cUMP hydrolysis by PDE3A
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- 作者：Stefan Berrisch ; Jessica Ostermeyer…
- 关键词：Cyclic UMP ; Phosphodiesterase ; PDE3A ; Neonatal rat cardiomyocytes ; HL ; 1 cells
- 刊名：Naunyn-Schmiedeberg's Archives of Pharmacology
- 出版年：2017
- 出版时间：March 2017
- 年：2017
- 卷：390
- 期：3
- 页码：269-280
- 全文大小：
- 刊物类别：Biomedical and Life Sciences
- 刊物主题：Pharmacology/Toxicology; Neurosciences;
- 出版者：Springer Berlin Heidelberg
- ISSN：1432-1912
- 卷排序：390
文摘
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 KM value of ~143 μM and a Vmax value of ~42 μmol/min/mg. PDE3A hydrolyzed cAMP with a KM value of ~0.7 μM and a Vmax of ~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.