title: cUMP hydrolysis by PDE3A.
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 KMvalue of ~143 μM and a Vmaxvalue of ~42 μmol/min/mg. PDE3A hydrolyzed cAMP with a KMvalue of ~0.7 μM and a Vmaxof ~1.2 μmol/min/mg (determination of AMP formation). The PDE3 inhibitor milrinone inhibited cUMP hydrolysis (determination of UMP formation) by PDE3A (Ki= 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.
authors.affiliation: Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625, Hannover, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Berrisch
authors.affiliation: Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625, Hannover, Germany.
authors.forename: Jessica
authors.initials: J
authors.lastname: Ostermeyer
authors.affiliation: Research Core Unit Metabolomics, Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625, Hannover, Germany.
authors.forename: Volkhard
authors.initials: V
authors.lastname: Kaever
authors.affiliation: Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625, Hannover, Germany.
authors.forename: Solveig
authors.initials: S
authors.lastname: Kälble
authors.affiliation: Molecular Cardiology Research Group, Department of Cardiology and Angiology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625, Hannover, Germany.
authors.forename: Denise
authors.initials: D
authors.lastname: Hilfiker-Kleiner
authors.affiliation: Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625, Hannover, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Seifert
authors.affiliation: Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625, Hannover, Germany. schneider.erich@mh-hannover.de.
authors.forename: Erich H
authors.initials: EH
authors.lastname: Schneider
mesh_terms: D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D008954:Models, Biological; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology*; D009712:Nucleotides, Cyclic / Q000378:metabolism*; D058987:Phosphodiesterase 3 Inhibitors / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014542:Uridine Monophosphate / Q000378:metabolism*
chemical_list: D009712:Nucleotides, Cyclic; D058987:Phosphodiesterase 3 Inhibitors; D011994:Recombinant Proteins; C045338:cyclic 3',5'-uridine monophosphate; D000242:Cyclic AMP; D014542:Uridine Monophosphate; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517287:PDE3A protein, human; C517289:Pde3a protein, mouse; C517290:Pde3a protein, rat
keywords: Cyclic UMP; HL-1 cells; Neonatal rat cardiomyocytes; PDE3A; Phosphodiesterase