cUMP hydrolysis by PDE3B.

Previous results indicate that the phosphodiesterase PDE3B hydrolyzes cUMP. Also, almost 50 years ago, cUMP-hydrolytic activity was observed in rat adipose tissue. We intended to characterize the enzyme kinetics of PDE3B-mediated cUMP hydrolysis, to determine the PDE3B binding mode of cUMP, and to analyze cUMP hydrolysis in adipocyte preparations. Educts (cNMPs) and products (NMPs) of the PDE reactions as well as intracellular cNMPs were quantitated by HPLC-coupled tandem mass spectrometry. PDE3B expression was determined by qPCR and Western blot. Docking studies were performed with the PDE3B crystal structure PDB ID 1SO2 (complex with a dihydropyridazine inhibitor). PDE3B hydrolyzed cUMP (Km ~ 550 µM, Vmax ~ 76 µmol/min/mg) and cAMP (Km ~ 0.7 µM, Vmax ~ 4.3 µmol/min/mg) in a milrinone (PDE3-selective inhibitor)-sensitive manner (Ki for inhibition of cUMP hydrolysis: 205 nM). cUMP forms one hydrogen bond with PDE3B (uracil 3-NH with side chain oxygen of Q988). Two hydrogen bonds stabilize cAMP binding. cCMP does not interact with PDE3B. Possibly, the cytosine base cannot form hydrogen bonds with PDE3B, and the 4-NH2 group clashes with L987 of the enzyme. Adipocyte differentiation of 3T3-L1 MBX cells increased mRNA of PDE3B, but not of PDE3A. Significant amounts of cUMP were detected in differentiated and undifferentiated 3T3-L1 MBX cells. 3T3-L1 MBX adipocyte lysates and rat epididymal adipose tissue membranes contained milrinone-sensitive cUMP-hydrolytic activity. PDE3B is a low-affinity and high-velocity phosphodiesterase for cUMP. The cUMP-hydrolyzing activity described almost 50 years ago for rat adipose tissue is caused by PDE3, probably by the isoform PDE3B.

cUMP hydrolysis by PDE3A.

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.