2. Academic Validation
  3. cUMP hydrolysis by PDE3A

cUMP hydrolysis by PDE3A

  • Naunyn Schmiedebergs Arch Pharmacol. 2017 Mar;390(3):269-280. doi: 10.1007/s00210-016-1328-1.
Stefan Berrisch 1,  Jessica Ostermeyer 1,  Volkhard Kaever 2,  Solveig Kälble 1,  Denise Hilfiker-Kleiner 3,  Roland Seifert 1,  Erich H Schneider 4
Affiliations

Affiliations

  • 1 Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625, Hannover, Germany.
  • 2 Research Core Unit Metabolomics, Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625, Hannover, Germany.
  • 3 Molecular Cardiology Research Group, Department of Cardiology and Angiology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625, Hannover, Germany.
  • 4 Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625, Hannover, Germany. [email protected].
PMID: 27975297 DOI: 10.1007/s00210-016-1328-1
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.

Keywords

Cyclic UMP; HL-1 cells; Neonatal rat cardiomyocytes; PDE3A; Phosphodiesterase.

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