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Mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. It is unknown whether these tentative new second messenger molecules occur in vivo. We used high performance liquid chromatography quadrupole tandem mass spectrometry to quantitate nucleoside 3',5'-cyclic monophosphates. cCMP was detected in all organs studied, most notably pancreas, spleen and the female reproductive system. cUMP was not detected in organs, probably due to the intrinsically low sensitivity of mass spectrometry to detect this molecule and organ matrix effects. Intratracheal infection of mice with recombinant Pseudomonas aeruginosa harboring the nucleotidyl cyclase toxin ExoY massively increased cUMP in lung. The identity of cCMP and cUMP in organs was confirmed by high performance liquid chromatography quadrupole time of flight mass spectrometry. cUMP also appeared in serum, urine and faeces following infection. Taken together, this report unequivocally shows for the first time that cCMP and cUMP occur in vivo., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)