PDE1 or PDE5 inhibition augments NO-dependent hypoxic constriction of porcine coronary artery via elevating inosine 3',5'-cyclic monophosphate level.

Hypoxic coronary vasospasm may lead to myocardial ischaemia and cardiac dysfunction. Inosine 3',5'-cyclic monophosphate (cIMP) is a putative second messenger to mediate this pathological process. Nevertheless, it remains unclear as to whether levels of cIMP can be regulated in living tissue such as coronary artery and if so, what is the consequence of this regulation on hypoxia-induced vasoconstriction. In the present study, we found that cIMP was a key determinant of hypoxia-induced constriction but not that of the subsequent relaxation response in porcine coronary arteries. Subsequently, coronary arteries were treated with various phosphodiesterase (PDE) inhibitors to identify PDE types that are capable of regulating cIMP levels. We found that inhibition of PDE1 and PDE5 substantially elevated cIMP content in endothelium-denuded coronary artery supplemented with exogenous purified cIMP. However, cGMP levels were far lower than their levels in intact coronary arteries and lower than cIMP levels measured in endothelium-denuded coronary arteries supplemented with exogenous cIMP. The increased cIMP levels induced by PDE1 or PDE5 inhibition further led to augmented hypoxic constriction without apparently affecting the relaxation response. In intact coronary artery, PDE1 or PDE5 inhibition up-regulated cIMP levels under hypoxic condition. Concomitantly, cGMP level increased to a comparable level. Nevertheless, the hypoxia-mediated constriction was enhanced in this situation that was largely compromised by an even stronger inhibition of PDEs. Taken together, these data suggest that cIMP levels in coronary arteries are regulated by PDE1 and PDE5, whose inhibition at a certain level leads to increased cIMP content and enhanced hypoxic constriction.

Hypoxic augmentation: The tale of a strange contraction.

Almost fifty years ago, experiments on isolated veins showed that acute hypoxia augments venoconstrictor responses in vitro and that such facilitation relied on anaerobic glycolysis. Over the years, this phenomenon was extended to a number of arterial preparations of different species and revisited, from a mechanistic point of view, with the successive demonstration that it depends on calcium handling in the vascular smooth muscle cells, is endothelium-dependent and requires the production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) and the activation of soluble guanylyl cyclase (sGC). However, rather than the vasodilator cyclic nucleotide 3',5'-cyclic guanosine monophosphate (cGMP), its canonical product, the latter enzyme produces 3',5'-cyclic inosine monophosphate (cIMP) instead during acute hypoxia; this non-canonical cyclic nucleotide facilitates the contractile process in the vascular smooth muscle cells. This 'biased' activity of soluble guanylyl cyclase appears to involve stimulation of NAD(P)H:quinone oxidoreductase 1 (NQO-1). The exact interactions between hypoxia, anaerobic metabolism and NQO-1 leading to biased activity of soluble guanylyl cyclase remain to be established.

3',5'-cIMP as Potential Second Messenger in the Vascular Wall.

Traditionally, only the 3',5'-cyclic monophosphates of adenosine and guanosine (produced by adenylyl cyclase and guanylyl cyclase, respectively) are regarded as true "second messengers" in the vascular wall, despite the presence of other cyclic nucleotides in different tissues. Among these noncanonical cyclic nucleotides, inosine 3',5'-cyclic monophosphate (cIMP) is synthesized by soluble guanylyl cyclase in porcine coronary arteries in response to hypoxia, when the enzyme is activated by endothelium-derived nitric oxide. Its production is associated with augmentation of vascular contraction mediated by stimulation of Rho kinase. Based on these findings, cIMP appears to meet most, if not all, of the criteria required for it to be accepted as a "second messenger," at least in the vascular wall.

Design, Synthesis, and Biological Evaluation of Novel Cyclic Adenosine-Inosine Monophosphate (cAIMP) Analogs That Activate Stimulator of Interferon Genes (STING).

We describe novel STING-activating cyclic dinucleotides whose constituent nucleosides are adenosine and inosine and that vary by ribose substitution, internucleotide linkage position, and phosphate modification. In mammalian cells in vitro, some of these cAIMP analogs induce greater STING-dependent IRF and NF-κB pathway signaling than do the reference agonists for murine (DMXAA) or human (2',3'-cGAMP) STING. In human blood ex vivo, they induce type I interferons (IFNs) and proinflammatory cytokines: for the former, 3',3'-cAIMP (9; EC50 of 6.4 µM) and analogs 52-56 (EC50 of 0.4-4.7 µM), which contain one or two 2'-fluoro-2'-deoxyriboses and/or bis-phosphorothioate linkages, are more potent than 2',3'-cGAMP (EC50 of 19.6 µM). Interestingly, 9 induces type I IFNs more strongly than do its linkage isomers 2',3'-cAIMP (10), 3',2'-cAIMP (23), and 2',2'-cAIMP (27). Lastly, some of the cAIMP analogs are more resistant than 2',3'-cGAMP to enzymatic cleavage in vitro. We hope to exploit our findings to develop STING-targeted immunotherapies.

Cyclic Purine and Pyrimidine Nucleotides Bind to the HCN2 Ion Channel and Variably Promote C-Terminal Domain Interactions and Opening.

Cyclic AMP is thought to facilitate the opening of the HCN2 channel by binding to a C-terminal domain and promoting or inhibiting interactions between subunits. Here, we correlated the ability of cyclic nucleotides to promote interactions of isolated HCN2 C-terminal domains in solution with their ability to facilitate channel opening. Cyclic IMP, a cyclic purine nucleotide, and cCMP, a cyclic pyrimidine nucleotide, bind to a C-terminal domain containing the cyclic nucleotide-binding domain but, in contrast to other cyclic nucleotides examined, fail to promote its oligomerization, and produce only modest facilitation of opening of the full-length channel. Comparisons between ligand bound structures identify a region between the sixth and seventh ß strands and the distal C helix as important for facilitation and tight binding. We propose that promotion of interactions between the C-terminal domains by a given ligand contribute to its ability to facilitate opening of the full-length channel.

Purine 3':5'-cyclic nucleotides with the nucleobase in a syn orientation: cAMP, cGMP and cIMP.

Purine 3':5'-cyclic nucleotides are very well known for their role as the secondary messengers in hormone action and cellular signal transduction. Nonetheless, their solid-state conformational details still require investigation. Five crystals containing purine 3':5'-cyclic nucleotides have been obtained and structurally characterized, namely adenosine 3':5'-cyclic phosphate dihydrate, C10H12N5O6P·2H2O or cAMP·2H2O, (I), adenosine 3':5'-cyclic phosphate 0.3-hydrate, C10H12N5O6P·0.3H2O or cAMP·0.3H2O, (II), guanosine 3':5'-cyclic phosphate pentahydrate, C10H12N5O7P·5H2O or cGMP·5H2O, (III), sodium guanosine 3':5'-cyclic phosphate tetrahydrate, Na(+)·C10H11N5O7P(-)·4H2O or Na(cGMP)·4H2O, (IV), and sodium inosine 3':5'-cyclic phosphate tetrahydrate, Na(+)·C10H10N4O7P(-)·4H2O or Na(cIMP)·4H2O, (V). Most of the cyclic nucleotide zwitterions/anions [two from four cAMP present in total in (I) and (II), cGMP in (III), cGMP(-) in (IV) and cIMP(-) in (V)] are syn conformers about the N-glycosidic bond, and this nucleobase arrangement is accompanied by Crib-H...Npur hydrogen bonds (rib = ribose and pur = purine). The base orientation is tuned by the ribose pucker. An analysis of data obtained from the Cambridge Structural Database made in the context of syn-anti conformational preferences has revealed that among the syn conformers of various purine nucleotides, cyclic nucleotides and dinucleotides predominate significantly. The interactions stabilizing the syn conformation have been indicated. The inter-nucleotide contacts in (I)-(V) have been systematized in terms of the chemical groups involved. All five structures display three-dimensional hydrogen-bonded networks.

Endothelium-Dependent Contractions of Isolated Arteries to Thymoquinone Require Biased Activity of Soluble Guanylyl Cyclase with Subsequent Cyclic IMP Production.

Preliminary experiments on isolated rat arteries demonstrated that thymoquinone, a compound widely used for its antioxidant properties and believed to facilitate endothelium-dependent relaxations, as a matter of fact caused endothelium-dependent contractions. The present experiments were designed to determine the mechanisms underlying this unexpected response. Isometric tension was measured in rings (with and without endothelium) of rat mesenteric arteries and aortae and of porcine coronary arteries. Precontracted preparations were exposed to increasing concentrations of thymoquinone, which caused concentration-dependent, sustained further increases in tension (augmentations) that were prevented by endothelium removal, Nω-nitro-L-arginine methyl ester [L-NAME; nitric oxide (NO) synthase inhibitor], and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; soluble guanylyl cyclase [sGC] inhibitor). In L-NAME-treated rings, the NO-donor diethylenetriamine NONOate restored the thymoquinone-induced augmentations; 5-[1-(phenylmethyl)-1H-indazol-3-yl]-2-furanmethanol (sGC activator) and cyclic IMP (cIMP) caused similar restorations. By contrast, in ODQ-treated preparations, the cell-permeable cGMP analog did not restore the augmentation by thymoquinone. The compound augmented the content (measured with ultra-high performance liquid chromatography-tandem mass spectrometry) of cIMP, but not that of cGMP; these increases in cIMP content were prevented by endothelium removal, L-NAME, and ODQ. The augmentation of contractions caused by thymoquinone was prevented in porcine arteries, but not in rat arteries, by 1-(5-isoquinolinylsulfonyl)homopiperazine dihydrochloride and trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride (Rho-kinase inhibitors); in the latter, but not in the former, it was reduced by 3,5-dichloro-N-[[(1α,5α,6-exo,6α)-3-(3,3-dimethylbutyl)-3-azabicyclo[3.1.0]hex-6-yl]methyl]-benzamide hydrochloride (T-type calcium channel inhibitor), demonstrating species/vascular bed differences in the impact of cIMP on calcium handling. Thymoquinone is the first pharmacological agent that causes endothelium-dependent augmentation of contractions of isolated arteries, which requires endothelium-derived NO and biased sGC activation, resulting in the augmented production of cIMP favoring the contractile process.

Conventional and Unconventional Mechanisms for Soluble Guanylyl Cyclase Signaling.

Soluble guanylyl cyclase (sGC) is the principal enzyme in mediating the biological actions of nitric oxide. On activation, sGC converts guanosine triphosphate to guanosine 3',5'-cyclic monophosphate (cGMP), which mediates diverse physiological processes including vasodilation, platelet aggregation, and myocardial functions predominantly by acting on cGMP-dependent protein kinases. Cyclic GMP has long been considered as the sole second messenger for sGC action. However, emerging evidence suggests that, in addition to cGMP, other nucleoside 3',5'-cyclic monophosphates (cNMPs) are synthesized by sGC in response to nitric oxide stimulation, and some of these nucleoside 3',5'-cyclic monophosphates are involved in various physiological activities. For example, inosine 3',5'-cyclic monophosphate synthesized by sGC may play a critical role in hypoxic augmentation of vasoconstriction. The involvement of cytidine 3',5'-cyclic monophosphate and uridine 3',5'-cyclic monophosphate in certain cardiovascular activities is also implicated.

Hypoxic Vasospasm Mediated by cIMP: When Soluble Guanylyl Cyclase Turns Bad.

In a number of isolated blood vessel types, hypoxia causes an acute contraction that is dependent on the presence of nitric oxide and activation of soluble guanylyl cyclase. It is more pronounced when the preparations are constricted and is therefore termed hypoxic augmentation of vasoconstriction. This hypoxic response is accompanied by increases in the intracellular level of inosine 5'-triphosphate and in the synthesis of inosine 3',5'-cyclic monophosphate (cIMP) by soluble guanylyl cyclase. The administration of exogenous cIMP or inosine 5'-triphosphate causes augmented vasoconstriction to hypoxia. Furthermore, the vasoconstriction evoked by hypoxia and cIMP is associated with increased activity of Rho kinase (ROCK), indicating that cIMP may mediate the hypoxic effect by sensitizing the myofilaments to Ca through ROCK. Hypoxia is implicated in exaggerated vasoconstriction in the pathogenesis of coronary artery disease, myocardial infarction, hypertension, and stroke. The newly found role of cIMP may help to identify unique therapeutic targets for certain cardiovascular disorders.

A facile and sensitive method for quantification of cyclic nucleotide monophosphates in mammalian organs: basal levels of eight cNMPs and identification of 2',3'-cIMP.

A sensitive, versatile and economical method to extract and quantify cyclic nucleotide monophosphates (cNMPs) using LC-MS/MS, including both 3',5'-cNMPs and 2',3'-cNMPs, in mammalian tissues and cellular systems has been developed. Problems, such as matrix effects from complex biological samples, are addressed and have been optimized. This protocol allows for comparison of multiple cNMPs in the same system and was used to examine the relationship between tissue levels of cNMPs in a panel of rat organs. In addition, the study reports the first identification and quantification of 2',3'-cIMP. The developed method will allow for quantification of cNMPs levels in cells and tissues with varying disease states, which will provide insight into the role(s) and interplay of cNMP signalling pathways.

cIMP synthesized by sGC as a mediator of hypoxic contraction of coronary arteries.

cGMP is considered the only mediator synthesized by soluble guanylyl cyclase (sGC) in response to nitric oxide (NO). However, purified sGC can synthesize several other cyclic nucleotides, including inosine 3',5'-cyclic monophosphate (cIMP). The present study was designed to determine the role of cIMP in hypoxic contractions of isolated porcine coronary arteries. Vascular responses were examined by measuring isometric tension. Cyclic nucleotides were assayed by HPLC tandem mass spectroscopy. Rho kinase (ROCK) activity was determined by measuring the phosphorylation of myosin phosphatase target subunit 1 using Western blot analysis and an ELISA kit. The level of cIMP, but not that of cGMP, was elevated by hypoxia in arteries with, but not in those without, endothelium [except if treated with diethylenetriamine (DETA) NONOate]; the increases in cIMP were inhibited by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). Hypoxia (Po2: 25-30 mmHg) augmented contractions of arteries with and without endothelium if treated with DETA NONOate; these hypoxic contractions were blocked by ODQ. In arteries without endothelium, hypoxic augmentation of contraction was also obtained with exogenous cIMP. In arteries with endothelium, hypoxic augmentation of contraction was further enhanced by inosine 5'-triphosphate, the precursor for cIMP. The augmentation of contraction caused by hypoxia or cIMP was accompanied by increased phosphorylation of myosin phosphatase target subunit 1 at Thr(853), which was prevented by the ROCK inhibitor Y-27632. ROCK activity in the supernatant of isolated arteries was stimulated by cIMP in a concentration-dependent fashion. These results demonstrate that cIMP synthesized by sGC is the likely mediator of hypoxic augmentation of coronary vasoconstriction, in part by activating ROCK.

Meat quality traits of four Chinese indigenous chicken breeds and one commercial broiler stock.

Meat quality traits of four genotypes of Chinese indigenous chicken [Ninghai chicken (NC), frizzle chicken (FC), Ninghai xiang chicken (XC), and Zhenning loquat chicken (LC)] and one genotype of commercial broiler [Arbor Acres plus broiler (AAB)] were analyzed. The indigenous chickens were raised before the commercial chickens in order to achieve the same final processed days. Indigenous chickens of NC, FC, XC, and LC showed significantly higher inosine-5'-monophosphate (IMP) content, shorter fiber diameter, and lower shear force than those of AAB (P<0.05). In the indigenous genotypes, NC and FC had significantly shorter fiber diameters and lower shear forces than XC and LC (P<0.05), and NC and XC had a higher IMP content than FC and LC (P<0.05). Moreover, the indigenous genotype of LC significantly displayed the highest protein content (P<0.05) in the five genotypes of birds, and no significant differences of protein content were found between the other genotypes of NC, FC, XC, and AAB (P>0.05). The indigenous chickens from FC displayed the highest total lipid content in the five bird genotypes (P<0.05). Significant differences of pH, color values of L* and a*, and drip loss for the five genotypes of birds were also observed. In conclusion, there were significant differences in the meat quality traits of the bird breeds selected in this study, and the indigenous chickens, especially the NC genotype, produced better quality meat as far as the IMP content, fiber diameters, and shear forces were concerned.

Discovery of a cAMP deaminase that quenches cyclic AMP-dependent regulation.

An enzyme of unknown function within the amidohydrolase superfamily was discovered to catalyze the hydrolysis of the universal second messenger, cyclic-3',5'-adenosine monophosphate (cAMP). The enzyme, which we have named CadD, is encoded by the human pathogenic bacterium Leptospira interrogans. Although CadD is annotated as an adenosine deaminase, the protein specifically deaminates cAMP to cyclic-3',5'-inosine monophosphate (cIMP) with a kcat/Km of 2.7 ± 0.4 × 10(5) M(-1) s(-1) and has no activity on adenosine, adenine, or 5'-adenosine monophosphate (AMP). This is the first identification of a deaminase specific for cAMP. Expression of CadD in Escherichia coli mimics the loss of adenylate cyclase in that it blocks growth on carbon sources that require the cAMP-CRP transcriptional activator complex for expression of the cognate genes. The cIMP reaction product cannot replace cAMP as the ligand for CRP binding to DNA in vitro and cIMP is a very poor competitor of cAMP activation of CRP for DNA binding. Transcriptional analyses indicate that CadD expression represses expression of several cAMP-CRP dependent genes. CadD adds a new activity to the cAMP metabolic network and may be a useful tool in intracellular study of cAMP-dependent processes.

Speciation studies of diorganotin(IV) complexes with 3,3-bis(1-methylimidazol-2-yl)propionate--displacement reaction by DNA constituents.

The interaction of 3,3-bis(1-methylimidazol-2-yl)propionate (BIMP) with dimethyltin(IV) dichloride (DMT), dibutyltin(IV) dichloride (DBT), and diphenyltin(IV) dichloride (DPT) is investigated at 25°C and 0.1 M ionic strength in water for dimethyltin(IV), and in a 50% dioxane-water mixture for dibutyltin(IV) and diphenyltin(IV). The stepwise formation constants of the 1 : 1 and 1 : 2 complexes formed in solution are calculated from potentiometric measurements using the nonlinear least-square program MINIQUAD-75. The concentration distribution of the various complex species is evaluated as a function of pH. Displacement reactions of the coordinated 3,3-bis(1-methylimidazol-2-yl)propionate by inosine and inosine-5'-monophosphate are investigated from calculations based upon equilibrium properties.

cCMP, cUMP, cTMP, cIMP and cXMP as possible second messengers: development of a hypothesis based on studies with soluble guanylyl cyclase α(1)ß(1).

Adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate are second messengers that regulate multiple physiological functions. The existence of additional cyclic nucleotides in mammalian cells was postulated many years ago, but technical problems hampered development of the field. Using highly specific and sensitive mass spectrometry methods, soluble guanylyl cyclase has recently been shown to catalyze the formation of several cyclic nucleotides in vitro. This minireview discusses the broad substrate-specificity of soluble guanylyl cyclase and the possible second messenger roles of cyclic nucleotides other than adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate. We hope that this article stimulates productive and critical research in an area that has been neglected for many years.

The first 3':5'-cyclic nucleotide-amino acid complex: L-His-cIMP.

In the crystal structure of the L-His-cIMP complex, i.e. L-histidinium inosine 3':5'-cyclic phosphate [systematic name: 5-(2-amino-2-carboxyethyl)-1H-imidazol-3-ium 7-hydroxy-2-oxo-6-(6-oxo-6,9-dihydro-1H-purin-9-yl)-4a,6,7,7a-tetrahydro-4H-1,3,5,2λ(5)-furo[3,2-d][1,3,2λ(5)]dioxaphosphinin-2-olate], C(6)H(10)N(3)O(2)(+)·C(10)H(10)N(4)O(7)P(-), the Hoogsteen edge of the hypoxanthine (Hyp) base of cIMP and the Hyp face are engaged in specific amino acid-nucleotide (His···cIMP) recognition, i.e. by abutting edge-to-edge and by π-π stacking, respectively. The Watson-Crick edge of Hyp and the cIMP phosphate group play a role in nonspecific His···cIMP contacts. The interactions between the cIMP anions (anti/C3'-endo/trans-gauche/chair conformers) are realized mainly between riboses and phosphate groups. The results for this L-His-cIMP complex, compared with those for the previously reported solvated L-His-IMP crystal structure, indicate a different nature of amino acid-nucleotide recognition and interactions upon the 3':5'-cyclization of the nucleotide phosphate group.

Nucleotidyl cyclase activity of soluble guanylyl cyclase α1ß1.

Soluble guanylyl cyclase (sGC) regulates several important physiological processes by converting GTP into the second-messenger cGMP. sGC has several structural and functional properties in common with adenylyl cyclases (ACs). Recently, we reported that membranous ACs and sGC are potently inhibited by 2',3'-O-(2,4,6-trinitrophenyl)-substituted purine and pyrimidine nucleoside 5'-triphosphates. Using a highly sensitive high-performance liquid chromatography-tandem mass spectrometry method, we report that highly purified recombinant sGC of rat possesses nucleotidyl cyclase activity. As opposed to GTP, ITP, XTP and ATP, the pyrimidine nucleotides UTP and CTP were found to be sGC substrates in the presence of Mn(2+). When Mg(2+) is used, sGC generates cGMP, cAMP, cIMP, and cXMP. In conclusion, soluble "guanylyl" cyclase possesses much broader substrate specificity than previously assumed. Our data have important implications for cyclic nucleotide-mediated signal transduction.