Abstract
As previously reported, the cardiac phosphodiesterase PDE3A hydrolyzes cUMP. Moreover, cUMP-degrading activity was detected in cow and dog hearts several decades ago. Our aim was to characterize the enzyme kinetic parameters of PDE3A-mediated cUMP hydrolysis and to investigate whether cUMP and cUMP-hydrolyzing PDEs are present in cardiomyocytes. PDE3A-mediated cUMP hydrolysis was characterized in time course, inhibitor, and Michaelis-Menten kinetics experiments. Intracellular cyclic nucleotide (cNMP) concentrations and the mRNAs of cUMP-degrading PDEs were quantitated in neonatal rat cardiomyocytes (NRCMs) and murine HL-1 cardiomyogenic cells. Moreover, we investigated cUMP degradation in HL-1 cell homogenates and intact cells. Educts (cNMPs) and products (NMPs) of the PDE reactions were detected by HPLC-coupled tandem mass spectrometry. PDE3A degraded cUMP (measurement of UMP formation) with a K M value of ~143 μM and a V max value of ~42 μmol/min/mg. PDE3A hydrolyzed cAMP with a K M value of ~0.7 μM and a V max of ~1.2 μmol/min/mg (determination of AMP formation). The PDE3 inhibitor milrinone inhibited cUMP hydrolysis (determination of UMP formation) by PDE3A (K i = 57 nM). Significant amounts of cUMP as well as of PDE3A mRNA (in addition to PDE3B and PDE9A transcripts) were detected in HL-1 cells and NRCMs. Although HL-1 cell homogenates contain a milrinone-sensitive cUMP-hydrolyzing activity, intact HL-1 cells may use additional PDE3-independent mechanisms for cUMP disposal. PDE3A is a low-affinity and high-velocity PDE for cUMP. Future studies should investigate biological effects of cUMP in cardiomyocytes and the role of PDE3A in detoxifying high intracellular cUMP concentrations under pathophysiological conditions.
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References
Beckert U, Wolter S, Hartwig C, Bähre H, Kaever V, Ladant D, Frank DW, Seifert R (2014) ExoY from Pseudomonas aeruginosa is a nucleotidyl cyclase with preference for cGMP and cUMP formation. Biochem Biophys Res Commun 450:870–874. doi:10.1016/j.bbrc.2014.06.088
Bender AT, Beavo JA (2006) Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol Rev 58:488–520. doi:10.1124/pr.58.3.5
Bähre H, Danker KY, Stasch JP, Kaever V, Seifert R (2014) Nucleotidyl cyclase activity of soluble guanylyl cyclase in intact cells. Biochem Biophys Res Commun 443:1195–1199. doi:10.1016/j.bbrc.2013.12.108
Bähre H, Hartwig C, Munder A, Wolter S, Stelzer T, Schirmer B, Beckert U, Frank DW, Tümmler B, Kaever V, Seifert R (2015) cCMP and cUMP occur in vivo. Biochem Biophys Res Commun 460:909–914. doi:10.1016/j.bbrc.2015.03.115
Cheng Y, Prusoff WH (1973) Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22:3099–3108
Chung YW, Lagranha C, Chen Y, Sun J, Tong G, Hockman SC, Ahmad F, Esfahani SG, Bae DH, Polidovitch N, Wu J, Rhee DK, Lee BS, Gucek M, Daniels MP, Brantner CA, Backx PH, Murphy E, Manganiello VC (2015) Targeted disruption of PDE3B, but not PDE3A, protects murine heart from ischemia/reperfusion injury. Proc Natl Acad Sci U S A 112:E2253–E2262. doi:10.1073/pnas.1416230112
Claycomb WC, Lanson NA, Stallworth BS, Egeland DB, Delcarpio JB, Bahinski A, Izzo NJ (1998) HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci U S A 95:2979–2984
Dazert P, Meissner K, Vogelgesang S, Heydrich B, Eckel L, Böhm M, Warzok R, Kerb R, Brinkmann U, Schaeffeler E, Schwab M, Cascorbi I, Jedlitschky G, Kroemer HK (2003) Expression and localization of the multidrug resistance protein 5 (MRP5/ABCC5), a cellular export pump for cyclic nucleotides, in human heart. Am J Pathol 163:1567–1577. doi:10.1016/S0002-9440(10)63513-4
Dittmar F, Abdelilah-Seyfried S, Tschirner SK, Kaever V, Seifert R (2015) Temporal and organ-specific detection of cNMPs including cUMP in the zebrafish. Biochem Biophys Res Commun 468:708–712. doi:10.1016/j.bbrc.2015.11.020
Dittmar F, Wolter S, Seifert R (2016) Regulation of apoptosis by cyclic nucleotides in human erythroleukemia (HEL) cells and human myelogenous leukemia (K-562) cells. Biochem Pharmacol 112:13–23. doi:10.1016/j.bcp.2016.04.018
Göttle M, Dove S, Kees F, Schlossmann J, Geduhn J, König B, Shen Y, Tang WJ, Kaever V, Seifert R (2010) Cytidylyl and uridylyl cyclase activity of Bacillus anthracis edema factor and Bordetella pertussis CyaA. Biochemistry 49:5494–5503. doi:10.1021/bi100684g
Hardman JG, Sutherland EW (1965) A cyclic 3′,5′-nucleotide phosphodiesterase from heart with specificity for uridine 3′,5′-phosphate. J Biol Chem 240:3704–3705
Hartwig C, Bähre H, Wolter S, Beckert U, Kaever V, Seifert R (2014) cAMP, cGMP, cCMP and cUMP concentrations across the tree of life: high cCMP and cUMP levels in astrocytes. Neurosci Lett 579:183–187. doi:10.1016/j.neulet.2014.07.019
Hasan A, Danker KY, Wolter S, Bähre H, Kaever V, Seifert R (2014) Soluble adenylyl cyclase accounts for high basal cCMP and cUMP concentrations in HEK293 and B103 cells. Biochem Biophys Res Commun 448:236–240. doi:10.1016/j.bbrc.2014.04.099
Hilfiker-Kleiner D, Kaminski K, Kaminska A, Fuchs M, Klein G, Podewski E, Grote K, Kiian I, Wollert KC, Hilfiker A, Drexler H (2004) Regulation of proangiogenic factor CCN1 in cardiac muscle: impact of ischemia, pressure overload, and neurohumoral activation. Circulation 109:2227–2233. doi:10.1161/01.CIR.0000127952.90508.9D
Ito M, Tanaka T, Saitoh M, Masuoka H, Nakano T, Hidaka H (1988) Selective inhibition of cyclic AMP phosphodiesterase from various human tissues by milrinone, a potent cardiac bipyridine. Biochem Pharmacol 37:2041–2044
Krawutschke C, Koesling D, Russwurm M (2015) Cyclic GMP in vascular relaxation: export is of similar importance as degradation. Arterioscler Thromb Vasc Biol 35:2011–2019. doi:10.1161/ATVBAHA.115.306133
Kuhn M (2015) Cardiology: a big-hearted molecule. Nature 519:416–417. doi:10.1038/nature14373
Laue S, Winterhoff M, Kaever V, van den Heuvel JJ, Russel FG, Seifert R (2014) cCMP is a substrate for MRP5. Naunyn Schmiedeberg’s Arch Pharmacol 387:893–895. doi:10.1007/s00210-014-1018-9
Lee DI, Zhu G, Sasaki T, Cho GS, Hamdani N, Holewinski R, Jo SH, Danner T, Zhang M, Rainer PP, Bedja D, Kirk JA, Ranek MJ, Dostmann WR, Kwon C, Margulies KB, Van Eyk JE, Paulus WJ, Takimoto E, Kass DA (2015) Phosphodiesterase 9A controls nitric-oxide-independent cGMP and hypertrophic heart disease. Nature 519:472–476. doi:10.1038/nature14332
Liu H, Maurice DH (1998) Expression of cyclic GMP-inhibited phosphodiesterases 3A and 3B (PDE3A and PDE3B) in rat tissues: differential subcellular localization and regulated expression by cyclic AMP. Br J Pharmacol 125:1501–1510. doi:10.1038/sj.bjp.0702227
Meacci E, Taira M, Moos M, Smith CJ, Movsesian MA, Degerman E, Belfrage P, Manganiello V (1992) Molecular cloning and expression of human myocardial cGMP-inhibited cAMP phosphodiesterase. Proc Natl Acad Sci U S A 89:3721–3725
Monzel M, Kuhn M, Bähre H, Seifert R, Schneider EH (2014) PDE7A1 hydrolyzes cCMP. FEBS Lett 588:3469–3474. doi:10.1016/j.febslet.2014.08.005
Niiya T, Osawa H, Onuma H, Suzuki Y, Taira M, Yamada K, Makino H (2001) Activation of mouse phosphodiesterase 3B gene promoter by adipocyte differentiation in 3T3-L1 cells. FEBS Lett 505:136–140
Omori K, Kotera J (2007) Overview of PDEs and their regulation. Circ Res 100:309–327. doi:10.1161/01.RES.0000256354.95791.f1
Reinecke D, Burhenne H, Sandner P, Kaever V, Seifert R (2011) Human cyclic nucleotide phosphodiesterases possess a much broader substrate-specificity than previously appreciated. FEBS Lett 585:3259–3262. doi:10.1016/j.febslet.2011.09.004
Ritter CA, Jedlitschky G, Meyerzu Schwabedissen H, Grube M, Köck K, Kroemer HK (2005) Cellular export of drugs and signaling molecules by the ATP-binding cassette transporters MRP4 (ABCC4) and MRP5 (ABCC5). Drug Metab Rev 37:253–278. doi:10.1081/DMR-200047984
Sassi Y, Abi-Gerges A, Fauconnier J, Mougenot N, Reiken S, Haghighi K, Kranias EG, Marks AR, Lacampagne A, Engelhardt S, Hatem SN, Lompre AM, Hulot JS (2012) Regulation of cAMP homeostasis by the efflux protein MRP4 in cardiac myocytes. FASEB J 26:1009–1017. doi:10.1096/fj.11-194027
Seifert R (2015) cCMP and cUMP: emerging second messengers. Trends Biochem Sci 40:8–15. doi:10.1016/j.tibs.2014.10.008
Seifert R, Schneider EH, Bähre H (2015) From canonical to non-canonical cyclic nucleotides as second messengers: pharmacological implications. Pharmacol Ther 148:154–184. doi:10.1016/j.pharmthera.2014.12.002
Sousa C, Botelho C, Rodrigues D, Azeredo J, Oliveira R (2012) Infective endocarditis in intravenous drug abusers: an update. Eur J Clin Microbiol Infect Dis 31:2905–2910. doi:10.1007/s10096-012-1675-x
Stangherlin A, Zoccarato A (2015) Relax: it’s not all about degradation. Arterioscler Thromb Vasc Biol 35:1907–1909. doi:10.1161/ATVBAHA.115.306217
Taira M, Hockman SC, Calvo JC, Belfrage P, Manganiello VC (1993) Molecular cloning of the rat adipocyte hormone-sensitive cyclic GMP-inhibited cyclic nucleotide phosphodiesterase. J Biol Chem 268:18573–18579
Wechsler J, Choi YH, Krall J, Ahmad F, Manganiello VC, Movsesian MA (2002) Isoforms of cyclic nucleotide phosphodiesterase PDE3A in cardiac myocytes. J Biol Chem 277:38072–38078. doi:10.1074/jbc.M203647200
Wolter S, Golombek M, Seifert R (2011) Differential activation of cAMP- and cGMP-dependent protein kinases by cyclic purine and pyrimidine nucleotides. Biochem Biophys Res Commun 415:563–566. doi:10.1016/j.bbrc.2011.10.093
Wolter S, Kloth C, Golombek M, Dittmar F, Försterling L, Seifert R (2015) cCMP causes caspase-dependent apoptosis in mouse lymphoma cell lines. Biochem Pharmacol 98:119–131. doi:10.1016/j.bcp.2015.08.096
Zong X, Krause S, Chen CC, Krüger J, Gruner C, Cao-Ehlker X, Fenske S, Wahl-Schott C, Biel M (2012) Regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel activity by cCMP. J Biol Chem 287:26506–26512. doi:10.1074/jbc.M112.357129
Acknowledgements
We thank Prof. Dr. Ralf Gerhard (Institute of Toxicology, MHH) and Dr. Sabine Wolter (Institute of Pharmacology, MHH) for excellent scientific discussions as well as Ms. Annette Garbe (Research Core Unit Metabolomics, MHH) and Ms. Martina Kasten (Molecular Cardiology Research Group, Department of Cardiology and Angiology, MHH) for outstanding technical support. Moreover, we thank the reviewers for their constructive comments.
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Berrisch, S., Ostermeyer, J., Kaever, V. et al. cUMP hydrolysis by PDE3A. Naunyn-Schmiedeberg's Arch Pharmacol 390, 269–280 (2017). https://doi.org/10.1007/s00210-016-1328-1
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DOI: https://doi.org/10.1007/s00210-016-1328-1