Hydrolysis of the non-canonical cyclic nucleotide cUMP by PDE9A: kinetics and binding mode.

The non-canonical cyclic nucleotide cUMP and the phosphodiesterase PDE9A both occur in neuronal cells. Using HPLC-coupled tandem mass spectrometry, we characterized the kinetics of PDE9A-mediated cUMP hydrolysis. PDE9A is a low-affinity and high-velocity enzyme for cUMP (Vmax = ~ 6 µmol/min/mg; Km = ~ 401 µM). The PDE9 inhibitor BAY 73-6691 inhibited PDE9A-catalyzed cUMP hydrolysis (Ki = 590 nM). Docking studies indicate two H-bonds between the cUMP uridine moiety and Gln453/Asn405 of PDE9A. By contrast, the guanosine moiety of cGMP forms three H-bonds with Gln453. cCMP is not hydrolyzed at a concentration of 3 µM, but inhibits the PDE9A-catalyzed cUMP hydrolysis at concentrations of 100 µM or more. The probable main reason is that the cytosine moiety cannot act as H-bond acceptor for Gln453. A comparison of PDE9A with PDE7A suggests that the preference of the former for cGMP and cUMP and of the latter for cAMP and cCMP is due to stabilized alternative conformations of the side chain amide of Gln453 and Gln413, respectively. This so-called glutamine switch is known to be involved in the regulation of cAMP/cGMP selectivity of some PDEs.

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.

Temporal and organ-specific detection of cNMPs including cUMP in the zebrafish.

The cyclic pyrimidine nucleotides cCMP and cUMP occur in mammalian cell lines. Recently, cCMP was also identified in mouse organs. Due to technical difficulties, it has not been possible to detect cUMP in organs or tissues yet. Here, we have generated a temporal profile of the occurrence of nucleoside 3',5'-cyclic monophosphates during different developmental stages of embryogenesis and in different organs of the adult zebrafish Danio rerio. Cyclic nucleotides were quantified by high performance liquid chromatography quadrupole tandem mass spectrometry. The identity of cCMP and cUMP in the zebrafish was confirmed by high performance liquid chromatography quadrupole time-of-flight mass spectrometry. We show for the first time that cUMP can be detected during embryogenesis and in adult organs of this vertebrate model system.

cAMP, cGMP, cCMP and cUMP concentrations across the tree of life: High cCMP and cUMP levels in astrocytes.

Adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) are well-established second messengers, whereas the physiological role of the cyclic pyrimidine nucleotides cytidine 3',5'-cyclic monophosphate (cCMP) and uridine 3',5'-cyclic monophosphate (cUMP) is poorly understood. Certain mammalian nucleotidyl cyclases (NCs) and bacterial NC toxins can generate cCMP and cUMP. Human HEK293 cells and rat B103 neuroblastoma cells are of neuronal origin and possess high basal concentrations of cCMP and cUMP that can be attributed to soluble adenylyl cyclase activity. These data prompted us to conduct a systematic analysis of basal nucleoside 3',5'-cyclic monophosphate (cNMP) concentrations across the tree of life. cCMP and cUMP were identified in many mammalian cell lines and primary cells. cNMP patterns varied broadly among cells, and in several systems, cCMP and cUMP concentrations were quite high. Prokaryotes, fungi, amoeba and invertebrates lacked cCMP and cUMP, whereas cAMP was found across the tree of life. High cCMP and cUMP concentrations were found in astrocytes. The distinct cNMP patterns support specific second messenger roles of cCMP and cUMP, specifically in astrocytes.

ExoY from Pseudomonas aeruginosa is a nucleotidyl cyclase with preference for cGMP and cUMP formation.

In addition to the well known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. Soluble guanylyl cyclase and soluble adenylyl cyclase produce all four cNMPs. Several bacterial toxins exploit mammalian cyclic nucleotide signaling. The type III secretion protein ExoY from Pseudomonas aeruginosa induces severe lung damage and effectively produces cGMP. Here, we show that transfection of mammalian cells with ExoY or infection with ExoY-expressing P. aeruginosa not only massively increases cGMP but also cUMP levels. In contrast, the structurally related CyaA from Bordetella pertussis and edema factor from Bacillus anthracis exhibit a striking preference for cAMP increases. Thus, ExoY is a nucleotidyl cyclase with preference for cGMP and cUMP production. The differential effects of bacterial toxins on cNMP levels suggest that cUMP plays a distinct second messenger role.

Soluble adenylyl cyclase accounts for high basal cCMP and cUMP concentrations in HEK293 and B103 cells.

Intact HEK293 cells and B103 neuroblastoma cells possess high basal concentrations of the established second messengers cAMP and cGMP and of the emerging second messengers cCMP and cUMP. We asked the question which nucleotidyl cyclase accounts for the high basal cNMP concentrations. Activators and inhibitors of soluble guanylyl cyclase had no major effects on cNMPs, and the activator of membranous adenylyl cyclase forskolin increased only cAMP. Addition of bicarbonate to medium increased, whereas removal of bicarbonate decreased levels of all four cNMPs. The inhibitor of soluble adenylyl cyclase, 2-(1H-benzo[d]imidazol-2-ylthio)-N'-(5-bromo-2-hydroxybenzylidene) propanehydrazide (KH7), reduced bicarbonate-stimulated cNMPs. In conclusion, bicarbonate-stimulated soluble adenylyl cyclase plays an important role in the regulation of basal cellular cNMP levels, most notably cCMP and cUMP.

Nucleotidyl cyclase activity of soluble guanylyl cyclase in intact cells.

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and generates the second messenger cyclic GMP (cGMP). Recently, purified sGC α1ß1 has been shown to additionally generate the cyclic pyrimidine nucleotides cCMP and cUMP. However, since cyclic pyrimidine nucleotide formation occurred only the presence of Mn(2+) but not Mg(2+), the physiological relevance of these in vitro findings remained unclear. Therefore, we studied cyclic nucleotide formation in intact cells. We observed NO-dependent cCMP- and cUMP formation in intact HEK293 cells overexpressing sGC α1ß1 and in RFL-6 rat fibroblasts endogenously expressing sGC, using HPLC-tandem mass spectrometry. The identity of cCMP and cUMP was unambiguously confirmed by HPLC-time-of-flight mass spectrometry. Our data indicate that cCMP and cUMP play second messenger roles and that Mn(2+) is a physiological sGC cofactor.