tsai LC, Shimizu-Albergine M, and Beavo JA
- Subjects
- 3',5'-Cyclic-AMP Phosphodiesterases deficiency, 3',5'-Cyclic-AMP Phosphodiesterases metabolism, Adrenal Cortex metabolism, Animals, Cells, Cultured, Cyclic AMP physiology, Mice, Mice, Inbred C57BL, Mice, Knockout, Protein Binding genetics, 3',5'-Cyclic-AMP Phosphodiesterases physiology, Adrenal Cortex enzymology, Steroids biosynthesis
- Abstract
The functions of the phosphodiesterase 8B (PDE8) family of phosphodiesterases have been largely unexplored because of the unavailability of selective pharmacological inhibitors. Here, we report a novel function of PDE8B as a major regulator of adrenal steroidogenesis using a genetically ablated PDE8B mouse model as well as cell lines treated with either a new PDE8-selective inhibitor or a short hairpin RNA (shRNA) construct against PDE8B. We demonstrate that PDE8B is highly enriched in mouse adrenal fasciculata cells, and show that PDE8B knockout mice have elevated urinary corticosterone as a result of adrenal hypersensitivity toward adrenocorticotropin. Likewise, ablation of PDE8B mRNA transcripts by an shRNA construct potentiates steroidogenesis in the commonly used Y-1 adrenal cell line. We also observed that the PDE8-selective inhibitor (PF-04957325) potentiates adrenocorticotropin stimulation of steroidogenesis by increasing cAMP-dependent protein kinase activity in both primary isolated adrenocortical cells and Y-1 cells. It is noteworthy that PDE8s have their greatest control under low adrenocorticotropin-stimulated conditions, whereas other higher K(m) PDE(s) modulate steroidogenesis more effectively when cells are fully stimulated. Finally, both genetic ablation of PDE8B and long-term pharmacological inhibition of PDE8s cause increased expression of steroidogenic enzymes. We conclude that PDE8B is a major regulator of one or more pools of cAMP that promote steroidogenesis via both short- and long-term mechanisms. These findings further suggest PDE8B as a potential therapeutic target for the treatment of several different adrenal diseases.
- Published
- 2011
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20. Regulation of endothelial barrier function by cyclic nucleotides: the role of phosphodiesterases.
- Author
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Surapisitchat J and Beavo JA
- Subjects
- Animals, Humans, Permeability, Signal Transduction, Cyclic AMP physiology, Cyclic GMP physiology, Endothelium, Vascular metabolism, Phosphoric Diester Hydrolases physiology
- Abstract
The endothelium plays an important role in maintaining normal vascular function. Endothelial barrier dysfunction leading to increased permeability and vascular leakage is associated with several pathological conditions such as edema and sepsis. Thus, the development of drugs that improve endothelial barrier function is an active area of research. In this chapter, the current knowledge concerning the signaling pathways regulating endothelial barrier function is discussed with a focus on cyclic nucleotide second messengers (cAMP and cGMP) and cyclic nucleotide phosphodiesterases (PDEs). Both cAMP and cGMP have been shown to have differential effects on endothelial permeability in part due to the various effector molecules, crosstalk, and compartmentalization of cyclic nucleotide signaling. PDEs, by controlling the amplitude, duration, and localization of cyclic nucleotides, have been shown to play a critical role in regulating endothelial barrier function. Thus, PDEs are attractive drug targets for the treatment of disease states involving endothelial barrier dysfunction.
- Published
- 2011
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21. Evaluation of the therapeutic utility of phosphodiesterase 5A inhibition in the mdx mouse model of duchenne muscular dystrophy.
- Author
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Percival JM, Adamo CM, Beavo JA, and Froehner SC
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- Animals, Cyclic GMP physiology, Disease Models, Animal, Mice, Mice, Inbred mdx, Muscle, Skeletal enzymology, Muscular Dystrophy, Duchenne etiology, Myocytes, Cardiac enzymology, Nitric Oxide physiology, Nitric Oxide Synthase Type I physiology, Signal Transduction, Cyclic Nucleotide Phosphodiesterases, Type 5 physiology, Muscular Dystrophy, Duchenne drug therapy, Phosphodiesterase 5 Inhibitors therapeutic use
- Abstract
Duchenne muscular dystrophy (DMD) is a devastating and ultimately fatal disease characterized by progressive muscle wasting and weakness. DMD is caused by the absence of a functional dystrophin protein, which in turn leads to reduced expression and mislocalization of dystrophin-associated proteins including neuronal nitric oxide (NO) synthase mu (nNOSμ). Disruption of nNOSμ signaling results in muscle fatigue and unopposed sympathetic vasoconstriction during exercise, thereby increasing contraction-induced damage in dystrophin-deficient muscles. The loss of normal nNOSμ signaling during exercise is central to the vascular dysfunction proposed over 40 years ago to be an important pathogenic mechanism in DMD. Recent preclinical studies focused on circumventing defective nNOSμ signaling in dystrophic skeletal and cardiac muscle by inhibiting phosphodiesterase 5A (PDE5A) have shown promising results. This review addresses nNOS signaling in normal and dystrophin-deficient muscles and the potential of PDE5A inhibition as a therapeutic approach for the treatment of cardiovascular deficits in DMD.
- Published
- 2011
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22. Cyclic nucleotides and phosphodiesterases in monocytic differentiation.
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Hertz AL and Beavo JA
- Subjects
- Animals, Cell Differentiation drug effects, Dendritic Cells cytology, Granulocyte-Macrophage Colony-Stimulating Factor pharmacology, Humans, Macrophage Colony-Stimulating Factor pharmacology, Osteoclasts cytology, Phosphodiesterase Inhibitors pharmacology, Monocytes cytology, Nucleotides, Cyclic physiology, Phosphoric Diester Hydrolases physiology
- Abstract
Monocytes are immune cells that can differentiate into a number of cell types including macrophages, dendritic cells, and osteoclasts upon exposure to various cytokines. The phenotypes of these differentiated cells are highly heterogeneous and their differentiation can be affected by the cyclic nucleotides, 3'-5'-cyclic adenosine monophosphate (cAMP) and 3'-5'-cyclic guanosine monophosphate (cGMP). The intracellular levels of cAMP and cGMP are controlled through regulation of production by adenylyl and guanylyl cyclases and through degradation by cyclic nucleotide phosphodiesterases (PDEs). PDE inhibition and subsequent changes in cyclic nucleotide levels can alter the final phenotype of a differentiating monocyte with regards to surface marker expression, gene expression, or changes in secreted chemokine and cytokine levels. The differentiation process itself can also be either inhibited or augmented by changes in cyclic nucleotide levels, depending on the system being studied and the timing of cyclic nucleotide elevation. This chapter explores the effects of PDE inhibition and increases in cGMP and cAMP on monocytic differentiation into osteoclasts, dendritic cells, and macrophages.
- Published
- 2011
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23. Sildenafil reverses cardiac dysfunction in the mdx mouse model of Duchenne muscular dystrophy.
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Adamo CM, Dai DF, Percival JM, Minami E, Willis MS, Patrucco E, Froehner SC, and Beavo JA
- Subjects
- Animals, Cardiomyopathies enzymology, Cardiomyopathies etiology, Cardiomyopathies genetics, Cyclic Nucleotide Phosphodiesterases, Type 5 metabolism, Disease Models, Animal, Dystrophin genetics, Mice, Mice, Inbred mdx, Muscular Dystrophy, Duchenne complications, Muscular Dystrophy, Duchenne enzymology, Muscular Dystrophy, Duchenne genetics, Purines pharmacology, Sildenafil Citrate, Cardiomyopathies drug therapy, Cardiomyopathies physiopathology, Muscular Dystrophy, Duchenne physiopathology, Phosphodiesterase 5 Inhibitors pharmacology, Piperazines pharmacology, Sulfones pharmacology
- Abstract
Duchenne muscular dystrophy (DMD) is a progressive and fatal genetic disorder of muscle degeneration. Patients with DMD lack expression of the protein dystrophin as a result of mutations in the X-linked dystrophin gene. The loss of dystrophin leads to severe skeletal muscle pathologies as well as cardiomyopathy, which manifests as congestive heart failure and arrhythmias. Like humans, dystrophin-deficient mice (mdx mice) show cardiac dysfunction as evidenced by a decrease in diastolic function followed by systolic dysfunction later in life. We have investigated whether sildenafil citrate (Viagra), a phosphodiesterase 5 (PDE5) inhibitor, can be used to ameliorate the age-related cardiac dysfunction present in the mdx mice. By using echocardiography, we show that chronic sildenafil treatment reduces functional deficits in the cardiac performance of aged mdx mice, with no effect on normal cardiac function in WT controls. More importantly, when sildenafil treatment was started after cardiomyopathy had developed, the established symptoms were rapidly reversed within a few days. It is recognized that PDE5 inhibitors can have cardioprotective effects in other models of cardiac damage, but the present study reports a prevention and reversal of pathological cardiac dysfunction as measured by functional analysis in a mouse model of DMD. Overall, the data suggest that PDE5 inhibitors may be a useful treatment for the cardiomyopathy affecting patients with DMD at early and late stages of the disease.
- Published
- 2010
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24. Phosphodiesterase 8A (PDE8A) regulates excitation-contraction coupling in ventricular myocytes.
- Author
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Patrucco E, Albergine MS, Santana LF, and Beavo JA
- Subjects
- Animals, Ion Channel Gating drug effects, Isoproterenol pharmacology, Mice, Myocardium enzymology, Myocytes, Cardiac drug effects, Sarcoplasmic Reticulum drug effects, Sarcoplasmic Reticulum metabolism, 3',5'-Cyclic-AMP Phosphodiesterases metabolism, Excitation Contraction Coupling drug effects, Heart Ventricles cytology, Myocytes, Cardiac enzymology
- Abstract
In ventricular myocytes, activation of protein kinase A (PKA) by 3'-5' cyclic adenosine monophosphate (cAMP) increases the force of contraction by increasing L-type Ca(2+) channel currents (I(Ca)) and sarcoplasmic reticulum (SR) Ca(2+) release during excitation-contraction coupling. Cyclic-nucleotide phosphodiesterases (PDEs) comprise a large family of enzymes whose role in the cell is to regulate the spatial and temporal profile of cAMP signals by controlling the degradation of this second messenger. At present, however, the molecular identity and functional roles of the PDEs expressed in ventricular myocytes are incompletely understood. Here, we tested the hypothesis that PDE8A plays a critical role in the modulation of at least one compartment of cAMP and hence PKA activity during beta-adrenergic receptor (betaAR) activation in ventricular myocytes. Consistent with this hypothesis, we found that PDE8A transcript and protein are expressed in ventricular myocytes. Our data indicate that evoked [Ca(2+)](i) transients and I(Ca) increased to a much larger extent in PDE8A null (PDE8A(-/-)) than in wild-type (WT) myocytes during beta-adrenergic signaling activation. In addition, Ca(2+) spark activity was higher in PDE8A(-/-) than in WT myocytes. Our data indicate that PDE8A is a novel cardiac PDE that controls one or more pools of cAMP implicated in regulation of Ca(2+) movement through cardiomyocyte.
- Published
- 2010
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25. Cardiac hypertrophy is not amplified by deletion of cGMP-dependent protein kinase I in cardiomyocytes.
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Lukowski R, Rybalkin SD, Loga F, Leiss V, Beavo JA, and Hofmann F
- Subjects
- Animals, Base Sequence, Cardiomegaly genetics, Cardiomegaly pathology, Cells, Cultured, Cyclic GMP-Dependent Protein Kinase Type I, Cyclic GMP-Dependent Protein Kinases genetics, DNA Primers genetics, Gene Expression, Isoproterenol pharmacology, Mice, Mice, Knockout, Models, Cardiovascular, Myocytes, Cardiac pathology, Myocytes, Smooth Muscle enzymology, RNA, Messenger genetics, RNA, Messenger metabolism, Signal Transduction, Cardiomegaly enzymology, Cardiomegaly etiology, Cyclic GMP-Dependent Protein Kinases deficiency, Myocytes, Cardiac enzymology
- Abstract
It has been suggested that cGMP kinase I (cGKI) dampens cardiac hypertrophy. We have compared the effect of isoproterenol (ISO) and transverse aortic constriction (TAC) on hypertrophy in WT [control (CTR)] mice, total cGKI-KO mice, and cGKIbeta rescue mice (betaRM) lacking cGKI specifically in cardiomyocytes (CMs). Infusion of ISO did not change the expression of cGKI in the hearts of CTR mice or betaRM but raised the heart weight by approximately 20% in both. An identical hypertrophic growth response was measured in CMs from CTR mice and betaRM and in isolated adult CMs cultured with or without 1 muM ISO. In both genotypes, ISO infusion induced similar changes in the expression of hypertrophy-associated cardiac genes and significant elevation of serum atrial natriuretic peptide and total cardiac cGMP. No differences in cardiac hypertrophy were obtained by 7-day ISO infusion in 4- to 6-week-old conventional cGKI-KO and CTR mice. Furthermore, TAC-induced hypertrophy of CTR mice and betaRM was not different and did not result in changes of the cGMP-hydrolyzing phosphodiesterase activities in hypertropic hearts or CMs. These results strongly suggest that cardiac myocyte cGKI does not affect the development of heart hypertrophy induced by pressure overload or chronic ISO infusion.
- Published
- 2010
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26. Elevated cyclic AMP and PDE4 inhibition induce chemokine expression in human monocyte-derived macrophages.
- Author
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Hertz AL, Bender AT, Smith KC, Gilchrist M, Amieux PS, Aderem A, and Beavo JA
- Subjects
- Activating Transcription Factor 3 physiology, Chemokines genetics, Humans, Macrophages metabolism, Oligonucleotide Array Sequence Analysis, Transcription, Genetic physiology, Chemokines metabolism, Cyclic AMP metabolism, Macrophages drug effects, Monocytes cytology, Phosphodiesterase 4 Inhibitors, Phosphodiesterase Inhibitors pharmacology
- Abstract
Macrophages are central mediators of the innate immune system that can be differentiated from monocytes upon exposure to cytokines. While increased cyclic adenosine monophosphate (cAMP) levels are known to inhibit many lipopolysaccharide-elicited macrophage inflammatory responses, the effects of elevated cAMP on monocyte/macrophage differentiation are not as well understood. We show here that during differentiation, cAMP agonists can cause a large increase in the mRNA and protein levels of several of the pro-inflammatory CXCL and CCL chemokines. The cAMP mediator-exchange protein activated by cAMP (Epac) contributes substantially to the increase in these chemokines. These chemokines are known to play an important role in the regulation of immune responses, particularly regarding the pathogenesis of asthma and chronic obstructive pulmonary disorder. We also found that a selective cAMP-degrading phosphodiesterase (PDE) 4 inhibitor can potentiate the chemokine expression elicited by low-dose forskolin or Prostaglandin E2 (PGE(2)). These data suggest that chemokine receptor antagonists administered in conjunction with a PDE4 inhibitor may improve both the efficacy and safety of PDE4-inhibitor therapy for chronic inflammatory disorders.
- Published
- 2009
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27. Role of Ca2+/calmodulin-stimulated cyclic nucleotide phosphodiesterase 1 in mediating cardiomyocyte hypertrophy.
- Author
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Miller CL, Oikawa M, Cai Y, Wojtovich AP, Nagel DJ, Xu X, Xu H, Florio V, Rybalkin SD, Beavo JA, Chen YF, Li JD, Blaxall BC, Abe J, and Yan C
- Subjects
- Angiotensin II metabolism, Animals, Calcium Signaling drug effects, Cardiomegaly chemically induced, Cardiotonic Agents adverse effects, Cardiotonic Agents pharmacology, Cells, Cultured, Cyclic GMP metabolism, Enzyme Inhibitors adverse effects, Enzyme Inhibitors pharmacology, Gene Expression Regulation, Enzymologic drug effects, Gene Expression Regulation, Enzymologic physiology, Heart Ventricles enzymology, Humans, Isoproterenol adverse effects, Isoproterenol pharmacology, Male, Mice, Rats, Rats, Sprague-Dawley, Second Messenger Systems drug effects, Calcium metabolism, Calcium Signaling radiation effects, Calmodulin metabolism, Cardiomegaly enzymology, Cyclic Nucleotide Phosphodiesterases, Type 1 biosynthesis, Myocytes, Cardiac enzymology, Second Messenger Systems physiology
- Abstract
Rationale: Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca(2+)/calmodulin (CaM)-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca(2+)/CaM and cGMP signaling; however, its function in cardiomyocytes is unknown., Objective: Herein, we investigate the role of Ca(2+)/CaM-stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes and in the heart in vivo., Methods and Results: Inhibition of PDE1 activity using a PDE1-selective inhibitor, IC86340, or downregulation of PDE1A using siRNA prevented phenylephrine induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal and adult rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic isoproterenol infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts; however, PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated neonatal and adult rat ventricular myocytes treated with neurohumoral stimuli such as angiotensin II (Ang II) and isoproterenol. Furthermore, PDE1A plays a critical role in phenylephrine-induced reduction of intracellular cGMP- and cGMP-dependent protein kinase (PKG) activity and thereby cardiomyocyte hypertrophy in vitro., Conclusions: These results elucidate a novel role for Ca(2+)/CaM-stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG-dependent mechanism, thereby demonstrating Ca(2+) and cGMP signaling cross-talk during cardiac hypertrophy.
- Published
- 2009
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28. The structure of the GAF A domain from phosphodiesterase 6C reveals determinants of cGMP binding, a conserved binding surface, and a large cGMP-dependent conformational change.
- Author
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Martinez SE, Heikaus CC, Klevit RE, and Beavo JA
- Subjects
- Allosteric Site, Amino Acid Sequence, Animals, Chickens, Crystallography, X-Ray, Molecular Conformation, Molecular Sequence Data, Protein Binding, Protein Conformation, Protein Folding, Protein Structure, Secondary, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Cyclic GMP chemistry, Cyclic Nucleotide Phosphodiesterases, Type 6 chemistry
- Abstract
The photoreceptor phosphodiesterase (PDE6) regulates the intracellular levels of the second messenger cGMP in the outer segments of cone and rod photoreceptor cells. PDE6 contains two regulatory GAF domains, of which one (GAF A) binds cGMP and regulates the activity of the PDE6 holoenzyme. To increase our understanding of this allosteric regulation mechanism, we present the 2.6A crystal structure of the cGMP-bound GAF A domain of chicken cone PDE6. Nucleotide specificity appears to be provided in part by the orientation of Asn-116, which makes two hydrogen bonds to the guanine ring of cGMP but is not strictly conserved among PDE6 isoforms. The isolated PDE6C GAF A domain is monomeric and does not contain sufficient structural determinants to form a homodimer as found in full-length PDE6C. A highly conserved surface patch on GAF A indicates a potential binding site for the inhibitory subunit Pgamma. NMR studies reveal that the apo-PDE6C GAF A domain is structured but adopts a significantly altered structural state indicating a large conformational change with rearrangement of secondary structure elements upon cGMP binding. The presented crystal structure will help to define the cGMP-dependent regulation mechanism of the PDE6 holoenzyme and its inhibition through Pgamma binding.
- Published
- 2008
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29. Solution structure of the cGMP binding GAF domain from phosphodiesterase 5: insights into nucleotide specificity, dimerization, and cGMP-dependent conformational change.
- Author
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Heikaus CC, Stout JR, Sekharan MR, Eakin CM, Rajagopal P, Brzovic PS, Beavo JA, and Klevit RE
- Subjects
- Binding Sites, Dimerization, Humans, Kinetics, Magnetic Resonance Spectroscopy, Models, Molecular, Protein Conformation, Substrate Specificity, Cyclic GMP metabolism, Cyclic Nucleotide Phosphodiesterases, Type 5 chemistry, Cyclic Nucleotide Phosphodiesterases, Type 5 metabolism
- Abstract
Phosphodiesterase 5 (PDE5) controls intracellular levels of cGMP through its regulation of cGMP hydrolysis. Hydrolytic activity of the C-terminal catalytic domain is increased by cGMP binding to the N-terminal GAF A domain. We present the NMR solution structure of the cGMP-bound PDE5A GAF A domain. The cGMP orientation in the buried binding pocket was defined through 37 intermolecular nuclear Overhauser effects. Comparison with GAF domains from PDE2A and adenylyl cyclase cyaB2 reveals a conserved overall domain fold of a six-stranded beta-sheet and four alpha-helices that form a well defined cGMP binding pocket. However, the nucleotide coordination is distinct with a series of altered binding contacts. The structure suggests that nucleotide binding specificity is provided by Asp-196, which is positioned to form two hydrogen bonds to the guanine ring of cGMP. An alanine mutation of Asp-196 disrupts cGMP binding and increases cAMP affinity in constructs containing only GAF A causing an altered cAMP-bound structural conformation. NMR studies on the tandem GAF domains reveal a flexible GAF A domain in the absence of cGMP, and indicate a large conformational change upon ligand binding. Furthermore, we identify a region of approximately 20 residues directly N-terminal of GAF A as critical for tight dimerization of the tandem GAF domains. The features of the PDE5 regulatory domain revealed here provide an initial structural basis for future investigations of the regulatory mechanism of PDE5 and the design of GAF-specific regulators of PDE5 function.
- Published
- 2008
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30. Cyclic nucleotide analogs as probes of signaling pathways.
- Author
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Poppe H, Rybalkin SD, Rehmann H, Hinds TR, Tang XB, Christensen AE, Schwede F, Genieser HG, Bos JL, Doskeland SO, Beavo JA, and Butt E
- Subjects
- Blood Platelets drug effects, Blood Platelets enzymology, Blood Platelets metabolism, Cells, Cultured, Cyclic AMP-Dependent Protein Kinases metabolism, Cyclic GMP-Dependent Protein Kinases metabolism, Humans, Hydrolysis, Phosphoric Diester Hydrolases metabolism, Substrate Specificity, Cyclic AMP analogs & derivatives, Cyclic AMP pharmacology, Cyclic GMP analogs & derivatives, Cyclic GMP pharmacology, Molecular Probes pharmacology, Signal Transduction drug effects
- Published
- 2008
- Full Text
- View/download PDF
31. Phosphodiesterase type 5: expanding roles in cardiovascular regulation.
- Author
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Kass DA, Champion HC, and Beavo JA
- Subjects
- Animals, Cardiovascular Diseases drug therapy, Cardiovascular Diseases enzymology, Cyclic Nucleotide Phosphodiesterases, Type 5 chemistry, Cyclic Nucleotide Phosphodiesterases, Type 5 genetics, Humans, Phosphodiesterase Inhibitors pharmacology, Phosphodiesterase Inhibitors therapeutic use, Cardiovascular System enzymology, Cyclic Nucleotide Phosphodiesterases, Type 5 physiology
- Abstract
Phosphodiesterase type 5A (PDE5A) selectively hydrolyzes cyclic GMP. Inhibitors of PDE5A such as sildenafil are widely used to treat erectile dysfunction, but growing evidence supports important roles for the enzyme in both the vasculature and heart. In disorders such as cardiac failure, PDE5A upregulation may contribute to a decline in cGMP and protein kinase G signaling, exacerbating dysfunction. PDE5A plays an important role in the pulmonary vasculature where its inhibition benefits patients with pulmonary hypertension. In the heart, PDE5A signaling appears compartmentalized, and its inhibition is cardioprotective against ischemia-reperfusion and antracycline toxicity, blunts acute adrenergic contractile stimulation, and can suppress chronic hypertrophy and dysfunction attributable to pressure-overload. In this review, we discuss the molecular biology, pharmacology, and physiology of PDE5A, mechanisms of vascular and cardiac regulation, and recent evidence supporting the utility of selective PDE5A inhibition for the treatment of cardiovascular disorders.
- Published
- 2007
- Full Text
- View/download PDF
32. Differential regulation of endothelial cell permeability by cGMP via phosphodiesterases 2 and 3.
- Author
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Surapisitchat J, Jeon KI, Yan C, and Beavo JA
- Subjects
- 3',5'-Cyclic-AMP Phosphodiesterases biosynthesis, 3',5'-Cyclic-AMP Phosphodiesterases genetics, Cell Membrane Permeability drug effects, Cyclic GMP genetics, Cyclic Nucleotide Phosphodiesterases, Type 2, Cyclic Nucleotide Phosphodiesterases, Type 3, Endothelial Cells cytology, Endothelium, Vascular cytology, Endothelium, Vascular metabolism, Humans, Phosphoric Diester Hydrolases biosynthesis, Phosphoric Diester Hydrolases genetics, 3',5'-Cyclic-AMP Phosphodiesterases physiology, Cell Membrane Permeability physiology, Cyclic GMP physiology, Endothelial Cells metabolism, Phosphoric Diester Hydrolases physiology
- Abstract
Endothelial barrier dysfunction leading to increased permeability and vascular leakage is an underlying cause of several pathological conditions, including edema and sepsis. Whereas cAMP has been shown to decrease endothelial permeability, the role of cGMP is controversial. Endothelial cells express cGMP-inhibited phosphodiesterase (PDE)3A and cGMP-stimulated PDE2A. Thus we hypothesized that the effect of cGMP on endothelial permeability is dependent on the concentration of cGMP present and on the relative expression levels of PDE2A and PDE3A. When cAMP synthesis was slightly elevated with a submaximal concentration of 7-deacetyl-7-(O-[N-methylpiperazino]-gamma-butyryl)-dihydrochloride-forskolin (MPB-forskolin), we found that low doses of either atrial natriuretic peptide (ANP) or NO donors potentiated the inhibitory effects of MPB-forskolin on thrombin-induced permeability. However, this inhibitory effect of forskolin was reversed at higher doses of ANP or NO. These data suggest that cGMP at lower concentrations inhibits PDE3A and thereby increases a local pool of cAMP, whereas higher concentrations cGMP activates PDE2A, reversing the effect. Inhibitors of PDE3A mimicked the effect of low-dose ANP on thrombin-induced permeability, and inhibition of PDE2A reversed the stimulation of permeability seen with higher doses of ANP. Finally, increasing PDE2A expression with tumor necrosis factor-alpha reversed the inhibition of permeability caused by low doses of ANP. As predicted, the effect of tumor necrosis factor-alpha on permeability was reversed by a PDE2A inhibitor. These findings suggest that the effect of increasing concentrations of cGMP on endothelial permeability is biphasic, which, in large part, is attributable to the relative amounts of PDE2A and PDE3A in endothelial cells.
- Published
- 2007
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33. Cyclic nucleotide signaling mechanisms in trypanosomes: possible targets for therapeutic agents.
- Author
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Laxman S and Beavo JA
- Subjects
- Adenylyl Cyclases chemistry, Adenylyl Cyclases metabolism, Animals, Carrier Proteins metabolism, Chagas Disease drug therapy, Chagas Disease parasitology, Cyclic AMP chemistry, Humans, Models, Molecular, Molecular Structure, Phosphoric Diester Hydrolases classification, Phosphoric Diester Hydrolases genetics, Phosphoric Diester Hydrolases metabolism, Phylogeny, Protein Conformation, Protozoan Proteins chemistry, Protozoan Proteins metabolism, RNA Interference, Trypanosoma pathogenicity, Trypanosomiasis, African drug therapy, Trypanosomiasis, African parasitology, Cyclic AMP metabolism, Second Messenger Systems physiology, Trypanocidal Agents pharmacology, Trypanocidal Agents therapeutic use, Trypanosoma drug effects, Trypanosoma metabolism
- Abstract
Trypanosome infections cause several major human diseases, including sleeping sickness and Chagas disease, which affect millions of people in Africa and South America, respectively. Although adenosine 3',5'-monophosphate (cAMP) signaling and regulation have been widely studied in mammalian systems, and these pathways provide targets for the treatment of numerous pathologies, a molecular understanding of cAMP signaling in trypanosomes remains incomplete. Recent studies in these parasites, however, have revealed diverse families of adenylyl cyclase and phosphodiesterase that regulate cAMP concentrations. Importantly, these enzymes differ pharmacologically and biochemically from their mammalian counterparts. In this review, we discuss recent developments, emerging ideas, and gaps in knowledge in this area of research, highlighting aspects of enzymes in the cAMP signaling pathway that may be good targets for antitrypanosomal drug therapy.
- Published
- 2007
- Full Text
- View/download PDF
34. Rosiglitazone inhibits acyl-CoA synthetase activity and fatty acid partitioning to diacylglycerol and triacylglycerol via a peroxisome proliferator-activated receptor-gamma-independent mechanism in human arterial smooth muscle cells and macrophages.
- Author
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Askari B, Kanter JE, Sherrid AM, Golej DL, Bender AT, Liu J, Hsueh WA, Beavo JA, Coleman RA, and Bornfeldt KE
- Subjects
- Amino Acid Sequence, Animals, Aorta, Coenzyme A Ligases genetics, DNA Primers, Humans, Hypoglycemic Agents pharmacology, Macrophages drug effects, Macrophages, Peritoneal drug effects, Macrophages, Peritoneal physiology, Male, Mice, Mice, Inbred C57BL, Molecular Sequence Data, Monocytes physiology, Muscle, Smooth, Vascular drug effects, Peptide Fragments chemistry, Reverse Transcriptase Polymerase Chain Reaction, Rosiglitazone, Coenzyme A Ligases antagonists & inhibitors, Diglycerides metabolism, Fatty Acids metabolism, Macrophages physiology, Muscle, Smooth, Vascular physiology, PPAR gamma physiology, Thiazolidinediones pharmacology, Triglycerides metabolism
- Abstract
Rosiglitazone is an insulin-sensitizing agent that has recently been shown to exert beneficial effects on atherosclerosis. In addition to peroxisome proliferator-activated receptor (PPAR)-gamma, rosiglitazone can affect other targets, such as directly inhibiting recombinant long-chain acyl-CoA synthetase (ACSL)-4 activity. Because it is unknown if ACSL4 is expressed in vascular cells involved in atherosclerosis, we investigated the ability of rosiglitazone to inhibit ACSL activity and fatty acid partitioning in human and murine arterial smooth muscle cells (SMCs) and macrophages. Human and murine SMCs and human macrophages expressed Acsl4, and rosiglitazone inhibited Acsl activity in these cells. Furthermore, rosiglitazone acutely inhibited partitioning of fatty acids into phospholipids in human SMCs and inhibited fatty acid partitioning into diacylglycerol and triacylglycerol in human SMCs and macrophages through a PPAR-gamma-independent mechanism. Conversely, murine macrophages did not express ACSL4, and rosiglitazone did not inhibit ACSL activity in these cells, nor did it affect acute fatty acid partitioning into cellular lipids. Thus, rosiglitazone inhibits ACSL activity and fatty acid partitioning in human and murine SMCs and in human macrophages through a PPAR-gamma-independent mechanism likely to be mediated by ACSL4 inhibition. Therefore, rosiglitazone might alter the biological effects of fatty acids in these cells and in atherosclerosis.
- Published
- 2007
- Full Text
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35. Foxp3-dependent programme of regulatory T-cell differentiation.
- Author
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Gavin MA, Rasmussen JP, Fontenot JD, Vasta V, Manganiello VC, Beavo JA, and Rudensky AY
- Subjects
- 3',5'-Cyclic-AMP Phosphodiesterases genetics, 3',5'-Cyclic-AMP Phosphodiesterases metabolism, Animals, Cell Lineage, Cyclic Nucleotide Phosphodiesterases, Type 3, Female, Forkhead Transcription Factors genetics, Gene Expression Regulation, Homeostasis, Interleukin-12 immunology, Interleukin-12 metabolism, Male, Mice, Signal Transduction, T-Lymphocytes, Regulatory immunology, Cell Differentiation, Forkhead Transcription Factors metabolism, T-Lymphocytes, Regulatory cytology, T-Lymphocytes, Regulatory metabolism
- Abstract
Regulatory CD4+ T cells (Tr cells), the development of which is critically dependent on X-linked transcription factor Foxp3 (forkhead box P3), prevent self-destructive immune responses. Despite its important role, molecular and functional features conferred by Foxp3 to Tr precursor cells remain unknown. It has been suggested that Foxp3 expression is required for both survival of Tr precursors as well as their inability to produce interleukin (IL)-2 and independently proliferate after T-cell-receptor engagement, raising the possibility that such 'anergy' and Tr suppressive capacity are intimately linked. Here we show, by dissociating Foxp3-dependent features from those induced by the signals preceding and promoting its expression in mice, that the latter signals include several functional and transcriptional hallmarks of Tr cells. Although its function is required for Tr cell suppressor activity, Foxp3 to a large extent amplifies and fixes pre-established molecular features of Tr cells, including anergy and dependence on paracrine IL-2. Furthermore, Foxp3 solidifies Tr cell lineage stability through modification of cell surface and signalling molecules, resulting in adaptation to the signals required to induce and maintain Tr cells. This adaptation includes Foxp3-dependent repression of cyclic nucleotide phosphodiesterase 3B, affecting genes responsible for Tr cell homeostasis.
- Published
- 2007
- Full Text
- View/download PDF
36. Modulation of Leydig cell function by cyclic nucleotide phosphodiesterase 8A.
- Author
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Vasta V, Shimizu-Albergine M, and Beavo JA
- Subjects
- 3',5'-Cyclic-AMP Phosphodiesterases chemistry, 3',5'-Cyclic-AMP Phosphodiesterases deficiency, 3',5'-Cyclic-AMP Phosphodiesterases genetics, Animals, In Situ Hybridization, Luteinizing Hormone blood, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Models, Molecular, Protein Structure, Tertiary, 3',5'-Cyclic-AMP Phosphodiesterases metabolism, Leydig Cells enzymology
- Abstract
Leydig cells produce testosterone in the testes under the pulsatile control of pituitary luteinizing hormone (LH). cAMP is the intracellular messenger for LH action on steroidogenesis, and pharmacological evidence indicates that the response to LH can be modulated by cyclic nucleotide phosphodiesterases (PDEs). However the types and roles of the PDEs present in Leydig cells have not been fully defined. We report here that PDE8A is expressed in Leydig cells, and using PDE8A knockout mice we provide evidence that PDE8A is a key regulator of LH signaling and steroidogenesis. A 4-fold increase in the sensitivity to LH for testosterone production was detected in Leydig cells isolated from PDE8A knockout mice. In Leydig cells from wild-type mice, 3-isobutyl-1-methylxanthine, a compound that inhibits all cAMP PDEs except PDE8A, elicited only a small increase in the sensitivity of testosterone production to LH. However, in the PDE8-null mice, the effect of this inhibitor is much more pronounced. These observations indicate that PDE8A and at least one other PDE control the same or a complementary pool of cAMP that mediates LH-regulated steroidogenesis. Overall, these results suggest that pharmacological manipulation of PDE8A, alone or in combination with other PDEs present in Leydig cells, may be exploited to modulate testosterone synthesis and possibly to treat various conditions where the local levels of this androgen need to be altered.
- Published
- 2006
- Full Text
- View/download PDF
37. Hydrolysis products of cAMP analogs cause transformation of Trypanosoma brucei from slender to stumpy-like forms.
- Author
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Laxman S, Riechers A, Sadilek M, Schwede F, and Beavo JA
- Subjects
- Animals, Cell Membrane Permeability drug effects, Etazolate pharmacology, Hydrolysis, Phosphodiesterase Inhibitors chemistry, Phosphodiesterase Inhibitors pharmacology, Phosphoric Diester Hydrolases metabolism, Cyclic AMP analogs & derivatives, Cyclic AMP pharmacology, Trypanosoma brucei brucei drug effects, Trypanosoma brucei brucei growth & development
- Abstract
African sleeping sickness is a disease caused by Trypanosoma brucei. T. brucei proliferate rapidly in the mammalian bloodstream as long, slender forms, but at higher population densities they transform into nondividing, short, stumpy forms. This is thought to be a mechanism adopted by T. brucei to establish a stable host-parasite relationship and to allow a transition into the insect stage of its life cycle. Earlier studies have suggested a role for cAMP in mediating this transformation. In this study, using membrane-permeable nucleotide analogs, we show that it is not the cAMP analogs themselves but rather the hydrolyzed products of membrane-permeable cAMP analogs that prevent proliferation of T. brucei. The metabolic products are more potent than the cAMP analogs, and hydrolysis-resistant cAMP analogs are not antiproliferative. We further show that the antiproliferative effect of these membrane-permeable adenosine analogs is caused by transformation into forms resembling short, stumpy bloodstream forms. These data suggest that the slender-to-stumpy transformation of T. brucei may not be mediated directly by cAMP and also raise the possibility of using such adenosine analogs as antitrypanosomal drugs.
- Published
- 2006
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38. Characterization of a novel cAMP-binding, cAMP-specific cyclic nucleotide phosphodiesterase (TcrPDEB1) from Trypanosoma cruzi.
- Author
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Díaz-Benjumea R, Laxman S, Hinds TR, Beavo JA, and Rascón A
- Subjects
- 3',5'-Cyclic-AMP Phosphodiesterases chemistry, 3',5'-Cyclic-AMP Phosphodiesterases genetics, Amino Acid Sequence, Animals, Catalytic Domain, Cells, Cultured, Cyclic GMP metabolism, Gene Amplification genetics, Gene Expression Regulation, Enzymologic, Humans, Hydrolysis, Kinetics, Molecular Sequence Data, Open Reading Frames genetics, Parasites enzymology, Protein Binding, Protein Structure, Tertiary, Protozoan Proteins metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Sequence Alignment, Sequence Homology, ortho-Aminobenzoates metabolism, 3',5'-Cyclic-AMP Phosphodiesterases metabolism, Cyclic AMP metabolism, Trypanosoma cruzi enzymology
- Abstract
Trypanosoma cruzi, the causative agent of Chagas disease, encodes a number of different cAMP-specific PDE (phosphodiesterase) families. Here we report the identification and characterization of TcrPDEB1 and its comparison with the previously identified TcrPDEB2 (formerly known as TcPDE1). These are two different PDE enzymes of the TcrPDEB family, named in accordance with the recent recommendations of the Nomenclature Committee for Kinetoplast PDEs [Kunz, Beavo, D'Angelo, Flawia, Francis, Johner, Laxman, Oberholzer, Rascon, Shakur et al. (2006) Mol. Biochem. Parasitol. 145, 133-135]. Both enzymes show resistance to inhibition by many mammalian PDE inhibitors, and those that do inhibit do so with appreciable differences in their inhibitor profiles for the two enzymes. Both enzymes contain two GAF (cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA) domains and a catalytic domain highly homologous with that of the T. brucei TbPDE2/TbrPDEB2 family. The N-terminus+GAF-A domains of both enzymes showed significant differences in their affinities for cyclic nucleotide binding. Using a calorimetric technique that allows accurate measurements of low-affinity binding sites, the TcrPDEB2 N-terminus+GAF-A domain was found to bind cAMP with an affinity of approximately 500 nM. The TcrPDEB1 N-terminus+GAF-A domain bound cAMP with a slightly lower affinity of approximately 1 muM. The N-terminus+GAF-A domain of TcrPDEB1 did not bind cGMP, whereas the N-terminus+GAF-A domain of TcrPDEB2 bound cGMP with a low affinity of approximately 3 muM. GAF domains homologous with those found in these proteins were also identified in related trypanosomatid parasites. Finally, a fluorescent cAMP analogue, MANT-cAMP [2'-O-(N-methylanthraniloyl)adenosine-3',5'-cyclic monophosphate], was found to be a substrate for the TcPDEB1 catalytic domain, opening the possibility of using this molecule as a substrate in non-radioactive, fluorescence-based PDE assays, including screening for trypanosome PDE inhibitors.
- Published
- 2006
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39. Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use.
- Author
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Bender AT and Beavo JA
- Subjects
- Animals, Humans, Models, Biological, Models, Molecular, Multigene Family physiology, Phosphodiesterase Inhibitors, Nucleotides, Cyclic metabolism, Phosphoric Diester Hydrolases metabolism, Phosphoric Diester Hydrolases physiology
- Abstract
Cyclic nucleotide phosphodiesterases (PDEs) are enzymes that regulate the cellular levels of the second messengers, cAMP and cGMP, by controlling their rates of degradation. There are 11 different PDE families, with each family typically having several different isoforms and splice variants. These unique PDEs differ in their three-dimensional structure, kinetic properties, modes of regulation, intracellular localization, cellular expression, and inhibitor sensitivities. Current data suggest that individual isozymes modulate distinct regulatory pathways in the cell. These properties therefore offer the opportunity for selectively targeting specific PDEs for treatment of specific disease states. The feasibility of these enzymes as drug targets is exemplified by the commercial and clinical successes of the erectile dysfunction drugs, sildenafil (Viagra), tadalafil (Cialis), and vardenafil (Levitra). PDE inhibitors are also currently available or in development for treatment of a variety of other pathological conditions. In this review the basic biochemical properties, cellular regulation, expression patterns, and physiological functions of the different PDE isoforms will be discussed. How these properties relate to the current and future development of PDE inhibitors as pharmacological agents is especially considered. PDEs hold great promise as drug targets and recent research advances make this an exciting time for the field of PDE research.
- Published
- 2006
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40. PDE1B2 regulates cGMP and a subset of the phenotypic characteristics acquired upon macrophage differentiation from a monocyte.
- Author
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Bender AT and Beavo JA
- Subjects
- Antigens, CD metabolism, Cell Line, Tumor, Cyclic AMP-Dependent Protein Kinases metabolism, Cyclic Nucleotide Phosphodiesterases, Type 1, Humans, Phenotype, Phosphoric Diester Hydrolases genetics, Phosphorylation, RNA Interference, Substrate Specificity, Cell Differentiation, Cyclic GMP metabolism, Macrophages cytology, Macrophages metabolism, Monocytes cytology, Phosphoric Diester Hydrolases metabolism
- Abstract
Monocyte-to-macrophage differentiation with the cytokine granulocyte-macrophage colony-stimulating factor induces expression of the cyclic nucleotide phosphodiesterase PDE1B2. However, what role PDE1B2 plays in macrophage biology has not been elucidated. We have addressed this question by inhibiting PDE1B2 induction by using RNA interference. Using a retrovirus-based system, we created HL-60 stable cell lines that express a short-hairpin RNA targeting PDE1B2. HL-60 cells treated with phorbol-12-myristate-13-acetate differentiate to a macrophage-like phenotype and up-regulate PDE1B2. However, expression of PDE1B2 short hairpin RNA effectively suppresses PDE1B2 mRNA, protein, and activity up-regulation. Using the HL-60 PDE1B2 knockdown cells and agonists for either adenylyl or guanylyl cyclase, it was found that PDE1B2 predominantly regulates cGMP and plays a lesser role in cAMP regulation in response to cyclase agonists. Furthermore, in intact HL-60 cells, PDE1B2 activity can be regulated by changes in Ca+2 levels. Inhibiting PDE1B2 up-regulation does not prevent HL-60 cell differentiation, because several markers of macrophage differentiation are unaffected. However, suppression of PDE1B2 expression alters some aspects of the macrophage-like phenotype, because cell spreading, phagocytic ability, and CD11b expression are augmented. The cAMP analog 8-Bromo-cAMP reverses the changes caused by PDE1B2 knockdown. Also, PDE1B2 knockdown cells have lower basal levels of cAMP and alterations in the phosphorylation state of several probable PKA substrate proteins. Thus, the effects of PDE1B2 on differentiation may ultimately be mediated through decreased cAMP. In conclusion, PDE1B2 regulates a subset of phenotypic changes that occur upon phorbol-12-myristate-13-acetate-induced differentiation and likely also plays a role in differentiated macrophages by regulating agonist-stimulated cGMP levels.
- Published
- 2006
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41. Cyclic nucleotide specific phosphodiesterases of the kinetoplastida: a unified nomenclature.
- Author
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Kunz S, Beavo JA, D'Angelo MA, Flawia MM, Francis SH, Johner A, Laxman S, Oberholzer M, Rascon A, Shakur Y, Wentzinger L, Zoraghi R, and Seebeck T
- Subjects
- Animals, Cyclic AMP, Cyclic GMP, Phosphoric Diester Hydrolases metabolism, Kinetoplastida enzymology, Nucleotides, Cyclic, Phosphoric Diester Hydrolases classification, Terminology as Topic
- Published
- 2006
- Full Text
- View/download PDF
42. Identification of a new variant of PDE1A calmodulin-stimulated cyclic nucleotide phosphodiesterase expressed in mouse sperm.
- Author
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Vasta V, Sonnenburg WK, Yan C, Soderling SH, Shimizu-Albergine M, and Beavo JA
- Subjects
- Alternative Splicing, Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Cloning, Molecular, Cyclic Nucleotide Phosphodiesterases, Type 1, Genetic Variation, Humans, Isoenzymes genetics, Isoenzymes metabolism, Male, Mice, Mice, Inbred C57BL, Molecular Sequence Data, Phosphoric Diester Hydrolases metabolism, Sequence Homology, Amino Acid, Sperm Motility physiology, Sperm Tail enzymology, Testis enzymology, Phosphoric Diester Hydrolases genetics, Spermatozoa enzymology
- Abstract
In mature sperm, cAMP plays an important role as a second messenger regulating functions that include capacitation, the acrosome reaction, motility, and, in some cases, chemosensing. We have cloned from mouse testis a novel calmodulin-stimulated cyclic nucleotide phosphodiesterase 1A isoform, Pde1a_v7 (mmPDE1A7), which arises from an alternative transcription start in the cyclic nucleotide phosphodiesterase 1A gene. The open reading frame is predicted to encode a polypeptide with a molecular mass of 52 kDa. Two further variants of this form, which contain two additional new exons, arise from alternative splicing. Analysis of testis cDNA by real-time polymerase chain reaction (PCR) indicates that the Pde1A_v7 transcript variant is the most abundant. The PDE1A_v7 protein uniquely lacks the first amino-terminal calmodulin-binding domain, but does possess an inhibitory domain and a second calmodulin-binding site shared with other variants. In vitro translation of the corresponding Pde1a_v7 cDNA produced a 52-kDa polypeptide having cyclic nucleotide hydrolytic activity, which was stimulated threefold by calcium-bound calmodulin. Immunoprecipitation of cyclic nucleotide phosphodiesterase 1 activity from detergent extracts of mouse sperm revealed a major protein of the size expected for PDE1A_v7, and the immunocytochemical staining for cyclic nucleotide phosphodiesterase 1A in mouse sperm showed intense immunoreactivity in the tail only. These observations, along with the PCR data, strongly suggest that this new variant PDE1A_v7 is the major form of cyclic nucleotide phosphodiesterase 1A expressed in mature sperm and is therefore likely to play an important role in cyclic nucleotide regulation of mature sperm function.
- Published
- 2005
- Full Text
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43. The cyanobacterial tandem GAF domains from the cyaB2 adenylyl cyclase signal via both cAMP-binding sites.
- Author
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Bruder S, Linder JU, Martinez SE, Zheng N, Beavo JA, and Schultz JE
- Subjects
- Adenylyl Cyclases physiology, Amino Acid Sequence, Binding Sites, Cyclic Nucleotide Phosphodiesterases, Type 2, Dimerization, Molecular Sequence Data, Phosphoric Diester Hydrolases chemistry, Signal Transduction, Adenylyl Cyclases chemistry, Anabaena enzymology, Cyclic AMP metabolism
- Abstract
The tandem GAF domains from the cyanobacterium Anabaena PCC7120 cyaB2 adenylyl cyclase form an antiparallel dimer with cAMP bound to all four binding sites. cAMP binding causes highly cooperative allosteric enzyme activation (>500-fold; EC(50) = 1 microM; Hill coefficient >2.0). The cyaB2 GAF domains, like those of the cyclic nucleotide phosphodiesterases (PDEs), contain conserved NKFDE motifs that when mutated in the PDEs abrogate cyclic nucleotide binding. We mutated the aspartic acids within this motif in cyaB2 to determine which domains were required for signaling. Constructs containing an Asp/Ala mutation in either GAF domain still showed positive cooperative cAMP stimulation but with reduced Hill coefficients. The cyaB2 GAF domain NKFDE motifs contain inserts of 14 (GAF-A) and 19 (GAF-B) amino acids not present in PDE2 or cyaB1. Constructs having these inserts deleted could still be activated by cAMP (23- to 100-fold) but lost all positive cooperative activation, suggesting that the inserts play an important role in domain interaction and/or stabilization of the cAMP-binding pockets. In the shortened constructs, even those with a single Asp/Ala mutation in the NKFDE motifs could still be activated by cAMP. However, in a double Asp/Ala mutant of the shortened construct, stimulation by cAMP was almost completely lost, and the EC(50) shifted far to the right. Overall, the data suggest that in GAF domains without these inserts, only the canonical lysine:aspartate salt bridge keeps the alpha4-helix and the alpha4-beta5 linker that close over the cyclic nucleotide properly oriented, thereby stabilizing the binding pocket. The cyaB2 GAF ensemble appears to be an evolutionary intermediate where both GAF domains still participate in allosteric activation by cAMP.
- Published
- 2005
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44. Crystal structure of the tandem GAF domains from a cyanobacterial adenylyl cyclase: modes of ligand binding and dimerization.
- Author
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Martinez SE, Bruder S, Schultz A, Zheng N, Schultz JE, Beavo JA, and Linder JU
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Binding Sites, Conserved Sequence, Crystallization, Cyclic AMP metabolism, Cyclic GMP metabolism, Dimerization, Enzyme Activation, Hydrogen Bonding, Ligands, Models, Molecular, Molecular Sequence Data, Protein Folding, Adenylyl Cyclases chemistry, Anabaena enzymology
- Abstract
In several species, GAF domains, which are widely expressed small-molecule-binding domains that regulate enzyme activity, are known to bind cyclic nucleotides. However, the molecular mechanism by which cyclic nucleotide binding affects enzyme activity is not known for any GAF domain. In the cyanobacterium, Anabaena, the cyaB1 and cyaB2 genes encode adenylyl cyclases that are stimulated by binding of cAMP to their N-terminal GAF domains. Replacement of the tandem GAF-A/B domains in cyaB1 with the mammalian phosphodiesterase 2A GAF-A/B tandem domains allows regulation of the chimeric protein by cGMP, suggesting a highly conserved mechanism of activation. Here, we describe the 1.9-A crystal structure of the tandem GAF-A/B domains of cyaB2 with bound cAMP and compare it to the previously reported structure of the PDE2A GAF-A/B. Unexpectedly, the cyaB2 GAF-A/B dimer is antiparallel, unlike the parallel dimer of PDE2A. Moreover, there is clear electron density for cAMP in both GAF-A and -B, whereas in PDE2A, cGMP is found only in GAF-B. Phosphate and ribose group contacts are similar to those in PDE2A. However, the purine-binding pockets appear very different from that in PDE2A GAF-B. Differences in the beta2-beta3 loop suggest that this loop confers much of the ligand specificity in this and perhaps in many other GAF domains. Finally, a conserved asparagine appears to be a new addition to the signature NKFDE motif, and a mechanism for this motif to stabilize the cNMP-binding pocket is proposed.
- Published
- 2005
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45. Trypanosome cyclic nucleotide phosphodiesterase 2B binds cAMP through its GAF-A domain.
- Author
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Laxman S, Rascón A, and Beavo JA
- Subjects
- Amino Acid Sequence, Ammonium Sulfate, Animals, Binding Sites, Cell Line, Cyclic GMP metabolism, Cyclic Nucleotide Phosphodiesterases, Type 2, Humans, Kidney cytology, Molecular Sequence Data, Phosphoric Diester Hydrolases chemistry, Point Mutation, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Transfection, Cyclic AMP metabolism, Phosphoric Diester Hydrolases genetics, Phosphoric Diester Hydrolases metabolism, Trypanosoma brucei brucei enzymology
- Abstract
Trypanosoma brucei, the causative agent of sleeping sickness in humans and livestock, expresses at least three cAMP-specific class I phosphodiesterases (PDEs), all of which are essential for survival of the parasite. These PDEs have either one or two N-terminal GAF domains, which in other proteins function as signaling domains. However, neither the functional roles nor ligands for these domains in trypanosome PDEs are known. The present study shows that TbPDE2B, which contains two tandem GAF domains, binds cAMP with high affinity through its GAF-A domain. A purified recombinant N terminus + GAF-A domain binds cAMP with an affinity (Ki) of approximately 16 nM. It also binds cGMP but with a 15-fold lower affinity of approximately 275 nM. The TbPDE2B holoenzyme has a somewhat lower affinity (approximately 55 nM) for cAMP but a greatly lower affinity (approximately 10 microM) for cGMP. This suggests that both the selectivity and affinity for a ligand can be determined not only by the nature of the binding domain but also by the adjacent domains. Additionally, binding of cAMP to the holoenzyme showed positive cooperativity, with a Hill coefficient value of 1.75. However, binding of cGMP to the holoenzyme did not show any cooperativity, suggesting differences in the conformational changes caused by binding of these two cyclic nucleotides with the protein. Point mutation of a key predicted binding site residue (T317A) resulted in a complete loss of high affinity cAMP binding. This mutation increased the apparent Km of the mutant enzyme for substrate without altering the Vmax. A truncated catalytic domain construct of TbPDE2B also exhibited an increased Km, strongly suggesting that cAMP binding to the GAF-A domain can regulate TbPDE2B by allowing the full activity of the enzyme to be expressed. These properties of the GAF-A domain of TbPDE2B thus suggest that it could be a new target for anti-trypanosomal drugs.
- Published
- 2005
- Full Text
- View/download PDF
46. Selective up-regulation of PDE1B2 upon monocyte-to-macrophage differentiation.
- Author
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Bender AT, Ostenson CL, Wang EH, and Beavo JA
- Subjects
- Cell Differentiation, Cells, Cultured, Cyclic Nucleotide Phosphodiesterases, Type 1, Dendritic Cells physiology, Humans, Interleukin-4 pharmacology, Kinetics, Phosphoric Diester Hydrolases genetics, Promoter Regions, Genetic, RNA, Messenger analysis, Transcription Initiation Site, Up-Regulation, Macrophages cytology, Monocytes cytology, Phosphoric Diester Hydrolases physiology
- Abstract
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a major regulator of monocyte to macrophage differentiation. In both humans and mice, the main phenotype of decreased GM-CSF function is pulmonary proteinosis due to aberrant function of alveolar macrophages. Recently, this cytokine has been shown to up-regulate a cyclic nucleotide phosphodiesterase, PDE1B. Two PDE1B variants with unique N-terminal sequences, PDE1B1 and PDE1B2, have been identified. Here, we report that the previously uncharacterized PDE1B2 is selectively increased by GM-CSF by stimulation of transcription at a previously unknown transcriptional start site. Analysis of the exon and intron organization of the PDE1B gene reveals that PDE1B2 has a different N-terminal sequence because of a separate first exon that is located 11.5 kb downstream from the PDE1B1 first exon. By using 5'-RACE, alignment of EST sequences, and a luciferase-reporter system, we provide evidence that PDE1B2 has a separate transcriptional start site from PDE1B1 that can be activated by monocyte differentiation. Furthermore, IL-4 treatment in the presence of GM-CSF, which shifts the differentiation from a macrophage to a dendritic cell phenotype, suppresses the up-regulation of PDE1B2. Induction of PDE1B2 is also found in T cells upon activation by PHA. Therefore, PDE1B2 may have a regulatory role in multiple immune cell types. Last, characterization of the catalytic properties of recombinant PDE1B2 shows that it prefers cGMP over cAMP as a substrate and, thus, is likely to regulate cGMP in macrophages. Also, PDE1B2 has a nearly 3-fold lower EC(50) for activation by calmodulin than PDE1B1.
- Published
- 2005
- Full Text
- View/download PDF
47. Molecular determinants of cGMP binding to chicken cone photoreceptor phosphodiesterase.
- Author
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Huang D, Hinds TR, Martinez SE, Doneanu C, and Beavo JA
- Subjects
- 3',5'-Cyclic-AMP Phosphodiesterases chemistry, 3',5'-Cyclic-AMP Phosphodiesterases genetics, 3',5'-Cyclic-AMP Phosphodiesterases metabolism, 3',5'-Cyclic-GMP Phosphodiesterases chemistry, 3',5'-Cyclic-GMP Phosphodiesterases isolation & purification, Amino Acid Sequence, Animals, Cattle, Chickens, Cyclic Nucleotide Phosphodiesterases, Type 2, Eye Proteins chemistry, Eye Proteins genetics, Eye Proteins isolation & purification, Eye Proteins metabolism, Mice, Models, Molecular, Molecular Sequence Data, Mutation, Protein Binding, Protein Structure, Tertiary, Protein Subunits chemistry, Protein Subunits genetics, Protein Subunits metabolism, Sequence Alignment, 3',5'-Cyclic-GMP Phosphodiesterases genetics, 3',5'-Cyclic-GMP Phosphodiesterases metabolism, Cyclic GMP metabolism, Protein Conformation, Retinal Cone Photoreceptor Cells enzymology
- Abstract
Structural studies on photoreceptor phosphodiesterases type 6 (PDE6s) have been hampered by an inability to express and purify substantial amounts of enzyme. Here we describe bacterial expression and characterization of the chicken cone PDE6 regulatory GAF-A and GAF-B domains. High affinity cGMP binding was found only for GAF-A as predicted from sequence alignments with the GAF domains of PDE2 and PDE5. A homology model of the GAF-A domain of chicken cone PDE6 based on the crystal structure of mouse PDE2A GAF-B was used to identify residues likely to make contact with cGMP. Alanine mutagenesis of 4 of these residues (F123A, D169A, T172A, and T176A) showed that each was absolutely required for cGMP binding. Three of these residues map to the H4 helical structure of the GAF-A domain indicating this region as a key structural component for cGMP binding. Mutagenesis of another residue, S97A, decreased cGMP binding affinity 5-fold. Finally mutagenesis of Glu-124 indicated that it is responsible for part but not all of the high specificity for cGMP binding to PDE6 GAF-A. Since little data is available on the properties of the chicken cone PDE6 holoenzyme, we also characterized the native PDEs of chicken retina. Two histone-activated PDE6 peaks were separated by ion exchange chromatography and identified by mass spectrometry as cone and rod photoreceptor PDE6s, respectively. Both of these PDEs had cGMP binding and kinetic properties similar to their corresponding bovine photoreceptor PDEs. Moreover the cGMP binding properties of chicken cone PDE6 holoenzyme were very similar to those of the bacterially expressed individual GAF-A or GAF-A/B domains.
- Published
- 2004
- Full Text
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48. Specific localized expression of cGMP PDEs in Purkinje neurons and macrophages.
- Author
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Bender AT and Beavo JA
- Subjects
- Animals, Cyclic Nucleotide Phosphodiesterases, Type 5, Cytokines physiology, Humans, Microglia enzymology, Phosphodiesterase I metabolism, Phosphoric Diester Hydrolases metabolism, 3',5'-Cyclic-GMP Phosphodiesterases biosynthesis, Macrophages enzymology, Purkinje Cells enzymology
- Abstract
As cGMP hydrolyzing cyclic nucleotide phosphodiesterases (PDEs) have diverse regulatory and catalytic properties, the specific cGMP PDEs a cell expresses will determine the duration and intensity of a cGMP signal. This, in turn, results in different cellular responses between cell types and tissues. Therefore, identifying which cGMP PDEs are expressed in different tissues and cell types could increase our understanding of physiological and pathological processes. The brain is one area where large numbers of diverse cGMP PDEs are expressed in specific regions and cell types. A case in point is differential expression of cGMP PDEs in neuronal cells. For example, we have recently found that PDE5 is expressed in all Purkinje neurons while PDE1B is expressed in only a subset of these neurons. The expression of PDE2 has also been found to be selective for discrete populations of neurons. Another example of selective cGMP PDE expression is seen with cytokine-induced differentiation of monocytes to macrophages. We have recently discovered that monocyte differentiation with the cytokine macrophage colony-stimulating factor (M-CSF) causes an upregulation of PDE2 and a small increase in PDE1B while granulocyte-macrophage colony-stimulating factor (GM-CSF) causes a large increase in PDE1B but a decrease in PDE2. These same cytokines can influence the phenotype of microglial cells and are likely to affect their expression of cGMP PDEs. In this report, we present recent results from our laboratory and review earlier findings illustrating the concept of highly specific expression of cGMP PDEs and discuss how this may be important for understanding brain function and dysfunction.
- Published
- 2004
- Full Text
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49. A-kinase anchoring proteins interact with phosphodiesterases in T lymphocyte cell lines.
- Author
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Asirvatham AL, Galligan SG, Schillace RV, Davey MP, Vasta V, Beavo JA, and Carr DW
- Subjects
- A Kinase Anchor Proteins, Binding Sites, Cyclic AMP physiology, Cyclic AMP-Dependent Protein Kinases physiology, Humans, Isoenzymes metabolism, Jurkat Cells, Phosphoproteins genetics, Repressor Proteins genetics, Tumor Suppressor Proteins genetics, Adaptor Proteins, Signal Transducing, Carrier Proteins metabolism, Membrane Proteins metabolism, Phosphoric Diester Hydrolases metabolism, Proteins metabolism, T-Lymphocytes enzymology
- Abstract
The cAMP protein kinase A (PKA) pathway in T cells conveys an inhibitory signal to suppress inflammation. This study was performed to understand the mechanisms involved in cAMP-mediated signaling in T lymphocytes. A-kinase anchoring proteins (AKAPs) bind and target PKA to various subcellular locations. AKAPs also bind other signaling molecules such as cyclic nucleotide phosphodiesterases (PDEs) that hydrolyze cAMP in the cell. PDE4 and PDE7 have important roles in T cell activation. Based on this information, we hypothesized that AKAPs associate with PDEs in T lymphocytes. Immunoprecipitation of Jurkat cell lysates with Abs against both the regulatory subunit of PKA (RIIalpha) and specific AKAPs resulted in increased PDE activity associated with RIIalpha and AKAP95, AKAP149, and myeloid translocation gene (MTG) compared with control (IgG). Immunoprecipitation and pull-down analyses demonstrate that PDE4A binds to AKAP149, AKAP95, and MTG, but not AKAP79, whereas PDE7A was found to bind only MTG. Further analysis of MTG/PDE association illustrated that PDE4A and PDE7A bind residues 1-344 of MTG16b. Confocal analysis of HuT 78 cells stained with anti-PDE7A showed overlapping staining patterns with the Golgi marker GM130, suggesting that PDE7A is located in the Golgi. The staining pattern of PDE7A also showed similarity to the staining pattern of MTG, supporting the immunoprecipitation data and suggesting that MTG may interact with PDE7A in the Golgi. In summary, these data suggest that AKAPs interact with both PKA and PDE in T lymphocytes and thus are a key component of the signaling complex regulating T cell activation.
- Published
- 2004
- Full Text
- View/download PDF
50. Molecular determinants for cyclic nucleotide binding to the regulatory domains of phosphodiesterase 2A.
- Author
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Wu AY, Tang XB, Martinez SE, Ikeda K, and Beavo JA
- Subjects
- Animals, Base Sequence, Binding, Competitive, Catalysis, Cloning, Molecular, Cyclic Nucleotide Phosphodiesterases, Type 2, DNA Primers, Mice, Models, Molecular, Mutagenesis, Site-Directed, 3',5'-Cyclic-AMP Phosphodiesterases metabolism, Cyclic AMP metabolism, Cyclic GMP metabolism
- Abstract
Binding of cGMP to the GAF-B domain of phosphodiesterase 2A allosterically activates catalytic activity. We report here a series of mutagenesis studies on the GAF-B domain of PDE2A that support a novel mechanism for molecular recognition of cGMP. Alanine mutations of Phe-438, Asp-439, and Thr-488, amino acids that interact with the pyrimidine ring, decrease cGMP affinity slightly but increase cAMP affinity by up to 8-fold. Each interaction is required to provide for cAMP/cGMP specificity. Mutations of any of the residues that interact with the phosphate-ribose moiety or the imidazole ring abolish cGMP binding. Thus, residues that interact with the pyrimidine ring collectively control cAMP/cGMP specificity, whereas residues that bind the phosphate-ribose moiety and imidazole ring are critical for high affinity binding. Similar decreases in binding were found for mutations made in a bacterially expressed GAF-A/B plus catalytic domain construct. Because these constructs had very high catalytic activity, it appears that these mutations did not cause a global denaturation. The affinities of cAMP and cGMP for wild-type GAF-B alone were approximately 4-fold greater than for the holoenzyme, suggesting that the presence of neighboring domains alters the conformation of GAF-B. More importantly, the PDE2A GAF-B, GAF-A/B, GAF-A/B+C domains, and holoenzyme all bind cGMP with much higher affinity than has previously been reported. This high affinity suggests that cGMP binding to PDE2 GAF-B activates the enzyme rapidly, stoichiometrically, and in an all or none fashion, rather than variably over a large range of cyclic nucleotide concentrations.
- Published
- 2004
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