cyclic-gmp and cyclic-3--5--uridine-monophosphate

The cyclic nucleotides cAMP and cGMP are well-characterized second messenger molecules regulating many important intracellular processes, such as differentiation, proliferation, and apoptosis. The latter is a highly regulated process of programmed cell death wherein several regulatory proteins, like those belonging to the Bcl-2 family, are involved. The initiation of apoptosis is regulated by three different pathways: the intrinsic or mitochondrial, the extrinsic, and the ER stress pathway. Recently, it has been published that the pyrimidine cyclic nucleotides cCMP and cUMP also function as second messenger molecules, and additionally have an effect on apoptosis signaling pathways. cCMP induced PKA-independent apoptosis via the intrinsic and ER-stress pathway in S49 mouse lymphoma cells, and cCMP as well as cUMP induced apoptosis in human HEL cells via the intrinsic pathway. However, in human K-562 cells, which are known to be multidrug-resistant, cCMP and cUMP had no effect. Summarized in this chapter are the initiation of apoptosis by cCMP and cUMP regarding the various apoptotic pathways, the enzymes involved in apoptosis, as well as the most relevant methods for the detection and examination of apoptosis and the corresponding signaling pathways.

Topics: Adenylyl Cyclases; Animals; Apoptosis; Biological Assay; Blotting, Western; Cell Cycle; Cell Line; Cell Proliferation; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic CMP; Cyclic GMP; Flow Cytometry; Fluorometry; Humans; Membrane Potential, Mitochondrial; Nucleotides, Cyclic; Second Messenger Systems; Soluble Guanylyl Cyclase; Uridine Monophosphate

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and generates the second messenger cyclic GMP (cGMP). Recently, purified sGC α1β1 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 α1β1 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.

Topics: Animals; Cell Line; Cyclic AMP; Cyclic CMP; Cyclic GMP; Guanylate Cyclase; HEK293 Cells; Humans; Manganese; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Nucleotides, Cyclic; Rats; Receptors, Cytoplasmic and Nuclear; Recombinant Proteins; Second Messenger Systems; Soluble Guanylyl Cyclase; Transfection; Uridine Monophosphate

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.

Topics: Apoptosis; Bacterial Proteins; Cell Survival; Cyclic GMP; Glucosyltransferases; Nucleotides, Cyclic; Nucleotidyltransferases; Pseudomonas aeruginosa; Pseudomonas Infections; Uridine Monophosphate

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.

Topics: Animals; Astrocytes; Cells, Cultured; Cricetinae; Cyclic AMP; Cyclic CMP; Cyclic GMP; Fungi; Haplorhini; Humans; Invertebrates; Nucleotides, Cyclic; Plants; Prokaryotic Cells; Rats; Species Specificity; Uridine Monophosphate

In addition to the well-known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. The Pseudomonas aeruginosa toxin ExoY massively increases cGMP and cUMP in cells, whereas the Bordetella pertussis toxin CyaA increases cAMP and, to a lesser extent, cCMP. To mimic and dissect toxin effects, we synthesized cNMP-acetoxymethylesters as prodrugs. cNMP-AMs rapidly and effectively released the corresponding cNMP in cells. The combination of cGMP-AM plus cUMP-AM mimicked cytotoxicity of ExoY. cUMP-AM and cGMP-AM differentially activated gene expression. Certain cCMP and cUMP effects were independent of the known cNMP effectors protein kinases A and G and guanine nucleotide exchange factor Epac. In conclusion, cNMP-AMs are useful tools to mimic and dissect bacterial nucleotidyl cyclase toxin effects.

Topics: Adenylate Cyclase Toxin; Animals; Bacterial Proteins; Bacterial Toxins; Cyclic GMP; Glucosyltransferases; Nucleotides, Cyclic; Rats; Second Messenger Systems; Tumor Cells, Cultured; Uridine Monophosphate

The cyclic purine nucleotides cAMP and cGMP are well-characterized second messengers and activators of PKA and PKG, respectively. In contrast, the functions of the cyclic pyrimidine nucleotides cCMP and cUMP are poorly understood. cCMP induces relaxation of smooth muscle via PKGI, and phosphodiesterases differentially hydrolyze cNMPs. Here, we report that cNMPs differentially activate PKA isoforms and PKGIα. The combination of cCMP with cAMP reduced the EC(50) of cAMP for PKA. PKGIα exhibited higher specificity for the cognate cNMP than PKA. Our data support a role of cCMP and cUMP as second messengers.

Topics: Cyclic AMP; Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Cyclic GMP-Dependent Protein Kinases; Enzyme Activation; Nucleotides, Cyclic; Uridine Monophosphate

Glucose transport in isolated rat cardiomyocytes is stimulated by insulin, catecholamines, and anoxia approximately 2- to 3-fold over basal rates. The molecular mechanisms controlling these responses are unknown. In our search for possible cellular mediators of glucose transport stimulation, we examined the effects of a number of nucleotides on 3-O-methylglucose transport in heart cells. The nucleotides and/or permeable analogs (monosuccinyl, 8-bromo, and dibutyryl derivatives) included cUMP, cIMP, cCMP, cAMP, and cGMP at concentrations ranging from 10 nM to 1 mM. Of all the nucleotides tested only cGMP analogs induced a significant stimulation of transport at concentrations as low as 100 nM. This effect was observed in both the 8-bromo- and dibutyryl derivatives and with 1 mM cGMP itself. The effect was concentration dependent for both analogs and produced a maximal response equivalent to that of 100 nM insulin. This insulinomimetic effect of cGMP was examined in more detail in order to evaluate its role as a potential mediator of this response. Agents that are known to stimulate guanylate cyclase in the heart produced a clear stimulation of transport when added to cardiomyocytes. These include insulin, aminophylline, histamine, beta-estradiol, and biotin-nitrophenyl ester. Methylene blue, an inhibitor of guanylate cyclase, blocked the insulin response when added to cells before insulin, but was ineffective when added after insulin. In addition, agents that raise intracellular cGMP levels by inhibiting cyclic nucleotide phosphodiesterases were also examined for effects on glucose transport. Out of several phosphodiesterase inhibitors tested, only Zaprinast (which selectively increases cGMP in heart) stimulated transport in a concentration-dependent manner to within 80% of the maximal insulin effect. These results are consistent with the notion that cGMP may be involved in glucose transport stimulation.

Topics: Aminophylline; Animals; Cyclic AMP; Cyclic CMP; Cyclic GMP; Cyclic IMP; Dose-Response Relationship, Drug; Estradiol; Female; Glucose; Insulin; Methylene Blue; Myocardium; Nucleotides, Cyclic; Phosphodiesterase Inhibitors; Rats; Rats, Inbred Strains; Uridine Monophosphate