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Sample records for adenosine monophosphate amp

  1. Adsorption of nucleotides on biomimetic apatite: The case of adenosine 5‧ monophosphate (AMP)

    NASA Astrophysics Data System (ADS)

    Hammami, K.; Feki, H. El; Marsan, O.; Drouet, C.

    2015-10-01

    This work investigates the interaction between the nucleotide adenosine 5‧ monophosphate molecule (AMP) and a biomimetic nanocrystalline carbonated apatite as a model for bone mineral. The analogy of the apatite phase used in this work with biological apatite was first pointed out by complementary techniques. AMP adsorption isotherms were then investigated. Obtained data were fitted to a Sips isotherm with an exponent greater than one suggesting positive cooperativity among adsorbed molecules. The data were compared to a previous study relative to the adsorption of another nucleotide, cytidine monophosphate (CMP) onto a similar substrate, evidencing some effect of the chemical nature of the nucleic base. An enhanced adsorption was observed under acidic (pH 6) conditions as opposed to pH 7.4, which parallels the case of DNA adsorption on biomimetic apatite. An estimated standard Gibbs free energy associated to the adsorption process (ΔG°ads ≅ -22 kJ/mol) intermediate between "physisorption" and "chemisorption" was found. The analysis of the solids after adsorption pointed to the preservation of the main characteristics of the apatite substrate but shifts or enhancements of Raman bands attributed to AMP showed the existence of chemical interactions involving both the phosphate and adenine parts of AMP. This contribution adds to the works conducted in view of better understanding the interaction of DNA/RNA and their constitutive nucleotides and the surface of biomimetic apatites. It could prove helpful in disciplines such as bone diagenesis (DNA/apatite interface in aged bones) or nanomedicine (setup of DNA- or RNA-loaded apatite systems). Also, the adsorption of nucleic acids on minerals like apatites could have played a role in the preservation of such biomolecules in the varying conditions known to exist at the origin of life on Earth, underlining the importance of dedicated adsorption studies.

  2. Selective Phosphonylation of 5'-Adenosine Monophosphate (5'-AMP) via Pyrophosphite [PPi(III)

    NASA Astrophysics Data System (ADS)

    Kaye, Karl; Bryant, David E.; Marriott, Katie E. R.; Ohara, Shohei; Fishwick, Colin W. G.; Kee, Terence P.

    2016-11-01

    We describe here experiments which demonstrate the selective phospho-transfer from a plausibly prebiotic condensed phosphorus (P) salt, pyrophosphite [H2P2O5 2-; PPi(III)], to the phosphate group of 5'-adenosine mono phosphate (5'-AMP). We show further that this P-transfer process is accelerated both by divalent metal ions (M2+) and by organic co-factors such as acetate (AcO-). In this specific case of P-transfer from PPi(III) to 5'-AMP, we show a synergistic enhancement of transfer in the combined presence of M2+ & AcO-. Isotopic labelling studies demonstrate that hydrolysis of the phosphonylated 5'-AMP, [P(III)P(V)-5'-AMP], proceeds via nuceophilic attack of water at the Pi(III) terminus.

  3. Selective Phosphonylation of 5'-Adenosine Monophosphate (5'-AMP) via Pyrophosphite [PPi(III)

    NASA Astrophysics Data System (ADS)

    Kaye, Karl; Bryant, David E.; Marriott, Katie E. R.; Ohara, Shohei; Fishwick, Colin W. G.; Kee, Terence P.

    2016-05-01

    We describe here experiments which demonstrate the selective phospho-transfer from a plausibly prebiotic condensed phosphorus (P) salt, pyrophosphite [H2P2O5 2-; PPi(III)], to the phosphate group of 5'-adenosine mono phosphate (5'-AMP). We show further that this P-transfer process is accelerated both by divalent metal ions (M2+) and by organic co-factors such as acetate (AcO-). In this specific case of P-transfer from PPi(III) to 5'-AMP, we show a synergistic enhancement of transfer in the combined presence of M2+ & AcO-. Isotopic labelling studies demonstrate that hydrolysis of the phosphonylated 5'-AMP, [P(III)P(V)-5'-AMP], proceeds via nuceophilic attack of water at the Pi(III) terminus.

  4. Isolation of cyclic adenosine 3':5'-monophosphate (cAMP) from lakes of differing trophic status: Correlation with planktonic metabolic variables

    SciTech Connect

    Francko, D.A.; Wetzel, R.G.

    1982-01-01

    The seasonal dynamics of particulate and dissolved cyclic adenosine 3':5'-monophosphate (cAMP) were examined in the epilimnia and littoral zones of two trophically dissimilar lakes. Each cAMP fraction was found in quantities comparable to those reported for cultured phytoplankton species. Both cAMP fractions varied greatly in concentration during the season and between the oligotrophic and hypereutrophic lakes. Increases in phytoplankton community densities were paralleled by weight-specific changes in particulate cAMP levels, depending on the phytoplankton species present. A linear relationship between cellular cAMP levels and in situ primary productivity rates was found in the oligotrophic lake. In both lakes, certain phytoplanktonic associations had particulate cAMP levels linearly related to chlorophyll a content and specific activity of alkaline phosphatase.

  5. Genetics Home Reference: adenosine monophosphate deaminase deficiency

    MedlinePlus

    Skip to main content Your Guide to Understanding Genetic Conditions Enable Javascript for addthis links to activate. ... Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Home Health Conditions adenosine monophosphate deaminase deficiency adenosine ...

  6. A continuous spectrophotometric assay for monitoring adenosine 5'-monophosphate production.

    PubMed

    First, Eric A

    2015-08-15

    A number of biologically important enzymes release adenosine 5'-monophosphate (AMP) as a product, including aminoacyl-tRNA synthetases, cyclic AMP (cAMP) phosphodiesterases, ubiquitin and ubiquitin-like ligases, DNA ligases, coenzyme A (CoA) ligases, polyA deadenylases, and ribonucleases. In contrast to the abundance of assays available for monitoring the conversion of adenosine 5'-triphosphate (ATP) to ADP, there are relatively few assays for monitoring the conversion of ATP (or cAMP) to AMP. In this article, we describe a homogeneous assay that continuously monitors the production of AMP. Specifically, we have coupled the conversion of AMP to inosine 5'-monophosphate (IMP) (by AMP deaminase) to the oxidation of IMP (by IMP dehydrogenase). This results in the reduction of oxidized nicotine adenine dinucleotide (NAD(+)) to reduced nicotine adenine dinucleotide (NADH), allowing AMP formation to be monitored by the change in the absorbance at 340 nm. Changes in AMP concentrations of 5 μM or more can be reliably detected. The ease of use and relatively low expense make the AMP assay suitable for both high-throughput screening and kinetic analyses. PMID:25957126

  7. 5'-Adenosine monophosphate and adenosine metabolism, and adenosine responses in mouse, rat and guinea pig heart.

    PubMed

    Headrick, J P; Peart, J; Hack, B; Garnham, B; Matherne, G P

    2001-11-01

    We examined myocardial 5'-adenosine monophosphate (5'-AMP) catabolism, adenosine salvage and adenosine responses in perfused guinea pig, rat and mouse heart. MVO(2) increased from 71+/-8 microl O(2)/min per g in guinea pig to 138+/-17 and 221+/-15 microl O(2)/min per g in rat and mouse. VO(2)/beat was 0.42+/-0.03, 0.50+/-0.03 and 0.55+/-0.04 microl O(2)/g in guinea pig, rat and mouse, respectively. Resting and peak coronary flows were highest in mouse vs. rat and guinea pig, and peak ventricular pressures and Ca(2+) sensitivity declined as heart mass increased. Net myocardial 5'-AMP dephosphorylation increased significantly as mass declined (3.8+/-0.5, 9.0+/-1.4 and 11.0+/-1.6 nmol/min per g in guinea pig, rat and mouse, respectively). Despite increased 5'-AMP catabolism, coronary venous [adenosine] was similar in guinea pig, rat and mouse (45+/-8, 69+/-10 and 57+/-14 nM, respectively). Comparable venous [adenosine] was achieved by increased salvage vs. deamination: 64%, 41% and 39% of adenosine formed was rephosphorylated while 23%, 46%, and 50% was deaminated in mouse, rat and guinea pig, respectively. Moreover, only 35-45% of inosine and its catabolites derive from 5'-AMP (vs. IMP) dephosphorylation in all species. Although post-ischemic purine loss was low in mouse (due to these adaptations), functional tolerance to ischemia decreased with heart mass. Cardiovascular sensitivity to adenosine also differed between species, with A(1) receptor sensitivity being greatest in mouse while A(2) sensitivity was greatest in guinea pig. In summary: (i) cardiac 5'-AMP dephosphorylation, VO(2), contractility and Ca(2+) sensitivity all increase as heart mass falls; (ii) adaptations in adenosine salvage vs. deamination limit purine loss and yield similar adenosine levels across species; (iii) ischemic tolerance declines with heart mass; and (iv) cardiovascular sensitivity to adenosine varies, with increasing A(2) sensitivity relative to A(1) sensitivity in larger hearts.

  8. Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphoregulation of mitochondrial complex I is inhibited by nucleoside reverse transcriptase inhibitors

    SciTech Connect

    Lund, Kaleb C. Wallace, Kendall B.

    2008-01-01

    Nucleoside analog reverse transcriptase inhibitors (NRTIs) are known to directly inhibit mitochondrial complex I activity as well as various mitochondrial kinases. Recent observations that complex I activity and superoxide production are modulated through cAMP-dependent phosphorylation suggests a mechanism through which NRTIs may affect mitochondrial respiration via kinase-dependent protein phosphorylation. In the current study, we examine the potential for NRTIs to inhibit the cAMP-dependent phosphorylation of complex I and the associated NADH:CoQ oxidoreductase activities and rates of superoxide production using HepG2 cells. Phosphoprotein staining of immunocaptured complex I revealed that 3'-azido-3'-deoxythymidine (AZT; 10 and 50 {mu}M), AZT monophosphate (150 {mu}M), and 2',3'-dideoxycytidine (ddC; 1 {mu}M) prevented the phosphorylation of the NDUFB11 subunit of complex I. This was associated with a decrease in complex I activity with AZT and AZT monophosphate only. In the presence of succinate, superoxide production was increased with 2',3'-dideoxyinosine (ddI; 10 {mu}M) and ddC (1 {mu}M). In the presence of succinate + cAMP, AZT showed an inverse dose-dependent effect on superoxide production. None of the NRTIs examined inhibit PKA activity suggesting that the observed effects are due to a direct interaction with complex I. These data demonstrate a direct effect of NRTIs on cAMP-dependent regulation of mitochondrial bioenergetics independent of DNA polymerase-{gamma} activity; in the case of AZT, these observations may provide a mechanism for the observed long-term toxicity with this drug.

  9. Adenosine Monophosphate-Based Detection of Bacterial Spores

    NASA Technical Reports Server (NTRS)

    Kern, Roger G.; Chen, Fei; Venkateswaran, Kasthuri; Hattori, Nori; Suzuki, Shigeya

    2009-01-01

    A method of rapid detection of bacterial spores is based on the discovery that a heat shock consisting of exposure to a temperature of 100 C for 10 minutes causes the complete release of adenosine monophosphate (AMP) from the spores. This method could be an alternative to the method described in the immediately preceding article. Unlike that method and related prior methods, the present method does not involve germination and cultivation; this feature is an important advantage because in cases in which the spores are those of pathogens, delays involved in germination and cultivation could increase risks of infection. Also, in comparison with other prior methods that do not involve germination, the present method affords greater sensitivity. At present, the method is embodied in a laboratory procedure, though it would be desirable to implement the method by means of a miniaturized apparatus in order to make it convenient and economical enough to encourage widespread use.

  10. Supplementation of chitosan alleviates high-fat diet-enhanced lipogenesis in rats via adenosine monophosphate (AMP)-activated protein kinase activation and inhibition of lipogenesis-associated genes.

    PubMed

    Chiu, Chen-Yuan; Chan, Im-Lam; Yang, Tsung-Han; Liu, Shing-Hwa; Chiang, Meng-Tsan

    2015-03-25

    This study investigated the role of chitosan in lipogenesis in high-fat diet-induced obese rats. The lipogenesis-associated genes and their upstream regulatory proteins were explored. Diet supplementation of chitosan efficiently decreased the increased weights in body, livers, and adipose tissues in high-fat diet-fed rats. Chitosan supplementation significantly raised the lipolysis rate; attenuated the adipocyte hypertrophy, triglyceride accumulation, and lipoprotein lipase activity in epididymal adipose tissues; and decreased hepatic enzyme activities of lipid biosynthesis. Chitosan supplementation significantly activated adenosine monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation and attenuated high-fat diet-induced protein expressions of lipogenic transcription factors (PPAR-γ and SREBP1c) in livers and adipose tissues. Moreover, chitosan supplementation significantly inhibited the expressions of downstream lipogenic genes (FAS, HMGCR, FATP1, and FABP4) in livers and adipose tissues of high-fat diet-fed rats. These results demonstrate for the first time that chitosan supplementation alleviates high-fat diet-enhanced lipogenesis in rats via AMPK activation and lipogenesis-associated gene inhibition.

  11. Protective mechanisms of adenosine 5'-monophosphate in platelet activation and thrombus formation.

    PubMed

    Fuentes, E; Badimon, L; Caballero, J; Padró, T; Vilahur, G; Alarcón, M; Pérez, P; Palomo, I

    2014-03-01

    Platelet activation is relevant to a variety of acute thrombotic events. We sought to examine adenosine 5'-monophosphate (AMP) mechanisms of action in preventing platelet activation, thrombus formation and platelet-related inflammatory response. We assessed the effect of AMP on 1) P-selectin expression and GPIIb/IIIa activation by flow cytometry; 2) Platelet aggregation and ATP secretion induced by ADP, collagen, TRAP-6, convulxin and thrombin; 3) Platelet rolling and firm adhesion, and platelet-leukocyte interactions under flow-controlled conditions; and, 4) Platelet cAMP levels, sP-selectin, sCD40L, IL-1β, TGF-β1 and CCL5 release, PDE3A activity and PKA phosphorylation. The effect of AMP on in vivo thrombus formation was also evaluated in a murine model. The AMP docking with respect to A2 adenosine receptor was determined by homology. AMP concentration-dependently (0.1 to 3 mmol/l) inhibited P-selectin expression and GPIIb/IIIa activation, platelet secretion and aggregation induced by ADP, collagen, TRAP-6 and convulxin, and diminished platelet rolling and firm adhesion. Furthermore, AMP induced a marked increase in the rolling speed of leukocytes retained on the platelet surface. At these concentrations AMP significantly decreased inflammatory mediator from platelet, increased intraplatelet cAMP levels and inhibited PDE3A activity. Interestingly, SQ22536, ZM241385 and SCH58261 attenuated the antiplatelet effect of AMP. Docking experiments revealed that AMP had the same orientation that adenosine inside the A2 adenosine receptor binding pocket. These in vitro antithrombotic properties were further supported in an in vivo model of thrombosis. Considering the successful use of combined antiplatelet therapy, AMP may be further developed as a novel antiplatelet agent. PMID:24306059

  12. Histone deacetylases 6 increases the cyclic adenosine monophosphate level and promotes renal cyst growth.

    PubMed

    Wu, Ming; Mei, Changlin

    2016-07-01

    Autosomal dominant polycystic kidney disease (ADPKD) is characterized by abnormal enhanced cell proliferation and fluid secretion, which are triggered by increased levels of cyclic adenosine monophosphate (cAMP). Cebotaru et al. showed that a HDAC6 inhibitor reduced the cAMP level and inhibited cyst formation in Pkd1 knockout mice, which may become a new potential therapeutic agent for ADPKD. This study also raised several intriguing questions that might advance our understanding of the molecular pathogenesis of ADPKD. PMID:27312442

  13. Inhibition of Cyclic Adenosine Monophosphate (cAMP)-response Element-binding Protein (CREB)-binding Protein (CBP)/β-Catenin Reduces Liver Fibrosis in Mice.

    PubMed

    Osawa, Yosuke; Oboki, Keisuke; Imamura, Jun; Kojika, Ekumi; Hayashi, Yukiko; Hishima, Tsunekazu; Saibara, Toshiji; Shibasaki, Futoshi; Kohara, Michinori; Kimura, Kiminori

    2015-11-01

    Wnt/β-catenin is involved in every aspect of embryonic development and in the pathogenesis of many human diseases, and is also implicated in organ fibrosis. However, the role of β-catenin-mediated signaling on liver fibrosis remains unclear. To explore this issue, the effects of PRI-724, a selective inhibitor of the cAMP-response element-binding protein-binding protein (CBP)/β-catenin interaction, on liver fibrosis were examined using carbon tetrachloride (CCl4)- or bile duct ligation (BDL)-induced mouse liver fibrosis models. Following repetitive CCl4 administrations, the nuclear translocation of β-catenin was observed only in the non-parenchymal cells in the liver. PRI-724 treatment reduced the fibrosis induced by CCl4 or BDL. C-82, an active form of PRI-724, inhibited the activation of isolated primary mouse quiescent hepatic stellate cells (HSCs) and promoted cell death in culture-activated HSCs. During the fibrosis resolution period, an increase in F4/80(+) CD11b(+) and Ly6C(low) CD11b(+) macrophages was induced by CCl4 and was sustained for two weeks thereafter, even after having stopped CCl4 treatment. PRI-724 accelerated the resolution of CCl4-induced liver fibrosis, and this was accompanied by increased matrix metalloproteinase (MMP)-9, MMP-2, and MMP-8 expression in intrahepatic leukocytes. In conclusion, targeting the CBP/β-catenin interaction may become a new therapeutic strategy in treating liver fibrosis. PMID:26870800

  14. Adenosine 3',5'-monophosphate waves in dictyostelium discoideum: a demonstration by isotope dilution-fluorography

    SciTech Connect

    Tomchik, K.J.; Devreotes, P.N.

    1981-04-24

    The distribution of adenosine 3',5'-monophosphate (cyclic AMP) in fields of aggregating amoebae of Dictyostelium discoidenum was examined by a novel isotope dilution-fluorographic technique. Cellular cyclic AMP was visualized by its competition with exogenous /sup 3/H-labeled cyclic AMP for high-affinity binding sites on protein kinase immobilized on a Millipore filter used to blot the monolayer. The cyclic AMP was distributed in spiral or concentric circular wave patterns which centered on the foci of the aggregations. These patterns were correlated with those of cell shape change that propagate through the monolayers. These observations support the hypothesis that the aggregation process in Dictyostelium is mediated by the periodic relay of cyclic AMP signals and suggest a simple scheme for the dynamics of the aggregation process.

  15. Cyclic adenosine 3',5'-monophosphate and germination of sporangiospores from the fungus Mucor.

    PubMed

    Orlowski, M

    1980-06-01

    Cyclic adenosine 3',5'-monophosphate (cAMP) metabolism was examined in germinating sporangiospores of Mucor genevensis and Mucor mucedo. Exogenous cAMP prevented normal hyphal development from sporangiospores. Internal pools of cAMP fluctuated profoundly during development. Spherical growth of the spores was characterized by large pools of cAMP whereas germ tube emergence and hyphal elongation were characterized by small pools of cAMP. These observations suggest a possible role for cAMP in sporangiospore germination. Adenylate cyclase activities fluctuated significantly during germination with maximum values attained during spherical growth. In contrast, cAMP phosphodiesterase activities remained constant throughout germination. Internal cAMP levels may therefore be regulated by adjustment of adenylate cyclase activities. The binding of cAMP by soluble cell proteins was measured. cAMP-binding activity changed greatly during germination. Dormant and spherically growing spores possessed the highest activities. Developing hyphae contained the lowest activities. Use of the photoaffinity label, 8-azido-[32P]cAMP, in conjunction with sodium dodecyl sulfate-polyacrylamide-gel electrophoresis allowed the identification of a small population of morphogenetic-stage-specific proteins which bind cAMP and may be of regulatory significance to development.

  16. [Cyclic adenosine monophosphate and atherogenic factors].

    PubMed

    Gerasimova, E N

    1977-01-01

    Hypercholesterolemia caused a decrease in the activity of adenylcyclase in rabbit liver tissue and in thrombocytes; hypertriglyceridemia, which developed after administration of hydrocortisone, led to an increase in the activity of adenylcyclase and in the content of 3,5-AMP in adipose tissue. Activities of adenylcyclase, phosphodiesterase and content of prostaglandines E1 and F2alpha were measured in thrombocytes of 39 healthy men without any symptoms of of ischemic heart impairment, in 52 patients with coronary atherosclerosis of the III degree (by Myasnikov's classification) as well as in 12 patients during the period of rehabilitation after myocardial infarction. The activity of adenylate cyclase system was impaired in atherosclerosis. This phenomenon might be caused by alteration in concentration of glucocorticoids in the organism.

  17. Effects of adenosine on polymorphonuclear leucocyte function, cyclic 3': 5'-adenosine monophosphate, and intracellular calcium.

    PubMed Central

    Nielson, C. P.; Vestal, R. E.

    1989-01-01

    1. Inhibition of human polymorphonuclear leucocyte (PMN) function by adenosine was studied with respect to effects of adenosine on intracellular cyclic AMP and calcium during the PMN respiratory burst. 2. The adenosine analogue 5'-N-ethylcarboxamide-adenosine (NECA) and L-N6-phenyl-isopropyl-adenosine (L-PIA) inhibited PMN oxygen metabolite generation with relative potencies (NECA greater than adenosine greater than L-PIA) characteristic of an A2 receptor. 3. The respiratory burst was inhibited by adenosine when PMN were activated by calcium ionophore or chemotactic peptide but not when cells where activated by oleoyl-acetyl-glycerol (OAG). 4. Adenosine increased intracellular cyclic AMP during the PMN respiratory burst regardless of whether cells were stimulated by ionophore, chemotactic peptide or OAG. 5. To determine whether the differences in cell inhibition by adenosine were related to differences in intracellular calcium mobilization by each activating agent, calcium was evaluated with the fluorescent probe, indo-1. Adenosine suppressed the increase in intracellular calcium following PMN activation by calcium ionophore or chemotactic peptide. In contrast, calcium did not increase in PMN activated by OAG and adenosine did not affect intracellular calcium changes following this stimulus. 6. These results demonstrate that physiological concentrations of adenosine inhibit the PMN respiratory burst in association with an increase in intracellular cyclic AMP and reduction of intracellular calcium. PMID:2547490

  18. Role of CNPase in the oligodendrocytic extracellular 2',3'-cAMP-adenosine pathway.

    PubMed

    Verrier, Jonathan D; Jackson, Travis C; Gillespie, Delbert G; Janesko-Feldman, Keri; Bansal, Rashmi; Goebbels, Sandra; Nave, Klaus-Armin; Kochanek, Patrick M; Jackson, Edwin K

    2013-10-01

    Extracellular adenosine 3',5'-cyclic monophosphate (3',5'-cAMP) is an endogenous source of localized adenosine production in many organs. Recent studies suggest that extracellular 2',3'-cAMP (positional isomer of 3',5'-cAMP) is also a source of adenosine, particularly in the brain in vivo post-injury. Moreover, in vitro studies show that both microglia and astrocytes can convert extracellular 2',3'-cAMP to adenosine. Here, we examined the ability of primary mouse oligodendrocytes and neurons to metabolize extracellular 2',3'-cAMP and their respective adenosine monophosphates (2'-AMP and 3'-AMP). Cells were also isolated from mice deficient in 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase). Oligodendrocytes metabolized 2',3'-cAMP to 2'-AMP with 10-fold greater efficiency than did neurons (and also more than previously examined microglia and astrocytes); whereas, the production of 3'-AMP was minimal in both oligodendrocytes and neurons. The production of 2'-AMP from 2',3'-cAMP was reduced by 65% in CNPase -/- versus CNPase +/+ oligodendrocytes. Oligodendrocytes also converted 2'-AMP to adenosine, and this was also attenuated in CNPase -/- oligodendrocytes. Inhibition of classic 3',5'-cAMP-3'-phosphodiesterases with 3-isobutyl-1-methylxanthine did not block metabolism of 2',3'-cAMP to 2'-AMP and inhibition of classic ecto-5'-nucleotidase (CD73) with α,β-methylene-adenosine-5'-diphosphate did not attenuate the conversion of 2'-AMP to adenosine. These studies demonstrate that oligodendrocytes express the extracellular 2',3'-cAMP-adenosine pathway (2',3'-cAMP → 2'-AMPadenosine). This pathway is more robustly expressed in oligodendrocytes than in all other CNS cell types because CNPase is the predominant enzyme that metabolizes 2',3'-cAMP to 2-AMP in CNS cells. By reducing levels of 2',3'-cAMP (a mitochondrial toxin) and increasing levels of adenosine (a neuroprotectant), oligodendrocytes may protect axons from injury. PMID:23922219

  19. Adenosine 3', 5'-cyclic monophosphate levels in Thermomonospora curvata during cellulase biosynthesis

    SciTech Connect

    Fennington, G.; Neubauer, D.; Stutzenberger, F.

    1983-01-01

    The enzymatic degradation of cellulose requires the synergistic activity of at least three enzymes: exo-beta-1,4-glucanase (EC3.2.1.91), endo-beta-1,4-glucanase (EC3.2.1.4), and beta-glucosidase (EC3.2.1.21). Despite extensive studies on a variety of cellulolytic bacteria and fungi, the mechanism(s) regulating the biosynthesis of this inducible catabolic enzyme complex remains unknown. The intracellular concentrations of cyclic nucleotides such as adenosine 3',5'-cyclic monophosphate (cAMP) have been shown to play a major role in mediating catabolite repression of enzyme biosynthesis. The cAMP acts through a cAMP receptor protein (termed CRP or CAP) which is a dimer having two identical subunits each capable of binding one molecule of cAMP. The N-terminal domain of the CRP binds the cAMP while the C-terminal domain binds to DNA at the promotor region of a cAMP-dependent operon and stimulates transcription by promoting the formation of a preinitiation complex between RNA polymerase and the DNA. Intracellular cAMP levels in E. coli (the prototype organism for such studies) are influenced by the type and availability of carbon source used for growth. High intracellular cAMP levels should lead to higher concentrations of cAMP-CRP complexes which should increase the transcription rates for cAMP-dependent operons (such as the lac operon of beta-galactosidase) and indeed the differential rate of beta-galactosidase biosynthesis correlates to intracellular cAMP levels. In the case of cellulase, catabolite repression by glucose or other readily metabolizable compounds closely controls production in an apparently similar manner and therefore a correlation may exist between enzyme biosynthesis and intracellular cAMP levels. This communication describes the fluctuation in cAMP levels during cellulase induction and repression in the thermophilic actinomycete, Thermomonospora curvata.

  20. Adenosine 5′-monophosphate ameliorates D-galactosamine/lipopolysaccharide-induced liver injury through an adenosine receptor-independent mechanism in mice

    PubMed Central

    Zhan, Y; Wang, Z; Yang, P; Wang, T; Xia, L; Zhou, M; Wang, Y; Wang, S; Hua, Z; Zhang, J

    2014-01-01

    D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced lethality and acute liver failure is dependent on endogenously produced inflammatory cytokines. Adenosine has been proven to be a central role in the regulation of inflammatory response. It is not entirely clear that which adenosine action is actually crucial to limiting inflammatory tissue destruction. Here we showed that GalN/LPS challenge elevated hepatic adenosine and induced lethality in adenosine receptor-deficient mice with equal efficiency as wild-type mice. In GalN/LPS-treated mice, pretreatment with adenosine 5′-monophosphate (5′-AMP) significantly elevated hepatic adenosine level and reduced mortality through decreasing cytokine and chemokine production. In RAW264.7 cells, 5′-AMP treatment inhibited the production of inflammatory cytokines, which is not mediated through adenosine receptors. 5′-AMP failed to attenuate LPS-induced nuclear factor-κB (NF-κB) p65 nuclear translocation, but reduced LPS-induced recruitment of NF-κB p65 to inflammatory gene promoters and decreased LPS-induced enrichment of H3K4 dimethylation at the tumor necrosis factor-α (TNF-α) promoter, which was involved in 5′-AMP-induced elevation of cellular adenosine and a decline of methylation potential. In vitro biochemical analysis revealed that adenosine directly attenuated recruitment of NF-κB to the TNF-α and interleukin-6 promoters. Our findings demonstrate that 5′-AMP-inhibiting inflammatory response is not mediated by adenosine receptors and it may represent a potential protective agent for amelioration of LPS-induced liver injury. PMID:24407238

  1. Attempts to detect cyclic adenosine 3':5'-monophosphate in higher plants by three assay methods.

    PubMed

    Bressan, R A; Ross, C W

    1976-01-01

    Endogenous levels of cyclic adenosine-3':5'-monophosphate in coleoptile first leaf segments of oat (Avena sativa L.), potato (Solanum tuberosum L.) tubers, tobacco (Nicotiana tabacum L.) callus, and germinating seeds of lettuce (Lactuca sativa L.) were measured with a modified Gilman binding assay and a protein kinase activation assay. The incorporation of adenosine-8-(14)C into compounds with properties similar to those of cyclic AMP was also measured in studies with germinating lettuce seeds. The binding assay proved reliable for mouse and rat liver analyses, but was nonspecific for plant tissues. It responded to various components from lettuce and potato tissues chromatographically similar to but not identical with cyclic AMP. The protein kinase activation assay was much more specific, but it also exhibited positive responses in the presence of compounds not chromatographically identical to cyclic AMP. The concentrations of cyclic AMP in the plant tissues tested were at the lower limits of detection and characterization obtainable with these assays. The estimates of maximal levels were much lower than reported in many previous studies. PMID:16659419

  2. Measuring the dynamics of cyclic adenosine monophosphate level in living cells induced by low-level laser irradiation using bioluminescence resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Huang, Yimei; Zheng, Liqin; Yang, Hongqin; Chen, Jiangxu; Wang, Yuhua; Li, Hui; Xie, Shusen; Zeng, Haishan

    2015-05-01

    Several studies demonstrated that the cyclic adenosine monophosphate (cAMP), an important second messenger, is involved in the mechanism of low-level laser irradiation (LLLI) treatment. However, most of these studies obtained the cAMP level in cell culture extracts or supernatant. In this study, the cAMP level in living cells was measured with bioluminescence resonance energy transfer (BRET). The effect of LLLI on cAMP level in living cells with adenosine receptors blocked was explored to identify the role of adenosine receptors in LLLI. The results showed that LLLI increased the cAMP level. Moreover, the rise of cAMP level was light dose dependent but wavelength independent for 658-, 785-, and 830-nm laser light. The results also exhibited that the adenosine receptors, a class of G protein-coupled receptor (GPCR), modulated the increase of cAMP level induced by LLLI. The cAMP level increased more significantly when the A3 adenosine receptors (A3R) were blocked by A3R antagonist compared with A1 adenosine receptor or A2a adenosine receptor blocked in HEK293T cells after LLLI, which was in good agreement with the adenosine receptors' expressions. All these results suggested that measuring the cAMP level with BRET could be a useful technique to study the role of GPCRs in living cells under LLLI.

  3. Control of cyclic adenosine 3',5'-monophosphate levels by depolarizing agents in fungi.

    PubMed

    Trevillyan, J M; Pall, M L

    1979-05-01

    It has been reported that diverse treatments which depolarize the plasma membrane of Neurospora crassa produce rapid increases in cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels. In the current study, membrane active antibiotics, which are known or putative depolarizing agents, were found to produce similar cyclic AMP increases, not only in N. crassa, but also in the distantly related fungi Saccharomyces cerevisiae and Mucor racemosus. Uncouplers of oxidative phosphorylation, which have been found to depolarize Neurospora, also produced cyclic AMP increases in all three fungi. The time course of the cyclic AMP response to these various treatments was similar in all three fungi. The fungal studies and studies on depolarized central nervous tissue suggest that cyclic AMP increases may be produced in response to plasma membrane depolarization in diverse eucaryotic cells. A model is proposed for eucaryotic microorganisms in which membrane depolarization serves as a signal of breakdown of the plasma membrane integrity. The subsequent cyclic AMP increase, in turn, may mediate cellular response to help protect the plasma membrane from chemical and mechanical threats to its integrity.

  4. Novel adenosine 3 prime ,5 prime -cyclic monophosphate dependent protein kinases in a marine diatom

    SciTech Connect

    Lin, P.P.C.; Volcani, B.E. )

    1989-08-08

    Two novel adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) dependent protein kinases have been isolated from the diatom Cylindrotheca fusiformis. The kinases, designated I and II, are eluted from DEAE-Sephacel at 0.10 and 0.15 M NaCl. They have a high affinity for cAMP and are activated by micromolar cAMP. They exhibit maximal activity at 5 mM Mg{sup 2+} and pH 8 with the preferred phosphate donor ATP and phosphate acceptor histone H1. They phosphorylate sea urchin sperm histone H1 on a single serine site in the sequence Arg-Lys-Gly-Ser({sup 32}P)-Ser-Asn-Ala-Arg and have an apparent M{sub r} of 75,000 as determined by gel filtration and sucrose density sedimentation. In the kinase I preparation a single protein band with an apparent M{sub r} of about 78,000 is photolabeled with 8-azido({sup 32}P)cAMP and is also phosphorylated with ({gamma}-{sup 32}P)ATP in a cAMP-dependent manner, after autoradiography following sodium dodecyl sulfate gel electrophoresis. The rate of phosphorylation of the 78,000-dalton band is independent of the enzyme concentration. The results indicate that (i) these diatom cAMP-dependent protein kinases are monomeric proteins, possessing both the cAMP-binding regulatory and catalytic domains on the same polypeptide chain, (ii) the enzymes do not dissociate into smaller species upon activation by binding cAMP, and (iii) self-phosphorylation of the enzymes by an intrapeptide reaction is cAMP dependent. The two diatom cAMP kinases are refractory to the heat-stable protein kinase modulator from rabbit muscle, but they respond differently to proteolytic degradation and to inhibition by arachidonic acid and several microbial alkaloids.

  5. Photo protection of RNA building blocks: Adenosine 5‧-monophosphate, cytidine 5‧-monophosphate and cytosine

    NASA Astrophysics Data System (ADS)

    Nielsen, Jakob Brun; Thøgersen, Jan; Jensen, Svend Knak; Keiding, Søren Rud

    2013-04-01

    Photoprotection of the RNA nucleotides adenosine 5'-monophosphate and cytidine 5'-monophosphate, and the nucleobase cytosine was studied using UV pump, IR probe femtosecond transient absorption spectroscopy. The excitation energy is contained in the aromatic ring system, protecting the RNA backbone. All three molecules dissipate the excitation energy by internal conversion and subsequent vibrational relaxation to the electronic ground state in less than 10 ps. In addition, a second deactivation channel is found in cytidine 5'-monophosphate, illustrated by a signal at 1563 cm-1 with a lifetime of 33 ps assigned to an nπ∗ state in agreement with observations in the UV region.

  6. Adenosine 3′:5′-Cyclic Monophosphate in Chlamydomonas reinhardtii: Isolation and Characterization

    PubMed Central

    Amrhein, Nikolaus; Filner, Philip

    1973-01-01

    Chlamydomonas reinhardtii contains a factor that can replace adenosine 3′:5′-cyclic monophosphate (cAMP) in the stimulation of rabbit-muscle protein kinase. The factor cochromatographs and coelectrophoreses with authentic cAMP, and is inactivated by beef heart cyclic nucleotide phosphodiesterase. When C. reinhardtii is exposed to aminophylline (theophylline2 ethylenediamine), the concentration of the factor in the cells increases within 1 hr, from about 25 pmol of cAMP equivalents per g dry weight to more than 250 pmol. Cyclic nucleotide phosphodiesterase activity is present in crude extract of C. reinhardtii and is inhibited by theophylline. We conclude that cAMP occurs in C. reinhardtii and that the endogenous concentration is governed at least in part by a theophylline-sensitive cyclic nucleotide phosphodiesterase. These findings provide a sound basis for attributing the effects of methylxanthines on flagellar function and regeneration in C. reinhardtii to the resultant elevation of endogenous cAMP. PMID:16592076

  7. Intracellular adenosine 5'-triphosphate, adenosine 5'-diphosphate, and adenosine 5'-monophosphate detection by short-end injection capillary electrophoresis using methylcellulose as the effective electroosmostic flow suppressor.

    PubMed

    Zinellu, Angelo; Sotgia, Salvatore; Pasciu, Valeria; Madeddu, Manuela; Leoni, Giovanni Giuseppe; Naitana, Salvatore; Deiana, Luca; Carru, Ciriaco

    2008-07-01

    We present a new rapid CE method to measure adenine nucleotides adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) in cells. The short-end injection mode allows a decrease in the analysis time by injecting samples at the outlet end of a silica capillary closest to the detection window, reducing the migration distance. Moreover, the use of methylcellulose (MC) as run buffer additive to suppress EOF permits to further reduce the migration times of analytes. Thus, when a capillary with an effective length of 10.2 cm was used with a 60 mmol/L sodium acetate buffer pH 3.80 in the presence of 0.01% of MC, the migration time of analytes were 1.35 min for ATP, 1.85 min for ADP, and 4.64 min for AMP. These conditions gave a good reproducibility for intra- and interassay (CV <4 and 8%, respectively) and all the procedure demonstrated an excellent analytical recovery (from 98.3 to 99 %). The method suitability was proved both on red blood cells and in spermatozoa. We compared our proposed method to a spectrophotometric assay, by measuring ATP levels in 40 spermatozoa samples. The obtained data were analyzed by the Passing and Bablok regression and Bland-Altman test. PMID:18551716

  8. Development of Potent Adenosine Monophosphate Activated Protein Kinase (AMPK) Activators.

    PubMed

    Dokla, Eman M E; Fang, Chun-Sheng; Lai, Po-Ting; Kulp, Samuel K; Serya, Rabah A T; Ismail, Nasser S M; Abouzid, Khaled A M; Chen, Ching-Shih

    2015-11-01

    Previously, we reported the identification of a thiazolidinedione-based adenosine monophosphate activated protein kinase (AMPK) activator, compound 1 (N-[4-({3-[(1-methylcyclohexyl)methyl]-2,4-dioxothiazolidin-5-ylidene}methyl)phenyl]-4-nitro-3-(trifluoromethyl)benzenesulfonamide), which provided a proof of concept to delineate the intricate role of AMPK in regulating oncogenic signaling pathways associated with cell proliferation and epithelial-mesenchymal transition (EMT) in cancer cells. In this study, we used 1 as a scaffold to conduct lead optimization, which generated a series of derivatives. Analysis of the antiproliferative and AMPK-activating activities of individual derivatives revealed a distinct structure-activity relationship and identified 59 (N-(3-nitrophenyl)-N'-{4-[(3-{[3,5-bis(trifluoromethyl)phenyl]methyl}-2,4-dioxothiazolidin-5-ylidene)methyl]phenyl}urea) as the optimal agent. Relative to 1, compound 59 exhibits multifold higher potency in upregulating AMPK phosphorylation in various cell lines irrespective of their liver kinase B1 (LKB1) functional status, accompanied by parallel changes in the phosphorylation/expression levels of p70S6K, Akt, Foxo3a, and EMT-associated markers. Consistent with its predicted activity against tumors with activated Akt status, orally administered 59 was efficacious in suppressing the growth of phosphatase and tensin homologue (PTEN)-null PC-3 xenograft tumors in nude mice. Together, these findings suggest that 59 has clinical value in therapeutic strategies for PTEN-negative cancer and warrants continued investigation in this regard.

  9. Development of Novel Adenosine Monophosphate-Activated Protein Kinase Activators

    PubMed Central

    Guh, Jih-Hwa; Chang, Wei-Ling; Yang, Jian; Lee, Su-Lin; Wei, Shuo; Wang, Dasheng; Kulp, Samuel K.; Chen, Ching-Shih

    2010-01-01

    In light of the unique ability of thiazolidinediones to mediate peroxisome proliferator-activated receptor (PPAR)γ-independent activation of adenosine monophosphate-activated protein kinase (AMPK) and suppression of interleukin (IL)-6 production, we conducted a screening of an in-house, thiazolidinedione-based focused compound library to identify novel agents with these dual pharmacological activities. Cell-based assays pertinent to the activation status of AMPK and mammalian homolog of target of rapamycin (i.e., phosphorylation of AMPK and p70 ribosomal protein S6 kinase, respectively), and IL-6/IL-6 receptor signaling (i.e., IL-6 production and signal transducer and activator of transcription 3 phosphorylation, respectively) in lipopolysaccharide (LPS)-stimulated THP-1 human macrophages were used to screen this compound library, which led to the identification of compound 53 (N-{4-[3-(1-Methylcyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidene-methyl]-phenyl}-4-nitro-3-trifluoromethyl-benzenesulfonamide) as the lead agent. Evidence indicates that this drug-induced suppression of LPS-stimulated IL-6 production was attributable to AMPK activation. Furthermore, compound 53-mediated AMPK activation was demonstrated in C-26 colon adenocarcinoma cells, indicating that it is not a cell line-specific event. PMID:20170185

  10. Lymphocyte beta 2-adrenoceptors and adenosine 3':5'-cyclic monophosphate during and after normal pregnancy.

    PubMed Central

    von Mandach, U.; Gubler, H. P.; Engel, G.; Huch, R.; Huch, A.

    1993-01-01

    1. The beta 2-sympathomimetics, used to inhibit preterm labour, bind predominantly to beta 2-adrenoceptors, activating adenylate cyclase to form adenosine 3':5'-cyclic monophosphate (cyclic AMP), a messenger substance which inhibits the enzyme cascade triggering smooth muscle contraction. beta 2-Adrenoceptor density and cyclic AMP formation can be used as markers of beta 2-adrenergic effect. 2. The present study addresses the influence of pregnancy on the beta-adrenoceptor system. beta 2-Adrenoceptor density and cyclic AMP concentrations (basal and evoked by isoprenaline) in circulating lymphocytes were determined at three points in gestation (16, 29 and 37 weeks) and 9 weeks post partum in 22 normal pregnancies. (-)-[125Iodo]-cyanopindolol was used as the ligand to identify a homogeneous population of beta 2-adrenoceptors on lymphocytes. B- and T-cell fractions were estimated from the same samples. 3. beta 2-Adrenoceptor density decreased significantly during gestation until week 37 (P < 0.01), then increased post partum (P < 0.005). Cyclic AMP concentrations (basal and evoked by isoprenaline) were significantly lower after 16 weeks of gestation than post partum (P < 0.05). 4. The results, which cannot be explained in terms of a shift in the lymphocyte (B- and T-cell) ratio, indicate that beta-adrenoceptor density and function are reduced in normal pregnancy and only return to normal post partum. These findings may be of significance in devising future tocolytic therapy with beta 2-adrenoceptor agonists. PMID:8383562

  11. The effect of p,p'-dichlorodiphenyltrichloroethane on levels of guanosine 3',5'-cyclic monophosphate and adenosine 3',5'-cyclic monophosphate in two species of insects.

    PubMed

    Bodnaryk, R P

    1976-11-01

    Within 1 h after topical application of a convulsive dose (4 mug per fly, 47 mg/kg) of p,p'-dichlorodiphenyltrichloroethane (DDT) to the adult male of Sarcophaga bullata Parker, guanosine 3',5'-cyclic monophosphate (cyclic GMP) levels rose by 71.5% (P less than 0.05) in the head, 159.5% (P less than 0.01) in the thorax, and 23.4% (P greater than 0.05) in the abdomen compared to controls. Adenosine 3',5'-cyclic monophosphate (cyclic AMP) levels were not significantly affected by the DDT treatment. A convulsive dose (100 mug per larva, 250 mg/kg) of DDT applied to larvae of Mamestra configurata Wlk. caused the whole body level of cyclic GMP to rise by 81.6% (P less than 0.01) after 1 h, and by 95.9% (P less than 0.01) after 3 h. Levels of cyclic AMP were not affected. A hypothesis is advanced suggesting that an abnormally high rate of discharge of acetylcholine (and in the later stages of poisoning, its actual accumulation) at central cholinergic synapses causes cyclic GMP levels to rise, perhaps in post-synaptic cells. The elevated cyclic GMP-cyclic AMP ratio found in DDT-poisoned insects may be of fundamental importance in the complex sequence of events leading to tremor, hyperexcitability, paralysis, and death.

  12. Investigation on the occurrence and significance of cyclic adenosine 3':5'-monophosphate in phytoplankton and natural aquatic communities

    SciTech Connect

    Francko, D.A.

    1980-01-01

    This study demonstrates, on the basis of several analyanalytical criteria, that the production and extracellular release of cyclic adenosine 3':5'-monophosphate (cAMP) is widespread among phytoplankton species. The production and release of CAMP varied markedly among different species grown under similar environmental conditions, and intraspecifically during the life cycle of a given algal species. This investigation marks the first time cAMP has been investigated in natural aquatic systems. An examination of epilimnetic lakewater samples from Lawrence Lake, a hardwater oligotrophic lake, and Wintergreen Lake, a hardwater hypereutrophic lake, both in southwestern Michigan, demonstrated that cAMP existed in both particulate-associated and dissolved forms in these systems.

  13. Repetitive mechanical strain suppresses macrophage uptake of immunoglobulin G complexes and enhances cyclic adenosine monophosphate synthesis.

    PubMed Central

    Mattana, J.; Sankaran, R. T.; Singhal, P. C.

    1995-01-01

    Uptake of immunoglobulin G (IgG) complexes by macrophages (M phi) may play an important role in disease states characterized by increased levels of circulating immune complexes. In sites such as the glomerular mesangium M phi may be subjected to repetitive mechanical strain, although in vitro studies of M phi endocytosis are typically carried out with cells grown on rigid surfaces. We undertook the present study to determine whether repetitive mechanical strain could modulate M phi endocytosis of IgG complexes. IgG complex uptake was significantly diminished in M phi that were subjected to repetitive mechanical strain using parameters corresponding to peak and minimal intraglomerular pressures compared with control, and uptake varied according to the amount of mechanical strain applied. There was no significant difference in surface binding of IgG between M phi subjected to strain and those not. Mechanical strain did not significantly influence the rate of IgG complex degradation. Inhibition of nitric oxide synthase and guanylate cyclase activity did not alter the effect of mechanical strain, although this effect was potentiated by 3-isobutyl-1-methylxanthine (IBMX). Angiotensin II, which has been shown to reduce adenosine 3',5'-cyclic monophosphate (cAMP) production in M phi, significantly attenuated the suppressive effect of mechanical strain on IgG complex uptake as well as another inhibitor of cAMP generation, indomethacin. Enzyme immunoassay demonstrated significantly enhanced levels of cAMP in M phi that were subjected to mechanical strain compared with control, an effect that was potentiated by IBMX and attenuated by angiotensin II and indomethacin. These results demonstrate that repetitive mechanical strain significantly reduces IgG complex uptake by M phi, most likely by enhancing cAMP synthesis. Such an effect might play a significant role in macromolecule handling by M phi in sites in which they are subjected to repetitive mechanical deformation such as

  14. Induction of phosphodiesterase by cyclic adenosine 3':5'-monophosphate in differentiating Dictyostelium discoideum amoebae.

    PubMed

    Klein, C

    1975-09-25

    Cyclic adenosine 3':5'-monophosphate added to the starvation media of Dictyostelium discoideum amoebae induces both intracellular and extracellular phosphodiesterase activities of these cells. The induced enzyme activity appears several hours earlier than that in starved cells which have not been induced with cyclic nucleotide. In both cases, the appearance of enzyme is inhibited by cycloheximide, and actinomycin D, and daunomycin. The KmS for the extracellular enzyme(s) of nucleotide-induced and uninduced control cells are identical. The induction of enzyme activity seems specific for cyclic adenosine 3':5'-monophosphate since cyclic guanosine 3':5'-monophosphate, as well as other nucleotides, have no effect. No differences in the activity or excretion of either N-acetylglucosaminidase or the inhibitory of the extracellular phosphodiesterase are observed between cyclic adenosine 3':5'-monophosphate-induced and control cells. A direct activation of phosphodiesterase by cyclic adenosine 3':5'-monophosphate can be excluded, since the addition of this nucleotide to cell lysates has no effect on the enzyme activity. PMID:170256

  15. Estradiol and chlordecone (Kepone) decrease adenosine 3'5'-cyclic monophosphate concentrations in the ovariectomized immature rat uterus.

    PubMed

    Johnson, D C; Banerjee, S; Chatterjee, S

    1995-10-01

    Adenosine 3'5'-cyclic monophosphate (cAMP) has been repeatedly shown to mimic some actions of estrogen in the rat uterus. However, the relationship between estrogens and uterine cAMP remains controversial. The effect of chronic exposure (3 days) to a biologically potent, long-acting estrogen, estradiol benzoate (EB), or the xenoestrogen chlordecone (Kepone), which has a long half-life in the circulation, was examined in ovariectomized immature rats. Both compounds, when administered in doses that provided equal increases in uterine weight, produced equivalent decreases in uterine cAMP content. Although the decrease in cAMP was apparent within 48 hr, it was more pronounced at 72 hr. There was no reduction in cAMP produced in response to direct stimulation of uterine adenylyl cyclase by forskolin, indicating that loss of the enzyme was not a factor in the lowering of cAMP content. The pure anti-estrogen ICI-182,780, in a dose-dependent fashion, prevented the action the estradiol benzoate and chlordecone, suggesting that the lowering of cAMP was dependent on an estrogen receptor. The physiological significance of reduced uterine cAMP with chronic estrogen treatment remains to be determined. PMID:7545817

  16. Adenosine 3′:5′-cyclic monophosphate- and guanosine 3′:5′-cyclic monophosphate-dependent protein kinases: Possible homologous proteins

    PubMed Central

    Lincoln, Thomas M.; Corbin, Jackie D.

    1977-01-01

    The properties of purified mammalian adenosine 3′:5′-cyclic monophosphate (cAMP)- and guanosine 3′:5′-cyclic monophosphate (cGMP)-dependent protein kinases were compared. Several physical characteristics of the two enzymes were similar, including size, shape, affinity for cyclic nucleotide binding, and Km for ATP. In addition, the amino acid composition of the two proteins indicated a close composition homology (70-90%). Both cyclic nucleotide-dependent protein kinases catalyzed phosphorylation of rat liver pyruvate kinase (EC 2.7.1.40) and fructose 1,6-diphosphatase (EC 3.1.3.11), rabbit skeletal muscle glycogen synthase (EC 2.4.1.11) and phosphorylase b kinase (EC 2.7.1.38), and calf thymus histone H2b. The phosphorylation of several synthetic peptides and of trypsin-sensitive and trypsin-insensitive sites in glycogen synthase suggested similar recognition sites on the protein substrates for the two kinases. The cAMP-dependent protein kinase was the better catalyst with each protein or peptides substrate. The results suggest that the two enzymes evolved from a common ancestral protein. Images PMID:198777

  17. An evaluation of short-term corticosteroid response in perennial allergic rhinitis using histamine and adenosine monophosphate nasal challenge

    PubMed Central

    Wilson, Andrew M; Sims, Erika J; Orr, Linda C; Robb, Fiona; Lipworth, Brian J

    2003-01-01

    Aims To evaluate the role of AMP nasal challenge as a measure of short-term treatment response in patients receiving intranasal corticosteroids. Adenosine monophosphate (AMP) challenge has been shown to be a good inflammatory surrogate in the lower airways, but it has not been properly evaluated as a nasal challenge test. Methods Fourteen patients with perennial allergic rhinitis (PAR) were randomized to receive 2 weeks treatment with placebo (PL) or 200 µg intranasal mometasone furoate (MF) once daily in a randomized single-blind crossover study. AMP (25–800 mg ml−1) and histamine (0.25–8 mg ml−1) nasal challenge testing were performed after each treatment period with 30% decrease in minimal cross-sectional area (MCA). Domiciliary symptom data were collected. Results There was a significant (P < 0.05) improvement in PC30 MCA and nasal volume with AMP but not with histamine comparing MF vs PL. This amounted to a 2.8 (95% CI 1.5, 4.0) and 0.7 (95% CI −0.5, 1.9) doubling-dose change for AMP and histamine challenges, respectively. There were significant (P < 0.05) improvements in nasal symptoms and quality of life. Conclusions AMP nasal challenge using acoustic rhinometry may be a useful test to assess short-term treatment response in patient with PAR. PMID:12680883

  18. Investigation on the occurrence and significance of cyclic adenosine 3':5'-monophosphate in phytoplankton and natural aquatic communities

    SciTech Connect

    Francko, D.A.

    1980-01-01

    This study is an investigation into the occurrence and potential functions of cyclic adenosine 3':5'-monophosphate (cAMP), a potent and ubiquitous metabolic regulatory molecule in heterotrophic organisms, in phytoplankton and in natural aquatic communities. Laboratory-cultured phytoplankton were grown under both optimal and suboptimal nutrient regimes under constant temperature and illumination regimes. Cellular and extracellular cAMP production, characterized by a number of biochemical techniques, was correlated with growth rate dynamics, chlorophyll a synthesis, /sup 14/C-bicarbonate uptake, alkaline phosphatase activity, and heterocyst formation. The blue-green alga Anabaena flos-aquae was used as a model system in the examination of these metabolic variables. Additionally, this alga was used to test the effects of perturbation of cAMP levels on the aforementioned metabolic variables. Investigations on the occurrence and seasonal dynamics of cAMP in aquatic systems were conducted on Lawrence Lake, a hardwater oligotrophic lake, and on Wintergreen Lake, a hardwater hypereutrophic lake, both in southwestern Michigan. Putative cAMP from both systems was characterized by several biochemical techniques. Weekly sampling of particulate and dissolved cAMP in the epilimnia of both lakes was correlated with data on the rates of primary productivity, alkaline phosphatase activity, chlorophyll a synthesis and changes in phytoplankton community structure.

  19. The effect of polystyrene beads on cyclic 3′,5′-adenosine monophosphate concentration in leukocytes

    PubMed Central

    Manganiello, Vincent; Evans, Warren H.; Stossel, Thomas P.; Mason, Robert J.; Vaughan, Martha

    1971-01-01

    After incubation with polystyrene latex beads for 5 min. the cyclic 3′,5′-adenosine monophosphate (cyclic AMP) content of human peripheral blood leukocyte suspensions was increased severalfold. Preparations enriched in mononuclear cells and containing only 0-20% polymorphonuclear leukocytes (PMN) and no visible platelets exhibited a quantitatively similar response. Purified fractions of cells containing 85-90% PMN responded to polystyrene beads with a much smaller increase in cyclic AMP content. Phagocytosis of paraffin oil emulsion in the unfractionated mixed human leukocyte preparation was associated with little or no change in cyclic AMP levels. There was no change in cyclic AMP content of rabbit alveolar macrophages or guinea pig PMN during phagocytosis of polystyrene beads. All of these observations are consistent with the view that particle uptake per se does not increase cyclic AMP levels in phagocytic cells. It seems probable that the increase in cyclic AMP concentration that results when unfractionated human blood leukocytes are incubated with polystyrene beads occurs in cells other than PMN. PMID:4331596

  20. Cyclic Adenosine Monophosphate Accumulation and beta-Adrenergic Binding in Unweighted and Denervated Rat Soleus Muscle

    NASA Technical Reports Server (NTRS)

    Kirby, Christopher R.; Woodman, Christopher R.; Woolridge, Dale; Tischler, Marc E.

    1992-01-01

    Unweighting, but not denervation, of muscle reportedly "spares" insulin receptors, increasing insulin sensitivity. Unweighting also increases beta-adrenergic responses of carbohydrate metabolism. These differential characteristics were studied further by comparing cyclic adenosine monophosphate (cAMP) accumulation and beta-adrenergic binding in normal and 3-day unweighted or denervated soleus muscle. Submaximal amounts of isoproterenol, a p-agonist, increased cAMP accumulation in vitro and in vivo (by intramuscular (IM) injection) to a greater degree (P less than .05) in unweighted muscles. Forskolin or maximal isoproterenol had similar in vitro effects in all muscles, suggesting increased beta-adrenergic sensitivity following unweighting. Increased sensitivity was confirmed by a greater receptor density (B(sub max)) for iodo-125(-)-pindolol in particulate preparations of unweighted (420 x 10(exp -18) mol/mg muscle) than of control or denervated muscles (285 x 10(exp-18) mol/mg muscle). The three dissociation constant (Kd) values were similar (20.3 to 25.8 pmol/L). Total binding capacity (11.4 fmol/muscle) did not change during 3 days of unweighting, but diminished by 30% with denervation. This result illustrates the "sparing" and loss of receptors, respectively, in these two atrophy models. In diabetic animals, IM injection of insulin diminished CAMP accumulation in the presence of theophylline in unweighted muscle (-66% +/- 2%) more than in controls (-42% +'- 6%, P less than .001). These results show that insulin affects CAMP formation in muscle, and support a greater in vivo insulin response following unweighting atrophy. These various data support a role for lysosomal proteolysis in denervation, but not in unweighting, atrophy.

  1. Directed breeding of an Arthrobacter mutant for high-yield production of cyclic adenosine monophosphate by N + ion implantation

    NASA Astrophysics Data System (ADS)

    Song, He; Chen, Xiaochun; Cao, Jiaming; Fang, Ting; Bai, Jianxin; Xiong, Jian; Ying, Hanjie

    2010-08-01

    To obtain a cyclic adenosine monophosphate (cAMP) high-yield production strain, Arthrobacter NG-1 was mutated by N + ion implantation with an energy level of 10 keV and dose of 7×10 15 ions/cm 2. Combined with directed screening methods, a xanthine-defective and 8-azaguanine (8-AG)-resistant mutant Arthrobacter A302 was selected. The concentration of cAMP produced by this mutant was 41.7% higher than that of the original strain and reached 9.78 g/L. Through ten-generation investigation, the capability of cAMP production of A302 was found to be stable. Compared with the original strain, the special activities of key enzymes in A302, which influenced the cAMP biosynthesis, was analyzed. IMP dehydrogenase activity was defective, whereas PRPP amidotransferase, sAMP synthetase and adenylate cyclase activities were increased by 61.5%, 147% and 21.7%, respecitively, which might explain the mutagenesis mechanism by N + ions implantation under the enzymatic level.

  2. Cyclic adenosine monophosphate phosphodiesterase activity in peripheral blood mononuclear leucocytes from patients with atopic dermatitis: correlation with respiratory atopy.

    PubMed

    Sawai, T; Ikai, K; Uehara, M

    1998-05-01

    We determined the cyclic adenosine monophosphate phosphodiesterase (cAMP-PDE) activity in peripheral blood mononuclear leucocytes from 100 patients with atopic dermatitis (AD) aged 13-57 years (mean +/- SD, 29.8 +/- 17.7 years). The correlation between cAMP-PDE activity and clinical parameters such as the severity of eczema and a personal or family predisposition to atopic respiratory diseases (ARD) (asthma or allergic rhinitis) was examined. Although the enzymic activity varied from normal to very high in the AD patients, cAMP-PDE activity was significantly (P < 0.005) elevated in AD patients (42.1 +/- 22.0 units) as compared with the normal controls (12.4 +/- 5.6) and clinical control subjects (13.4 +/- 9.5). In contrast, we found no correlation between cAMP-PDE activity and the severity of eczema when AD patients were classified into four categories (remission, mild, moderate and severe) according to the extent of their skin involvement. Furthermore, we found that systemic corticosteroid therapy in severe AD patients did not alter the cAMP-PDE activity. cAMP-PDE activity was significantly (P < 0.01) higher in those AD patients who had a personal history of ARD (47.2 +/- 11.2) than in AD patients with a family history of ARD (37.2 +/- 17.4) and those without a personal or family history ('pure' AD) (34.4 +/- 19.8). Nevertheless, the cAMP-PDE activity was significantly higher even in 'pure' AD patients than in the controls. These results suggest that an elevation of cAMP-PDE activity is closely related to a predisposition to respiratory atopy, and does not follow inflammation in AD patients. PMID:9666832

  3. [Concentration of prostaglandins and cyclic adenosine-3',5'-monophosphate in the tissues of rats].

    PubMed

    Komissarenko, V P; Slavnov, V N; Epsheĭn, E V; Malinkovich, V D

    1977-04-01

    The content of prostaglandines (PG) and cyclic 3',5'-adenosine monphosphate (cAMP) was investigated in rat tissues by the radioisotopic method of competitive binding. Maximum quantities of both PG and cAMP were revealed in the same most actively functioning organs: the brain, incretory glands, small intestine. Fatty tissue showed minimum quantities of these substances. Results indicate a close functional relationship between the PG synthesis and adenylatecyclase activity in the body tissues.

  4. Enhanced Production of Adenosine Triphosphate by Pharmacological Activation of Adenosine Monophosphate-Activated Protein Kinase Ameliorates Acetaminophen-Induced Liver Injury.

    PubMed

    Hwang, Jung Hwan; Kim, Yong-Hoon; Noh, Jung-Ran; Choi, Dong-Hee; Kim, Kyoung-Shim; Lee, Chul-Ho

    2015-10-01

    The hepatic cell death induced by acetaminophen (APAP) is closely related to cellular adenosine triphosphate (ATP) depletion, which is mainly caused by mitochondrial dysfunction. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a key sensor of low energy status. AMPK regulates metabolic homeostasis by stimulating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. We found that the decrease in active phosphorylation of AMPK in response to APAP correlates with decreased ATP levels, in vivo. Therefore, we hypothesized that the enhanced production of ATP via AMPK stimulation can lead to amelioration of APAP-induced liver failure. A769662, an allosteric activator of AMPK, produced a strong synergistic effect on AMPK Thr172 phosphorylation with APAP in primary hepatocytes and liver tissue. Interestingly, activation of AMPK by A769662 ameliorated the APAP-induced hepatotoxicity in C57BL/6N mice treated with APAP at a dose of 400 mg/kg intraperitoneally. However, mice treated with APAP alone developed massive centrilobular necrosis, and APAP increased their serum alanine aminotransferase and aspartate aminotransferase levels. Furthermore, A769662 administration prevented the loss of intracellular ATP without interfering with the APAP-mediated reduction of mitochondrial dysfunction. In contrast, inhibition of glycolysis by 2-deoxy-glucose eliminated the beneficial effects of A769662 on APAP-mediated liver injury. In conclusion, A769662 can effectively protect mice against APAP-induced liver injury through ATP synthesis by anaerobic glycolysis. Furthermore, stimulation of AMPK may have potential therapeutic application for APAP overdose. PMID:26434492

  5. Enhanced Production of Adenosine Triphosphate by Pharmacological Activation of Adenosine Monophosphate-Activated Protein Kinase Ameliorates Acetaminophen-Induced Liver Injury.

    PubMed

    Hwang, Jung Hwan; Kim, Yong-Hoon; Noh, Jung-Ran; Choi, Dong-Hee; Kim, Kyoung-Shim; Lee, Chul-Ho

    2015-10-01

    The hepatic cell death induced by acetaminophen (APAP) is closely related to cellular adenosine triphosphate (ATP) depletion, which is mainly caused by mitochondrial dysfunction. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a key sensor of low energy status. AMPK regulates metabolic homeostasis by stimulating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. We found that the decrease in active phosphorylation of AMPK in response to APAP correlates with decreased ATP levels, in vivo. Therefore, we hypothesized that the enhanced production of ATP via AMPK stimulation can lead to amelioration of APAP-induced liver failure. A769662, an allosteric activator of AMPK, produced a strong synergistic effect on AMPK Thr172 phosphorylation with APAP in primary hepatocytes and liver tissue. Interestingly, activation of AMPK by A769662 ameliorated the APAP-induced hepatotoxicity in C57BL/6N mice treated with APAP at a dose of 400 mg/kg intraperitoneally. However, mice treated with APAP alone developed massive centrilobular necrosis, and APAP increased their serum alanine aminotransferase and aspartate aminotransferase levels. Furthermore, A769662 administration prevented the loss of intracellular ATP without interfering with the APAP-mediated reduction of mitochondrial dysfunction. In contrast, inhibition of glycolysis by 2-deoxy-glucose eliminated the beneficial effects of A769662 on APAP-mediated liver injury. In conclusion, A769662 can effectively protect mice against APAP-induced liver injury through ATP synthesis by anaerobic glycolysis. Furthermore, stimulation of AMPK may have potential therapeutic application for APAP overdose.

  6. Enhanced Production of Adenosine Triphosphate by Pharmacological Activation of Adenosine Monophosphate-Activated Protein Kinase Ameliorates Acetaminophen-Induced Liver Injury

    PubMed Central

    Hwang, Jung Hwan; Kim, Yong-Hoon; Noh, Jung-Ran; Choi, Dong-Hee; Kim, Kyoung-Shim; Lee, Chul-Ho

    2015-01-01

    The hepatic cell death induced by acetaminophen (APAP) is closely related to cellular adenosine triphosphate (ATP) depletion, which is mainly caused by mitochondrial dysfunction. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a key sensor of low energy status. AMPK regulates metabolic homeostasis by stimulating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. We found that the decrease in active phosphorylation of AMPK in response to APAP correlates with decreased ATP levels, in vivo. Therefore, we hypothesized that the enhanced production of ATP via AMPK stimulation can lead to amelioration of APAP-induced liver failure. A769662, an allosteric activator of AMPK, produced a strong synergistic effect on AMPK Thr172 phosphorylation with APAP in primary hepatocytes and liver tissue. Interestingly, activation of AMPK by A769662 ameliorated the APAP-induced hepatotoxicity in C57BL/6N mice treated with APAP at a dose of 400 mg/kg intraperitoneally. However, mice treated with APAP alone developed massive centrilobular necrosis, and APAP increased their serum alanine aminotransferase and aspartate aminotransferase levels. Furthermore, A769662 administration prevented the loss of intracellular ATP without interfering with the APAP-mediated reduction of mitochondrial dysfunction. In contrast, inhibition of glycolysis by 2-deoxy-glucose eliminated the beneficial effects of A769662 on APAP-mediated liver injury. In conclusion, A769662 can effectively protect mice against APAP-induced liver injury through ATP synthesis by anaerobic glycolysis. Furthermore, stimulation of AMPK may have potential therapeutic application for APAP overdose. PMID:26434492

  7. Role of K(V)LQT1 in cyclic adenosine monophosphate-mediated Cl(-) secretion in human airway epithelia.

    PubMed

    Mall, M; Wissner, A; Schreiber, R; Kuehr, J; Seydewitz, H H; Brandis, M; Greger, R; Kunzelmann, K

    2000-09-01

    Ion transport defects underlying cystic fibrosis (CF) lung disease are characterized by impaired cyclic adenosine monophosphate (cAMP)-dependent Cl(-) conductance. Activation of Cl(-) secretion in airways depends on simultaneous activation of luminal Cl(-) channels and basolateral K(+) channels. We determined the role of basolateral K(+) conductance in cAMP- dependent Cl(-) secretion in native human airway epithelium obtained from non-CF and CF patients. CF tissues showed typical alterations of short-circuit currents with enhanced amiloride-sensitive Na(+) conductance and defective cAMP-mediated Cl(-) conductance. In non-CF tissues, Cl(-) secretion was significantly inhibited by the chromanol 293B (10 micromol/liter), a specific inhibitor of K(V)LQT1 K(+) channels. Inhibition was increased after cAMP-dependent stimulation. Similar effects were obtained with Ba(2+) (5 mmol/liter). In patch-clamp experiments with a human bronchial epithelial cell line, stimulation with forskolin (10 micromol/liter) simultaneously activated Cl(-) and K(+) conductance. The K(+) conductance was reversibly inhibited by Ba(2+) and 293B. Analysis of reverse-transcribed messenger RNA from non-CF and CF airways showed expression of human K(V)LQT1. We conclude that the K(+) channel K(V)LQT1 is important in maintaining cAMP-dependent Cl(-) secretion in human airways. Activation of K(V)LQT1 in CF airways in parallel with stimulation of residual CF transmembrane conductance regulator Cl(-) channel activity or alternative Cl(-) channels could help to circumvent the secretory defect.

  8. Dietary effects of adenosine monophosphate to enhance growth, digestibility, innate immune responses and stress resistance of juvenile red sea bream, Pagrus major.

    PubMed

    Hossain, Md Sakhawat; Koshio, Shunsuke; Ishikawa, Manabu; Yokoyama, Saichiro; Sony, Nadia Mahjabin

    2016-09-01

    Our study explored the dietary effects of adenosine monophosphate (AMP) to enhance growth, digestibility, innate immune responses and stress resistance of juvenile red sea bream. A semi-purified basal diet supplemented with 0% (Control), 0.1% (AMP-0.1), 0.2% (AMP-0.2), 0.4% (AMP-0.4) and 0.8% (AMP-0.8) purified AMP to formulate five experimental diets. Each diet was randomly allocated to triplicate groups of fish (mean initial weight 3.4 g) for 56 days. The results indicated that dietary AMP supplements tended to improve growth performances. One of the best ones was found in diet group AMP-0.2, followed by diet groups AMP-0.1, AMP-0.4 and AMP-0.8. The Apparent digestibility coefficients (dry matter, protein and lipid) also improved by AMP supplementation and the significantly highest dry matter digestibility was observed in diet group AMP-0.2. Fish fed diet groups AMP-0.2 and AMP-0.4 had significantly higher peroxidase and bactericidal activities than fish fed the control diet. Nitro-blue-tetrazolium (NBT) activity was found to be significantly (P < 0.05) greater in fish fed diet groups AMP-0.4 and AMP-0.8. Total serum protein, lysozyme activity and agglutination antibody titer were also increased (P > 0.05) by dietary supplementation. In contrast, catalase activity decreased with AMP supplementation. Moreover, the fish fed AMP supplemented diets had better improvement (P < 0.05) in body lipid contents, condition factor, hematocrit content and glutamyl oxaloacetic transaminase (GOT) level than the control group. Supplementation also improved both freshwater and oxidative stress resistances. Interestingly, the fish fed diet groups AMP-0.2 and AMP-0.4 showed the least oxidative stress condition. Finally it is concluded that, dietary AMP supplementation enhanced the growth, digestibility, immune response and stress resistance of red sea bream. The regression analysis revealed that a dietary AMP supplementation between 0.2 and 0.4% supported weight gain and

  9. Muscle A-Kinase Anchoring Protein-α is an Injury-Specific Signaling Scaffold Required for Neurotrophic- and Cyclic Adenosine Monophosphate-Mediated Survival.

    PubMed

    Wang, Yan; Cameron, Evan G; Li, Jinliang; Stiles, Travis L; Kritzer, Michael D; Lodhavia, Rahul; Hertz, Jonathan; Nguyen, Tu; Kapiloff, Michael S; Goldberg, Jeffrey L

    2015-12-01

    Neurotrophic factor and cAMP-dependent signaling promote the survival and neurite outgrowth of retinal ganglion cells (RGCs) after injury. However, the mechanisms conferring neuroprotection and neuroregeneration downstream to these signals are unclear. We now reveal that the scaffold protein muscle A-kinase anchoring protein-α (mAKAPα) is required for the survival and axon growth of cultured primary RGCs. Although genetic deletion of mAKAPα early in prenatal RGC development did not affect RGC survival into adulthood, nor promoted the death of RGCs in the uninjured adult retina, loss of mAKAPα in the adult increased RGC death after optic nerve crush. Importantly, mAKAPα was required for the neuroprotective effects of brain-derived neurotrophic factor and cyclic adenosine-monophosphate (cAMP) after injury. These results identify mAKAPα as a scaffold for signaling in the stressed neuron that is required for RGC neuroprotection after optic nerve injury. PMID:26844267

  10. Adenosine monophosphate deaminase 3 activation shortens erythrocyte half-life and provides malaria resistance in mice.

    PubMed

    Hortle, Elinor; Nijagal, Brunda; Bauer, Denis C; Jensen, Lora M; Ahn, Seong Beom; Cockburn, Ian A; Lampkin, Shelley; Tull, Dedreia; McConville, Malcolm J; McMorran, Brendan J; Foote, Simon J; Burgio, Gaetan

    2016-09-01

    The factors that determine red blood cell (RBC) lifespan and the rate of RBC aging have not been fully elucidated. In several genetic conditions, including sickle cell disease, thalassemia, and G6PD deficiency, erythrocyte lifespan is significantly shortened. Many of these diseases are also associated with protection from severe malaria, suggesting a role for accelerated RBC senescence and clearance in malaria resistance. Here, we report a novel, N-ethyl-N-nitrosourea-induced mutation that causes a gain of function in adenosine 5'-monophosphate deaminase (AMPD3). Mice carrying the mutation exhibit rapid RBC turnover, with increased erythropoiesis, dramatically shortened RBC lifespan, and signs of increased RBC senescence/eryptosis, suggesting a key role for AMPD3 in determining RBC half-life. Mice were also found to be resistant to infection with the rodent malaria Plasmodium chabaudi. We propose that resistance to P. chabaudi is mediated by increased RBC turnover and higher rates of erythropoiesis during infection. PMID:27465915

  11. Amp Synthesis in Aqueous Solution of Adenosine and Phosphorus Pentoxide

    NASA Astrophysics Data System (ADS)

    Yamagata, Y.; Kojima, H.; Ejiri, K.; Inomata, K.

    1982-12-01

    Possible formation of a P4O10 molecule in magma, the stability of the molecule in hydrous volcanic gas at high temperatures and a possible prebiotic phosphate cycle were discussed in relation to chemical evolution. To demonstrate the utility of phosphorus pentoxide as a phosphorylating agent, aqueous solutions of adenosine (0.02M) and phosphorus pentoxide (0.2M) were incubated at 37°C for 5 months. The pH of the solutions was adjusted every day or every few days to each fixed value (9.0, 10.5, 11.5, 12.5) with 10 N NaOH. The HPLC analysis showed the formation of 2'-AMP, 3'-AMP, 5'-AMP, cyclic (2' 3')-AMP and cyclic (3' 5')-AMP. The main components of the products were 2'- and 3'-AMP, though cyclic (2' 3')-AMP was the main component in the early period of the incubation at pH 9.0. The yields (conversion rate of adenosine to AMPs) were increased almost linearly with the incubation time for 5 months in the case of pH 9.0. The final yields were about 3% (pH 9.0), 6% (pH 9.0, 1 M NaCl), 5% (pH 9.0, 0.01 M CaCl2, 0.01 M MgCl2), 7% (pH 9.0, 0.5 M NaCl, 0.01 M CaCl2, 0.01 M MgCl2), 9% (pH 9.0, 1 M NaCl, 0.01 M CaCl2, 0.01 M MgCl2), 32% (pH 10.5), 43% (pH 11.5), 35% (pH 12.5).

  12. Study of the Renal Tubular Interactions of Thyrocalcitonin, Cyclic Adenosine 3′, 5′ -Monophosphate, 25-Hydroxycholecalciferol, and Calcium Ion

    PubMed Central

    Puschett, Jules B.; Beck, William S.; Jelonek, Adam; Fernandez, Pedro C.

    1974-01-01

    Acute clearance studies were performed in thyroparathyroidectomized animals to determine the actions and interactions of thyrocalcitonin (TCT), cyclic adenosine 3′5′-monophosphate (cAMP), 25-hydroxycholecalciferol (25HCC), and calcium ion on the reabsorption of phosphate, calcium, sodium, and potassium by the kidney. The infusion of 25HCC in a dosage of 60 U/h to moderately saline-expanded animals (2.5% body weight) induced a fall in the excretion of all of the ions under study after 90-120 min similar to that observed in previous experiments from this laboratory. Mean decrements in fractional excretion were: phosphate, 42.0% (P < 0.005); calcium, 25.0% (P < 0.005); sodium, 23.4% (P < 0.001); and potassium, 14.7% (P < 0.005). The superimposition of either porcine or salmon TCT (1-100 MRC U/h for 2 h) resulted in no further alterations in electrolyte excretion. However, the infusion of TCT during steady-state saline expansion, before the administration of 25HCC, obviated the renal transport effects of the vitamin D metabolite. Both in the latter studies, as well as those in which similar doses of TCT were given to hydropenic animals, the hormone itself failed to induce any consistent alteration in electrolyte excretion. Cyclic AMP (50 mg/h) caused an increase in the excretion of phosphate, sodium, and potassium and no change in calcium excretion. Like TCT, the nucleotide blocked the action of 25HCC on the kidney. Raising the mean level of serum ultrafilterable calcium to 3.02±0.25 mEq/liter from 1.62±0.17 mEq/liter likewise prevented enhanced ionic reabsorption due to 25HCC. PMID:4359939

  13. The role of cyclic AMP and its protein kinase in mediating acetylcholine release and the action of adenosine at frog motor nerve endings.

    PubMed Central

    Hirsh, J. K.; Silinsky, E. M.; Solsona, C. S.

    1990-01-01

    1. The importance of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and its protein kinase (protein kinase A, PKA) in promoting acetylcholine (ACh) release was studied at frog motor nerve endings. The effects of cyclic AMP-dependent protein phosphorylation on the action of adenosine receptor agonists were also investigated. 2. Cyclic AMP was delivered to a local region of the cytoplasm just beneath the plasma membrane of motor nerve endings using phospholipid vesicles (liposomes) as a vehicle. Cyclic AMP in liposomes produced a parallel reduction in the mean level of evoked ACh release (m) and spontaneous ACh release (miniature endplate potential frequency; m.e.p.p.f) in most experiments. These inhibitory effects of cyclic AMP on quantal ACh release resemble the action of adenosine. 3. The effects of global increases in cytoplasmic cyclic AMP concentrations using lipophilic cyclic AMP analogues were generally different from those observed with cyclic AMP. 8-(4-Chlorophenylthio) cyclic AMP (CPT cyclic AMP) produced approximately two fold increases in m and m.e.p.p.f. Dibutyryl cyclic AMP (db cyclic AMP) also increased m and m.e.p.p.f, with the effect on m being smaller and more variable. 4. All three cyclic AMP analogues reduced the effects of adenosine receptor agonists on spontaneous and evoked ACh release. 5. The roles of protein phosphorylation in mediating ACh release and the inhibitory effects of adenosine were studied with the protein kinase inhibitor H7. H7 (30-100 microM) produced no consistent effect on evoked or spontaneous ACh release. At these concentrations, however, H7 exerted an unfortunate inhibitory action on the nicotinic ACh receptor/ion channel.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2175231

  14. Developmental Competence of Vitrified-Warmed Bovine Oocytes at the Germinal-Vesicle Stage is Improved by Cyclic Adenosine Monophosphate Modulators during In Vitro Maturation

    PubMed Central

    Ezoe, Kenji; Yabuuchi, Akiko; Tani, Tetsuya; Mori, Chiemi; Miki, Tetsuya; Takayama, Yuko; Beyhan, Zeki; Kato, Yoko; Okuno, Takashi; Kobayashi, Tamotsu; Kato, Keiichi

    2015-01-01

    Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV) oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP) modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK) or 3-isobutyl-1-methylxanthine (IBMX) to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF) activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming. PMID:25965267

  15. Medium optimization for the production of cyclic adenosine 3',5'-monophosphate by Microbacterium sp. no. 205 using response surface methodology.

    PubMed

    Chen, Xiao-Chun; Bai, Jian-Xin; Cao, Jia-Ming; Li, Zhen-Jiang; Xiong, Jian; Zhang, Lei; Hong, Yuan; Ying, Han-Jie

    2009-01-01

    Response surface methodology was employed to optimize medium composition for the production of cyclic adenosine 3',5'-monophosphate (cAMP) with Microbacterium sp. no. 205. A fractional factorial design (2(11-7)) was applied to evaluate the effects of different components in the medium. K(2)HPO(4), MgSO(4) and NaF were found to significantly influence on the cAMP production. The steepest ascent method was used to access the optimal region of the medium composition. The concentrations of the three factors were optimized subsequently using central composite design and response surface methodology. The optimal medium composition to achieve the optimal cAMP production was determined (g/L): K(2)HPO(4), 12.78; MgSO(4), 3.53 and NaF, 0.18. The corresponding cAMP concentration was 8.50 g/L, which was about 1.8-fold increase compared with that using the original medium. Validation experiments were also carried out to prove the adequacy and the accuracy of the model obtained. The cAMP fermentation in 5L fermenter reached 9.87 g/L. PMID:18778935

  16. Vasoactive intestinal peptide: A potent stimulator of adenosine 3′:5′-cyclic monophosphate accumulation in gut carcinoma cell lines in culture*

    PubMed Central

    Laburthe, M.; Rousset, M.; Boissard, C.; Chevalier, G.; Zweibaum, A.; Rosselin, G.

    1978-01-01

    Vasoactive intestinal peptide (VIP) is a potent and efficient stimulator of adenosine 3′:5′-cyclic monophosphate (cAMP) accumulation in a human colon carcinoma cell line, HT 29. cAMP accumulation is sensitive to a concentration of VIP as low as 3×10-12 M. Maximum VIP-induced cAMP levels were observed with 10-9 M VIP and are about 200 times above the basal levels. Half-maximum cAMP production was obtained at 3×10-10 M VIP. 125I-Labeled VIP was found to bind to HT 29 cells; this binding was competitively inhibited by concentrations of unlabeled VIP between 10-10 and 10-7 M. Half-maximum inhibition of binding was observed with 2×10-9 M VIP. Secretin also stimulated cAMP accumulation in HT 29 cells, but its effectiveness was 1/1000 that of VIP. The other peptides tested at 10-7 M, such as insulin, glucagon, bovine pancreatic polypeptide, somatostatin, octapeptide of cholecystokinin, neurotensin, and substance P, did not stimulate cAMP accumulation. Prostaglandin E1 and catecholamines stimulated cAMP production but were 1/2.3 and 1/5.5 as efficient as VIP, respectively. Another malignant cell line from the gut, the human rectal tumor cell line HRT 18, is also sensitive to VIP. In HRT 18 cells, VIP stimulated cAMP accumulation with a maximal effect at 10-8 M; half-maximum stimulation was observed at about 10-9 M. These results demonstrate the presence of VIP receptors in two malignant human intestinal cell lines (HT 29 and HRT 18) in culture and provide a model for studying the action of VIP on cell proliferation. PMID:208077

  17. Regulation of insulin-like growth factor I transcription by cyclic adenosine 3',5'-monophosphate (cAMP) in fetal rat bone cells through an element within exon 1: protein kinase A-dependent control without a consensus AMP response element

    NASA Technical Reports Server (NTRS)

    McCarthy, T. L.; Thomas, M. J.; Centrella, M.; Rotwein, P.

    1995-01-01

    Insulin-like growth factor I (IGF-I) is a locally synthesized anabolic growth factor for bone. IGF-I synthesis by primary fetal rat osteoblasts (Ob) is stimulated by agents that increase the intracellular cAMP concentration, including prostaglandin E2 (PGE2). Previous studies with Ob cultures demonstrated that PGE2 enhanced IGF-I transcription through selective use of IGF-I promoter 1, with little effect on IGF-I messenger RNA half-life. Transient transfection of Ob cultures with an array of promoter 1-luciferase reporter fusion constructs has now allowed localization of a potential cis-acting promoter element(s) responsible for cAMP-stimulated gene expression to the 5'-untranslated region (5'-UTR) of IGF-I exon 1, within a segment lacking a consensus cAMP response element. Our evidence derives from three principal observations: 1) a transfection construct containing only 122 nucleotides (nt) of promoter 1 and 328 nt of the 5'-UTR retained full PGE2-stimulated reporter expression; 2) maximal PGE2-driven reporter expression required the presence of nt 196 to 328 of exon 1 when tested within the context of IGF-I promoter 1; 3) cotransfection of IGF-I promoter-luciferase-reporter constructs with a plasmid encoding the alpha-isoform of the catalytic subunit of murine cAMP-dependent protein kinase (PKA) produced results comparable to those seen with PGE2 treatment, whereas cotransfection with a plasmid encoding a mutant regulatory subunit of PKA that cannot bind cAMP blocked PGE2-induced reporter expression. Deoxyribonuclease I footprinting of the 5'-UTR of exon 1 demonstrated protected sequences at HS3A, HS3B, and HS3D, three of six DNA-protein binding sites previously characterized with rat liver nuclear extracts. Of these three regions, only the HS3D binding site is located within the functionally identified hormonally responsive segment of IGF-I exon 1. These results directly implicate PKA in the control of IGF-I gene transcription by PGE2 and identify a segment of

  18. Xylazine Activates Adenosine Monophosphate-Activated Protein Kinase Pathway in the Central Nervous System of Rats.

    PubMed

    Shi, Xing-Xing; Yin, Bai-Shuang; Yang, Peng; Chen, Hao; Li, Xin; Su, Li-Xue; Fan, Hong-Gang; Wang, Hong-Bin

    2016-01-01

    Xylazine is a potent analgesic extensively used in veterinary and animal experimentation. Evidence exists that the analgesic effect can be inhibited using adenosine 5'-monophosphate activated protein kinase (AMPK) inhibitors. Considering this idea, the aim of this study was to investigate whether the AMPK signaling pathway is involved in the central analgesic mechanism of xylazine in the rat. Xylazine was administrated via the intraperitoneal route. Sprague-Dawley rats were sacrificed and the cerebral cortex, cerebellum, hippocampus, thalamus and brainstem were collected for determination of liver kinase B1 (LKB1) and AMPKα mRNA expression using quantitative real-time polymerase chain reaction (qPCR), and phosphorylated LKB1 and AMPKα levels using western blot. The results of our study showed that compared with the control group, xylazine induced significant increases in AMPK activity in the cerebral cortex, hippocampus, thalamus and cerebellum after rats received xylazine (P < 0.01). Increased AMPK activities were accompanied with increased phosphorylation levels of LKB1 in corresponding regions of rats. The protein levels of phosphorylated LKB1 and AMPKα in these regions returned or tended to return to control group levels. However, in the brainstem, phosphorylated LKB1 and AMPKα protein levels were decreased by xylazine compared with the control (P < 0.05). In conclusion, our data indicates that xylazine alters the activities of LKB1 and AMPK in the central nervous system of rats, which suggests that xylazine affects the regulatory signaling pathway of the analgesic mechanism in the rat brain. PMID:27049320

  19. Hydrogen peroxide induces murine macrophage chemokine gene transcription via extracellular signal-regulated kinase- and cyclic adenosine 5'-monophosphate (cAMP)-dependent pathways: involvement of NF-kappa B, activator protein 1, and cAMP response element binding protein.

    PubMed

    Jaramillo, Maritza; Olivier, Martin

    2002-12-15

    Hydrogen peroxide (H(2)O(2)) has been shown to act as a second messenger that activates chemokine expression. In the present study, we investigated the mechanisms underlying this cellular regulation in the murine macrophage cell line B10R. We report that H(2)O(2) increases mRNA expression of various chemokines, macrophage-inflammatory protein (MIP)-1alpha/CC chemokine ligand (CCL)3, MIP-1beta/CCL4, MIP-2/CXC chemokine ligand 2, and monocyte chemoattractant protein-1/CCL2, by activating the extracellular signal-regulated kinase (ERK) pathway and the nuclear translocation of the transcription factors NF-kappaB, AP-1, and CREB. Blockage of the ERK pathway with specific inhibitors against mitogen-activated protein kinase kinase 1/2 and ERK1/ERK2 completely abolished both the H(2)O(2)-mediated chemokine up-regulation and the activation of all NF studied. Similarly, selective inhibition of cAMP and NF-kappaB strongly down-regulated the induction of all chemokine transcripts as well as CREB and NF-kappaB activation, respectively. Of interest, we detected a significant decrease of NF-kappaB, AP-1, and CREB DNA binding activities by reciprocal competition for these binding sites when either specific cold oligonucleotides (NF-kappaB, AP-1, and CREB) or Abs against various transcription factor subunits (p50, p65, c-Fos, Jun B, c-Jun, and CREB-1) were added. These findings indicate that cooperation between ERK- and cAMP-dependent pathways seems to be required to achieve the formation of an essential transcriptional factor complex for maximal H(2)O(2)-dependent chemokine modulation. Finally, experiments performed with actinomycin D suggest that H(2)O(2)-mediated MIP-1beta mRNA up-regulation results from transcriptional control, whereas that of MIP-1alpha, MIP-2, and monocyte chemoattractant protein-1 is due to both gene transcription activation and mRNA posttranscriptional stabilization.

  20. Xylazine Activates Adenosine Monophosphate-Activated Protein Kinase Pathway in the Central Nervous System of Rats

    PubMed Central

    Shi, Xing-Xing; Yin, Bai-Shuang; Yang, Peng; Chen, Hao; Li, Xin; Su, Li-Xue; Fan, Hong-Gang; Wang, Hong-Bin

    2016-01-01

    Xylazine is a potent analgesic extensively used in veterinary and animal experimentation. Evidence exists that the analgesic effect can be inhibited using adenosine 5’-monophosphate activated protein kinase (AMPK) inhibitors. Considering this idea, the aim of this study was to investigate whether the AMPK signaling pathway is involved in the central analgesic mechanism of xylazine in the rat. Xylazine was administrated via the intraperitoneal route. Sprague-Dawley rats were sacrificed and the cerebral cortex, cerebellum, hippocampus, thalamus and brainstem were collected for determination of liver kinase B1 (LKB1) and AMPKα mRNA expression using quantitative real-time polymerase chain reaction (qPCR), and phosphorylated LKB1 and AMPKα levels using western blot. The results of our study showed that compared with the control group, xylazine induced significant increases in AMPK activity in the cerebral cortex, hippocampus, thalamus and cerebellum after rats received xylazine (P < 0.01). Increased AMPK activities were accompanied with increased phosphorylation levels of LKB1 in corresponding regions of rats. The protein levels of phosphorylated LKB1 and AMPKα in these regions returned or tended to return to control group levels. However, in the brainstem, phosphorylated LKB1 and AMPKα protein levels were decreased by xylazine compared with the control (P < 0.05). In conclusion, our data indicates that xylazine alters the activities of LKB1 and AMPK in the central nervous system of rats, which suggests that xylazine affects the regulatory signaling pathway of the analgesic mechanism in the rat brain. PMID:27049320

  1. Control and localization of rat adrenal cyclic guanosine 3', 5'-monophosphate. Comparison with adrenal cyclic adenosine 3', 5'-monophosphate.

    PubMed Central

    Whitley, T H; Stowe, N W; Ong, S H; ey, R L; Steiner, A L

    1975-01-01

    Cyclic AMP and cyclic GMP were measured in rat adrenal glands after either hypophysectomy alone or after hypophysectomy and treatment with ACTH. Adrenal cyclic GMP levels rise in acutely hypophysectomized rats to a maximum at 1 h of approximately 200% of control levels; there is a return to base line at 4-12 h after hypophysectomy. In contrast, adrenal cyclic AMP falls immediately to about 50% of control levels after hypophysectomy and remains at approximately 1 pmol per mg tissue. Doses of ACTH beyond the physiological range markedly suppress adrenal cyclic GMP while producing a 50-fold or greater rise in cyclic AMP in hypophysectomized rats. This pattern of adrenal cyclic GMP rise was unchanged in acutely hypophysectomized animals treated with desamethasone. N-6-2'-0 dibutyryl cyclic AMP acted similarly to the effect of ACTH in bringing about a suppression of adrenal cyclic GMP levels. Physiological i.v. pulse doses of ACTH produced a rapid dose related increase in adrenal cyclic GMP. In vitro incubation of quartered adrenal pairs with 500 mU ACTH produced elevated cyclic AMP levels and suppression of cyclic GMP. Whereas adrenal cyclic AMP fell rapidly to 50% of control levels after hypophysectomy and remained at about 1 pmol per mg tissue for 7 days, adrenal cyclic GMP showed a biphasic rhythm in long-term hypophysectomized animals. After an initial peak at 1 h after hypophysectomy, adrenal cyclic GMP declined to baseline at 4-12 h but thereafter progressively rose with time, eventually reaching levels over 1 pmol per mg tissue. Fluorescent immunocytochemical staining of rat adrenal zona fasciculata showed cyclic AMP largely confined to cytoplasmic elements with little fluorescence contained in nuclei. In constant, cyclic GMP was found discretely positioned in nuclei with prominent fluorescence in nucleoli in addition to cytoplasmic localization. It is concluded that in hypophysectomized rats ACTH, either directly or in conjunction with altertion of adrenal

  2. Adenosine 3':5'-cyclic monophosphate in higher plants: Isolation and characterization of adenosine 3':5'-cyclic monophosphate from Kalanchoe and Agave.

    PubMed

    Ashton, A R; Polya, G M

    1977-07-01

    1.3':5'-Cyclic AMP was extensively purified from Kalanchoe daigremontiana and Agave americana by neutral alumina and anion- and cation-exchange column chromatography. Inclusion of 3':5'-cyclic [8-3H]AMP from the point of tissue extraction permitted calculation of yields. The purification procedure removed contaminating material that was shown to interfere with the 3':5'-cyclic AMP estimation and characterization procedures. 2. The partially purified 3':5'-cyclic AMP was quantified by means of a radiochemical saturation assay using an ox heart 3':5'-cyclic AMP-binding protein and by an assay involving activation of a mammalian protein kinase. 3. The plant 3':5'-cyclic AMP co-migrated with 3':5'-cyclic [8-3H]AMP on cellulose chromatography, poly(ethyleneimine)-cellulose chromatography and silica-gel t.l.c. developed with several solvent systems. 4. The plant 3':5'-cyclic AMP was degraded by ox heart 3':5'-cyclic nucleotide phosphodiesterase at the same rates as authentic 3':5'-cyclic AMP. 1-Methyl-3-isobutylxanthine (1 mM), a specific inhibitor of the 3':5'-cyclic nucleotide phosphodieterase, completely inhibited such degradation. 5. The concentrations of 3':5'-cyclic AMP satisfying the above criteria in Kalanchoe and Agave were 2-6 and 1 pmol/g fresh wt. respectively. Possible bacterial contribution to these analyses was estimated to be less than 0.002pmol/g fresh wt. Evidence for the occurrence of 3':5'-cyclic AMP in plants is discussed.

  3. Role of adenosine deaminase, ecto-(5'-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes.

    PubMed Central

    Newby, A C

    1980-01-01

    1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable ( less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells. PMID:6249264

  4. Dielectric spectra broadening as a signature for dipole-matrix interaction. III. Water in adenosine monophosphate/adenosine-5'-triphosphate solutions.

    PubMed

    Puzenko, Alexander; Levy, Evgeniya; Shendrik, Andrey; Talary, Mark S; Caduff, Andreas; Feldman, Yuri

    2012-11-21

    In this, the third part of our series on the dielectric spectrum symmetrical broadening of water, we consider the nucleotide aqueous solutions. Where in Parts I [E. Levy et al., J. Chem. Phys. 136, 114502 (2012)] and II [E. Levy et al., J. Chem. Phys. 136, 114503 (2012)], the dipole-dipole or ion-dipole interaction had a dominant feature, now the interplay between these two types of dipole-matrix interactions will be considered. We present the results of high frequency dielectric measurements of different concentrations of adenosine monophosphate/adenosine-5'-triphosphate aqueous solutions. We observed the Cole-Cole broadening of the main relaxation peak of the solvent in the solutions. Moreover, depending on the nucleotide concentration, we observed both types of dipole-matrix interaction. The 3D trajectory approach (described in detail in Part I) is applied in order to highlight the differences between the two types of interaction.

  5. Gas-Phase Conformations and Energetics of Protonated 2^'-DEOXYADENOSINE-5^'-MONOPHOSPHATE and ADENOSINE-5^'-MONOPHOSPHATE: Irmpd Action Spectroscopy and Theoretical Studies

    NASA Astrophysics Data System (ADS)

    Wu, Ranran; Nei, Y.-W.; He, Chenchen; Hamlow, Lucas; Berden, Giel; Oomens, J.; Rodgers, M. T.

    2015-06-01

    Nature uses protonation to alter the structures and reactivities of molecules to facilitate various biological functions and chemical transformations. For example, in nucleobase repair and salvage processes, protonation facilitates nucleobase removal by lowering the activation barrier for glycosidic bond cleavage. Systematic studies of the structures of protonated 2'-deoxyribonucleotides and ribonucleotides may provide insight into the roles protonation plays in altering the nucleobase orientation relative to the glycosidic bond and sugar puckering. In this study, infrared multiple photon dissociation (IRMPD) action spectroscopy experiments in conjunction with electronic structure calculations are performed to probe the effects of protonation on the structures and stabilities of 2^'-deoxyadenosine-5^'-monophosphate (pdAdo) and adenosine-5^'-monophosphate (pAdo). Photodissociation as a function of IR wavelength is measured to generate the IRMPD action spectra. Geometry optimizations and frequency analyses performed at the B3LYP/6-311+G(d,p) level of theory are used to characterize the stable low-energy structures and to generate their linear IR spectra. Single point energy calculations performed at the B3LYP/6-311+G(2d,2p) and MP2(full)/6-311+G(2d,2p) levels of theory provide relative stabilities of the optimized conformations. The structures accessed in the experiments are determined by comparing the calculated linear IR spectra for the stable low-energy conformers computed to the measured IRMPD action spectra. The effects of the 2^'-hydroxyl moiety are elucidated by comparing the structures and IRMPD spectra of [pAdo+H]+ to those of its DNA analogue. Comparisons are also made to the deprotonated forms of these nucleotides and the protonated forms of the analogous nucleosides to elucidate the effects of protonation and the phosphate group on the structures.

  6. Airway hyperresponsiveness to methacholine, adenosine 5-monophosphate, mannitol, eucapnic voluntary hyperpnoea and field exercise challenge in elite cross-country skiers

    PubMed Central

    Sue-Chu, Malcolm; Brannan, John D; Anderson, Sandra D; Chew, Nora; Bjermer, Leif

    2010-01-01

    Background Methacholine hyperresponsiveness is prevalent in elite athletes. Comparative studies have hitherto been limited to methacholine, eucapnic voluntary hyperpnoea and exercise. This study investigated airway responsiveness to these stimuli as well as to adenosine 5′-monophosphate (AMP) and mannitol, in 58 cross-country ski athletes. Methods Exhaled nitric oxide concentration (FENO), spirometry and bronchial challenge in random order with methacholine, AMP and mannitol were consecutively performed on three study days in the autumn. Specific IgE to eight aeroallergens and a self-completed questionnaire about respiratory symptoms, allergy and asthmatic medication were also performed on day 1. Eucapnic voluntary hyperventilation (EVH) and field exercise tests were randomly performed in 33 of the skiers on two study days in the following winter. Results Of 25 (43%) skiers with airway hyperresponsiveness (AHR), 23, five and three skiers were hyperresponsive to methacholine, AMP and mannitol, respectively. Methacholine hyperresponsiveness was more prevalent in subjects without asthma-like symptoms. The FENO was not significantly different in skiers with and without methacholine hyperresponsiveness. Four of 14 skiers with and four of 19 skiers without methacholine hyperresponsiveness were hyperresponsive to EVH or exercise challenge. AHR to any stimulus was present in 16 asymptomatic and nine symptomatic skiers. Asthma-like symptoms were not correlated with AHR to any stimulus. Conclusions Methacholine hyperresponsiveness is more common in asymptomatic skiers and is a poor predictor of hyperresponsiveness to mannitol and hyperpnoea. The low prevalence of hyperresponsiveness to indirect stimuli may suggest differences in the pathogenesis of methacholine hyperresponsiveness in elite skiers and non-athletes. PMID:20460257

  7. Effects of single or combined histamine H1-receptor and leukotriene CysLT1-receptor antagonism on nasal adenosine monophosphate challenge in persistent allergic rhinitis

    PubMed Central

    Lee, Daniel K C; Jackson, Catherine M; Soutar, Patricia C; Fardon, Thomas C; Lipworth, Brian J

    2004-01-01

    Background The effects of single or combined histamine H1-receptor and leukotriene CysLT1-receptor antagonism on nasal adenosine monophosphate (AMP) challenge in allergic rhinitis are unknown. Objective We elected to study the effects of usual clinically recommended doses of fexofenadine (FEX), montelukast (ML) and FEX + ML combination, compared with placebo (PL), on nasal AMP challenge in patients with persistent allergic rhinitis. Methods Twelve patients with persistent allergic rhinitis (all skin prick positive to house dust mite) were randomized in a double-blind cross-over fashion to receive for 1 week either FEX 180 mg, ML 10 mg, FEX 180 mg +ML 10 mg combination, or PL, with nasal AMP challenge performed 12 h after dosing. There was a 1-week washout period between each randomized treatment. The primary outcome measure was the maximum percentage peak nasal inspiratory flow (PNIF) fall from baseline over a 60-min period after nasal challenge with a single 400 mg ml−1 dose of AMP. The area under the 60-min time–response curve (AUC) and nasal symptoms were measured as secondary outcomes. Results There was significant attenuation (P < 0.05) of the mean maximum percentage PNIF fall from baseline after nasal AMP challenge vs. PL, 48; with FEX, 37; 95% confidence interval for difference 2, 20; ML, 35 (4, 22); and FEX + ML, 32 (7, 24). The AUC (%.min) was also significantly attenuated (P < 0.05) vs. PL, 1893; with FEX, 1306 (30, 1143); ML, 1246 (214, 1078); and FEX + ML, 1153 (251, 1227). There were no significant differences for FEX vs. ML vs. FEX + ML comparing either the maximum or AUC response. The total nasal symptom score (out of 12) was also significantly improved (P < 0.05) vs. PL, 3.3; with FEX, 2.1 (0.3, 2.0); ML, 2.0 (0.5, 1.9); and FEX + ML, 2.5 (0.1, 1.4). Conclusion FEX and ML as monotherapy significantly attenuated the response to nasal AMP challenge and improved nasal symptoms compared with PL, while combination therapy conferred no additional

  8. In Silico Design for Adenosine Monophosphate-Activated Protein Kinase Agonist from Traditional Chinese Medicine for Treatment of Metabolic Syndromes

    PubMed Central

    Tang, Hsin-Chieh

    2014-01-01

    Adenosine monophosphate-activated protein kinase (AMPK) acts as a master mediator of metabolic homeostasis. It is considered as a significant millstone to treat metabolic syndromes including obesity, diabetes, and fatty liver. It can sense cellular energy or nutrient status by switching on the catabolic pathways. Investigation of AMPK has new findings recently. AMPK can inhibit cell growth by the way of autophagy. Thus AMPK has become a hot target for small molecular drug design of tumor inhibition. Activation of AMPK must undergo certain extent change of the structure. Through the methods of structure-based virtual screening and molecular dynamics simulation, we attempted to find out appropriate small compounds from the world's largest TCM Database@Taiwan that had the ability to activate the function of AMPK. Finally, we found that two TCM compounds, eugenyl_beta-D-glucopyranoside and 6-O-cinnamoyl-D-glucopyranose, had the qualification to be AMPK agonist. PMID:24899913

  9. Adenosine modulates light responses of rat retinal ganglion cell photoreceptors througha cAMP-mediated pathway

    PubMed Central

    Sodhi, Puneet; Hartwick, Andrew T E

    2014-01-01

    Adenosine is an established neuromodulator in the mammalian retina, with A1 adenosine receptors being especially prevalent in the innermost ganglion cell layer. Activation of A1 receptors causes inhibition of adenylate cyclase, decreases in intracellular cyclic AMP (cAMP) levels and inhibition of protein kinase A (PKA). In this work, our aim was to characterize the effects of adenosine on the light responses of intrinsically photosensitive retinal ganglion cells (ipRGCs) and to determine whether these photoreceptors are subject to neuromodulation through intracellular cAMP-related signalling pathways. Using multielectrode array recordings from postnatal and adult rat retinas, we demonstrated that adenosine significantly shortened the duration of ipRGC photoresponses and reduced the number of light-evoked spikes fired by these neurons. The effects were A1 adenosine receptor-mediated, and the expression of this receptor on melanopsin-containing ipRGCs was confirmed by calcium imaging experiments on isolated cells in purified cultures. While inhibition of the cAMP/PKA pathway by adenosine shortened ipRGC light responses, stimulation of this pathway with compounds such as forskolin had the opposite effect and lengthened the duration of ipRGC spiking. Our findings reveal that the modification of ipRGC photoresponses through a cAMP/PKA pathway is a general feature of rat ganglion cell photoreceptors, and this pathway can be inhibited through activation of A1 receptors by adenosine. As adenosine levels in the retina rise at night, adenosinergic modulation of ipRGCs may serve as an internal regulatory mechanism to limit transmission of nocturnal photic signals by ipRGCs to the brain. Targeting retinal A1 adenosine receptors for ipRGC inhibition represents a potential therapeutic target for sleep disorders and migraine-associated photophobia. PMID:25038240

  10. Role of adenosine monophosphate in regulation of metabolic pathways of perfused rat liver

    PubMed Central

    Hunter, A. R.; Jefferson, L. S.

    1969-01-01

    1. By perfusion of rat livers with 3mm-AMP in the perfusion medium we obtain increased intracellular concentrations of AMP. 2. These high intracellular concentrations of AMP lead to an increased output of glucose and urea into the perfusion medium. 3. The increased output of glucose in livers from fed rats is brought about primarily by an AMP-stimulated breakdown of liver glycogen. In livers from starved rats the increase in glucose output is not as great, reflecting the low contents of glycogen in livers from starved rats. 4. AMP inhibits gluconeogenesis from lactate in perfused livers. In the presence of high concentrations of lactate, however, the counteracting effects of AMP to increase glycogenolysis and to inhibit gluconeogenesis result in little change in the net glucose output. 5. The increased urea output is brought about by increased breakdown of amino acids that are present in the perfusion medium. In livers from starved rats the overall urea production is much higher, indicating increased catabolism of amino acids and other nitrogenous substrates in the absence of carbohydrate substrates. 6. AMP causes an inhibition of incorporation of labelled precursors into protein and nucleic acid. This may result from increased catabolism of precursors of proteins and nucleic acids as reflected by the more rapid breakdown of nitrogenous compounds. In support of this hypothesis, cell-free systems for amino acid incorporation isolated from livers perfused with and without AMP are equally capable of supporting protein synthesis. 7. The labelling pattern of RNA in perfused livers corresponds very closely to those found by pulse-labelling in vivo. AMP in no way alters the qualitative nature of the labelling patterns. 8. We consider these results as supporting evidence for the role of the concentration ratio of AMP to ATP in controlling the metabolic pathways that lead to the formation of ATP. PMID:5774478

  11. Vasoactive intestinal peptide attenuates liver ischemia/reperfusion injury in mice via the cyclic adenosine monophosphate-protein kinase a pathway.

    PubMed

    Ji, Haofeng; Zhang, Yu; Liu, Yuanxing; Shen, Xiu-Da; Gao, Feng; Nguyen, Terry T; Busuttil, Ronald W; Waschek, James A; Kupiec-Weglinski, Jerzy W

    2013-09-01

    Hepatic ischemia/reperfusion injury (IRI), an exogenous, antigen-independent, local inflammation response, occurs in multiple clinical settings, including liver transplantation, hepatic resection, trauma, and shock. The nervous system maintains extensive crosstalk with the immune system through neuropeptide and peptide hormone networks. This study examined the function and therapeutic potential of the vasoactive intestinal peptide (VIP) neuropeptide in a murine model of liver warm ischemia (90 minutes) followed by reperfusion. Liver ischemia/reperfusion (IR) triggered an induction of gene expression of intrinsic VIP; this peaked at 24 hours of reperfusion and coincided with a hepatic self-healing phase. Treatment with the VIP neuropeptide protected livers from IRI; this was evidenced by diminished serum alanine aminotransferase levels and well-preserved tissue architecture and was associated with elevated intracellular cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling. The hepatocellular protection rendered by VIP was accompanied by diminished neutrophil/macrophage infiltration and activation, reduced hepatocyte necrosis/apoptosis, and increased hepatic interleukin-10 (IL-10) expression. Strikingly, PKA inhibition restored liver damage in otherwise IR-resistant VIP-treated mice. In vitro, VIP not only diminished macrophage tumor necrosis factor α/IL-6/IL-12 expression in a PKA-dependent manner but also prevented necrosis/apoptosis in primary mouse hepatocyte cultures. In conclusion, our findings document the importance of VIP neuropeptide-mediated cAMP-PKA signaling in hepatic homeostasis and cytoprotection in vivo. Because the enhancement of neural modulation differentially regulates local inflammation and prevents hepatocyte death, these results provide the rationale for novel approaches to managing liver IRI in transplant patients. PMID:23744729

  12. Rapid quantification of adenosine cyclic 3',5'-monophosphate by competitive enzyme-linked immunosorbent assay.

    PubMed

    Hsieh, M S; Jap, T S; Chiang, H

    1993-01-01

    A reliable and rapid enzyme-linked immunosorbent assay (ELISA) for cyclic AMP determination is described. Succinyl cyclic AMP, coupled to human albumin, was injected into rabbit to elicit antibodies to cyclic nucleotide hapten. Succinyl cyclic nucleotide to human albumin as immunogen or the cyclic AMP to porcine thyroglobulin as coating antigen was conjugated by a carbodiimide coupling procedure. The latter conjugate, captured to microplate with coating buffer and blocked with 0.8% gelatin for 30 minutes, was bound to antibody in inverse proportion to free cyclic AMP in a sample or standard. Bound antibody was then quantified with horseradish peroxidase-labelled goat antirabbit immunoglobulin and ABTS (2, 2'-Azinobis (3-ethylbenzthiazolinesulfonic Acid). Our results showed that concentration of both standard and sample cyclic AMP could be measured as low as 2.5 fmol/well (0.05 pmol/ml). The intra- and inter-assay coefficients of variation for samples were 6.0-8.0% and 8.9-9.5%, respectively. In addition, there was no cross-reaction of the antisera with ADP, ATP, 5'-AMP or cyclic GMP. Short period of incubation at room temperature seems as good as long period of incubation at 4 degrees C. The biological study demonstrated a consistency between increase in platelet-cyclic AMP generation after prostaglandin E1 stimulation and its biological effects. Our approach to ELISA is validated by showing agreement in levels, obtained in parallel by ELISA and RIA, of cyclic AMP content in extracts of prostaglandin E1-stimulated platelet cells.

  13. 3',5'-cyclic adenosine monophosphate and adenylate cyclase in phototransduction by limulus ventral photoreceptors.

    PubMed Central

    Brown, J E; Kaupp, U B; Malbon, C C

    1984-01-01

    Biochemical and electrophysiological measurements were made on photoreceptor cells from Limulus ventral eyes to investigate the possible role of cyclic AMP and adenylate cyclase in the visual transduction mechanism. Cyclic AMP content in a photoreceptor-enriched fraction (the end organs) of Limulus ventral eyes was approximately 15 pmol/mg protein. The cyclic AMP content was increased by bathing eyes in 1-methyl-3-isobutyl xanthine or forskolin and was increased almost 100-fold when bathed in both. Illumination did not change cyclic AMP content significantly in any of these conditions. Discrete events that can be recorded electrophysiologically occur spontaneously in darkness. An increase in the frequency of discrete events is evoked by dim illumination. The discrete events are a sign of excitation of Limulus photoreceptor cells. Drug-induced changes in the rate of occurrence of discrete events recorded electrophysiologically in darkness were not correlated with changes in cyclic AMP content. Adenylate cyclase activity measured from a small number of pooled photoreceptor clusters was stimulated by fluoride and vanadate ions, hydrolysis-resistant analogues of GTP, cholera toxin and forskolin. The Limulus enzyme is similar pharmacologically to mammalian and avian adenylate cyclases. Activation of adenylate cyclase by drugs was not correlated with changes in the rate of occurrence of discrete events recorded electrophysiologically in darkness. A heat-treated Lubrol extract of membranes from Limulus ventral eyes reconstituted the adenylate cyclase activity of membranes from S49 mouse lymphoma cyc- mutant cells which lack a functional regulatory protein. These findings suggest that Limulus ventral eye photoreceptors contain a regulatory protein that mediates the activation of adenylate cyclase by guanine nucleotides, fluoride or cholera toxin. This regulatory protein is homologous with that found in mammalian and avian adenylate cyclases. Our findings suggest that

  14. A new crystal form of human histidine triad nucleotide-binding protein 1 (hHINT1) in complex with adenosine 5′-monophosphate at 1.38 Å resolution

    PubMed Central

    Dolot, Rafał; Ozga, Magdalena; Włodarczyk, Artur; Krakowiak, Agnieszka; Nawrot, Barbara

    2012-01-01

    Histidine triad nucleotide-binding protein 1 (HINT1) represents the most ancient and widespread branch of the histidine triad protein superfamily. HINT1 plays an important role in various biological processes and has been found in many species. Here, the structure of the human HINT1–adenosine 5′-monophosphate (AMP) complex at 1.38 Å resolution obtained from a new monoclinic crystal form is reported. The final structure has R cryst = 0.1207 (R free = 0.1615) and the model exhibits good stereochemical quality. Detailed analysis of the high-resolution data allowed the details of the protein structure to be updated in comparison to the previously published data. PMID:22869114

  15. Chronic intermittent hypoxia induces cardiac hypertrophy by impairing autophagy through the adenosine 5'-monophosphate-activated protein kinase pathway.

    PubMed

    Xie, Sheng; Deng, Yan; Pan, Yue-Ying; Ren, Jie; Jin, Meng; Wang, Yu; Wang, Zhi-Hua; Zhu, Die; Guo, Xue-Ling; Yuan, Xiao; Shang, Jin; Liu, Hui-Guo

    2016-09-15

    Autophagy is tightly regulated to maintain cardiac homeostasis. Impaired autophagy is closely associated with pathological cardiac hypertrophy. However, the relationship between autophagy and cardiac hypertrophy induced by chronic intermittent hypoxia (CIH) is not known. In the present study, we measured autophagy-related genes and autophagosomes during 10 weeks of CIH in rats, and 6 days in H9C2 cardiomyocytes, and showed that autophagy was impaired. This conclusion was confirmed by the autophagy flux assay. We detected significant hypertrophic changes in myocardium with impaired autophagy. Rapamycin, an autophagy enhancer, attenuated the cardiac hypertrophy induced by CIH. Moreover, silencing autophagy-related gene 5 (ATG5) exerted the opposite effect. The role of adenosine monophosphate-activated protein kinase (AMPK) in regulating autophagy under CIH was confirmed using AICAR to upregulate this enzyme and restore autophagy flux. Restoring autophagy by AICAR or rapamycin significantly reversed the hypertrophic changes in cardiomyocytes. To investigate the mechanism of autophagy impairment, we compared phospho (p)-AMPK, p-Akt, cathepsin D, and NFAT3 levels, along with calcineurin activity, between sham and CIH groups. CIH activated calcineurin, and inhibited AMPK and AMPK-mediated autophagy in an Akt- and NFAT3-independent manner. Collectively, these data demonstrated that impaired autophagy induced by CIH through the AMPK pathway contributed to cardiac hypertrophy. PMID:27412517

  16. Chronic intermittent hypoxia induces cardiac hypertrophy by impairing autophagy through the adenosine 5'-monophosphate-activated protein kinase pathway.

    PubMed

    Xie, Sheng; Deng, Yan; Pan, Yue-Ying; Ren, Jie; Jin, Meng; Wang, Yu; Wang, Zhi-Hua; Zhu, Die; Guo, Xue-Ling; Yuan, Xiao; Shang, Jin; Liu, Hui-Guo

    2016-09-15

    Autophagy is tightly regulated to maintain cardiac homeostasis. Impaired autophagy is closely associated with pathological cardiac hypertrophy. However, the relationship between autophagy and cardiac hypertrophy induced by chronic intermittent hypoxia (CIH) is not known. In the present study, we measured autophagy-related genes and autophagosomes during 10 weeks of CIH in rats, and 6 days in H9C2 cardiomyocytes, and showed that autophagy was impaired. This conclusion was confirmed by the autophagy flux assay. We detected significant hypertrophic changes in myocardium with impaired autophagy. Rapamycin, an autophagy enhancer, attenuated the cardiac hypertrophy induced by CIH. Moreover, silencing autophagy-related gene 5 (ATG5) exerted the opposite effect. The role of adenosine monophosphate-activated protein kinase (AMPK) in regulating autophagy under CIH was confirmed using AICAR to upregulate this enzyme and restore autophagy flux. Restoring autophagy by AICAR or rapamycin significantly reversed the hypertrophic changes in cardiomyocytes. To investigate the mechanism of autophagy impairment, we compared phospho (p)-AMPK, p-Akt, cathepsin D, and NFAT3 levels, along with calcineurin activity, between sham and CIH groups. CIH activated calcineurin, and inhibited AMPK and AMPK-mediated autophagy in an Akt- and NFAT3-independent manner. Collectively, these data demonstrated that impaired autophagy induced by CIH through the AMPK pathway contributed to cardiac hypertrophy.

  17. DNA sequence polymorphism within the bovine adenosine monophosphate deaminase 1 (AMPD1) is associated with production traits in Chinese cattle.

    PubMed

    Wei, C-B; Wang, J-Q; Chen, F-Y; Niu, H; Li, K

    2015-02-06

    The objectives of the present study were to detect an 18-bp deletion mutation in the bovine adenosine monophosphate deaminase 1 (AMPD1) gene and analyze its effect on growth traits in 2 Chinese cattle breeds using DNA sequencing and agarose electrophoresis. The five 19-bp polymerase chain reaction products of the AMPD1 gene exhibited 3 genotypes and 2 alleles: WW: homozygote genotype (wild-type); DD: homozygote genotype (mutant-type); WD: heterozygote genotype. Frequencies of the W allele varied from 66.15-70.35%. The associations between the 18-bp deletion mutation in the AMPD1 gene with production traits in 226 Jia-Xian red cattle was analyzed. The animals with genotype WW showed significantly higher heart girth and body weight than those with genotypes WD and DD at 24 months (P < 0.01). Our results indicate that the deletion mutation in the AMPD1 gene is associated with production traits, and may be used for marker-assisted selection in beef cattle breeding programs.

  18. The expression of cyclic adenosine monophosphate responsive element modulator in rat sertoli cells following seminal extract administration

    PubMed Central

    Akmal, Muslim; Siregar, Tongku Nizwan; Wahyuni, Sri; Hamny; Nasution, Mustafa Kamal; Indriati, Wiwik; Panjaitan, Budianto; Aliza, Dwinna

    2016-01-01

    Aim: This study aims to determine the effect of seminal vesicle extract on cyclic adenosine monophosphate responsive element modulator (CREM) expression in rat Sertoli cells. Materials and Methods: This study examined the expression of CREM on 20 male rats (Rattus norvegicus) at 4 months of age, weighing 250-300 g. The rats were divided into four groups: K0, KP1, KP2, and KP3. K0 group was injected with 0.2 ml normal saline; KP1 was injected with 25 mg cloprostenol (Prostavet C, Virbac S. A); KP2 and KP3 were injected with 0.2 and 0.4 ml seminal vesicle extract, respectively. The treatments were conducted 5 times within 12-day interval. At the end of the study, the rats were euthanized by cervical dislocation; then, the testicles were necropsied and processed for histology observation using immunohistochemistry staining. Results: CREM expression in rat Sertoli cells was not altered by the administration of either 0.2 or 0.4 ml seminal vesicle extract. Conclusion: The administration of seminal vesicle extract is unable to increase CREM expression in rat Sertoli cells. PMID:27733803

  19. Adenosine monophosphate-activated protein kinase (AMPK) activators for the prevention, treatment and potential reversal of pathological pain

    PubMed Central

    Price, Theodore J.; Das, Vaskar; Dussor, Gregory

    2015-01-01

    Pathological pain is an enormous medical problem that places a significant burden on patients and can result from an injury that has long since healed or be due to an unidentifiable cause. Although treatments exist, they often either lack efficacy or have intolerable side effects. More importantly, they do not reverse the changes in the nervous system mediating pathological pain, and thus symptoms often return when therapies are discontinued. Consequently, novel therapies are urgently needed that have both improved efficacy and disease-modifying properties. Here we highlight an emerging target for novel pain therapies, adenosine monophosphate-activated protein kinase (AMPK). AMPK is capable of regulating a variety of cellular processes including protein translation, activity of other kinases, and mitochondrial metabolism, many of which are thought to contribute to pathological pain. Consistent with these properties, preclinical studies show positive, and in some cases disease-modifying effects of either pharmacological activation or genetic regulation of AMPK in models of nerve injury, chemotherapy-induced peripheral neuropathy (CIPN), postsurgical pain, inflammatory pain, and diabetic neuropathy. Given the AMPK-activating ability of metformin, a widely prescribed and well-tolerated drug, these preclinical studies provide a strong rationale for both retrospective and prospective human pain trials with this drug. They also argue for the development of novel AMPK activators, whether orthosteric, allosteric, or modulators of events upstream of the kinase. Together, this review will present the case for AMPK as a novel therapeutic target for pain and will discuss future challenges in the path toward development of AMPK-based pain therapeutics. PMID:26521775

  20. Estradiol regulation of hypothalamic astrocyte adenosine 5'-monophosphate-activated protein kinase activity: role of hindbrain catecholamine signaling.

    PubMed

    Tamrakar, Pratistha; Briski, Karen P

    2015-01-01

    Recent work challenges the conventional notion that metabolic monitoring in the brain is the exclusive function of neurons. This study investigated the hypothesis that hypothalamic astrocytes express the ultra-sensitive energy gauge adenosine 5'-monophosphate-activated protein kinase (AMPK), and that the ovarian hormone estradiol (E) controls activation of this sensor by insulin-induced hypoglycemia (IIH). E- or oil (O)-implanted ovariectomized (OVX) rats were pretreated by caudal fourth ventricular administration of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA) prior to sc insulin or vehicle injection. Individual astrocytes identified in situ by glial fibrillary acidic protein immunolabeling were laser-microdissected from the ventromedial (VMH), arcuate (ARH), and paraventricular (PVH) nuclei and the lateral hypothalamic area (LHA), and pooled within each site for Western blot analysis of AMPK and phosphoAMPK (pAMPK) protein expression. In the VMH, baseline astrocyte AMPK and pAMPK levels were respectively increased or decreased in OVX+E versus OVX+O; these profiles did not differ between E and O rats in other hypothalamic loci. In E animals, astrocyte AMPK protein was reduced [VMH] or augmented [PVH; LHA] in response to either 6-OHDA or IIH. IIH increased astrocyte pAMPK expression in each structure in vehicle-, but not 6-OHDA-pretreated E rats. Results provide novel evidence for hypothalamic astrocyte AMPK expression and hindbrain catecholamine-dependent activation of this cell-specific sensor by hypoglycemia in the presence of estrogen. Further research is needed to determine the role of astrocyte AMPK in reactivity of these glia to metabolic imbalance and contribution to restoration of neuro-metabolic stability.

  1. Effects of dibutyryl cyclic adenosine monophosphate on hypercapnic depression of diaphragmatic contractility in pentobarbital-anesthetized dogs

    PubMed Central

    Fujii, Yoshitaka; Uemura, Aki

    2010-01-01

    Background: Hypercapnia is associated with diaphragm muscle dysfunction that causes a reduction of diaphragmatic force generated for a constant elective myographic activity. No published data are available concerning hypercapnic depression of diaphragmatic contractility during dibutyryl cyclic adenosine monophospate (DBcAMP) administration. Objective: The aim of this study was to assess the effects of DBcAMP on hypercapnic depression of diaphragmatic contractility in pentobarbital-anesthetized dogs. Methods: This experimental study was conducted from July to December 2008 at the Department of Anesthesiology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Japan. Adult (aged >5 years) mongrel dogs weighing 10 to 15 kg were randomly divided into 3 equal groups. Hypercapnia (80–90 mm Hg) was induced with 10% carbon dioxide added to the inspired gas. When hypercapnia was established, group 1 was infused with low-dose DBcAMP (0.05 mg/kg/min); group 2 was infused with high-dose DBcAMP (0.2 mg/kg/min); and group 3 received placebo (saline). Study drug was administered intravenously for 60 minutes. Diaphragmatic contractility was assessed by transdiaphragmatic pressure (Pdi) at baseline, induction of hypercapnia, and study drug administration. Results: Twenty-one dogs were divided into 3 groups of 7. There were no significant differences observed at baseline. In the presence of hypercapnia, Pdi (mean [SD], cm H2O) at low- (20-Hz) and high-frequency (100-Hz) stimulation was significantly decreased from baseline in each group (all, P = 0.001). In groups 1 and 2, Pdi at both stimuli was significantly increased during DBcAMP administration compared with hypercapnia-induced values (group 1: 20-Hz, 13.5 [2.2] vs 15.0 [2.4], respectively, P = 0.001, 100-Hz, 21.2 [1.6] vs 22.5 [1.6], P = 0.001; group 2: 20-Hz, 13.7 [1.4] vs 19.2 [1.7], P = 0.001, 100-Hz, 21.0 [2.4] vs 27.2 [2.5], P = 0.001). The Pdi at both stimuli during DBcAMP administration was significantly

  2. Dietary adenine controls adult lifespan via adenosine nucleotide biosynthesis and AMPK, and regulates the longevity benefit of caloric restriction

    PubMed Central

    Stenesen, Drew; Suh, Jae Myoung; Seo, Jin; Yu, Kweon; Lee, Kyu-Sun; Kim, Jong-Seok; Min, Kyung-Jin; Graff, Jonathan M.

    2012-01-01

    SUMMARY A common thread among conserved lifespan regulators lies within intertwined roles in metabolism and energy homeostasis. We show that heterozygous mutations of adenosine monophosphate (AMP) biosynthetic enzymes extend Drosophila lifespan. The lifespan benefit of these mutations depends upon increased AMP to adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to ATP ratios and adenosine monophosphate-activated protein kinase (AMPK). Transgenic expression of AMPK in adult fat body or adult muscle, key metabolic tissues, extended lifespan, while AMPK RNAi reduced lifespan. Supplementing adenine, a substrate for AMP biosynthesis, to the diet of long-lived AMP biosynthesis mutants reversed lifespan extension. Remarkably, this simple change in diet also blocked the pro-longevity effects of dietary restriction. These data establish AMP biosynthesis, adenosine nucleotide ratios, and AMPK as determinants of adult lifespan, provide a mechanistic link between cellular anabolism and energy sensing pathways, and indicate that dietary adenine manipulations might alter metabolism to influence animal lifespan. PMID:23312286

  3. Basal and adenosine receptor-stimulated levels of cAMP are reduced in lymphocytes from alcoholic patients

    SciTech Connect

    Diamond, I.; Wrubel, B.; Estrin, W.; Gordon, A.

    1987-03-01

    Alcoholism causes serious neurologic disease that may be due, in part, to the ability of ethanol to interact with neural cell membranes and change neuronal function. Adenosine receptors are membrane-bound proteins that appear to mediate some of the effects of ethanol in the brain. Human lymphocytes also have adenosine receptors, and their activation causes increases in cAMP levels. To test the hypothesis that basal and adenosine receptor-stimulated cAMP levels in lymphocytes might be abnormal in alcoholism, the authors studied lymphocytes from 10 alcoholic subjects, 10 age- and sex-matched normal individuals, and 10 patients with nonalcoholic liver disease. Basal and adenosine receptor-stimulated cAMP levels were reduced 75% in lymphocytes from alcoholic subjects. Also, there was a 76% reduction in ethanol stimulation of cAMP accumulation in lymphocytes from alcoholics. Similar results were demonstrable in isolated T cells. Unlike other laboratory tests examined, these measurements appeared to distinguish alcoholics from normal subjects and from patients with nonalcoholic liver disease. Reduced basal and adenosine receptor-stimulated levels of cAMP in lymphocytes from alcoholics may reflect a change in cell membranes due either to chronic alcohol abuse or to a genetic predisposition unique to alcoholic subjects.

  4. [The secretion of adenosin 3',5'-monophosphate after hydrokinetic and ecbolic stimulation in the canine pancreas (author's transl)].

    PubMed

    Teufel, H; Boeckmann, U

    1981-05-01

    The secretion of cAMP is studied in vivo and in the isolated perfused canine pancreas after administration of secretin and CCK or caerulein in comparison with hydrokinetic or ecbolic secretory events as well as with the magnitude and time course of changes in tissue cAMP. 1) The total output of cAMP and pancreatic juice shows a significant and positive correlation after stimulation with secretin. The linear correspondence between cAMP concentration and secretory rates of pancreatic juice beyond 3 ml/5 min and their non-linear, reciprocal correlation at lower rates of fluid secretion point to an active as well as to a passive secretory mechanism for cAMP. 2) CCK and caerulein increase secretion of cAMP too. The output of cAMP however neither corresponds to the time course of protein secretion nor correlates quantitatively with the latter. 3) The behaviour of cAMP secretion and concentration in the pancreatic juice after administration of secretin and CCK or caerulein as well as differs from the changes in tissue cAMP levels. The respective maximum of cAMP output after addition of secretin or ecbolic secretagogues during the greatest decrease in cellular cAMP levels yields on the average about 1% of the estimated reduction in total tissue cAMP content. The results indicate a functional coherence in secretion of pancreatic juice and cAMP but oppose the assumption, that essential amounts of cAMP are released during exocytosis of zymogen granules. The secretion of cAMP may be possibly influenced by cytoplasmatic cAMP levels, but neither reflects the present changes in cellular cAMP nor seems to be of a regulatory importance for the latter.

  5. [The secretion of adenosin 3',5'-monophosphate after hydrokinetic and ecbolic stimulation in the canine pancreas (author's transl)].

    PubMed

    Teufel, H; Boeckmann, U

    1981-05-01

    The secretion of cAMP is studied in vivo and in the isolated perfused canine pancreas after administration of secretin and CCK or caerulein in comparison with hydrokinetic or ecbolic secretory events as well as with the magnitude and time course of changes in tissue cAMP. 1) The total output of cAMP and pancreatic juice shows a significant and positive correlation after stimulation with secretin. The linear correspondence between cAMP concentration and secretory rates of pancreatic juice beyond 3 ml/5 min and their non-linear, reciprocal correlation at lower rates of fluid secretion point to an active as well as to a passive secretory mechanism for cAMP. 2) CCK and caerulein increase secretion of cAMP too. The output of cAMP however neither corresponds to the time course of protein secretion nor correlates quantitatively with the latter. 3) The behaviour of cAMP secretion and concentration in the pancreatic juice after administration of secretin and CCK or caerulein as well as differs from the changes in tissue cAMP levels. The respective maximum of cAMP output after addition of secretin or ecbolic secretagogues during the greatest decrease in cellular cAMP levels yields on the average about 1% of the estimated reduction in total tissue cAMP content. The results indicate a functional coherence in secretion of pancreatic juice and cAMP but oppose the assumption, that essential amounts of cAMP are released during exocytosis of zymogen granules. The secretion of cAMP may be possibly influenced by cytoplasmatic cAMP levels, but neither reflects the present changes in cellular cAMP nor seems to be of a regulatory importance for the latter. PMID:6265337

  6. Investigating real-time activation of adenosine receptors by bioluminescence resonance energy transfer technique

    NASA Astrophysics Data System (ADS)

    Huang, Yimei; Yang, Hongqin; Zheng, Liqin; Chen, Jiangxu; Wang, Yuhua; Li, Hui; Xie, Shusen

    2013-02-01

    Adenosine receptors play important roles in many physiological and pathological processes, for example regulating myocardial oxygen consumption and the release of neurotransmitters. The activations of adenosine receptors have been studied by some kinds of techniques, such as western blot, immunohistochemistry, etc. However, these techniques cannot reveal the dynamical response of adenosine receptors under stimulation. In this paper, bioluminescence resonance energy transfer technique was introduced to study the real-time activation of adenosine receptors by monitoring the dynamics of cyclic adenosine monophosphate (cAMP) level. The results showed that there were significant differences between adenosine receptors on real-time responses under stimulation. Moreover, the dynamics of cAMP level demonstrated that competition between adenosine receptors existed. Taken together, our study indicates that monitoring the dynamics of cAMP level using bioluminescence resonance energy transfer technique could be one potential approach to investigate the mechanism of competitions between adenosine receptors.

  7. Adenosine A1 receptors mediate inhibition of cAMP formation in vitro in the pontine, REM sleep induction zone.

    PubMed

    Marks, Gerald A; Birabil, Christian G; Speciale, Samuel G

    2005-11-01

    Microinjection of adenosine A1 receptor agonist or an inhibitor of adenylyl cyclase into the caudal, oral pontine reticular formation (PnOc) of the rat induces a long-lasting increase in REM sleep. Here, we report significant inhibition of forskolin-stimulated cAMP in dissected pontine tissue slices containing the PnOc incubated with the A1 receptor agonist, cyclohexaladenosine (10(-8) M). These data are consistent with adenosine A1 receptor agonist actions on REM sleep mediated through inhibition of cAMP.

  8. [Influence of ADP-ribose, AMP and adenosine on bioelectric activity of hibernating ground squirrel atrium and papillary muscle].

    PubMed

    Kuz'min, V S; Abramochkin, D V; Sukhova, G S; Rozenshtraukh, L V

    2008-01-01

    The aim of work was to investigate effects of adenosine, AMP and ADP-ribose (1x10(-5)) on bioelectric activity of atrium and papillary muscle of nonhibernating (rat) and hibernating (Yakutian ground squirrel) animals. Action potential (AP) was registered with use of standard microelectrode technique. AP duration (APD) at level of 90% repolarisation in rat atrium in control experiments was 30+/-5 ms, APD at level of 50% repolarisation was 12+/-2 ms. APD at level of 90% repolarisation in rat papillary muscle was 56+/-7 ms, at level of 50% repolarisation was 18+/-2 ms. APD at level of 90% repolarisation in ground squirrel atrium was 77+/-6, APD at level of 50% repolarisation was 38+/-6 ms. APD at level of 90% repolarisation in ground squirrel papillary muscle was 105+/-9 ms, APD at level of 50% repolarisation was 42+/-8 ms. Purine nucleotides and nucleoside, that were tested in work, except ADP-ribose, act as inhibitory factors and decrease APD both in rat and hibernating ground squirrel heart. ADP-ribose decreases APD in papillary muscle of hibernator but did not in its atrium. In ground squirrel atrium AMP and adenosine decrease APD at level of 50% repolarisation by 10+/-3% and 18+/-3% respectively. AMP and adenosine decrease APD at level of 90% repolarisation by 9+/-2% and 11+/-2% respectively. In ground squirrel papillary muscle ADP-ribose, AMP and adenosine decrease APD at level of 50% repolarisation by 26+/-8%, 23+/-8% and 26+/-7%. ADP-ribose, AMP and adenosine decrease APD at level of 90% repolarisation by 12+/-3%, 10+/-3%, 13+/-3%. Thus, decrease of APD in ground squirrel papillary muscle at level of 90% repolarisation during nucleotides and adenosine action was 2-2.5 fold less, than the rat.

  9. The reversal of glucose repressed prodigiosin production in Serratia marcescens by the cyclic 3'5'-adenosine monophosphate inhibitor theophylline.

    PubMed

    Clements-Jewery, S

    1976-04-15

    Glucose was found to cause severe repression of prodigiosin production in Serratia marcescens and a dose related partial reversal was demonstrated by theophylline. It is suggested that this reversal is due to the inhibition of cAMP phosphodiesterase and the concomitant increase in cellular cAMP concentration.

  10. Assay of adenosine 3',5' cyclic monophosphate by stimulation of protein kinase: a method not involving radioactivity

    SciTech Connect

    Handa, A.K.; Bressan, R.A.

    1980-03-01

    In order to meet a need for a cAMP assay which is not subject to interference by compounds in plant extracts, and which is suitable for use on occasions separated by many /sup 32/P half-lives, an assay based on cAMP-dependent protein kinase has been developed which does not require the use of (..gamma..-/sup 32/P)ATP. Instead of measuring the cAMP-stimulated increase in the rate of transfer of (..gamma..-/sup 32/P) phosphate from (..gamma..-/sup 32/P)ATP to protein, the rate of loss of ATP from the reaction mixture is determined. The ATP remaining after the protein kinase reaction is assayed by ATP-dependent chemiluminescence of the firefly luciferin-luciferase system. Under conditions of the protein kinase reaction in which a readily measurable decrease in ATP concentration occurs, the logarithm of the concentration of ATP decreases in proportion to the cAMP concentration, i.e., the reaction can be described by the equation: (ATP) = (ATP)/sub 0/ e/sup -(cAMP)kt/. The assay based on this relationship can detect less than 1 pmol of cAMP. The levels of cAMP found with this assay after partial purification of the cAMP from rat tissue, algal cells, and the media in which the cells were grown agreed with measurements made by the cAMP binding-competition assay of Gilman, and the potein kinase stimulation assay based on transfer of (/sup 32/P) phosphate from (..gamma..-/sup 32/P)ATP to protein. All of the enzymes and chemicals required for the assay of cAMP by protein kinase catalyzed loss of ATP can be stored frozen for months, making the assay suitable for occasional use.

  11. A long-acting β2-adrenergic agonist increases the expression of muscarine cholinergic subtype‑3 receptors by activating the β2-adrenoceptor cyclic adenosine monophosphate signaling pathway in airway smooth muscle cells.

    PubMed

    Liu, Yuan-Hua; Wu, Song-Ze; Wang, Gang; Huang, Ni-Wen; Liu, Chun-Tao

    2015-06-01

    The persistent administration of β2‑adrenergic (β2AR) agonists has been demonstrated to increase the risk of severe asthma, partly due to the induction of tolerance to bronchoprotection via undefined mechanisms. The present study investigated the potential effect of the long‑acting β2‑adrenergic agonist, formoterol, on the expression of muscarinic M3 receptor (M3R) in rat airway smooth muscle cells (ASMCs). Primary rat ASMCs were isolated and characterized following immunostaining with anti‑α‑smooth muscle actin antibodies. The protein expression levels of M3R and phospholipase C‑β1 (PLCβ1) were characterized by western blot analysis and the production of inositol 1,4,5‑trisphosphate (IP3) was determined using an enzyme‑linked immunosorbent assay. Formoterol increased the protein expression of M3R in rat ASMCs in a time‑ and dose‑dependent manner, which was significantly inhibited by the β2AR antagonist, ICI118,551 and the cyclic adenosine monophosphate (cAMP) inhibitor, SQ22,536. The increased protein expression of M3R was positively correlated with increased production of PLCβ1 and IP3. Furthermore, treatment with the glucocorticoid, budesonide, and the PLC inhibitor, U73,122, significantly suppressed the formoterol‑induced upregulated protein expression levels of M3R and PLCβ1 and production of IP3. The present study demonstrated that formoterol mediated the upregulation of M3R in the rat ASMCs by activating the β2AR‑cAMP signaling pathway, resulting in increased expression levels of PLCβ1 and IP3, which are key to inducing bronchoprotection tolerance. Administration of glucocorticoids or a PLC antagonist prevented formoterol‑induced bronchoprotection tolerance by suppressing the protein expression of M3R. PMID:25672589

  12. A long-acting β2-adrenergic agonist increases the expression of muscarine cholinergic subtype-3 receptors by activating the β2-adrenoceptor cyclic adenosine monophosphate signaling pathway in airway smooth muscle cells

    PubMed Central

    LIU, YUAN-HUA; WU, SONG-ZE; WANG, GANG; HUANG, NI-WEN; LIU, CHUN-TAO

    2015-01-01

    The persistent administration of β2-adrenergic (β2AR) agonists has been demonstrated to increase the risk of severe asthma, partly due to the induction of tolerance to bronchoprotection via undefined mechanisms. The present study investigated the potential effect of the long-acting β2-adrenergic agonist, formoterol, on the expression of muscarinic M3 receptor (M3R) in rat airway smooth muscle cells (ASMCs). Primary rat ASMCs were isolated and characterized following immunostaining with anti-α-smooth muscle actin antibodies. The protein expression levels of M3R and phospholipase C-β1 (PLCβ1) were characterized by western blot analysis and the production of inositol 1,4,5-trisphosphate (IP3) was determined using an enzyme-linked immunosorbent assay. Formoterol increased the protein expression of M3R in rat ASMCs in a time- and dose-dependent manner, which was significantly inhibited by the β2AR antagonist, ICI118,551 and the cyclic adenosine monophosphate (cAMP) inhibitor, SQ22,536. The increased protein expression of M3R was positively correlated with increased production of PLCβ1 and IP3. Furthermore, treatment with the glucocorticoid, budesonide, and the PLC inhibitor, U73,122, significantly suppressed the formoterol-induced upregulated protein expression levels of M3R and PLCβ1 and production of IP3. The present study demonstrated that formoterol mediated the upregulation of M3R in the rat ASMCs by activating the β2AR-cAMP signaling pathway, resulting in increased expression levels of PLCβ1 and IP3, which are key to inducing bronchoprotection tolerance. Administration of glucocorticoids or a PLC antagonist prevented formoterol-induced bronchoprotection tolerance by suppressing the protein expression of M3R. PMID:25672589

  13. Nature of the effect of adenosine 5'-monophosphate on the cyanide-insensitive respiration in mitochondria of Moniliella tomentosa.

    PubMed Central

    Vanderleyden, J; Van Den Eynde, E; Verachtert, H

    1980-01-01

    The alternative oxidase of Moniliella tomentosa mitochondria is stimulated by 5'-AMP. This effect may be masked, depending on the isolation procedure of the mitochondria. The preparation of submitochondrial particles results in the expression of the 5'-AMP effect. Two more methods are now described to reveal the 5'-AMP effect whenever it would be masked: (1) switching on the myokinase activity of the mitochondria to deplete them of endogenous 5'-AMP; (2) using detergents (sodium dodecyl sulphate, sodium deoxycholate) in a controlled detergent:protein ratio, or chloroform. The alternative oxidase of detergent-solubilized mitochondria was somewhat less selective towards nucleotides than were intact mitochondria. The effect of nucleotides on quinol oxidation by mitochondrial preparations and on quinol autoxidation was also studied. Mitochondrial oxidation of succinate by the alternative oxidase and autoxidation of quinols behaved similarly in the presence of certain nucleotides. Both reactions were stimulated. Both reactions were also inhibited by salicylhydroxamic acid. These effects on quinol oxidation disappeared when bovine serum albumin or mitochondrial proteins were present. From the results obtained it is not possible to exclude quinol autoxidation as a final step of the alternative oxidase. PMID:7189398

  14. [Isolation of inosine-5'-monophosphate from fish muscles].

    PubMed

    Tugaĭ, V A; Akulin, V N; Epshteĭn, L M

    1987-01-01

    Conditions for transformation of tissue adenosine-5'-monophosphate (AMP) into inosine-5'-monophosphate (IMP) with the aid of endogenic AMP-aminohydrolase are developed resting on the studied properties of AMP-aminohydrolase (EC 3.5.4.6) from saltwater fish muscles (one of the enzymes participating in the nucleotide metabolism). Sorption of the nucleotide is performed on the activated charcoals A gamma-3 A gamma-5 which eluate IMP from acid solutions. It reduces the process of isolation, permits application of the acid wash solutions to remove salts; the alkaline ethyl alcohol-aid elution at the subsequent stages accelerates the process of nucleotide concentration by means of vacuum evaporation. The suggested approaches allow developing a simple method of IMP production from fish tissues which diminishes the cost of preparation.

  15. 3'5'-cyclic adenosine monophosphate-dependent up-regulation of phosphodiesterase type 3A in porcine cumulus cells.

    PubMed

    Sasseville, Maxime; Côté, Nancy; Vigneault, Christian; Guillemette, Christine; Richard, François J

    2007-04-01

    The means by which cumulus cells react to gonadotropin stimulation and regulate the subsequent production and degradation of cAMP are largely unknown. In this article, we report that cyclic nucleotide phosphodiesterase (PDE) type 3A (Pde3a) is transcriptionally regulated in porcine cumulus cells by a cAMP-dependent pathway during in vitro maturation (IVM). cAMP-PDE activity was increased in the cumulus-oocyte complex (COC) after 10 h of IVM, and 78% of this increase was sensitive to a Pde3-specific inhibitor, cilostamide. Although no variation was observed in the oocyte, cilostamide-sensitive cAMP-PDE activity increased in the cumulus cells after IVM. This was supported by Western blotting, which showed that the intensity of a 135-kDa anti-Pde3a immunoreactive band was increased in COC after IVM. The Pde3a mRNA level was up-regulated 28-fold in the COC after 4 h of IVM and remained high up to 12 h. The mRNA up-regulation and increased activity were inhibited by an RNA synthesis inhibitor, alpha-amanitin. The cilostamide-sensitive increase in PDE activity was inhibited by a protein synthesis inhibitor, cycloheximide. Pregnant mare serum gonadotropin (PMSG) caused dose-dependent activation of Pde3. The PMSG-dependent increase in Pde3 activity and Pde3a mRNA were mimicked by the adenylyl cyclase activator forskolin or prostaglandin E2. PMSG-dependent Pde3 activation was inhibited by the protein kinase A-specific inhibitor H89. Collectively, our results show for the first time that degradation of the intracellular cyclic nucleotide by Pde3a is transcriptionally up-regulated via a cAMP-dependent pathway in cumulus cells, suggesting that it has a functional role during the ovulatory gonadotropin surge.

  16. Role of 3', 5' cyclic adenosine monophosphate and protein kinase C in the regulation of insulin-like growth factor-binding protein secretion by thyroid-stimulating hormone in isolated ovine thyroid cells.

    PubMed

    Wang, J F; Hill, D J; Becks, G P

    1994-05-01

    Isolated sheep thyroid follicles release insulin-like growth factors (IGF)-I and -II together with IGF-binding proteins (IGFBPs). We previously showed that TSH suppresses the biosynthesis and release of IGFBPs in vitro which may increase the tissue availability of IGFs, allowing a synergy with TSH which potentiates both thyroid growth and function. Many of the actions of TSH on thyroid cell function are dependent upon activation of adenylate cyclase, although increased synthesis of inositol trisphosphate and activation of protein kinase C (PKC) have also been implicated. We have now examined whether probable changes in intracellular cyclic adenosine monophosphate (cAMP) or PKC are involved in TSH-mediated suppression of IGFBP release. Confluent primary cultures of ovine thyroid cells were maintained in serum-free Ham's modified F-12M medium containing transferrin, somatostatin and glycyl-histidyl-lysine (designated 3H), and further supplemented with sodium iodide (10(-8)-10(-3) mol/l), dibutyryl cAMP (0.25-1 mmol/l), forskolin (5-20 mumol/l) or 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-11)-10(-6) mol/l), with or without exposure to TSH (200 microU/ml). The uptake and organification of Na [125I] by cells was examined after test incubations of up to 48 h, and IGFBPs in conditioned media were analysed by ligand blot using 125I-labelled IGF-II. The PKC activity in the cytosol and plasma membrane fractions of cells was measured by phosphorylation of histone using [gamma-32P]ATP, and PKC immunoreactivity was visualized by Western immunoblot analysis. While dibutyryl cAMP or forskolin largely reproduced the stimulatory effect of TSH on iodine organification, they did not mimic the inhibitory effect of TSH on the secretion of IGFBPs of 43, 34, 28 and 19 kDa. Incubation with physiological or pharmacological concentrations of iodide (10(-6)-10(-3) mol/l) for up to 48 h significantly decreased TSH action on iodide uptake and organification but did not alter the

  17. Adenylyl cyclase activation underlies intracellular cyclic AMP accumulation, cyclic AMP transport, and extracellular adenosine accumulation evoked by beta-adrenergic receptor stimulation in mixed cultures of neurons and astrocytes derived from rat cerebral cortex.

    PubMed

    Rosenberg, P A; Li, Y

    1995-09-18

    We have previously shown that stimulation of cortical cultures containing both neurons and astrocytes with the beta-adrenergic agonist isoproterenol (ISO) results in transport of cAMP from astrocytes followed by extracellular hydrolysis to adenosine [Rosenberg et al. J. Neurosci. 14 (1994) 2953-2965]. In this study we found that the endogenous catecholamines epinephrine (EPI) and norepinephrine (NE), but not dopamine, serotonin, or histamine, all at 10 microM, significantly stimulated intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation in cortical cultures. Detailed dose-response experiments were performed for NE and EPI, as well as ISO. For each catecholamine, the potencies in evoking intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation were similar. These data provide additional evidence that a single common mechanism, namely beta-adrenergic mediated activation of adenylyl cyclase, underlies intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation. It appears that regulation of extracellular adenosine levels via cAMP transport and extracellular hydrolysis to adenosine may be a final common pathway of neuromodulation in cerebral cortex for catecholamines, and, indeed, any substance whose receptors are coupled to adenylyl cyclase.

  18. A Temporal-Specific and Transient cAMP Increase Characterizes Odorant Classical Conditioning

    ERIC Educational Resources Information Center

    Cui, Wen; Smith, Andrew; Darby-King, Andrea; Harley, Carolyn W.; McLean, John H.

    2007-01-01

    Increases in cyclic adenosine monophosphate (cAMP) are proposed to initiate learning in a wide variety of species. Here, we measure changes in cAMP in the olfactory bulb prior to, during, and following a classically conditioned odor preference trial in rat pups. Measurements were taken up to the point of maximal CREB phosphorylation in olfactory…

  19. Mulberry leaf polyphenol extract induced apoptosis involving regulation of adenosine monophosphate-activated protein kinase/fatty acid synthase in a p53-negative hepatocellular carcinoma cell.

    PubMed

    Yang, Tzi-Peng; Lee, Huei-Jane; Ou, Ting-Tsz; Chang, Ya-Ju; Wang, Chau-Jong

    2012-07-11

    The polyphenols in mulberry leaf possess the ability to inhibit cell proliferation, invasion, and metastasis of tumors. It was reported that the p53 status plays an important role in switching apoptosis and the cell cycle following adenosine monophosphate-activated protein kinase (AMPK) activation. In this study, we aimed to detect the effect of the mulberry leaf polyphenol extract (MLPE) on inducing cell death in p53-negative (Hep3B) and p53-positive (Hep3B with transfected p53) hepatocellular carcinoma cells and also to clarify the role of p53 in MLPE-treated cells. After treatment of the Hep3B cells with MLPE, apoptosis was induced via the AMPK/PI3K/Akt and Bcl-2 family pathways. Transient transfection of p53 into Hep3B cells led to switching autophagy instead of apoptosis by MLPE treatment. We demonstrated that acridine orange staining and protein expressions of LC-3 and beclin-1 were increased in p53-transfected cells. These results implied induction of apoptosis or autophagy in MLPE-treated hepatocellular carcinoma cells can be due to the p53 status. We also found MLPE can not only activate AMPK but also diminish fatty acid synthase, a molecular target for cancer inhibition. At present, our results indicate MLPE can play an active role in mediating the cell death of hepatocellular carcinoma cells and the p53 might play an important role in regulating the death mechanisms.

  20. Uric acid induces oxidative stress and growth inhibition by activating adenosine monophosphate-activated protein kinase and extracellular signal-regulated kinase signal pathways in pancreatic β cells.

    PubMed

    Zhang, Yongneng; Yamamoto, Tetsuya; Hisatome, Ichiro; Li, Youfeng; Cheng, Weijie; Sun, Ning; Cai, Bozhi; Huang, Tianliang; Zhu, Yuzhang; Li, Zhi; Jing, Xubin; Zhou, Rui; Cheng, Jidong

    2013-08-15

    Hyperuricaemia is a disorder of purine metabolism, and is strongly associated with insulin resistance and abnormal glucose metabolism. As the producer of insulin, pancreatic β cells might be affected by elevated serum uric acid levels and contribute to the disregulated glucose metabolism. In this study, we investigated the effect of high uric acid on rat pancreatic β cell function. Under high uric acid condition, proliferation of pancreatic β cells was inhibited, production of reactive oxygen species increased, and glucose stimulated insulin secretion was also compromised. Further examination on signal transduction pathways revealed that uric acid-induced ROS is involved in the activation of adenosine monophosphate-activated protein kinase (AMPK), and extracellular signal-regulated kinase (ERK). Pharmacological inhibition of ERK activation rescued β cells from growth inhibition. More importantly, activation of ERK induced by uric acid is significantly diminished by AMPK inhibitor, indicating ERK as a downstream target of AMPK in response to high uric acid condition. We also investigated the transportation channel for uric acid into pancreatic β cells. While major urate transporter URAT1 is not expressed in β cells, organic anion transporter (OAT) inhibitor successfully blocked the activation of ERK by uric acid. Our data indicate that high uric acid levels induce oxidative damage and inhibit growth of rat pancreatic β cells by activating the AMPK and ERK signal pathways. Hyperuricemia may contribute to abnormal glucose metabolism by causing oxidative damage and function inhibition of pancreatic β cells.

  1. Estrogen regulates energy metabolic pathway and upstream adenosine 5'-monophosphate-activated protein kinase and phosphatase enzyme expression in dorsal vagal complex metabolosensory neurons during glucostasis and hypoglycemia.

    PubMed

    Tamrakar, Pratistha; Ibrahim, Baher A; Gujar, Amit D; Briski, Karen P

    2015-02-01

    The ability of estrogen to shield the brain from the bioenergetic insult hypoglycemia is unclear. Estradiol (E) prevents hypoglycemic activation of the energy deficit sensor adenosine 5'-monophosphate-activated protein kinase (AMPK) in hindbrain metabolosensory A2 noradrenergic neurons. This study investigates the hypothesis that estrogen regulates A2 AMPK through control of fuel metabolism and/or upstream protein kinase/phosphatase enzyme expression. A2 cells were harvested by laser microdissection after insulin or vehicle (V) injection of E- or oil (O)-implanted ovariectomized female rats. Cell lysates were evaluated by immunoblot for glycolytic, tricarboxylic acid cycle, respiratory chain, and acetyl-CoA-malonyl-CoA pathway enzymes. A2 phosphofructokinase (PFKL), isocitrate dehydrogenase, pyruvate dehydrogenase, and ATP synthase subunit profiles were elevated in E/V vs. O/V; hypoglycemia augmented PFKL and α-ketoglutarate dehydrogenase expression in E only. Hypoglycemia increased A2 Ca(2+) /calmodulin-dependent protein kinase-β in O and reduced protein phosphatase in both groups. A2 phospho-AMPK levels were equivalent in O/V vs. E/V but elevated during hypoglycemia in O only. These results implicate E in compensatory upregulation of substrate catabolism and corresponding maintenance of energy stability of A2 metabolosensory neurons during hypoglycemia, outcomes that support the potential viability of molecular substrates for hormone action as targets for therapies alleviating hypoglycemic brain injury.

  2. Cross-talk between glucagon- and adenosine-mediated signalling systems in rat hepatocytes: effects on cyclic AMP-phosphodiesterase activity.

    PubMed Central

    Robles-Flores, M; Allende, G; Piña, E; García-Sáinz, J A

    1995-01-01

    The effect of adenosine analogues on glucagon-stimulated cyclic AMP accumulation in rat hepatocytes was explored. N6-Cyclopentyladenosine (CPA), 5'-N-ethylcarboxamidoadenosine and N6-(R-phenylisopropyl)adenosine inhibited in a dose-dependent manner the cyclic AMP accumulation induced by glucagon. This effect seems to be mediated through A1 adenosine receptors. Pertussis toxin completely abolished the effect of CPA on glucagon-stimulated cyclic AMP accumulation in whole cells which suggested that a pertussis-toxin-sensitive G-protein was involved. On the other hand, this action of adenosine analogues on glucagon-induced cyclic AMP accumulation was reverted by the selective low-Km cyclic AMP-phosphodiesterase inhibitor Ro 20-1724. Analysis of cyclic AMP-phosphodiesterase activity in purified hepatocyte plasma membranes showed that glucagon in the presence of GTP inhibited basal PDE activity by 45% and that CPA reverted this inhibition in dose-dependent manner. In membranes derived from pertussis-toxin-treated rats, we observed no inhibition of cyclic AMP-phosphodiesterase activity by glucagon in the absence or presence of CPA. Our results indicate that in hepatocyte plasma membranes, stimulation of adenylate cyclase activity and inhibition of a low-Km cyclic AMP phosphodiesterase activity are co-ordinately regulated by glucagon, and that A1 adenosine receptors can inhibit glucagon-stimulated cyclic AMP accumulation by blocking glucagon's effect on phosphodiesterase activity. Images Figure 2 PMID:8554517

  3. Alkaline Phosphatase, Soluble Extracellular Adenine Nucleotides, and Adenosine Production after Infant Cardiopulmonary Bypass

    PubMed Central

    Davidson, Jesse A.; Urban, Tracy; Tong, Suhong; Twite, Mark; Woodruff, Alan

    2016-01-01

    -operative alkaline phosphatase activity leads to impaired capacity to clear adenosine monophosphate. AP supplementation improves serum clearance of adenosine monophosphate to adenosine. These findings represent a potential therapeutic mechanism for alkaline phosphatase infusion during cardiac surgery. New and Noteworthy We identify alkaline phosphatase (AP) as the primary soluble ectonucleotidase in infants undergoing cardiopulmonary bypass and show decreased capacity to clear AMP when AP activity decreases post-bypass. Supplementation of AP ex vivo improves this capacity and may represent the beneficial therapeutic mechanism of AP infusion seen in phase 2 studies. PMID:27384524

  4. Raman spectroscopic measurement of base stacking in solutions of adenosine, AMP, ATP, and oligoadenylates.

    PubMed

    Weaver, J L; Williams, R W

    1988-12-13

    Measurements of the colligative properties of nucleosides and their derivatives have shown that bases form transient aggregates in solution [Ts'o (1967) J. Am. Chem. Soc. 89, 3612-3622]. Aggregation of nucleotides cannot be measured by osmometry due to the presence of counterions. Sedimentation measurements are difficult to obtain and have been complicated by differences in pH [Ferguson et al. (1974) Biophys. Chem. 1, 325-337]. Raman studies of oligonucleotides have shown that the intensities due to base vibrational modes depend on the extent of base stacking, but this dependence has not been quantitated. We have measured this dependence by relating changes in the Raman spectra of nucleotides and nucleosides with previous measurements of colligative properties. Visible Raman spectra of ATP, AMP, and adenosine, taken over a range of concentrations from 1 to 1000 mM, show that the peak intensity ratio (I1305 + I1380)/I1340 varies linearly with the log of the concentration for all three bases. This concentration-dependent change correlates with published molal osmotic coefficient data for functionally similar bases with a correlation coefficient of 0.99. In contrast, UV resonance Raman spectra of the same bases show changes that vary linearly with concentration.

  5. Role of 3'-5'-cyclic adenosine monophosphate on the epidermal growth factor dependent survival in mammary epithelial cells.

    PubMed

    Grinman, Diego Y; Romorini, Leonardo; Presman, Diego M; Rocha-Viegas, Luciana; Coso, Omar A; Davio, Carlos; Pecci, Adali

    2016-01-01

    Epidermal growth factor (EGF) has been suggested to play a key role in the maintenance of epithelial cell survival during lactation. Previously, we demonstrated that EGF dependent activation of PI3K pathway prevents apoptosis in confluent murine HC11 cells cultured under low nutrient conditions. The EGF protective effect is associated with increased levels of the antiapoptotic protein Bcl-XL. Here, we identify the EGF-dependent mechanism involved in cell survival that converges in the regulation of bcl-X expression by activated CREB. EGF induces Bcl-XL expression through activation of a unique bcl-X promoter, the P1; being not only the PI3K/AKT signaling pathway but also the increase in cAMP levels and the concomitant PKA/CREB activation necessary for both bcl-XL upregulation and apoptosis avoidance. Results presented in this work suggest the existence of a novel connection between the EGF receptor and the adenylate cyclase that would have an impact in preventing apoptosis under low nutrient conditions.

  6. Role of adenosine 5'-monophosphate-activated protein kinase in α-linolenic acid-induced intestinal lipid metabolism.

    PubMed

    Zhou, Xihong; Chen, Jingqing; Wu, Weiche; Wang, Xinxia; Wang, Yizhen

    2015-09-28

    n-3 Long-chain PUFA up-regulate intestinal lipid metabolism. However, whether these metabolic effects of PUFA on intestine are mediated by AMP-activated protein kinase (AMPK) remains to be elucidated. To determine the effects of α-linolenic acid (ALA) on intestinal fatty acid (FA) metabolism and whether these effects were affected by AMPK deletion, mice deficient in the catalytic subunit of AMPKα1 or AMPKα2 and wild-type (WT) mice were fed either a high-fat diet (HF) or HF supplemented with ALA (HF-A). The results showed that ALA supplementation decreased serum TAG content in WT mice. ALA also increased mRNA expression of genes (carnitine palmitoyltransferase 1a, acyl-CoA oxidase 1, medium-chain acyl-CoA dehydrogenase, cytochrome P450 4A10 and pyruvate dehydrogenase kinase isoenzyme 4a) involved in intestinal lipid oxidation and mRNA expression of TAG synthesis-related genes (monoacylglycerol O-acyltransferase 2, diacylglycerol O-acyltransferases 1 and 2) in WT mice. Consistent with these, expression levels of phosphorylated AMPKα1 and AMPKα2 were also increased in WT mice after ALA addition. However, in the absence of either AMPKα1 or AMPKα2, ALA supplementation failed to increase intestinal lipid oxidation. In addition, no significant effects of either diet (HF and HF-A) or genotype (WT, AMPKα1(-/-) and AMPKα2(-/-)) on FA uptake in the intestine and faecal TAG output were observed. Our results suggest that AMPK is indispensable for the effects of ALA on intestinal lipid oxidation. PMID:26268732

  7. Adenosine monophosphate-activated protein kinase activation, substrate transporter translocation, and metabolism in the contracting hyperthyroid rat heart.

    PubMed

    Heather, Lisa C; Cole, Mark A; Atherton, Helen J; Coumans, Will A; Evans, Rhys D; Tyler, Damian J; Glatz, Jan F C; Luiken, Joost J F P; Clarke, Kieran

    2010-01-01

    Thyroid hormones can modify cardiac metabolism via multiple molecular mechanisms, yet their integrated effect on overall substrate metabolism is poorly understood. Here we determined the effect of hyperthyroidism on substrate metabolism in the isolated, perfused, contracting rat heart. Male Wistar rats were injected for 7 d with T(3) (0.2 mg/kg x d ip). Plasma free fatty acids increased by 97%, heart weights increased by 33%, and cardiac rate pressure product, an indicator of contractile function, increased by 33% in hyperthyroid rats. Insulin-stimulated glycolytic rates and lactate efflux rates were increased by 33% in hyperthyroid rat hearts, mediated by an increased insulin-stimulated translocation of the glucose transporter GLUT4 to the sarcolemma. This was accompanied by a 70% increase in phosphorylated AMP-activated protein kinase (AMPK) and a 100% increase in phosphorylated acetyl CoA carboxylase, confirming downstream signaling from AMPK. Fatty acid oxidation rates increased in direct proportion to the increased heart weight and rate pressure product in the hyperthyroid heart, mediated by synchronized changes in mitochondrial enzymes and respiration. Protein levels of the fatty acid transporter, fatty acid translocase (FAT/CD36), were reduced by 24% but were accompanied by a 19% increase in the sarcolemmal content of fatty acid transport protein 1 (FATP1). Thus, the relationship between fatty acid metabolism, cardiac mass, and contractile function was maintained in the hyperthyroid heart, associated with a sarcolemmal reorganization of fatty acid transporters. The combined effects of T(3)-induced AMPK activation and insulin stimulation were associated with increased sarcolemmal GLUT4 localization and glycolytic flux in the hyperthyroid heart. PMID:19940039

  8. Activation of 5' adenosine monophosphate-activated protein kinase blocks cumulus cell expansion through inhibition of protein synthesis during in vitro maturation in Swine.

    PubMed

    Santiquet, Nicolas; Sasseville, Maxime; Laforest, Martin; Guillemette, Christine; Gilchrist, Robert B; Richard, François J

    2014-08-01

    The serine/threonine kinase 5' adenosine monophosphate-activated protein kinase (AMPK), a heterotrimeric protein known as a metabolic switch, is involved in oocyte nuclear maturation in mice, cattle, and swine. The present study analyzed AMPK activation in cumulus cell expansion during in vitro maturation (IVM) of porcine cumulus-oocyte complexes (COC). 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) is a well-known activator of AMPK. It inhibited oocyte meiotic resumption in COC. Moreover, cumulus cell expansion did not occur in the presence of AICAR, demonstrating its marked impact on cumulus cells. Activation of AMPK was supported by AICAR-mediated phosphorylation of alpha AMPK subunits. Furthermore, the presence of AICAR increased glucose uptake, a classical response to activation of this metabolic switch in response to depleted cellular energy levels. Neither nuclear maturation nor cumulus expansion was reversed by glucosamine, an alternative substrate in hyaluronic acid synthesis, through the hexosamine biosynthetic pathway, which ruled out possible depletion of substrates. Both increased gap junction communication and phosphodiesterase activity in COC are dependent on protein synthesis during the initial hours of IVM; however, both were inhibited in the presence of AICAR, which supports the finding that activation of AMPK by AICAR mediated inhibition of protein synthesis. Moreover, this protein synthesis inhibition was equivalent to that of the well-known protein synthesis inhibitor cycloheximide, as observed on cumulus expansion and protein concentration. Finally, the phosphorylation level of selected kinases was investigated. The pattern of raptor phosphorylation is supportive of activation of AMPK-mediated inhibition of protein synthesis. In conclusion, AICAR-mediated AMPK activation in porcine COC inhibited cumulus cell expansion and protein synthesis. These results bring new considerations to the importance of this kinase in ovarian

  9. Targeting Energy Metabolic and Oncogenic Signaling Pathways in Triple-negative Breast Cancer by a Novel Adenosine Monophosphate-activated Protein Kinase (AMPK) Activator*

    PubMed Central

    Lee, Kuen-Haur; Hsu, En-Chi; Guh, Jih-Hwa; Yang, Hsiao-Ching; Wang, Dasheng; Kulp, Samuel K.; Shapiro, Charles L.; Chen, Ching-Shih

    2011-01-01

    The antitumor activities of the novel adenosine monophosphate-activated protein kinase (AMPK) activator, OSU-53, were assessed in in vitro and in vivo models of triple-negative breast cancer. OSU-53 directly stimulated recombinant AMPK kinase activity (EC50, 0.3 μm) and inhibited the viability and clonogenic growth of MDA-MB-231 and MDA-MB-468 cells with equal potency (IC50, 5 and 2 μm, respectively) despite lack of LKB1 expression in MDA-MB-231 cells. Nonmalignant MCF-10A cells, however, were unaffected. Beyond AMPK-mediated effects on mammalian target of rapamycin signaling and lipogenesis, OSU-53 also targeted multiple AMPK downstream pathways. Among these, the protein phosphatase 2A-dependent dephosphorylation of Akt is noteworthy because it circumvents the feedback activation of Akt that results from mammalian target of rapamycin inhibition. OSU-53 also modulated energy homeostasis by suppressing fatty acid biosynthesis and shifting the metabolism to oxidation by up-regulating the expression of key regulators of mitochondrial biogenesis, such as a peroxisome proliferator-activated receptor γ coactivator 1α and the transcription factor nuclear respiratory factor 1. Moreover, OSU-53 suppressed LPS-induced IL-6 production, thereby blocking subsequent Stat3 activation, and inhibited hypoxia-induced epithelial-mesenchymal transition in association with the silencing of hypoxia-inducible factor 1a and the E-cadherin repressor Snail. In MDA-MB-231 tumor-bearing mice, daily oral administration of OSU-53 (50 and 100 mg/kg) suppressed tumor growth by 47–49% and modulated relevant intratumoral biomarkers of drug activity. However, OSU-53 also induced protective autophagy that attenuated its antiproliferative potency. Accordingly, cotreatment with the autophagy inhibitor chloroquine increased the in vivo tumor-suppressive activity of OSU-53. OSU-53 is a potent, orally bioavailable AMPK activator that acts through a broad spectrum of antitumor activities. PMID

  10. Metformin inhibits growth of human non-small cell lung cancer cells via liver kinase B-1-independent activation of adenosine monophosphate-activated protein kinase

    PubMed Central

    GUO, QIANQIAN; LIU, ZHIYAN; JIANG, LILI; LIU, MENGJIE; MA, JIEQUN; YANG, CHENGCHENG; HAN, LILI; NAN, KEJUN; LIANG, XUAN

    2016-01-01

    Metformin, the most widely administered oral anti-diabetic therapeutic agent, exerts its glucose-lowering effect predominantly via liver kinase B1 (LKB1)-dependent activation of adenosine monophosphate-activated protein kinase (AMPK). Accumulating evidence has demonstrated that metformin possesses potential antitumor effects. However, whether the antitumor effect of metformin is via the LKB1/AMPK signaling pathway remains to be determined. In the current study, the effects of metformin on proliferation, cell cycle progression, and apoptosis of human non-small cell lung cancer (NSCLC) H460 (LKB1-null) and H1299 (LKB1-positive) cells were assessed, and the role of LKB1/AMPK signaling in the anti-growth effects of metformin were investigated. Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell cycle distribution and apoptosis were assessed by flow cytometry, and protein expression levels were measured by western blotting. Metformin inhibited proliferation, induced significant cell cycle arrest at the G0–G1 phase and increased apoptosis in NSCLC cells in a time- and concentration-dependent manner, regardless of the level of LKB1 protein expression. Furthermore, knockdown of LKB1 with short hairpin RNA (shRNA) did not affect the antiproliferative effect of metformin in the H1299 cells. Metformin stimulated AMPK phosphorylation and subsequently suppressed the phosphorylation of mammalian target of rapamycin and its downstream effector, 70-kDa ribosomal protein S6 kinase in the two cell lines. These effects were abrogated by silencing AMPK with small interfering RNA (siRNA). In addition, knockdown of AMPK with siRNA inhibited the effect of metformin on cell proliferation in the two cell lines. These results provide evidence that the growth inhibition of metformin in NSCLC cells is mediated by LKB1-independent activation of AMPK, indicating that metformin may be a potential therapeutic agent for the treatment of

  11. Involvement of adenosine monophosphate-activated protein kinase in the influence of timed high-fat evening diet on the hepatic clock and lipogenic gene expression in mice.

    PubMed

    Huang, Yan; Zhu, Zengyan; Xie, Meilin; Xue, Jie

    2015-09-01

    A high-fat diet may result in changes in hepatic clock gene expression, but potential mechanisms are not yet elucidated. Adenosine monophosphate-activated protein kinase (AMPK) is a serine/threonine protein kinase that is recognized as a key regulator of energy metabolism and certain clock genes. Therefore, we hypothesized that AMPK may be involved in the alteration of hepatic clock gene expression under a high-fat environment. This study aimed to examine the effects of timed high-fat evening diet on the activity of hepatic AMPK, clock genes, and lipogenic genes. Mice with hyperlipidemic fatty livers were induced by orally administering high-fat milk via gavage every evening (19:00-20:00) for 6 weeks. Results showed that timed high-fat diet in the evening not only decreased the hepatic AMPK protein expression and activity but also disturbed its circadian rhythm. Accordingly, the hepatic clock genes, including clock, brain-muscle-Arnt-like 1, cryptochrome 2, and period 2, exhibited prominent changes in their expression rhythms and/or amplitudes. The diurnal rhythms of the messenger RNA expression of peroxisome proliferator-activated receptorα, acetyl-CoA carboxylase 1α, and carnitine palmitoyltransferase 1 were also disrupted; the amplitude of peroxisome proliferator-activated receptorγcoactivator 1α was significantly decreased at 3 time points, and fatty liver was observed. These findings demonstrate that timed high-fat diet at night can change hepatic AMPK protein levels, activity, and circadian rhythm, which may subsequently alter the circadian expression of several hepatic clock genes and finally result in the disorder of hepatic lipogenic gene expression and the formation of fatty liver.

  12. A high isoflavone diet decreases 5' adenosine monophosphate-activated protein kinase activation and does not correct selenium-induced elevations in fasting blood glucose in mice.

    PubMed

    Stallings, Michael T; Cardon, Brandon R; Hardman, Jeremy M; Bliss, Tyler A; Brunson, Scott E; Hart, Chris M; Swiss, Maria D; Hepworth, Squire D; Christensen, Merrill J; Hancock, Chad R

    2014-04-01

    Selenium (Se) has been implicated as a micronutrient that decreases adenosine monophosphate-activated protein kinase (AMPK) signaling and may increase diabetes risk by reducing insulin sensitivity. Soy isoflavones (IF) are estrogen-like compounds that have been shown to attenuate insulin resistance, hyperglycemia, adiposity, and increased AMPK activation. We hypothesized that a high IF (HIF) diet would prevent the poor metabolic profile associated with high Se intake. The purpose of this study was to examine changes in basal glucose metabolism and AMPK signaling in response to an HIF diet and/or supplemental Se in a mouse model. Male FVB mice were divided into groups receiving either a control diet with minimal IF (low IF) or an HIF diet. Each dietary group was further subdivided into groups receiving either water or Se at a dose of 3 mg Se/kg body weight daily, as Se-methylselenocysteine (SMSC). After 5 months, mice receiving SMSC had elevated fasting glucose (P < .05) and a tendency for glucose intolerance (P = .08). The increase in dietary IF did not result in improved fasting blood glucose. Interestingly, after 6 months, HIF-fed mice had decreased basal AMPK activation in liver and skeletal muscle tissue (P < .05). Basal glucose metabolism was changed by SMSC supplementation as evidenced by increased fasting blood glucose and glucose intolerance. High dietary IF levels did not protect against aberrant blood glucose. In FVB mice, decreased basal AMPK activation is not the mechanism through which Se exerts its effect. These results suggest that more research must be done to elucidate the role of Se and IF in glucose metabolism.

  13. cAMP-independent dilation of coronary arterioles to adenosine : role of nitric oxide, G proteins, and K(ATP) channels.

    PubMed

    Hein, T W; Kuo, L

    1999-10-01

    Adenosine is known to play an important role in the regulation of coronary blood flow during metabolic stress. However, there is sparse information on the mechanism of adenosine-induced dilation at the microcirculatory levels. In the present study, we examined the role of endothelial nitric oxide (NO), G proteins, cyclic nucleotides, and potassium channels in coronary arteriolar dilation to adenosine. Pig subepicardial coronary arterioles (50 to 100 microm in diameter) were isolated, cannulated, and pressurized to 60 cm H(2)O without flow for in vitro study. The arterioles developed basal tone and dilated dose dependently to adenosine. Disruption of endothelium, blocking of endothelial ATP-sensitive potassium (K(ATP)) channels by glibenclamide, and inhibition of NO synthase by N(G)-nitro-L-arginine methyl ester and of soluble guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one produced identical attenuation of vasodilation to adenosine. Combined administration of these inhibitors did not further attenuate the vasodilatory response. Production of NO from coronary arterioles was significantly increased by adenosine. Pertussis toxin, but not cholera toxin, significantly inhibited vasodilation to adenosine, and this inhibitory effect was only evident in vessels with an intact endothelium. Tetraethylammonium, glibenclamide, and a high concentration of extraluminal KCl abolished vasodilation of denuded vessels to adenosine; however, inhibition of calcium-activated potassium channels by iberiotoxin had no effect on this dilation. Rp-8-Br-cAMPS, a cAMP antagonist, inhibited vasodilation to cAMP analog 8-Br-cAMP but failed to block adenosine-induced dilation. Furthermore, vasodilations to 8-Br-cAMP and sodium nitroprusside were not inhibited by glibenclamide, indicating that cAMP- and cGMP-induced dilations are not mediated by the activation of K(ATP) channels. These results suggest that adenosine activates both endothelial and smooth muscle pathways to exert

  14. Adenosine dry powder inhalation for bronchial challenge testing, part 2: proof of concept in asthmatic subjects.

    PubMed

    Lexmond, Anne J; van der Wiel, Erica; Hagedoorn, Paul; Bult, Wouter; Frijlink, Henderik W; ten Hacken, Nick H T; de Boer, Anne H

    2014-09-01

    Adenosine is an indirect stimulus to assess bronchial hyperresponsiveness (BHR(2)) in asthma. Bronchial challenge tests are usually performed with nebulised solutions of adenosine 5'-monophosphate (AMP(3)). The nebulised AMP test has several disadvantages, like long administration times and a restrictive maximum concentration that does not result in BHR in all patients. In this study, we investigated the applicability of dry powder adenosine for assessment of BHR in comparison to nebulised AMP. Dry powder adenosine was prepared in doubling doses (0.01-80 mg) derived from the nebulised AMP test with addition of two higher doses. Five asthmatic subjects performed two bronchial challenge tests, one with nebulised AMP following the 2-min tidal breathing method; the second with dry powder adenosine administered with an investigational inhaler and single slow inhalations (inspiratory flow rate 30-40 L/min). All subjects reached a 20% fall in FEV₁(4) with the new adenosine test (PD20(5)) compared to four subjects with the AMP test (PC₂₀(6)). Dry powder adenosine was well tolerated by all subjects and better appreciated than nebulised AMP. In conclusion, this new bronchial challenge test appears to be a safe and convenient alternative to the nebulised AMP test to assess BHR in asthmatic subjects.

  15. Post-meal responses of elongation factor 2 (eEF2) and adenosine monophosphate-activated protein kinase (AMPK) to leucine and carbohydrate supplements for regulating protein synthesis duration and energy homeostasis in rat skeletal muscle.

    PubMed

    Wilson, Gabriel J; Moulton, Christopher J; Garlick, Peter J; Anthony, Tracy G; Layman, Donald K

    2012-11-13

    Previous research demonstrates that the anabolic response of muscle protein synthesis (MPS) to a meal is regulated at the level of translation initiation with signals derived from leucine (Leu) and insulin to activate mTORC1 signaling. Recent evidence suggests that the duration of the meal response is limited by energy status of the cell and inhibition of translation elongation factor 2 (eEF2). This study evaluates the potential to extend the anabolic meal response with post-meal supplements of Leu or carbohydrates. Adult (~256 g) male Sprague-Dawley rats were food deprived for 12 h, then either euthanized before a standard meal (time 0) or at 90 or 180 min post-meal. At 135 min post-meal, rats received one of five oral supplements: 270 mg leucine (Leu270), 80:40:40 mg leucine, isoleucine, and valine (Leu80), 2.63 g carbohydrates (CHO2.6), 1 g carbohydrates (CHO1.0), or water (Sham control). Following the standard meal, MPS increased at 90 min then declined to pre-meal baseline at 180 min. Rats administered Leu270, Leu80, CHO2.6, or CHO1.0 maintained elevated rates of MPS at 180 min, while Sham controls declined from peak values. Leu80 and CHO1.0 treatments maintained MPS, but with values intermediate between Sham controls and Leu270 and CHO2.6 supplements. Consistent with MPS findings, the supplements maintained elongation activity and cellular energy status by preventing increases in AMP/ATP and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), acetyl-CoA carboxylase ACC and eEF2. The impact of the supplements on MPS and cellular energy status was in proportion to the energy content within the individual treatments (i.e., Leu270 > Leu80; CHO2.6 > CHO1.0), but the Leu supplements produced a disproportionate anabolic stimulation of MPS, eEF2 and energy status with significantly lower energy content. In summary, the incongruity between MPS and translation initiation at 180 min reflects a block in translation elongation due to reduced

  16. New perspectives in signaling mediated by receptors coupled to stimulatory G protein: the emerging significance of cAMP efflux and extracellular cAMP-adenosine pathway.

    PubMed

    Godinho, Rosely O; Duarte, Thiago; Pacini, Enio S A

    2015-01-01

    G protein-coupled receptors (GPCRs) linked to stimulatory G (Gs) proteins (GsPCRs) mediate increases in intracellular cyclic AMP as consequence of activation of nine adenylyl cyclases , which differ considerably in their cellular distribution and activation mechanisms. Once produced, cyclic AMP may act via distinct intracellular signaling effectors such as protein kinase A and the exchange proteins activated by cAMP (Epacs). More recently, attention has been focused on the efflux of cAMP through a specific transport system named multidrug resistance proteins that belongs to the ATP-binding cassette transporter superfamily. Outside the cell, cAMP is metabolized into adenosine, which is able to activate four distinct subtypes of adenosine receptors, members of the GPCR family: A1, A2A, A2B, and A3. Taking into account that this phenomenon occurs in numerous cell types, as consequence of GsPCR activation and increment in intracellular cAMP levels, in this review, we will discuss the impact of cAMP efflux and the extracellular cAMP-adenosine pathway on the regulation of GsPCR-induced cell response.

  17. New perspectives in signaling mediated by receptors coupled to stimulatory G protein: the emerging significance of cAMP efflux and extracellular cAMP-adenosine pathway

    PubMed Central

    Godinho, Rosely O.; Duarte, Thiago; Pacini, Enio S. A.

    2015-01-01

    G protein-coupled receptors (GPCRs) linked to stimulatory G (Gs) proteins (GsPCRs) mediate increases in intracellular cyclic AMP as consequence of activation of nine adenylyl cyclases , which differ considerably in their cellular distribution and activation mechanisms. Once produced, cyclic AMP may act via distinct intracellular signaling effectors such as protein kinase A and the exchange proteins activated by cAMP (Epacs). More recently, attention has been focused on the efflux of cAMP through a specific transport system named multidrug resistance proteins that belongs to the ATP-binding cassette transporter superfamily. Outside the cell, cAMP is metabolized into adenosine, which is able to activate four distinct subtypes of adenosine receptors, members of the GPCR family: A1, A2A, A2B, and A3. Taking into account that this phenomenon occurs in numerous cell types, as consequence of GsPCR activation and increment in intracellular cAMP levels, in this review, we will discuss the impact of cAMP efflux and the extracellular cAMP-adenosine pathway on the regulation of GsPCR-induced cell response. PMID:25859216

  18. Conservation and divergence of the cyclic adenosine monophosphate-protein kinase A (cAMP–PKA) pathway in two plant-pathogenic fungi: Fusarium graminearum and F. verticillioides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cyclic AMP (cAMP)-PKA pathway is a central signaling cascade that transmits extracellular stimuli and governs cell responses through the second messenger cAMP. The importance of cAMP signaling in fungal biology has been well documented. Two key conserved components, adenylate cyclase (AC) and ca...

  19. Cardiac cAMP: production, hydrolysis, modulation and detection

    PubMed Central

    Boularan, Cédric; Gales, Céline

    2015-01-01

    Cyclic adenosine 3′,5′-monophosphate (cAMP) modulates a broad range of biological processes including the regulation of cardiac myocyte contractile function where it constitutes the main second messenger for β-adrenergic receptors' signaling to fulfill positive chronotropic, inotropic and lusitropic effects. A growing number of studies pinpoint the role of spatial organization of the cAMP signaling as an essential mechanism to regulate cAMP outcomes in cardiac physiology. Here, we will briefly discuss the complexity of cAMP synthesis and degradation in the cardiac context, describe the way to detect it and review the main pharmacological arsenal to modulate its availability. PMID:26483685

  20. Relaxation of isolated taenia coli of guinea-pig by enantiomers of 2-azido analogues of adenosine and adenine nucleotides.

    PubMed Central

    Cusack, N. J.; Planker, M.

    1979-01-01

    1 2-Azido photoaffinity analogues of adenosine 5'triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), and adenosine have been synthesized and tested on guinea-pig taenia coli. 2 2-Azido-ATP and 2-azido-ADP were approximately 20 times more potent than ATP as relaxants of taenia coli, and required prolonged washout times before recovery of the muscle. 3 2-Azido-AMP and 2-azidoadenosine were 2 to 12 times more potent than ATP, but took much longer (up to 100 s) to reach maximal relaxation. This behaviour is different from that of AMP and adenosine which were much less potent than ATP. 4 L-Enantiomers of adenosine and adenine nucleotides were also tested. L-ATP and L-ADP were 3 to 6 times less potent than ATP and ADP, and L-AMP and L-adenosine were inactive. 2-Azido-L-ATP and 2-azido-L-ADP were approximately 120 times less potent than 2-Azido-ATP and 6 times less potent than ATP as relaxants of taenia coli. 2-Azido-L-AMP and 2-azidio-L-adenosine were almost inactive. 5 2-Azido derivatives are photolysed by u.v. irradiation to reactive intermediates. 2-Azido-ATP and 2-azidoadenosine might be suitable photoaffinity ligands for labelling putative P2 and P1 purine receptors respectively. 2-Azido-L-ATP and 2-azido-L-adenosine could be useful controls for nonspecific labelling. PMID:497519

  1. Adenosine 3':5'-cyclic monophosphate-dependent protein kinase in brown fat from newborn rabbits. Changes in the binding of adenosine 3':5'-cyclic monophosphate after preincubation of the tissue with noradrenaline or incubation of the enzyme with adenosine triphosphate.

    PubMed Central

    Knight, B L

    1975-01-01

    The equilibrium binding of cyclic AMP to a 150-fold purified preparation of protein kinase, when expressed as the reciprocal of bound against the reciprocal of free cyclic AMP, gave a plot consisting of two straight lines. The values of apparent Kb given by these lines were lowered by preincubating the intact tissue with noradrenaline or incubating the enzyme preparation with Mg2+ plus ATP. This effect was reversed by incubating the preparation (which contained some phosphatase impurities) with Mg2+ alone. None of these procedures affected the maximal binding of cyclic AMP. During incubation of the enzyme with Mg2+ plus ATP, the terminal phosphoryl group was incorporated into protein, over 40% being present in the kinase itself. This phosphate was removed during incubation of the preparation with Mg2+ alone. The validity of expressing cyclic AMP binding as a double-reciprocal plot is discussed, and the experimental plots are compared with those derived theoretically. The results suggest that protein kinase in brown fat is present in two forms, one with an apparent Kb for cyclic AMP or approx. 250 nM (dephosphorylation) and one with an apparent Kb of approx. 14 nM (phosphorylated). Preincubation of the tissue with noradrenaline results in phosphorylation of the kinase and an increase from 15 to 45% in the proportion of the higher-affinity form. PMID:179526

  2. Stimulation of cartilage amino acid uptake by growth hormone-dependent factors in serum. Mediation by adenosine 3':5'-monophosphate.

    PubMed

    Drezner, M K; Eisenbarth, G S; Neelon, F A; Lebovitz, H E

    1975-02-13

    The effects of growth hormone-dependent serum factors on amino acid transport and on cartilage cyclic AMP levels in embryonic chicken cartilage were studied in vitro. Cartilages incubated in medium containing rat serum showed a significantly greater uptake of alpha-amino [1-14C] isobutyrate or [1-14C] cycloleucine than control cartilages incubated in medium alone. Normal rat serum (5%) added to the incubation medium also caused an increase in cartilage cyclic AMP content (from as little as 23% to as much as 109%). The factors in serum which increase cartilage cyclic AMP and amino acid uptake are growth hormone dependent, since neither growth hormone itself nor serum from hypophysectomized rats restores these serum factors. Studies comparing the ability of sera with varying amounts of growth hormone-dependent factors to stimulate amino-aminoisobutyrate transport and to increase cartilage cyclic AMP show a striking linear correlation between the two effects (r=0.977). Theophylline and prostaglandin E1, WHICH RAISE CARTILAGE CYCLIC AMP also increase amino-aminoisobutyrate transport. Exogenous cyclic AMP, N6-monobutyryl cyclic AMP and n6, 02'-dibutyryl cyclic AMP increase cartilage amino-aminoisobutyrate transport. The data are compatible with the thesis that growth hormone-dependent serum factors increase cartilage amino acid transport by elevating cartilage cyclic AMP.

  3. Fluorometric Determination of Adenosine Nucleotide Derivatives as Measures of the Microfouling, Detrital, and Sedimentary Microbial Biomass and Physiological Status

    PubMed Central

    Davis, William M.; White, David C.

    1980-01-01

    Adenosine, adenine, cyclic adenosine monophosphate (AMP), AMP, nicotinamide adenine dinucleotide, adenosine diphosphate, and adenosine triphosphate (ATP) were recovered quantitatively from aqueous portions of lipid extracts of microfouling, detrital, and sedimentary microbial communities. These could be detected quantitatively in the picomolar range by forming their 1-N6-etheno derivatives and analyzing by high-pressure liquid chromatography with fluorescence detection. Lipid extraction and subsequent analysis allowed the simultaneous measurement of the microbial community structure, total microbial biomass with the quantitative recovery of the adenine-containing cellular components, which were protected from enzymatic destruction. This extraction and fluorescent derivatization method showed equivalency with the luciferin-luciferase method for bacterial ATP measurements. Quick-freezing samples in the field with dry ice-acetone preserved the ATP and energy charge (a ratio of adenosine nucleotides) for analysis at remote laboratories. The metabolic lability of ATP in estuarine detrital and microfouling communities, as well as bacterial monocultures of constant biomass, showed ATP to be a precarious measure of biomass under some conditions. Combinations of adenosine and adenine nucleotides gave better correlations with microbial biomass measured as extractable lipid phosphate in the detrital and microfouling microbial communities than did ATP alone. Stresses such as anoxia or filtration are reflected in the rapid accumulation of intracellular adenosine and the excretion of adenosine and AMP into the surrounding milieu. Increases in AMP and adenosine may prove to be more sensitive indicators of metabolic status than the energy charge. PMID:16345633

  4. Differences in responsiveness of intrapulmonary artery and vein to arachidonic acid: mechanism of arterial relaxation involves cyclic guanosine 3':5'-monophosphate and cyclic adenosine 3':5'-monophosphate

    SciTech Connect

    Ignarro, L.J.; Harbison, R.G.; Wood, K.S.; Wolin, M.S.; McNamara, D.B.; Hyman, A.L.; Kadowitz, P.J.

    1985-06-01

    The objective of this study was to examine the relationship between responses of bovine intrapulmonary artery and vein to arachidonic acid and cyclic nucleotide levels in order to better understand the mechanism of relaxation elicited by arachidonic acid and acetylcholine. Arachidonic acid relaxed phenylephrine-precontracted arterial rings and elevated both cyclic GMP and cyclic AMP levels in arteries with intact endothelium. In contrast, endothelium-damaged arterial rings contracted to arachidonic acid without demonstrating significant changes in cyclic nucleotide levels. Indomethacin partially inhibited endothelium-dependent relaxation and abolished cyclic AMP accumulation whereas methylene blue, a guanylate cyclase inhibitor, partially inhibited relaxation and abolished cyclic GMP accumulation in response to arachidonic acid. All vessel responses were blocked by a combination of the two inhibitors. Prostaglandin (PG) I2 relaxed arterial rings and elevated cyclic AMP levels whereas PGE2 and PGF2 alpha caused contraction, suggesting that the indomethacin-sensitive component of arachidonic acid-elicited relaxation is due to PGI2 formation and cyclic AMP accumulation. The methylene blue-sensitive component is attributed to an endothelium-dependent but cyclooxygenase-independent generation of a substance causing cyclic GMP accumulation. Intrapulmonary veins contracted to arachidonic acid with no changes in cyclic nucleotide levels and PGI2 was without effect. Homogenates of intrapulmonary artery and vein formed 6-keto-PGF1 alpha, PGF2 alpha and PGE2 from (/sup 14/C)arachidonic acid, which was inhibited by indomethacin. Thus, bovine intrapulmonary vein may not possess receptors for PGI2.

  5. Glial Restricted Precursor Cell Transplant with Cyclic Adenosine Monophosphate Improved Some Autonomic Functions but Resulted in a Reduced Graft Size after Spinal Cord Contusion Injury in Rats

    PubMed Central

    Nout, Yvette S.; Culp, Esther; Schmidt, Markus H.; Tovar, C. Amy; Pröschel, Christoph; Mayer-Pröschel, Margot; Noble, Mark D.; Beattie, Michael S.; Bresnahan, Jacqueline C.

    2010-01-01

    Transplantation of glial restricted precursor (GRP) cells has been shown to reduce glial scarring after spinal cord injury (SCI) and, in combination with neuronal restricted precursor (NRP) cells or enhanced expression of neurotrophins, to improve recovery of function after SCI. We hypothesized that combining GRP transplants with rolipram and cAMP would improve functional recovery, similar to that seen after combining Schwann cell transplants with increasing cAMP. A short term study, 1)uninjured control, 2)SCI+vehicle, and 3)SCI+cAMP, showed that spinal cord [cAMP] were increased 14 days after SCI. We used 51 male rats subjected to a thoracic SCI for a 12-week survival study: 1)SCI+vehicle, 2)SCI+GRP, 3)SCI+cAMP, 4)SCI+GRP+cAMP, and 5)uninjured endpoint age-matched control (AM). Rolipram was administered for 2 weeks after SCI. At 9 days after SCI, GRP transplantation and injection of dibutyryl-cAMP into the spinal cord were performed. GRP cells survived, differentiated, and formed extensive transplants that were well integrated with host tissue. Presence of GRP cells increased the amount of tissue in the lesion; however, cAMP reduced the graft size. White matter sparing at the lesion epicenter was not affected. Serotonergic input to the lumbosacral spinal cord was not affected by treatment, but the amount of serotonin immediately caudal to the lesion was reduced in the cAMP groups. Using telemetric monitoring of corpus spongiosum penis pressure we show that the cAMP groups regained the same number of micturitions per 24 hrs when compared to the AM group, however, the frequency of peak pressures was increased in these groups compared to the AM group. In contrast, the GRP groups had similar frequency of peak pressures compared to baseline and the AM group. Animals that received GRP cells regained the same number of erectile events per 24 hrs compared to baseline and the AM group. Since cAMP reduced the GRP transplant graft, and some modest positive effects were seen

  6. Biphasic effects of dibutyryl cyclic adenosine 3',5'-monophosphate on synergistic stimulation of DNA synthesis by diacylglycerol, and the ionophore A23187 in guinea pig lymphocytes

    SciTech Connect

    Otani, S.; Matsui-Yuasa, I.; Morisawa, S.

    1987-06-22

    When guinea pig lymphocytes were cultured with 1-oleoyl-2-acetylglycerol (OAG) and the ionophore A23187 for 8 h, (TH-thymidine incorporation into the acid-insoluble fraction of the cells was stimulated synergistically. Further addition of dibutyryl cAMP caused a biphasic effect on the synergistic stimulation. Dibutyryl cAMP augmented the synergistic stimulation when A23187 was at the concentration of 0.075 g/ml, but inhibited it when the ionophore was at 0.25 g/ml. At the higher concentration A23187, dibutyryl cAMP stimulated the (TH)thymidine incorporation when culture was for 4 h, but inhibited it when culture was for 8 h. The results were the same when 12-0-tetradecanoylphorbol-13-acetate (TPA) was used instead of OAG. Butyrate could replace dibutyryl cAMP for stimulation of (TH)thymidine incorporation in combination with TPA and A23187, but not with OAG and A23187 at the lower ionophore concentration. Dibutyryl cAMP but not butyrate stimulated ornithine decarboxylase induction caused by TPA and A23187. These results suggest that the effect of dibutyryl cAMP on DNA synthesis induced by OAG and A23187 was biphasic and depended on the concentration of A23187 and on the time of culture, and that the stimulation mechanism of butyrate is different from that of dibutyryl cAMP.

  7. Effects of limited exposure of rabbit chondrocyte cultures to parathyroid hormone and dibutyryl adenosine 3',5'-monophosphate on cartilage-characteristic proteoglycan synthesis

    SciTech Connect

    Kato, Y.; Koike, T.; Iwamoto, M.; Kinoshita, M.; Sato, K.; Hiraki, Y.; Suzuki, F.

    1988-05-01

    Treatment of rabbit chondrocyte cultures with PTH or (Bu)2cAMP for 30 h increased by 2- to 3-fold the incorporation of (35S)sulfate and 3H radioactivity with glucosamine as the precursor into large chondroitin sulfate proteoglycans characteristically found in cartilage matrix. However, PTH and (Bu)2cAMP did not increase either (35S)sulfate incorporation into small proteoglycans or the incorporation of 3H radioactivity into hyaluronic acid and other glycosaminoglycans. PTH and (Bu)2cAMP also increased the incorporation of (3H) serine into both proteoglycans and total protein. In all cultures described above, the stimulation of (3H)serine incorporation into proteoglycans exceeded that of (3H)serine incorporation into total protein. These data indicate that PTH and (Bu)2cAMP selectively stimulate cartilage proteoglycan synthesis while they increase total protein synthesis. Since cAMP seems to play a mediatory role in the action of PTH, we elected to examine the effects of a limited exposure of chondrocytes to PTH or (Bu)2cAMP on the synthesis of proteoglycans. Treatment with PTH or (Bu)2cAMP for only the initial 2-7 h did not increase the rates of incorporation of (35S)sulfate, the 3H radioactivity with glucosamine, and (3H)serine into proteoglycans, as measured at 30 h, despite the fact that this treatment brought about a rapid and transient rise in the cAMP level. Furthermore, the application of prostaglandin I2 at concentrations that increased cAMP levels in a similar fashion as did PTH did not affect (35S) sulfate incorporation into proteoglycans.

  8. Enzymatic production of 5'-inosinic acid by AMP deaminase from a newly isolated Aspergillus oryzae.

    PubMed

    Li, Shubo; Chen, Leitao; Hu, Yangjun; Fang, Guohui; Zhao, Mouming; Guo, Yuan; Pang, Zongwen

    2017-02-01

    5'-adenylic acid deaminase (AMP deaminase), an important enzyme for the food industry, can catalyze the irreversible hydrolysis of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia. In this study, a new strain was screened that efficiently produces 3191.6U/g of AMP deaminase at 32°C. After purification, the optimal temperature and pH of the AMP deaminase were found to be 40°C and 6.0, respectively, but it was partially inhibited by Fe(3+), Cu(2+), Al(3+), and Zn(2+). With amplification of the AMP deaminase production system, 6mL of crude enzyme could produce 2.00mg/g of IMP from 2.04mg/g of dried yeast with an 84.8% molar yield after 40min. These results provide a new insight into AMP deaminase production and offer a potential platform for producing 5'-IMP. PMID:27596420

  9. Enzymatic production of 5'-inosinic acid by AMP deaminase from a newly isolated Aspergillus oryzae.

    PubMed

    Li, Shubo; Chen, Leitao; Hu, Yangjun; Fang, Guohui; Zhao, Mouming; Guo, Yuan; Pang, Zongwen

    2017-02-01

    5'-adenylic acid deaminase (AMP deaminase), an important enzyme for the food industry, can catalyze the irreversible hydrolysis of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia. In this study, a new strain was screened that efficiently produces 3191.6U/g of AMP deaminase at 32°C. After purification, the optimal temperature and pH of the AMP deaminase were found to be 40°C and 6.0, respectively, but it was partially inhibited by Fe(3+), Cu(2+), Al(3+), and Zn(2+). With amplification of the AMP deaminase production system, 6mL of crude enzyme could produce 2.00mg/g of IMP from 2.04mg/g of dried yeast with an 84.8% molar yield after 40min. These results provide a new insight into AMP deaminase production and offer a potential platform for producing 5'-IMP.

  10. Cyclic 3'-5'-adenosine monophosphate binds to annexin I and regulates calcium-dependent membrane aggregation and ion channel activity.

    PubMed

    Cohen, B E; Lee, G; Arispe, N; Pollard, H B

    1995-12-27

    The annexin (Anx) gene family comprises a set of calcium-dependent membrane binding proteins, which have been implicated in a wide variety of cellular processes including membrane fusion and calcium channel activity. We report here that cAMP activates Ca(2+)-dependent aggregation of both phosphatidylserine (PS) liposomes and bovine chromaffin granules driven by [des 1-12]annexin I (lipocortin I, Anx1). The mechanism of cAMP action involves an increase in AnxI-dependent cooperativity on the rate of such a reaction without affecting the corresponding k1/2 values. Cyclic AMP causes the values of the Hill coefficient (nH) for AnxI to change from 3 to 6 in both PS liposomes and chromaffin granules. By contrast, ATP inhibits the rate of aggregation activity without affecting the cooperativity or the extent of aggregation process. We were also able to photolabel Anx1 specifically with an 8-azido analogue of cAMP by a calcium-independent process. Such a process is saturable, yielding a Kd = 0.8 microM by Scatchard analysis. Specific displacement occurs in the presence of cAMP and ATP. Finally, we found that cAMP alters the conductance of calcium channels formed by AnxI in planar lipid bilayers. We interpret these data to indicate that AnxI binds both calcium and cAMP independently, and that both actions have functional consequences. This is the first report of a nucleotide binding function for a member of the annexin gene family.

  11. Mechanisms involved in 3',5'-cyclic adenosine monophosphate-mediated inhibition of the ubiquitin-proteasome system in skeletal muscle.

    PubMed

    Gonçalves, Dawit A P; Lira, Eduardo C; Baviera, Amanda M; Cao, Peirang; Zanon, Neusa M; Arany, Zoltan; Bedard, Nathalie; Tanksale, Preeti; Wing, Simon S; Lecker, Stewart H; Kettelhut, Isis C; Navegantes, Luiz C C

    2009-12-01

    Although it is well known that catecholamines inhibit skeletal muscle protein degradation, the molecular underlying mechanism remains unclear. This study was undertaken to investigate the role of beta(2)-adrenoceptors (AR) and cAMP in regulating the ubiquitin-proteasome system (UPS) in skeletal muscle. We report that increased levels of cAMP in isolated muscles, promoted by the cAMP phosphodiesterase inhibitor isobutylmethylxanthine was accompanied by decreased activity of the UPS, levels of ubiquitin-protein conjugates, and expression of atrogin-1, a key ubiquitin-protein ligase involved in muscle atrophy. In cultured myotubes, atrogin-1 induction after dexamethasone treatment was completely prevented by isobutylmethylxanthine. Furthermore, administration of clenbuterol, a selective beta(2)-agonist, to mice increased muscle cAMP levels and suppressed the fasting-induced expression of atrogin-1 and MuRF-1, atrogin-1 mRNA being much more responsive to clenbuterol. Moreover, clenbuterol increased the phosphorylation of muscle Akt and Foxo3a in fasted rats. Similar responses were observed in muscles exposed to dibutyryl-cAMP. The stimulatory effect of clenbuterol on cAMP and Akt was abolished in muscles from beta(2)-AR knockout mice. The suppressive effect of beta(2)-agonist on atrogin-1 was not mediated by PGC-1alpha (peroxisome proliferator-activated receptor-gamma coactivator 1alpha known to be induced by beta(2)-agonists and previously shown to inhibit atrogin-1 expression), because food-deprived PGC-1alpha knockout mice were still sensitive to clenbuterol. These findings suggest that the cAMP increase induced by stimulation of beta(2)-AR in skeletal muscles from fasted mice is possibly the mechanism by which catecholamines suppress atrogin-1 and the UPS, this effect being mediated via phosphorylation of Akt and thus inactivation of Foxo3.

  12. Dual specificity and novel structural folding of yeast phosphodiesterase-1 for hydrolysis of second messengers cyclic adenosine and guanosine 3',5'-Monophosphate

    DOE PAGES

    Tian, Yuanyuan; Cui, Wenjun; Huang, Manna; Robinson, Howard; Wan, Yiqian; Wang, Yousheng; Ke, Hengming

    2014-08-05

    Cyclic nucleotide phosphodiesterases (PDEs) decompose second messengers cAMP and cGMP that play critical roles in many physiological processes. PDE1 of Saccharomyces cerevisiae has been subcloned and expressed in Escherichia coli. Recombinant yPDE1 has a KM of 110 μM and a kcat of 16.9 s⁻¹ for cAMP and a KM of 105 μM and a kcat of 11.8 s₅⁻¹ for cGMP. Thus, the specificity constant (kcat/KMcAMP)/(kcat/KMcGMP) of 1.4 indicates a dual specificity of yPDE1 for hydrolysis of both cAMP and cGMP. The crystal structures of unliganded yPDE1 and its complex with GMP at 1.31 Å resolution reveal a new structural foldingmore » that is different from those of human PDEs but is partially similar to that of some other metalloenzymes such as metallo-β-lactamase. In spite of their different structures and divalent metals, yPDE1 and human PDEs may share a common mechanism for hydrolysis of cAMP and cGMP.« less

  13. Arginine vasopressin increases cellular free calcium concentration and adenosine 3',5'-monophosphate production in rat renal papillary collecting tubule cells in culture

    SciTech Connect

    Ishikawa, S.; Okada, K.; Saito, T.

    1988-09-01

    The role of calcium (Ca) in the cellular action of arginine vasopressin (AVP) was examined in rat renal papillary collecting tubule cells in culture. AVP increased both the cellular free Ca concentration ((Ca2+)i) using fura-2, and cAMP production in a dose-dependent manner. AVP-induced cellular Ca mobilization was totally blocked by the antagonist to the antidiuretic action of AVP, and somewhat weakened by the antagonist to the vascular action of AVP. 1-Deamino-8-D-AVP (dDAVP). an antidiuretic analog of AVP, also increased (Ca2+) significantly. Cellular Ca mobilization was not obtained with cAMP, forskolin (a diterpene activator of adenylate cyclase), or phorbol-12-myristate-13-acetate. The early phase of (Ca2+)i depended on the intracellular Ca pool, since an AVP-induced rise in (Ca2+)i was obtained in cells pretreated with Ca-free medium containing 1 mM EGTA, verapamil, or cobalt, which blocked cellular Ca uptake. Also, AVP increased /sup 45/Ca2+ influx during the initial 10 min, which initiated the sustained phase of cellular Ca mobilization. However, cellular cAMP production induced by AVP during the 10-min observation period was diminished in the cells pretreated with Ca-free medium, verapamil, or cobalt, but was still significantly higher than the basal level. This was also diminished by a high Ca concentration in medium. These results indicate that 1) AVP concomitantly regulates cellular free Ca as well as its second messenger cAMP production; 2) AVP-induced elevation of cellular free Ca is dependent on both the cellular Ca pool and extracellular Ca; and 3) there is an optimal level of extracellular Ca to modulate the AVP action in renal papillary collecting tubule cells.

  14. Corticotropin-releasing factor binding to peripheral tissue and activation of the adenylate cyclase-adenosine 3',5'-monophosphate system

    SciTech Connect

    Dave, J.R.; Eiden, L.E.; Eskay, R.L.

    1985-06-01

    Specific binding sites for rat corticotropin-releasing factor (rCRF) are present in rat adrenal medulla, ventral prostate, spleen, liver, kidney, and testis and bovine chromaffin cells in culture. Maximal binding of (/sup 125/I)rCRF occurred within 25 min at 4 C and was saturable. Scatchard analysis of rCRF binding to rat adrenal membranes and bovine chromaffin cells revealed the existence of two classes of binding sites. One class had a relatively higher apparent affinity and lower number of binding sites, whereas the other class had a relatively lower affinity and higher number of binding sites. CRF induced a dose-related increase in rat adrenal membrane adenylate cyclase activity and cAMP levels in bovine chromaffin cells. Nanomolar concentrations of rCRF maximally stimulated adenylate cyclase activity in rat adrenal membranes and maximally increased cAMP levels in bovine chromaffin cells to 86% and 130% above control values, respectively. The demonstration of specific CRF-binding sites in a variety of peripheral tissues and the finding that activation of specific CRF-binding sites in adrenal tissue stimulates the adenylate cyclase-cAMP system suggest that CRF may have an important regulatory role in various peripheral tissues.

  15. The effect of glucose, insulin and noradrenaline on lipolysis and on the concentrations of adenosine 3′:5′-cyclic monophosphate and adenosine 5′-triphosphate in adipose tissue

    PubMed Central

    Knight, Brian L.; Iliffe, Jill

    1973-01-01

    Glycerol release and tissue concentrations of ATP and cyclic AMP were followed during the incubation of adipose tissue with or without glucose, insulin and noradrenaline. Glucose plus insulin or, to a lesser extent, glucose alone increased the accumulation of glycerol during incubations both with and without noradrenaline by slowing the decline in the rate of glycerol release with time. Insulin alone decreased the accumulation by accelerating the fall in glycerol release. In the absence of noradrenaline, ATP and cyclic AMP concentrations were not significantly affected by insulin or glucose. With noradrenaline or noradrenaline plus insulin the ATP concentration gradually fell. With noradrenaline plus glucose the ATP concentration fell rapidly and then stabilized, or, if insulin was also present, returned to the control value. In the presence of noradrenaline, the concentration of cyclic AMP rose during the first 20min and then fell. Insulin lowered the peak concentration of cyclic AMP, but glucose had no effect either on the peak value or the fall in the concentration of the nucleotide. The increase and fall in the concentration of cyclic AMP with noradrenaline or noradrenaline plus insulin bore similarities to the increase and decline in the lipolytic rate in incubations without glucose. It is proposed that glucose stimulates ATP production by furnishing glycerol 1-phosphate and thus removing free fatty acids, but that it can influence lipolysis by a mechanism which is distinct from any which is mediated by free fatty acids, possibly by inhibiting the inactivation of the lipase. PMID:4353001

  16. Diabetic complications within the context of aging: Nicotinamide adenine dinucleotide redox, insulin C-peptide, sirtuin 1-liver kinase B1-adenosine monophosphate-activated protein kinase positive feedback and forkhead box O3.

    PubMed

    Ido, Yasuo

    2016-07-01

    Recent research in nutritional control of aging suggests that cytosolic increases in the reduced form of nicotinamide adenine dinucleotide and decreasing nicotinamide adenine dinucleotide metabolism plays a central role in controlling the longevity gene products sirtuin 1 (SIRT1), adenosine monophosphate-activated protein kinase (AMPK) and forkhead box O3 (FOXO3). High nutrition conditions, such as the diabetic milieu, increase the ratio of reduced to oxidized forms of cytosolic nicotinamide adenine dinucleotide through cascades including the polyol pathway. This redox change is associated with insulin resistance and the development of diabetic complications, and might be counteracted by insulin C-peptide. My research and others' suggest that the SIRT1-liver kinase B1-AMPK cascade creates positive feedback through nicotinamide adenine dinucleotide synthesis to help cells cope with metabolic stress. SIRT1 and AMPK can upregulate liver kinase B1 and FOXO3, key factors that help residential stem cells cope with oxidative stress. FOXO3 directly changes epigenetics around transcription start sites, maintaining the health of stem cells. 'Diabetic memory' is likely a result of epigenetic changes caused by high nutritional conditions, which disturb the quiescent state of residential stem cells and impair tissue repair. This could be prevented by restoring SIRT1-AMPK positive feedback through activating FOXO3. PMID:27181414

  17. Diabetic complications within the context of aging: Nicotinamide adenine dinucleotide redox, insulin C-peptide, sirtuin 1-liver kinase B1-adenosine monophosphate-activated protein kinase positive feedback and forkhead box O3.

    PubMed

    Ido, Yasuo

    2016-07-01

    Recent research in nutritional control of aging suggests that cytosolic increases in the reduced form of nicotinamide adenine dinucleotide and decreasing nicotinamide adenine dinucleotide metabolism plays a central role in controlling the longevity gene products sirtuin 1 (SIRT1), adenosine monophosphate-activated protein kinase (AMPK) and forkhead box O3 (FOXO3). High nutrition conditions, such as the diabetic milieu, increase the ratio of reduced to oxidized forms of cytosolic nicotinamide adenine dinucleotide through cascades including the polyol pathway. This redox change is associated with insulin resistance and the development of diabetic complications, and might be counteracted by insulin C-peptide. My research and others' suggest that the SIRT1-liver kinase B1-AMPK cascade creates positive feedback through nicotinamide adenine dinucleotide synthesis to help cells cope with metabolic stress. SIRT1 and AMPK can upregulate liver kinase B1 and FOXO3, key factors that help residential stem cells cope with oxidative stress. FOXO3 directly changes epigenetics around transcription start sites, maintaining the health of stem cells. 'Diabetic memory' is likely a result of epigenetic changes caused by high nutritional conditions, which disturb the quiescent state of residential stem cells and impair tissue repair. This could be prevented by restoring SIRT1-AMPK positive feedback through activating FOXO3.

  18. Glycolytic potential and activity of adenosine monophosphate kinase (AMPK), glycogen phosphorylase (GP) and glycogen debranching enzyme (GDE) in steer carcasses with normal (<5.8) or high (>5.9) 24h pH determined in M. longissimus dorsi.

    PubMed

    Apaoblaza, A; Galaz, A; Strobel, P; Ramírez-Reveco, A; Jeréz-Timaure, N; Gallo, C

    2015-03-01

    Muscle glycogen concentration (MGC) and lactate (LA), activity of glycogen debranching enzyme (GDE), glycogen phosphorylase (GP) and adenosine monophosphate kinase (AMPK) were determined at 0.5h (T0) and 24h (T24) post-mortem in Longissimus dorsi samples from 38 steers that produced high pH (>5.9) and normal pH (<5.8) carcasses at 24h postmortem. MGC, LA and glycolytic potential were higher (P<0.05) in normal pH carcasses. GDE activity was similar (P>0.05) in both pH categories. GP activity increased between T0 and T24 only in normal pH carcasses. AMPK activity was four times higher in normal pH v/s high pH carcasses, without changing its activity over time. Results reinforce the idea that differences in postmortem glycogenolytic/glycolytic flow in L. dorsi of steers showing normal v/s high muscle pH at 24h, could be explained not only by the higher initial MGC in normal pH carcasses, but also by a high and sustained activity of AMPK and an increased GP activity at 24h postmortem.

  19. Cloning of the cDNA encoding adenosine 5'-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

    NASA Astrophysics Data System (ADS)

    Jiang, Keyong; Sun, Shujuan; Liu, Mei; Wang, Baojie; Meng, Xiaolin; Wang, Lei

    2013-01-01

    AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5'-UTR of 78 bp, a 3'-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) ( P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) ( P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals.

  20. Structural Studies of Potassium Transport Protein KtrA Regulator of Conductance of K+ (RCK) C Domain in Complex with Cyclic Diadenosine Monophosphate (c-di-AMP).

    PubMed

    Kim, Henna; Youn, Suk-Jun; Kim, Seong Ok; Ko, Junsang; Lee, Jie-Oh; Choi, Byong-Seok

    2015-06-26

    Although it was only recently identified as a second messenger, c-di-AMP was found to have fundamental importance in numerous bacterial functions such as ion transport. The potassium transporter protein, KtrA, was identified as a c-di-AMP receptor. However, the co-crystallization of c-di-AMP with the protein has not been studied. Here, we determined the crystal structure of the KtrA RCK_C domain in complex with c-di-AMP. The c-di-AMP nucleotide, which adopts a U-shaped conformation, is bound at the dimer interface of RCK_C close to helices α3 and α4. c-di-AMP interacts with KtrA RCK_C mainly by forming hydrogen bonds and hydrophobic interactions. c-di-AMP binding induces the contraction of the dimer, bringing the two monomers of KtrA RCK_C into close proximity. The KtrA RCK_C was able to interact with only c-di-AMP, but not with c-di-GMP, 3',3-cGAMP, ATP, and ADP. The structure of the KtrA RCK_C domain and c-di-AMP complex would expand our understanding about the mechanism of inactivation in Ktr transporters governed by c-di-AMP.

  1. A tyrosine-phosphorylated 55-kilodalton motility-associated bovine sperm protein is regulated by cyclic adenosine 3',5'-monophosphates and calcium.

    PubMed

    Vijayaraghavan, S; Trautman, K D; Goueli, S A; Carr, D W

    1997-06-01

    Sperm motility is regulated by protein phosphorylation. We have recently shown that a serine/threonine phosphatase system is involved in motility regulation. Two of the components of the phosphatase system, GSK-3 and PP1gamma2, are regulated by tyrosine phosphorylation. During our investigation of sperm tyrosine-phosphorylated proteins we discovered a 55-kDa protein whose tyrosine phosphorylation correlates closely to the motility state of sperm. This protein is tyrosine phosphorylated to a much higher degree in motile caudal than in immotile caput epididymal sperm. Motility inhibition of caudal epididymal sperm by protein kinase A (PKA) anchoring inhibition or by ionomycin-induced calcium overload led to the virtual disappearance of tyrosine phosphorylation of the 55-kDa protein. Conversely, treatment of sperm with motility activators, isobutylmethylxanthine or 8-bromo-cAMP, resulted in increased tyrosine phosphorylation of the protein. The protein was present in the soluble 100 000 x g supernatants of sperm extracts and was heat labile. Chromatography through diethylaminoethyl-cellulose and Western blot analysis showed that this 55-kDa protein is not a regulatory subunit of PKA or alpha-tubulin. Our results represent the identification of a soluble protein whose tyrosine phosphorylation varies directly with motility and suggest that motility regulation may involve cross talk between PKA, calcium, and tyrosine kinase pathways. PMID:9166697

  2. Killer Cell Lectin-like Receptor G1 Inhibits NK Cell Function through Activation of Adenosine 5'-Monophosphate-Activated Protein Kinase.

    PubMed

    Müller-Durovic, Bojana; Lanna, Alessio; Polaco Covre, Luciana; Mills, Rachel S; Henson, Sian M; Akbar, Arne N

    2016-10-01

    NK cells are the first line of defense against infected and transformed cells. Defective NK cell activity was shown to increase susceptibility for viral infections and reduce tumor immune-surveillance. With age, the incidence of infectious diseases and malignancy rises dramatically, suggesting that impaired NK cell function might contribute to disease in these individuals. We found an increased frequency of NK cells with high expression of the inhibitory killer cell lectin-like receptor G1 (KLRG1) in individuals >70 y. The role of KLRG1 in ageing is not known, and the mechanism of KLRG1-induced inhibition of NK cell function is not fully understood. We report that NK cells with high KLRG1 expression spontaneously activate the metabolic sensor AMP-activated protein kinase (AMPK) and that activation of AMPK negatively regulates NK cell function. Pre-existing AMPK activity is further amplified by ligation of KLRG1 in these cells, which leads to internalization of the receptor and allows interaction with AMPK. We show that KLRG1 activates AMPK by preventing its inhibitory dephosphorylation by protein phosphatase-2C rather than inducing de novo kinase activation. Finally, inhibition of KLRG1 or AMPK prevented KLRG1-induced activation of AMPK and reductions in NK cell cytotoxicity, cytokine secretion, proliferation, and telomerase expression. This novel signaling pathway links metabolic sensing, effector function, and cell differentiation with inhibitory receptor signaling that may be exploited to enhance NK cell activity during ageing.

  3. The cAMP Pathway as Therapeutic Target in Autoimmune and Inflammatory Diseases

    PubMed Central

    Raker, Verena Katharina; Becker, Christian; Steinbrink, Kerstin

    2016-01-01

    Nucleotide signaling molecules contribute to the regulation of cellular pathways. In the immune system, cyclic adenosine monophosphate (cAMP) is well established as a potent regulator of innate and adaptive immune cell functions. Therapeutic strategies to interrupt or enhance cAMP generation or effects have immunoregulatory potential in autoimmune and inflammatory disorders. Here, we provide an overview of the cyclic AMP axis and its role as a regulator of immune functions and discuss the clinical and translational relevance of interventions with these processes. PMID:27065076

  4. Intermedin1-53 attenuates vascular smooth muscle cell calcification by inhibiting endoplasmic reticulum stress via cyclic adenosine monophosphate/protein kinase A pathway.

    PubMed

    Chang, Jin-Rui; Duan, Xiao-Hui; Zhang, Bao-Hong; Teng, Xu; Zhou, Ye-Bo; Liu, Yue; Yu, Yan-Rong; Zhu, Yi; Tang, Chao-Shu; Qi, Yong-Fen

    2013-10-01

    We previously reported that endoplasmic reticulum (ER) stress-mediated apoptosis participated in vascular calcification. Importantly, a novel paracrine/autocrine peptide intermedin1-53 (IMD1-53) in the vasculature inhibited vascular calcification in rats. But the mechanisms needed to be fully elucidated. Vascular smooth muscle cells (VSMCs) calcification was induced by CaCl2 and β-glycerophosphate. Tunicamycin (Tm) or dithiothreitol (DTT) was used to induce ER stress. We found that IMD1-53 (10(-7)mol/L) treatment significantly alleviated the protein expression of ER stress hallmarks activating transcription factor 4 (ATF4), ATF6, glucose-regulated protein 78 (GRP78) and GRP94 induced by Tm or DTT. ER stress occurred in early and late calcification of VSMCs but was inhibited by IMD1-53. These inhibitory effects of IMD1-53 were abolished by treatment with the protein kinase A (PKA) inhibitor H89. Pretreatment with IMD1-53 decreased the number of apoptotic VSMCs and downregulated protein expression of cleaved caspase 12 and C/EBP homologous protein (CHOP) in calcified VSMCs. Concurrently, IMD1-53 restored the loss of VSMC lineage markers and ameliorated calcium deposition and alkaline phosphatase activity in calcified VSMCs as well. The observation was further verified by Alizarin Red S staining, which showed that IMD1-53 reduced positive red nodules among calcified VSMCs. In conclusion, IMD1-53 attenuated VSMC calcification by inhibiting ER stress through cAMP/PKA signalling.

  5. 3,3'-Diindolylmethane attenuates cardiac H9c2 cell hypertrophy through 5'-adenosine monophosphate-activated protein kinase-α.

    PubMed

    Zong, Jing; Wu, Qing-Qing; Zhou, Heng; Zhang, Jie-Yu; Yuan, Yuan; Bian, Zhou-Yan; Deng, Wei; Dai, Jia; Li, Fang-Fang; Xu, Man; Fang, Yi; Tang, Qi-Zhu

    2015-07-01

    3,3'-Diindolylmethane (DIM) is the major product of the acid-catalyzed condensation of indole-3-carbinol (I3C), a component of extracts of Brassica food plants. Numerous studies have suggested that DIM has several beneficial biological activities, including elimination of free radicals, antioxidant and anti-angiogenic effects and activation of apoptosis of various tumor cells. In the present study, an in vitro model was established, using 1 µM angiotensin II (Ang II) in cultured rat cardiac H9c2 cells, to observe the effects of DIM on cardiac hypertrophy. Following 24 h stimulation with DIM (1, 5, and 10 µM) with or without Ang II, cells were characterized by immunofluorescence to analyze cardiac α-actinin expression. Cardiomyocyte hypertrophy and molecular markers of cardiac hypertrophy were assessed by quantitative polymerase chain reaction. Atrial natriuretic peptide, brain natriuretic peptide and myosin heavy chain β mRNA expression were induced by Ang II in H9c2 cells treated with the optimal concentration of DIM for 6, 12, and 24 h. The levels of phosphorylated and total proteins of the 5' AMP-activated protein kinase α (AMPKα)/mitogen-activated protein kinase (MAPK)/mechanistic target of rapamycin (mTOR) signaling pathways in H9c2 cells treated with DIM for 0, 15, 30, and 60 min induced by Ang II were determined by western blot analysis. The results showed that DIM attenuated cellular hypertrophy in vitro, enhanced the phosphorylation of AMPKα and inhibited the MAPK‑mTOR signaling pathway in response to hypertrophic stimuli. PMID:25816057

  6. Direct biochemical measurements of microtubule assembly and disassembly in Chinese hamster ovary cells. The effect of intercellular contact, cold, D2O, and N6,O2'-dibutyryl cyclic adenosine monophosphate

    PubMed Central

    1975-01-01

    A study was undertaken to develop a means of quantitating the amount of tubulin present as a soluble pool and as intact microtubules in cultured Chinese hamster ovary cells. A procedure was developed in which these cells grown on monolayer culture in Petri dishes were placed in a "microtubule stabilizing medium" (MTM) consisting of 50% glycerol, 10% dimethylsulfoxide and sodium phosphate magnesium buffer, as described previously by Filner and Behnke. These cells then were homogenized and the homogenate was spun in the ultracentrifuge. Colchicine binding activity was then determined in the supernates and the pellets. The values, when compared with total colchicine binding activity present in replicate homogenates, were used to determine the percentage of tubulin present as intact microtubules. A statistical analysis of thin sections of cells treated with MTM revealed no statistically significant difference between MTM-treated cells and untreated controls. It was further discovered that the relative amount of colchicine binding activity recovered in the high speed pellet varied dramatically, depending upon the cell number of the culture being studied. Preconfluent cultures showed very low colchicine binding activity averaging less than 5%, while confluent and postconfluent cultures often possessed as high as 25% of their total colchicine binding activity in pelletable material. Although cold and D2O treatment had little or no effect on these values, N6,O2'-dibutyryl cyclic adenosine monophosphate increased them. It is hoped that this study will serve as the basis for a reliable quantitative procedure for measuring microtubule polymerization and depolymerization in vivo. PMID:162792

  7. Berberine treatment prevents cardiac dysfunction and remodeling through activation of 5'-adenosine monophosphate-activated protein kinase in type 2 diabetic rats and in palmitate-induced hypertrophic H9c2 cells.

    PubMed

    Chang, Wenguang; Zhang, Ming; Meng, Zhaojie; Yu, Yang; Yao, Fan; Hatch, Grant M; Chen, Li

    2015-12-15

    Diabetic cardiomyopathy is the major cause of death in type 2 diabetic patients. Berberine is an isoquinoline alkaloid extract from traditional chinese herbs and its hypoglycemic and hypolipidemic effects make it a promising drug for treatment of type 2 diabetes. We examined if berberine improved cardiac function and attenuated cardiac hypertrophy and fibrosis in high fat diet and streptozotocin induced-type 2 diabetic rats in vivo and reduced expression of hypertrophy markers in palmitate-induced hypertrophic H9c2 cells in vitro. Treatment of diabetic animals with berberine partially improved cardiac function and restored fasting blood insulin, fasting blood glucose, total cholesterol, and triglyceride levels to that of control. In addition, berberine treatment of diabetic animals increased cardiac 5'-adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (AKT) activation and reduced glycogen synthase kinase 3 beta (GSK3β) activation compared to control. Palmitate incubation of H9c2 cells resulted in cellular hypertrophy and decreased expression of alpha-myosin heavy chain (α-MHC) and increased expression of beta-myosin heavy chain (β-MHC) compared to controls. Berberine treatment of palmitate-incubated H9c2 cells reduced hypertrophy, increased α-MHC expression and decreased β-MHC expression. In addition, berberine treatment of palmitate-incubated H9c2 cells increased AMPK and AKT activation and reduced GSK3β activation. The presence of the AMPK inhibitor Compound C attenuated the effects of berberine. The results strongly indicate that berberine treatment may be protective against the development of diabetic cardiomyopathy. PMID:26522928

  8. Berberine treatment prevents cardiac dysfunction and remodeling through activation of 5'-adenosine monophosphate-activated protein kinase in type 2 diabetic rats and in palmitate-induced hypertrophic H9c2 cells.

    PubMed

    Chang, Wenguang; Zhang, Ming; Meng, Zhaojie; Yu, Yang; Yao, Fan; Hatch, Grant M; Chen, Li

    2015-12-15

    Diabetic cardiomyopathy is the major cause of death in type 2 diabetic patients. Berberine is an isoquinoline alkaloid extract from traditional chinese herbs and its hypoglycemic and hypolipidemic effects make it a promising drug for treatment of type 2 diabetes. We examined if berberine improved cardiac function and attenuated cardiac hypertrophy and fibrosis in high fat diet and streptozotocin induced-type 2 diabetic rats in vivo and reduced expression of hypertrophy markers in palmitate-induced hypertrophic H9c2 cells in vitro. Treatment of diabetic animals with berberine partially improved cardiac function and restored fasting blood insulin, fasting blood glucose, total cholesterol, and triglyceride levels to that of control. In addition, berberine treatment of diabetic animals increased cardiac 5'-adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (AKT) activation and reduced glycogen synthase kinase 3 beta (GSK3β) activation compared to control. Palmitate incubation of H9c2 cells resulted in cellular hypertrophy and decreased expression of alpha-myosin heavy chain (α-MHC) and increased expression of beta-myosin heavy chain (β-MHC) compared to controls. Berberine treatment of palmitate-incubated H9c2 cells reduced hypertrophy, increased α-MHC expression and decreased β-MHC expression. In addition, berberine treatment of palmitate-incubated H9c2 cells increased AMPK and AKT activation and reduced GSK3β activation. The presence of the AMPK inhibitor Compound C attenuated the effects of berberine. The results strongly indicate that berberine treatment may be protective against the development of diabetic cardiomyopathy.

  9. Early glycogen synthase kinase-3β and protein phosphatase 2A independent tau dephosphorylation during global brain ischaemia and reperfusion following cardiac arrest and the role of the adenosine monophosphate kinase pathway.

    PubMed

    Majd, Shohreh; Power, John H T; Koblar, Simon A; Grantham, Hugh J M

    2016-08-01

    Abnormal tau phosphorylation (p-tau) has been shown after hypoxic damage to the brain associated with traumatic brain injury and stroke. As the level of p-tau is controlled by Glycogen Synthase Kinase (GSK)-3β, Protein Phosphatase 2A (PP2A) and Adenosine Monophosphate Kinase (AMPK), different activity levels of these enzymes could be involved in tau phosphorylation following ischaemia. This study assessed the effects of global brain ischaemia/reperfusion on the immediate status of p-tau in a rat model of cardiac arrest (CA) followed by cardiopulmonary resuscitation (CPR). We reported an early dephosphorylation of tau at its AMPK sensitive residues, Ser(396) and Ser(262) after 2 min of ischaemia, which did not recover during the first two hours of reperfusion, while the tau phosphorylation at GSK-3β sensitive but AMPK insensitive residues, Ser(202) /Thr(205) (AT8), as well as the total amount of tau remained unchanged. Our data showed no alteration in the activities of GSK-3β and PP2A during similar episodes of ischaemia of up to 8 min and reperfusion of up to 2 h, and 4 weeks recovery. Dephosphorylation of AMPK followed the same pattern as tau dephosphorylation during ischaemia/reperfusion. Catalase, another AMPK downstream substrate also showed a similar pattern of decline to p-AMPK, in ischaemic/reperfusion groups. This suggests the involvement of AMPK in changing the p-tau levels, indicating that tau dephosphorylation following ischaemia is not dependent on GSK-3β or PP2A activity, but is associated with AMPK dephosphorylation. We propose that a reduction in AMPK activity is a possible early mechanism responsible for tau dephosphorylation.

  10. Luciferase-based assay for adenosine: application to S-adenosyl-L-homocysteine hydrolase.

    PubMed

    Burgos, Emmanuel S; Gulab, Shivali A; Cassera, María B; Schramm, Vern L

    2012-04-17

    S-Adenosyl-L-homocysteine hydrolase (SAHH) catalyzes the reversible conversion of S-adenosyl-L-homocysteine (SAH) to adenosine (ADO) and L-homocysteine, promoting methyltransferase activity by relief of SAH inhibition. SAH catabolism is linked to S-adenosylmethionine metabolism, and the development of SAHH inhibitors is of interest for new therapeutics with anticancer or cholesterol-lowering effects. We have developed a continuous enzymatic assay for adenosine that facilitates high-throughput analysis of SAHH. This luciferase-based assay is 4000-fold more sensitive than former detection methods and is well suited for continuous monitoring of ADO formation in a 96-well-plate format. The high-affinity adenosine kinase from Anopheles gambiae efficiently converts adenosine to adenosine monophosphate (AMP) in the presence of guanosine triphosphate. AMP is converted to adenosine triphosphate and coupled to firefly luciferase. With this procedure, kinetic parameters (K(m), k(cat)) for SAHH were obtained, in good agreement with literature values. Assay characteristics include sustained light output combined with ultrasensitive detection (10(-7) unit of SAHH). The assay is documented with the characterization of slow-onset inhibition for inhibitors of the hydrolase. Application of this assay may facilitate the development of SAHH inhibitors and provide an ultrasensitive detection for the formation of adenosine from other biological reactions.

  11. Discovery and Preclinical Characterization of 6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic Acid (PF-06409577), a Direct Activator of Adenosine Monophosphate-activated Protein Kinase (AMPK), for the Potential Treatment of Diabetic Nephropathy.

    PubMed

    Cameron, Kimberly O; Kung, Daniel W; Kalgutkar, Amit S; Kurumbail, Ravi G; Miller, Russell; Salatto, Christopher T; Ward, Jessica; Withka, Jane M; Bhattacharya, Samit K; Boehm, Markus; Borzilleri, Kris A; Brown, Janice A; Calabrese, Matthew; Caspers, Nicole L; Cokorinos, Emily; Conn, Edward L; Dowling, Matthew S; Edmonds, David J; Eng, Heather; Fernando, Dilinie P; Frisbie, Richard; Hepworth, David; Landro, James; Mao, Yuxia; Rajamohan, Francis; Reyes, Allan R; Rose, Colin R; Ryder, Tim; Shavnya, Andre; Smith, Aaron C; Tu, Meihua; Wolford, Angela C; Xiao, Jun

    2016-09-01

    Adenosine monophosphate-activated protein kinase (AMPK) is a protein kinase involved in maintaining energy homeostasis within cells. On the basis of human genetic association data, AMPK activators were pursued for the treatment of diabetic nephropathy. Identification of an indazole amide high throughput screening (HTS) hit followed by truncation to its minimal pharmacophore provided an indazole acid lead compound. Optimization of the core and aryl appendage improved oral absorption and culminated in the identification of indole acid, PF-06409577 (7). Compound 7 was advanced to first-in-human trials for the treatment of diabetic nephropathy.

  12. Overexpression of human selenoprotein H in neuronal cells enhances mitochondrial biogenesis and function through activation of protein kinase A, protein kinase B, and cyclic adenosine monophosphate response element-binding protein pathway.

    PubMed

    Mehta, Suresh L; Mendelev, Natalia; Kumari, Santosh; Andy Li, P

    2013-03-01

    Mitochondrial biogenesis is activated by nuclear encoded transcription co-activator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which is regulated by several upstream factors including protein kinase A and Akt/protein kinase B. We have previously shown that selenoprotein H enhances the levels of nuclear regulators for mitochondrial biogenesis, increases mitochondrial mass and improves mitochondrial respiratory rate, under physiological condition. Furthermore, overexpression of selenoprotein H protects neuronal HT22 cells from ultraviolet B irradiation-induced cell damage by lowering reactive oxygen species production, and inhibiting activation of caspase-3 and -9, as well as p53. The objective of this study is to identify the cell signaling pathways by which selenoprotein H initiates mitochondrial biogenesis. We first confirmed our previous observation that selenoprotein H transfected HT22 cells increased the protein levels of nuclear-encoded mitochondrial biogenesis factors, peroxisome proliferator-activated receptor γ coactivator-1α, nuclear respiratory factor 1 and mitochondrial transcription factor A. We then observed that total and phosphorylation of protein kinase A, Akt/protein kinase B and cyclic adenosine monophosphate response element-binding protein (CREB) were significantly increased in selenoprotein H transfected cells compared to vector transfected HT22 cells. To verify whether the observed stimulating effects on mitochondrial biogenesis pathways are caused by selenoprotein H and mediated through CREB, we knocked down selenoprotein H mRNA level using siRNA and inhibited CREB with napthol AS-E phosphate in selenoprotein H transfected cells and repeated the measurements of the aforementioned biomarkers. Our results revealed that silencing of selenoprotein H not only decreased the protein levels of PGC-1α, nuclear respiratory factor 1 and mitochondrial transcription factor A, but also decreased the total and

  13. Cyclic AMP efflux inhibitors as potential therapeutic agents for leukemia

    PubMed Central

    Perez, Dominique R.; Smagley, Yelena; Garcia, Matthew; Carter, Mark B.; Evangelisti, Annette; Matlawska-Wasowska, Ksenia; Winter, Stuart S.; Sklar, Larry A.; Chigaev, Alexandre

    2016-01-01

    Apoptotic evasion is a hallmark of cancer. We propose that some cancers may evade cell death by regulating 3′-5′-cyclic adenosine monophosphate (cAMP), which is associated with pro-apoptotic signaling. We hypothesize that leukemic cells possess mechanisms that efflux cAMP from the cytoplasm, thus protecting them from apoptosis. Accordingly, cAMP efflux inhibition should result in: cAMP accumulation, activation of cAMP-dependent downstream signaling, viability loss, and apoptosis. We developed a novel assay to assess cAMP efflux and performed screens to identify inhibitors. In an acute myeloid leukemia (AML) model, several identified compounds reduced cAMP efflux, appropriately modulated pathways that are responsive to cAMP elevation (cAMP-responsive element-binding protein phosphorylation, and deactivation of Very Late Antigen-4 integrin), and induced mitochondrial depolarization and caspase activation. Blocking adenylyl cyclase activity was sufficient to reduce effects of the most potent compounds. These compounds also decreased cAMP efflux and viability of B-lineage acute lymphoblastic leukemia (B-ALL) cell lines and primary patient samples, but not of normal primary peripheral blood mononuclear cells. Our data suggest that cAMP efflux is a functional feature that could be therapeutically targeted in leukemia. Furthermore, because some of the identified drugs are currently used for treating other illnesses, this work creates an opportunity for repurposing. PMID:27129155

  14. New insight into the binding modes of TNP-AMP to human liver fructose-1,6-bisphosphatase

    NASA Astrophysics Data System (ADS)

    Han, Xinya; Huang, Yunyuan; Zhang, Rui; Xiao, San; Zhu, Shuaihuan; Qin, Nian; Hong, Zongqin; Wei, Lin; Feng, Jiangtao; Ren, Yanliang; Feng, Lingling; Wan, Jian

    2016-08-01

    Human liver fructose-1,6-bisphosphatase (FBPase) contains two binding sites, a substrate fructose-1,6-bisphosphate (FBP) active site and an adenosine monophosphate (AMP) allosteric site. The FBP active site works by stabilizing the FBPase, and the allosteric site impairs the activity of FBPase through its binding of a nonsubstrate molecule. The fluorescent AMP analogue, 2‧,3‧-O-(2,4,6-trinitrophenyl)adenosine 5‧-monophosphate (TNP-AMP) has been used as a fluorescent probe as it is able to competitively inhibit AMP binding to the AMP allosteric site and, therefore, could be used for exploring the binding modes of inhibitors targeted on the allosteric site. In this study, we have re-examined the binding modes of TNP-AMP to FBPase. However, our present enzyme kinetic assays show that AMP and FBP both can reduce the fluorescence from the bound TNP-AMP through competition for FBPase, suggesting that TNP-AMP binds not only to the AMP allosteric site but also to the FBP active site. Mutagenesis assays of K274L (located in the FBP active site) show that the residue K274 is very important for TNP-AMP to bind to the active site of FBPase. The results further prove that TNP-AMP is able to bind individually to the both sites. Our present study provides a new insight into the binding mechanism of TNP-AMP to the FBPase. The TNP-AMP fluorescent probe can be used to exam the binding site of an inhibitor (the active site or the allosteric site) using FBPase saturated by AMP and FBP, respectively, or the K247L mutant FBPase.

  15. Targeting cAMP/PKA pathway for glycemic control and type 2 diabetes therapy.

    PubMed

    Yang, Haihua; Yang, Linghai

    2016-08-01

    In mammals, cyclic adenosine monophosphate (cAMP) is an intracellular second messenger that is usually elicited by binding of hormones and neurotransmitters to G protein-coupled receptors (GPCRs). cAMP exerts many of its physiological effects by activating cAMP-dependent protein kinase (PKA), which in turn phosphorylates and regulates the functions of downstream protein targets including ion channels, enzymes, and transcription factors. cAMP/PKA signaling pathway regulates glucose homeostasis at multiple levels including insulin and glucagon secretion, glucose uptake, glycogen synthesis and breakdown, gluconeogenesis, and neural control of glucose homeostasis. This review summarizes recent genetic and pharmacological studies concerning the regulation of glucose homeostasis by cAMP/PKA in pancreas, liver, skeletal muscle, adipose tissues, and brain. We also discuss the strategies for targeting cAMP/PKA pathway for research and potential therapeutic treatment of type 2 diabetes mellitus (T2D). PMID:27194812

  16. Ontogeny of catecholamine and adenosine receptor-mediated cAMP signaling of embryonic red blood cells: role of cGMP-inhibited phosphodiesterase 3 and hemoglobin.

    PubMed

    Baumann, R; Blass, C; Götz, R; Dragon, S

    1999-12-15

    We have previously shown that the cAMP signaling pathway controls major aspects of embryonic red blood cell (RBC) function in avian embryos (Glombitza et al, Am J Physiol 271:R973, 1996; and Dragon et al, Am J Physiol 271:R982, 1996) that are important for adaptation of the RBC gas transport properties to the progressive hypercapnia and hypoxia of later stages of avian embryonic development. Data about the ontogeny of receptor-mediated cAMP signaling are lacking. We have analyzed the response of primitive and definitive chick embryo RBC harvested from day 3 to 18 of development towards forskolin, beta-adrenergic, and A2 receptor agonists. The results show a strong response of immature definitive and primitive RBC to adenosine A2 and beta-adrenergic receptor agonists, which is drastically reduced in the last stage of development, coincident with the appearance of mature, transcriptionally inactive RBC. Modulation of cGMP-inhibited phosphodiesterase 3 (PDE3) has a controlling influence on cAMP accumulation in definitive RBC. Under physiological conditions, PDE3 is inhibited due to activation of soluble guanylyl cyclase (sGC). Inhibition of sGC with the specific inhibitor ODQ decreases receptor-mediated stimulation of cAMP production; this effect is reversed by the PDE3 inhibitor milrinone. sGC is acitivated by nitric oxide (NO), but we found no evidence for production of NO by erythrocyte NO-synthase. However, embryonic hemoglobin releases NO in an oxygen-linked manner that may activate guanylyl cyclase.

  17. Rotation around the glycosidic bond as driving force of proton transfer in protonated 2‧-deoxyriboadenosine monophosphate (dAMP)

    NASA Astrophysics Data System (ADS)

    Shishkin, Oleg V.; Dopieralski, Przemyslaw; Palamarchuk, Gennady V.; Latajka, Zdzislaw

    2010-04-01

    Theoretical investigation of the conformation of 2'-deoxyriboadenosine monophosphate protonated at the N7 atom and stabilized by a very strong C8-H⋯O-P hydrogen bond indicates that this hydrogen bond may be disrupted by rotation of the adenine moiety around the glycosidic bond. A B3LYP/aug-cc-pVDZ scan of the relaxed potential energy surface demonstrates that this rotation is a multi-stage process, accompanying proton transfer from the N7 atom of adenine to the oxygen atom of the phosphate group with a change of conformation of the nucleotide from south/anti to north/syn conformation. Car-Parrinello molecular dynamics simulation indicates that rotation around the glycosidic bond is the preferred way for relaxation of the molecular geometry of this conformer. Both processes, i.e. conformational transition and proton transfer, are strongly coupled. However, the conformer containing a strong C-H⋯O hydrogen bond also corresponds to a local minimum on the Gibbs free energy surface. A specific property of this hydrogen bond is the large variation of the H⋯O distance (ranging from 1.3 to 2.2 Å), which is not caused by proton movement between the carbon and oxygen atoms, but rather by relative motions of the adenine and phosphate moieties.

  18. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5'-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa).

    PubMed

    Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E; Gallo-Reynoso, Juan P

    2015-01-01

    Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5'-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5'-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5'-monophosphate (IMP), adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), ATP, guanosine 5'-diphosphate (GDP), guanosine 5'-triphosphate (GTP), and xanthosine 5'-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts.

  19. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5'-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa).

    PubMed

    Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E; Gallo-Reynoso, Juan P

    2015-01-01

    Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5'-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5'-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5'-monophosphate (IMP), adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), ATP, guanosine 5'-diphosphate (GDP), guanosine 5'-triphosphate (GTP), and xanthosine 5'-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts. PMID:26283971

  20. Purine metabolism in adenosine deaminase deficiency.

    PubMed Central

    Mills, G C; Schmalstieg, F C; Trimmer, K B; Goldman, A S; Goldblum, R M

    1976-01-01

    Purine and pyrimidine metabolites were measured in erythrocytes, plasma, and urine of a 5-month-old infant with adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) deficiency. Adenosine and adenine were measured using newly devised ion exchange separation techniques and a sensitive fluorescence assay. Plasma adenosine levels were increased, whereas adenosine was normal in erythrocytes and not detectable in urine. Increased amounts of adenine were found in erythrocytes and urine as well as in the plasma. Erythrocyte adenosine 5'-monophosphate and adenosine diphosphate concentrations were normal, but adenosine triphosphate content was greatly elevated. Because of the possibility of pyrimidine starvation, pyrimidine nucleotides (pyrimidine coenzymes) in erythrocytes and orotic acid in urine were measured. Pyrimidine nucleotide concentrations were normal, while orotic acid was not detected. These studies suggest that the immune deficiency associated with adenosine deaminase deficiency may be related to increased amounts of adenine, adenosine, or adenine nucleotides. PMID:1066699

  1. AMP-Conjugated Quantum Dots: Low Immunotoxicity Both In Vitro and In Vivo

    NASA Astrophysics Data System (ADS)

    Dai, Tongcheng; Li, Na; Liu, Lu; Liu, Qin; Zhang, Yuanxing

    2015-11-01

    Quantum dots (QDs) are engineered nanoparticles that possess special optical and electronic properties and have shown great promise for future biomedical applications. In this work, adenosine 5'-monophosphate (AMP), a small biocompatible molecular, was conjugated to organic QDs to produce hydrophilic AMP-QDs. Using macrophage J774A.1 as the cell model, AMP-QDs exhibited both prior imaging property and low toxicity, and more importantly, triggered limited innate immune responses in macrophage, indicating low immunotoxicity in vitro. Using BALB/c mice as the animal model, AMP-QDs were found to be detained in immune organs but did not evoke robust inflammation responses or obvious histopathological abnormalities, which reveals low immunotoxicity in vivo. This work suggests that AMP is an excellent surface ligand with low immunotoxicity, and potentially used in surface modification for more extensive nanoparticles.

  2. [Cyclic AMP level in the muscle tissue of cattle and the physico-chemical properties of meat].

    PubMed

    Górna, M; Wieckowski, W

    1982-01-01

    The purpose of this study was to determine quantitative changes of cyclic adenosine mono-phosphate (c-AMP) level in meat of slaughter bulls. Meat samples were taken from the muscle longissimus dorsi (LD) of bulls overstressed by transport, which were stunned with electricity without rest. The obtained data did not show statistical correlations between c-AMP level and pI-values but showed statistical correlations between c-AMP level and some sensory properties of beef. The highest concentration of c-AMP was detected in the muscle about 90 minutes after slaughter. Individual differences in the concentration of c-AMP in the LD were observed in 30 min. after slaughter. In this time the high c-AMP level was correlated with the colour and sensory properties of meat and bouillon.

  3. Neurochemical Measurement of Adenosine in Discrete Brain Regions of Five Strains of Inbred Mice

    PubMed Central

    Pani, Amar K.; Jiao, Yun; Sample, Kenneth J.; Smeyne, Richard J.

    2014-01-01

    Adenosine (ADO), a non-classical neurotransmitter and neuromodulator, and its metabolites adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP), have been shown to play an important role in a number of biochemical processes. Although their signaling is well described, it has been difficult to directly, accurately and simultaneously quantitate these purines in tissue or fluids. Here, we describe a novel method for measuring adenosine (ADO) and its metabolites using high performance liquid chromatography with electrochemical detection (HPLC-ECD). Using this chromatographic technique, we examined baseline levels of ADO and ATP, ADP and AMP in 6 different brain regions of the C57BL/6J mouse: stratum, cortex, hippocampus, olfactory bulb, substantia nigra and cerebellum and compared ADO levels in 5 different strains of mice (C57BL/6J, Swiss-Webster, FVB/NJ, 129P/J, and BALB/c). These studies demonstrate that baseline levels of purines vary significantly among the brain regions as well as between different mouse strains. These dissimilarities in purine concentrations may explain the variable phenotypes among background strains described in neurological disease models. PMID:24642754

  4. Selective activation of adenosine A2A receptors on immune cells by a CD73-dependent prodrug suppresses joint inflammation in experimental rheumatoid arthritis.

    PubMed

    Flögel, Ulrich; Burghoff, Sandra; van Lent, Peter L E M; Temme, Sebastian; Galbarz, Lisa; Ding, Zhaoping; El-Tayeb, Ali; Huels, Sandra; Bönner, Florian; Borg, Nadine; Jacoby, Christoph; Müller, Christa E; van den Berg, Wim B; Schrader, Jürgen

    2012-08-01

    Adenosine A(2A) receptor (A(2A)R) agonists are both highly effective anti-inflammatory agents and potent vasodilators. To separate these two activities, we have synthesized phosphorylated A(2A)R agonists (prodrugs) that require the presence of ecto-5'-nucleotidase (CD73) to become activated. In the model of collagen-induced arthritis, 2-(cyclohexylethylthio)adenosine 5'-monophosphate (chet-AMP), but not 2-(cyclohexylethylthio)adenosine (chet-adenosine), potently reduced inflammation as assessed by fluorine-19 ((19)F) magnetic resonance imaging and by histology. The prodrug effect was blunted by inhibition of CD73 and A(2A)R. The selectivity of drug action is due to profound up-regulation of CD73 and adenosine A(2A)R expression in neutrophils and inflammatory monocytes as found in recovered cells from the synovial fluid of arthritic mice. Plasma chet-adenosine was in the subnanomolar range when chet-AMP was applied, whereas concentrations required for vasodilation were about 100 times higher. Thus, chet-AMP is a potent immunosuppressant with negligible vasodilatory activity. These data suggest that phosphorylated A(2A)R agonists may serve as a promising new group of drugs for targeted immunotherapy of inflammation. PMID:22875828

  5. Selective activation of adenosine A2A receptors on immune cells by a CD73-dependent prodrug suppresses joint inflammation in experimental rheumatoid arthritis.

    PubMed

    Flögel, Ulrich; Burghoff, Sandra; van Lent, Peter L E M; Temme, Sebastian; Galbarz, Lisa; Ding, Zhaoping; El-Tayeb, Ali; Huels, Sandra; Bönner, Florian; Borg, Nadine; Jacoby, Christoph; Müller, Christa E; van den Berg, Wim B; Schrader, Jürgen

    2012-08-01

    Adenosine A(2A) receptor (A(2A)R) agonists are both highly effective anti-inflammatory agents and potent vasodilators. To separate these two activities, we have synthesized phosphorylated A(2A)R agonists (prodrugs) that require the presence of ecto-5'-nucleotidase (CD73) to become activated. In the model of collagen-induced arthritis, 2-(cyclohexylethylthio)adenosine 5'-monophosphate (chet-AMP), but not 2-(cyclohexylethylthio)adenosine (chet-adenosine), potently reduced inflammation as assessed by fluorine-19 ((19)F) magnetic resonance imaging and by histology. The prodrug effect was blunted by inhibition of CD73 and A(2A)R. The selectivity of drug action is due to profound up-regulation of CD73 and adenosine A(2A)R expression in neutrophils and inflammatory monocytes as found in recovered cells from the synovial fluid of arthritic mice. Plasma chet-adenosine was in the subnanomolar range when chet-AMP was applied, whereas concentrations required for vasodilation were about 100 times higher. Thus, chet-AMP is a potent immunosuppressant with negligible vasodilatory activity. These data suggest that phosphorylated A(2A)R agonists may serve as a promising new group of drugs for targeted immunotherapy of inflammation.

  6. Cyclic AMP system in muscle tissue during prolonged hypokinesia

    NASA Technical Reports Server (NTRS)

    Antipenko, Y. A.; Bubeyev, Y. A.; Korovkin, B. F.; Mikhaleva, N. P.

    1980-01-01

    Components of the cyclic Adenosine-cyclic-35-monophosphate (AMP) system in the muscle tissue of white rats were studied during 70-75 days of hypokinesia, created by placing the animals in small booths which restricted their movements, and during the readaptation period. In the initial period, cyclic AMP levels and the activities of phosphodiesterase and adenylate cyclase in muscle tissue were increased. The values for these indices were roughly equal for controls and experimental animals during the adaptation period, but on the 70th day of the experiment cAMP levels dropped, phosphodiesterase activity increased, and the stimulative effect of epinephrine on the activity of adenylate cyclase decreased. The indices under study normalized during the readaptation period.

  7. Studies of the cAMP mediated aggregation in Dictyostelium discoideum: receptor mediated activation of the adenylate cyclase

    SciTech Connect

    Theibert, W.E.A.B.

    1985-01-01

    Dictyostelium discoideum, a eukaryotic amoeba of the cellular slime mold family, provides an interesting paradigm in developmental biology. During development, hundreds of thousands of cells aggregate to form a multicellular aggregate. Aggregation is mediated by chemotaxis and chemical signaling. Waves of adenosine 3'-5' cyclic monophosphate (cAMP) propagate through the monolayer and provide transient gradients for chemotaxis. The author has used a reversible inhibitor of the cAMP signaling response to demonstrate that adaptation to cAMP is independent of the activation of the adenylate cyclase and therefore is not caused by the rise in intracellular cAMP. Next, it is shown that adenosine inhibits the cAMP signaling response. Inhibition is rapid, reversible, and depends on the cAMP stimulus concentration. Then the specificity of the cAMP receptors which mediates signaling is determined and compared with the receptors which mediate chemotaxis, the cGMP response, and cAMP binding antagonism. The cAMP surface receptor has been identified by photoaffinity labeling intact cells with (/sup 32/P)-8-N/sub 3/-cAMP using an ammonium sulfate binding stabilization technique. The photoactivated ligand specifically labels a polypeptide, localized to the membrane fraction, which migrates as a closely spaced doublet on SDS Page.

  8. Drugs elevating extracellular adenosine promote regeneration of haematopoietic progenitor cells in severely myelosuppressed mice: their comparison and joint effects with the granulocyte colony-stimulating factor.

    PubMed

    Hofer, Michal; Pospísil, Milan; Znojil, Vladimír; Vacek, Antonín; Weiterova, Lenka; Holá, Jirina; Vácha, Jirí

    2002-01-01

    We tested capabilities of drugs elevating extracellular adenosine and of granulocyte colony-stimulating factor (G-CSF) given alone or in combination to modulate regeneration from severe myelosuppression resulting from combined exposure of mice to ionizing radiation and carboplatin. Elevation of extracellular adenosine was induced by joint administration of dipyridamole (DP), a drug inhibiting the cellular uptake of adenosine, and adenosine monophosphate (AMP), serving as an adenosine prodrug. DP+AMP, G-CSF or all these drugs in combination were administered in a 4-d treatment regimen starting on day 3 after induction of myelosuppression. Comparable enhancements of haematopoietic regeneration due to elevation of extracellular adenosine or to action of G-CSF were demonstrated as shown by elevated numbers of haematopoietic progenitor cells for granulocytes/macrophages (GM-CFC) and erythrocytes (BFU-E) in the bone marrow and spleen in early time intervals after termination of the drug treatment, i.e. on days 7 and 10 after induction of myelosuppression. Coadministration of all the drugs further potentiated the restoration of progenitor cell pools in the haematopoietic organs. The effects of the drug treatments on progenitor cells were reflected in the peripheral blood in later time intervals of days 15 and 20 after induction of myelosuppression, especially as significantly elevated numbers of granulocytes and less pronounced elevation of lymphocytes and erythrocytes. The results substantiate the potential of drugs elevating extracellular adenosine for clinical utilization in myelosuppressive states, e.g. those accompanying oncological radio- and chemotherapy.

  9. Intracellular tortuosity underlies slow cAMP diffusion in adult ventricular myocytes

    PubMed Central

    Richards, Mark; Lomas, Oliver; Jalink, Kees; Ford, Kerrie L.; Vaughan-Jones, Richard D.; Lefkimmiatis, Konstantinos; Swietach, Pawel

    2016-01-01

    Aims 3′,5′-Cyclic adenosine monophosphate (cAMP) signals in the heart are often confined to concentration microdomains shaped by cAMP diffusion and enzymatic degradation. While the importance of phosphodiesterases (degradative enzymes) in sculpting cAMP microdomains is well established in cardiomyocytes, less is known about cAMP diffusivity (DcAMP) and factors affecting it. Many earlier studies have reported fast diffusivity, which argues against sharply defined microdomains. Methods and results [cAMP] dynamics in the cytoplasm of adult rat ventricular myocytes were imaged using a fourth generation genetically encoded FRET-based sensor. The [cAMP]-response to the addition and removal of isoproterenol (β-adrenoceptor agonist) quantified the rates of cAMP synthesis and degradation. To obtain a read out of DcAMP, a stable [cAMP] gradient was generated using a microfluidic device which delivered agonist to one half of the myocyte only. After accounting for phosphodiesterase activity, DcAMP was calculated to be 32 µm2/s; an order of magnitude lower than in water. Diffusivity was independent of the amount of cAMP produced. Saturating cAMP-binding sites with the analogue 6-Bnz-cAMP did not accelerate DcAMP, arguing against a role of buffering in restricting cAMP mobility. cAMP diffused at a comparable rate to chemically unrelated but similar sized molecules, arguing for a common physical cause of restricted diffusivity. Lower mitochondrial density and order in neonatal cardiac myocytes allowed for faster diffusion, demonstrating the importance of mitochondria as physical barriers to cAMP mobility. Conclusion In adult cardiac myocytes, tortuosity due to physical barriers, notably mitochondria, restricts cAMP diffusion to levels that are more compatible with microdomain signalling. PMID:27089919

  10. The Pseudomonas aeruginosa Chp Chemosensory System Regulates Intracellular cAMP Levels by Modulating Adenylate Cyclase Activity

    PubMed Central

    Fulcher, Nanette B.; Holliday, Phillip M.; Klem, Erich; Cann, Martin J.; Wolfgang, Matthew C.

    2010-01-01

    Summary Multiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signaling molecule adenosine 3’, 5’-cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis-like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP-dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP-dependent twitching motility is cAMP-independent. Overall, our data define a novel function for a chemotaxis-like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP-dependent virulence systems. PMID:20345659

  11. Presence of free cyclic AMP receptor protein and regulation of its level by cyclic AMP in neuroblastoma-glioma hybrid cells.

    PubMed Central

    Walter, U; Costa, M R; Breakefield, X O; Greengard, P

    1979-01-01

    Neuroblastoma-glioma hybrid cells of line 108CC-5 were found to contain high levels of soluble adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase activity and high levels of two specific cAMP receptor proteins, RI and RII. Treatment of the hybrid cells with dibutyryl cAMP increased the level of RI but did not significantly affect the level either of RII or of cAMP-dependent protein kinase activity. The effect of dibutyryl cAMP could be mimicked by prostaglandin E1 and 3-isobutyl-1-methylxanthine, both of which are known to raise cAMP levels in neuroblastoma-glioma hybrid cells. Both in control as well as in dibutyryl cAMP-treated cells, RII but not RI was associated with cAMP-dependent protein kinase. Several lines of evidence suggest that RI represents the free regulatory subunit of type I cAMP-dependent protein kinase. The presence of this regulatory subunit as free cAMP receptor protein in neuroblastoma-glioma hybrid cells may be of significance with respect to the regulation of growth and differentiation in tumor cells. Images PMID:226964

  12. Effects of different concentrations of metal ions on degradation of adenosine triphosphate in common carp (Cyprinus carpio) fillets stored at 4°C: An in vivo study.

    PubMed

    Li, Dapeng; Qin, Na; Zhang, Longteng; Lv, Jian; Li, Qingzheng; Luo, Yongkang

    2016-11-15

    The impact of different concentrations of Na(+), K(+), Ca(2+), Mg(2+), Fe(2+), and Zn(2+) on the degradation of adenosine triphosphate (ATP) and the influence of these ions on the activity of adenosine monophosphate deaminase (AMP-deaminase) and acid phosphatase (ACP) in common carp fillets (in vivo) during 4°C storage was examined. The content of ATP, inosine monophosphate (IMP), and hypoxanthine (Hx), and the activity of AMP-deaminase and ACP were determined. Results indicated that the effects of different concentrations of six kinds of metal ions on AMP-deaminase and ACP were not the same. Na(+), K(+), Fe(2+), and Zn(2+) enhanced AMP-deaminase activity, which led to the rapid degradation of ATP and to the generation of a large quantity of IMP within a short time. Ca(2+) and Mg(2+) delayed the change in AMP-deaminase and ACP activity in carp and caused a further delay in the degradation of ATP. Fe(2+) and Zn(2+) inhibited ACP activity, which reduced the decomposition of IMP and the formation of Hx. PMID:27283700

  13. A new traveling wave phenomenon of Dictyostelium in the presence of cAMP

    NASA Astrophysics Data System (ADS)

    Ševčíková, Hana; Čejková, Jitka; Krausová, Lenka; Přibyl, Michal; Štěpánek, František; Marek, Miloš

    2010-06-01

    The emergence of wave patterns in chemical and biological systems is of interest for the understanding of development, differentiation, signaling, and other phenomena. In this work we report a new type of wave pattern - called the “global wave” - which was observed in populations of Dictyostelium discoideum cells exposed to an excess of cyclic adenosine- 3‧, 5‧- monophosphate (cAMP) added to the supporting agar. It has been found that the addition of different amounts of cAMP to the agar leads to important deviations from the standard course of aggregation: (i) the formation and propagation of a global wave that has not been observed before; (ii) the delayed onset or absence of cAMP waves patterning; (iii) an atypical mechanism of cells clustering; and (iv) a faster or incomplete developmental cycle. We suggest that the global wave is a chemotactic response of the Dictyostelium cells to a wave of the cAMP concentration.

  14. Role of the cAMP Pathway in Glucose and Lipid Metabolism.

    PubMed

    Ravnskjaer, Kim; Madiraju, Anila; Montminy, Marc

    2016-01-01

    3'-5'-Cyclic adenosine monophosphate (cyclic AMP or cAMP) was first described in 1957 as an intracellular second messenger mediating the effects of glucagon and epinephrine on hepatic glycogenolysis (Berthet et al., J Biol Chem 224(1):463-475, 1957). Since this initial characterization, cAMP has been firmly established as a versatile molecular signal involved in both central and peripheral regulation of energy homeostasis and nutrient partitioning. Many of these effects appear to be mediated at the transcriptional level, in part through the activation of the transcription factor CREB and its coactivators. Here we review current understanding of the mechanisms by which the cAMP signaling pathway triggers metabolic programs in insulin-responsive tissues.

  15. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5′-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa)

    PubMed Central

    Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E.; Gallo-Reynoso, Juan P.

    2015-01-01

    Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5′-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5′-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5′-monophosphate (IMP), adenosine 5′-monophosphate (AMP), adenosine 5′-diphosphate (ADP), ATP, guanosine 5′-diphosphate (GDP), guanosine 5′-triphosphate (GTP), and xanthosine 5′-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts. PMID:26283971

  16. Uptake of 3H-cAMP by retinal pigment epithelium isolated from bluegill sunfish (Lepomis macrochirus)

    PubMed Central

    Keith, Thomas A; Radhakrishnan, Varsha; Moredock, Steve; García, Dana M

    2006-01-01

    Background In bluegill sunfish, the melanin-containing pigment granules of the retinal pigment epithelium undergo cyclic movements in response both to ambient lighting and circadian cues. Pigment granules aggregate into the cell body at night (in the dark), and disperse into apical processes during the day (in the light). Regulation of pigment granule aggregation in a number of fishes depends on modulating the intracellular levels of cyclic adenosine monophosphate. Results Here we show isolated RPE takes up cyclic adenosine monophosphate (cAMP) in a saturable manner, exogenously applied cAMP induces pigment granule aggregation in retinal pigment epithelium isolated from bluegill, and aggregation induced in this manner is inhibited by treatment with probenecid, an organic anion transport inhibitor. Conclusion Our results raise the possibility that cAMP functions as a messenger secreted from the neural retina to signal darkness to the RPE, which takes it up. It further suggests that organic anion transport systems are the route by which cAMP crosses RPE cell membranes since probenecid inhibits extracellular cAMP from causing pigment granule aggregation. PMID:17196104

  17. The Popeye Domain Containing Genes and cAMP Signaling

    PubMed Central

    Brand, Thomas; Poon, Kar Lai; Simrick, Subreena; Schindler, Roland F.R.

    2016-01-01

    3'-5'-cyclic adenosine monophosphate (cAMP) is a second messenger, which plays an important role in the heart. It is generated in response to activation of G-protein-coupled receptors (GPCRs). Initially, it was thought that protein kinase A (PKA) exclusively mediates cAMP-induced cellular responses such as an increase in cardiac contractility, relaxation, and heart rate. With the identification of the exchange factor directly activated by cAMP (EPAC) and hyperpolarizing cyclic nucleotide-gated (HCN) channels as cAMP effector proteins it became clear that a protein network is involved in cAMP signaling. The Popeye domain containing (Popdc) genes encode yet another family of cAMP-binding proteins, which are prominently expressed in the heart. Loss-of-function mutations in mice are associated with cardiac arrhythmia and impaired skeletal muscle regeneration. Interestingly, the cardiac phenotype, which is present in both, Popdc1 and Popdc2 null mutants, is characterized by a stress-induced sinus bradycardia, suggesting that Popdc proteins participate in cAMP signaling in the sinuatrial node. The identification of the two-pore channel TREK-1 and Caveolin 3 as Popdc-interacting proteins represents a first step into understanding the mechanisms of heart rate modulation triggered by Popdc proteins. PMID:27500161

  18. The combined inhibitory effect of the adenosine A1 and cannabinoid CB1 receptors on cAMP accumulation in the hippocampus is additive and independent of A1 receptor desensitization.

    PubMed

    Serpa, André; Correia, Sara; Ribeiro, Joaquim A; Sebastião, Ana M; Cascalheira, José F

    2015-01-01

    Adenosine A1 and cannabinoid CB1 receptors are highly expressed in hippocampus where they trigger similar transduction pathways. We investigated how the combined acute activation of A1 and CB1 receptors modulates cAMP accumulation in rat hippocampal slices. The CB1 agonist WIN55212-2 (0.3-30 μM) decreased forskolin-stimulated cAMP accumulation with an EC50 of 6.6±2.7 μM and an Emax of 31%±2%, whereas for the A1 agonist, N6-cyclopentyladenosine (CPA, 10-150 nM), an EC50 of 35±19 nM, and an Emax of 29%±5 were obtained. The combined inhibitory effect of WIN55212-2 (30 μM) and CPA (100 nM) on cAMP accumulation was 41%±6% (n=4), which did not differ (P>0.7) from the sum of the individual effects of each agonist (43%±8%) but was different (P<0.05) from the effects of CPA or WIN55212-2 alone. Preincubation with CPA (100 nM) for 95 min caused desensitization of adenosine A1 activity, which did not modify the effect of WIN55212-2 (30 μM) on cAMP accumulation. In conclusion, the combined effect of CB1 and A1 receptors on cAMP formation is additive and CB1 receptor activity is not affected by short-term A1 receptor desensitization.

  19. Schwann Cells Metabolize Extracellular 2',3'-cAMP to 2'-AMP.

    PubMed

    Verrier, Jonathan D; Kochanek, Patrick M; Jackson, Edwin K

    2015-08-01

    The 3',5'-cAMP-adenosine pathway (3',5'-cAMP→5'-AMP→adenosine) and the 2',3'-cAMP-adenosine pathway (2',3'-cAMP→2'-AMP/3'-AMP→adenosine) are active in the brain. Oligodendrocytes participate in the brain 2',3'-cAMP-adenosine pathway via their robust expression of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase; converts 2',3'-cAMP to 2'-AMP). Because Schwann cells also express CNPase, it is conceivable that the 2',3'-cAMP-adenosine pathway exists in the peripheral nervous system. To test this and to compare the 2',3'-cAMP-adenosine pathway to the 3',5'-cAMP-adenosine pathway in Schwann cells, we examined the metabolism of 2',3'-cAMP, 2'-AMP, 3'-AMP, 3',5'-cAMP, and 5'-AMP in primary rat Schwann cells in culture. Addition of 2',3'-cAMP (3, 10, and 30 µM) to Schwann cells increased levels of 2'-AMP in the medium from 0.006 ± 0.002 to 21 ± 2, 70 ± 3, and 187 ± 10 nM/µg protein, respectively; in contrast, Schwann cells had little ability to convert 2',3'-cAMP to 3'-AMP or 3',5'-cAMP to either 3'-AMP or 5'-AMP. Although Schwann cells slightly converted 2',3'-cAMP and 2'-AMP to adenosine, they did so at very modest rates (e.g., 5- and 3-fold, respectively, more slowly compared with our previously reported studies in oligodendrocytes). Using transected myelinated rat sciatic nerves in culture medium, we observed a time-related increase in endogenous intracellular 2',3'-cAMP and extracellular 2'-AMP. These findings indicate that Schwann cells do not have a robust 3',5'-cAMP-adenosine pathway but do have a 2',3'-cAMP-adenosine pathway; however, because the pathway mostly involves 2'-AMP formation rather than 3'-AMP, and because the conversion of 2'-AMP to adenosine is slow, metabolism of 2',3'-cAMP mostly results in the accumulation of 2'-AMP. Accumulation of 2'-AMP in peripheral nerves postinjury could have pathophysiological consequences. PMID:25998049

  20. Revisiting cAMP signaling in the carotid body

    PubMed Central

    Nunes, Ana R.; Holmes, Andrew P.; Conde, Sílvia V.; Gauda, Estelle B.; Monteiro, Emília C.

    2014-01-01

    Chronic carotid body (CB) activation is now recognized as being essential in the development of hypertension and promoting insulin resistance; thus, it is imperative to characterize the chemotransduction mechanisms of this organ in order to modulate its activity and improve patient outcomes. For several years, and although controversial, cyclic adenosine monophosphate (cAMP) was considered an important player in initiating the activation of the CB. However, its relevance was partially displaced in the 90s by the emerging role of the mitochondria and molecules such as AMP-activated protein kinase and O2-sensitive K+ channels. Neurotransmitters/neuromodulators binding to metabotropic receptors are essential to chemotransmission in the CB, and cAMP is central to this process. cAMP also contributes to raise intracellular Ca2+ levels, and is intimately related to the cellular energetic status (AMP/ATP ratio). Furthermore, cAMP signaling is a target of multiple current pharmacological agents used in clinical practice. This review (1) provides an outline on the classical view of the cAMP-signaling pathway in the CB that originally supported its role in the O2/CO2 sensing mechanism, (2) presents recent evidence on CB cAMP neuromodulation and (3) discusses how CB activity is affected by current clinical therapies that modify cAMP-signaling, namely dopaminergic drugs, caffeine (modulation of A2A/A2B receptors) and roflumilast (PDE4 inhibitors). cAMP is key to any process that involves metabotropic receptors and the intracellular pathways involved in CB disease states are likely to involve this classical second messenger. Research examining the potential modification of cAMP levels and/or interactions with molecules associated with CB hyperactivity is currently in its beginning and this review will open doors for future explorations. PMID:25389406

  1. A cAMP-Regulated Chloride Channel in Lymphocytes that is Affected in Cystic Fibrosis

    NASA Astrophysics Data System (ADS)

    Chen, Jennifer H.; Schulman, Howard; Gardner, Phyllis

    1989-02-01

    A defect in regulation of a chloride channel appears to be the molecular basis for cystic fibrosis (CF), a common lethal genetic disease. It is shown here that a chloride channel with kinetic and regulatory properties similar to those described for secretory epithelial cells is present in both T and B lymphocyte cell lines. The regulation of the channels by adenosine 3',5'-monophosphate (cAMP)--dependent protein kinase in transformed B cells from CF patients is defective. Thus, lymphocytes may be an accessible source of CF tissue for study of this defect, for cloning of the chloride channel complex, and for diagnosis of the disease.

  2. cAMP enhances BMP2-signaling through PKA and MKP1-dependent mechanisms

    SciTech Connect

    Ghayor, Chafik; Ehrbar, Martin; Miguel, Blanca San; Graetz, Klaus W.; Weber, Franz E.

    2009-04-03

    Recent studies suggest that the elevation of intracellular cyclic adenosine monophosphate (cAMP) and the activation of the protein kinase A regulate BMP-induced osteogenesis. However, the precise mechanisms underlying the enhancing effect of cAMP on BMP2 signaling were not completely revealed. In this study we investigated the effect of elevated cAMP level and PKA activation on the BMP2-induced osteoblastic differentiation in pluripotent C2C12 cells. Alkaline phosphatase activity and its mRNA were consistently induced by BMP2 treatment. The pretreatment of C2C12 cells with Forskolin, a cAMP generating agent, dbcAMP, an analogue of cAMP, or IBMX (3-isobutyl 1-methyl xanthine), and a nonspecific inhibitor of phosphodiesterases elicited further activation of alkaline phosphatase. Furthermore, elevated intracellular cAMP level increased BMP2-induced MKP1. On the other hand, BMP2-induced Erk phosphorylation (p44/p42) and cell proliferation were suppressed in the presence of cAMP. Thus, cAMP might enhance BMP2-induced osteoblastic differentiation by a MKP1-Erk-dependent mechanism.

  3. Bacterial Cyclic AMP-Phosphodiesterase Activity Coordinates Biofilm Formation

    PubMed Central

    Kalivoda, Eric J.; Brothers, Kimberly M.; Stella, Nicholas A.; Schmitt, Matthew J.; Shanks, Robert M. Q.

    2013-01-01

    Biofilm-related infections are a major contributor to human disease, and the capacity for surface attachment and biofilm formation are key attributes for the pathogenesis of microbes. Serratia marcescens type I fimbriae-dependent biofilms are coordinated by the adenylate cyclase, CyaA, and the cyclic 3′,5′-adenosine monophosphate (cAMP)-cAMP receptor protein (CRP) complex. This study uses S. marcescens as a model system to test the role of cAMP-phosphodiesterase activity in controlling biofilm formation. Herein we describe the characterization of a putative S. marcescens cAMP-phosphodiesterase gene (SMA3506), designated as cpdS, and demonstrated to be a functional cAMP-phosphodiesterase both in vitro and in vivo. Deletion of cpdS resulted in defective biofilm formation and reduced type I fimbriae production, whereas multicopy expression of cpdS conferred a type I fimbriae-dependent hyper-biofilm. Together, these results support a model in which bacterial cAMP-phosphodiesterase activity modulates biofilm formation. PMID:23923059

  4. Somatostatin-14 and somatostatin-28 pretreatment down-regulate somatostatin-14 receptors and have biphasic effects on forskolin-stimulated cyclic adenosine, 3',5'-monophosphate synthesis and adrenocorticotropin secretion in mouse anterior pituitary tumor cells.

    PubMed

    Heisler, S; Srikant, C B

    1985-07-01

    Activation of somatostatin-14 (S-14) receptors on mouse AtT-20 pituitary tumor cells by S-14 or somatostatin-28 (S-28) inhibits forskolin-stimulated cAMP synthesis and ACTH secretion. In this study, the effects of prolonged exposure of cells to S-14 or S-28 was found to reduce, in a time- and concentration-dependent fashion, the density of S-14 receptors without affecting the affinity of these sites for [125I]Tyr11-S-14. This response was rapidly reversible after removal of peptide from incubation media. Additionally, S-14 and S-28 pretreatment also resulted in a time-dependent sensitizing effect on forskolin-stimulated cAMP formation and ACTH secretion which preceded S-14 receptor down-regulation. Enhancement of the forskolin response was concentration dependent, with maximal effects observed at 10(-8) M with either peptide. Higher pretreatment concentrations of S-14 resulted in an abolition of the enhanced biological response to forskolin; pretreatment with S-28 (10(-6) M) depressed forskolin- and (-)isoproterenol-induced cAMP formation below levels observed in nonpretreated cells. The enhancing effect of S-14 and S-28 required new protein synthesis, since it was partially blocked by cycloheximide; the depressor effect was independent of new protein synthesis. Both the enhanced and depressed forskolin responses after peptide pretreatment were reversible after withdrawal of S-14 or S-28; normalization of the forskolin response (cAMP formation and ACTH secretion) followed the return to control levels of S-14 receptor density. Pretreatment of cells with 10(-8) M or 10(-6) M S-28 increased and decreased, respectively, the ACTH secretory response to agonists which act in the absence of prior cAMP synthesis such as 8-bromo-cAMP, A-23187, and phorbol ester. The data suggest that S-14 receptor down-regulation is not causally associated with the sensitizing effects of S-14 and S-28 on adenylate cyclase and that the S-14 receptor may be also coupled to other effector

  5. Involvement of the second messenger cAMP in gravity-signal transduction in physarum

    NASA Astrophysics Data System (ADS)

    Block, I.; Rabien, H.; Ivanova, K.

    The aim of the investigation was to clarify, whether cellular signal processing following graviperception involves second messenger pathways. The test object was a most gravisensitive free-living ameboid cell, the myxomycete (acellular slime mold) Physarum polycephalum. It was demonstrated that the motor response is related to acceleration-dependent changes in the levels of the cellular second messenger cyclic adenosine monophosphate (cAMP). Rotating Physarum plasmodia in the gravity field of the Earth about a horizontal axis increased their cAMP concentration. Depriving the cells for a few days of the acceleration stimulus (near weightlessness in a space experiment on STS-69) slightly lowered plasmodial cAMP levels. Thus, the results provide first indications that the acceleration-stimulus signal transduction chain of Physarum uses an ubiquitous second messenger pathway.

  6. The cyclic AMP signaling pathway: Exploring targets for successful drug discovery (Review)

    PubMed Central

    YAN, KUO; GAO, LI-NA; CUI, YUAN-LU; ZHANG, YI; ZHOU, XIN

    2016-01-01

    During development of disease, complex intracellular signaling pathways regulate an intricate series of events, including resistance to external toxins, the secretion of cytokines and the production of pathological phenomena. Adenosine 3′,5′-cyclic monophosphate (cAMP) is a nucleotide that acts as a key second messenger in numerous signal transduction pathways. cAMP regulates various cellular functions, including cell growth and differentiation, gene transcription and protein expression. This review aimed to provide an understanding of the effects of the cAMP signaling pathway and the associated factors on disease occurrence and development by examining the information from a new perspective. These novel insights aimed to promote the development of novel therapeutic approaches and aid in the development of new drugs. PMID:27035868

  7. Adenosine 5'-(gamma-thio) triphosphate (ATPgammaS) stimulates both P2Y receptors linked to inositol phosphates production and cAMP accumulation in bovine adrenocortical fasciculata cells.

    PubMed

    Nishi, Haruhisa; Hori, Seiji; Niitsu, Akiyoshi; Kawamura, Masahiro

    2004-01-16

    The study was aimed to investigate the existence of at least two kinds of P2Y receptors linked to steroidogenesis in bovine adrenocortical fasciculata cells (BAFCs). Extracellular nucleotides facilitated steroidogenesis in BAFCs. The potency order was UTP > adenosine 5'-(gamma-thio) triphosphate (ATPgammaS) > ATP > 2-methylthio ATP (2MeSATP) > adenosine 5'-(beta-thio) diphosphate (ADPbetaS) > alpha,beta-methylene ATP (alpha,beta-me-ATP), beta,gamma-methylene ATP (beta,gamma -me-ATP). ATPgammaS (10-100 microM) remarkably stimulated both total inositol phosphates (IPs) production and cyclic AMP (cAMP) accumulation. Competitive displacement experiments by using [35S]ATPgammaS as a radioactive ligand in BAFCs showed that the potency under these unlabelled ligands was ATPgammaS > ATP > ADPbetaS > 2MeSATP > UTP > alpha,beta-me-ATP, beta,gamma-me-ATP. These suggest that two different binding sites of [35S]ATPgammaS, namely P2Y receptors, exist in BAFCs, and that these receptors are linked to steroidogenesis via distinct second messenger systems in the cells.

  8. Cyclic adenosine 5'-diphosphoribose (cADPR) cyclic guanosine 3',5'-monophosphate positively function in Ca(2+) elevation in methyl jasmonate-induced stomatal closure, cADPR is required for methyl jasmonate-induced ROS accumulation NO production in guard cells.

    PubMed

    Hossain, M A; Ye, W; Munemasa, S; Nakamura, Y; Mori, I C; Murata, Y

    2014-11-01

    Methyl jasmonate (MeJA) signalling shares several signal components with abscisic acid (ABA) signalling in guard cells. Cyclic adenosine 5'-diphosphoribose (cADPR) and cyclic guanosine 3',5'-monophosphate (cGMP) are second messengers in ABA-induced stomatal closure. In order to clarify involvement of cADPR and cGMP in MeJA-induced stomatal closure in Arabidopsis thaliana (Col-0), we investigated effects of an inhibitor of cADPR synthesis, nicotinamide (NA), and an inhibitor of cGMP synthesis, LY83583 (LY, 6-anilino-5,8-quinolinedione), on MeJA-induced stomatal closure. Treatment with NA and LY inhibited MeJA-induced stomatal closure. NA inhibited MeJA-induced reactive oxygen species (ROS) accumulation and nitric oxide (NO) production in guard cells. NA and LY suppressed transient elevations elicited by MeJA in cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) in guard cells. These results suggest that cADPR and cGMP positively function in [Ca(2+)]cyt elevation in MeJA-induced stomatal closure, are signalling components shared with ABA-induced stomatal closure in Arabidopsis, and that cADPR is required for MeJA-induced ROS accumulation and NO production in Arabidopsis guard cells.

  9. Developmental and diurnal dynamics of Pax4 expression in the mammalian pineal gland: nocturnal down-regulation is mediated by adrenergic-cyclic adenosine 3',5'-monophosphate signaling.

    PubMed

    Rath, Martin F; Bailey, Michael J; Kim, Jong-So; Ho, Anthony K; Gaildrat, Pascaline; Coon, Steven L; Møller, Morten; Klein, David C

    2009-02-01

    Pax4 is a homeobox gene that is known to be involved in embryonic development of the endocrine pancreas. In this tissue, Pax4 counters the effects of the related protein, Pax6. Pax6 is essential for development of the pineal gland. In this study we report that Pax4 is strongly expressed in the pineal gland and retina of the rat. Pineal Pax4 transcripts are low in the fetus and increase postnatally; Pax6 exhibits an inverse pattern of expression, being more strongly expressed in the fetus. In the adult the abundance of Pax4 mRNA exhibits a diurnal rhythm in the pineal gland with maximal levels occurring late during the light period. Sympathetic denervation of the pineal gland by superior cervical ganglionectomy prevents the nocturnal decrease in pineal Pax4 mRNA. At night the pineal gland is adrenergically stimulated by release of norepinephrine from the sympathetic innervation; here, we found that treatment with adrenergic agonists suppresses pineal Pax4 expression in vivo and in vitro. This suppression appears to be mediated by cAMP, a second messenger of norepinephrine in the pineal gland, based on the observation that treatment with a cAMP mimic reduces pineal Pax4 mRNA levels. These findings suggest that the nocturnal decrease in pineal Pax4 mRNA is controlled by the sympathetic neural pathway that controls pineal function acting via an adrenergic-cAMP mechanism. The daily changes in Pax4 expression may influence gene expression in the pineal gland.

  10. Prostaglandin E2-induced up-regulation of c-fos messenger ribonucleic acid is primarily mediated by 3',5'-cyclic adenosine monophosphate in MC3T3-E1 osteoblasts

    NASA Technical Reports Server (NTRS)

    Fitzgerald, J.; Dietz, T. J.; Hughes-Fulford, M.

    2000-01-01

    The mechanism by which the proto-oncogene, c-fos, is up-regulated in response to PGE2 in the mouse osteoblastic (MC3T3-E1) cell line was investigated using RT-PCR. c-fos messenger RNA up-regulation by dmPGE2 is rapid, starting 10 min post stimulation, and transient. The specific protein kinase A (PKA) inhibitor, H89, inhibited c-fos induction. Moreover, down-regulation of protein kinase C (PKC) activity by chronic TPA treatment had no effect on the induction of c-fos by dmPGE2. We conclude that up-regulation of c-fos by dmPGE2 is primarily dependent on PKA in MC3T3-E1 osteoblasts. In S49 lymphoma wild-type but not S49 cyc- cells, which are deficient in cAMP signaling, dmPGE2 up-regulates c-fos and increases cell growth compared with unstimulated cells. Thus in S49 lymphoma cells, c-fos induction by PGE2 is also dependent on cAMP signaling. The minimal c-fos promoter region required for dmPGE2-induced expression was identified by transfecting c-fos promoter deletion constructs coupled to the chloramphenicol acetyltransferase (CAT) reporter gene into Vero cells. Transfection of a plasmid containing 99 bp c-fos proximal promoter was sufficient to direct c-fos/CAT expression following stimulation with dmPGE2. Because induction of c-fos is mediated by cAMP, these data are consistent with activation of c-fos via the CRE/ATF cis element.

  11. Targeting brain tumor cAMP: the case for sex-specific therapeutics

    PubMed Central

    Warrington, Nicole M.; Sun, Tao; Rubin, Joshua B.

    2015-01-01

    A relationship between cyclic adenosine 3′, 5′-monophosphate (cAMP) levels and brain tumor biology has been evident for nearly as long as cAMP and its synthetase, adenylate cyclase (ADCY) have been known. The importance of the pathway in brain tumorigenesis has been demonstrated in vitro and in multiple animal models. Recently, we provided human validation for a cooperating oncogenic role for cAMP in brain tumorigenesis when we found that SNPs in ADCY8 were correlated with glioma (brain tumor) risk in individuals with Neurofibromatosis type 1 (NF1). Together, these studies provide a strong rationale for targeting cAMP in brain tumor therapy. However, the cAMP pathway is well-known to be sexually dimorphic, and SNPs in ADCY8 affected glioma risk in a sex-specific fashion, elevating the risk for females while protecting males. The cAMP pathway can be targeted at multiple levels in the regulation of its synthesis and degradation. Sex differences in response to drugs that target cAMP regulators indicate that successful targeting of the cAMP pathway for brain tumor patients is likely to require matching specific mechanisms of drug action with patient sex. PMID:26283963

  12. Changes in the cyclic AMP content during growth and development of Acetabularia.

    PubMed

    Minder, C; Vanden Driessche, T

    1978-05-26

    The 3',5'-adenosine monophosphate (cyclic-AMP) content of the unicellular alga Acetabularia has been examined at various developmental stages. It has been found that very young algae, less than 10mm in length, have a high cAMP content [more than 7 pmoles per 100 mg wet weight (WW)], but that with the growth of the algae, the cAMP content decreases rapidly, reaching the low level of 0.5--1.0 pmoles per 100mg WW. The cAMP content remains at this level until cap differentiation, after which an increase in cAMP content accompanies cap enlargement. It has been shown that these results are unlikely to be affected by changes in the cAMP content induced by variations in circadian rhythm. Treatment with theophylline (2.10(-3) M), a phosphodieterase inhibitor, results in an increase in the cAMP content and delays growth and cap formation. Experiments on the effects of theophylline upon the circadian rhythm of oxygen evolution have shown that the continuous presence of theophylline in the culture medium does not induce a phase shift in the rhythm. The cAMP content of anucleate Acetabularia shows development stage variations parallel to that of the whole algae.

  13. Role of adenosine kinase in the control of Streptomyces differentiations: Loss of adenosine kinase suppresses sporulation and actinorhodin biosynthesis while inducing hyperproduction of undecylprodigiosin in Streptomyces lividans.

    PubMed

    Rajkarnikar, Arishma; Kwon, Hyung-Jin; Suh, Joo-Won

    2007-11-16

    Adenosine kinase (ADK) catalyses phosphorylation of adenosine (Ado) and generates adenosine monophosphate (AMP). ADK gene (adk(Sli), an ortholog of SCO2158) was disrupted in Streptomyces lividans by single crossover-mediated vector integration. The adk(Sli) disruption mutant (Deltaadk(Sli)) was devoid of sporulation and a plasmid copy of adk(Sli) restored sporulation ability in Deltaadk(Sli), thus indicating that loss of adk(Sli) abolishes sporulation in S. lividans. Ado supplementation strongly suppressed sporulation ability in S. lividans wild-type (wt), supporting that disruption of adk(Sli) resulted in Ado accumulation, which in turn suppressed sporulation. Cell-free experiments demonstrated that Deltaadk(Sli) lacked ADK activity and in vitro characterization confirms that adk(Sli) encodes ADK. The intracellular level of Ado was highly elevated while the AMP level was significantly reduced after loss of adk(Sli) while Deltaadk(Sli) displayed no significant derivation from wt in the levels of S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM). Notably, Ado supplementation to wt lowered AMP content, albeit not to the level of Deltaadk(Sli), implying that the reduction of AMP level is partially forced by Ado accumulation in Deltaadk(Sli). In Deltaadk(Sli), actinorhodin (ACT) production was suppressed and undecylprodigiosin (RED) production was dramatically enhanced; however, Ado supplementation failed to exert this differential control. A promoter-probe assay verified repression of actII-orf4 and induction of redD in Deltaadk(Sli), substantiating that unknown metabolic shift(s) of ADK-deficiency evokes differential genetic control on secondary metabolism in S. lividans. The present study is the first report revealing the suppressive role of Ado in Streptomyces development and the differential regulatory function of ADK activity in Streptomyces secondary metabolism, although the underlying mechanism has yet to be elucidated.

  14. Use of potato tuber nucleotide pyrophosphatase to synthesize adenosine 5'-monophosphate methyl ester: evidence that the solvolytic preferences of the enzyme are regulated by pH and temperature.

    PubMed

    Agudo, A; Ribeiro, J M; Canales, J; Cameselle, J C

    1998-07-01

    Nucleotide alkyl esters are pharmacologically important as potential (ant)agonists of purinoceptors and inhibitors of enzymes. Potato nucleotide pyrophosphatase (PNP) was compared with snake venom phosphodiesterase (SVP) as a catalyst to synthesize nucleotide alkyl esters. In methanol-water mixtures, the methanolysis/hydrolysis ratio of PNP, but not SVP, changed with pH and temperature, being optimal at high pH and low temperature. In a semi-preparative experiment, a crude PNP preparation produced 0.17 mM AMP-O-methyl ester (AMP-OMe) from 1 mM diadenosine 5',5"'-P1,P2-diphosphate (AppA) and 5M methanol, at pH 9 and 0 degrees C. Drawbacks to large-scale use are: low rates inherent to low temperatures, ATP unsuitability as a substrate for alcoholysis, and high cost of AppA. Advantages of PNP vs. SVP are cheapness, non-toxicity, and availability of the enzyme source.

  15. Effect of cholera toxin on cAMP levels and Na/sup +/ influx in isolated intestinal epithelial cells

    SciTech Connect

    Hyun, C.S.; Kimmich, G.A.

    1982-09-01

    Freshly isolated chicken intestinal cells contain approximately 20 pmol adenosine 3',5'-cyclic monophosphate (cAMP)/mg cellular protein. Incubation with 3 ..mu..g/ml cholera toxin (CT) at 37/sup 0/C induces an elevation of cellular cAMP beginning 10-15 min after initial exposure. The response is linear with time for 40-50 min and causes a six- to eightfold increase over control levels at steady state. Dibutyryl cAMP and agents that increase cAMP production inhibit Na/sup +/ influx into the isolated enterocytes. Chlorpromazine completely abolishes the toxin-induced elevation of cAMP in the isolated cells and also reverses the effect on Na/sup +/ entry. The data provide evidence for a cAMP-mediated control of intestinal cell Na/sup +/ uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT on Na/sup +/ during induction of intestinal secretory activity. Studies on the time-dependent effects of chlorpromazine on both intracellular cAMP concentration and Na/sup +/ influx suggest that the reactivation of the Na/sup +/ transport system after cAMP-induced inhibition is slow relative to the disappearance of cAMP.

  16. Time- and dose-related interactions between glucocorticoid and cyclic adenosine 3',5'-monophosphate on CCAAT/enhancer-binding protein-dependent insulin-like growth factor I expression by osteoblasts

    NASA Technical Reports Server (NTRS)

    McCarthy, T. L.; Ji, C.; Chen, Y.; Kim, K.; Centrella, M.

    2000-01-01

    Glucocorticoid has complex effects on osteoblasts. Several of these changes appear to be related to steroid concentration, duration of exposure, or specific effects on growth factor expression or activity within bone. One important bone growth factor, insulin-like growth factor I (IGF-I), is induced in osteoblasts by hormones such as PGE2 that increase intracellular cAMP levels. In this way, PGE2 activates transcription factor CCAAT/enhancer-binding protein-delta (C/EBPdelta) and enhances its binding to a specific control element found in exon 1 in the IGF-I gene. Our current studies show that preexposure to glucocorticoid enhanced C/EBPdelta and C/EBPbeta expression by osteoblasts and thereby potentiated IGF-I gene promoter activation in response to PGE2. Importantly, this directly contrasts with inhibitory effects on IGF-I expression that result from sustained or pharmacologically high levels of glucocorticoid exposure. Consistent with the stimulatory effect of IGF-I on bone protein synthesis, pretreatment with glucocorticoid sensitized osteoblasts to PGE2, and in this context significantly enhanced new collagen and noncollagen protein synthesis. Therefore, pharmacological levels of glucocorticoid may reduce IGF-I expression by osteoblasts and cause osteopenic disease, whereas physiological transient increases in glucocorticoid may permit or amplify the effectiveness of hormones that regulate skeletal tissue integrity. These events appear to converge on the important role of C/EBPdelta and C/EBPbeta on IGF-I expression by osteoblasts.

  17. Dual activity of certain HIT-proteins: A. thaliana Hint4 and C. elegans DcpS act on adenosine 5'-phosphosulfate as hydrolases (forming AMP) and as phosphorylases (forming ADP).

    PubMed

    Guranowski, Andrzej; Wojdyła, Anna Maria; Zimny, Jarosław; Wypijewska, Anna; Kowalska, Joanna; Jemielity, Jacek; Davis, Richard E; Bieganowski, Paweł

    2010-01-01

    Histidine triad (HIT)-family proteins interact with different mono- and dinucleotides and catalyze their hydrolysis. During a study of the substrate specificity of seven HIT-family proteins, we have shown that each can act as a sulfohydrolase, catalyzing the liberation of AMP from adenosine 5'-phosphosulfate (APS or SO(4)-pA). However, in the presence of orthophosphate, Arabidopsis thaliana Hint4 and Caenorhabditis elegans DcpS also behaved as APS phosphorylases, forming ADP. Low pH promoted the phosphorolytic and high pH the hydrolytic activities. These proteins, and in particular Hint4, also catalyzed hydrolysis or phosphorolysis of some other adenylyl-derivatives but at lower rates than those for APS cleavage. A mechanism for these activities is proposed and the possible role of some HIT-proteins in APS metabolism is discussed. PMID:19896942

  18. AMP/GMP Analogs as Affinity ESIPT Probes for Highly Selective Sensing of Alkaline Phosphatase Activity in Living Systems.

    PubMed

    Jia, Yan; Li, Peng; Han, Keli

    2015-11-01

    Current probes for alkaline phosphatase (ALP) detection had been developed mainly by adding a phosphate group to a dye, which would lead to indistinct performance when implemented in a living system as several phosphatases exist together. In this study, the nucleotides adenosine monophosphate (AMP) and guanosine monophosphate (GMP) were introduced into 2'-(2'-hydroxyphenyl)-benzothiazole-based probes, and highly fluorescent turn-on probes with good selectivity towards ALP over several phosphatases, as well as high affinity and low toxicity were obtained. In the presence of L-phenylalanine, an ALP inhibitor, a strong decrease in fluorescence recovery was observed. These probes allowed for real-time imaging of endogenous ALP activity in living cells as well as in a zebrafish model.

  19. Central role of soluble adenylyl cyclase and cAMP in sperm physiology

    PubMed Central

    Buffone, Mariano G.; Wertheimer, Eva V.; Visconti, Pablo E.; Krapf, Dario

    2014-01-01

    Cyclic adenosine 3′,5′-monophosphate (cAMP), the first second messenger to be described, plays a central role in cell signaling in a wide variety of cell types. Over the last decades, a wide body of literature addressed the different roles of cAMP in cell physiology, mainly in response to neurotransmitters and hormones. cAMP is synthesized by a wide variety of adenylyl cylases that can generally be grouped in two types: transmembrane adenylyl cyclase and soluble adenylyl cyclases. In particular, several aspects of sperm physiology are regulated by cAMP produced by a single atypical adenylyl cyclase (Adcy10, aka sAC, SACY). The signature that identifies sAC among other ACs, is their direct stimulation by bicarbonate. The essential nature of cAMP in sperm function has been demonstrated using gain of function as well as loss of function approaches. This review unifies state of the art knowledge of the role of cAMP and those enzymes involved in cAMP signaling pathways required for the acquisition of fertilizing capacity of mammalian sperm. PMID:25066614

  20. The mechanisms of action of cAMP. A quantum chemical study.

    PubMed

    van Ool, P J; Buck, H M

    1982-01-01

    Quantum chemical calculations were performed on the formation of intermediates with trigonal bipyramidal (TBP) configurations in the hydrolysis of adenosine 3',5'-monophosphate (cAMP) with phosphodiesterases and the activation of protein kinases by cAMP. The results show that in the reaction sequence concerning the hydrolysis of cAMP with phosphodiesterase the TBP intermediate must possess an equatorial-apical cyclic phosphate ring with the 3'-oxygen atom in the apical position. This could be an additional reason for the sensitivity of the 3' position in cAMP towards modifications in comparison with the 5' position. According to the calculations, a mechanistic model is presented for the enzymatic hydrolysis of cAMP with the involvement of a covalently bonded enzyme-nucleotide intermediate. Also a model is offered for the activation of protein kinase by cAMP. The activation of protein kinase is assumed to proceed via diequatorial-ring-positioned TBP intermediates resulting in the formation of a covalent bond between cAMP and the protein kinase with retention of the cyclic phosphate ring. It seems likely that the enzyme-nucleotide intermediate enforces a conformational change in the enzyme, which causes the dissociation of the regulatory and catalytic subunit of the protein kinase, necessary for a physiological response.

  1. Isolation of novel ribozymes that ligate AMP-activated RNA substrates

    NASA Technical Reports Server (NTRS)

    Hager, A. J.; Szostak, J. W.

    1997-01-01

    BACKGROUND: The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5'-5'-pyrophosphate 'capped' RNA or DNA intermediate. The activation of nucleic acid substrates by adenosine 5'-monophosphate (AMP) may be a vestige of 'RNA world' catalysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and specifically binding AMP has previously been isolated. RESULTS: We used in vitro selection and directed evolution to explore the ability of ribozymes to catalyze the template-directed ligation of AMP-activated RNAs. We subjected a pool of 10(15) RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5'-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of approximately 5 x10(5) over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3'-5'-phosphodiester bonds and were highly specific for activation by AMP at the ligation site. CONCLUSIONS: The existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases resulted from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-activated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates.

  2. Effects of 4-week treatment with lithium and olanzapine on levels of brain-derived neurotrophic factor, B-cell CLL/lymphoma 2 and phosphorylated cyclic adenosine monophosphate response element-binding protein in the sub-regions of the hippocampus.

    PubMed

    Hammonds, Michael D; Shim, Seong S

    2009-08-01

    A large body of evidence indicates that lithium, the prototype mood stabilizer in the treatment of bipolar disorder, has diverse neuroprotective and neurotrophic actions, and the actions are associated with its efficacy in treating bipolar disorder. It has been suggested that up-regulation of neurotrophic and neuroprotective factors including brain-derived neurotrophic factor (BDNF) and B-cell CLL/lymphoma 2 (Bcl-2) may underlie these neuroplastic actions of the drug. Olanzapine, an atypical anti-psychotic drug, has been shown to be an effective mood stabilizer. Olanzapine also has neurotrophic and neuroprotective actions, and these actions may underlie the efficacy of the drug for bipolar disorder and schizophrenia. However, the molecular mechanism by which the drug produces the neuroplastic actions is poorly understood. To understand a common molecular mechanism underlying the neuroplastic actions of lithium and olanzapine, we assessed the effect of 4-week lithium and olanzapine treatment on the levels of BDNF, Bcl-2 and cyclic adenosine monophosphate response element-binding protein (CREB), a transcription factor involved in expression of BDNF and Bcl-2, in the dentate gyrus and hippocampal area CA1. Our results show that 4-week treatment with both olanzapine and lithium increases the levels of Bcl-2 and CREB in the dentate gyrus and hippocampal area CA1. Four-week lithium treatment up-regulates BDNF in the dentate gyrus, and 4-week olanzapine treatment marginally did so. Neither drug altered BDNF levels in area CA1. These results suggest that the up-regulation of Bcl-2 and CREB may underlie the neuroplastic actions of olanzapine and lithium.

  3. Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 83, 127 and 128 of the cyclic nucleotide binding pocket.

    PubMed Central

    Lee, E J; Glasgow, J; Leu, S F; Belduz, A O; Harman, J G

    1994-01-01

    The cyclic 3', 5' adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli was mutagenized to substitute cysteine or glycine for serine 83; cysteine, glycine, isoleucine, or serine for threonine 127; and threonine or alanine for serine 128. Cells that expressed the binding pocket residue-substituted forms of CRP were characterized by measurements of beta-galactosidase activity. Purified wild-type and mutant CRP preparations were characterized by measurement of cAMP binding activity and by their capacity to support lacP activation in vitro. CRP structure was assessed by measurement of sensitivity to protease and DTNB-mediated subunit crosslinking. The results of this study show that cAMP interactions with serine 83, threonine 127 and serine 128 contribute to CRP activation and have little effect on cAMP binding. Amino acid substitutions that introduce hydrophobic amino acid side chain constituents at either position 127 or 128 decrease CRP discrimination of cAMP and cGMP. Finally, cAMP-induced CRP structural change(s) that occur in or near the CRP hinge region result from cAMP interaction with threonine 127; substitution of threonine 127 by cysteine, glycine, isoleucine, or serine produced forms of CRP that contained, independently of cAMP binding, structural changes similar to those of the wild-type CRP:cAMP complex. Images PMID:8065899

  4. Identification of cholinergic and non-cholinergic neurons in the pons expressing phosphorylated cyclic adenosine monophosphate response element-binding protein as a function of rapid eye movement sleep.

    PubMed

    Datta, S; Siwek, D F; Stack, E C

    2009-09-29

    Recent studies have shown that in the pedunculopontine tegmental nucleus (PPT), increased neuronal activity and kainate receptor-mediated activation of intracellular protein kinase A (PKA) are important physiological and molecular steps for the generation of rapid eye movement (REM) sleep. In the present study performed on rats, phosphorylated cyclic AMP response element-binding protein (pCREB) immunostaining was used as a marker for increased intracellular PKA activation and as a reflection of increased neuronal activity. To identify whether activated cells were either cholinergic or noncholinergic, the PPT and laterodorsal tegmental nucleus (LDT) cells were immunostained for choline acetyltransferase (ChAT) in combination with pCREB or c-Fos. The results demonstrated that during high rapid eye movement sleep (HR, approximately 27%), significantly higher numbers of cells expressed pCREB and c-Fos in the PPT, of which 95% of pCREB-expressing cells were ChAT-positive. With HR, the numbers of pCREB-positive cells were also significantly higher in the medial pontine reticular formation (mPRF), pontine reticular nucleus oral (PnO), and dorsal subcoeruleus nucleus (SubCD) but very few in the locus coeruleus (LC) and dorsal raphe nucleus (DRN). Conversely, with low rapid eye movement sleep (LR, approximately 2%), the numbers of pCREB expressing cells were very few in the PPT, mPRF, PnO, and SubCD but significantly higher in the LC and DRN. The results of regression analyses revealed significant positive relationships between the total percentages of REM sleep and numbers of ChAT+/pCREB+ (Rsqr=0.98) cells in the PPT and pCREB+ cells in the mPRF (Rsqr=0.88), PnO (Rsqr=0.87), and SubCD (Rsqr=0.84); whereas significantly negative relationships were associated with the pCREB+ cells in the LC (Rsqr=0.70) and DRN (Rsqr=0.60). These results provide evidence supporting the hypothesis that during REM sleep, the PPT cholinergic neurons are active, whereas the LC and DRN neurons are

  5. Mucosal adenosine stimulates chloride secretion in canine tracheal epithelium

    SciTech Connect

    Pratt, A.D.; Clancy, G.; Welsh, M.J.

    1986-08-01

    Adenosine is a local regulator of a variety of physiological functions in many tissues and has been observed to stimulate secretion in several Cl-secreting epithelia. In canine tracheal epithelium the authors found that adenosine stimulates Cl secretion from both the mucosal and submucosal surfaces. Addition of adenosine, or its analogue 2-chloroadenosine, to the mucosal surface potently stimulated Cl secretion with no effect on the rate of Na absorption. Stimulation resulted from an interaction of adenosine with adenosine receptors, because it was blocked by the adenosine receptor blocker, 8-phenyltheophylline. The adenosine receptor was a stimulatory receptor as judged by the rank-order potency of adenosine and its analogues and by the increase in cellular adenosine 3',5'-cyclic monophosphate levels produced by 2-chloroadenosine. Adenosine also stimulated Cl secretion when it was added to the submucosal surface, although the maximal increase in secretion was less and it was much less potent. The observation that mucosal 8-phenyletheophylline blocked the effect of submucosal 2-chloroadenosine, whereas submucosal 8-phenyltheophylline did not prevent a response to mucosal or submucosal 2-chloroadenosine, suggests that adenosine receptors are located on the mucosal surface. Thus submucosal adenosine may stimulate secretion by crossing the epithelium and interacting with receptors located on the mucosal surface. Because adenosine can be released from mast cells located in the airway lumen in response to inhaled material, and because adenosine stimulated secretion from the mucosal surface, it may be in a unique position to control the epithelium on a regional level.

  6. Hepatic gene expression profiling of 5′-AMP-induced hypometabolism in mice

    PubMed Central

    Miki, Takao; Van Oort-Jansen, Anita; Matsumoto, Tomoko; Loose, David S.; Lee, Cheng Chi

    2011-01-01

    There is currently much interest in clinical applications of therapeutic hypothermia. Hypothermia can be a consequence of hypometabolism. We have recently established a procedure for the induction of a reversible deep hypometabolic state in mice using 5′-adenosine monophosphate (5′-AMP) in conjunction with moderate ambient temperature. The current study aims at investigating the impact of this technology at the gene expression level in a major metabolic organ, the liver. Our findings reveal that expression levels of the majority of genes in liver are not significantly altered by deep hypometabolism. However, among those affected by hypometabolism, more genes are differentially upregulated than downregulated both in a deep hypometabolic state and in the early arousal state. These altered gene expression levels during 5′-AMP induced hypometabolism are largely restored to normal levels within 2 days of the treatment. Our data also suggest that temporal control of circadian genes is largely stalled during deep hypometabolism. PMID:21224422

  7. Aging of the rat adrenocortical cell: response to ACTH and cyclic AMP in vitro.

    PubMed

    Malamed, S; Carsia, R V

    1983-03-01

    To study intrinsic age-related changes in adrenocortical steroid production, cells isolated from rats of different ages (3 to 24 months) were used. Acute (2 hour) corticosterone production in response to stimulation by adrenocorticotrophic hormone (ACTH) and adenosine 3':5'-cyclic monophosphate (cAMP) was measured by radioimmunoassay. With age, adrenocortical cells lose much of their ability to produce corticosterone in the absence or presence of ACTH or cAMP. The loss is progressive from 6 to 24 months of age. Analysis of the data suggests that from 6 to 12 months, an intracellular steroidogenic lesion develops; in addition there may be a loss in ACTH receptors on the plasma membrane. After 12 months these defects increase and are accompanied by a decrease in receptor sensitivity to ACTH.

  8. Genetically-encoded tools for cAMP probing and modulation in living systems

    PubMed Central

    Paramonov, Valeriy M.; Mamaeva, Veronika; Sahlgren, Cecilia; Rivero-Müller, Adolfo

    2015-01-01

    Intracellular 3′-5′-cyclic adenosine monophosphate (cAMP) is one of the principal second messengers downstream of a manifold of signal transduction pathways, including the ones triggered by G protein-coupled receptors. Not surprisingly, biochemical assays for cAMP have been instrumental for basic research and drug discovery for decades, providing insights into cellular physiology and guiding pharmaceutical industry. However, despite impressive track record, the majority of conventional biochemical tools for cAMP probing share the same fundamental shortcoming—all the measurements require sample disruption for cAMP liberation. This common bottleneck, together with inherently low spatial resolution of measurements (as cAMP is typically analyzed in lysates of thousands of cells), underpin the ensuing limitations of the conventional cAMP assays: (1) genuine kinetic measurements of cAMP levels over time in a single given sample are unfeasible; (2) inability to obtain precise information on cAMP spatial distribution and transfer at subcellular levels, let alone the attempts to pinpoint dynamic interactions of cAMP and its effectors. At the same time, tremendous progress in synthetic biology over the recent years culminated in drastic refinement of our toolbox, allowing us not only to bypass the limitations of conventional assays, but to put intracellular cAMP life-span under tight control—something, that seemed scarcely attainable before. In this review article we discuss the main classes of modern genetically-encoded tools tailored for cAMP probing and modulation in living systems. We examine the capabilities and weaknesses of these different tools in the context of their operational characteristics and applicability to various experimental set-ups involving living cells, providing the guidance for rational selection of the best tools for particular needs. PMID:26441653

  9. Intraoperative urinary cyclic AMP monitoring in primary hyperparathyroidism.

    PubMed Central

    Schenk, W G; Wills, M; MacLeod, M S; Hanks, J B

    1993-01-01

    OBJECTIVE: This study examined the utility of intraoperative urinary cyclic 3'5' adenosine monophosphate (UcAMP), an indicator of parathyroid (PTH) hormone end-organ activity, as a "biochemical frozen section," signaling the real-time resolution of PTH hyperactivity during surgery for primary hyperparathyroidism. SUMMARY BACKGROUND DATA: The unsuccessful initial neck exploration for primary hyperparathyroidism, leaving the patient with persistent hyperfunctioning parathyroid tissue, results in part from the surgeon's inability intraoperatively to correlate a gland's gross appearance and size estimation with physiologic function. Preoperative imaging, intraoperative imaging, and intraoperative histologic/cytologic surveillance have not resolved this dilemma. METHODS: Twenty-seven patients underwent a prospective intraoperative UcAMP monitoring protocol. The patients all had a clinical diagnosis of primary hyperparathyroidism and an average preoperative serum calcium of 12.0 +/- 0.3 mg/dl. UcAMP was assayed intraoperatively using 20-minute nonequilibrium radioimmunoassay providing real-time feedback to the operating team. RESULTS: All patients had an elevated UcAMP confirming PTh hyperactivity at the beginning of the procedure. One patient, subsequently found to have an supernumerary ectopic adenoma, had four normal glands identified intraoperatively, and his intraoperative UcAMP values corroborated persistent hyperparathyroidism, the UcAMP of the remaining 26 patients decreased from 7.0 +/- 1.1 to 2.7 +/- 0.7 nm.dl GF (p < .00005) after complete adenoma excision, and they remain normocalcemic. The protocol provided useful and relevant information to the operating team, and aided in surgical decision-making, in 10 of the 27 cases (37%). CONCLUSION: Intraoperative biochemical surveillance with ucAMP monitoring reliably signals resolution of PTH hyperfunction. It is a useful adjunct to the surgeon's skill, judgment, and experience in parathyroid surgery. PMID:8387765

  10. Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 72 and 82 of the cyclic nucleotide binding pocket.

    PubMed Central

    Belduz, A O; Lee, E J; Harman, J G

    1993-01-01

    The 3', 5' cyclic adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli was mutagenized to substitute leucine, glutamine, or aspartate for glutamate 72; and lysine, histidine, leucine, isoleucine, or glutamine for arginine 82. Substitutions were made in wild-type CRP and in a CRP*, or cAMP-independent, form of the protein to assess the effects of the amino acid substitutions on CRP structure. Cells containing the binding pocket residue-substituted forms of CRP were characterized through beta-galactosidase activity and by measurement of cAMP binding activity. This study confirms a role for both glutamate 72 and arginine 82 in cAMP binding and activation of CRP. Glutamine or leucine substitution of glutamate 72 produced forms of CRP having low affinity for the cAMP and unresponsive to the nucleotide. Aspartate substituted for glutamate 72 produced a low affinity cAMP-responsive form of CRP. CRP has a stringent requirement for the positioning of the position 72 glutamate carboxyl group within the cyclic nucleotide binding pocket. Results of this study also indicate that there are differences in the binding requirements of cAMP and cGMP, a competitive inhibitor of cAMP binding to CRP. PMID:8388097

  11. CREB modulates calcium signaling in cAMP-induced bone marrow stromal cells (BMSCs).

    PubMed

    Zhang, Linxia; Liu, Li; Thompson, Ryan; Chan, Christina

    2014-10-01

    Calcium signaling has a versatile role in many important cellular functions. Despite its importance, regulation of calcium signaling in bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) has not been explored extensively. Our previous study revealed that cyclic adenosine monophosphate (cAMP) enabled BMSCs to generate calcium signal upon stimulation by dopamine, KCl and glutamate. Concurrently, cAMP transiently activated the transcription factor cAMP response element binding protein (CREB) in BMSCs. Activity of CREB can be modulated by the calcium/calmodulin-dependent kinase signaling pathway, however, whether the calcium signaling observed in cAMP-induced BMSCs requires CREB has not been investigated. In an effort to uncover the role of CREB in the generation of calcium signaling in response to modulators such as dopamine and KCl, we knocked down CREB activity in BMSCs. Our study indicated that BMSCs, but not its close relative fibroblasts, are responsive to dopamine and KCl after cAMP treatment. Calcium signal elicited by dopamine depends, in part, on calcium influx whereas that elicited by KCl depends completely on calcium influx. Knock-down of CREB activity significantly reduced or abolished the cAMP-induced calcium response, and reintroducing a constitutively active CREB partially restored the calcium response.

  12. CREB modulates calcium signaling in cAMP-induced bone marrow stromal cells (BMSCs)

    PubMed Central

    Zhang, Linxia; Liu, Li; Thompson, Ryan; Chan, Christina

    2014-01-01

    Calcium signaling has a versatile role in many important cellular functions. Despite its importance, regulation of calcium signaling in bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) has not been explored extensively. Our previous study revealed that cyclic adenosine monophosphate (cAMP) enabled BMSCs to generate calcium signal upon stimulation by dopamine, KCl and glutamate. Concurrently, cAMP transiently activated the transcription factor cAMP response element binding protein (CREB) in BMSCs. Activity of CREB can be modulated by the calcium/calmodulin-dependent kinase signaling pathway, however, whether the calcium signaling observed in cAMP-induced BMSCs requires CREB has not been investigated. In an effort to uncover the role of CREB in the generation of calcium signaling in response to modulators such as dopamine and KCl, we knocked down CREB activity in BMSCs. Our study indicated that BMSCs, but not its close relative fibroblasts, are responsive to dopamine and KCl after cAMP treatment. Calcium signal elicited by dopamine depends, in part, on calcium influx whereas that elicited by KCl depends completely on calcium influx. Knock-down of CREB activity significantly reduced or abolished the cAMP-induced calcium response, and reintroducing a constitutively active CREB partially restored the calcium response. PMID:25154887

  13. Electron transfer between the QmoABC membrane complex and adenosine 5'-phosphosulfate reductase.

    PubMed

    Duarte, Américo G; Santos, André A; Pereira, Inês A C

    2016-04-01

    The dissimilatory adenosine 5'-phosphosulfate reductase (AprAB) is a key enzyme in the sulfate reduction pathway that catalyzes the reversible two electron reduction of adenosine 5'-phosphosulfate (APS) to sulfite and adenosine monophosphate (AMP). The physiological electron donor for AprAB is proposed to be the QmoABC membrane complex, coupling the quinone-pool to sulfate reduction. However, direct electron transfer between these two proteins has never been observed. In this work we demonstrate for the first time direct electron transfer between the Desulfovibrio desulfuricans ATCC 27774 QmoABC complex and AprAB. Cyclic voltammetry conducted with the modified Qmo electrode and AprAB in the electrolyte solution presented the Qmo electrochemical signature with two additional well-defined one electron redox processes, attributed to the AprAB FAD redox behavior. Moreover, experiments performed under catalytic conditions using the QmoABC modified electrode, with AprAB and APS in solution, show a catalytic current peak develop in the cathodic wave, attributed to substrate reduction, and which is not observed in the absence of QmoABC. Substrate dependence conducted with different electrode preparations (with and without immobilized Qmo) demonstrated that the QmoABC complex is essential for efficient electron delivery to AprAB, in order to sustain catalysis. These results confirm the role of Qmo in electron transfer to AprAB. PMID:26768116

  14. The adenosine salvage pathway as an alternative to mitochondrial production of ATP in maturing mammalian oocytes.

    PubMed

    Scantland, Sara; Tessaro, Irene; Macabelli, Carolina H; Macaulay, Angus D; Cagnone, Gaël; Fournier, Éric; Luciano, Alberto M; Robert, Claude

    2014-09-01

    Although the oocyte is the largest cell in the body and an unavoidable phase in life, its physiology is still poorly understood, and other cell types provide little insight into its unique nature. Even basic cellular functions in the oocyte such as energy metabolism are not yet fully understood. It is known that the mitochondria of the female gamete exhibit an immature form characterized by limited energy production from glucose and oxidative phosphorylation. We show that the bovine oocyte uses alternative means to maintain ATP production during maturation, namely, the adenosine salvage pathway. Meiosis resumption is triggered by destruction of cyclic AMP by phosphodiesterases producing adenosine monophosphate that is converted into ATP by adenylate kinases and creatine kinases. Inhibition of these enzymes decreased ATP production, and addition of their substrates restored ATP production in denuded oocytes. Addition of phosphocreatine to the oocyte maturation medium influenced the phenotype of the resulting blastocysts. We propose a model in which adenylate kinases and creatine kinases act as drivers of ATP production from added AMP during oocyte maturation.

  15. Changes in the Arabidopsis thaliana Proteome Implicate cAMP in Biotic and Abiotic Stress Responses and Changes in Energy Metabolism

    PubMed Central

    Alqurashi, May; Gehring, Chris; Marondedze, Claudius

    2016-01-01

    The second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) is increasingly recognized as having many different roles in plant responses to environmental stimuli. To gain further insights into these roles, Arabidopsis thaliana cell suspension culture was treated with 100 nM of cell permeant 8-bromo-cAMP for 5 or 10 min. Here, applying mass spectrometry and comparative proteomics, 20 proteins were identified as differentially expressed and we noted a specific bias in proteins with a role in abiotic stress, particularly cold and salinity, biotic stress as well as proteins with a role in glycolysis. These findings suggest that cAMP is sufficient to elicit specific stress responses that may in turn induce complex changes to cellular energy homeostasis. PMID:27258261

  16. Dynamics of cellular and extracellular cAmp in Anabaena flos-aquae (cyanophyta): intrinsic culture variability and correlation with metabolic variables

    SciTech Connect

    Francko, D.A.; Wetzel, R.G.

    1981-01-01

    The production and extracellular release of cyclic adenosine 3':5'-monophosphate (cAMP) by the blue-green alga Anabaena flos-aquae (Lyngb.) Breb. varied greatly within and between active growth phase and stationary phase and under differing nutrient regimes. Enhanced cellular cAMP production was found in actively growing Anabaena inoculated into media deficient in nitrate or phosphate, or into fresh media containing non-limiting nutrient concentrations. In stationary phase Anabaena, but not actively growing cells, the concentrations of intracellular cAMP present in cells grown under a variety of nutrient regimes could be significantly correlated to (/sup 14/C)-bicarbonate uptake by an exponential relationship.

  17. Dynamics of cellular and extracellular cAMP in Anabaena flos-aquae (Cyanophyta): intrinsic culture variability and correlation with metabolic variables

    SciTech Connect

    Francko, D.A.; Wetzel, R.G.

    1981-06-01

    The production and extracellular release of cyclic adenosine 3':5'-monophosphate (cAMP) by the blue-green alga Anabaena flos-aquae (Lyngb.) Breb. varied greatly within and between active growth phase and stationary phase and under differing nutrient regimes. Enhanced cellular cAMP production was found in actively growing Anaebaena inoculated into media deficient in nitrate or phosphate, or into fresh media containing non-limiting nutrient concentrations. In stationary phase Anabaena, but not actively growing cells, the concentrations of intracellular cAMP present in cells grown under a variety of nutrient regimes could be significantly correlated to (/sup 14/C)-bicarbonate uptake by an exponential relationship.

  18. cAMP-binding proteins in medullary tubules from rat kidney: effect of ADH

    SciTech Connect

    Gapstur, S.M.; Homma, S.; Dousa, T.P.

    1988-08-01

    Little is known of the regulatory steps in the cellular action of vasopressin (AVP) on the renal epithelium, subsequent to the cAMP generation. We studied cAMP-binding proteins in the medullary collecting tubule (MCT) and the thick ascending limb of Henle's loop (MTAL) microdissected from the rat kidney by use of photoaffinity labeling. Microdissected tubules were homogenized and photoaffinity labeled by incubation with 1 microM 32P-labeled 8-azido-adenosine 3',5'-cyclic monophosphate (N3-8-(32P)-cAMP); the incorporated 32P was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Both in MCT and MTAL preparations, the analyses showed incorporation of N3-8-(32P)cAMP into two bands (Mr = 49,000 and Mr = 55,000) that comigrated with standards of the cAMP-dependent protein kinase regulatory subunits RI and RII. In MCT, most of the 32P (80%) was incorporated into RI, whereas in MTAL the 32P incorporated into RI and RII was equivalent. When freshly dissected MCT segments were incubated with 10(-12)-10(-6) M AVP, the subsequent photoaffinity labeling of RI with N3-8-(32P)cAMP was markedly diminished in a dose-dependent manner compared with controls. Our results suggest that cAMP binds in MCT and MTAL to regulatory subunits RI and RII of cAMP-dependent protein kinase. However, in MCT the dominant type of cAMP-dependent protein kinase appears to be type I. The outlined procedure is suitable to indirectly measure the occupancy of RI by endogenous cAMP generated in MCT cells in response to physiological levels (10(-12) M) of AVP.

  19. DBcAMP stimulates vesicle transport and HRP excretion in isolated perfused rat liver.

    PubMed

    Hayakawa, T; Bruck, R; Ng, O C; Boyer, J L

    1990-11-01

    To clarify the effect of adenosine 3',5'-cyclic monophosphate (cAMP) on the transcytotic vesicle pathway, we measured the biliary excretion of bile acid, phospholipid, and horseradish peroxidase (HRP) in the isolated perfused rat liver (IPRL) with or without infusion of N6,2'-O-dibutyryl-cAMP (DBcAMP). A linear relationship between bile flow and bile acid excretion was observed in both control and DBcAMP-infused livers. DBcAMP increased the y-axis intercept from 1.10 +/- 0.16 to 1.48 +/- 0.19 microliters.min-1.g liver-1 (P less than 0.01) and the slope from 6.5 +/- 1.99 to 10.77 +/- 1.71 microliters/mumol bile acid (P less than 0.01). DBcAMP also increased the biliary excretion of bile acid and phospholipid during a 1.0 mumol/min infusion of taurocholate. When HRP was pulse loaded for 1 min, HRP appeared in bile in early (4-6 min) and late (20-25 min) peaks. DBcAMP markedly increased the late peak of HRP from 0.33 +/- 0.08 to 1.15 +/- 0.32 ng.min-1.g liver-1 (P less than 0.01), a phenomenon blocked by colchicine. An electron-microscopic morphometric analysis indicated that DBcAMP increased both the density and %area of HRP-containing vesicles in the pericanalicular area, compared with controls, 18 min after a 1-min pulse of HRP. DBcAMP had no effect on the uptake rate of HRP in 4-h primary hepatocyte cultures but stimulated biliary excretion of HRP when preloaded in the IPRL. These findings indicate that cAMP regulates excretory function in part by stimulating the microtubule-dependent transcytotic vesicle transport system.

  20. The cyclic di-nucleotide c-di-AMP is an allosteric regulator of metabolic enzyme function

    PubMed Central

    Precit, Mimi; Delince, Matthieu; Pensinger, Daniel; Huynh, TuAnh Ngoc; Jurado, Ashley R.; Goo, Young Ah; Sadilek, Martin; Iavarone, Anthony T.; Sauer, John-Demian; Tong, Liang; Woodward, Joshua J.

    2014-01-01

    SUMMARY Cyclic di-adenosine monophosphate (c-di-AMP) is a broadly conserved second messenger required for bacterial growth and infection. However, the molecular mechanisms of c-di-AMP signaling are still poorly understood. Using a chemical proteomics screen for c-di-AMP interacting proteins in the pathogen Listeria monocytogenes, we identified several broadly conserved protein receptors, including the central metabolic enzyme pyruvate carboxylase (LmPC). Biochemical and crystallographic studies of the LmPC-c-di-AMP interaction revealed a previously unrecognized allosteric regulatory site 25 Å from the active site. Mutations in this site disrupted c-di-AMP binding and affected enzyme catalysis of LmPC as well as PC from pathogenic Enterococcus faecalis. C-di-AMP depletion resulted in altered metabolic activity in L. monocytogenes. Correction of this metabolic imbalance rescued bacterial growth, reduced bacterial lysis, and resulted in enhanced bacterial burdens during infection. These findings greatly expand the c-di-AMP signaling repertoire and reveal a central metabolic regulatory role for a cyclic di-nucleotide. PMID:25215494

  1. Transcriptomic analysis of cyclic AMP response in bovine cumulus cells.

    PubMed

    Khan, D R; Guillemette, C; Sirard, M A; Richard, F J

    2015-09-01

    Acquisition of oocyte developmental competence needs to be understood to improve clinical outcomes of assisted reproduction. The stimulation of cumulus cell concentration of cyclic adenosine 3'5'-monophosphate (cAMP) by pharmacological agents during in vitro maturation (IVM) participates in improvement of oocyte quality. However, precise coordination and downstream targets of cAMP signaling in cumulus cells are largely unknown. We have previously demonstrated better embryo development after cAMP stimulation for first 6 h during IVM. Using this model, we investigated cAMP signaling in cumulus cells through in vitro culture of cumulus-oocyte complexes (COCs) in the presence of cAMP raising agents: forskolin, IBMX, and dipyridamole (here called FID treatment). Transcriptomic analysis of cumulus cells indicated that FID-induced differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism, and oocyte competence. Functional genomic analysis revealed that protein kinase-A (PKA), extracellular signal regulated kinases (ERK1/2), and calcium (Ca(2+)) pathways as key regulators of FID signaling. Inhibition of PKA (H89) in FID-supplemented COCs or substitution of FID with calcium ionophore (A23187) demonstrated that FID activated primarily the PKA pathway which inhibited ERK1/2 phosphorylation and was upstream of calcium signaling. Furthermore, inhibition of ERK1/2 phosphorylation by FID supported a regulation by dual specific phosphatase (DUSP1) via PKA. Our findings imply that cAMP (FID) regulates cell metabolism, steroidogenesis, intracellular signaling and cumulus expansion through PKA which modulates these functions through optimization of ERK1/2 phosphorylation and coordination of calcium signaling. These findings have implications for development of new strategies for improving oocyte in vitro maturation leading to better developmental competence.

  2. Transcriptomic analysis of cyclic AMP response in bovine cumulus cells

    PubMed Central

    Khan, D. R.; Guillemette, C.; Sirard, M. A.

    2015-01-01

    Acquisition of oocyte developmental competence needs to be understood to improve clinical outcomes of assisted reproduction. The stimulation of cumulus cell concentration of cyclic adenosine 3′5′-monophosphate (cAMP) by pharmacological agents during in vitro maturation (IVM) participates in improvement of oocyte quality. However, precise coordination and downstream targets of cAMP signaling in cumulus cells are largely unknown. We have previously demonstrated better embryo development after cAMP stimulation for first 6 h during IVM. Using this model, we investigated cAMP signaling in cumulus cells through in vitro culture of cumulus-oocyte complexes (COCs) in the presence of cAMP raising agents: forskolin, IBMX, and dipyridamole (here called FID treatment). Transcriptomic analysis of cumulus cells indicated that FID-induced differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism, and oocyte competence. Functional genomic analysis revealed that protein kinase-A (PKA), extracellular signal regulated kinases (ERK1/2), and calcium (Ca2+) pathways as key regulators of FID signaling. Inhibition of PKA (H89) in FID-supplemented COCs or substitution of FID with calcium ionophore (A23187) demonstrated that FID activated primarily the PKA pathway which inhibited ERK1/2 phosphorylation and was upstream of calcium signaling. Furthermore, inhibition of ERK1/2 phosphorylation by FID supported a regulation by dual specific phosphatase (DUSP1) via PKA. Our findings imply that cAMP (FID) regulates cell metabolism, steroidogenesis, intracellular signaling and cumulus expansion through PKA which modulates these functions through optimization of ERK1/2 phosphorylation and coordination of calcium signaling. These findings have implications for development of new strategies for improving oocyte in vitro maturation leading to better developmental competence. PMID:26082143

  3. An HD-domain phosphodiesterase mediates cooperative hydrolysis of c-di-AMP to affect bacterial growth and virulence

    PubMed Central

    Huynh, TuAnh Ngoc; Luo, Shukun; Pensinger, Daniel; Sauer, John-Demian; Tong, Liang; Woodward, Joshua J.

    2015-01-01

    The nucleotide cyclic di-3′,5′- adenosine monophosphate (c-di-AMP) was recently identified as an essential and widespread second messenger in bacterial signaling. Among c-di-AMP–producing bacteria, altered nucleotide levels result in several physiological defects and attenuated virulence. Thus, a detailed molecular understanding of c-di-AMP metabolism is of both fundamental and practical interest. Currently, c-di-AMP degradation is recognized solely among DHH-DHHA1 domain-containing phosphodiesterases. Using chemical proteomics, we identified the Listeria monocytogenes protein PgpH as a molecular target of c-di-AMP. Biochemical and structural studies revealed that the PgpH His-Asp (HD) domain bound c-di-AMP with high affinity and specifically hydrolyzed this nucleotide to 5′-pApA. PgpH hydrolysis activity was inhibited by ppGpp, indicating a cross-talk between c-di-AMP signaling and the stringent response. Genetic analyses supported coordinated regulation of c-di-AMP levels in and out of the host. Intriguingly, a L. monocytogenes mutant that lacks c-di-AMP phosphodiesterases exhibited elevated c-di-AMP levels, hyperinduced a host type-I IFN response, and was significantly attenuated for infection. Furthermore, PgpH homologs, which belong to the 7TMR-HD family, are widespread among hundreds of c-di-AMP synthesizing microorganisms. Thus, PgpH represents a broadly conserved class of c-di-AMP phosphodiesterase with possibly other physiological functions in this crucial signaling network. PMID:25583510

  4. Opposing roles of PKA and EPAC in the cAMP-dependent regulation of schwann cell proliferation and differentiation [corrected].

    PubMed

    Bacallao, Ketty; Monje, Paula V

    2013-01-01

    In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) not only induces differentiation into a myelinating SC-related phenotype, but also synergistically enhances the mitogenic action of growth factors such as neuregulin. To better understand the molecular mechanism by which cAMP exerts these apparently contradictory functions, we investigated the role of the two main effectors of cAMP, protein kinase A (PKA) and the exchange protein activated by cAMP (EPAC), on the proliferation and differentiation of both isolated and axon-related SCs. For these studies, a variety of PKA and EPAC agonists and antagonists were used, including pathway-selective analogs of cAMP and pharmacological inhibitors. Our studies indicated that the activity of PKA rather than EPAC was required for the adjuvant effect of cAMP on S-phase entry, whereas the activity of EPAC rather than PKA was required for SC differentiation and myelin formation. Even though selective EPAC activation had an overall anti-proliferative effect in SCs, it failed to drive the expression of Krox-20, a master regulator of myelination, and that of myelin-specific proteins and lipids, suggesting that EPAC activation was insufficient to drive a full differentiating response. Interestingly, inhibition of EPAC activity resulted in a drastic impairment of SC differentiation and myelin formation but not Krox-20 expression, which indicates an independent mechanism of Krox-20 regulation in response to cAMP. In conclusion, our data supports the idea that the outcome of cAMP signaling in SCs depends on the particular set of effectors activated. Whereas the mitogenic action of cAMP relies exclusively on PKA activity, the differentiating action of cAMP requires a PKA-independent (non-canonical) cAMP-specific pathway that is partially transduced by EPAC.

  5. Puerarin, an isoflavonoid derived from Radix puerariae, potentiates endothelium-independent relaxation via the cyclic AMP pathway in porcine coronary artery.

    PubMed

    Yeung, Dennis K Y; Leung, Susan W S; Xu, Yan Chun; Vanhoutte, Paul M; Man, Ricky Y K

    2006-12-15

    Puerarin, an isoflavonoid derived from the Chinese medicinal herb Radix puerariae, has been suggested to be useful in the management of various cardiovascular disorders. The present study examined the effect of acute exposure (30 min) to puerarin on vascular relaxation. Rings from porcine coronary artery of either sex were used. The highest concentration of puerarin (100 microM) produced a small but statistically significant relaxation of U46619-contracted rings. Vascular relaxations were also studied in the presence of lower concentrations of puerarin (0.1, 1 and 10 microM) which had no direct relaxation effect. Puerarin enhanced vasorelaxation to endothelium-independent relaxing agents, sodium nitroprusside and cromakalim. However, puerarin had no effect on vasorelaxation induced by endothelium-dependent relaxing agents, bradykinin and calcium ionophore A23187. The potentiating action of puerarin (10 microM) on sodium nitroprusside-mediated relaxation was not affected by the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME; 300 microM), or by the disruption of the endothelium with Triton X-100. The effect of puerarin was reversible following a washout period. The potentiating effects were comparable with the 3'-5'-cyclic adenosine monophosphate (cyclic AMP) analogues, 8-bromoadenosine-3'-5'-cyclic monophosphate (8-Br-cyclic AMP; 10 muM) and Sp-isomer [S nomenclature refers to phosphorus] of adenosine-3', 5'-cyclic monophosphorothioate (Sp-cyclic AMPS; 3 microM), but not the 3'-5'-cyclic guanosine monophosphate (cyclic GMP) analogue, 8-bromoguanosine-3'-5'-cyclic monophosphate (8-Br-cyclic GMP; 3 microM). The cyclic AMP antagonist, Rp-isomer [R nomenclature refers to phosphorus] of 8-bromoadenosine-3', 5'-cyclic monophosphorothioate (Rp-8-Br-cyclic AMPS; 10 microM), but not cyclic GMP antagonist, Rp-isomer of 8-bromoguanosine-3', 5'-cyclic monophosphorothioate (Rp-8-Br-cyclic GMPS; 10 microM), reversed the effects of puerarin (10

  6. Modulation of dihydropyridine-sensitive calcium channels in Drosophila by a cAMP-mediated pathway.

    PubMed

    Bhattacharya, A; Gu, G G; Singh, S

    1999-06-15

    Drosophila has proved to be a valuable system for studying the structure and function of ion channels. However, relatively little is known about the regulation of ion channels, particularly that of Ca2+ channels, in Drosophila. Physiological and pharmacological differences between invertebrate and mammalian L-type Ca2+ channels raise questions on the extent of conservation of Ca2+ channel modulatory pathways. We have examined the role of cyclic adenosine monophosphate (cAMP) cascade in modulating the dihydropyridine (DHP)-sensitive Ca2+ channels in the larval muscles of Drosophila, using mutations and drugs that disrupt specific steps in this pathway. The L-type (DHP-sensitive) Ca2+ channel current was increased in the dunce mutants, which have high cAMP concentration owing to cAMP-specific phosphodiesterase (PDE) disruption. The current was decreased in the rutabaga mutants, where adenylyl cyclase (AC) activity is altered thereby decreasing the cAMP concentration. The dunce effect was mimicked by 8-Br-cAMP, a cAMP analog, and IBMX, a PDE inhibitor. The rutabaga effect was rescued by forskolin, an AC activator. H-89, an inhibitor of protein kinase-A (PKA), reduced the current and inhibited the effect of 8-Br-cAMP. The data suggest modulation of L-type Ca2+ channels of Drosophila via a cAMP-PKA mediated pathway. While there are differences in L-type channels, as well as in components of cAMP cascade, between Drosophila and vertebrates, main features of the modulatory pathway have been conserved. The data also raise questions on the likely role of DHP-sensitive Ca2+ channel modulation in synaptic plasticity, and learning and memory, processes disrupted by the dnc and the rut mutations. PMID:10380071

  7. Influence of cAMP and protein kinase A on neurite length from spiral ganglion neurons

    PubMed Central

    Xu, Ningyong; Engbers, Jonathan; Khaja, Sobia; Xu, Linjing; Clark, J. Jason; Hansen, Marlan R.

    2011-01-01

    Regrowth of peripheral spiral ganglion neuron (SGN) fibers is a primary objective in efforts to improve cochlear implant outcomes and to potentially reinnervate regenerated hair cells. Cyclic adenosine monophosphate (cAMP) regulates neurite growth and guidance via activation of protein kinase A (PKA) and Exchange Protein directly Activated by Cylic AMP (Epac). Here we explored the effects of cAMP signaling on SGN neurite length in vitro. We find that the cAMP analog, cpt-cAMP, exerts a biphasic effect on neurite length; increasing length at lower concentrations and reducing length at higher concentrations. This biphasic response occurs in cultures plated on laminin, fibronectin, or tenascin C suggesting that it is not substrate dependent. cpt-cAMP also reduces SGN neurite branching. The Epac-specific agonist, 8-pCPT-2’-O-Me-cAMP, does not alter SGN neurite length. Constitutively active PKA isoforms strongly inhibit SGN neurite length similar to higher levels of cAMP. Chronic membrane depolarization activates PKA in SGNs and also inhibits SGN neurite length. However, inhibition of PKA fails to rescue neurite length in depolarized cultures implying that activation of PKA is not necessary for the inhibition of SGN neurite length by chronic depolarization. Expression of constitutively active phosphatidylinositol 3-kinase, but not c-Jun N-terminal kinase, isoforms partially rescues SGN neurite length in the presence of activated PKA. Taken together, these results suggest that activation of cAMP/PKA represents a potential strategy to enhance SGN fiber elongation following deafness; however such therapies will likely require careful titration so as to simultaneously promote rather than inhibit nerve fiber regeneration. PMID:22154930

  8. A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death

    PubMed Central

    Wang, Z; Liu, D; Varin, A; Nicolas, V; Courilleau, D; Mateo, P; Caubere, C; Rouet, P; Gomez, A-M; Vandecasteele, G; Fischmeister, R; Brenner, C

    2016-01-01

    Although cardiac cytosolic cyclic 3′,5′-adenosine monophosphate (cAMP) regulates multiple processes, such as beating, contractility, metabolism and apoptosis, little is known yet on the role of this second messenger within cardiac mitochondria. Using cellular and subcellular approaches, we demonstrate here the local expression of several actors of cAMP signaling within cardiac mitochondria, namely a truncated form of soluble AC (sACt) and the exchange protein directly activated by cAMP 1 (Epac1), and show a protective role for sACt against cell death, apoptosis as well as necrosis in primary cardiomyocytes. Upon stimulation with bicarbonate (HCO3−) and Ca2+, sACt produces cAMP, which in turn stimulates oxygen consumption, increases the mitochondrial membrane potential (ΔΨm) and ATP production. cAMP is rate limiting for matrix Ca2+ entry via Epac1 and the mitochondrial calcium uniporter and, as a consequence, prevents mitochondrial permeability transition (MPT). The mitochondrial cAMP effects involve neither protein kinase A, Epac2 nor the mitochondrial Na+/Ca2+ exchanger. In addition, in mitochondria isolated from failing rat hearts, stimulation of the mitochondrial cAMP pathway by HCO3− rescued the sensitization of mitochondria to Ca2+-induced MPT. Thus, our study identifies a link between mitochondrial cAMP, mitochondrial metabolism and cell death in the heart, which is independent of cytosolic cAMP signaling. Our results might have implications for therapeutic prevention of cell death in cardiac pathologies. PMID:27100892

  9. Dimethyl fumarate activates the prostaglandin EP2 receptor and stimulates cAMP signaling in human peripheral blood mononuclear cells.

    PubMed

    Fiedler, Sarah E; Kerns, Amelia R; Tsang, Catherine; Tsang, Vivian; Bourdette, Dennis; Salinthone, Sonemany

    2016-06-17

    Dimethyl fumarate (DMF) was recently approved by the FDA for the treatment of relapsing remitting MS. The pathology of MS is a result of both immune dysregulation and oxidative stress induced damage, and DMF is believed to have therapeutic effects on both of these processes. However, the mechanisms of action of DMF are not fully understood. To determine if DMF is able to activate signaling cascades that affect immune dysregulation, we treated human peripheral blood mononuclear cells with DMF. We discovered that DMF stimulates cyclic adenosine monophosphate (cAMP) production after 1 min treatment in vitro. cAMP is a small molecule second messenger that has been shown to modulate immune response. Using pharmacological inhibitors, we determined that adenylyl cyclase mediates DMF induced cAMP production; DMF activated the prostaglandin EP2 receptor to produce cAMP. This response was not due to increased endogenous production of prostaglandin E2 (PGE2), but was enhanced by addition of exogenous PGE2. Furthermore, we determined that the bioactive metabolite of DMF, monomethyl fumarate (MMF), also stimulates cAMP production. These novel findings suggest that DMF may provide protection against MS by inhibiting immune cell function via the cAMP signaling pathway. PMID:27157139

  10. Spinal Activation of the cAMP-PKA Pathway Induces Respiratory Motor Recovery Following High Cervical Spinal Cord Injury

    PubMed Central

    Kajana, S.; Goshgarian, H.G.

    2008-01-01

    The present study investigated the involvement of the adenosine 3’5’-cyclic monophosphate-dependent protein kinase A (cAMP-PKA) pathway in the activation of the crossed phrenic pathways after left C2 spinal cord hemisection. Experiments were conducted on left C2 spinal cord hemisected, anesthetized, vagotomized, pancuronium paralyzed, and artificially ventilated male Sprague-Dawley rats. One week post-injury, the ipsilateral phrenic nerve exhibited no respiratory-related activity indicating a functionally complete hemisection. Intrathecal spinal cord administration of the cAMP analog, 8-Br-cAMP at the level of the phrenic nucleus resulted in an enhancement of contralateral phrenic nerve output and a restoration of respiratory-related activity in the phrenic nerve ipsilateral to the hemisection. Furthermore, pretreatment with Rp-8-Br-cAMP, a PKA inhibitor, abolished the effects of 8-Br-cAMP. These results suggest that PKA activation is necessary for the cAMP-mediated respiratory recovery following high cervical spinal cord injury and that activation of intracellular signaling cascades may represent an important strategy for improving respiratory function after spinal cord injury. PMID:18656458

  11. Cyclic AMP in cervical mucus.

    PubMed

    Póvoa, H; Figueira, D R; Campos da Paz, A; Spichler, E R; Lopes, E R

    1981-01-01

    Cyclic adenosine monophosphate normally stimulates motility of spermatozoa. Its concentration in cervical mucus was studied by an isotopic competitive method in 15 normal women aged between 20 and 50 years. Values were very high, particularly in the periovulatory period, with a mean (+/-SD) value of 167.90 +/- 154.96 nmol/l. These are very high when compared with values in other biological fluids (blood serum and urine).

  12. Changes in phosphorylation of adenosine phosphate and redox state of nicotinamide-adenine dinucleotide (phosphate) in Geobacter sulfurreducens in response to electron acceptor and anode potential variation.

    PubMed

    Rose, Nicholas D; Regan, John M

    2015-12-01

    Geobacter sulfurreducens is one of the dominant bacterial species found in biofilms growing on anodes in bioelectrochemical systems. The intracellular concentrations of reduced and oxidized forms of nicotinamide-adenine dinucleotide (NADH and NAD(+), respectively) and nicotinamide-adenine dinucleotide phosphate (NADPH and NADP(+), respectively) as well as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were measured in G. sulfurreducens using fumarate, Fe(III)-citrate, or anodes poised at different potentials (110, 10, -90, and -190 mV (vs. SHE)) as the electron acceptor. The ratios of CNADH/CNAD+ (0.088±0.022) and CNADPH/CNADP+ (0.268±0.098) were similar under all anode potentials tested and with Fe(III)-citrate (reduced extracellularly). Both ratios significantly increased with fumarate as the electron acceptor (0.331±0.094 for NAD and 1.96±0.37 for NADP). The adenylate energy charge (the fraction of phosphorylation in intracellular adenosine phosphates) was maintained near 0.47 under almost all conditions. Anode-growing biofilms demonstrated a significantly higher molar ratio of ATP/ADP relative to suspended cultures grown on fumarate or Fe(III)-citrate. These results provide evidence that the cellular location of reduction and not the redox potential of the electron acceptor controls the intracellular redox potential in G. sulfurreducens and that biofilm growth alters adenylate phosphorylation.

  13. Polyphosphate, cyclic AMP, guanosine tetraphosphate, and c-di-GMP reduce in vitro Lon activity.

    PubMed

    Osbourne, Devon O; Soo, Valerie W C; Konieczny, Igor; Wood, Thomas K

    2014-01-01

    Lon protease is conserved from bacteria to humans and regulates cellular processes by degrading different classes of proteins including antitoxins, transcriptional activators, unfolded proteins, and free ribosomal proteins. Since we found that Lon has several putative cyclic diguanylate (c-di-GMP) binding sites and since Lon binds polyphosphate (polyP) and lipid polysaccharide, we hypothesized that Lon has an affinity for phosphate-based molecules that might regulate its activity. Hence we tested the effect of polyP, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), guanosine tetraphosphate (ppGpp), c-di-GMP, and GMP on the ability of Lon to degrade α-casein. Inhibition of in vitro Lon activity occurred for polyP, cAMP, ppGpp, and c-di-GMP. We also demonstrated by HPLC that Lon is able to bind c-di-GMP. Therefore, four cell signals were found to regulate the activity of Lon protease.

  14. Polyphosphate, cyclic AMP, guanosine tetraphosphate, and c-di-GMP reduce in vitro Lon activity

    PubMed Central

    Osbourne, Devon O; Soo, Valerie WC; Konieczny, Igor; Wood, Thomas K

    2014-01-01

    Lon protease is conserved from bacteria to humans and regulates cellular processes by degrading different classes of proteins including antitoxins, transcriptional activators, unfolded proteins, and free ribosomal proteins. Since we found that Lon has several putative cyclic diguanylate (c-di-GMP) binding sites and since Lon binds polyphosphate (polyP) and lipid polysaccharide, we hypothesized that Lon has an affinity for phosphate-based molecules that might regulate its activity. Hence we tested the effect of polyP, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), guanosine tetraphosphate (ppGpp), c-di-GMP, and GMP on the ability of Lon to degrade α-casein. Inhibition of in vitro Lon activity occurred for polyP, cAMP, ppGpp, and c-di-GMP. We also demonstrated by HPLC that Lon is able to bind c-di-GMP. Therefore, four cell signals were found to regulate the activity of Lon protease. PMID:24874800

  15. Compartmentalisation of cAMP-dependent signalling in blood platelets: The role of lipid rafts and actin polymerisation.

    PubMed

    Raslan, Zaher; Naseem, Khalid M

    2015-01-01

    Prostacyclin (PGI2) inhibits blood platelets through the activation of membrane adenylyl cyclases (ACs) and cyclic adenosine 3',5'-monophosphate (cAMP)-mediated signalling. However, the molecular mechanism controlling cAMP signalling in blood platelet remains unclear, and in particular how individual isoforms of AC and protein kinase A (PKA) are coordinated to target distinct substrates in order to modulate platelet activation. In this study, we demonstrate that lipid rafts and the actin cytoskeleton may play a key role in regulating platelet responses to cAMP downstream of PGI2. Disruption of lipid rafts with methyl-beta-cyclodextrin (MβCD) increased platelet sensitivity to PGI2 and forskolin, a direct AC cyclase activator, resulting in greater inhibition of collagen-stimulated platelet aggregation. In contrast, platelet inhibition by the direct activator of PKA, 8-CPT-6-Phe-cAMP was unaffected by MβCD treatment. Consistent with the functional data, lipid raft disruption increased PGI2-stimulated cAMP formation and proximal PKA-mediated signalling events. Platelet inhibition, cAMP formation and phosphorylation of PKA substrates in response to PGI2 were also increased in the presence of cytochalasin D, indicating a role for actin cytoskeleton in signalling in response to PGI2. A potential role for lipid rafts in cAMP signalling is strengthened by our finding that a pool of ACV/VI and PKA was partitioned into lipid rafts. Our data demonstrate partial compartmentalisation of cAMP signalling machinery in platelets, where lipid rafts and the actin cytoskeleton regulate the inhibitory effects induced by PGI2. The increased platelet sensitivity to cAMP-elevating agents signalling upon raft and cytoskeleton disruption suggests that these compartments act to restrain basal cAMP signalling.

  16. AMP-activated protein kinase mediates mitochondrial fission in response to energy stress

    PubMed Central

    Courchet, Julien; Lewis, Tommy L.; Losón, Oliver C.; Hellberg, Kristina; Young, Nathan P.; Chen, Hsiuchen; Polleux, Franck; Chan, David C.; Shaw, Reuben J.

    2016-01-01

    Mitochondria undergo fragmentation in response to electron transport chain (ETC) poisons and mitochondrial DNA–linked disease mutations, yet how these stimuli mechanistically connect to the mitochondrial fission and fusion machinery is poorly understood. We found that the energy-sensing adenosine monophosphate (AMP)–activated protein kinase (AMPK) is genetically required for cells to undergo rapid mitochondrial fragmentation after treatment with ETC inhibitors. Moreover, direct pharmacological activation of AMPK was sufficient to rapidly promote mitochondrial fragmentation even in the absence of mitochondrial stress. A screen for substrates of AMPK identified mitochondrial fission factor (MFF), a mitochondrial outer-membrane receptor for DRP1, the cytoplasmic guanosine triphosphatase that catalyzes mitochondrial fission. Nonphosphorylatable and phosphomimetic alleles of the AMPK sites in MFF revealed that it is a key effector of AMPK-mediated mitochondrial fission. PMID:26816379

  17. Adenosine Kinase: Exploitation for Therapeutic Gain

    PubMed Central

    2013-01-01

    Adenosine kinase (ADK; EC 2.7.1.20) is an evolutionarily conserved phosphotransferase that converts the purine ribonucleoside adenosine into 5′-adenosine-monophosphate. This enzymatic reaction plays a fundamental role in determining the tone of adenosine, which fulfills essential functions as a homeostatic and metabolic regulator in all living systems. Adenosine not only activates specific signaling pathways by activation of four types of adenosine receptors but it is also a primordial metabolite and regulator of biochemical enzyme reactions that couple to bioenergetic and epigenetic functions. By regulating adenosine, ADK can thus be identified as an upstream regulator of complex homeostatic and metabolic networks. Not surprisingly, ADK dysfunction is involved in several pathologies, including diabetes, epilepsy, and cancer. Consequently, ADK emerges as a rational therapeutic target, and adenosine-regulating drugs have been tested extensively. In recent attempts to improve specificity of treatment, localized therapies have been developed to augment adenosine signaling at sites of injury or pathology; those approaches include transplantation of stem cells with deletions of ADK or the use of gene therapy vectors to downregulate ADK expression. More recently, the first human mutations in ADK have been described, and novel findings suggest an unexpected role of ADK in a wider range of pathologies. ADK-regulating strategies thus represent innovative therapeutic opportunities to reconstruct network homeostasis in a multitude of conditions. This review will provide a comprehensive overview of the genetics, biochemistry, and pharmacology of ADK and will then focus on pathologies and therapeutic interventions. Challenges to translate ADK-based therapies into clinical use will be discussed critically. PMID:23592612

  18. Intracellular calcium measurements with arsenazo III during cyclic AMP injections into molluscan neurons.

    PubMed

    Hockberger, P; Connor, J A

    1983-02-18

    Injections of cyclic adenosine monophosphate into molluscan neurons often produce a transient membrane depolarization. By using the calcium indicator dye arsenazo III, it was found that cyclic nucleotide injections into neurons of Archidoris montereyensis resulted in elevation of internal calcium concentrations. However, this was demonstrated to be a secondary consequence of an induced increase in membrane sodium permeability, and not due to any direct effect of cyclic adenosine monophosphate on cellular calcium influx or internal calcium regulating processes.

  19. Effects of papaverine and vasointestinal polypeptide on penile and vascular cAMP and cGMP in control and diabetic animals: an in vitro study.

    PubMed

    Miller, M A; Morgan, R J; Thompson, C S; Mikhailidis, D P; Jeremy, J Y

    1995-06-01

    Adenosine 3'5'-cyclic monophosphate (cAMP) and guanosine 3'5'-cyclic monophosphate (cGMP) mediate penile erection. We have previously established that adenylate and guanylate cyclase activity is elevated in the diabetic rat penis and aorta. This study investigates the action of papaverine and vasoactive intestinal polypeptide (VIP) on these cyclases. The aortae and penes of Sprague Dawley rats (n = 7) were stimulated with VIP and papaverine. Diabetes mellitus (DM) was induced in Sprague Dawley rats (n = 7) with streptozotocin and the penile and aortic tissues were treated with VIP. The penes, aortae and carotid arteries of New Zealand White rabbits were similarly processed. cAMP and cGMP generation was measured by radioimmunoassay. In all tissues: VIP stimulated cAMP synthesis; VIP did not increase cGMP levels; papaverine was without effect on either cAMP or cGMP synthesis. VIP-stimulated cAMP was significantly enhanced in the diabetic rat penis and aorta; there was also a significant elevation in the basal levels of cGMP in these tissues. These data: (1) consolidate that cAMP is a mediator of penile erection, (2) indicate that papaverine and VIP elicit erection by different mechanisms, (3) suggest that an enhanced penile capacity to generate cAMP in DM may constitute an adaptive response to counteract the previously reported reduction in VIP content and VIP receptors, and (4) indicate that the penile and vascular tissues of the rabbit respond in a similar manner to VIP and papaverine. PMID:7496446

  20. cAMP/PKA signaling defects in tumors: genetics and tissue-specific pluripotential cell-derived lesions in human and mouse.

    PubMed

    Stratakis, Constantine A

    2013-05-22

    In the last few years, bench and clinical studies led to significant new insight into how cyclic adenosine monophosphate (cAMP) signaling, the molecular pathway that had been identified in the early 2000s as the one involved in most benign cortisol-producing adrenal hyperplasias, affects adrenocortical growth and development, as well as tumor formation. A major discovery was the identification of tissue-specific pluripotential cells (TSPCs) as the culprit behind tumor formation not only in the adrenal, but also in bone. Discoveries in animal studies complemented a number of clinical observations in patients. Gene identification continued in parallel with mouse and other studies on the cAMP signaling and other pathways. PMID:23485729

  1. Selective enhancement of wnt4 expression by cyclic AMP-associated cooperation between rat central astrocytes and microglia.

    PubMed

    Ohnishi, Masatoshi; Urasaki, Tomoka; Ochiai, Hiroyuki; Matsuoka, Kohei; Takeo, Shin; Harada, Tomoki; Ohsugi, Yoshihito; Inoue, Atsuko

    2015-11-13

    The wnt protein family has important members involved in cell differentiation, proliferation and plasticity expression; however, little is known about its biosynthesis processes. On the other hand, an increase in the intracerebral cyclic adenosine 3', 5'-monophosphate (cAMP) level leads to synaptic plasticity via the de novo synthesis of any protein. Here, the effect of dibutyryl cAMP (dbcAMP), a membrane permeability cAMP analog, on the wnt family was investigated in rat primary-cultured glial cells containing astrocytes and microglia. Among wnt3a, 4, 5a, 7a and 11 mRNA, only wnt4 expression was increased by longer treatment (24 h), compared with short treatment (2 h), with dbcAMP in a concentration-dependent manner, and its effect reached statistical significance at 1 mM. In cultures of isolated astrocytes or microglia, wnt4 expression was not affected by 1 mM dbcAMP for 24 h, and microglial wnt4 protein was undetectable even when cells were treated with the drug. Mixed glial cells treated for 24 h with 1 mM dbcAMP showed significantly increased wnt4 protein, as well as mRNA. Immunofluorescence manifested that cells that expressed wnt4 protein were astrocytes, but not microglia. Intraperitoneal injection of 1.25 mg/kg rolipram, a phosphodiesterase (PDE) IV inhibitor that can pass through the blood brain barrier and inhibits cAMP degradation specifically, showed a tendency to increase wnt4 expression in the adult rat brain after 24 h, and the increases in wnt4 mRNA and protein levels reached statistical significance in the hippocampus and striatum, respectively. This is the first finding to help elucidate the selective biosynthesis of central wnt4 through cAMP-stimulated microglia and astrocytes interaction.

  2. Reduced expression of cytochrome oxidases largely explains cAMP inhibition of aerobic growth in Shewanella oneidensis

    PubMed Central

    Yin, Jianhua; Meng, Qiu; Fu, Huihui; Gao, Haichun

    2016-01-01

    Inhibition of bacterial growth under aerobic conditions by elevated levels of cyclic adenosine 3′,5′-monophosphate (cAMP), first revealed more than 50 years ago, was attributed to accumulation of toxic methylglyoxal (MG). Here, we report a Crp-dependent mechanism rather than MG accumulation that accounts for the phenotype in Shewanella oneidensis, an emerging research model for the bacterial physiology. We show that a similar phenotype can be obtained by removing CpdA, a cAMP phosphodiesterase that appears more effective than its Escherichia coli counterpart. Although production of heme c and cytochromes c is correlated well with cAMP levels, neither is sufficient for the retarded growth. Quantities of overall cytochromes c increased substantially in the presence of elevated cAMP, a phenomenon resembling cells respiring on non-oxygen electron acceptors. In contrast, transcription of Crp-dependent genes encoding both cytochromes bd and cbb3 oxidases is substantially repressed under the same condition. Overall, our results suggest that cAMP of elevated levels drives cells into a low-energetic status, under which aerobic respiration is inhibited. PMID:27076065

  3. Increases in cAMP, MAPK Activity and CREB Phosphorylation during REM Sleep: Implications for REM Sleep and Memory Consolidation

    PubMed Central

    Luo, Jie; Phan, Trongha X.; Yang, Yimei; Garelick, Michael G.; Storm, Daniel R.

    2013-01-01

    The cyclic adenosine monophosphate (cAMP), mitogen-activated protein kinase (MAPK) and cAMP response element-binding protein (CREB) transcriptional pathway is required for consolidation of hippocampus-dependent memory. In mice, this pathway undergoes a circadian oscillation required for memory persistence that reaches a peak during the daytime. Since mice exhibit polyphasic sleep patterns during the day, this suggested the interesting possibility that cAMP, MAPK activity and CREB phosphorylation may be elevated during sleep. Here, we report that cAMP, phospho-p44/42 MAPK and phospho-CREB are higher in rapid eye movement (REM) sleep compared to awake mice but are not elevated in non-rapid eye movement (NREM) sleep. This peak of activity during REM sleep does not occur in mice lacking calmodulin-stimulated adenylyl cyclases, a mouse strain that learns but cannot consolidate hippocampus-dependent memory. We conclude that a preferential increase in cAMP, MAPK activity and CREB phosphorylation during REM sleep may contribute to hippocampus-dependent memory consolidation. PMID:23575844

  4. Increases in cAMP, MAPK activity, and CREB phosphorylation during REM sleep: implications for REM sleep and memory consolidation.

    PubMed

    Luo, Jie; Phan, Trongha X; Yang, Yimei; Garelick, Michael G; Storm, Daniel R

    2013-04-10

    The cyclic adenosine monophosphate (cAMP), mitogen-activated protein kinase (MAPK), and cAMP response element-binding protein (CREB) transcriptional pathway is required for consolidation of hippocampus-dependent memory. In mice, this pathway undergoes a circadian oscillation required for memory persistence that reaches a peak during the daytime. Because mice exhibit polyphasic sleep patterns during the day, this suggested the interesting possibility that cAMP, MAPK activity, and CREB phosphorylation may be elevated during sleep. Here, we report that cAMP, phospho-p44/42 MAPK, and phospho-CREB are higher in rapid eye movement (REM) sleep compared with awake mice but are not elevated in non-REM sleep. This peak of activity during REM sleep does not occur in mice lacking calmodulin-stimulated adenylyl cyclases, a mouse strain that learns but cannot consolidate hippocampus-dependent memory. We conclude that a preferential increase in cAMP, MAPK activity, and CREB phosphorylation during REM sleep may contribute to hippocampus-dependent memory consolidation. PMID:23575844

  5. cAMP levels in fast- and slow-twitch skeletal muscle after an acute bout of aerobic exercise

    NASA Technical Reports Server (NTRS)

    Sheldon, A.; Booth, F. W.; Kirby, C. R.

    1993-01-01

    The present study examined whether exercise duration was associated with elevated and/or sustained elevations of postexercise adenosine 3',5'-cyclic monophosphate (cAMP) by measuring cAMP levels in skeletal muscle for up to 4 h after acute exercise bouts of durations that are known to either produce (60 min) or not produce (10 min) mitochondrial proliferation after chronic training. Treadmill-acclimatized, but untrained, rats were run at 22 m/min for 0 (control), 10, or 60 min and were killed at various postexercise (0, 0.5, 1, 2, and 4 h) time points. Fast-twitch white and red (quadriceps) and slow-twitch (soleus) muscles were quickly excised, frozen in liquid nitrogen, and assayed for cAMP with a commercial kit. Unexpectedly, cAMP contents in all three muscles were similar to control (nonexercise) at most (21 of 30) time points after a single 10- or 60-min run. Values at 9 of 30 time points were significantly different from control (P < 0.05); i.e., 3 time points were significantly higher than control and 6 were significantly less than control. These data suggest that the cAMP concentration of untrained skeletal muscle after a single bout of endurance-type exercise is not, by itself, associated with exercise duration.

  6. Reduced expression of cytochrome oxidases largely explains cAMP inhibition of aerobic growth in Shewanella oneidensis.

    PubMed

    Yin, Jianhua; Meng, Qiu; Fu, Huihui; Gao, Haichun

    2016-01-01

    Inhibition of bacterial growth under aerobic conditions by elevated levels of cyclic adenosine 3',5'-monophosphate (cAMP), first revealed more than 50 years ago, was attributed to accumulation of toxic methylglyoxal (MG). Here, we report a Crp-dependent mechanism rather than MG accumulation that accounts for the phenotype in Shewanella oneidensis, an emerging research model for the bacterial physiology. We show that a similar phenotype can be obtained by removing CpdA, a cAMP phosphodiesterase that appears more effective than its Escherichia coli counterpart. Although production of heme c and cytochromes c is correlated well with cAMP levels, neither is sufficient for the retarded growth. Quantities of overall cytochromes c increased substantially in the presence of elevated cAMP, a phenomenon resembling cells respiring on non-oxygen electron acceptors. In contrast, transcription of Crp-dependent genes encoding both cytochromes bd and cbb3 oxidases is substantially repressed under the same condition. Overall, our results suggest that cAMP of elevated levels drives cells into a low-energetic status, under which aerobic respiration is inhibited. PMID:27076065

  7. RNA initiation with dinucleoside monophosphates during transcription of bacteriophage T4 DNA with RNA polymerase of Escherichia coli.

    PubMed

    Hoffman, D J; Niyogi, S K

    1973-02-01

    The effects of dinucleoside monophosphates on the transcription of phage T4 DNA by E. coli RNA polymerase have been examined at various concentrations of the sigma subunit and extremely low concentration of ribonucleoside triphosphate. The following conclusions were reached: (i) Labeled specific dinucleoside monophosphates are incorporated as chain initiators. (ii) When the ratio of sigma factor to core enzyme is small, there is a general stimulation by most 5'-guanosyl dinucleoside monophosphates. (iii) When the ratio is increased or holoenzyme is present, ApU, CpA, UpA, and GpU are the most effective stimulators. (iv) At high concentrations of sigma factor, only certain adenosine-containing dinucleoside monophosphates (ApU, CpA, UpA, and ApA) stimulate the reaction. (v) Competition hybridization studies indicate that the RNAs stimulated by dinucleoside monophosphates (ApU, CpA, UpA, and GpU) are of the T4 "early" type. (vi) Studies involving both combinations of stimulatory dinucleoside monophosphates and competitive effects of these compounds on chain initiation by ATP and GTP suggest that the stimulatory dinucleoside monophosphates act as chain initiators and may recognize part of a continuous sequence in a promoter region. Studies based on the incorporation of (3)H-labeled stimulatory dinucleoside monophosphates support the above conclusions.

  8. Expression of nitric oxide synthase in rat glomerular mesangial cells mediated by cyclic AMP.

    PubMed Central

    Mühl, H.; Kunz, D.; Pfeilschifter, J.

    1994-01-01

    1. Treatment of rat mesangial cells with interleukin 1 beta (IL-1 beta) or tumour necrosis factor alpha (TNF alpha) has been shown to induce a macrophage-type of nitric oxide (NO) synthase. Here we report that adenosine 3':5'-cyclic monophosphate (cyclic AMP) is another mediator that triggers induction of NO synthase in mesangial cells. 2. Incubation of mesangial cells with the beta-adrenoceptor agonist, salbutamol, forskolin or cholera toxin, which all activate adenylate cyclase and increase intracellular cyclic AMP concentration, increased nitrite formation in a dose-dependent manner. Likewise, the addition of the membrane-permeable cyclic AMP analogue, N6, 0-2'-dibutyryladenosine 3',5'-phosphate (Bt2 cyclic AMP) or the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine enhanced NO synthase activity in a dose-dependent manner. 3. There was a lag period of about 8 h before a significantly enhanced secretion of nitrite could be detected upon exposure of cells to forskolin and for maximal stimulation, forskolin had to be present during the whole incubation period. 4. Treatment of mesangial cells with actinomycin D, cycloheximide or dexamethasone completely suppressed forskolin-stimulated NO-synthase activity, thus demonstrating that transcription and protein synthesis are necessary for nitrite formation. 5. Bt2 cyclic AMP, the most potent inducer of nitrite production, increased NO synthase mRNA levels in mesangial cells in a time- and dose-dependent fashion. Dexamethasone completely inhibited the increase of NO synthase mRNA in response to Bt2 cyclic AMP. 6. Combination of Bt2 cyclic AMP and IL-1 beta or TNF alpha revealed a strong synergy in terms of nitrite formation.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 7 Figure 8 Figure 9 PMID:7518300

  9. Effect of the dB-c-AMP and forskolin on /sup 45/Ca influx, net Ca uptake and tension on rabbit aortic smooth muscle

    SciTech Connect

    Not Available

    1986-03-01

    The effect of dibutiryl-adenosine-3',5'-cyclic-monophosphate (dB-c-AMP) and forskolin on aortic tension and /sup 45/Ca influx were measured. dB-c-AMP reduced both the rate of force development and the maximal tension achieved in solutions containing various K/sup +/ concentrations. Stimulated /sup 45/Ca influx was also reduced however to a lesser extent than was the tension. Forskolin showed more marked effects of a similar nature. Thus, both these agents which increase intracellular c-AMP caused a rightward shift in the curve expressing force(ordinate) as a function of Ca influx (abscissa). Consequently, they found that dB-c-AMP stimulated more net Ca to be taken up by the sarcoplasmic reticulum(SR) at the same influx rate. The conclusion that c-AMP produced these effects by stimulating Ca uptake into the superficial SR was supported by the finding that dB-c-AMP increased the amount of Ca taken up into a caffeine releasable fraction.

  10. Dynamic coupling between the LID and NMP domain motions in the catalytic conversion of ATP and AMP to ADP by adenylate kinase

    NASA Astrophysics Data System (ADS)

    Jana, Biman; Adkar, Bharat V.; Biswas, Rajib; Bagchi, Biman

    2011-01-01

    The catalytic conversion of adenosine triphosphate (ATP) and adenosine monophosphate (AMP) to adenosine diphosphate (ADP) by adenylate kinase (ADK) involves large amplitude, ligand induced domain motions, involving the opening and the closing of ATP binding domain (LID) and AMP binding domain (NMP) domains, during the repeated catalytic cycle. We discover and analyze an interesting dynamical coupling between the motion of the two domains during the opening, using large scale atomistic molecular dynamics trajectory analysis, covariance analysis, and multidimensional free energy calculations with explicit water. Initially, the LID domain must open by a certain amount before the NMP domain can begin to open. Dynamical correlation map shows interesting cross-peak between LID and NMP domain which suggests the presence of correlated motion between them. This is also reflected in our calculated two-dimensional free energy surface contour diagram which has an interesting elliptic shape, revealing a strong correlation between the opening of the LID domain and that of the NMP domain. Our free energy surface of the LID domain motion is rugged due to interaction with water and the signature of ruggedness is evident in the observed root mean square deviation variation and its fluctuation time correlation functions. We develop a correlated dynamical disorder-type theoretical model to explain the observed dynamic coupling between the motion of the two domains in ADK. Our model correctly reproduces several features of the cross-correlation observed in simulations.

  11. Dynamic coupling between the LID and NMP domain motions in the catalytic conversion of ATP and AMP to ADP by adenylate kinase.

    PubMed

    Jana, Biman; Adkar, Bharat V; Biswas, Rajib; Bagchi, Biman

    2011-01-21

    The catalytic conversion of adenosine triphosphate (ATP) and adenosine monophosphate (AMP) to adenosine diphosphate (ADP) by adenylate kinase (ADK) involves large amplitude, ligand induced domain motions, involving the opening and the closing of ATP binding domain (LID) and AMP binding domain (NMP) domains, during the repeated catalytic cycle. We discover and analyze an interesting dynamical coupling between the motion of the two domains during the opening, using large scale atomistic molecular dynamics trajectory analysis, covariance analysis, and multidimensional free energy calculations with explicit water. Initially, the LID domain must open by a certain amount before the NMP domain can begin to open. Dynamical correlation map shows interesting cross-peak between LID and NMP domain which suggests the presence of correlated motion between them. This is also reflected in our calculated two-dimensional free energy surface contour diagram which has an interesting elliptic shape, revealing a strong correlation between the opening of the LID domain and that of the NMP domain. Our free energy surface of the LID domain motion is rugged due to interaction with water and the signature of ruggedness is evident in the observed root mean square deviation variation and its fluctuation time correlation functions. We develop a correlated dynamical disorder-type theoretical model to explain the observed dynamic coupling between the motion of the two domains in ADK. Our model correctly reproduces several features of the cross-correlation observed in simulations.

  12. Rolipram stimulates angiogenesis and attenuates neuronal apoptosis through the cAMP/cAMP-responsive element binding protein pathway following ischemic stroke in rats

    PubMed Central

    HU, SHOUYE; CAO, QINGWEN; XU, PENG; JI, WENCHEN; WANG, GANG; ZHANG, YUELIN

    2016-01-01

    Rolipram, a phosphodiesterase-4 inhibitor, can activate the cyclic adenosine monophosphate (cAMP)/cAMP-responsive element binding protein (CREB) pathway to facilitate functional recovery following ischemic stroke. However, to date, the effects of rolipram on angiogenesis and cerebral ischemia-induced neuronal apoptosis are yet to be fully elucidated. In this study, the aim was to reveal the effect of rolipram on the angiogenesis and neuronal apoptosis following brain cerebral ischemia. Rat models of ischemic stroke were established following transient middle cerebral artery occlusion and rolipram was administered for three, seven and 14 days. The results were examined using behavioral tests, triphenyl tetrazolium chloride staining, immunostaining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) to evaluate the effects of rolipram therapy on functional outcome, angiogenesis and apoptosis. Western blot analysis was used to show the phosphorylated- (p-)CREB protein level in the ischemic hemisphere. The rolipram treatment group exhibited a marked reduction in infarct size and modified neurological severity score compared with the vehicle group, and rolipram treatment significantly promoted the microvessel density in the ischemic boundary region and increased p-CREB protein levels in the ischemic hemisphere. Furthermore, a significant reduction in the number of TUNEL-positive cells was observed in the rolipram group compared with the vehicle group. These findings suggest that rolipram has the ability to attenuate cerebral ischemic injury, stimulate angiogenesis and reduce neuronal apoptosis though the cAMP/CREB pathway. PMID:26998028

  13. Cyclic AMP synergizes with butyrate in promoting β-defensin 9 expression in chickens.

    PubMed

    Sunkara, Lakshmi T; Zeng, Xiangfang; Curtis, Amanda R; Zhang, Guolong

    2014-02-01

    Host defense peptides (HDP) have both microbicidal and immunomodulatory properties. Specific induction of endogenous HDP synthesis has emerged as a novel approach to antimicrobial therapy. Cyclic adenosine monophosphate (cAMP) and butyrate have been implicated in HDP induction in humans. However, the role of cAMP signaling and the possible interactions between cAMP and butyrate in regulating HDP expression in other species remain unknown. Here we report that activation of cAMP signaling induces HDP gene expression in chickens as exemplified by β-defensin 9 (AvBD9). We further showed that, albeit being weak inducers, cAMP agonists synergize strongly with butyrate or butyrate analogs in AvBD9 induction in macrophages and primary jejunal explants. Additionally, oral supplementation of forskolin, an adenylyl cyclase agonist in the form of a Coleus forskohlii extract, was found to induce AvBD9 expression in the crop of chickens. Furthermore, feeding with both forskolin and butyrate showed an obvious synergy in triggering AvBD9 expression in the crop and jejunum of chickens. Surprisingly, inhibition of the MEK-ERK mitogen-activated protein kinase (MAPK) pathway augmented the butyrate-FSK synergy, whereas blocking JNK or p38 MAPK pathway significantly diminished AvBD9 induction in chicken macrophages and jejunal explants in response to butyrate and FSK individually or in combination. Collectively, these results suggest the potential for concomitant use of butyrate and cAMP signaling activators in enhancing HDP expression, innate immunity, and disease resistance in both animals and humans.

  14. RNA aptamer based electrochemical biosensor for sensitive and selective detection of cAMP.

    PubMed

    Zhao, Fulin; Xie, Qingyun; Xu, Mingfei; Wang, Shouyu; Zhou, Jiyong; Liu, Fei

    2015-04-15

    Cyclic adenosine monophosphate (cAMP) is an important small biological molecule associated with the healthy state of living organism. In order to realize highly sensitive and specific detection of cAMP, here an RNA aptamer and electrochemical impedance spectroscopy (EIS) based biosensor enhanced by gold nanoparticles electrodeposited on the surface of gold electrode is designed. The designed aptasensor has a wide effective measuring range from 50pM to 250pM with a detection limit of 50pM in PBS buffer, and an effective measuring range from 50nM to 1μM with a detection limit of 50nM in serum. The designed biosensor is also able to detect cAMP with high sensitivity, specificity, and stability. Since the biosensor can be easily fabricated with low cost and repeatedly used for at least two times, it owns great potential in wide application fields such as clinical test and food inspection, etc.

  15. Regulation of Mct1 by cAMP-dependent internalization in rat brain endothelial cells.

    PubMed

    Smith, Jeffrey P; Uhernik, Amy L; Li, Lun; Liu, Zejian; Drewes, Lester R

    2012-10-22

    In the cerebrovascular endothelium, monocarboxylic acid transporter 1 (Mct1) controls blood-brain transport of short chain monocarboxylic and keto acids, including pyruvate and lactate, to support brain energy metabolism. Mct1 function is acutely decreased in rat brain cerebrovascular endothelial cells by β-adrenergic signaling through cyclic adenosine monophosphate (cAMP); however, the mechanism for this acute reduction in transport capacity is unknown. In this report, we demonstrate that cAMP induces the dephosphorylation and internalization of Mct1 from the plasma membrane into caveolae and early endosomes in the RBE4 rat brain cerebrovascular endothelial cell line. Additionally, we provide evidence that Mct1 constitutively cycles through clathrin vesicles and recycling endosomes in a pathway that is not dependent upon cAMP signaling in these cells. Our results are important because they show for the first time the regulated and unregulated vesicular trafficking of Mct1 in cerebrovascular endothelial cells; processes which have significance for better understanding normal brain energy metabolism, and the etiology and potential therapeutic approaches to treating brain diseases, such as stroke, in which lactic acidosis is a key component.

  16. cAMP-Inhibits Cytoplasmic Phospholipase A₂ and Protects Neurons against Amyloid-β-Induced Synapse Damage.

    PubMed

    Bate, Clive; Williams, Alun

    2015-01-01

    A key event in Alzheimer's disease (AD) is the production of amyloid-β (Aβ) peptides and the loss of synapses. In cultured neurons Aβ triggered synapse damage as measured by the loss of synaptic proteins. α-synuclein (αSN), aggregates of which accumulate in Parkinson's disease, also caused synapse damage. Synapse damage was associated with activation of cytoplasmic phospholipase A₂ (cPLA₂), an enzyme that regulates synapse function and structure, and the production of prostaglandin (PG) E₂. In synaptosomes PGE₂ increased concentrations of cyclic adenosine monophosphate (cAMP) which suppressed the activation of cPLA₂ demonstrating an inhibitory feedback system. Thus, Aβ/αSN-induced activated cPLA₂ produces PGE₂ which increases cAMP which in turn suppresses cPLA₂ and, hence, its own production. Neurons pre-treated with pentoxifylline and caffeine (broad spectrum phosphodiesterase (PDE) inhibitors) or the PDE4 specific inhibitor rolipram significantly increased the Aβ/αSN-induced increase in cAMP and consequently protected neurons against synapse damage. The addition of cAMP analogues also inhibited cPLA₂ and protected neurons against synapse damage. These results suggest that drugs that inhibit Aβ-induced activation of cPLA₂ and cross the blood-brain barrier may reduce synapse damage in AD. PMID:26389963

  17. cAMP-Inhibits Cytoplasmic Phospholipase A2 and Protects Neurons against Amyloid-β-Induced Synapse Damage

    PubMed Central

    Bate, Clive; Williams, Alun

    2015-01-01

    A key event in Alzheimer’s disease (AD) is the production of amyloid-β (Aβ) peptides and the loss of synapses. In cultured neurons Aβ triggered synapse damage as measured by the loss of synaptic proteins. α-synuclein (αSN), aggregates of which accumulate in Parkinson’s disease, also caused synapse damage. Synapse damage was associated with activation of cytoplasmic phospholipase A2 (cPLA2), an enzyme that regulates synapse function and structure, and the production of prostaglandin (PG) E2. In synaptosomes PGE2 increased concentrations of cyclic adenosine monophosphate (cAMP) which suppressed the activation of cPLA2 demonstrating an inhibitory feedback system. Thus, Aβ/αSN-induced activated cPLA2 produces PGE2 which increases cAMP which in turn suppresses cPLA2 and, hence, its own production. Neurons pre-treated with pentoxifylline and caffeine (broad spectrum phosphodiesterase (PDE) inhibitors) or the PDE4 specific inhibitor rolipram significantly increased the Aβ/αSN-induced increase in cAMP and consequently protected neurons against synapse damage. The addition of cAMP analogues also inhibited cPLA2 and protected neurons against synapse damage. These results suggest that drugs that inhibit Aβ-induced activation of cPLA2 and cross the blood–brain barrier may reduce synapse damage in AD. PMID:26389963

  18. Mapping the Free Energy Landscape of PKA Inhibition and Activation: A Double-Conformational Selection Model for the Tandem cAMP-Binding Domains of PKA RIα.

    PubMed

    Akimoto, Madoka; McNicholl, Eric Tyler; Ramkissoon, Avinash; Moleschi, Kody; Taylor, Susan S; Melacini, Giuseppe

    2015-01-01

    Protein Kinase A (PKA) is the major receptor for the cyclic adenosine monophosphate (cAMP) secondary messenger in eukaryotes. cAMP binds to two tandem cAMP-binding domains (CBD-A and -B) within the regulatory subunit of PKA (R), unleashing the activity of the catalytic subunit (C). While CBD-A in RIα is required for PKA inhibition and activation, CBD-B functions as a "gatekeeper" domain that modulates the control exerted by CBD-A. Preliminary evidence suggests that CBD-B dynamics are critical for its gatekeeper function. To test this hypothesis, here we investigate by Nuclear Magnetic Resonance (NMR) the two-domain construct RIα (91-379) in its apo, cAMP2, and C-bound forms. Our comparative NMR analyses lead to a double conformational selection model in which each apo CBD dynamically samples both active and inactive states independently of the adjacent CBD within a nearly degenerate free energy landscape. Such degeneracy is critical to explain the sensitivity of CBD-B to weak interactions with C and its high affinity for cAMP. Binding of cAMP eliminates this degeneracy, as it selectively stabilizes the active conformation within each CBD and inter-CBD contacts, which require both cAMP and W260. The latter is contributed by CBD-B and mediates capping of the cAMP bound to CBD-A. The inter-CBD interface is dispensable for intra-CBD conformational selection, but is indispensable for full activation of PKA as it occludes C-subunit recognition sites within CBD-A. In addition, the two structurally homologous cAMP-bound CBDs exhibit marked differences in their residual dynamics profiles, supporting the notion that conservation of structure does not necessarily imply conservation of dynamics.

  19. Mapping the Free Energy Landscape of PKA Inhibition and Activation: A Double-Conformational Selection Model for the Tandem cAMP-Binding Domains of PKA RIα

    PubMed Central

    Akimoto, Madoka; McNicholl, Eric Tyler; Ramkissoon, Avinash; Moleschi, Kody; Taylor, Susan S.; Melacini, Giuseppe

    2015-01-01

    Protein Kinase A (PKA) is the major receptor for the cyclic adenosine monophosphate (cAMP) secondary messenger in eukaryotes. cAMP binds to two tandem cAMP-binding domains (CBD-A and -B) within the regulatory subunit of PKA (R), unleashing the activity of the catalytic subunit (C). While CBD-A in RIα is required for PKA inhibition and activation, CBD-B functions as a “gatekeeper” domain that modulates the control exerted by CBD-A. Preliminary evidence suggests that CBD-B dynamics are critical for its gatekeeper function. To test this hypothesis, here we investigate by Nuclear Magnetic Resonance (NMR) the two-domain construct RIα (91–379) in its apo, cAMP2, and C-bound forms. Our comparative NMR analyses lead to a double conformational selection model in which each apo CBD dynamically samples both active and inactive states independently of the adjacent CBD within a nearly degenerate free energy landscape. Such degeneracy is critical to explain the sensitivity of CBD-B to weak interactions with C and its high affinity for cAMP. Binding of cAMP eliminates this degeneracy, as it selectively stabilizes the active conformation within each CBD and inter-CBD contacts, which require both cAMP and W260. The latter is contributed by CBD-B and mediates capping of the cAMP bound to CBD-A. The inter-CBD interface is dispensable for intra-CBD conformational selection, but is indispensable for full activation of PKA as it occludes C-subunit recognition sites within CBD-A. In addition, the two structurally homologous cAMP-bound CBDs exhibit marked differences in their residual dynamics profiles, supporting the notion that conservation of structure does not necessarily imply conservation of dynamics. PMID:26618408

  20. Suppression of LPS-Induced TNF-α Production in Macrophages by cAMP Is Mediated by PKA-AKAP95-p105

    PubMed Central

    Wall, Estelle A.; Zavzavadjian, Joelle R.; Chang, Mi Sook; Randhawa, Baljinder; Zhu, Xiaocui; Hsueh, Robert C.; Liu, Jamie; Driver, Adrienne; Bao, Xiaoyan Robert; Sternweis, Paul C.; Simon, Melvin I.; Fraser, Iain D. C.

    2009-01-01

    The activation of macrophages through Toll-like receptor (TLR) pathways leads to the production of a broad array of cytokines and mediators that coordinate the immune response. The inflammatory potential of this response can be reduced by compounds, such as prostaglandin E2, that induce the production of cyclic adenosine monophosphate (cAMP). Through experiments with cAMP analogs and multigene RNA interference (RNAi), we showed that key anti-inflammatory effects of cAMP were mediated specifically by cAMP-dependent protein kinase (PKA). Selective inhibitors of PKA anchoring, time-lapse microscopy, and RNAi screening suggested that differential mechanisms of PKA action existed. We showed a specific role for A kinase-anchoring protein 95 in suppressing the expression of the gene encoding tumor necrosis factor-α, which involved phosphorylation of p105 (also known as Nfkb1) by PKA at a site adjacent to the region targeted by inhibitor of nuclear factor κB kinases. These data suggest that crosstalk between the TLR4 and cAMP pathways in macrophages can be coordinated through PKA-dependent scaffolds that localize specific pools of the kinase to distinct substrates. PMID:19531803

  1. Effect of adenosine and adenosine analogs on ( sup 14 C)aminopyrine accumulation by rabbit parietal cells

    SciTech Connect

    Ota, S.; Hiraishi, H.; Terano, A.; Mutoh, H.; Kurachi, Y.; Shimada, T.; Ivey, K.J.; Sugimoto, T. )

    1989-12-01

    Adenosine receptors that modulate adenylate cyclase activity have been identified recently in a number of tissues. Adenosine A2 receptor is stimulatory to adenylate cyclase, whereas adenosine A1 receptor is inhibitory to adenylate cyclase. We investigated the effect of adenosine and its analogs on (14C)aminopyrine accumulation by rabbit parietal cells. Rabbit gastric mucosal cells were isolated by enzyme digestion. Parietal cells were enriched by nonlinear percoll gradients. (14C)Aminopyrine accumulation was used as an indicator of acid secretion. The effect of 2-chloroadenosine on histamine-stimulated (14C)aminopyrine accumulation was studied. The effects of N-ethylcarboxamideadenosine, 2-chloroadenosine, stable analogs of adenosine, and adenosine on (14C)aminopyrine accumulation were assessed. Cyclic AMP content of parietal cells was determined by radioimmunoassay. Histamine and carbachol, known secretagogues, stimulated (14C)aminopyrine accumulation. 2-Chloroadenosine did not suppress histamine-stimulated (14C)aminopyrine accumulation. 2-Chloroadenosine, N-ethylcarboxamideadenosine, and adenosine dose dependently increased (14C)aminopyrine accumulation. The order of potency was N-ethylcarboxamideadenosine greater than 2-chloroadenosine greater than adenosine. 8-Phenyltheophylline and theophylline, adenosine-receptor antagonists, or cimetidine did not have significant effects on the increase of AP uptake induced by 2-chloroadenosine. Coadministration of dipyridamole, and adenosine uptake inhibitor, augmented the effect of adenosine on (14C)aminopyrine accumulation. 2-Chloroadenosine, N-ethylcarboxamideadenosine, and adenosine each induced a significant increase in cellular cyclic AMP. We conclude that there may be adenosine A2 receptors on rabbit parietal cells which modulate gastric acid secretion.

  2. p85 regulatory subunit of PI3K mediates cAMP-PKA and estrogens biological effects on growth and survival.

    PubMed

    Cosentino, C; Di Domenico, M; Porcellini, A; Cuozzo, C; De Gregorio, G; Santillo, M R; Agnese, S; Di Stasio, R; Feliciello, A; Migliaccio, A; Avvedimento, E V

    2007-03-29

    Cyclic adenosine 3'5' monophosphate (cAMP) and protein kinase A (PKA) cooperate with phosphatidylinositol 3' kinase (PI3K) signals in the control of growth and survival. To determine the molecular mechanism(s) involved, we identified and mutagenized a specific serine (residue 83) in p85alpha(PI3K), which is phosphorylated in vivo and in vitro by PKA. Expression of p85alpha(PI3K) mutants (alanine or aspartic substitutions) significantly altered the biological responses of the cells to cAMP. cAMP protection from anoikis was reduced in cells expressing the alanine version p85alpha(PI3K). These cells did not arrest in G1 in the presence of cAMP, whereas cells expressing the aspartic mutant p85D accumulated in G1 even in the absence of cAMP. S phase was still efficiently inhibited by cAMP in cells expressing both mutants. The binding of PI3K to Ras p21 was greatly reduced in cells expressing p85A in the presence or absence of cAMP. Conversely, expression of the aspartic mutant stimulated robustly the binding of PI3K to p21 Ras in the presence of cAMP. Mutation in the Ser 83 inhibited cAMP, but not PDGF stimulation of PI3K. Conversely, the p85D aspartic mutant amplified cAMP stimulation of PI3K activity. Phosphorylation of Ser 83 by cAMP-PKA in p85alpha(PI3K) was also necessary for estrogen signaling as expression of p85A or p85D mutants inhibited or amplified, respectively, the binding of estrogen receptor to p85alpha and AKT phosphorylation induced by estrogens. The data presented indicate that: (1) phosphorylation of Ser 83 in p85alpha(PI3K) is critical for cAMP-PKA induced G1 arrest and survival in mouse 3T3 fibroblasts; (2) this site is necessary for amplification of estrogen signals by cAMP-PKA and related receptors. Finally, these data suggest a general mechanism of PI3K regulation by cAMP, operating in various cell types and under different conditions. PMID:17016431

  3. Role of adenosine receptors in caffeine tolerance

    SciTech Connect

    Holtzman, S.G.; Mante, S.; Minneman, K.P. )

    1991-01-01

    Caffeine is a competitive antagonist at adenosine receptors. Receptor up-regulation during chronic drug treatment has been proposed to be the mechanism of tolerance to the behavioral stimulant effects of caffeine. This study reassessed the role of adenosine receptors in caffeine tolerance. Separate groups of rats were given scheduled access to drinking bottles containing plain tap water or a 0.1% solution of caffeine. Daily drug intake averaged 60-75 mg/kg and resulted in complete tolerance to caffeine-induced stimulation of locomotor activity, which could not be surmounted by increasing the dose of caffeine. 5'-N-ethylcarboxamidoadenosine (0.001-1.0 mg/kg) dose dependently decreased the locomotor activity of caffeine-tolerant rats and their water-treated controls but was 8-fold more potent in the latter group. Caffeine (1.0-10 mg/kg) injected concurrently with 5-N-ethylcarboxamidoadenosine antagonized the decreases in locomotor activity comparably in both groups. Apparent pA2 values for tolerant and control rats also were comparable: 5.05 and 5.11. Thus, the adenosine-antagonist activity of caffeine was undiminished in tolerant rats. The effects of chronic caffeine administration on parameters of adenosine receptor binding and function were measured in cerebral cortex. There were no differences between brain tissue from control and caffeine-treated rats in number and affinity of adenosine binding sites or in receptor-mediated increases (A2 adenosine receptor) and decreases (A1 adenosine receptor) in cAMP accumulation. These results are consistent with theoretical arguments that changes in receptor density should not affect the potency of a competitive antagonist. Experimental evidence and theoretical considerations indicate that up-regulation of adenosine receptors is not the mechanism of tolerance to caffeine-induced stimulation of locomotor activity.

  4. cAMP-synthesis in a medullary thyroid carcinoma cell line: response to adrenergic agents and prostaglandines.

    PubMed

    Mertens, P R; Goretzki, P E; Keck, E

    1999-01-01

    Calcitonin secretion by C-cells is mediated through intracellular 3'5'-cyclic adenosine monophosphate (cAMP) and calcium signaling. Calcitonin release stimulation tests may take advantage of both signaling cascades in screening for medullary thyroid carcinomas (MTC). To elucidate the regulation of the adenylyl cyclase system we have determined cAMP levels of a calcitonin-expressing MTC cell line (RG) after exposure to adrenergic agents and prostaglandines. In early passages (20-30) cAMP concentrations were significantly elevated in RG cells after exposure to beta-adrenergic agents and prostaglandines E1 and E2. In advanced passages (60-80) the beta-adrenergic response was no longer detectable and adrenergic receptors were uncoupled from the adenylyl cyclase complex; while the effect of prostaglandines E1 and E2 remained unaffected. Preincubation with dexamethasone, in a process requiring protein new synthesis, re-established the adrenergic response in later passages, indicating that RG cells dedifferentiated in culture over time. Our in vitro findings suggest that MTC cell dedifferentiation may be accompanied by adrenergic receptor-uncoupling from the adenylate cyclase system and that this process may be reversed by dexamethasone incubation.

  5. Effect of Electrical Stimulation on Beta-Adrenergic Receptor Population and Cyclic AMP Production in Chicken and Rat Skeletal Muscle Cell Cultures

    NASA Technical Reports Server (NTRS)

    Young, Ronald B.; Bridge, Kristin Y.; Strietzel, Catherine J.

    2000-01-01

    Expression of the beta-adrenergic receptor (PAR) and its coupling to Adenosine 3'5' Cyclic Monophosphate (cAMP) synthesis are important components of the signaling system that controls muscle atrophy and hypertrophy and the goal of this study was to determine if electrical stimulation in a pattern simulating slow muscle contraction would alter the PAR response in primary cultures of avian and mammalian skeletal muscle cells. Specifically chicken skeletal muscle cells and rat skeletal muscle cells that had been grown for 7 d in culture, were subjected to electrical stimulation for an additional 2 d at a pulse frequency of 0.5 pulses/sec and a pulse duration of 200 msec. In chicken skeletal muscle cells, the PAR population was not significantly affected by electrical stimulation; however, the ability, of these cells to synthesize cyclic AMP was reduced by approximately one-half. In contrast, the PAR population in rat muscle cells was increased slightly but not significantly by electrical stimulation, and the ability of these cells to synthesize cyclic AMP was increased by almost twofold. The basal levels of intracellular cyclic AMP in neither rat muscle cells nor chicken muscle cells were affected by electrical stimulation.

  6. Effects of cold exposure on cyclic AMP concentration in plasma, liver, and brown and white adipose tissues in cold-acclimated rats

    NASA Astrophysics Data System (ADS)

    Habara, Yoshiaki

    1989-06-01

    Effects of acute cold exposure on plasma energy substrates and tissue 3',5'-adenosine monophosphate (cAMP) were analyzed in intact rats, to define an involvement of the nucleotide in nonshivering thermogenesis (NST) and resultant cold acclimation. After an acute cold exposure to -5°C, the plasma glucose level increased gradually in warm-kept control rats (C) while it decreased significantly in cold-acclimated rats (CA). However, it was increased considerably by an extreme cold exposure to -15°C in both C and CA. By contrast, plasma levels of free fatty acids (FFA) increased immediately after cold exposure and the release lasted during the period of exposure especially in C. The cold exposure also increased plasma cAMP concentration but no concomitant increase was found in the liver. In both brown (IBAT) and white (WAT) adipose tissues the nucleotide concentration showed a stepwise decrease. The observed correlation between lipolysis and plasma cAMP response after cold exposure suggests an involvement of the adenylate cyclase-cAMP system in NST via lipid metabolism, at least, in the early stages of cold acclimation.

  7. Immunomodulatory Effects of Lippia sidoides Extract: Induction of IL-10 Through cAMP and p38 MAPK-Dependent Mechanisms

    PubMed Central

    Rajgopal, Arun; Rebhun, John F.; Burns, Charlie R.; Scholten, Jeffrey D.; Balles, John A.

    2015-01-01

    Abstract Lippia sidoides is an aromatic shrub that grows wild in the northeastern region of Brazil. In local traditional medicine, the aerial portions of this species are used as anti-infectives, antiseptics, spasmolytics, sedatives, hypotensives, and anti-inflammatory agents. In this research, we evaluate the potential immunological properties of Lippia extract through in vitro analysis of its ability to modulate intracellular cyclic adenosine monophosphate (cAMP) levels and interleukin-10 (IL-10) production. These results show that Lippia extract increases intracellular cAMP through the inhibition of phosphodiesterase activity. They also demonstrate that Lippia extract increases IL-10 production in THP-1 monocytes through both an increase in intracellular cAMP and the activation of p38 MAPK. These results suggest that the Lippia-mediated inhibition of phosphodiesterase activity and the subsequent increase in intracellular cAMP may explain some of the biological activities associated with L. sidoides. In addition, the anti-inflammatory activity of L. sidoides may also be due, in part, to its ability to induce IL-10 production through the inhibition of cyclic nucleotide-dependent phosphodiesterase activity and by its activation of the p38 MAPK pathway. PMID:25599252

  8. Immunomodulatory effects of Lippia sidoides extract: induction of IL-10 through cAMP and p38 MAPK-dependent mechanisms.

    PubMed

    Rajgopal, Arun; Rebhun, John F; Burns, Charlie R; Scholten, Jeffrey D; Balles, John A; Fast, David J

    2015-03-01

    Lippia sidoides is an aromatic shrub that grows wild in the northeastern region of Brazil. In local traditional medicine, the aerial portions of this species are used as anti-infectives, antiseptics, spasmolytics, sedatives, hypotensives, and anti-inflammatory agents. In this research, we evaluate the potential immunological properties of Lippia extract through in vitro analysis of its ability to modulate intracellular cyclic adenosine monophosphate (cAMP) levels and interleukin-10 (IL-10) production. These results show that Lippia extract increases intracellular cAMP through the inhibition of phosphodiesterase activity. They also demonstrate that Lippia extract increases IL-10 production in THP-1 monocytes through both an increase in intracellular cAMP and the activation of p38 MAPK. These results suggest that the Lippia-mediated inhibition of phosphodiesterase activity and the subsequent increase in intracellular cAMP may explain some of the biological activities associated with L. sidoides. In addition, the anti-inflammatory activity of L. sidoides may also be due, in part, to its ability to induce IL-10 production through the inhibition of cyclic nucleotide-dependent phosphodiesterase activity and by its activation of the p38 MAPK pathway.

  9. Fluorescence decay studies of modified dinucleoside monophosphates containing 1-N6-ethenoadenosine.

    PubMed

    Kubota, Y; Motoda, Y; Fujisaki, Y; Steiner, R F

    1983-10-01

    Five dinucleoside monophosphates containing 1-N6-ethenoadenosine (epsilon A) have been studied using fluorescence measurements. The fluorescence spectra of these dinucleoside monophosphates are almost the same as the fluorescence spectrum of epsilon AMP. Fluorescence quantum yields of these dimers are greatly reduced compared to that of epsilon AMP. Intramolecular base-base interactions may be responsible for fluorescence quenching. It is found that the fluorescence decay kinetics does not obey a simple decay law but that the decay data can be well described as a sum of three exponentials. This implies that these dimers cannot be characterized as a two-state system, but can be described as systems consisting of three or more conformational states. Sequence effects upon the fluorescence behavior are observed. The fluorescence quenching and decay parameters of Gp epsilon A and Up epsilon A indicate a higher degree of base-base interaction than in their epsilon ApG and epsilon ApU counterparts.

  10. Rapid glucocorticoid inhibition of vasoactive intestinal peptide-induced cyclic AMP accumulation and prolactin release in rat pituitary cells in culture.

    PubMed Central

    Rotsztejn, W H; Dussaillant, M; Nobou, F; Rosselin, G

    1981-01-01

    Vasoactive intestinal peptide (VIP) stimulates both adenosine 3',5'-cyclic monophosphate (cAMP) accumulation and prolactin release in normal rat pituitary cells in culture. cAMP accumulation is significant (P less than 0.01) at VIP concentrations as low as 1 nM and reaches a maximum with 0.1 microM. Addition of dexamethasone as early as 15 min before VIP inhibits VIP stimulation of both cAMP production and PRL secretion. The rapid inhibition is dose-dependent: it appears at doses as low as 0.01 pM and is complete at 1 pM dexamethasone. Increasing concentrations of dexamethasone induce a noncompetitive type of inhibition, as shown by the decrease in Vmax with no change in the apparent Km for VIP. Cycloheximide (1 mM) counteracts the inhibitory effect of dexamethasone on VIP-induced cAMP production, which suggests the involvement of a rapid protein synthesis mechanism. Ru-26988, a specific glucocorticoid devoid of any mineralocorticoid activity and which does not bind to intracellular transcortin-like component, also produces an inhibition of VIP-induced cAMP accumulation. Corticosterone also inhibits VIP-induced cAMP production but at concentrations higher than those of dexamethasone. In contrast, aldosterone, progesterone, estradiol, and testosterone have no effect. These results demonstrate that, in normal rat pituitary cells in culture, glucocorticoids at physiological concentrations rapidly inhibit the cAMP production and prolactin release induced by VIP by acting through specific glucocorticoid receptors. PMID:6278481

  11. Reduced rate of adenosine triphosphate synthesis by in vivo 31P nuclear magnetic resonance spectroscopy and downregulation of PGC-1beta in distal skeletal muscle following burn.

    PubMed

    Tzika, A Aria; Mintzopoulos, Dionyssios; Padfield, Katie; Wilhelmy, Julie; Mindrinos, Michael N; Yu, Hongue; Cao, Haihui; Zhang, Qunhao; Astrakas, Loukas G; Zhang, Jiangwen; Yu, Yong-Ming; Rahme, Laurence G; Tompkins, Ronald G

    2008-02-01

    Using a mouse model of burn trauma, we tested the hypothesis that severe burn trauma corresponding to 30% of total body surface area (TBSA) causes reduction in adenosine triphosphate (ATP) synthesis in distal skeletal muscle. We employed in vivo 31P nuclear magnetic resonance (NMR) in intact mice to assess the rate of ATP synthesis, and characterized the concomitant gene expression patterns in skeletal muscle in burned (30% TBSA) versus control mice. Our NMR results showed a significantly reduced rate of ATP synthesis and were complemented by genomic results showing downregulation of the ATP synthase mitochondrial F1 F0 complex and PGC-1beta gene expression. Our findings suggest that inflammation and muscle atrophy in burns are due to a reduced ATP synthesis rate that may be regulated upstream by PGC-1beta. These findings implicate mitochondrial dysfunction in distal skeletal muscle following burn injury. That PGC-1beta is a highly inducible factor in most tissues and responds to common calcium and cyclic adenosine monophosphate (cAMP) signaling pathways strongly suggests that it may be possible to develop drugs that can induce PGC-1beta.

  12. One-step isolation of adenosine triphosphate from crude fermentation broth of Saccharomyces cerevisiae by anion-exchange chromatography using supermacroporous cryogel.

    PubMed

    Yun, Junxian; Shen, Shaochuan; Chen, Fang; Yao, Kejian

    2007-12-01

    Adenosine triphosphate (ATP) is an important high-energy compound widely used in biological and therapeutic fields. It can be produced by phosphorylation of adenosine monophosphate (AMP) with microbial cells in industrial scale and the effective isolation of ATP from microbial fermentation broth is a challenging work. In this work, we develop a novel one-step method to directly separate ATP from fermentation broth of Saccharomyces cerevisiae by anion-exchange chromatography using supermacroporous cryogel. The cryogel bed with tertiary amine groups was prepared by grafting N,N-dimethylaminoethyl methacrylate (DMAEMA) monomer chains onto the matrix of a polyacrylamide-based cryogel in a glass column and its properties of liquid dispersion, water permeability, porosity as well as the ligand density were measured. Chromatographic separation of ATP from the fermentation broth by the cryogel was carried out using deionised water and 0.01 M HCl as running buffer, respectively. The breakthrough characteristics and elution performance in the cryogel bed were revealed and analyzed. The purities of the obtained ATP were analyzed quantitatively by high performance liquid chromatography (HPLC). The maximal purity of ATP by the one-step separation method was 95.5% using 0.01 M HCl as running buffer in this work. The corresponding chromatographic behaviors were investigated and analyzed.

  13. One-step isolation of adenosine triphosphate from crude fermentation broth of Saccharomyces cerevisiae by anion-exchange chromatography using supermacroporous cryogel.

    PubMed

    Yun, Junxian; Shen, Shaochuan; Chen, Fang; Yao, Kejian

    2007-12-01

    Adenosine triphosphate (ATP) is an important high-energy compound widely used in biological and therapeutic fields. It can be produced by phosphorylation of adenosine monophosphate (AMP) with microbial cells in industrial scale and the effective isolation of ATP from microbial fermentation broth is a challenging work. In this work, we develop a novel one-step method to directly separate ATP from fermentation broth of Saccharomyces cerevisiae by anion-exchange chromatography using supermacroporous cryogel. The cryogel bed with tertiary amine groups was prepared by grafting N,N-dimethylaminoethyl methacrylate (DMAEMA) monomer chains onto the matrix of a polyacrylamide-based cryogel in a glass column and its properties of liquid dispersion, water permeability, porosity as well as the ligand density were measured. Chromatographic separation of ATP from the fermentation broth by the cryogel was carried out using deionised water and 0.01 M HCl as running buffer, respectively. The breakthrough characteristics and elution performance in the cryogel bed were revealed and analyzed. The purities of the obtained ATP were analyzed quantitatively by high performance liquid chromatography (HPLC). The maximal purity of ATP by the one-step separation method was 95.5% using 0.01 M HCl as running buffer in this work. The corresponding chromatographic behaviors were investigated and analyzed. PMID:18024244

  14. Studies on adenosine triphosphate transphosphorylases. XVIII. Synthesis and preparation of peptides and peptide fragments of rabbit muscle ATP-AMP transphosphorylase (adenylate kinase) and their nucleotide-binding properties.

    PubMed

    Kuby, S A; Hamada, M; Johnson, M S; Russell, G A; Manship, M; Palmieri, R H; Fleming, G; Bredt, D S; Mildvan, A S

    1989-08-01

    Two peptide fragments, derived from the head and tail of rabbit muscle myokinase, were found to possess remarkable and specific ligand-binding properties (Hamada et al., 1979). By initiating systematic syntheses and measurements of equilibrium substrate-binding properties of these two sets of peptides, or portions thereof, which encompass the binding sites for (a) the magnesium complexes of the nucleotide substrates (MgATP2- and MgADP-) and (b) the uncomplexed nucleotide substrates (ADP3- and AMP2-) of rabbit muscle myokinase, some of the requirements for binding of the substrates to ATP-AMP transphosphorylase are being deduced and chemically outlined. One requirement for tight nucleotide binding appears to be a minimum peptide length of 15-25 residues. In addition, Lys-172 and/or Lys-194 may be involved in the binding of epsilon AMP. The syntheses are described as a set of peptides corresponding to residues 31-45, 20-45, 5-45, and 1-45, and a set of peptides corresponding to residues 178-192, 178-194, and 172-194 of rabbit muscle adenylate kinase. The ligand-binding properties of the first set of synthetic peptides to the fluorescent ligands: epsilon MgATP/epsilon ATP and epsilon MgADP/epsilon ADP are quantitatively presented in terms of their intrinsic dissociation constants (K'd) and values of N (maximal number of moles bound per mole of peptide); and compared with the peptide fragment MT-I (1-44) obtained from rabbit muscle myokinase (Kuby et al., 1984) and with the native enzyme (Hamada et al., 1979). In addition, the values of N and K'd are given for the second set of synthetic peptides to the fluorescent ligands epsilon AMP and epsilon ADP as well as for the peptide fragments MT-XII(172-194) and CB-VI(126-194) (Kuby et al., 1984) and, in turn, compared with the native enzyme. A few miscellaneous dissociation constants which had been derived kinetically are also given for comparison (e.g., the Ki for epsilon AMP and the value of KMg epsilon ATP obtained for

  15. Activation of the cAMP-PKA signaling pathway in rat dorsal root ganglion and spinal cord contributes toward induction and maintenance of bone cancer pain.

    PubMed

    Zhu, Gui-Qin; Liu, Su; He, Duan-Duan; Liu, Yue-Peng; Song, Xue-Jun

    2014-08-01

    The objective of this study was to explore the role of cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signaling in the development of bone cancer pain in rats. Female Sprague-Dawley rats (N=48) were divided randomly into four groups: sham (n=8), tumor cell implantation (TCI) (n=16), TCI+saline (n=8), and TCI+PKA inhibitor (n=16). Bone cancer-induced pain behaviors - thermal hyperalgesia and mechanical allodynia - were tested at postoperative days -3, -1, 1, 3, 5, 7, 10, and 14. A PKA inhibitor, Rp-cAMPS (1 mmol/l/20 μl), was injected intrathecally on postoperative days 3, 4, and 5 (early phase) or 7, 8, and 9 postoperative days (late phase). The expression of PKA mRNA in dorsal root ganglia (DRG) was detected by reverse transcription-PCR. The concentration of cAMP and activity of PKA in DRG and spinal cord were measured by enzyme-linked immunosorbent assay. TCI treatment induced significant pain behaviors, manifested as thermal hyperalgesia and mechanical allodynia. Spinal administration of the PKA inhibitor Rp-cAMPS during the early phase and late phase significantly delayed or reversed, respectively, TCI-induced thermal hyperalgesia and mechanical allodynia. TCI treatment also led to obvious tumor growth and bone destruction. The level of PKA mRNA in the DRG, as well as the concentration of cAMP and the activity of PKA, in both the DRG and spinal cord were significantly increased after TCI treatment (P<0.01). We conclude that the inhibition of the cAMP-PKA signaling pathway may reduce bone cancer pain. PMID:24978483

  16. cAMP/PKA/CREB/GLT1 signaling involved in the antidepressant-like effects of phosphodiesterase 4D inhibitor (GEBR-7b) in rats

    PubMed Central

    Liu, Xu; Guo, Haibiao; Sayed, Mohammad Daud SOM; Lu, Yang; Yang, Ting; Zhou, Dongsheng; Chen, Zhongming; Wang, Haitao; Wang, Chuang; Xu, Jiangping

    2016-01-01

    Objectives GEBR-7b, a potential phosphodiesterase 4D inhibitor, has been shown to have memory-enhancing effects in rodents. However, it is still unknown whether GEBR-7b also has the antidepressant-like effects in rats. Herein, we examined the potential of GEBR-7b to attenuate depression-like behaviors in the rat model of depression induced by chronic unpredictable stress (CUS). Next, we also investigated the alterations of cyclic adenosine monophosphate (cAMP), protein kinase A (PKA) catalytic subunit (PKAca), cAMP response element-binding (CREB), and glutamate transporter 1 (GLT1) levels produced by GEBR-7b in the rats model of depression. Methods Effects of GEBR-7b on CUS (35 days)-induced depression-like behaviors were examined by measuring immobility time in the forced swimming test (FST). Hippocampal cAMP levels were examined by enzyme-linked immunosorbent assay, whereas PKAca, phosphorylation of CREB (pCREB), CREB, and GLT1 in the hippocampus of rats were subjected to Western blot analysis. Results CUS exposure caused a depression-like behavior evidenced by the increased immobility time in FST. Depression-like behavior induced by CUS was accompanied by a significant increased GLT, decreased cAMP, PKAca, pCREB activities in hippocampus. However, repeated GEBR-7b administration significantly reversed CUS-induced depression-like behavior and changes of cAMP/PKA/CREB/GLT1 signaling. No alteration was observed in locomotor activity in open field test. Conclusion These findings indicate that GEBR-7b reversed the depression-like behaviors induced by CUS in rats, which is at least in part mediated by modulating cAMP, PKAca, pCREB, and GLT1 levels in the hippocampus of rats, supporting its neuroprotective potential against behavioral and biochemical dysfunctions induced by CUS. PMID:26855578

  17. Mechanisms involved in increased sensitivity to adenosine A(2A) receptor activation and hypoxia-induced vasodilatation in porcine coronary arteries.

    PubMed

    Hedegaard, Elise R; Nielsen, Berit D; Mogensen, Susie; Rembold, Christopher M; Frøbert, Ole; Simonsen, Ulf

    2014-01-15

    Hypoxia-induced coronary vasorelaxation is a compensatory mechanism increasing blood flow. We hypothesized that hypoxia shares pathways with adenosine and causes vasorelaxation through the adenosine A(2A) receptor and force suppression by increasing cAMP and phosphorylated heat shock protein (HSP)20. Adenosine receptors in porcine left anterior descending coronary arteries (LAD) were examined by RT-PCR and isometric tension recording in myographs. Vasorelaxation was induced by adenosine, 1% oxygen, or both in the absence or presence of ZM241385, an adenosine A(2A) receptor antagonist. cAMP was determined by ELISA and p-HSP20/HSP20 and p-MLC/MLC were determined by immunoblotting and densitometric analyses. In coronary arteries exposed to 1% oxygen, there was increased sensitivity to adenosine, the adenosine A2 selective agonist NECA, and the adenosine A(2A) selective receptor agonist CGS21680. ZM241385 shifted concentration-response curves for CGS21680 to the right, whereas the adenosine A1 antagonist DPCPX, the adenosine A2B receptor antagonist MRS1754 and the adenosine A3 receptor antagonist MRS1523 failed to reduce vasodilatation induced by CGS21680. 1% oxygen or adenosine increased cAMP accumulation and HSP20 phosphorylation without changing T850-MYPT1 and MLC phosphorylation. ZM241385 failed to change 1% oxygen-induced vasodilation, cAMP accumulation, HSP20 phosphorylation and MLC phosphorylation. The PKA inhibitor Rp-8-CPT-cAMPS significantly reduced vasorelaxation induced by 1% oxygen or CGS21680. Our findings suggest that the increased sensitivity to adenosine, NECA, and CGS21680 at 1% oxygen involves adenosine A(2A) receptors. Adenosine and 1% oxygen induce vasorelaxation in PGF2α-contracted porcine coronary arteries partly by force suppression caused by increased cAMP and phosphorylation of HSP20.

  18. Adaptations in adenosine signaling in drug dependence: therapeutic implications.

    PubMed

    Hack, Stephen P; Christie, Macdonald J

    2003-01-01

    Adenosine is an important endogenous purine neuromodulator in the central nervous system that modulates many important cellular processes in neurons. The physiological effects of adenosine are transduced through four pharmacologically classified receptor types i.e., A1, A2A, A2B and A3. All adenosine receptors are G-protein coupled receptors (GPCR) of the type 1 variety. Adaptations in adenosine signaling have been implicated in a wide range of pathophysiological processes, such as epilepsies, sleep disorders, pain, and drug addictions. Knowledge relating to the etiology of addictive processes is far from complete, and as a result the therapeutic options to deal with drug dependence issues are limited. Drugs of abuse mediate their effects through many distinct cellular effectors, such as neurotransmitter transporters, ion channels, and receptor proteins. However, a unifying feature of the major drugs of abuse-i.e., opiates, cocaine, and alcohol-is that they all directly or indirectly modulate adenosine signaling in neurons. Agents targeting adenosine receptors may therefore offer novel avenues for the development of therapies to manage or treat addictions. A consistent cellular adaptation to long-term drug use is the up- or down-regulation of signaling pathways driven by adenylyl cyclase/cyclic AMP (cAMP) in several brain regions linked to addiction. Withdrawal from mu-opioids or cocaine following their chronic administration leads to an upregulation of adenylyl cyclase-mediated signaling, resulting in high levels of cAMP. Cyclic AMP produced in this way acts as a substrate for the endogenous production of adenosine. Increased levels of endogenous adenosine interact with presynaptic A1 receptors to inhibit the excessive neuronal excitation often seen during morphine/cocaine withdrawal. These pre-clinical findings fit well with other data indicating that drugs which boost endogenous adenosine levels or directly interact with inhibitory A1 receptors can alleviate

  19. Role played by the adenylcyclase-cAMP system of the rat septal area on Na+, K+ and water renal excretion.

    PubMed

    Camargo, L A; Saad, W A; Graeff, F G; Silva Neto, C R; Antunes-Rodrigues, J; Covian, M R

    1977-08-01

    In this study, the participation of the adenylcyclase--cAMP system of the rat septal area in the mediation of the natriuretic, kaliuretic and diuretic effects of noradrenaline (NA) was investigated. The intraseptal injection of 20 nmol of NA caused a significant increase in the urinary excretion of Na+ and K+ as well as in the urinary volume during the 2 hr period following the intracerebral injection which was blocked by 40 nmol of phentolamine, locally injected, 30 min before the catecholamine. In contrast, pretreatment with propranolol (100 nmol) potentiated the effects of NA on salt and water renal excretion. The intraseptal injection of 3.12 to 50 nmol of dibutryrl cyclic adenosine monophosphate (db cAMP) caused dose-dependent increase in natriuresis and kaliuresis, but a decrease in urinary volume. Under the same experimental conditions, caffeine administration (6.25 to 100 nmol) also induced dose-dependent increases in Na+ and K+ urinary output. These results indicate that the saluretic effect of NA may be mediated by an alpha receptor-induced activation of the adenylcyclase--cAMP system in the septal area. PMID:199910

  20. The PAMP c-di-AMP Is Essential for Listeria monocytogenes Growth in Rich but Not Minimal Media due to a Toxic Increase in (p)ppGpp. [corrected].

    PubMed

    Whiteley, Aaron T; Pollock, Alex J; Portnoy, Daniel A

    2015-06-10

    Cyclic di-adenosine monophosphate (c-di-AMP) is a widely distributed second messenger that appears to be essential in multiple bacterial species, including the Gram-positive facultative intracellular pathogen Listeria monocytogenes. In this study, the only L. monocytogenes diadenylate cyclase gene, dacA, was deleted using a Cre-lox system activated during infection of cultured macrophages. All ΔdacA strains recovered from infected cells harbored one or more suppressor mutations that allowed growth in the absence of c-di-AMP. Suppressor mutations in the synthase domain of the bi-functional (p)ppGpp synthase/hydrolase led to reduced (p)ppGpp levels. A genetic assay confirmed that dacA was essential in wild-type but not strains lacking all three (p)ppGpp synthases. Further genetic analysis suggested that c-di-AMP was essential because accumulated (p)ppGpp altered GTP concentrations, thereby inactivating the pleiotropic transcriptional regulator CodY. We propose that c-di-AMP is conditionally essential for metabolic changes that occur in growth in rich medium and host cells but not minimal medium.

  1. Inotropic responses of the frog ventricle to adenosine triphosphate and related changes in endogenous cyclic nucleotides.

    PubMed Central

    Flitney, F W; Singh, J

    1980-01-01

    1. A study has been made of a well documented but poorly understood response of the isolated frog ventricle to treatment with exogenous adenosine 5' triphosphate (ATP). Measurements of membrane potential, isometric twitch tension and levels of endogenous 3',5'-cyclic nucleotides have been made at various times during the ATP-induced response. 2. ATP elicits a characteristic triphasic response, which comprises an initial, abrupt increase in contractility, rising to a maximum within a few beats (first phase); followed by a period when the twitch amplitude falls, sometimes to below the control level (second phase); and superceded by a more slowly developing and longer-lasting increase in contractile force (third phase). The response is unaffected by atropine, propranolol or phentolamine. However, the prostaglandin synthetase inhibitor indomethacin depresses the first phase and entirely suppresses the third phase. 3. The inotropic effects of ATP are accompanied by changes in the shape of the action potential. These effects are dose-related. The duration of the action potential (D-30mV) and its positive overshoot (O) are increased during all phases of the response, for [ATP]o's up to 10(-5) M. However, at higher [ATP]o's, D-30mV and O ar both reduced during the second phase (but not the first or third phase), when isometric twitch tension is also depressed. The relationship between action potential duration and twitch tension (P) for different [ATP]o's is linear for all three phases of the response, but the slopes of the curves (delta P/delta D) are markedly different, indicating that the sensitivity of the contractile system to membrane depolarization is not constant, but varies continuously throughout the response. 4. ATP has a potent stimulatory effect on the metabolism of endogenous 3',5'-cyclic nucleotides. The time courses of the changes in adenosine 3','5-cyclic monophosphate (3',5'-cyclic AMP) and guanosine 3',5'-cyclic monophosphate (3',5'-cyclic GMP) are

  2. Lipopolysaccharide Compromises Human Sperm Function by Reducing Intracellular cAMP.

    PubMed

    Li, Zhongyuan; Zhang, Dahu; He, Yuanqiao; Ding, Zhiyong; Mao, Fei; Luo, Tao; Zhang, Xiaoping

    2016-01-01

    A worldwide decline in the quality of human semen is currently occurring. In mammals, sperm are produced from diploid stem-cell spermatogonia by spermatogenesis in testes and become mature in epididymis. Nevertheless, these biological processes can be affected by Gram-negative bacterial infection mediated by lipopolysaccharide (LPS), the major endotoxin of Gram-negative bacteria. It is well known that LPS can disturb spermatogenesis and affect sperm maturation and quality in vivo. However, the effect of LPS on the ejaculated mature sperm in vitro remains unclear. Thus, this study aimed to assess the in vitro toxicity of LPS on human sperm function and to elucidate the underlying mechanism. Human sperm were incubated with LPS (0.1-100 μg/ml) for 1-12 h in vitro and, subsequently, sperm viability, motility and capacitation, and the acrosome reaction were examined. LPS dose-dependently inhibited total and progressive motility and the ability to move through a viscous medium of the sperm but did not affect sperm viability, capacitation, and the acrosome reaction. To explore the underlying mechanism of LPS's actions, we examined the effects of LPS on the intracellular concentrations of cyclic adenosine monophosphate (cAMP) and calcium ([Ca(2+)]i) and protein-tyrosine phosphorylation of human sperm, which are key regulators of human sperm function. LPS decreased intracellular cAMP dose-dependently but had no effect on [Ca(2+)]i and protein-tyrosine phosphorylation of human sperm. These findings suggest that LPS inhibits human sperm motility by decreasing intracellular cAMP. PMID:26782775

  3. Lipopolysaccharide Compromises Human Sperm Function by Reducing Intracellular cAMP.

    PubMed

    Li, Zhongyuan; Zhang, Dahu; He, Yuanqiao; Ding, Zhiyong; Mao, Fei; Luo, Tao; Zhang, Xiaoping

    2016-01-01

    A worldwide decline in the quality of human semen is currently occurring. In mammals, sperm are produced from diploid stem-cell spermatogonia by spermatogenesis in testes and become mature in epididymis. Nevertheless, these biological processes can be affected by Gram-negative bacterial infection mediated by lipopolysaccharide (LPS), the major endotoxin of Gram-negative bacteria. It is well known that LPS can disturb spermatogenesis and affect sperm maturation and quality in vivo. However, the effect of LPS on the ejaculated mature sperm in vitro remains unclear. Thus, this study aimed to assess the in vitro toxicity of LPS on human sperm function and to elucidate the underlying mechanism. Human sperm were incubated with LPS (0.1-100 μg/ml) for 1-12 h in vitro and, subsequently, sperm viability, motility and capacitation, and the acrosome reaction were examined. LPS dose-dependently inhibited total and progressive motility and the ability to move through a viscous medium of the sperm but did not affect sperm viability, capacitation, and the acrosome reaction. To explore the underlying mechanism of LPS's actions, we examined the effects of LPS on the intracellular concentrations of cyclic adenosine monophosphate (cAMP) and calcium ([Ca(2+)]i) and protein-tyrosine phosphorylation of human sperm, which are key regulators of human sperm function. LPS decreased intracellular cAMP dose-dependently but had no effect on [Ca(2+)]i and protein-tyrosine phosphorylation of human sperm. These findings suggest that LPS inhibits human sperm motility by decreasing intracellular cAMP.

  4. Absence of a cAMP-mediated antiabsorptive effect in an undifferentiated jejunal epithelium.

    PubMed

    MacLeod, R J; Hamilton, J R

    1987-06-01

    In the relatively undifferentiated jejunal mucosa occurring in piglet viral enteritis, we measured the response of transepithelial Na+ and Cl- fluxes in vitro to raised intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels. At the acute 40-h stage of transmissible gastroenteritis (TGE), luminal membrane markers, sucrase and lactase, and a basolateral jejunal epithelial membrane marker Na+-K+-ATPase, were significantly decreased in activity, while a proliferative marker, thymidine kinase, was significantly enriched; these enzyme characteristics are typical of enterocytes isolated from crypts of other species. As expected, control piglet jejunum in short-circuited Ussing chambers after theophylline (10 mM) developed significant net secretory Na and Cl fluxes primarily due to significant antiabsorptive effects (delta JNa m----s = 3.48 +/- 0.52, delta JCl m----s = 2.59 +/- 0.28). Furosemide (10(-4) M), an inhibitor of electroneutral NaCl cotransport, produced antiabsorptive effects (delta JNa m----s = 2.53 +/- 0.31, delta JCl m----s = 2.58 +/- 0.28) in control jejunum that were not significantly different from those seen in response to theophylline. TGE jejunum, however, responded to theophylline not by an antiabsorptive effect but by significant electrogenic Cl- secretion (delta JCl s----m = 1.59 +/- 0.48); furosemide had no effect on ion fluxes in TGE tissue. Control and TGE jejunal mucosal homogenates did not differ in their basal or theophylline-stimulated levels of cAMP. We conclude that the relatively undifferentiated small intestine occurring in acute TGE does not generate either a cAMP-mediated antiabsorptive effect or a furosemide-mediated antiabsorptive effect.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Rapid activation by 3,5,3'-L-triiodothyronine of adenosine 5'-monophosphate-activated protein kinase/acetyl-coenzyme a carboxylase and akt/protein kinase B signaling pathways: relation to changes in fuel metabolism and myosin heavy-chain protein content in rat gastrocnemius muscle in vivo.

    PubMed

    de Lange, Pieter; Senese, Rosalba; Cioffi, Federica; Moreno, Maria; Lombardi, Assunta; Silvestri, Elena; Goglia, Fernando; Lanni, Antonia

    2008-12-01

    T3 stimulates metabolic rate in many tissues and induces changes in fuel use. The pathways by which T3 induces metabolic/structural changes related to altered fuel use in skeletal muscle have not been fully clarified. Gastrocnemius muscle (isolated at different time points after a single injection of T3 into hypothyroid rats), displayed rapid inductions of AMP-activated protein kinase (AMPK) phosphorylation (threonine 172; within 6 h) and acetyl-coenzyme A carboxylase phosphorylation (serine 79; within 12 h). As a consequence, increases occurred in mitochondrial fatty acid oxidation and carnitine palmitoyl transferase activity. Concomitantly, T3 stimulated signaling toward increased glycolysis through a rapid increase in Akt/protein kinase B (serine 473) phosphorylation (within 6 h) and a directly related increase in the activity of phosphofructokinase. The kinase specificity of the above effects was verified by treatment with inhibitors of AMPK and Akt activity (compound C and wortmannin, respectively). In contrast, glucose transporter 4 translocation to the membrane (activated by T3 within 6 h) was maintained when either AMPK or Akt activity was inhibited. The metabolic changes were accompanied by a decline in myosin heavy-chain Ib protein [causing a shift toward the fast-twitch (glycolytic) phenotype]. The increases in AMPK and acetyl-coenzyme A carboxylase phosphorylation were transient events, both levels declining from 12 h after the T3 injection, but Akt phosphorylation remained elevated until at least 48h after the injection. These data show that in skeletal muscle, T3 stimulates both fatty acid and glucose metabolism through rapid activations of the associated signaling pathways involving AMPK and Akt/protein kinase B.

  6. Vasopressin V2 receptor mRNA expression and cAMP accumulation in aging rat kidney.

    PubMed

    Klingler, C; Preisser, L; Barrault, M B; Lluel, P; Horgen, L; Teillet, L; Ancellin, N; Corman, B

    1997-06-01

    The ability of the kidney to regulate water balance is impaired with age, although the secretion of vasopressin is maintained in senescent animals. This suggests that the cellular response to antidiuretic hormone is reduced in aging kidney. To test this hypothesis, the relationship between the expression of the vasopressin. V2 receptor mRNA and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was investigated in the medullary thick ascending limb of Henle's loop (MTAL) of adult and aging rats. Tubular suspensions of MTAL were prepared from 10- and 30-mo-old female WAG/Rij rats. The accumulation of cAMP for maximal concentration of vasopressin was 34% larger in adult than in old animals (9.5 +/- 0.5 pmol/4 min, n = 16, and 7.1 +/- 0.6 pmol/4 min, n = 12, respectively). The concentration of vasopressin corresponding to half-maximal stimulation was similar in the two groups (0.66 +/- 0.20 and 0.52 +/- 0.09 nmol, n = 5, in adult and old animals), indicating comparable sensitivity of the renal cells with age. The age-related impaired response to vasopressin of the V2 receptor was specific for females and was not observed in males. Direct stimulation of adenylyl cyclase by forskolin induced a comparable accumulation of cAMP in adult and senescent rats. The V2 receptor mRNA level in the MTAL was constant between 10 and 30 mo whether the animals were normally hydrated or dehydrated for 2 days. These data indicate that, in MTAL, the age-related impaired cAMP accumulation by vasopressin would be linked to a change either in the translation of V2 mRNA or in posttranslational processing mechanisms or in the coupling between the V2 receptor and adenylyl cyclase. PMID:9227590

  7. Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells

    PubMed Central

    Kanazawa, Ippei; Yamaguchi, Toru; Yano, Shozo; Yamauchi, Mika; Yamamoto, Masahiro; Sugimoto, Toshitsugu

    2007-01-01

    Background Adiponectin is a key mediator of the metabolic syndrome that is caused by visceral fat accumulation. Adiponectin and its receptors are known to be expressed in osteoblasts, but their actions with regard to bone metabolism are still unclear. In this study, we investigated the effects of adiponectin on the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells. Results Adiponectin receptor type 1 (AdipoR1) mRNA was detected in the cells by RT-PCR. The adenosine monophosphate-activated protein kinase (AMP kinase) was phosphorylated by both adiponectin and a pharmacological AMP kinase activator, 5-amino-imidazole-4-carboxamide-riboside (AICAR), in the cells. AdipoR1 small interfering RNA (siRNA) transfection potently knocked down the receptor mRNA, and the effect of this knockdown persisted for as long as 10 days after the transfection. The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA, as determined by real-time PCR, and reduced ALP activity and mineralization, as determined by von Kossa and Alizarin red stainings. In contrast, AMP kinase activation by AICAR (0.01–0.5 mM) in wild-type MC3T3-E1 cells augmented their proliferation, differentiation, and mineralization. BrdU assay showed that the addition of adiponectin (0.01–1.0 μg/ml) also promoted their proliferation. Osterix, but not Runx-2, appeared to be involved in these processes because AdipoR1 siRNA transfection and AICAR treatments suppressed and enhanced osterix mRNA expression, respectively. Conclusion Taken together, this study suggests that adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions. PMID:18047638

  8. A High-Affinity Adenosine Kinase from Anopheles Gambiae

    SciTech Connect

    M Cassera; M Ho; E Merino; E Burgos; A Rinaldo-Matthis; S Almo; V Schramm

    2011-12-31

    Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (K{sub m} = 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site, but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap{sub 4}A (2.0 {angstrom} resolution) reveals interactions for adenosine and ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg{sup 2+} ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layer {alpha}/{beta}/{alpha} sandwich, and a small cap domain in contact with adenosine. The specificity and tight binding for adenosine arise from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168, and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64, and Asn68 and the ribosyl 2'- and 3'-hydroxyl groups. The structure is more similar to that of human adenosine kinase (48% identical) than to that of AK from Toxoplasma gondii (31% identical). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role for this enzyme in the maintenance of the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects.

  9. Liver AMP/ATP ratio and fructokinase expression are related to gender differences in AMPK activity and glucose intolerance in rats ingesting liquid fructose.

    PubMed

    Vilà, Laia; Roglans, Núria; Perna, Victoria; Sánchez, Rosa M; Vázquez-Carrera, Manuel; Alegret, Marta; Laguna, Juan C

    2011-08-01

    Women, but not men, show an association between fructose consumption and an increased risk of Type 2 diabetes mellitus. As rats are considered a model for human fructose metabolism, we sought to determine whether such a gender-related difference is present in Sprague-Dawley rats and to analyze the molecular mechanism behind. Male and female Sprague-Dawley rats had free access to water or to a 10% w/v fructose solution for 14 days. Plasma analytes, liver triglycerides and enzyme activities and the expression of enzymes and transcription factors related to fatty acid metabolism, insulin signaling and glucose tolerance were determined. Fructose-fed rats had hypertriglyceridemia, steatosis and reduced fatty acid oxidation activity, although the metabolic pattern of fructose-fed female rats was different to that observed for male rats. Fructose-fed female, but not male rats, showed no change in plasma leptin; they had hyperinsulinemia, an altered glucose tolerance test and less liver insulin receptor substrate-2. Further, only fructose-fed female rats had increased adenosine 5'-monophosphate (AMP)-activated protein kinase activity, resulting in a decreased expression of hepatic nuclear factor 4 and sterol response element binding protein 1. These differences were related to the fact that liver expression of the enzyme fructokinase, controlling fructose metabolism, was markedly induced by fructose ingestion in female, but not in male rats, resulting in a significant increase in the AMP/adenosine 5'-triphosphate (ATP) ratio and, thus, AMP-activated protein kinase activation, in female rats only. The difference in fructokinase induction could explain the higher metabolic burden produced by fructose ingestion in the livers of female Sprague-Dawley rats.

  10. Identification of possible adenosine receptors in vascular smooth muscle

    SciTech Connect

    Doctrow, S.R.

    1985-01-01

    Adenosine is a vasodilator and has been implicated in increased blood flow in tissues that undergo energy deficiency. During conditions such as hypoxia and ischemia, adenosine is produced and is said to increase blood flow by relaxing the vascular smooth muscle (VSM) lining the resistance vessels. The goal of this research was to identify receptors that might be responsible for adenosine-mediated VSM relaxation. When an insoluble fraction from calf aortic VSM was incubated with /sup 32/P-ATP, two components were phosphorylated. One was identified as myosin light chain by MW, pl, and immunoprecipitation. The other product was identified as phosphatidylinositol-4-phosphate (DPI) by tic. Both phosphorylations were inhibited by adenosine and by 5'-chloro-5'-deoxyadenosine (Cl-Ado). DPI production was much more sensitive to the nucleosides than was myosin phosphorylation. Neither inhibition involved change in cAMP production. Phosphatidylinositol (Pl) kinase in the VSM membranes required magnesium, was activated and solubilized by Triton X-100, and phosphorylated both endogenous and exogenous Pl. Cl-Ado inhibited Pl kinase in a manner competitive with respect to ATP and noncompetitive with respect to Pl. Adenosine and adenosine analogs modified in the ribose ring were inhibitors with potencies comparable to that of Cl-Ado. Adenine nucleotides and purine-modified adenosine analogs were weaker inhibitors than Cl-Ado.

  11. Calcium, cyclic AMP, and MAP kinases: dysregulation in polycystic kidney disease.

    PubMed

    Cowley, B D

    2008-02-01

    Low intracellular calcium, present in untreated polycystic kidney epithelia, results in a proliferative response to cyclic adenosine monophosphate. Treatment with a calcium channel blocker (CCB) caused exacerbation of autosomal dominant polycystic kidney disease in rats. Data regarding use of CCBs in human polycystic kidney disease (PKD) are limited and mixed. Thus, it is premature to extrapolate these findings to human PKD.

  12. Cyclic AMP reduces adhesion of isolated neuronal growth cones from developing rat forebrain to an astrocytic cell line from embryonic mouse striatum.

    PubMed

    Lockerbie, R O; Autillo-Touati, A; Araud, D; Seite, R; Chneiweiss, H; Glowinski, J; Prochiantz, A

    1989-01-01

    We have recently shown that isolated neuronal growth cones from developing rat forebrain possess an appreciable activity of adenylate cyclase, producing cyclic adenosine monophosphate, which can be stimulated by various neurotransmitter receptor agonists and by forskolin [Lockerbie R. O., Hervé D., Blanc G., Tassin J. P. and Glowinski J. (1988) Devl Brain Res. 38, 19-25]. In the present study, we have investigated the effect of cyclic adenosine monophosphate in an in vitro adhesion assay established between [3H]GABA-labelled isolated growth cones and a Simian virus-40 transformed astrocytic cell line from embryonic mouse striatum. Adhesion of the isolated growth cones onto the astrocytic clone increased steadily up to about 45 min before it began to level off at ca 16-18% of total [3H]GABA-labelled isolated growth cones added. Adhesion of the isolated growth cones onto the astrocytic clone was much superior to that seen on polyornithine and, in particular, on non-treated tissue culture wells. Adhesion "at plateau" was independent of both temperature and extracellular Ca2+ and was markedly reduced (ca 50%) by trypsin pre-treatment of the isolated growth cones. Pre-treatment of the isolated growth cones with either forskolin or lipophilic analogues of cyclic adenosine monophosphate attenuated adhesion in a time- and concentration-dependent manner. Approximately 30% reduction in adhesion to the astrocytic clone "at plateau" was observed after a 15 min pre-treatment of the isolated growth cones with forskolin at 10(-4) M or cyclic adenosine monophosphate analogues at 10(-3) M. A cyclic guanosine monophosphate analogue was without effect on adhesion of isolated growth cones. Scanning electron microscope analysis showed that isolated growth cones pre-treated with either cyclic adenosine monophosphate analogues or forskolin had a simpler morphology when attached to the astrocytic clone than isolated growth cones under control conditions. Pre-treatment of the isolated

  13. Role of LRP1 and ERK and cAMP Signaling Pathways in Lactoferrin-Induced Lipolysis in Mature Rat Adipocytes

    PubMed Central

    Ikoma-Seki, Keiko; Nakamura, Kanae; Morishita, Satoru; Ono, Tomoji; Sugiyama, Keikichi; Nishino, Hoyoku; Hirano, Hisashi; Murakoshi, Michiaki

    2015-01-01

    Lactoferrin (LF) is a multifunctional glycoprotein present in milk. A clinical study showed that enteric-coated bovine LF tablets decrease visceral fat accumulation. Furthermore, animal studies revealed that ingested LF is partially delivered to mesenteric fat, and in vitro studies showed that LF promotes lipolysis in mature adipocytes. The aim of the present study was to determine the mechanism underlying the induction of lipolysis in mature adipocytes that is induced by LF. To address this question, we used proteomics techniques to analyze protein expression profiles. Mature adipocytes from primary cultures of rat mesenteric fat were collected at various times after exposure to LF. Proteomic analysis revealed that the expression levels of hormone-sensitive lipase (HSL), which catalyzes the rate-limiting step of lipolysis, were upregulated and that HSL was activated by protein kinase A within 15 min after the cells were treated with LF. We previously reported that LF increases the intracellular concentration of cyclic adenosine monophosphate (cAMP), suggesting that LF activates the cAMP signaling pathway. In this study, we show that the expression level and the activity of the components of the extracellular signal-regulated kinase (ERK) signaling pathway were upregulated. Moreover, LF increased the activity of the transcription factor cAMP response element binding protein (CREB), which acts downstream in the cAMP and ERK signaling pathways and regulates the expression levels of adenylyl cyclase and HSL. Moreover, silencing of the putative LF receptor low-density lipoprotein receptor-related protein 1 (LRP1) attenuated lipolysis in LF-treated adipocytes. These results suggest that LF promoted lipolysis in mature adipocytes by regulating the expression levels of proteins involved in lipolysis through controlling the activity of cAMP/ERK signaling pathways via LRP1. PMID:26506094

  14. Abnormal activity of the MAPK- and cAMP-associated signaling pathways in frontal cortical areas in postmortem brain in schizophrenia.

    PubMed

    Funk, Adam J; McCullumsmith, Robert E; Haroutunian, Vahram; Meador-Woodruff, James H

    2012-03-01

    Recent evidence suggests that schizophrenia may result from alterations of integration of signaling mediated by multiple neurotransmitter systems. Abnormalities of associated intracellular signaling pathways may contribute to the pathophysiology of schizophrenia. Proteins and phospho-proteins comprising mitogen activated protein kinase (MAPK) and 3'-5'-cyclic adenosine monophosphate (cAMP)-associated signaling pathways may be abnormally expressed in the anterior cingulate (ACC) and dorsolateral prefrontal cortex (DLPFC) in schizophrenia. Using western blot analysis we examined proteins of the MAPK- and cAMP-associated pathways in these two brain regions. Postmortem samples were used from a well-characterized collection of elderly patients with schizophrenia (ACC=36, DLPFC=35) and a comparison (ACC=33, DLPFC=31) group. Near-infrared intensity of IR-dye labeled secondary antisera bound to targeted proteins of the MAPK- and cAMP-associated signaling pathways was measured using LiCor Odyssey imaging system. We found decreased expression of Rap2, JNK1, JNK2, PSD-95, and decreased phosphorylation of JNK1/2 at T183/Y185 and PSD-95 at S295 in the ACC in schizophrenia. In the DLPFC, we found increased expression of Rack1, Fyn, Cdk5, and increased phosphorylation of PSD-95 at S295 and NR2B at Y1336. MAPK- and cAMP-associated molecules constitute ubiquitous intracellular signaling pathways that integrate extracellular stimuli, modify receptor expression and function, and regulate cell survival and neuroplasticity. These data suggest abnormal activity of the MAPK- and cAMP-associated pathways in frontal cortical areas in schizophrenia. These alterations may underlie the hypothesized hypoglutamatergic function in this illness. Together with previous findings, these data suggest that abnormalities of intracellular signaling pathways may contribute to the pathophysiology of schizophrenia. PMID:22048463

  15. Opposing needling promotes behavior recovery and exerts neuroprotection via the cAMP/PKA/CREB signal transduction pathway in transient MCAO rats

    PubMed Central

    JIANG, YIJING; YANG, SHANLI; TAO, JING; LIN, ZHICHENG; YE, XIAOQIAN; YOU, YONGMEI; PENG, JUN; HONG, ZHENFENG; CHEN, LIDIAN

    2016-01-01

    The aim of the present study was to investigate whether the cyclic adenosine 3′,5′-monophosphate (cAMP)/protein kinase A(PKA)/cAMP-responsive element binding protein (CREB) signal transduction pathway triggered by γ-aminobutyric acid class B (GABAB) receptor activation is involved in neuroprotection against ischemia and behavioral recovery induced by opposing needling (ON). A total of 80 rats were randomly divided into four groups: A sham operation group, an ischemia group, an ON group and an ON group effectively inhibited by the GABAB receptor antagonist, CGP35384 (n=20/group). The behavior of the rats was assessed by their neurological deficit score, whereas the impairment of gait was examined using the CatWalk system. The volume of cerebral infarction was examined upon treatment with 2,3,5-triphenyltetrazolium chloride. The expression levels of CREB, GABAB1 and GABAB2 were examined by western blotting and reverse transcription-quantitative polymerase chain reaction, and the activity of adenylyl cyclase (AC), cAMP and PKA in the serum was detected using an enzyme-linked immunosorbent assay. In the present study, in comparison with other groups, the ON group exhibited a reduced score for the neurological deficit, the stride length and swing speed were improved, and the volume of infarction was reduced. However, these effects were reversed upon administration of CGP35384. Additionally, the expression levels of CREB, GABAB1 and GABAB2 were increased in the ON group. The levels of AC, cAMP and PKA in the serum were also increased in the ON group, whereas the addition of CGP35384 reversed these effects. The results of the present study demonstrated that ON markedly protected the brain against transient cerebral ischemic injury, and this effect was possibly mediated by the activation of the GABAB/cAMP/PKA/CREB signal transduction pathway. These findings implied that ON may be a potential therapeutic method for treating stroke. PMID:26780954

  16. Dysregulation of hepatic cAMP levels via altered Pde4b expression plays a critical role in alcohol-induced steatosis.

    PubMed

    Avila, Diana V; Barker, David F; Zhang, JingWen; McClain, Craig J; Barve, Shirish; Gobejishvili, Leila

    2016-09-01

    Alcohol-induced hepatic steatosis is a significant risk factor for progressive liver disease. Cyclic adenosine monophosphate (cAMP) signalling has been shown to significantly regulate lipid metabolism; however, the role of altered cAMP homeostasis in alcohol-mediated hepatic steatosis has never been studied. Our previous work demonstrated that increased expression of hepatic phosphodiesterase 4 (Pde4), which specifically hydrolyses and decreases cAMP levels, plays a pathogenic role in the development of liver inflammation/injury. The aim of this study was to examine the role of PDE4 in alcohol-induced hepatic steatosis. C57BL/6 wild-type and Pde4b knockout (Pde4b(-/-) ) mice were pair-fed control or ethanol liquid diets. One group of wild-type mice received rolipram, a PDE4-specific inhibitor, during alcohol feeding. We demonstrate for the first time that an early increase in PDE4 enzyme expression and a resultant decrease in hepatic cAMP levels are associated with the significant reduction in carnitine palmitoyltransferase 1A (Cpt1a) expression. Notably, alcohol-fed (AF) Pde4b(-/-) mice and AF wild-type mice treated with rolipram had significantly lower hepatic free fatty acid content compared with AF wild-type mice. Importantly, PDE4 inhibition in alcohol-fed mice prevented the decrease in hepatic Cpt1a expression via the Pparα/Sirt1/Pgc1α pathway. These results demonstrate that the alcohol- induced increase in hepatic Pde4, specifically Pde4b expression, and compromised cAMP signalling predispose the liver to impaired fatty acid oxidation and the development of steatosis. Moreover, these data also suggest that hepatic PDE4 may be a clinically relevant therapeutic target for the treatment of alcohol-induced hepatic steatosis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:27287961

  17. Modification of human placental alkaline phosphatase by periodate-oxidized 1,N6-ethenoadenosine monophosphate.

    PubMed Central

    Chang, G G; Shiao, M S; Lee, K R; Wu, J J

    1990-01-01

    Oxidation of 1,N6-ethenoadenosine monophosphate (epsilon AMP) with periodate cleaved the cis-diol of the ribose ring and resulted in the formation of a dialdehyde derivative (epsilon AMP-dial). At room temperature epsilon AMP-dial was unstable and underwent beta-elimination to give 4',5'-anhydro-1,N6-ethenoadenosine dialdehyde acetal (A epsilon Ado-dial). These nucleotide analogues were found to inactivate human placental alkaline phosphatase in a time- and concentration-dependent manner. epsilon AMP-dial was shown to be an affinity label for the enzyme on the basis of the following criteria. (a) Kinetics of the enzyme activity loss over a wide range of epsilon AMP-dial concentration showed a saturating phenomenon. Removal of the phosphate group made the reagent (A epsilon Ado-dial) become a general chemical modifying reagent. (b) The artificial substrate p-nitrophenyl phosphate gave substantial protection of the enzyme against inactivation. (c) epsilon AMP-dial was a substrate and a partial mixed-type inhibitor for the enzyme. Results of the inhibition and protection studies indicated that the reagent and substrate could combine with the enzyme simultaneously. Besides the phosphate-binding domain, an induced hydrophobic region is proposed for the substrate-binding site for human placental alkaline phosphatase. PMID:2176472

  18. Evolutionary paths of the cAMP-dependent protein kinase (PKA) catalytic subunits.

    PubMed

    Søberg, Kristoffer; Jahnsen, Tore; Rognes, Torbjørn; Skålhegg, Bjørn S; Laerdahl, Jon K

    2013-01-01

    3',5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits Cα and Cβ, encoded by the two genes PRKACA and PRKACB, respectively, are among the best understood and characterized human kinases. Here we have studied the evolution of this gene family in chordates, arthropods, mollusks and other animals employing probabilistic methods and show that Cα and Cβ arose by duplication of an ancestral PKA catalytic subunit in a common ancestor of vertebrates. The two genes have subsequently been duplicated in teleost fishes. The evolution of the PRKACG retroposon in simians was also investigated. Although the degree of sequence conservation in the PKA Cα/Cβ kinase family is exceptionally high, a small set of signature residues defining Cα and Cβ subfamilies were identified. These conserved residues might be important for functions that are unique to the Cα or Cβ clades. This study also provides a good example of a seemingly simple phylogenetic problem which, due to a very high degree of sequence conservation and corresponding weak phylogenetic signals, combined with problematic nonphylogenetic signals, is nontrivial for state-of-the-art probabilistic phylogenetic methods. PMID:23593352

  19. cAmp activation of apical membrane Cl(-) channels: theoretical considerations for impedance analysis.

    PubMed

    Păunescu, T G; Helman, S I

    2001-08-01

    Transepithelial electrical impedance analysis provides a sensitive method to evaluate the conductances and capacitances of apical and basolateral plasma membranes of epithelial cells. Impedance analysis is complicated, due not only to the anatomical arrangement of the cells and their paracellular shunt pathways, but also in particular to the existence of audio frequency-dependent capacitances or dispersions. In this paper we explore implications and consequences of anatomically related Maxwell-Wagner and Cole-Cole dielectric dispersions that impose limitations, approximations, and pitfalls of impedance analysis when tissues are studied under widely ranging spontaneous rates of transport, and in particular when apical membrane sodium and chloride channels are activated by adenosine 3',5'-cyclic monophosphate (cAMP) in A6 epithelia. We develop the thesis that capacitive relaxation processes of any origin lead not only to dependence on frequency of the impedance locus, but also to the appearance of depressed semicircles in Nyquist transepithelial impedance plots, regardless of the tightness or leakiness of the paracellular shunt pathways. Frequency dependence of capacitance precludes analysis of data in traditional ways, where capacitance is assumed constant, and is especially important when apical and/or basolateral membranes exhibit one or more dielectric dispersions.

  20. Reciprocal roles between caffeine and estrogen on bone via differently regulating cAMP/PKA pathway: the possible mechanism for caffeine-induced osteoporosis in women and estrogen's antagonistic effects.

    PubMed

    Zhou, Yi; Zhu, Zhuo-Li; Guan, Xiao-Xu; Hou, Wei-Wei; Yu, Hai-Yang

    2009-07-01

    Caffeine is consumed by most people in Europe and North America. As a risk factor for osteoporosis, caffeine has been reported to decrease bone mineral density, negatively influence calcium absorption and increase the risk of bone fracture in women. Except for the epidemiological observations and several studies which proved caffeine's unfavorable effects on osteoblast proliferation and impaired ability to form bone, little mechanism is known for the caffeine-induced osteoporosis. Since our unpublished studies showed that the precursor cells of osteoblasts, bone marrow-derived mesenchymal stem cells (BMSCs), were more sensitive than osteoblasts when exposed to the same dose of caffeine. We herein hypothesize that MSCs may be the primary target cells for caffeine-induced osteoporosis. It is well established that increasing cyclic 3',5'-adenosine monophosphate (cAMP) can regulate the expression of key genes involved in bone metabolism, including Cbfa1, PPARgamma, RANKL and OPG. We thereby propose the hypothesis that caffeine, a known inhibitor of cAMP phosphodiesterase, may affect bone metabolism by activating cAMP-dependent protein kinase A (PKA) pathway. In addition, considering the fact observed in epidemiology that caffeine's negative effects on bone only occurred in postmenopausal women and the inverse roles of caffeine and estrogen on bone metabolism, we postulate that caffeine may exert its undesirable influences on bone only in absence or low level of estrogen in vivo and estrogen may antagonize the adverse effect of caffeine on bone. Since several studies have demonstrated that estrogen may have ability to temper the biological effects of cAMP stimulators' roles on bone through cAMP to regulate some important genes' expression in bone metabolism. We assume that estrogen may block cAMP-dependent PKA pathway which is shared by caffeine, to exhibit its antagonistic roles. PMID:19278793

  1. Rat fat-cells have three types of adenosine receptors (Ra, Ri and P). Differential effects of pertussis toxin.

    PubMed Central

    García-Sáinz, J A; Torner, M L

    1985-01-01

    Activation of rat adipocyte R1 adenosine receptors by phenylisopropyladenosine (PIA) decreased cyclic AMP and lipolysis; this effect was blocked in cells from pertussis-toxin-treated rats. In contrast, the ability of 2',5'-dideoxyadenosine to decrease cyclic AMP was not affected by pertussis-toxin treatment. Addition of adenosine deaminase to the medium in which adipocytes from control animals were incubated resulted in activation of lipolysis. Interestingly, adipocytes from toxin-treated rats (which had an already increased basal lipolysis) responded in an opposite fashion to the addition of adenosine deaminase, i.e. the enzyme decreased lipolysis, which suggested that adenosine might be increasing lipolysis in these cells. Studies with the selective agonists N-ethylcarboxamidoadenosine (NECA) and PIA indicated that adenosine increases lipolysis and cyclic AMP accumulation in these cells and that these actions are mediated through Ra adenosine receptors. Adenosine-mediated accumulation of cyclic AMP was also observed in cells preincubated with pertussis toxin (2 micrograms/ml) for 3 h. In these studies NECA was also more effective than PIA. Our results indicate that there are three types of adenosine receptors in fat-cells, whose actions are affected differently by pertussis toxin, i.e. Ri-mediated actions are abolished, Ra-mediated actions are revealed and P-mediated actions are not affected. PMID:3004405

  2. Phosphorylation of adenosine with trimetaphosphate under simulated prebiotic conditions.

    PubMed

    Cheng, Changmei; Fan, Chang; Wan, Rong; Tong, Chunyuan; Miao, Zhiwei; Chen, Jing; Zhao, Yufen

    2002-06-01

    The phosphorylation of adenosine with trimetaphosphate in solution, in solid phase and using wet-dry cycles was carried out and it was found that wet-dry cycles were the most efficient. The catalytic effects of some metal ions on the phosphorylation were also studied and it was discovered that Ni(II) is the most effective. The combination of wet-dry cycles (4 cycles) and catalysis by Ni(II) led to an unprecedented high conversion of adenosine to phosphorylated products (30%) near neutral pH. The main phosphorylated products were 2',3'-cyclic AMP (10.4%) and 5'-ATP (13.0%). PMID:12227426

  3. A selective adenosine sensor derived from a triplex DNA aptamer.

    PubMed

    Patel, Mayurbhai; Dutta, Avishek; Huang, Haidong

    2011-07-01

    The aim of this study is to develop a selective adenosine aptamer sensor using a rational approach. Unlike traditional RNA aptamers developed from SELEX, duplex DNA containing an abasic site can function as a general scaffold to rationally design aptamers for small aromatic molecules. We discovered that abasic site-containing triplex DNA can also function as an aptamer and provide better affinity than duplex DNA aptamers. A novel adenosine aptamer sensor was designed using such a triplex. The aptamer is modified with furano-dU in the binding site to sense the binding. The sensor bound adenosine has a dissociation constant of 400 nM, more than tenfold stronger than the adenosine aptamer developed from SELEX. The binding quenched furano-dU fluorescence by 40%. It was also demonstrated in this study that this sensor is selective for adenosine over uridine, cytidine, guanosine, ATP, and AMP. The detection limit of this sensor is about 50 nM. The sensor can be used to quantify adenosine concentrations between 50 nM and 2 μM. PMID:21547431

  4. Intranasal Delivery of Influenza rNP Adjuvanted with c-di-AMP Induces Strong Humoral and Cellular Immune Responses and Provides Protection against Virus Challenge

    PubMed Central

    Sanchez, Maria Victoria; Ebensen, Thomas; Schulze, Kai; Cargnelutti, Diego; Blazejewska, Paulina; Scodeller, Eduardo A.; Guzmán, Carlos A.

    2014-01-01

    There is a critical need for new influenza vaccines able to protect against constantly emerging divergent virus strains. This will be sustained by the induction of vigorous cellular responses and humoral immunity capable of acting at the portal of entry of this pathogen. In this study we evaluate the protective efficacy of intranasal vaccination with recombinant influenza nucleoprotein (rNP) co-administrated with bis-(3′,5′)-cyclic dimeric adenosine monophosphate (c-di-AMP) as adjuvant. Immunization of BALB/c mice with two doses of the formulation stimulates high titers of NP-specific IgG in serum and secretory IgA at mucosal sites. This formulation also promotes a strong Th1 response characterized by high secretion of INF-γ and IL-2. The immune response elicited promotes efficient protection against virus challenge. These results suggest that c-di-AMP is a potent mucosal adjuvant which may significantly contribute towards the development of innovative mucosal vaccines against influenza. PMID:25140692

  5. Binding of adenosine and receptor-specific analogues to lymphocytes from control subjects and patients with common variable immunodeficiency.

    PubMed Central

    Shah, T; Simpson, R J; Webster, A D; Peters, T J

    1987-01-01

    Studies were performed on the binding of tritiated adenosine and its analogues, 5'-N-ethylcarboxamide adenosine (NECA) and N6-phenylisopropyladenosine (PIA), to human peripheral blood lymphocytes. These revealed binding only of adenosine (Kd, 1-10 microM, 14,000 binding sites/cell), which was abolished by dipyridamole, a specific adenosine transport inhibitor, suggesting that the binding is to the nucleoside transporter. The absence of high affinity (Kd less than or equal to 1 microM) binding of adenosine or of the two analogues. NECA and PIA suggests that the previously reported effects of adenosine on cAMP formation are not mediated by cell surface specific nucleoside receptors. Binding of adenosine to the carrier in lymphocytes from patients with common variable immunodeficiency was similar to those from control subjects. PMID:2958197

  6. Is a decrease in cyclic AMP a necessary and sufficient signal for maturation of amphibian oocytes

    SciTech Connect

    Gelerstein, S.; Shapira, H.; Dascal, N.; Yekuel, R.; Oron, Y.

    1988-05-01

    Acetylcholine rapidly lowered the intracellular levels of cyclic AMP in stage 5 and 6 Xenopus laevis oocytes. Acetylcholine alone did not induce oocyte maturation, though it did accelerate maturation induced by progesterone. The effect of acetylcholine on oocyte maturation was independent of extracellular calcium concentration. Adenosine increased cyclic AMP and abolished the progesterone-induced decrease in cyclic AMP levels in follicles and in denuded oocytes. This effect of adenosine was blocked by the Ra purinergic receptor antagonist, theophylline. Despite those effects, adenosine alone induced maturation in stage 6 oocytes and accelerated progesterone-induced maturation in both stage 5 and 6 cells. Adenosine also induced a significant increase in the rate of /sup 45/Ca efflux from oocytes in the presence and the absence of external calcium. We suggest that the activation of cell surface receptors involved in the release of calcium from cellular stores may induce or accelerate oocyte maturation independently of small changes in intracellular cyclic AMP concentration.

  7. cAMP- but not Ca(2+)-regulated Cl- conductance is lacking in cystic fibrosis mice epididymides and seminal vesicles.

    PubMed

    Leung, A Y; Wong, P Y; Yankaskas, J R; Boucher, R C

    1996-07-01

    Cystic fibrosis (CF) reflects the loss of adenosine 3',5'-cyclic monophosphate (cAMP)-regulated Cl- secretion consequent to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In humans, but not mice, with CF, the disease is associated with male infertility. The present study investigated the relative magnitudes of the cAMP pathways and an alternative Ca(2+)-regulated Cl- secretory pathway in primary cultures of the epididymides and the seminal vesicles of normal and CF mice. The basal equivalent short-circuit currents (Ieq) of cultures derived from the epididymides and the seminal vesicles from the CF mice were lower (6.0 +/- 0.6 and 4.0 +/- 1.0 muA/cm2, respectively) than those from normal mice (11.1 +/- 1.0 and 6.6 +/- 0.6 muA/cm2, respectively). Forskolin induced significant Ieq responses in both the epididymis (8.0 +/- 0.7 muA/cm2) and seminal vesicles (4.0 +/- 0.5 muA/cm2) from normal mice, whereas forskolin-induced changes in Ieq in CF mouse epididymis and seminal vesicles were absent, consistent with defective cAMP-CFTR-mediated Cl- secretion in CF mice. Ieq responses to agonists (ionomycin, ATP) that raise intracellular Ca2+ (Ca2+i) were larger than forskolin responses in normal animals (6.6 +/- 0.9 and 13.4 +/- 1.8 muA/cm2, respectively) and were preserved in CF (6.5 +/- 0.9 and 17.1 +/- 1.0 muA/cm2, respectively). We speculate that the fertility of male CF mice is maintained by persistent expression of the predominant alternative Ca(2+)-mediated Cl- transport system in the epididymides and seminal vesicles. PMID:8760045

  8. Intracellular pH in frog skin: Effects of Na sup + , volume, and cAMP

    SciTech Connect

    Civan, M.M.; Cragoe, E.J. Jr.; Peterson-Yantorno, K. Merck Sharp Dohme Research Laboratories, West Point, PA )

    1988-07-01

    Single skins were analyzed by {sup 31}P-nuclear magnetic resonance (NMR) spectroscopy during alternative perfusion with control and experimental solutions. Intracellular (pH{sub c}) and extracellular (pH{sub o}) pH were monitored by measuring the spectral frequencies of intracellular P{sub i} and external methylphosphonate, respectively. Base-line pH{sub c} was 7.20 {plus minus} 0.02 (SE) when pH{sub o} was 6.99 {plus minus} 0.02. A 4-acetamido-4{prime}-isothiocyanostilbene-2,2{prime}-disulfonic acid (SITS)-inhibitable, HCO{sup {minus}}{sub 3}-dependent alkaline shift in pH{sub c} can be elicited by replacing external Cl{sup {minus}} by gluconate or sulfate. The authors now report that this effect is observed even in sodium-free media. The substitution of gluconate for external Cl{sup {minus}} has also been reported to shrink cell volume. This shrinkage can be minimized by replacing Cl{sup {minus}} with gluconate during perfusion with hypotonic, rather than isotonic, media. Conducted in this manner, the anionic substitution produced a much smaller alkaline shift in pH{sub c}. In the presence of the Na-H antiport blocker 5-(N-methyl-N-isobutyl)amiloride (MIA), restoration of external Na{sup +} did not increase pH{sub c}. Adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) also alters pH{sub c}. The data suggest that frog skin regulates pH{sub c} by the parallel operation of Na-H and Na{sup +}-independent Cl-HCO{sub 3} antiports. Cell volume and cAMP may play regulating roles in this epithelium.

  9. Kinetic and mechanistic analysis of dinucleotide and oligonucleotide formation from the 5'-phosphorimidazolide of adenosine on Na(+)-montmorillonite

    NASA Technical Reports Server (NTRS)

    Kawamura, K.; Ferris, J. P.

    1994-01-01

    The rate constants for the condensation reaction of the 5'-phosphorimidazolide of adenosine (ImpA) to form dinucleotides and oligonucleotides have been measured in the presence of Na(+)-volclay (a Na(+)-montmorillonite) in pH 8 aqueous solution at 25 degrees C. The rates of the reaction of ImpA with an excess of adenosine 5'-monophosphoramidate (NH2pA), P1,P2-diadenosine 5',5'-pyrophosphate (A5'ppA), or adenosine 5'-monophosphate (5'-AMP or pA) in the presence of the montmorillonite to form NH2pA3'pA, A5'ppA3'pA, and pA3'pA, respectively, were measured. Only 3',5'-linked products were observed. The magnitude of the rate constants decrease in the order NH2pA3'pA > A5'-ppA3'pA > pA3'pA. The binding of ImpA to montmorillonite was measured, and the adsorption isotherm was determined. The binding of ImpA to montmorillonite and the formation of higher oligonucleotides is not observed in the absence of salts. Mg2+ enhances binding and oligonucleotide formation more than Ca2+ and Na+. The rate constants for the oligonucleotide formation were determined from the reaction products formed from 10 to 40 mM ImpA in the presence of Na(+)-montmorillonite using the computer program SIMFIT. The magnitudes of the rate constants for the formation of oligonucleotides increased in the order 2-mer < 3-mer < 4-mer ... 7-mer. The rate constants for dinucleotide and trinucleotide formation are more than 1000 times larger than those measured in the absence of montmorillonite. The rate constants for the formation of dinucleotide, trinucleotide, and tetranucleotide are 41,2.6, and 3.7 times larger than those for the formation of oligo(G)s with a poly(C) template. The hydrolysis of ImpA was accelerated 35 times in the presence of the montmorillonite. The catalytic ability of montmorillonite to form dinucleotides and oligonucleotides is quantitatively evaluated and possible pathways for oligo(A) formation are proposed.

  10. Modulation of dog atrial swelling-induced chloride current by cAMP: protein kinase A-dependent and -independent pathways.

    PubMed Central

    Du, X Y; Sorota, S

    1997-01-01

    1. The modulation of dog atrial swelling-induced chloride current (I(Cl,swelling)) by cAMP-elevating agents was studied. Forskolin (10 microM) or isoprenaline (1 microM) exerted multiple effects. Although the pattern between cells was variable, there was, in general, a stimulatory action and a more slowly developing inhibitory effect. 2. In any given cell, the response to forskolin or isoprenaline was qualitatively similar suggesting that all of the responses were dependent on stimulation of adenylyl cyclase. The effects of forskolin or isoprenaline on I(Cl,swelling) were inhibited by intracellular dialysis with a P-site inhibitor of adenylyl cyclase, 2'-deoxyadenosine 3'-monophosphate (300 microM). 3. Intracellular dialysis with a peptide inhibitor of protein kinase A (PKI(6-22); 100 microM) blocked the inhibitory response to forskolin or isoprenaline and all cells responded with a monophasic stimulation of I(Cl,swelling). 4. After intracellular dialysis of cells with PKI(6-22) (100 microM) and cAMP (100 microM), current amplitude was not further stimulated by forskolin. 5. After intracellular dialysis with PKI(6-22) and adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), forskolin stimulated I(Cl,swelling) and the effect of forskolin subsided after it was washed out. 6. In conclusion, there are dual pathways by which cAMP can modulate dog atrial cell I(Cl,swelling). Inhibition results from protein kinase A (PKA)-dependent phosphorylation. In addition, a stimulatory pathway exists that is independent of phosphorylation by PKA or other cellular kinases. Although alternative explanations are possible, the stimulatory effect of cAMP may represent a direct modulation of I(Cl,swelling). Images Figure 1 PMID:9097937

  11. Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms

    PubMed Central

    Francis, Heather L; DeMorrow, Sharon; Franchitto, Antonio; Venter, Julie K; Mancinelli, Romina A; White, Mellanie A; Meng, Fanyin; Ueno, Yoshiyuki; Carpino, Guido; Renzi, Anastasia; Baker, Kimberly K; Shine, Hannah E; Francis, Taylor C; Gaudio, Eugenio; Alpini, Gianfranco D; Onori, Paolo

    2012-01-01

    Although large cholangiocytes exert their functions by activation of cyclic adenosine 3′,5′-monophosphate (cAMP), Ca2+-dependent signaling regulates the function of small cholangiocytes. Histamine interacts with four receptors, H1–H4HRs. H1HR acts by Gαq activating IP3/Ca2+, whereas H2HR activates Gαs stimulating cAMP. We hypothesize that histamine increases biliary growth by activating H1HR on small and H2HR on large cholangiocytes. The expression of H1–H4HRs was evaluated in liver sections, isolated and cultured (normal rat intrahepatic cholangiocyte culture (NRIC)) cholangiocytes. In vivo, normal rats were treated with histamine or H1–H4HR agonists for 1 week. We evaluated: (1) intrahepatic bile duct mass (IBDM); (2) the effects of histamine, H1HR or H2HR agonists on NRIC proliferation, IP3 and cAMP levels and PKCα and protein kinase A (PKA) phosphorylation; and (3) PKCα silencing on H1HR-stimulated NRIC proliferation. Small and large cholangiocytes express H1–H4HRs. Histamine and the H1HR agonist increased small IBDM, whereas histamine and the H2HR agonist increased large IBDM. H1HR agonists stimulated IP3 levels, as well as PKCα phosphorylation and NRIC proliferation, whereas H2HR agonists increased cAMP levels, as well as PKA phosphorylation and NRIC proliferation. The H1HR agonist did not increase proliferation in PKCα siRNA-transfected NRICs. The activation of differential signaling mechanisms targeting small and large cholangiocytes is important for repopulation of the biliary epithelium during pathologies affecting different-sized bile ducts. PMID:22064319

  12. Adenosine and sleep

    SciTech Connect

    Yanik, G.M. Jr.

    1987-01-01

    Behavioral and biochemical approaches have been used to determine the relative contribution of endogenous adenosine and adenosine receptors to the sleep-wake cycle in the rat. Adenosine concentrations in specific areas of the rat brain were not affected by 24 hours of total sleep deprivation, or by 24 or 48 hours of REM sleep deprivation. In order to assess the effect of REM sleep deprivation on adenosine A/sub 1/ receptors, /sup 3/H-L-PIA binding was measured. The Bmax values for /sup 3/H-L-PIA binding to membrane preparations of the cortices and corpus striata from 48 hour REM sleep-deprived animals were increased 14.8% and 23%, respectively. These increases were not maintained following the cessation of sleep deprivation and recovered within 2 hours. The results of a 96 hour REM deprivation experiment were similar to those of the 48 hour REM sleep deprivation experiment. However, these increases were not evident in similar structures taken from stress control animals, and conclusively demonstrated that the changes in /sup 3/H-L-PIA binding resulted from REM sleep deprivation and not from stress.

  13. Effects of adenosine metabolism in astrocytes on central nervous system oxygen toxicity.

    PubMed

    Chen, Yu-liang; Zhang, Ya-nan; Wang, Zhong-zhuang; Xu, Wei-gang; Li, Run-ping; Zhang, Jun-dong

    2016-03-15

    Hyperbaric oxygen (HBO) is widely used in military operations, especially underwater missions. However, prolonged and continuous inhalation of HBO can cause central nervous system oxygen toxicity (CNS-OT), which greatly limits HBO's application. The regulation of astrocytes to the metabolism of adenosine is involved in epilepsy. In our study, we aimed to observe the effects of HBO exposure on the metabolism of adenosine in the brain. Furthermore, we aimed to confirm the possible mechanism underlying adenosine's mediation of the CNS-OT. Firstly, anesthetized rats exposed to 5 atm absolute HBO for 80 min. The concentrations of extracellular adenosine, ATP, ADP, and AMP were detected. Secondly, free-moving rats were exposed to HBO at the same pressure for 20 min, and the activities of 5'-nucleotidase and ADK in brain tissues were measured. For the mechanism studies, we observed the effects of a series of different doses of drugs related to adenosine metabolism on the latency of CNS-OT. Results showed HBO exposure could increase adenosine content by inhibiting ADK activity and improving 5'-nucleotidase activity. And adenosine metabolism during HBO exposure may be a protective response against HBO-induced CNS-OT. Moreover, the improvement of adenosine concentration, activation of adenosine A1R, or suppression of ADK and adenosine A2AR, which are involved in the prevention of HBO-induced CNS-OT. This is the first study to demonstrate HBO exposure regulated adenosine metabolism in the brain. Adenosine metabolism and adenosine receptors are related to HBO-induced CNS-OT development. These results will provide new potential targets for the termination or the attenuation of CNS-OT. PMID:26806404

  14. Viral keratitis-inhibitory effect of 9-beta-D-arabinofuranosylhypoxanthine 5'-monophosphate.

    PubMed

    Sidwell, R W; Allen, L B; Huffman, J H; Revankar, G R; Robins, R K; Tolman, R L

    1975-10-01

    Topical application of 9-beta-d-arabinofuranosylhypoxanthine 5'-monophosphate (ara-HxMP) significantly inhibited the development of keratitis induced by types 1 and 2 herpes simplex virus and vaccinia virus in the eyes of rabbits. Parameters for evaluation of efficacy were infectivity (corneal opacity, lesion size, and type), Draize (erythema, conjunctival swelling, and discharge), and reduction in titer of recoverable virus from the eye. When the relative efficacy of the related compounds 9-beta-d-arabinofuranosyladenine (ara-A), ara-A 5'-monophosphate (ara-AMP), and ara-Hx was determined against type 1 herpes simplex virus in a parallel experiment, the more water-soluble compounds (ara-HxMP, ara-AMP) were the most effective. The relative efficacy of ara-A was also determined against type 2 herpes and vaccinia virus-induced keratitis. Mortality in rabbits due to central nervous system involvement caused by types 1 and 2 herpes simplex virus was inhibited. Ara-HxMP was not discernibly toxic to the eye at concentrations of at least 20%; efficacy was still discernible with a 0.1% solution.

  15. Adenosine and the Auditory System

    PubMed Central

    Vlajkovic, Srdjan M; Housley, Gary D; Thorne, Peter R

    2009-01-01

    Adenosine is a signalling molecule that modulates cellular activity in the central nervous system and peripheral organs via four G protein-coupled receptors designated A1, A2A, A2B, and A3. This review surveys the literature on the role of adenosine in auditory function, particularly cochlear function and its protection from oxidative stress. The specific tissue distribution of adenosine receptors in the mammalian cochlea implicates adenosine signalling in sensory transduction and auditory neurotransmission although functional studies have demonstrated that adenosine stimulates cochlear blood flow, but does not alter the resting and sound-evoked auditory potentials. An interest in a potential otoprotective role for adenosine has recently evolved, fuelled by the capacity of A1 adenosine receptors to prevent cochlear injury caused by acoustic trauma and ototoxic drugs. The balance between A1 and A2A receptors is conceived as critical for cochlear response to oxidative stress, which is an underlying mechanism of the most common inner ear pathologies (e.g. noise-induced and age-related hearing loss, drug ototoxicity). Enzymes involved in adenosine metabolism, adenosine kinase and adenosine deaminase, are also emerging as attractive targets for controlling oxidative stress in the cochlea. Other possible targets include ectonucleotidases that generate adenosine from extracellular ATP, and nucleoside transporters, which regulate adenosine concentrations on both sides of the plasma membrane. Developments of selective adenosine receptor agonists and antagonists that can cross the blood-cochlea barrier are bolstering efforts to develop therapeutic interventions aimed at ameliorating cochlear injury. Manipulations of the adenosine signalling system thus hold significant promise in the therapeutic management of oxidative stress in the cochlea. PMID:20190966

  16. Structure of the DNA Ligase-Adenylate Intermediate: Lysine (ε-amino)-Linked Adenosine Monophosphoramidate*

    PubMed Central

    Gumport, Richard I.; Lehman, I. R.

    1971-01-01

    Proteolytic degradation of the Escherichia coli DNA ligase-adenylate intermediate releases adenosine 5′-monophosphate linked to the ε-amino group of lysine by a phosphoamide bond. Measurements of the rate of hydroxylaminolysis of the ligase-adenylate provide further support for a phosphoamide linkage in the native enzyme. Lysine (ε-amino)-linked adenosine monophosphoramidate has also been isolated from the T4 phage-induced ligase-adenylate intermediate. These results indicate that an initial step of the DNA ligase reaction consists of the nucleophilic attack of the ε-amino group of a lysine residue of the enzyme on the adenylyl phosphorus of DPN or ATP that leads to the formation of enzyme-bound lysine (εamino)-linked adenosine monophosphoramidate. PMID:4944632

  17. Smoke extract impairs adenosine wound healing: implications of smoke-generated reactive oxygen species.

    PubMed

    Allen-Gipson, Diane S; Zimmerman, Matthew C; Zhang, Hui; Castellanos, Glenda; O'Malley, Jennifer K; Alvarez-Ramirez, Horacio; Kharbanda, Kusum; Sisson, Joseph H; Wyatt, Todd A

    2013-05-01

    Adenosine concentrations are elevated in the lungs of patients with asthma and chronic obstructive pulmonary disease, where it balances between tissue repair and excessive airway remodeling. We previously demonstrated that the activation of the adenosine A2A receptor promotes epithelial wound closure. However, the mechanism by which adenosine-mediated wound healing occurs after cigarette smoke exposure has not been investigated. The present study investigates whether cigarette smoke exposure alters adenosine-mediated reparative properties via its ability to induce a shift in the oxidant/antioxidant balance. Using an in vitro wounding model, bronchial epithelial cells were exposed to 5% cigarette smoke extract, were wounded, and were then stimulated with either 10 μM adenosine or the specific A2A receptor agonist, 5'-(N-cyclopropyl)-carboxamido-adenosine (CPCA; 10 μM), and assessed for wound closure. In a subset of experiments, bronchial epithelial cells were infected with adenovirus vectors encoding human superoxide dismutase and/or catalase or control vector. In the presence of 5% smoke extract, significant delay was evident in both adenosine-mediated and CPCA-mediated wound closure. However, cells pretreated with N-acetylcysteine (NAC), a nonspecific antioxidant, reversed smoke extract-mediated inhibition. We found that cells overexpressing mitochondrial catalase repealed the smoke extract inhibition of CPCA-stimulated wound closure, whereas superoxide dismutase overexpression exerted no effect. Kinase experiments revealed that smoke extract significantly reduced the A2A-mediated activation of cyclic adenosine monophosphate-dependent protein kinase. However, pretreatment with NAC reversed this effect. In conclusion, our data suggest that cigarette smoke exposure impairs A2A-stimulated wound repair via a reactive oxygen species-dependent mechanism, thereby providing a better understanding of adenosine signaling that may direct the development of pharmacological

  18. Cadmium-induced decrement of the LH receptor expression and cAMP levels in the testis of rats.

    PubMed

    Gunnarsson, David; Nordberg, Gunnar; Lundgren, Per; Selstam, Gunnar

    2003-02-01

    Cadmium (Cd) is a widespread environmental pollutant, characterized by its ability to affect various organs. Adverse effect of Cd on the testis including decreased testosterone production are well-known phenomena, but the cellular events explaining these effects have not yet been established. In the present study the initial steps of gonadotropin mediated testosterone biosynthesis were examined in vivo in rats, in relation to Cd dose and time after injection. In the dose-response experiment Male Sprague-Dawley rats received a single subcutaneous (s.c.) injection of CdCl(2) (1, 5 or 10 micromol/kg body weight) and were sacrificed 48 h after injection. A statistically significant decrease in luteinizing hormone (LH) receptor mRNA level in the testicular tissue was demonstrated at the highest dose (10 micromol/kg). In the temporal-response experiment rats were given 10 micromol/kg of CdCl(2) s.c. and sacrificed 0.48, 4.8, 48 or 144 h after injection. LH receptor mRNA levels as well as cyclic adenosine monophosphate (cAMP) levels were found to be significantly lowered at 48 and 144 h. These observations of the mechanisms whereby Cd exerts its effect on the initial steps of testosterone biosynthesis are the first from in vivo experiments. PMID:12504342

  19. Role of coronary endothelium in cyclic AMP formation by the heart

    SciTech Connect

    Kroll, K.; Schrader, J.

    1986-03-01

    In order to quantify the activation of adenylate cyclase of the coronary endothelium in vivo, endothelial adenine nucleotides of isolated guinea pig hearts were selectively pre-labeled by intracoronary infusion of tritiated (H3)-adenosine, and the coronary efflux of H3-cAMP was measured. The adenosine receptor agonist, NECA (12 ..mu..M), increased total cAMP release 4 fold, and raised H3-cAMP release 22 fold. Several classes of coronary vasodilators (adenosine, L-PIA, D-PIA, the beta 2-adrenergic agonist procaterol, and PGE1) caused dose-dependent increases in endothelial-derived H3-cAMP release. These increases were accompanied by decreases in vascular resistance, at agonist doses without positive intropic effects. Hypoxic perfusion also raised H3-cAMP release, and this was antagonized by theophylline. It is concluded: (1) cyclic AMP formation by coronary endothelium can dominate total cAMP production by the heart; (2) coronary endothelial adenylate cyclase-coupled receptors for adenosine (A2), catecholamines (beta2) and prostaglandins are activated in parallel with coronary vasodilation; (3) endothelial adenylate cyclase can be activated by endogenous adenosine.

  20. Adenosine and Sleep

    PubMed Central

    Bjorness, Theresa E; Greene, Robert W

    2009-01-01

    Over the last several decades the idea that adenosine (Ado) plays a role in sleep control was postulated due in large part to pharmacological studies that showed the ability of Ado agonists to induce sleep and Ado antagonists to decrease sleep. A second wave of research involving in vitro cellular analytic approaches and subsequently, the use of neurochemical tools such as microdialysis, identified a population of cells within the brainstem and basal forebrain arousal centers, with activity that is both tightly coupled to thalamocortical activation and under tonic inhibitory control by Ado. Most recently, genetic tools have been used to show that Ado receptors regulate a key aspect of sleep, the slow wave activity expressed during slow wave sleep. This review will briefly introduce some of the phenomenology of sleep and then summarize the effect of Ado levels on sleep, the effect of sleep on Ado levels, and recent experiments using mutant mouse models to characterize the role for Ado in sleep control and end with a discussion of which Ado receptors are involved in such control. When taken together, these various experiments suggest that while Ado does play a role in sleep control, it is a specific role with specific functional implications and it is one of many neurotransmitters and neuromodulators affecting the complex behavior of sleep. Finally, since the majority of adenosine-related experiments in the sleep field have focused on SWS, this review will focus largely on SWS; however, the role of adenosine in REM sleep behavior will be addressed. PMID:20190965

  1. Rat cardiac myocyte adenosine transport and metabolism

    SciTech Connect

    Ford, D.A.; Rovetto, M.J.

    1987-01-01

    Based on the importance of myocardial adenosine and adenine nucleotide metabolism, the adenosine salvage pathway in ventricular myocytes was studied. Accurate estimates of transport rates, separate from metabolic fllux, were determined. Adenosine influx was constant between 3 and 60 s. Adenosine metabolism maintained intracellular adenosine concentrations < 10% of the extracellular adenosine concentrations and thus unidirectional influx could be measured. Myocytes transported adenosine via saturable and nonsaturable processes. A minimum estimate of the V/sub max/ of myocytic adenosine kinase indicated the saturable component of adenosine influx was independent of adenosine kinase activity. Saturable transport was inhibited by nitrobenzylthioinosine and verapamil. Extracellular adenosine taken up myocytes was rapidly phosphorylated to adenine taken up by myocytes was rapidly phosphorylated to adenine nucleotides. Not all extracellular adenosine, though, was phosphorylated on entering myocytes, since free, as opposed to protein-bound, intracellular adenosine was detected after digitonin extraction of cells in the presence of 1 mM ethylene-diaminetetraacetic acid.

  2. Adenosine deaminase from Streptomyces coelicolor: recombinant expression, purification and characterization.

    PubMed

    Pornbanlualap, Somchai; Chalopagorn, Pornchanok

    2011-08-01

    The sequencing of the genome of Streptomyces coelicolor A3(2) identified seven putative adenine/adenosine deaminases and adenosine deaminase-like proteins, none of which have been biochemically characterized. This report describes recombinant expression, purification and characterization of SCO4901 which had been annotated in data bases as a putative adenosine deaminase. The purified putative adenosine deaminase gives a subunit Mr=48,400 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 182,000 by comparative gel filtration. These values are consistent with the active enzyme being composed of four subunits with identical molecular weights. The turnover rate of adenosine is 11.5 s⁻¹ at 30 °C. Since adenine is deaminated ∼10³ slower by the enzyme when compared to that of adenosine, these data strongly show that the purified enzyme is an adenosine deaminase (ADA) and not an adenine deaminase (ADE). Other adenine nucleosides/nucleotides, including 9-β-D-arabinofuranosyl-adenine (ara-A), 5'-AMP, 5'-ADP and 5'-ATP, are not substrates for the enzyme. Coformycin and 2'-deoxycoformycin are potent competitive inhibitors of the enzyme with inhibition constants of 0.25 and 3.4 nM, respectively. Amino acid sequence alignment of ScADA with ADAs from other organisms reveals that eight of the nine highly conserved catalytic site residues in other ADAs are also conserved in ScADA. The only non-conserved residue is Asn317, which replaces Asp296 in the murine enzyme. Based on these data, it is suggested here that ADA and ADE proteins are divergently related enzymes that have evolved from a common α/β barrel scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism. PMID:21511036

  3. Adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate are synthesized by yeast acetyl coenzyme A synthetase.

    PubMed Central

    Guranowski, A; Günther Sillero, M A; Sillero, A

    1994-01-01

    Yeast (Saccharomyces cerevisiae) acetyl coenzyme A (CoA) synthetase (EC 6.2.1.1) catalyzes the synthesis of adenosine 5'-tetraphosphate (P4A) and adenosine 5'-pentaphosphate (p5A) from ATP and tri- or tetrapolyphosphate (P3 or P4), with relative velocities of 7:1, respectively. Of 12 nucleotides tested as potential donors of nucleotidyl moiety, only ATP, adenosine-5'-O-[3-thiotriphosphate], and acetyl-AMP were substrates, with relative velocities of 100, 62, and 80, respectively. The Km values for ATP, P3, and acetyl-AMP were 0.16, 4.7, and 1.8 mM, respectively. The synthesis of p4A could proceed in the absence of exogenous acetate but was stimulated twofold by acetate, with an apparent Km value of 0.065 mM. CoA did not participate in the synthesis of p4A (p5A) and inhibited the reaction (50% inhibitory concentration of 0.015 mM). At pH 6.3, which was optimum for formation of p4A (p5A), the rate of acetyl-CoA synthesis (1.84 mumol mg-1 min-1) was 245 times faster than the rate of synthesis of p4A measured in the presence of acetate. The known formation of p4A (p5A) in yeast sporulation and the role of acetate may therefore be related to acetyl-CoA synthetase. Images PMID:7910605

  4. Role of intragenic binding of cAMP responsive protein (CRP) in regulation of the succinate dehydrogenase genes Rv0249c-Rv0247c in TB complex mycobacteria

    PubMed Central

    Knapp, Gwendowlyn S.; Lyubetskaya, Anna; Peterson, Matthew W.; Gomes, Antonio L.C.; Ma, Zhuo; Galagan, James E.; McDonough, Kathleen A.

    2015-01-01

    Bacterial pathogens adapt to changing environments within their hosts, and the signaling molecule adenosine 3′, 5′-cyclic monophosphate (cAMP) facilitates this process. In this study, we characterized in vivo DNA binding and gene regulation by the cAMP-responsive protein CRP in M. bovis BCG as a model for tuberculosis (TB)-complex bacteria. Chromatin immunoprecipitation followed by deep-sequencing (ChIP-seq) showed that CRP associates with ∼900 DNA binding regions, most of which occur within genes. The most highly enriched binding region was upstream of a putative copper transporter gene (ctpB), and crp-deleted bacteria showed increased sensitivity to copper toxicity. Detailed mutational analysis of four CRP binding sites upstream of the virulence-associated Rv0249c-Rv0247c succinate dehydrogenase genes demonstrated that CRP directly regulates Rv0249c-Rv0247c expression from two promoters, one of which requires sequences intragenic to Rv0250c for maximum expression. The high percentage of intragenic CRP binding sites and our demonstration that these intragenic DNA sequences significantly contribute to biologically relevant gene expression greatly expand the genome space that must be considered for gene regulatory analyses in mycobacteria. These findings also have practical implications for an important bacterial pathogen in which identification of mutations that affect expression of drug target-related genes is widely used for rapid drug resistance screening. PMID:25940627

  5. Genetics Home Reference: adenosine deaminase 2 deficiency

    MedlinePlus

    ... Health Conditions adenosine deaminase 2 deficiency adenosine deaminase 2 deficiency Enable Javascript to view the expand/collapse ... PDF Open All Close All Description Adenosine deaminase 2 (ADA2) deficiency is a disorder characterized by abnormal ...

  6. An Adenosine-Mediated Glial-Neuronal Circuit for Homeostatic Sleep

    PubMed Central

    Bjorness, Theresa E.; Dale, Nicholas; Mettlach, Gabriel; Sonneborn, Alex; Sahin, Bogachan; Fienberg, Allen A.; Yanagisawa, Masashi; Bibb, James A.

    2016-01-01

    Sleep homeostasis reflects a centrally mediated drive for sleep, which increases during waking and resolves during subsequent sleep. Here we demonstrate that mice deficient for glial adenosine kinase (AdK), the primary metabolizing enzyme for adenosine (Ado), exhibit enhanced expression of this homeostatic drive by three independent measures: (1) increased rebound of slow-wave activity; (2) increased consolidation of slow-wave sleep; and (3) increased time constant of slow-wave activity decay during an average slow-wave sleep episode, proposed and validated here as a new index for homeostatic sleep drive. Conversely, mice deficient for the neuronal adenosine A1 receptor exhibit significantly decreased sleep drive as judged by these same indices. Neuronal knock-out of AdK did not influence homeostatic sleep need. Together, these findings implicate a glial-neuronal circuit mediated by intercellular Ado, controlling expression of homeostatic sleep drive. Because AdK is tightly regulated by glial metabolic state, our findings suggest a functional link between cellular metabolism and sleep homeostasis. SIGNIFICANCE STATEMENT The work presented here provides evidence for an adenosine-mediated regulation of sleep in response to waking (i.e., homeostatic sleep need), requiring activation of neuronal adenosine A1 receptors and controlled by glial adenosine kinase. Adenosine kinase acts as a highly sensitive and important metabolic sensor of the glial ATP/ADP and AMP ratio directly controlling intracellular adenosine concentration. Glial equilibrative adenosine transporters reflect the intracellular concentration to the extracellular milieu to activate neuronal adenosine receptors. Thus, adenosine mediates a glial-neuronal circuit linking glial metabolic state to neural-expressed sleep homeostasis. This indicates a metabolically related function(s) for this glial-neuronal circuit in the buildup and resolution of our need to sleep and suggests potential therapeutic targets

  7. Recognition of Nucleoside Monophosphate Substrates by Haemophilus influenzae Class C Acid Phosphatase

    SciTech Connect

    Singh, Harkewal; Schuermann, Jonathan P.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2010-12-08

    The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD{sup +} utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5{prime},3{prime}-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5{prime}-AMP, 3{prime}-AMP, and 2{prime}-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5{prime}-nucleotides and 3{prime}-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5{prime} substrates in an anti conformation and 3{prime} substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition.

  8. Recognition of nucleoside monophosphate substrates by Haemophilus influenzae class C acid phosphatase.

    PubMed

    Singh, Harkewal; Schuermann, Jonathan P; Reilly, Thomas J; Calcutt, Michael J; Tanner, John J

    2010-12-10

    The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD(+) utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5',3'-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5'-AMP, 3'-AMP, and 2'-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5'-nucleotides and 3'-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5' substrates in an anti conformation and 3' substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition. PMID:20934434

  9. Wnt Signaling Inhibits Osteoclast Differentiation by Activating Canonical and Noncanonical cAMP/PKA Pathways

    PubMed Central

    Weivoda, Megan M; Ruan, Ming; Hachfeld, Christine M; Pederson, Larry; Howe, Alan; Davey, Rachel A; Zajac, Jeffrey D; Kobayashi, Yasuhiro; Williams, Bart O; Westendorf, Jennifer J; Khosla, Sundeep; Oursler, Merry Jo

    2016-01-01

    Although there has been extensive characterization of the Wnt signaling pathway in the osteoblast lineage, the effects of Wnt proteins on the osteoclast lineage are less well studied. We found that osteoclast lineage cells express canonical Wnt receptors. Wnt3a reduced osteoclast formation when applied to early bone-marrow macrophage (BMM) osteoclast differentiation cultures, whereas late addition did not suppress osteoclast formation. Early Wnt3a treatment inactivated the crucial transcription factor NFATc1 in osteoclast progenitors. Wnt3a led to the accumulation of nuclear β-catenin, confirming activation of canonical Wnt signaling. Reducing low-density lipoprotein receptor-related proteins (Lrp) 5 and Lrp6 protein expression prevented Wnt3a-induced inactivation of NFATc1; however, deletion of β-catenin did not block Wnt3a inactivation of NFATc1, suggesting that this effect was mediated by a noncanonical pathway. Wnt3a rapidly activated the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and pharmacological stimulation of cAMP/PKA signaling suppressed osteoclast differentiation; Wnt3a-induced NFATc1 phosphorylation was blocked by inhibiting interactions between PKA and A-kinase anchoring proteins (AKAPs). These data indicate that Wnt3a directly suppresses osteoclast differentiation through both canonical (β-catenin) and noncanonical (cAMP/PKA) pathways in osteoclast precursors. In vivo reduction of Lrp5 and Lrp6 expressions in the early osteoclast lineage via Rank promoter Cre recombination reduced trabecular bone mass, whereas disruption of Lrp5/6 expression in late osteoclast precursors via cathepsin K (Ctsk) promoter Cre recombination did not alter the skeletal phenotype. Surprisingly, reduction of Lrp5/6 in the early osteoclast lineage decreased osteoclast numbers, as well as osteoblast numbers. Published studies have previously noted that β-catenin signaling is required for osteoclast progenitor proliferation. Our in vivo data

  10. Structural Studies of Thiamin Monophosphate Kinase in Complex with Substrates and Products.

    SciTech Connect

    McCulloch, K.M.; Kinsland, C.; Begley, T.P.; Ealick, S E.

    2008-06-03

    Thiamin monophosphate kinase (ThiL) catalyzes the ATP-dependent phosphorylation of thiamin monophosphate (TMP) to form thiamin pyrophosphate (TPP), the active form of vitamin B1. ThiL is a member of a small ATP binding superfamily that also includes the purine biosynthetic enzymes, PurM and PurL, NiFe hydrogenase maturation protein, HypE, and selenophosphate synthase, SelD. The latter four enzymes are believed to utilize phosphorylated intermediates during catalysis. To understand the mechanism of ThiL and its relationship to the other superfamily members, we determined the structure of Aquifex aeolicus ThiL (AaThiL) with nonhydrolyzable AMP-PCP and TMP, and also with the products of the reaction, ADP and TPP. The results suggest that AaThiL utilizes a direct, inline transfer of the {gamma}-phosphate of ATP to TMP rather than a phosphorylated enzyme intermediate. The structure of ThiL is compared to those of PurM, PurL, and HypE, and the ATP binding site is compared to that of PurL, for which nucleotide complexes are available.

  11. Dissociations in the Effects of β2-Adrenergic Receptor Agonists on cAMP Formation and Superoxide Production in Human Neutrophils: Support for the Concept of Functional Selectivity

    PubMed Central

    Brunskole Hummel, Irena; Reinartz, Michael T.; Kälble, Solveig; Burhenne, Heike; Schwede, Frank; Buschauer, Armin; Seifert, Roland

    2013-01-01

    In neutrophils, activation of the β2-adrenergic receptor (β2AR), a Gs-coupled receptor, inhibits inflammatory responses, which could be therapeutically exploited. The aim of this study was to evaluate the effects of various β2AR ligands on adenosine-3′,5′-cyclic monophosphate (cAMP) accumulation and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced superoxide anion (O2•−) production in human neutrophils and to probe the concept of ligand-specific receptor conformations (also referred to as functional selectivity or biased signaling) in a native cell system. This is an important question because so far, evidence for functional selectivity has been predominantly obtained with recombinant systems, due to the inherent difficulties to genetically manipulate human native cells. cAMP concentration was determined by HPLC/tandem mass spectrometry, and O2•− formation was assessed by superoxide dismutase-inhibitable reduction of ferricytochrome c. β2AR agonists were generally more potent in inhibiting fMLP-induced O2•− production than in stimulating cAMP accumulation. (−)-Ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the cAMP assay, but partially inhibited fMLP-induced O2•− production. Moreover, (−)-adrenaline was equi-efficacious in both assays whereas the efficacy of salbutamol was more than two-fold higher in the O2•− assay. Functional selectivity was visualized by deviations of ligand potencies and efficacies from linear correlations for various parameters. We obtained no evidence for involvement of protein kinase A in the inhibition of fMLP-induced O2•− production after β2AR-stimulation although cAMP-increasing substances inhibited O2•− production. Taken together, our data corroborate the concept of ligand-specific receptor conformations with unique signaling capabilities in native human cells and suggest that the β2AR inhibits O2•− production in a cAMP-independent manner. PMID

  12. Prostaglandin E2 induces vascular endothelial growth factor secretion in prostate cancer cells through EP2 receptor-mediated cAMP pathway.

    PubMed

    Wang, Xingya; Klein, Russell D

    2007-11-01

    Prostaglandin E2 (PGE2) has been shown to induce expression of vascular endothelial growth factor (VEGF) and other signaling molecules in several cancers. PGE2 elicits its functions though four G-protein coupled membrane receptors (EP1-4). In this study, we investigated the role of EP receptors in PGE2-induced molecular events in prostate cancer cells. qRT-PCR analysis revealed that PC-3 cells express a substantially higher level of EP2 and moderately higher EP4 than DU145 and LNCaP cells. LNCaP cells had virtually no detectable EP2 mRNA. EP1 and EP3 mRNAs were not detected in these cells. Treatment of prostate cancer cells with PGE2 (1 nM-10 microM) increased both VEGF secretion and cyclic adenosine monophosphate (cAMP) production. Levels of induction in PC-3 cells were greater than in DU145 and LNCaP cells. The selective EP2 agonist CAY10399 also significantly increased VEGF secretion and cAMP production in PC-3 cells, but not in DU145 and LNCaP cells. Moreover, PGE2 and CAY10399 increased mitogen activated protein kinase/extracellular signal regulated kinase (MAPK/Erk) and Akt phosphorylation in PC-3 and DU145 cells, but not in LNCaP cells. However, neither the MAPK/Erk inhibitor U0126 nor the PI3K/Akt inhibitor LY294002 abolished PGE2-induced VEGF secretion in PC-3 cells. We further demonstrated that the adenylate cyclase activator forskolin and the cAMP anologue 8-bromo-cAMP mimicked the effects of PGE2 on VEGF secretion in PC-3 cells. Meanwhile, the adenylate cyclase inhibitor 2'5'-dideoxyadenosine, at concentrations that inhibited PGE2-induced cAMP, significantly blocked PGE2-induced VEGF secretion in PC-3 cells. We conclude that PGE2-induced VEGF secretion in prostate cancer cells is mediated through EP2-, and possibly EP4-, dependent cAMP signaling pathways. PMID:17427962

  13. Exposure to Hydrogen Peroxide Induces Oxidation and Activation of AMP-activated Protein Kinase*

    PubMed Central

    Zmijewski, Jaroslaw W.; Banerjee, Sami; Bae, Hongbeom; Friggeri, Arnaud; Lazarowski, Eduardo R.; Abraham, Edward

    2010-01-01

    Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5′-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPKαβγ complex or HEK 293 cells to H2O2 was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPKα and AMPKβ subunits. In experiments using C-terminal truncation mutants of AMPKα (amino acids 1–312), we found that mutation of cysteine 299 to alanine diminished the ability of H2O2 to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H2O2 on kinase activity. Similar to the results obtained with H2O2-treated HEK 293 cells, activation and S-glutathionylation of the AMPKα subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H2O2 are increased. These results demonstrate that physiologically relevant concentrations of H2O2 can activate AMPK through oxidative modification of the AMPKα subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status. PMID:20729205

  14. Ionizing Radiation Activates AMP-Activated Kinase (AMPK): A Target for Radiosensitization of Human Cancer Cells

    SciTech Connect

    Sanli, Toran; Rashid, Ayesha; Liu Caiqiong

    2010-09-01

    Purpose: Adenosine monophosphate (AMP)-activated kinase (AMPK) is a molecular energy sensor regulated by the tumor suppressor LKB1. Starvation and growth factors activate AMPK through the DNA damage sensor ataxia-telangiectasia mutated (ATM). We explored the regulation of AMPK by ionizing radiation (IR) and its role as a target for radiosensitization of human cancer cells. Methods and Materials: Lung, prostate, and breast cancer cells were treated with IR (2-8 Gy) after incubation with either ATM or AMPK inhibitors or the AMPK activator metformin. Then, cells were subjected to either lysis and immunoblotting, immunofluorescence microscopy, clonogenic survival assays, or cell cycle analysis. Results: IR induced a robust phosphorylation and activation of AMPK in all tumor cells, independent of LKB1. IR activated AMPK first in the nucleus, and this extended later into cytoplasm. The ATM inhibitor KU-55933 blocked IR activation of AMPK. AMPK inhibition with Compound C or anti-AMPK {alpha} subunit small interfering RNA (siRNA) blocked IR induction of the cell cycle regulators p53 and p21{sup waf/cip} as well as the IR-induced G2/M arrest. Compound C caused resistance to IR, increasing the surviving fraction after 2 Gy, but the anti-diabetic drug metformin enhanced IR activation of AMPK and lowered the surviving fraction after 2 Gy further. Conclusions: We provide evidence that IR activates AMPK in human cancer cells in an LKB1-independent manner, leading to induction of p21{sup waf/cip} and regulation of the cell cycle and survival. AMPK appears to (1) participate in an ATM-AMPK-p21{sup waf/cip} pathway, (2) be involved in regulation of the IR-induced G2/M checkpoint, and (3) may be targeted by metformin to enhance IR responses.

  15. Involvement of adenosine A2a receptor in intraocular pressure decrease induced by 2-(1-octyn-1-yl)adenosine or 2-(6-cyano-1-hexyn-1-yl)adenosine.

    PubMed

    Konno, Takashi; Murakami, Akira; Uchibori, Takehiro; Nagai, Akihiko; Kogi, Kentaro; Nakahata, Norimichi

    2005-04-01

    The aim of the present study is to clarify the mechanism for the decrease in intraocular pressure by 2-alkynyladenosine derivatives in rabbits. The receptor binding analysis revealed that 2-(1-octyn-1-yl)adenosine (2-O-Ado) and 2-(6-cyano-1-hexyn-1-yl)adenosine (2-CN-Ado) selectively bound to the A(2a) receptor with a high affinity. Ocular hypotensive responses to 2-O-Ado and 2-CN-Ado were inhibited by the adenosine A(2a)-receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC), but not by the adenosine A(1)-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or the adenosine A(2b)-receptor antagonist alloxazine. In addition, 2-O-Ado and 2-CN-Ado caused an increase in outflow facility, which was inhibited by CSC, but not by DPCPX or alloxazine. Moreover, 2-O-Ado and 2-CN-Ado increased cAMP in the aqueous humor, and the 2-O-Ado-induced an increase in cAMP was inhibited by CSC. These results suggest that 2-O-Ado and 2-CN-Ado reduced intraocular pressure via an increase in outflow facility. The ocular hypotension may be mainly mediated through the activation of adenosine A(2a) receptor, although a possible involvement of adenosine A(1) receptor cannot be completely ruled out. 2-O-Ado and 2-CN-Ado are useful lead compounds for the treatment of glaucoma.

  16. Imaging Adenosine Triphosphate (ATP).

    PubMed

    Rajendran, Megha; Dane, Eric; Conley, Jason; Tantama, Mathew

    2016-08-01

    Adenosine triphosphate (ATP) is a universal mediator of metabolism and signaling across unicellular and multicellular species. There is a fundamental interdependence between the dynamics of ATP and the physiology that occurs inside and outside the cell. Characterizing and understanding ATP dynamics provide valuable mechanistic insight into processes that range from neurotransmission to the chemotaxis of immune cells. Therefore, we require the methodology to interrogate both temporal and spatial components of ATP dynamics from the subcellular to the organismal levels in live specimens. Over the last several decades, a number of molecular probes that are specific to ATP have been developed. These probes have been combined with imaging approaches, particularly optical microscopy, to enable qualitative and quantitative detection of this critical molecule. In this review, we survey current examples of technologies available for visualizing ATP in living cells, and identify areas where new tools and approaches are needed to expand our capabilities. PMID:27638696

  17. Imaging Adenosine Triphosphate (ATP).

    PubMed

    Rajendran, Megha; Dane, Eric; Conley, Jason; Tantama, Mathew

    2016-08-01

    Adenosine triphosphate (ATP) is a universal mediator of metabolism and signaling across unicellular and multicellular species. There is a fundamental interdependence between the dynamics of ATP and the physiology that occurs inside and outside the cell. Characterizing and understanding ATP dynamics provide valuable mechanistic insight into processes that range from neurotransmission to the chemotaxis of immune cells. Therefore, we require the methodology to interrogate both temporal and spatial components of ATP dynamics from the subcellular to the organismal levels in live specimens. Over the last several decades, a number of molecular probes that are specific to ATP have been developed. These probes have been combined with imaging approaches, particularly optical microscopy, to enable qualitative and quantitative detection of this critical molecule. In this review, we survey current examples of technologies available for visualizing ATP in living cells, and identify areas where new tools and approaches are needed to expand our capabilities.

  18. Sesamol decreases melanin biosynthesis in melanocyte cells and zebrafish: Possible involvement of MITF via the intracellular cAMP and p38/JNK signalling pathways.

    PubMed

    Baek, Seung-hwa; Lee, Sang-Han

    2015-10-01

    The development of antimelanogenic agents is important for the prevention of serious aesthetic problems such as melasma, freckles, age spots and chloasma. The aim of this study was to investigate the antimelanogenic effect of sesamol, an active lignan isolated from Sesamum indicum, in melan-a cells. Sesamol strongly inhibited melanin biosynthesis and the activity of intracellular tyrosinase by decreasing cyclic adenosine monophosphate (cAMP) accumulation. Sesamol significantly decreased the expression of melanogenesis-related genes, such as tyrosinase, tyrosinase-related protein-1,2 (TRP-1,2), microphthalmia-associated transcription factor (MITF) and melanocortin 1 receptor (MC1R). In addition, sesamol also induces phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK). Moreover, sesamol dose-dependently decreased zebrafish pigment formation, tyrosinase activity and expression of melanogenesis-related genes. These findings indicate that sesamol inhibited melanin biosynthesis by down-regulating tyrosinase activity and melanin production via regulation of gene expression of melanogenesis-related proteins through modulation of MITF activity, which promoted phosphorylation of p38 and JNK in melan-a cells. Together, these results suggest that sesamol strongly inhibits melanin biosynthesis, and therefore, sesamol represents a new skin-whitening agent for use in cosmetics.

  19. Inhibition of Platelet Activation and Thrombus Formation by Adenosine and Inosine: Studies on Their Relative Contribution and Molecular Modeling

    PubMed Central

    Fuentes, Eduardo; Pereira, Jaime; Mezzano, Diego; Alarcón, Marcelo; Caballero, Julio; Palomo, Iván

    2014-01-01

    Background The inhibitory effect of adenosine on platelet aggregation is abrogated after the addition of adenosine-deaminase. Inosine is a naturally occurring nucleoside degraded from adenosine. Objectives The mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated. Results Adenosine (0.5, 1 and 2 mmol/L) inhibited phosphatidylserine exposure from 52±4% in the control group to 44±4 (p<0.05), 29±2 (p<0.01) and 20±3% (p<0.001). P-selectin expression in the presence of adenosine 0.5, 1 and 2 mmol/L was inhibited from 32±4 to 27±2 (p<0.05), 14±3 (p<0.01) and 9±3% (p<0.001), respectively. At the concentrations tested, only inosine to 4 mmol/L had effect on platelet P-selectin expression (p<0.05). Adenosine and inosine inhibited platelet aggregation and ATP release stimulated by ADP and collagen. Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow. At the same concentrations adenosine inhibited platelet aggregation, decreased the levels of sCD40L and increased intraplatelet cAMP. In addition, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent adenosine receptor A2A antagonist) attenuated the effect of adenosine on platelet aggregation induced by ADP and intraplatelet level of cAMP. Adenosine and inosine significantly inhibited thrombosis formation in vivo (62±2% occlusion at 60 min [n = 6, p<0.01] and 72±1.9% occlusion at 60 min, [n = 6, p<0.05], respectively) compared with the control (98±2% occlusion at 60 min, n = 6). A2A is the adenosine receptor present in platelets; it is known that inosine is not an A2A ligand. Docking of adenosine and inosine inside A2A showed that the main difference is the formation by adenosine of an additional hydrogen bond between the NH2 of the adenine group and the residues Asn253 in H6 and Glu169 in EL2 of the A2A receptor. Conclusion Therefore

  20. Adenosine regulates the proinflammatory signaling function of thrombin in endothelial cells

    PubMed Central

    Hassanian, Seyed Mahdi; Dinarvand, Peyman; Rezaie, Alireza R.

    2014-01-01

    The plasma level of the regulatory metabolite adenosine increases during the activation of coagulation and inflammation. Here we investigated the effect of adenosine on modulation of thrombin-mediated proinflammatory responses in HUVECs. We found that adenosine inhibits the barrier-disruptive effect of thrombin in HUVECs by a concentration-dependent manner. Analysis of cell surface expression of adenosine receptors revealed that A2A and A2B are expressed at the highest level among the four receptor subtypes (A2B>A2A>A1>A3) on HUVECs. The barrier-protective effect of adenosine in response to thrombin was recapitulated by the A2A specific agonist, CGS 21680, and abrogated both by the siRNA knockdown of the A2A receptor and by the A2A-specific antagonists, ZM-241385 and SCH-58261. The thrombin-induced RhoA activation and its membrane translocation were both inhibited by adenosine in a cAMP-dependent manner, providing a molecular mechanism through which adenosine exerts a barrier-protective function. Adenosine also inhibited thrombin-mediated activation of NF-κB and decreased adhesion of monocytic THP-1 cells to stimulated HUVECs via down-regulation of expression of cell surface adhesion molecules, VCAM-1, ICAM-1 and E-selectin. Moreover, adenosine inhibited thrombin-induced elevated expression of proinflammatory cytokines, IL-6 and HMGB-1; and chemokines, MCP-1, CXCL-1 and CXCL-3. Taken together, these results suggest that adenosine may inhibit thrombin-mediated proinflammatory signaling responses, thereby protecting the endothelium from injury during activation of coagulation and inflammation. PMID:24477600

  1. Mechanism of A2 adenosine receptor activation. I. Blockade of A2 adenosine receptors by photoaffinity labeling

    SciTech Connect

    Lohse, M.J.; Klotz, K.N.; Schwabe, U.

    1991-04-01

    It has previously been shown that covalent incorporation of the photoreactive adenosine derivative (R)-2-azido-N6-p-hydroxy-phenylisopropyladenosine ((R)-AHPIA) into the A1 adenosine receptor of intact fat cells leads to a persistent activation of this receptor, resulting in a reduction of cellular cAMP levels. In contrast, covalent incorporation of (R)-AHPIA into human platelet membranes, which contain only stimulatory A2 adenosine receptors, reduces adenylate cyclase stimulation via these receptors. This effect of (R)-AHPIA is specific for the A2 receptor and can be prevented by the adenosine receptor antagonist theophylline. Binding studies indicate that up to 90% of A2 receptors can be blocked by photoincorporation of (R)-AHPIA. However, the remaining 10-20% of A2 receptors are sufficient to mediate an adenylate cyclase stimulation of up to 50% of the control value. Similarly, the activation via these 10-20% of receptors occurs with a half-life that is only 2 times longer than that in control membranes. This indicates the presence of a receptor reserve, with respect to both the extent and the rate of adenylate cyclase stimulation. These observations require a modification of the models of receptor-adenylate cyclase coupling.

  2. Adenosine-Associated Delivery Systems

    PubMed Central

    Kazemzadeh-Narbat, Mehdi; Annabi, Nasim; Tamayol, Ali; Oklu, Rahmi; Ghanem, Amyl; Khademhosseini, Ali

    2016-01-01

    Adenosine is a naturally occurring purine nucleoside in every cell. Many critical treatments such as modulating irregular heartbeat (arrhythmias), regulation of central nervous system (CNS) activity, and inhibiting seizural episodes can be carried out using adenosine. Despite the significant potential therapeutic impact of adenosine and its derivatives, the severe side effects caused by their systemic administration have significantly limited their clinical use. In addition, due to adenosine’s extremely short half-life in human blood (less than 10 s), there is an unmet need for sustained delivery systems to enhance efficacy and reduce side effects. In this paper, various adenosine delivery techniques, including encapsulation into biodegradable polymers, cell-based delivery, implantable biomaterials, and mechanical-based delivery systems, are critically reviewed and the existing challenges are highlighted. PMID:26453156

  3. Central or peripheral delivery of an adenosine A1 receptor agonist improves mechanical allodynia in a mouse model of painful diabetic neuropathy.

    PubMed

    Katz, N K; Ryals, J M; Wright, D E

    2015-01-29

    Diabetic peripheral neuropathy is a common complication of diabetes mellitus, and a significant proportion of individuals suffer debilitating pain that significantly affects their quality of life. Unfortunately, symptomatic treatment options have limited efficacy, and often carry significant risk of systemic adverse effects. Activation of the adenosine A1 receptor (A1R) by the analgesic small molecule adenosine has been shown to have antinociceptive benefits in models of inflammatory and neuropathic pain. The current study used a mouse model of painful diabetic neuropathy to determine the effect of diabetes on endogenous adenosine production, and if central or peripheral delivery of adenosine receptor agonists could alleviate signs of mechanical allodynia in diabetic mice. Diabetes was induced using streptozocin in male A/J mice. Mechanical withdrawal thresholds were measured weekly to characterize neuropathy phenotype. Hydrolysis of AMP into adenosine by ectonucleotidases was determined in the dorsal root ganglia (DRG) and spinal cord at 8 weeks post-induction of diabetes. AMP, adenosine and the specific A1R agonist, N(6)-cyclopentyladenosine (CPA), were administered both centrally (intrathecal) and peripherally (intraplantar) to determine the effect of activation of adenosine receptors on mechanical allodynia in diabetic mice. Eight weeks post-induction, diabetic mice displayed significantly decreased hydrolysis of extracellular AMP in the DRG; at this same time, diabetic mice displayed significantly decreased mechanical withdrawal thresholds compared to nondiabetic controls. Central delivery AMP, adenosine and CPA significantly improved mechanical withdrawal thresholds in diabetic mice. Surprisingly, peripheral delivery of CPA also improved mechanical allodynia in diabetic mice. This study provides new evidence that diabetes significantly affects endogenous AMP hydrolysis, suggesting that altered adenosine production could contribute to the development of

  4. Adenosine stimulates DNA fragmentation in human thymocytes by Ca(2+)-mediated mechanisms.

    PubMed

    Szondy, Z

    1994-12-15

    Incubation of human thymocytes with an optimum concentration of adenosine and its receptor site agonist, 2-chloroadenosine, induced increases in intracellular cyclic AMP (cAMP) (from a resting 0.6 +/- 0.1 to 4.1 +/- 0.2 pmol/10(7) cells within 5 min) and Ca2+ (from the resting 85 +/- 7 nM to a peak of 210 +/- 25 nM) levels and resulted in internucleosomal DNA fragmentation and cell death (apoptosis). Other adenosine analogues were also effective at inducing DNA fragmentation, the order of potency being 2-p-(carboxyethylphenylethylamino)-5'-carboxyamidoadenosine < 5'-(N-ethylcarboxamide)adenosine < or = cyclopentyladenosine < 2-chloroadenosine (2-CA). 2-CA treatment (with an optimum concentration of 40 microM) selectively depleted a thymocyte subpopulation (15-20% of the total cells) which expressed higher levels of the CD3 molecule and which was found mainly in the CD4+CD8+ double positive immature thymocyte population. DNA fragmentation was prevented by the addition of actinomycin D or cycloheximide to the thymocyte suspension, indicating that this process required both mRNA and protein synthesis. Endonuclease activation and cell killing were dependent on an early, sustained increase in cytosolic Ca2+ concentration, most of which was of extracellular origin and was a result of an adenosine-induced inositol trisphosphate release. Other agents known to elevate intracellular cAMP levels by different mechanisms failed to induce similar DNA fragmentation, but enhanced the effect of adenosine. This suggested a supporting role for cAMP in adenosine-induced DNA fragmentation. Phorbol dibutyrate, a protein kinase. C activator, previously shown to inhibit Ca(2+)-dependent DNA fragmentation and cell killing in human thymocytes [McConkey, Hartzell, Jondal and Orrenius (1989) J. Biol. Chem. 264, 13399-13402], at 60 ng/ml concentration also prevented adenosine-induced DNA fragmentation when added prior to adenosine. This suggested a complex cross-talk between the adenosine

  5. Evidence for the Calvin cycle and hexose monophosphate pathway in Thiobacillus ferrooxidans.

    PubMed

    Gale, N L; Beck, J V

    1967-10-01

    The enzymes of the Calvin reductive pentose phosphate cycle and the hexose monophosphate pathway have been demonstrated in cell-free extracts of Thiobacillus ferrooxidans. This, together with analyses of the products of CO(2) fixation in cell-free systems, suggests that these pathways are operative in whole cells of this microorganism. Nevertheless, the amount of CO(2) fixed in these cell-free systems was limited by the type and amount of compound added as substrate. The inability of cell extracts to regenerate pentose phosphates and to perpetuate the cyclic fixation of CO(2) is partially attributable to low activity of triose phosphate dehydrogenase under the experimental conditions found to be optimal for the enzymes involved in the utilization of ribose-5-phosphate or ribulose-1,5-diphosphate as substrate for CO(2) incorporation. With the exception of ribulose-1,5-diphosphate, all substrates required the addition of adenosine triphosphate (ATP) or adenosine diphosphate (ADP) for CO(2) fixation. Under optimal conditions, with ribose-5-phosphate serving as substrate, each micromole of ATP added resulted in the fixation of 1.5 mumoles of CO(2), whereas each micromole of ADP resulted in 0.5 mumole of CO(2) fixed. These values reflect the activity of adenylate kinase in the extract preparations. The K(m) for ATP in the phosphoribulokinase reaction was 0.91 x 10(-3)m. Kinetic studies conducted with carboxydismutase showed K(m) values of 1.15 x 10(-4)m and 5 x 10(-2)m for ribulose-1,5-diphosphate and bicarbonate, respectively.

  6. Enzymatic characterization of AMP phosphorylase and ribose-1,5-bisphosphate isomerase functioning in an archaeal AMP metabolic pathway.

    PubMed

    Aono, Riku; Sato, Takaaki; Yano, Ayumu; Yoshida, Shosuke; Nishitani, Yuichi; Miki, Kunio; Imanaka, Tadayuki; Atomi, Haruyuki

    2012-12-01

    AMP phosphorylase (AMPpase), ribose-1,5-bisphosphate (R15P) isomerase, and type III ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been proposed to constitute a novel pathway involved in AMP metabolism in the Archaea. Here we performed a biochemical examination of AMPpase and R15P isomerase from Thermococcus kodakarensis. R15P isomerase was specific for the α-anomer of R15P and did not recognize other sugar compounds. We observed that activity was extremely low with the substrate R15P alone but was dramatically activated in the presence of AMP. Using AMP-activated R15P isomerase, we reevaluated the substrate specificity of AMPpase. AMPpase exhibited phosphorylase activity toward CMP and UMP in addition to AMP. The [S]-v plot (plot of velocity versus substrate concentration) of the enzyme toward AMP was sigmoidal, with an increase in activity observed at concentrations higher than approximately 3 mM. The behavior of the two enzymes toward AMP indicates that the pathway is intrinsically designed to prevent excess degradation of intracellular AMP. We further examined the formation of 3-phosphoglycerate from AMP, CMP, and UMP in T. kodakarensis cell extracts. 3-Phosphoglycerate generation was observed from AMP alone, and from CMP or UMP in the presence of dAMP, which also activates R15P isomerase. 3-Phosphoglycerate was not formed when 2-carboxyarabinitol 1,5-bisphosphate, a Rubisco inhibitor, was added. The results strongly suggest that these enzymes are actually involved in the conversion of nucleoside monophosphates to 3-phosphoglycerate in T. kodakarensis.

  7. The NLRP3 inflammasome is activated by nanoparticles through ATP, ADP and adenosine

    PubMed Central

    Baron, L; Gombault, A; Fanny, M; Villeret, B; Savigny, F; Guillou, N; Panek, C; Le Bert, M; Lagente, V; Rassendren, F; Riteau, N; Couillin, I

    2015-01-01

    The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO2) and titanium dioxide (nano-TiO2) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO2 or nano-TiO2 caused a significant increase in P2Y1, P2Y2, A2A and/or A2B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2A or A2B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation. PMID:25654762

  8. 1,3-Dichloro-2-propanol inhibits progesterone production through the expression of steroidogenic enzymes and cAMP concentration in Leydig cells.

    PubMed

    Sun, Jianxia; Bai, Shun; Bai, Weibin; Zou, Feiyan; Zhang, Lei; Li, Guoqiang; Hu, Yunfeng; Li, Mingwei; Yan, Rian; Su, Zhijian; Huang, Yadong

    2014-07-01

    1,3-Dichloro-2-propanol (1,3-DCP) is a well-known food processing contaminant that has been shown to impede male reproductive function. However, its mechanism of action remains elusive. In this study, the effects of 1,3-DCP on progesterone production were investigated using the R2C Leydig cell model. 1,3-DCP significantly reduced cell viability from 7.48% to 97.4% at doses comprised between 0.5 and 6mM. Single cell gel/comet assays and atomic force microscopy assays showed that 1,3-DCP induced early phase cell apoptosis. In addition, 1,3-DCP significantly reduced progesterone production detected by radioimmunoassay (RIA). The results from quantitative polymerase chain reaction and western blotting demonstrated that the mRNA expression levels of steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase were significantly down-regulated in R2C cells. Particularly, the change rhythm of Star expression was highly consistent with progesterone production. Furthermore, the cyclic adenosine monophosphate (cAMP) and the mitochondrial membrane potential mediated by ROS, which are involved in regulating progesterone synthesis were also decreased in response to the 1,3-DCP treatment. Overall, the data presented here suggested that 1,3-DCP interferes with the male steroidogenic capacity mainly by down-regulating the level of cAMP and the key enzymes involved in the androgen synthesis pathway. PMID:24518350

  9. Beta-Adrenoceptor Activation Reduces Both Dermal Microvascular Endothelial Cell Migration via a cAMP-Dependent Mechanism and Wound Angiogenesis

    PubMed Central

    O'Leary, Andrew P; Fox, James M; Pullar, Christine E

    2015-01-01

    Angiogenesis is an essential process during tissue regeneration; however, the amount of angiogenesis directly correlates with the level of wound scarring. Angiogenesis is lower in scar-free foetal wounds while angiogenesis is raised and abnormal in pathophysiological scarring such as hypertrophic scars and keloids. Delineating the mechanisms that modulate angiogenesis and could reduce scarring would be clinically useful. Beta-adrenoceptors (β-AR) are G protein-coupled receptors (GPCRs) expressed on all skin cell-types. They play a role in wound repair but their specific role in angiogenesis is unknown. In this study, a range of in vitro assays (single cell migration, scratch wound healing, ELISAs for angiogenic growth factors and tubule formation) were performed with human dermal microvascular endothelial cells (HDMEC) to investigate and dissect mechanisms underpinning β-AR-mediated modulation of angiogenesis in chick chorioallantoic membranes (CAM) and murine excisional skin wounds. β-AR activation reduced HDMEC migration via cyclic adenosine monophosphate (cAMP)-dependent and protein kinase A (PKA)-independent mechanisms as demonstrated through use of an EPAC agonist that auto-inhibited the cAMP-mediated β-AR transduced reduction in HDMEC motility; a PKA inhibitor was, conversely, ineffective. ELISA studies demonstrated that β-AR activation reduced pro-angiogenic growth factor secretion from HDMECs (fibroblast growth factor 2) and keratinocytes (vascular endothelial growth factor A) revealing possible β-AR-mediated autocrine and paracrine anti-angiogenic mechanisms. In more complex environments, β-AR activation delayed HDMEC tubule formation and decreased angiogenesis both in the CAM assay and in murine excisional skin wounds in vivo. β-AR activation reduced HDMEC function in vitro and angiogenesis in vivo; therefore, β-AR agonists could be promising anti-angiogenic modulators in skin. J. Cell. Physiol. 230: 356–365, 2015. © 2014 The Authors. Journal

  10. Kisspeptin induction of somatolactin-α release in goldfish pituitary cells: functional role of cAMP/PKA-, PLC/PKC-, and Ca(2+)/calmodulin-dependent cascades.

    PubMed

    Jiang, Quan; He, Mulan; Ko, Wendy K W; Wong, Anderson O L

    2014-11-15

    Although the importance of kisspeptin in the pituitary is firmly established, the signaling mechanisms for the pituitary actions of kisspeptin are still largely unknown. Somatolactin (SL), a member of the growth hormone (GH)/prolactin (PRL) family, is a pituitary hormone with pleiotropic functions in fish, but its regulation by kisspeptin has not been examined. To investigate the functional role of kisspeptin in SL regulation, expression of two paralogues of goldfish Kiss1 receptors (Kiss1ra and Kiss1rb) were confirmed in immunoidentified SLα but not SLβ cells isolated by RT-PCR coupled with laser capture microdissection. In goldfish pituitary cells prepared from neurointermediate lobe (NIL), synthetic goldfish Kiss decapeptides (gKiss1-10 and gKiss2-10) could increase SLα release. Consistent with the lack of Kiss1r expression in SLβ cells, SLβ release was not altered by kisspeptin stimulation. In parallel experiments, goldfish gKiss1-10 could elevate cyclic adenosine monophosphate (cAMP) production, upregulate protein kinase A (PKA) and protein kinase C (PKC) activities, and trigger a rapid rise in intracellular Ca(2+) levels in goldfish NIL cells. Using a pharmacological approach, cAMP/PKA and phospholipase C (PLC)/PKC pathways and subsequent activation of Ca(2+)/calmodulin (CaM)-dependent cascades were shown to be involved in SLα release induced by gKiss1-10. Apparently, the Ca(2+)-dependent cascades were triggered by extracellular Ca(2+) entry via voltage-sensitive Ca(2+) channels and mobilization of inositol trisphosphate-sensitive intracellular Ca(2+) stores. Our results demonstrate that gKiss1-10 can act directly at the pituitary level to trigger SLα release via a complex network of post-receptor signaling mechanisms.

  11. Cyclic AMP in prokaryotes.

    PubMed Central

    Botsford, J L; Harman, J G

    1992-01-01

    Cyclic AMP (cAMP) is found in a variety of prokaryotes including both eubacteria and archaebacteria. cAMP plays a role in regulating gene expression, not only for the classic inducible catabolic operons, but also for other categories. In the enteric coliforms, the effects of cAMP on gene expression are mediated through its interaction with and allosteric modification of a cAMP-binding protein (CRP). The CRP-cAMP complex subsequently binds specific DNA sequences and either activates or inhibits transcription depending upon the positioning of the complex relative to the promoter. Enteric coliforms have provided a model to explore the mechanisms involved in controlling adenylate cyclase activity, in regulating adenylate cyclase synthesis, and in performing detailed examinations of CRP-cAMP complex-regulated gene expression. This review summarizes recent work focused on elucidating the molecular mechanisms of CRP-cAMP complex-mediated processes. For other bacteria, less detail is known. cAMP has been implicated in regulating antibiotic production, phototrophic growth, and pathogenesis. A role for cAMP has been suggested in nitrogen fixation. Often the only data that support cAMP involvement in these processes includes cAMP measurement, detection of the enzymes involved in cAMP metabolism, or observed effects of high concentrations of the nucleotide on cell growth. PMID:1315922

  12. Fluoroquinolones as potential photochemotherapeutic agents: covalent addition to guanosine monophosphate.

    PubMed

    Fasani, Elisa; Manet, Ilse; Capobianco, Massimo L; Monti, Sandra; Pretali, Luca; Albini, Angelo

    2010-08-21

    The triplet aryl cation photochemically generated from fluoroquinolones bearing a fluoro atom at position 8 attacks guanosine monophosphate (k(r) > 10(9) M(-1)s(-1)) and forms covalent adducts. The reaction is a model for the implementation of oxygen-independent photochemotherapy. PMID:20571620

  13. Studies on structures of lipid A-monophosphate clusters

    NASA Astrophysics Data System (ADS)

    Faunce, Chester A.; Reichelt, Hendrik; Paradies, Henrich H.

    2011-03-01

    Single crystalline clusters of lipid A-monophosphate were grown from organic dispersions containing 5-15% (v/v) water at various volume fractions, ϕ, and temperatures. The morphology of the single lipid A-monophosphate crystals was either rhombohedral or hexagonal. The hexagonal crystals were needlelike or cylindrical in shape, with the long dimension parallel to the c axis of the unit cell. The crystalline clusters were studied using electron microscopy and x-ray powder diffraction. Employing molecular location methods following a Rietveld refinement and whole-pattern refinement revealed two monoclinic crystal structures in the space groups P21 and C2, both converged with RF = 0.179. The two monoclinic crystal structures were packing (hydrocarbon chains) and conformational (sugar) polymorphs. Neither of these two structures had been encountered previously. Only intramolecular hydrogen bonding was observed for the polymorphs, which were located between the amide and the carboxyl groups. Another crystalline structure was found in the volume-fraction range 2.00 × 10-3 ≤ ϕ ≤ 2.50 × 10-3, which displayed hexagonal symmetry. The hexagonal symmetry of the self-assembled lipid A-monophosphate crystalline phase might be reconciled with the monoclinic symmetry found at low-volume-fractions. Therefore, lowering the symmetry from cubic, i.e., Ia overline 3d, to rhombohedral R overline 3 m, and finally to the monoclinic space group C2 was acceptable if the lipid A-monophosphate anion was completely orientationally ordered.

  14. Heat shock protein 70 and AMP-activated protein kinase contribute to 17-DMAG-dependent protection against heat stroke.

    PubMed

    Tsai, Yung-Chieh; Lam, Kwok-Keung; Peng, Yi-Jen; Lee, Yen-Mei; Yang, Chung-Yu; Tsai, Yi-Ju; Yen, Mao-Hsiung; Cheng, Pao-Yun

    2016-10-01

    Heat shock protein 70 (Hsp70) preconditioning induces thermotolerance, and adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a role in the process of autophagy. Here, we investigated whether 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) protected against heat stroke (HS) in rats by up-regulation of Hsp70 and phosphorylated AMPK (pAMPK). To produce HS, male Sprague-Dawley rats were placed in a chamber with an ambient temperature of 42°C. Physiological function (mean arterial pressure, heart rate and core temperature), hepatic and intestinal injury, inflammatory mediators and levels of Hsp70, pAMPK and light chain 3 (LC3B) in hepatic tissue were measured in HS rats or/and rats pre-treated with 17-DMAG. 17-DMAG pre-treatment significantly attenuated hypotension and organ dysfunction induced by HS in rats. The survival time during HS was also prolonged by 17-DMAG treatment. Hsp70 expression was increased, whereas pAMPK levels in the liver were significantly decreased in HS rats. Following pre-treatment with 17-DMAG, Hsp70 protein levels increased further, and pAMPK levels were enhanced. Treatment with an AMPK activator significantly increased the LC3BII/LC3BI ratio as a marker of autophagy in HS rats. Treatment with quercetin significantly suppressed Hsp70 and pAMPK levels and reduced the protective effects of 17-DMAG in HS rats. Both of Hsp70 and AMPK are involved in the 17-DMAG-mediated protection against HS. 17-DMAG may be a promising candidate drug in the clinical setting.

  15. Heat shock protein 70 and AMP-activated protein kinase contribute to 17-DMAG-dependent protection against heat stroke.

    PubMed

    Tsai, Yung-Chieh; Lam, Kwok-Keung; Peng, Yi-Jen; Lee, Yen-Mei; Yang, Chung-Yu; Tsai, Yi-Ju; Yen, Mao-Hsiung; Cheng, Pao-Yun

    2016-10-01

    Heat shock protein 70 (Hsp70) preconditioning induces thermotolerance, and adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a role in the process of autophagy. Here, we investigated whether 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) protected against heat stroke (HS) in rats by up-regulation of Hsp70 and phosphorylated AMPK (pAMPK). To produce HS, male Sprague-Dawley rats were placed in a chamber with an ambient temperature of 42°C. Physiological function (mean arterial pressure, heart rate and core temperature), hepatic and intestinal injury, inflammatory mediators and levels of Hsp70, pAMPK and light chain 3 (LC3B) in hepatic tissue were measured in HS rats or/and rats pre-treated with 17-DMAG. 17-DMAG pre-treatment significantly attenuated hypotension and organ dysfunction induced by HS in rats. The survival time during HS was also prolonged by 17-DMAG treatment. Hsp70 expression was increased, whereas pAMPK levels in the liver were significantly decreased in HS rats. Following pre-treatment with 17-DMAG, Hsp70 protein levels increased further, and pAMPK levels were enhanced. Treatment with an AMPK activator significantly increased the LC3BII/LC3BI ratio as a marker of autophagy in HS rats. Treatment with quercetin significantly suppressed Hsp70 and pAMPK levels and reduced the protective effects of 17-DMAG in HS rats. Both of Hsp70 and AMPK are involved in the 17-DMAG-mediated protection against HS. 17-DMAG may be a promising candidate drug in the clinical setting. PMID:27241357

  16. Gc protein-derived macrophage-activating factor (GcMAF) stimulates cAMP formation in human mononuclear cells and inhibits angiogenesis in chick embryo chorionallantoic membrane assay.

    PubMed

    Pacini, Stefania; Morucci, Gabriele; Punzi, Tiziana; Gulisano, Massimo; Ruggiero, Marco

    2011-04-01

    The effects of Gc protein-derived macrophage-activating factor (GcMAF) have been studied in cancer and other conditions where angiogenesis is deregulated. In this study, we demonstrate for the first time that the mitogenic response of human peripheral blood mononuclear cells (PBMCs) to GcMAF was associated with 3'-5'-cyclic adenosine monophosphate (cAMP) formation. The effect was dose dependent, and maximal stimulation was achieved using 0.1 ng/ml. Heparin inhibited the stimulatory effect of GcMAF on PBMCs. In addition, we demonstrate that GcMAF (1 ng/ml) inhibited prostaglandin E(1)- and human breast cancer cell-stimulated angiogenesis in chick embryo chorionallantoic membrane (CAM) assay. Finally, we tested different GcMAF preparations on CAM, and the assay proved to be a reliable, reproducible and inexpensive method to determine the relative potencies of different preparations and their stability; we observed that storage at room temperature for 15 days decreased GcMAF potency by about 50%. These data could prove useful for upcoming clinical trials on GcMAF. PMID:21170647

  17. Effects of fucoidan on insulin stimulation and pancreatic protection via the cAMP signaling pathway in vivo and in vitro.

    PubMed

    Jiang, Xiaoming; Yu, Jinfeng; Ma, Zhi; Zhang, Hong; Xie, Fengjie

    2015-09-01

    Diabetes is a global disease, in which pancreatic dysfunction is an important pathological process. In previous years, interest in the biological activities of seaweed has increased. Fucoidan is an extract of the seaweed Fucus vesiculosus, which has been widely investigated. The present study aimed to determine the effects of fucoidan on insulin stimulation and pancreatic protection in vivo and in vitro. Goto‑Kakizaki (GK) rats were provided with free access to standard food, with or without fucoidan, for 13 weeks, following which the body weights, and blood glucose and serum insulin levels of the rats were measured. Wistar rats were used as a control. In addition, the RIN‑5F rat insulin‑secreting cell line was treated with fucoidan in high glucose conditions, following which the dose‑dependent and time‑dependent effects of fucoidan were determined, and the concentration of insulin was measured. Glybenclamide was used as a positive control. In vivo, the body weight and serum insulin levels decreased, whereas blood glucose levels increased significantly in the GK rats, compared with the Wistar control rats. Although, fucoidan did not improve changes in body weight, the increased blood glucose levels were reduced and the decreased serum insulin levels were increased in the GK rats following oral administration of fucoidan. In vitro, fucoidan did not exhibit significant cytotoxicity towards the RIN‑5F cells, and the insulin secretion increased significantly in a dose‑ and time‑dependent manner. Treatment with amylin, an islet amyloid polypeptide and glybenclamide inhibitor, did not inhibit the stimulatory activity of fucoidan. The results of the present study also demonstrated that the concentration of cyclic adenosine monophosphate (cAMP) was significantly increased in the fucoidan‑treated RIN‑5F cells, and this increase was dose‑ and time‑dependent. In addition, treatment with a phosphodiesterase inhibitor, which decreases the degradation of

  18. Genetic and Functional Studies Implicate Synaptic Overgrowth and Ring Gland cAMP/PKA Signaling Defects in the Drosophila melanogaster Neurofibromatosis-1 Growth Deficiency

    PubMed Central

    Walker, James A.; Gouzi, Jean Y.; Long, Jennifer B.; Huang, Sidong; Maher, Robert C.; Xia, Hongjing; Khalil, Kheyal; Ray, Arjun; Van Vactor, David; Bernards, René; Bernards, André

    2013-01-01

    Neurofibromatosis type 1 (NF1), a genetic disease that affects 1 in 3,000, is caused by loss of a large evolutionary conserved protein that serves as a GTPase Activating Protein (GAP) for Ras. Among Drosophila melanogaster Nf1 (dNf1) null mutant phenotypes, learning/memory deficits and reduced overall growth resemble human NF1 symptoms. These and other dNf1 defects are relatively insensitive to manipulations that reduce Ras signaling strength but are suppressed by increasing signaling through the 3′-5′ cyclic adenosine monophosphate (cAMP) dependent Protein Kinase A (PKA) pathway, or phenocopied by inhibiting this pathway. However, whether dNf1 affects cAMP/PKA signaling directly or indirectly remains controversial. To shed light on this issue we screened 486 1st and 2nd chromosome deficiencies that uncover >80% of annotated genes for dominant modifiers of the dNf1 pupal size defect, identifying responsible genes in crosses with mutant alleles or by tissue-specific RNA interference (RNAi) knockdown. Validating the screen, identified suppressors include the previously implicated dAlk tyrosine kinase, its activating ligand jelly belly (jeb), two other genes involved in Ras/ERK signal transduction and several involved in cAMP/PKA signaling. Novel modifiers that implicate synaptic defects in the dNf1 growth deficiency include the intersectin-related synaptic scaffold protein Dap160 and the cholecystokinin receptor-related CCKLR-17D1 drosulfakinin receptor. Providing mechanistic clues, we show that dAlk, jeb and CCKLR-17D1 are among mutants that also suppress a recently identified dNf1 neuromuscular junction (NMJ) overgrowth phenotype and that manipulations that increase cAMP/PKA signaling in adipokinetic hormone (AKH)-producing cells at the base of the neuroendocrine ring gland restore the dNf1 growth deficiency. Finally, supporting our previous contention that ALK might be a therapeutic target in NF1, we report that human ALK is expressed in cells that give rise

  19. The cAMP-regulated and 293B-inhibited K+ conductance of rat colonic crypt base cells.

    PubMed

    Warth, R; Riedemann, N; Bleich, M; Van Driessche, W; Busch, A E; Greger, R

    1996-05-01

    We have shown previously that secretagogues acting via the second messenger adenosine 3',5'-cyclic monophosphate (cAMP) activate, besides their marked effect on the luminal Cl- conductance, a K+ conductance in the basolateral membrane of colonic crypt cells. This conductance is blocked by the chromanol 293B. This K+ conductance is examined here in more detail in cell-attached (c.a.) and cell-excised (c.e.) patch- clamp studies. Addition of forskolin (5 micromol/l) to the bath led to the activation of very small-conductance (probably < 3 pS) K+ channels in c.a. patches (n = 54). These channels were reversibly inhibited by the addition of 0.1 mmol/l of 293B to the bath (n = 21). Noise analysis revealed that these channels had fast kinetics and produced a Lorentzian noise component with a corner frequency (fc) of 308 +/- 10 Hz (n = 30). The current/voltage curves of this noise indicated that the underlying ion channels were K+ selective. 293B reduced the power density of the noise (So) to 46 +/- 8.7% of its control value and shifted fc from 291 +/- 26 to 468 +/- 54 Hz (n = 8). In c.e. patches from cells previously stimulated by forskolin, the same type of current persisted in 3 out of 18 experiments when the bath solution was a cytosolic-type solution without adenosine 5'-triphosphate (ATP) (CYT). In 15 experiments the addition of ATP (1 mmol/l) to CYT solution was necessary to induce or augment channel activity. In six experiments excision was performed into CYT + ATP solution and channel activity persisted. 293B exerted a reversible inhibitory effect. The channel activity was reduced by 5 mmol/l Ba2+ and was completely absent when K+ in the bath was replaced by Na+. These data suggest that forskolin activates a K+ channel of very small conductance which can be inhibited directly and reversibly by 293B.

  20. Features of adenosine metabolism of mouse heart.

    PubMed

    Deussen, Andreas; Weichsel, Johannes; Pexa, Annette

    2006-11-01

    Adenosine metabolism and transport were evaluated in the isolated perfused mouse heart and compared with the well-established model of isolated perfused guinea pig heart. Coronary venous release of adenosine under well-oxygenated conditions in the mouse exceeds that in the guinea pig threefold when related to tissue mass. Total myocardial adenosine production rate under this condition was approximately 2 nmol/min per gramme and similar in both species. Coronary resistance vessels of mice are highly sensitive to exogenous adenosine, and the threshold for adenosine-induced vasodilation is approximately 30 nmol/l. Adenosine membrane transport was largely insensitive to nitrobenzyl-thioinosine (NBTI) in mouse heart, which is in contrast to guinea pig and several other species. This indicates the dominance of NBTI-insensitive transporters in mouse heart. For future studies, the assessment of cytosolic and extracellular adenosine metabolism and its relationship with coronary flow will require the use of more effective membrane transport blockers.

  1. 28 kDa adenosine-binding proteins of brain and other tissues.

    PubMed Central

    Ravid, K; Rosenthal, R A; Doctrow, S R; Lowenstein, J M

    1989-01-01

    Membranes prepared from calf brain were solubilized and chromatographed on a column containing 5'-amino-5'-deoxyadenosine covalently linked to agarose through the 5'-amino group. When the column was eluted with adenosine, a pure protein emerged with subunit molecular mass of 28 kDa. The protein was extracted from the membranes with sodium cholate, but not with 100 microM-adenosine or 0.5 M-NaCl. A similar 28 kDa protein was isolated from the soluble fraction of calf brain. The yield of membrane-bound and soluble 28 kDa protein per gram of tissue was about the same. The 28 kDa protein was also found in membrane and soluble fractions of rabbit heart, rat liver and vascular smooth muscle from calf aorta. The yield per gram of tissue fell into the order brain greater than heart approximately vascular smooth muscle greater than liver for the 28 kDa protein from the membrane fraction, and brain approximately heart greater than vascular smooth muscle greater than liver for the 28 kDa protein from the soluble fraction. Polyclonal antibodies to pure 28 kDa protein from calf brain membranes cross-reacted with the 28 kDa protein from calf brain soluble fraction and with 28 kDa proteins isolated from other tissues. The 28 kDa protein from calf brain membranes was also eluted from the affinity column by AMP and 2',5'-dideoxyadenosine, but at a concentration higher than that at which adenosine eluted the protein, but N6-(R-phenylisopropyl)adenosine, 5'-N-ethylcarboxamidoadenosine, ADP, ATP, GTP, NAD+, cyclic AMP and inosine failed to elute the protein at concentrations up to 1 mM. The 28 kDa protein from the soluble fraction was not eluted by 3 mM-AMP or 1 mM-N6-(R-phenylisopropyl)adenosine,-5'-N-ethylcarboxamidoadenosine or -cyclic AMP. Unexpectedly, the soluble 28 kDa protein was eluted by AMP in the presence of sodium cholate. Soluble 28 kDa protein from calf brain had a KD for adenosine of 12 microM. Membrane 28 kDa protein from calf brain had a KD of 14 microM in the

  2. RECIPIENT PRETRANSPLANT INOSINE MONOPHOSPHATE DEHYDROGENASE ACTIVITY IN NONMYELOABLATIVE HCT

    PubMed Central

    Bemer, Meagan J.; Risler, Linda J.; Phillips, Brian R.; Wang, Joanne; Storer, Barry E.; Sandmaier, Brenda M.; Duan, Haichuan; Raccor, Brianne S.; Boeckh, Michael J.; McCune, Jeannine S.

    2014-01-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5’- monophosphate (IMP) to xanthosine 5’-monophosphate (XMP). We developed a highly sensitive liquid chromatography–mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNC) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T-cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation, but not with chronic GVHD, relapse, non-relapse mortality, or overall mortality. We conclude that quantitation of the recipient’s pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient’s sensitivity to MMF, but confirmatory studies are needed. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients. PMID:24923537

  3. A new caffeine biosynthetic pathway in tea leaves: utilisation of adenosine released from the S-adenosyl-L-methionine cycle.

    PubMed

    Koshiishi, C; Kato, A; Yama, S; Crozier, A; Ashihara, H

    2001-06-15

    The four-step caffeine biosynthetic pathway includes three methylation steps that utilise S-adenosyl-L-methionine (SAM) as the methyl donor. In the process SAM is converted to S-adenosyl-L-homocysteine (SAH) which in turn is hydrolysed to L-homocysteine and adenosine. Significant amounts of radioactivity from [methyl-(14)C]methionine and [methyl-(14)C]SAM were incorporated into theobromine and caffeine in young tea leaf segments, and very high SAH hydrolase activity was found in cell-free extracts from young tea leaves. Substantial amounts of radioactivity from [adenosyl-(14)C]SAH were also recovered as theobromine and caffeine in tea leaf segments, indicating that adenosine derived from SAH is utilised for the synthesis of the purine ring of caffeine. From the profiles of activity of related enzymes in tea leaf extracts, it is proposed that the major route from SAM to caffeine is a SAM-->SAH-->adenosine-->adenine-->AMP-->IMP-->XMP-->xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine pathway. In addition, direct adenosine kinase-catalysed formation of AMP from adenosine may participate as an alternative minor route. The activity of two of the three N-methyltransferase activities involved in caffeine biosynthesis and part of the activities of SAH hydrolase, adenosine nucleosidase, adenine phosphoribosyltransferase and adenosine kinase were located in tea chloroplasts. In contrast, no detectable activity of SAM synthetase was associated with the purified chloroplast fraction. This is a first demonstration that the purine skeleton of caffeine is synthesised from adenosine released from the SAM cycle.

  4. Fluorescent ligands for adenosine receptors.

    PubMed

    Kozma, Eszter; Jayasekara, P Suresh; Squarcialupi, Lucia; Paoletta, Silvia; Moro, Stefano; Federico, Stephanie; Spalluto, Giampiero; Jacobson, Kenneth A

    2013-01-01

    Interest is increasing in developing fluorescent ligands for characterization of adenosine receptors (ARs), which hold a promise of usefulness in the drug discovery process. The size of a strategically labeled AR ligand can be greatly increased after the attachment of a fluorophore. The choice of dye moiety (e.g. Alexa Fluor 488), attachment point and linker length can alter the selectivity and potency of the parent molecule. Fluorescent derivatives of adenosine agonists and antagonists (e.g. XAC and other heterocyclic antagonist scaffolds) have been synthesized and characterized pharmacologically. Some are useful AR probes for flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer, and scanning confocal microscopy. Thus, the approach of fluorescent labeled GPCR ligands, including those for ARs, is a growing dynamic research field.

  5. Quantitative effect and regulatory function of cyclic adenosine 5'-phosphate in Escherichia coli.

    PubMed

    Narang, Atul

    2009-09-01

    Cyclic adenosine 5'-phosphate (cAMP) is a global regulator of gene expression in Escherichia coli. Despite decades of intensive study, the quantitative effect and regulatory function of cAMP remain the subjects of considerable debate. Here, we analyse the data in the literature to show that: (a) In carbon-limited cultures (including cultures limited by glucose), cAMP is at near-saturation levels with respect to expression of several catabolic promoters (including lac, ara and gal). It follows that cAMP receptor protein (CRP) cAMP-mediated regulation cannot account for the strong repression of these operons in the presence of glucose. (b) The cAMP levels in carbon-excess cultures are substantially lower than those observed in carbon-limited cultures under these conditions, the expression of catabolic promoters is very sensitive to variation of cAMP levels. (c)=CRPcAMP invariably activates the expression of catabolic promoters, but it appears to inhibit the expression of anabolic promoters. (d) These results suggest that the physiological function of cAMP is to maintain homeostatic energy levels. In carbon-limited cultures, growth is limited by the supply of energy; the cAMP levels therefore increase to enhance energy accumulation by activating the catabolic promoters and inhibiting the anabolic promoters. Conversely, in carbonexcess cultures, characterized by the availability of excess energy, the cAMP levels decrease in order to depress energy accumulation by inhibiting the catabolic promoters and activating the anabolic promoters. PMID:19805906

  6. Nonnucleoside inhibitors of adenosine kinase.

    PubMed

    Gomtsyan, Arthur; Lee, Chih-Hung

    2004-01-01

    Adenosine (ADO) is an endogenous inhibitory neuromodulator that increases nociceptive thresholds in response to tissue trauma and inflammation. Adenosine kinase (AK) is a key intracellular enzyme regulating intra- and extracellular concentrations of ADO. AK inhibition selectively amplifies extracellular ADO levels at cell and tissue sites where accelerated release of ADO occurs. AK inhibitors have been shown to provide effective antinociceptive, antiinflammatory and anticonvulsant activity in animal models, thus suggesting their potential therapeutic utility for pain, inflammation, epilepsy and possibly other central and peripheral nervous system diseases associated with cellular trauma and inflammation. This beneficial outcome may potentially lack nonspecific effects associated with the systemic administration of ADO receptor agonists. Until recently all of the reported AK inhibitors contained adenosine-like structural motif. The present review will discuss design, synthesis and analgesic and antiinflammatory properties of the novel nonnucleoside AK inhibitors that do not have close structural resemblance with the natural substrate ADO. Two classes of the nonnucleoside AK inhibitors are built on pyridopyrimidine and alkynylpyrimidine cores.

  7. Nucleic acid molecules encoding isopentenyl monophosphate kinase, and methods of use

    DOEpatents

    Croteau, Rodney B.; Lange, Bernd M.

    2001-01-01

    A cDNA encoding isopentenyl monophosphate kinase (IPK) from peppermint (Mentha x piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of isopentenyl monophosphate kinase (SEQ ID NO:2), from peppermint (Mentha x piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for isopentenyl monophosphate kinase, or for a base sequence sufficiently complementary to at least a portion of isopentenyl monophosphate kinase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding isopentenyl monophosphate kinase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant isopentenyl monophosphate kinase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant isopentenyl monophosphate kinase may be used to obtain expression or enhanced expression of isopentenyl monophosphate kinase in plants in order to enhance the production of isopentenyl monophosphate kinase, or isoprenoids derived therefrom, or may be otherwise employed for the regulation or expression of isopentenyl monophosphate kinase, or the production of its products.

  8. Resonant Infrared Multiple Photon Dissociation Spectroscopy of Anionic Nucleotide Monophosphate Clusters.

    PubMed

    Ligare, Marshall R; Rijs, Anouk M; Berden, Giel; Kabeláč, Martin; Nachtigallova, Dana; Oomens, Jos; de Vries, Mattanjah S

    2015-06-25

    We report mid-infrared spectra and potential energy surfaces of four anionic, 2'-deoxynucleotide-5'-monophosphates (dNMPs) and the ionic DNA pairs [dGMP-dCMP-H](1-), [dAMP-dTMP-H](1-) with a total charge of the complex equal to -1. We recorded IR action spectra by resonant IR multiple-photon dissociation (IRMPD) using the FELIX free electron laser. The potential energy surface study employed an on-the-fly molecular dynamics quenching method (MD/Q), using a semiempirical AM1 method, followed by an optimization of the most stable structures using density functional theory. By employing infrared multiple-photon dissociation (IRMPD) spectroscopy in combination with high-level computational methods, we aim at a better understanding of the hydrogen bonding competition between the phosphate moieties and the nucleobases. We find that, unlike in multimer double stranded DNA structures, the hydrogen bonds in these isolated nucleotide pairs are predominantly formed between the phosphate groups. This intermolecular interaction appears to exceed the stabilization energy resulting from base pairing and directs the overall cluster structure and alignment.

  9. Optimization of benzoxazole-based inhibitors of Cryptosporidium parvum inosine 5'-monophosphate dehydrogenase.

    PubMed

    Gorla, Suresh Kumar; Kavitha, Mandapati; Zhang, Minjia; Chin, James En Wai; Liu, Xiaoping; Striepen, Boris; Makowska-Grzyska, Magdalena; Kim, Youngchang; Joachimiak, Andrzej; Hedstrom, Lizbeth; Cuny, Gregory D

    2013-05-23

    Cryptosporidium parvum is an enteric protozoan parasite that has emerged as a major cause of diarrhea, malnutrition, and gastroenteritis and poses a potential bioterrorism threat. C. parvum synthesizes guanine nucleotides from host adenosine in a streamlined pathway that relies on inosine 5'-monophosphate dehydrogenase (IMPDH). We have previously identified several parasite-selective C. parvum IMPDH (CpIMPDH) inhibitors by high-throughput screening. In this paper, we report the structure-activity relationship (SAR) for a series of benzoxazole derivatives with many compounds demonstrating CpIMPDH IC50 values in the nanomolar range and >500-fold selectivity over human IMPDH (hIMPDH). Unlike previously reported CpIMPDH inhibitors, these compounds are competitive inhibitors versus NAD(+). The SAR study reveals that pyridine and other small heteroaromatic substituents are required at the 2-position of the benzoxazole for potent inhibitory activity. In addition, several other SAR conclusions are highlighted with regard to the benzoxazole and the amide portion of the inhibitor, including preferred stereochemistry. An X-ray crystal structure of a representative E·IMP·inhibitor complex is also presented. Overall, the secondary amine derivative 15a demonstrated excellent CpIMPDH inhibitory activity (IC50 = 0.5 ± 0.1 nM) and moderate stability (t1/2 = 44 min) in mouse liver microsomes. Compound 73, the racemic version of 15a, also displayed superb antiparasitic activity in a Toxoplasma gondii strain that relies on CpIMPDH (EC50 = 20 ± 20 nM), and selectivity versus a wild-type T. gondii strain (200-fold). No toxicity was observed (LD50 > 50 μM) against a panel of four mammalian cells lines.

  10. Rp-cAMPS Prodrugs Reveal the cAMP Dependence of First-Phase Glucose-Stimulated Insulin Secretion

    PubMed Central

    Schwede, Frank; Chepurny, Oleg G.; Kaufholz, Melanie; Bertinetti, Daniela; Leech, Colin A.; Cabrera, Over; Zhu, Yingmin; Mei, Fang; Cheng, Xiaodong; Manning Fox, Jocelyn E.; MacDonald, Patrick E.; Genieser, Hans-G.; Herberg, Friedrich W.

    2015-01-01

    cAMP-elevating agents such as the incretin hormone glucagon-like peptide-1 potentiate glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells. However, a debate has existed since the 1970s concerning whether or not cAMP signaling is essential for glucose alone to stimulate insulin secretion. Here, we report that the first-phase kinetic component of GSIS is cAMP-dependent, as revealed through the use of a novel highly membrane permeable para-acetoxybenzyl (pAB) ester prodrug that is a bioactivatable derivative of the cAMP antagonist adenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In dynamic perifusion assays of human or rat islets, a step-wise increase of glucose concentration leads to biphasic insulin secretion, and under these conditions, 8-bromoadenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer, 4-acetoxybenzyl ester (Rp-8-Br-cAMPS-pAB) inhibits first-phase GSIS by up to 80%. Surprisingly, second-phase GSIS is inhibited to a much smaller extent (≤20%). Using luciferase, fluorescence resonance energy transfer, and bioluminescence resonance energy transfer assays performed in living cells, we validate that Rp-8-Br-cAMPS-pAB does in fact block cAMP-dependent protein kinase activation. Novel effects of Rp-8-Br-cAMPS-pAB to block the activation of cAMP-regulated guanine nucleotide exchange factors (Epac1, Epac2) are also validated using genetically encoded Epac biosensors, and are independently confirmed in an in vitro Rap1 activation assay using Rp-cAMPS and Rp-8-Br-cAMPS. Thus, in addition to revealing the cAMP dependence of first-phase GSIS from human and rat islets, these findings establish a pAB-based chemistry for the synthesis of highly membrane permeable prodrug derivatives of Rp-cAMPS that act with micromolar or even nanomolar potency to inhibit cAMP signaling in living cells. PMID:26061564

  11. Rp-cAMPS Prodrugs Reveal the cAMP Dependence of First-Phase Glucose-Stimulated Insulin Secretion.

    PubMed

    Schwede, Frank; Chepurny, Oleg G; Kaufholz, Melanie; Bertinetti, Daniela; Leech, Colin A; Cabrera, Over; Zhu, Yingmin; Mei, Fang; Cheng, Xiaodong; Manning Fox, Jocelyn E; MacDonald, Patrick E; Genieser, Hans-G; Herberg, Friedrich W; Holz, George G

    2015-07-01

    cAMP-elevating agents such as the incretin hormone glucagon-like peptide-1 potentiate glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells. However, a debate has existed since the 1970s concerning whether or not cAMP signaling is essential for glucose alone to stimulate insulin secretion. Here, we report that the first-phase kinetic component of GSIS is cAMP-dependent, as revealed through the use of a novel highly membrane permeable para-acetoxybenzyl (pAB) ester prodrug that is a bioactivatable derivative of the cAMP antagonist adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In dynamic perifusion assays of human or rat islets, a step-wise increase of glucose concentration leads to biphasic insulin secretion, and under these conditions, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, 4-acetoxybenzyl ester (Rp-8-Br-cAMPS-pAB) inhibits first-phase GSIS by up to 80%. Surprisingly, second-phase GSIS is inhibited to a much smaller extent (≤20%). Using luciferase, fluorescence resonance energy transfer, and bioluminescence resonance energy transfer assays performed in living cells, we validate that Rp-8-Br-cAMPS-pAB does in fact block cAMP-dependent protein kinase activation. Novel effects of Rp-8-Br-cAMPS-pAB to block the activation of cAMP-regulated guanine nucleotide exchange factors (Epac1, Epac2) are also validated using genetically encoded Epac biosensors, and are independently confirmed in an in vitro Rap1 activation assay using Rp-cAMPS and Rp-8-Br-cAMPS. Thus, in addition to revealing the cAMP dependence of first-phase GSIS from human and rat islets, these findings establish a pAB-based chemistry for the synthesis of highly membrane permeable prodrug derivatives of Rp-cAMPS that act with micromolar or even nanomolar potency to inhibit cAMP signaling in living cells. PMID:26061564

  12. Content of Adenosine Phosphates and Adenylate Energy Charge in Germinating Ponderosa Pine Seeds

    PubMed Central

    Ching, Te May; Ching, Kim K.

    1972-01-01

    An average of 540 picomoles of total adenosine phosphates was found in the embryo of mature seeds of ponderosa pine (Pinus ponderosa Laws.) and 1140 picomoles in the gametophyte. Adenylate energy charges were 0.44 and 0.26, respectively. After stratification, total adenosine phosphates increased 7-fold and 6-fold in embryo and gametophyte, respectively, and energy charges rose to 0.85 and 0.75. During germination, total adenosine phosphates increased to a 20-fold peak on the 9th day in gametophytic tissue, parallel with the peak of reserve regradation and organellar synthesis, and then decreased. In embryo and seedling, total adenosine phosphates elevated 80-fold with two distinct oscillating increases of AMP and ADP. The oscillating increases occurred before the emergence of radicle and cotyledons during which the highest mitotic index prevailed in all tissues. Energy charges fluctuated between 0.65 at the rapid cell dividing stage to 0.85 at the fully differentiated stage of the seedling, while energy charges remained around 0.75 in the gametophyte. These data indicated that the content of adenosine phosphates of germinating seeds reflects growth, organogenesis, and morphogenesis, and that a compartmentalized energy metabolism must exist in dividing and growing plant cells. PMID:16658212

  13. Caffeine exposure alters adenosine system and neurochemical markers during retinal development.

    PubMed

    Brito, Rafael; Pereira-Figueiredo, Danniel; Socodato, Renato; Paes-de-Carvalho, Roberto; Calaza, Karin C

    2016-08-01

    Evidence points to beneficial properties of caffeine in the adult central nervous system, but teratogenic effects have also been reported. Caffeine exerts most of its effects by antagonizing adenosine receptors, especially A1 and A2A subtypes. In this study, we evaluated the role of caffeine on the expression of components of the adenosinergic system in the developing avian retina and the impact of caffeine exposure upon specific markers for classical neurotransmitter systems. Caffeine exposure (5-30 mg/kg by in ovo injection) to 14-day-old chick embryos increased the expression of A1 receptors and concomitantly decreased A2A adenosine receptors expression after 48 h. Accordingly, caffeine (30 mg/kg) increased [(3) H]-8-cyclopentyl-1,3-dipropylxanthine (A1 antagonist) binding and reduced [(3) H]-ZM241385 (A2A antagonist) binding. The caffeine time-response curve demonstrated a reduction in A1 receptors 6 h after injection, but an increase after 18 and 24 h. In contrast, caffeine exposure increased the expression of A2A receptors from 18 and 24 h. Kinetic assays of [(3) H]-S-(4-nitrobenzyl)-6-thioinosine binding to the equilibrative adenosine transporter ENT1 revealed an increase in Bmax with no changes in Kd , an effect accompanied by an increase in adenosine uptake. Immunohistochemical analysis showed a decrease in retinal content of tyrosine hydroxylase, calbindin and choline acetyltransferase, but not Brn3a, after 48 h of caffeine injection. Furthermore, retinas exposed to caffeine had increased levels of phosphorylated extracellular signal-regulated kinase and cAMP-response element binding protein. Overall, we show an in vivo regulation of the adenosine system, extracellular signal-regulated kinase and cAMP-response element binding protein function and protein expression of specific neurotransmitter systems by caffeine in the developing retina. The beneficial or maleficent effects of caffeine have been demonstrated by the work of different studies. It

  14. X-ray absorption spectra of nucleotides (AMP, GMP, and CMP) in liquid water solutions near the nitrogen K-edge

    NASA Astrophysics Data System (ADS)

    Ukai, Masatoshi; Yokoya, Akinari; Fujii, Kentaro; Saitoh, Yuji

    2010-07-01

    The X-ray absorption of nucleotides (adenosine-5'-monophosphate, guanosine 5'-monophosphate, and cytidine 5'-monophosphate) are measured in both water solutions and thin solid films at X-ray energies near the nitrogen K-edge in the 'water-window' region. Each spectrum corresponds to the selective excitation of a nucleobase site in a nucleotide, and thus has features similar to the spectrum of the corresponding nucleobase. An additional new peak in the energy region of the nitrogen 1s → π* resonance is observed for each nucleotide. No significant difference between the water solutions and thin solid films is found, which might be attributable to the hydrophobic properties of a nucleobase in a nucleotide.

  15. Partial separation of platelet and placental adenosine receptors from adenosine A2-like binding protein

    SciTech Connect

    Zolnierowicz, S.; Work, C.; Hutchison, K.; Fox, I.H. )

    1990-04-01

    The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-({sup 3}H)ethylcarboxamidoadenosine (({sup 3}H)NECA). To solve this problem, we developed a rapid and simple method to separate adenosine receptors from the adenosine A2-like binding protein. Human platelet and placental membranes were solubilized with 1% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate. The soluble platelet extract was precipitated with polyethylene glycol and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. The adenosine A2-like binding protein was removed from the soluble placental extract with hydroxylapatite and adenosine receptors were precipitated with polyethylene glycol. The specificity of the ({sup 3}H)NECA binding is typical of an adenosine A2 receptor for platelets and an adenosine A1 receptor for placenta. This method leads to enrichment of adenosine A2 receptors for platelets and adenosine A1 receptors for placenta. This provides a useful preparation technique for pharmacologic studies of adenosine receptors.

  16. AMPED Program Overview

    ScienceCinema

    Gur, Ilan

    2016-07-12

    An overview presentation about ARPA-E's AMPED program. AMPED projects seek to develop advanced sensing, control, and power management technologies that redefine the way we think about battery management. Energy storage can significantly improve U.S. energy independence, efficiency, and security by enabling a new generation of electric vehicles. While rapid progress is being made in new battery materials and storage technologies, few innovations have emerged in the management of advanced battery systems. AMPED aims to unlock enormous untapped potential in the performance, safety, and lifetime of today's commercial battery systems exclusively through system-level innovations, and is thus distinct from existing efforts to enhance underlying battery materials and architectures.

  17. AMPED Program Overview

    SciTech Connect

    Gur, Ilan

    2014-03-04

    An overview presentation about ARPA-E's AMPED program. AMPED projects seek to develop advanced sensing, control, and power management technologies that redefine the way we think about battery management. Energy storage can significantly improve U.S. energy independence, efficiency, and security by enabling a new generation of electric vehicles. While rapid progress is being made in new battery materials and storage technologies, few innovations have emerged in the management of advanced battery systems. AMPED aims to unlock enormous untapped potential in the performance, safety, and lifetime of today's commercial battery systems exclusively through system-level innovations, and is thus distinct from existing efforts to enhance underlying battery materials and architectures.

  18. Adenosine Phosphates in Germinating Radish (Raphanus sativus L.) Seeds 1

    PubMed Central

    Moreland, Donald E.; Hussey, Griscelda G.; Shriner, Carole R.; Farmer, Fred S.

    1974-01-01

    Changes in concentrations of adenosine phosphates (AMP, ADP, and ATP), oxygen utilization, and fresh weights were measured during the first 48 hours after imbibition of water by quiescent radish seeds (Raphanus sativus L.) at 22.5 C. The changes in ATP concentrations, oxygen utilization, and fresh weights followed a triphasic time course, characterized by a rapid initial increase, which extended from 0 to approximately 1.5 hours, a lag phase from 1.5 to 16 hours, and a sharp linear increase from 16 to 48 hours. In unimbibed seeds, the concentrations of ATP, ADP, and AMP were <0.1, 0.9, and 2.2 nmoles/seed, respectively. After imbibition of water by the quiescent seeds, for 1 hour, the ATP concentration had increased to 2.5, and ADP and AMP concentrations had decreased to 0.3 and 0.1 nmole/seed, respectively. These early changes occurred also in seeds maintained under anaerobic conditions (argon), or when treated with either 5 mm fluoroacetate, or 5 mm iodoacetate. The concentrations of ADP and AMP did not change significantly from 1 to 48 hours. The termination of the lag phase at 16 hours correlated with radicle emergence. Cell division in the radicles was initiated at approximately 28 hours. ATP concentrations in seeds maintained under argon or treated with fluoroacetate remained relatively constant from approximately 2 to 48 hours. In contrast, the ATP concentration of iodoacetate-treated seeds decreased curvilinearly from 4 to 48 hours. Oxidative phosphorylation was estimated to have contributed 15, 20, and 65% of the pool ATP at 1.5, 16, and 48 hours, respectively. PMID:16658928

  19. PRODUCTION OF EXTRACELLULAR GUANOSINE-5'-MONOPHOSPHATE BY BACILLUS SUBTILIS

    PubMed Central

    Demain, A. L.; Miller, I. M.; Hendlin, D.

    1964-01-01

    Demain, A. L. (Merck Sharp & Dohme Research Laboratories, Rahway, N.J.), I. M. Miller, and D. Hendlin. Production of extracellular guanosine-5'-monophosphate by Bacillus subtilis. J. Bacteriol. 88:991–995. 1964.—Wild-type Bacillus subtilis colonies were found to feed purineless mutants. A strain with high feeding capacity was selected for study, with a guanineless mutant of B. subtilis used as the assay organism. The factor was excreted during its growth phase in a complex medium containing starch and soybean meal extract. Nutritional studies led to the development of a defined medium to be used for biochemical studies and to aid in the isolation of the factor. Starch was replaced by maltose and the soybean meal extract by Mn++. Production of the factor was sensitive to the pH of the medium during growth. Practically its entire extracellular accumulation occurred before visible lysis. The factor was identified as guanosine-5'-monophosphate derived by extracellular enzymatic hydrolysis of excreted ribonucleic acid. PMID:14219064

  20. Regulation of Cardiovascular Development by Adenosine and Adenosine-Mediated Embryo Protection

    PubMed Central

    Rivkees, Scott A.; Wendler, Christopher C.

    2012-01-01

    Few signaling molecules have the potential to influence the developing mammal as the nucleoside adenosine. Adenosine levels increase rapidly with tissue hypoxia and inflammation. Adenosine antagonists include the methlyxanthines caffeine and theophylline. The receptors that transduce adenosine action are the A1, A2a, A2b, and A3 adenosine receptors (ARs). We examined how adenosine acts via A1ARs to influence embryo development. Transgenic mice were studied along with embryo cultures. Embryos lacking A1ARs were markedly growth retarded following intrauterine hypoxia exposure. Studies of mice selectively lacking A1AR in the heart identify the heart as a key site of adenosines embryo protective effects. Studies of isolated embryos showed that adenosine plays a key role in modulating embryo cardiac function, especially in the setting of hypoxia. When pregnant mice were treated during embryogenesis with the adenosine antagonist caffeine, adult mice had abnormal heart function. Adenosine acts via A1ARs to play an essential role in protecting the embryo against intra uterine stress, and adenosine antagonists, including caffeine, may be an unwelcome exposure for the embryo. PMID:22423036

  1. Pyridopyrimidine analogues as novel adenosine kinase inhibitors.

    PubMed

    Zheng, G Z; Lee, C; Pratt, J K; Perner, R J; Jiang, M Q; Gomtsyan, A; Matulenko, M A; Mao, Y; Koenig, J R; Kim, K H; Muchmore, S; Yu, H; Kohlhaas, K; Alexander, K M; McGaraughty, S; Chu, K L; Wismer, C T; Mikusa, J; Jarvis, M F; Marsh, K; Kowaluk, E A; Bhagwat, S S; Stewart, A O

    2001-08-20

    A novel series of pyridopyrimidine analogues 9 was identified as potent adenosine kinase inhibitors based on the SAR and computational studies. Substitution of the C7 position of the pyridopyrimidino core with C2' substituted pyridino moiety increased the in vivo potency and enhanced oral bioavailability of these adenosine kinase inhibitors.

  2. Enzymatic regeneration of adenosine triphosphate cofactor

    NASA Technical Reports Server (NTRS)

    Marshall, D. L.

    1974-01-01

    Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

  3. Nucleoside monophosphate complex structures of the endonuclease domain from the influenza virus polymerase PA subunit reveal the substrate binding site inside the catalytic center.

    PubMed

    Zhao, Cong; Lou, Zhiyong; Guo, Yu; Ma, Ming; Chen, Yutao; Liang, Shuaiyi; Zhang, Liang; Chen, Shoudeng; Li, Xuemei; Liu, Yingfang; Bartlam, Mark; Rao, Zihe

    2009-09-01

    Highly pathogenic influenza virus strains currently in circulation pose a significant risk of a global pandemic. Following the reported crystal structure of the endonuclease domain from the avian influenza virus polymerase PA subunit, here we report the results of a systematic X-ray crystallographic analysis of its complex with adenosine, uridine, and thymidine nucleoside monophosphates (NMPs). Electron density corresponding to the monophosphate moiety of each nucleotide was apparent in each NMP complex and bound to the catalytic metal. A hydrophobic site was found to contribute to nucleoside binding. The NMP complex structures should represent the conformation of the bound product after nuclease cleavage. Moreover, one solvent molecule was found to occupy an equivalent position to the second reported Mn(2+) ion, where it mediates the interaction between bound NMPs and the N-terminal PA domain in the presence of the Mg(2+) ion. The results presented here indicate a possible cleavage mechanism and identify a distinct nucleotide binding pocket. The identification of this binding pocket opens a new avenue for anti-influenza drug discovery, targeting the cap-dependent endonuclease, in response to the worldwide threat of influenza. PMID:19587036

  4. Biosynthesis of 9-beta-D-arabinofuranosyladenine: hydrogen exchange at C-2' and oxygen exchange at C-3' of adenosine

    SciTech Connect

    Suhadolnik, R.J.; Pornbanlualap, S.; Wu, J.M.; Baker, D.C.; Hebbler, A.K.

    1989-04-01

    The data presented here describe new findings related to the bioconversion of adenosine to 9-beta-D-arabinofuranosyladenine (ara-A) by Streptomyces antibioticus by in vivo investigations and with a partially purified enzyme. First, in double label in vivo experiments with (2'-18O)- and (U-14C)adenosine, the 18O:14C ratio of the ara-A isolated does not change appreciably, indicating a stereospecific inversion of the C-2' hydroxyl of adenosine to ara-A with retention of the 18O at C-2'. In experiments with (3'-18O)- and (U-14C)-adenosine, (U-14C)ara-A was isolated; however, the 18O at C-3' is below detection. The adenosine isolated from the RNA from both double label experiments has essentially the same ratio of 18O:14C. Second, an enzyme has been isolated and partially purified from extracts of S. antibioticus that catalyzes the conversion of adenosine, but not AMP, ADP, ATP, inosine, guanosine, or D-ribose, to ara-A. In a single label enzyme-catalyzed experiment with (U-14C)adenosine, there was a 9.9% conversion to (U-14C)ara-A; with (2'-3H)-adenosine, there was a 8.9% release of the C-2' tritium from (2'-3H)adenosine which was recovered as 3H2O. Third, the release of 3H as 3H2O from (2'-3H)adenosine was confirmed by incubations of the enzyme with 3H2O and adenosine. Ninety percent of the tritium incorporated into the D-arabinose of the isolated ara-A was in C-2 and 8% was in C-3. The enzyme-catalyzed conversion of adenosine to ara-A occurs without added cofactors, displays saturation kinetics, a pH optimum of 6.8, a Km of 8 X 10(-4) M, and an inhibition by heavy metal cations. The enzyme also catalyzes the stereospecific inversion of the C-2' hydroxyl of the nucleoside antibiotic, tubercidin to form 7-beta-D-arabinofuranosyl-4-aminopyrrolo(2,3-d)pyrimidine. The nucleoside antibiotic, sangivamycin, in which the C-5 hydrogen is replaced with a carboxamide group, is not a substrate.

  5. Halobacterial adenosine triphosphatases and the adenosine triphosphatase from Halobacterium saccharovorum

    NASA Technical Reports Server (NTRS)

    Kristjansson, Hordur; Sadler, Martha H.; Hochstein, Lawrence I.

    1986-01-01

    Membranes prepared from various members of the genus Halobacterium contained a Triton X-l00 activated adenosine triphosphatase. The enzyme from Halobacterium saccharovorum was unstable in solutions of low ionic strength and maximally active in the presence of 3.5 M NaCl. A variety of nucleotide triphosphates was hydrolyzed. MgADP, the product of ATP hydrolysis, was not hydrolyzed and was a competitive inhibitor with respect to MgATP. The enzyme from H. saccharovorum was composed of at least 2 and possibly 4 subunits. The 83-kDa and 60-kDa subunits represented about 90 percent of total protein. The 60-kDa subunit reacted with dicyclohexyl-carbodiimide when inhibition was carried out in an acidic medium. The enzyme from H. saccharovorum, possesses properties of an F(1)F(0) as well as an E(1)E(2) ATPase.

  6. Poly(glycidyl methacrylate-co-N-methylolacrylamide-co-ethylene dimethacrylate) monolith coupled to high-performance liquid chromatography for the determination of adenosine phosphates in royal jelly.

    PubMed

    Liu, Dan; Zhang, Tianbin; Cheng, Yechun; Jia, Qiong

    2014-07-01

    A polymer monolith microextraction method coupled with high-performance liquid chromatography was developed for the determination of adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate. The monolithic column was synthesized inside fused-silica capillaries using thermal initiation free-radical polymerization with glycidyl methacrylate as the monomer, ethylene dimethacrylate as the cross-linker, cyclohexanol, and 1-dodecanol as the porogen. N-Methylolacrylamide, an important hydrophilic monomer, was incorporated into the polymerization mixture to enhance the hydrophilicity of the poly(glycidyl methacrylate-co-ethylene dimethacrylate) column. The obtained poly(glycidyl methacrylate-co-N-methylolacrylamide-co-ethylene dimethacrylate) monolith was characterized by scanning electron microscopy, Fourier-transform infrared spectra, and X-ray photoelectron spectroscopy. Optimum conditions for the preconcentration and separation of the target adenosines were also investigated. Under the optimum conditions, we obtained acceptable linearities, low limits of detection, and good relative standard deviations. The developed polymer monolith microextraction with high-performance liquid chromatography method exhibited a good performance with recovery values in the range of 76.9-104.7% when applied to the determination of the adenosines in five royal jelly samples.

  7. Probing biased/partial agonism at the G protein-coupled A(2B) adenosine receptor.

    PubMed

    Gao, Zhan-Guo; Balasubramanian, Ramachandran; Kiselev, Evgeny; Wei, Qiang; Jacobson, Kenneth A

    2014-08-01

    G protein-coupled A(2B) adenosine receptor (AR) regulates numerous important physiological functions, but its activation by diverse A(2B)AR agonists is poorly profiled. We probed potential partial and/or biased agonism in cell lines expressing variable levels of endogenous or recombinant A(2B)AR. In cAMP accumulation assays, both 5'-substituted NECA and C2-substituted MRS3997 are full agonists. However, only 5'-substituted adenosine analogs are full agonists in calcium mobilization, ERK1/2 phosphorylation and β-arrestin translocation. A(2B)AR overexpression in HEK293 cells markedly increased the agonist potency and maximum effect in cAMP accumulation, but less in calcium and ERK1/2. A(2B)AR siRNA silencing was more effective in reducing the maximum cAMP effect of non-nucleoside agonist BAY60-6583 than NECA's. A quantitative 'operational model' characterized C2-substituted MRS3997 as either balanced (cAMP accumulation, ERK1/2) or strongly biased agonist (against calcium, β-arrestin). N⁶-substitution biased against ERK1/2 (weakly) and calcium and β-arrestin (strongly) pathways. BAY60-6583 is ERK1/2-biased, suggesting a mechanism distinct from adenosine derivatives. BAY60-6583, as A(2B)AR antagonist in MIN-6 mouse pancreatic β cells expressing low A(2B)AR levels, induced insulin release. This is the first relatively systematic study of structure-efficacy relationships of this emerging drug target.

  8. Cloning and expression of an A1 adenosine receptor from rat brain

    SciTech Connect

    Mahan, L.C.; McVittie, L.D.; Smyk-Randall, E.M.; Nakata, H.; Monsma, F.J. Jr.; Gerfen, C.R.; Sibley, D.R. )

    1991-07-01

    The authors have used the polymerase chain reaction technique to selectively amplify guanine nucleotide-binding regulatory protein (G protein)-coupled receptor cDNA sequences from rat striatal mRNA, using sets of highly degenerate primers derived from transmembrane sequences of previously cloned G protein-coupled receptors. A novel cDNA fragment was identified, which exhibits considerable homology to various members of the G protein-coupled receptor family. This fragment was used to isolate a full-length cDNA from a rat striatal library. A 2.2-kilobase clone was obtained that encodes a protein of 326 amino acids with seven transmembrane domains, as predicted by hydropathy analysis. Stably transfected mouse A9-L cells and Chinese hamster ovary cells that expressed mRNA for this clone were screened with putative receptor ligands. Saturable and specific binding sites for the A1 adenosine antagonist (3H)-1,3-dipropyl-8-cyclopentylxanthine were identified on membranes from transfected cells. The rank order of potency and affinities of various adenosine agonist and antagonist ligands confirmed the identity of this cDNA clone as an A1 adenosine receptor. The high affinity binding of A1 adenosine agonists was shown to be sensitive to the nonhydrolyzable GTP analog guanylyl-5{prime}-imidodiphosphate. In adenylyl cyclase assays, adenosine agonists inhibited forskolin-stimulated cAMP production by greater than 50%, in a pharmacologically specific fashion. Northern blot and in situ hybridization analyses of receptor mRNA in brain tissues revealed two transcripts of 5.6 and 3.1 kilobases, both of which were abundant in cortex, cerebellum, hippocampus, and thalamus, with lower levels in olfactory bulb, striatum, mesencephalon, and retina. These regional distribution data are in good agreement with previous receptor autoradiographic studies involving the A1 adenosine receptor.

  9. [3H]-SCH 58261 labelling of functional A2A adenosine receptors in human neutrophil membranes

    PubMed Central

    Varani, Katia; Gessi, Stefania; Dionisotti, Silvio; Ongini, Ennio; Andrea Borea, Pier

    1998-01-01

    The present study describes the direct labelling of A2A adenosine receptors in human neutrophil membranes with the potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo[1,5-c]pyrimidine, ([3H]-SCH 58261). In addition, both receptor affinity and potency of a number of adenosine receptor agonists and antagonists were determined in binding, adenylyl cyclase and superoxide anion production assays.Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 1.34 nM and 75 fmol mg−1 protein, respectively. Adenosine receptor ligands competed for the binding of 1 nM [3H]-SCH 58261 to human neutrophil membranes, with a rank order of potency consistent with that typically found for interactions with the A2A adenosine receptors. In the adenylyl cyclase and in the superoxide anion production assays the same compounds exhibited a rank order of potency identical to that observed in binding experiments.Thermodynamic data indicated that [3H]-SCH 58261 binding to human neutrophils is entropy and enthalpy-driven. This finding is in agreement with the thermodynamic behaviour of antagonists binding to rat striatal A2A adenosine receptors.It was concluded that in human neutrophil membranes, [3H]-SCH 58261 directly labels binding sites with pharmacological properties similar to those of A2A adenosine receptors of other tissues. The receptors labelled by [3H]-SCH 58261 mediated the effects of adenosine and adenosine receptor agonists to stimulate cyclic AMP accumulation and inhibition of superoxide anion production in human neutrophils. PMID:9605581

  10. Optical Aptasensors for Adenosine Triphosphate

    PubMed Central

    Ng, Stella; Lim, Hui Si; Ma, Qian; Gao, Zhiqiang

    2016-01-01

    Nucleic acids are among the most researched and applied biomolecules. Their diverse two- and three-dimensional structures in conjunction with their robust chemistry and ease of manipulation provide a rare opportunity for sensor applications. Moreover, their high biocompatibility has seen them being used in the construction of in vivo assays. Various nucleic acid-based devices have been extensively studied as either the principal element in discrete molecule-like sensors or as the main component in the fabrication of sensing devices. The use of aptamers in sensors - aptasensors, in particular, has led to improvements in sensitivity, selectivity, and multiplexing capacity for a wide verity of analytes like proteins, nucleic acids, as well as small biomolecules such as glucose and adenosine triphosphate (ATP). This article reviews the progress in the use of aptamers as the principal component in sensors for optical detection of ATP with an emphasis on sensing mechanism, performance, and applications with some discussion on challenges and perspectives. PMID:27446501

  11. Effect of Serum from Chickens Treated with Clenbuterol on Myosin Accumulation, Beta-Adrenergic Receptor Population, and Cyclic AMP Synthesis in Embryonic Chicken Skeletal Muscle Cell Cultures

    NASA Technical Reports Server (NTRS)

    Young, Ronald B.; Bridge, Kristin Y.; Wuethrich, Andrew J.; Hancock, Deana L.

    2002-01-01

    Broiler chickens at 35 d of age were fed 1 ppm clenbuterol for 14 d. This level of dietary clenbuterol led to 5-7% increases in the weights of leg and breast muscle tissue. At the end of the 14-d period, serum was prepared from both control and clenbuterol-treated chickens, and was then employed as a component of cell culture media at a final concentration of 20% (v/v). Muscle cell cultures were prepared from both the leg and the breast muscle groups of 12-d chick embryos. Treatment groups included control chicken serum to which 10 nM, 50 nM, and 1 uM clenbuterol had been added, as well as cells grown in media containing 10% horse serum. Cultures were subjected to each treatment for 3 d, beginning on the seventh d in culture. Neither the percent fusion nor the number of nuclei in myotubes was significantly affected by any of the treatments. The quantity of myosin heavy chains (MHCs) was not increased by serum from clenbuterol-treated chickens in either breast or leg muscle cultures; however, the MHC quantity was 50-150% higher in cultures grown in control chicken serum to which 10 and 50 nM clenbuterol had also been added. The B-adrenergic receptor (betaAR) population was 4000-7000 betaARs per cell in cultures grown in chicken serum with leg muscle cultures having approximately 25-30% more receptors than breast muscle Culture. Receptor population was not significantly affected by the presence of clenbuterol or by the presence of serum from clenbuterol-treated chickens. In contrast, the betaAR Population in leg and breast muscle cultures grown in the presence of 10% horse serum was 16,000-18,000 betaARs per cell. Basal concentration of cyclic adenosine 3':5'monophosphate (cAMP) was not significantly affected by the treatments. When cultures grown in chicken serum were stimulated for 10 min with 1 uM isoproterenol, limited increases of 12-20% in cAMP Concentration above the. basal levels were observed. However, when cultures grown in the presence of horse serum were

  12. Vascular relaxation and cyclic guanosine monophosphate in hypertension

    SciTech Connect

    Otsuka, Y.; DiPiero, A.; Lockette, W.

    1986-03-01

    Isolated aortae from hypertensive rats have a decreased relaxation response to acetylcholine (Ach), A23187, and nitroprusside (SNP). Since cyclic guanosine monophosphate (cGMP) has been shown to increase in response to these vasodilators, the authors measured cGMP in response to these agents in isolated aortae from normotensive rats and DOCA, 1K1C, and coarctation induced hypertension. cGMP was measured by radioimmunoassay in vessels after exposure to phenylephrine followed by either Ach, A23187, or SNP. The aortae from the hypertensive rats had decreased basal levels of cGMP and attenuated increases in cGMP in response to Ach and A23187. Rises in cGMP in response to SNP were also attenuated in aortae from the hypertensive rats, even at concentrations which induced similar relaxation in normotensive and hypertensive blood vessels. The data suggest that changes in cGMP do not necessarily reflect changes in endothelium independent vascular relaxation in hypertension.

  13. The adenosine neuromodulation system in schizophrenia.

    PubMed

    Rial, Daniel; Lara, Diogo R; Cunha, Rodrigo A

    2014-01-01

    The management of schizophrenia endophenotypes, namely positive, negative, and cognitive symptoms is still an open goal, justifying the search of novel therapeutic avenues. We now review the evidence supporting the interest in targeting the adenosine modulation system to counteract the core features of schizophrenia. This interest is forwarded by the combined ability of strategies aimed at bolstering adenosine levels together with the increasingly recognized impact of adenosine A2A receptors to control dopaminergic signaling, working memory, and behavioral sensitization; this is further heralded by the suggested clinical effectiveness of therapies increasing extracellular adenosine such as dipyridamole and allopurinol and the emergent recognition of a role for adenosine in neurodevelopment. Finally, the combined role of A1 and A2A receptors in assisting the implementation of adaptive changes and encoding of information salience in neuronal circuits together with the adaptive alterations of A1 and A2A receptor density upon brain dysfunction prompts the novel working hypothesis that the parallel imbalance of adenosine formation and of A1 and A2A receptors blurs the adequate encoding of information salience in neuronal circuits, which we propose to be a core pathogenic feature in the development of schizophrenia endophenotypes. This proposal should also provide a rationale to assist the design of future therapeutic intervention targeting the adenosine modulation system to manage schizophrenia endophenotypes: these should not be based only on an attempt to target adenosine kinase-A1 receptors or only A2A receptors, but should instead simultaneously target these two arms of the adenosine modulation system. PMID:25175974

  14. Adenosine is required for sustained inflammasome activation via the A2A receptor and the HIF-1α pathway

    NASA Astrophysics Data System (ADS)

    Ouyang, Xinshou; Ghani, Ayaz; Malik, Ahsan; Wilder, Tuere; Colegio, Oscar Rene; Flavell, Richard Anthony; Cronstein, Bruce Neil; Mehal, Wajahat Zafar

    2013-12-01

    Inflammasome pathways are important in chronic diseases; however, it is not known how the signalling is sustained after initiation. Inflammasome activation is dependent on stimuli such as lipopolysaccharide (LPS) and ATP that provide two distinct signals resulting in rapid production of interleukin (IL)-1β, with the lack of response to repeat stimulation. Here we report that adenosine is a key regulator of inflammasome activity, increasing the duration of the inflammatory response via the A2A receptor. Adenosine does not replace signals provided by stimuli such as LPS or ATP but sustains inflammasome activity via a cAMP/PKA/CREB/HIF-1α pathway. In the setting of the lack of IL-1β responses after previous exposure to LPS, adenosine can supersede this tolerogenic state and drive IL-1β production. These data reveal that inflammasome activity is sustained, after initial activation, by A2A receptor-mediated signalling.

  15. Crystal structure of a c-di-AMP riboswitch reveals an internally pseudo-dimeric RNA.

    PubMed

    Jones, Christopher P; Ferré-D'Amaré, Adrian R

    2014-11-18

    Cyclic diadenosine monophosphate (c-di-AMP) is a second messenger that is essential for growth and homeostasis in bacteria. A recently discovered c-di-AMP-responsive riboswitch controls the expression of genes in a variety of bacteria, including important pathogens. To elucidate the molecular basis for specific binding of c-di-AMP by a gene-regulatory mRNA domain, we have determined the co-crystal structure of this riboswitch. Unexpectedly, the structure reveals an internally pseudo-symmetric RNA in which two similar three-helix-junction elements associate head-to-tail, creating a trough that cradles two c-di-AMP molecules making quasi-equivalent contacts with the riboswitch. The riboswitch selectively binds c-di-AMP and discriminates exquisitely against other cyclic dinucleotides, such as c-di-GMP and cyclic-AMP-GMP, via interactions with both the backbone and bases of its cognate second messenger. Small-angle X-ray scattering experiments indicate that global folding of the riboswitch is induced by the two bound cyclic dinucleotides, which bridge the two symmetric three-helix domains. This structural reorganization likely couples c-di-AMP binding to gene expression. PMID:25271255

  16. Folding Properties of Cytosine Monophosphate Kinase from E. coli Indicate Stabilization through an Additional Insert in the NMP Binding Domain

    PubMed Central

    Beitlich, Thorsten; Lorenz, Thorsten; Reinstein, Jochen

    2013-01-01

    The globular 25 kDa protein cytosine monophosphate kinase (CMPK, EC ID: 2.7.4.14) from E. coli belongs to the family of nucleoside monophosphate (NMP) kinases (NMPK). Many proteins of this family share medium to high sequence and high structure similarity including the frequently found α/β topology. A unique feature of CMPK in the family of NMPKs is the positioning of a single cis-proline residue in the CORE-domain (cis-Pro124) in conjunction with a large insert in the NMP binding domain. This insert is not found in other well studied NMPKs such as AMPK or UMP/CMPK. We have analyzed the folding pathway of CMPK using time resolved tryptophan and FRET fluorescence as well as CD. Our results indicate that unfolding at high urea concentrations is governed by a single process, whereas refolding in low urea concentrations follows at least a three step process which we interpret as follows: Pro124 in the CORE-domain is in cis in the native state (Nc) and equilibrates with its trans-isomer in the unfolded state (Uc - Ut). Under refolding conditions, at least the Ut species and possibly also the Uc species undergo a fast initial collapse to form intermediates with significant amount of secondary structure, from which the trans-Pro124 fraction folds to the native state with a 100-fold lower rate constant than the cis-Pro124 species. CMPK thus differs from homologous NMP kinases like UMP/CMP kinase or AMP kinase, where folding intermediates show much lower content of secondary structure. Importantly also unfolding is up to 100-fold faster compared to CMPK. We therefore propose that the stabilizing effect of the long NMP-domain insert in conjunction with a subtle twist in the positioning of a single cis-Pro residue allows for substantial stabilization compared to other NMP kinases with α/β topology. PMID:24205218

  17. Adenosine receptors as drug targets — what are the challenges?

    PubMed Central

    Chen, Jiang-Fan; Eltzschig, Holger K.; Fredholm, Bertil B.

    2014-01-01

    Adenosine signalling has long been a target for drug development, with adenosine itself or its derivatives being used clinically since the 1940s. In addition, methylxanthines such as caffeine have profound biological effects as antagonists at adenosine receptors. Moreover, drugs such as dipyridamole and methotrexate act by enhancing the activation of adenosine receptors. There is strong evidence that adenosine has a functional role in many diseases, and several pharmacological compounds specifically targeting individual adenosine receptors — either directly or indirectly — have now entered the clinic. However, only one adenosine receptor-specific agent — the adenosine A2A receptor agonist regadenoson (Lexiscan; Astellas Pharma) — has so far gained approval from the US Food and Drug Administration (FDA). Here, we focus on the biology of adenosine signalling to identify hurdles in the development of additional pharmacological compounds targeting adenosine receptors and discuss strategies to overcome these challenges. PMID:23535933

  18. Volume and ion effects of adenosine on MDCK cells

    SciTech Connect

    Coffey, A.K.; Allen, J.C.; Coutermarsh, B.A.; Mills, J.W.

    1986-03-01

    Adenosine (ADO) receptors, both internal inhibitory (P-site) and external stimulatory (A2) and inhibitory (Al) have been shown to modulate adenylate cyclase levels. Since the authors have previously shown that cAMP modulates ion content and volume of confluent monolayers of MDCK epithelial cells, the authors investigated the effects of ADO on these parameters. Exposure of MDCK cells to 0.1 mM ADO significantly increased cell volumes (2.35 +/- .10 pL/cell vs. control 2.08 +/- .01; p < .001) as determined by /sup 14/C-urea distribution space, and increased Na content (133 +/- 13 vs. 91 +/- 11 nmol/10/sup 6/ cells; p < .0002). Cl/sup -/ content also increased while K did not change. These effects were completely inhibited by treatment with the Na/sup +//H/sup +/ exchange inhibitor amiloride (0.1 mM) and by incubation in Na-free media. Together these results suggest that cell volume increased due to Na/sup +/ entry via the Na/sup +//H/sup +/ exchanger. However, 2-chloroadenosine (2CA), a potent analogue of ADO, caused a significant decrease in cell volume (1.99 +/- .86 vs. 2.21 +/- 0.13 pL/cell; p < .02) and Na (97 +/- 22 vs. 141 +/- 21 nmol/10/sup 6/ cells; p < .005). Measurement of cell cAMP by radioimmunoassay showed an increase in response to 2CA but not to ADO. Since the effects of 2CA mimic those of exogenous cAMP, MDCK cells appear to have a stimulatory (A2) receptor. However, ADO itself did not interact with this receptor and may have produced its effects by binding to a higher affinity, inhibitory receptor.

  19. Structural basis of the inhibition of class C acid phosphatases by adenosine 5;#8242;-phosphorothioate

    SciTech Connect

    Singh, Harkewal; Reilly, Thomas J.; Tanner, John J.

    2012-01-20

    The inhibition of phosphatases by adenosine 5'-phosphorothioate (AMPS) was first reported in the late 1960s; however, the structural basis for the inhibition has remained unknown. Here, it is shown that AMPS is a submicromolar inhibitor of class C acid phosphatases, a group of bacterial outer membrane enzymes belonging to the haloacid dehalogenase structural superfamily. Furthermore, the 1.35-{angstrom} resolution crystal structure of the inhibited recombinant Haemophilus influenzae class C acid phosphatase was determined; this is the first structure of a phosphatase complexed with AMPS. The conformation of AMPS is identical to that of the substrate 5'-AMP, except that steric factors force a rotation of the thiophosphoryl out of the normal phosphoryl-binding pocket. This conformation is catalytically nonproductive, because the P atom is not positioned optimally for nucleophilic attack by Asp64, and the O atom of the scissile O-P bond is too far from the Asp (Asp66) that protonates the leaving group. The structure of 5'-AMP complexed with the Asp64 {yields} Asn mutant enzyme was also determined at 1.35-{angstrom} resolution. This mutation induces the substrate to adopt the same nonproductive binding mode that is observed in the AMPS complex. In this case, electrostatic considerations, rather than steric factors, underlie the movement of the phosphoryl. The structures not only provide an explanation for the inhibition by AMPS, but also highlight the precise steric and electrostatic requirements of phosphoryl recognition by class C acid phosphatases. Moreover, the structure of the Asp64 {yields} Asn mutant illustrates how a seemingly innocuous mutation can cause an unexpected structural change.

  20. Mast Cell Adenosine Receptors Function: A Focus on the A3 Adenosine Receptor and Inflammation

    PubMed Central

    Rudich, Noam; Ravid, Katya; Sagi-Eisenberg, Ronit

    2012-01-01

    Adenosine is a metabolite, which has long been implicated in a variety of inflammatory processes. Inhaled adenosine provokes bronchoconstriction in asthmatics or chronic obstructive pulmonary disease patients, but not in non-asthmatics. This hyper responsiveness to adenosine appears to be mediated by mast cell activation. These observations have marked the receptor that mediates the bronchoconstrictor effect of adenosine on mast cells (MCs), as an attractive drug candidate. Four subtypes (A1, A2a, A2b, and A3) of adenosine receptors have been cloned and shown to display distinct tissue distributions and functions. Animal models have firmly established the ultimate role of the A3 adenosine receptor (A3R) in mediating hyper responsiveness to adenosine in MCs, although the influence of the A2b adenosine receptor was confirmed as well. In contrast, studies of the A3R in humans have been controversial. In this review, we summarize data on the role of different adenosine receptors in mast cell regulation of inflammation and pathology, with a focus on the common and distinct functions of the A3R in rodent and human MCs. The relevance of mouse studies to the human is discussed. PMID:22675325

  1. [Adenosine deaminase in experimental trypanosomiasis: future implications].

    PubMed

    Pérez-Aguilar, Mary Carmen; Rondón-Mercado, Rocío

    2015-09-01

    The adenosine deaminase represents a control point in the regulation of extracellular adenosine levels, thus playing a critical role in the modulation of purinergic responses to certain pathophysiological events. Several studies have shown that serum and plasma enzyme levels are elevated in some diseases caused by microorganisms, which may represent a compensatory mechanism due to the elevated levels of adenosine and the release of inflammatory mediators. Recent research indicates that adenosine deaminase activity decreases and affects hematological parameters of infected animals with Trypanosoma evansi, so that such alterations could have implications in the pathogenesis of the disease. In addition, the enzyme has been detected in this parasite; allowing the inference that it could be associated with the vital functions of the same, similar to what occurs in mammals. This knowledge may be useful in the association of chemotherapy with specific inhibitors of the enzyme in future studies.

  2. Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site.

    PubMed

    Adams, Julian; Chen, Zhi-Ping; Van Denderen, Bryce J W; Morton, Craig J; Parker, Michael W; Witters, Lee A; Stapleton, David; Kemp, Bruce E

    2004-01-01

    AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.

  3. Adenosine inhibits glutamatergic input to basal forebrain cholinergic neurons

    PubMed Central

    Hawryluk, J. M.; Ferrari, L. L.; Keating, S. A.

    2012-01-01

    Adenosine has been proposed as an endogenous homeostatic sleep factor that accumulates during waking and inhibits wake-active neurons to promote sleep. It has been specifically hypothesized that adenosine decreases wakefulness and promotes sleep recovery by directly inhibiting wake-active neurons of the basal forebrain (BF), particularly BF cholinergic neurons. We previously showed that adenosine directly inhibits BF cholinergic neurons. Here, we investigated 1) how adenosine modulates glutamatergic input to BF cholinergic neurons and 2) how adenosine uptake and adenosine metabolism are involved in regulating extracellular levels of adenosine. Our experiments were conducted using whole cell patch-clamp recordings in mouse brain slices. We found that in BF cholinergic neurons, adenosine reduced the amplitude of AMPA-mediated evoked glutamatergic excitatory postsynaptic currents (EPSCs) and decreased the frequency of spontaneous and miniature EPSCs through presynaptic A1 receptors. Thus we have demonstrated that in addition to directly inhibiting BF cholinergic neurons, adenosine depresses excitatory inputs to these neurons. It is therefore possible that both direct and indirect inhibition may synergistically contribute to the sleep-promoting effects of adenosine in the BF. We also found that blocking the influx of adenosine through the equilibrative nucleoside transporters or inhibiting adenosine kinase and adenosine deaminase increased endogenous adenosine inhibitory tone, suggesting a possible mechanism through which adenosine extracellular levels in the basal forebrain are regulated. PMID:22357797

  4. Applying Mathematical Processes (AMP)

    ERIC Educational Resources Information Center

    Kathotia, Vinay

    2011-01-01

    This article provides insights into the "Applying Mathematical Processes" resources, developed by the Nuffield Foundation. It features Nuffield AMP activities--and related ones from Bowland Maths--that were designed to support the teaching and assessment of key processes in mathematics--representing a situation mathematically, analysing,…

  5. The Catalytic Zinc Site and Mechanism of the Metalloenzyme PR-AMP Cyclohydrolase

    PubMed Central

    D'Ordine, Robert L.; Linger, Rebecca S.; Thai, Carolyn J.; Davisson, V. Jo

    2012-01-01

    The enzyme N1-(5'-phosphoribosyl) adenosine-5'-monophosphate cyclohydrolase (PR-AMP cyclohydrolase) is a Zn2+ metalloprotein encoded by the hisI gene. It catalyzes the third step of histidine biosynthesis (Scheme 1), an uncommon ring opening of a purine heterocycle for use in primary metabolism. A three dimensional structure of the enzyme from Methanobacterium thermoautotrophicum has revealed that three conserved cysteine residues occur at the dimer interface and likely form the catalytic site. To investigate of the functions of these cysteines in the enzyme from Methanococcus vannielii, a series of biochemical studies were pursued to test the basic hypothesis regarding their roles in catalysis. Inactivation of the enzyme activity by methyl methane thiosulfonate (MMTS) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) also compromised the Zn2+ binding properties of the protein inducing loss of up to 90% of the metal. Overall reaction stoichiometry and the potassium cyanide (KCN) induced cleavage of the protein suggested that all three cysteines were modified in the process. The enzyme was protected from DTNB-induced inactivation by inclusion of the substrate PR-AMP while Mg2+, a metal required for catalytic activity, enhanced the rate of inactivation. Site directed mutations of the conserved C93, C109, C116 and the double mutant C109/C116 were prepared and analyzed for catalytic activity, Zn2+ content, and reactivity with DTNB. Substitution of alanine for each of the conserved cysteines showed no measurable catalytic activity and only the C116A was still capable of binding Zn2+. Reactions of DTNB with the C109A/C116A double mutant showed that C93 is completely modified within 0.5 s. A model consistent with these data involves a DTNB-induced mixed disulfide linkage between C93 and C109 or C116, followed by ejection of the active site Zn2+ and provides further evidence that the Zn2+ coordination site involves the three conserved cysteine residues. The C93 reactivity is

  6. KW-3902, a selective high affinity antagonist for adenosine A1 receptors.

    PubMed Central

    Nonaka, H.; Ichimura, M.; Takeda, M.; Kanda, T.; Shimada, J.; Suzuki, F.; Kase, H.

    1996-01-01

    1. We demonstrate that 8-(noradamantan-3-yl)-1,3-dipropylxanthine (KW-3902) is a very potent and selective adenosine A1 receptor antagonist, assessed by radioligand binding and cyclic AMP response in cells. 2. In rat forebrain adenosine A1 receptors labelled with [3H]-cyclohexyladenosine (CHA), KW-3902 had a Ki value of 0.19 nM, whereas it showed a Ki value of 170 nM in rat striatal A2A receptors labelled with [3H]-2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoad enosine (CGS21680), indicating 890 fold A1 receptor selectivity versus the A2A receptor. KW-3902 at 10 microM showed no effect on recombinant rat A3 receptors expressed on CHO cells. 3. Saturation studies with [3H]-KW-3902 revealed that it bound with high affinity (Kd = 77 pM) and limited capacity (Bmax = 470 fmol mg-1 of protein) to a single class of recognition sites. A high positive correlation was observed between the pharmacological profile of adenosine ligands inhibiting the binding of [3H]-KW-3902 and that of [3H]-CHA. 4. KW-3902 showed potent A1 antagonism against the inhibition of forskolin-induced cyclic AMP accumulation in DDT1 MF-2 cells by the A1-selective agonist, cyclopentyladenosine with a dissociation constant (KB value) of 0.34 nM. KW-3902 antagonized 5'-N-ethylcarboxamidoadenosine-elicited cyclic AMP accumulation via A2B receptors with a KB value of 52 nM. 5. KW-3902 exhibited marked species-dependent differences in the binding affinities. The highest affinity was for the rat A1 receptor (ki = 0.19 nM) and these values for guinea-pig and dog A1 receptors were 1.3 and 10 nM, respectively. PMID:8732272

  7. (/sup 3/H)-8-cyclopentyl-1,3-dipropylxanthine binding to A1 adenosine receptors of intact rat ventricular myocytes

    SciTech Connect

    Martens, D.; Lohse, M.J.; Schwabe, U.

    1988-09-01

    The purpose of the present study was the identification of A1 adenosine receptors in intact rat ventricular myocytes, which are thought to mediate the negative inotropic effects of adenosine. The adenosine receptor antagonist (/sup 3/H)-8-cyclopentyl-1,3-dipropylxanthine was used as radioligand. Binding of the radioligand to intact myocytes was rapid, reversible, and saturable with a binding capacity of 40,000 binding sites per cell. The dissociation constant of the radioligand was 0.48 nM. The adenosine receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine, xanthine amine congener, and theophylline were competitive inhibitors with affinities in agreement with results obtained for A1 receptors in other tissues. Competition experiments using the adenosine receptor agonists R-N(6)-phenylisopropyladenosine, 5'-N-ethylcarboxamidoadenosine, and S-N(6)-phenylisopropyladenosine gave monophasic displacement curves with Ki values of 50 nM, 440 nM, and 4,300 nM, which agreed well with the GTP-inducible low affinity state in cardiac membranes. The low affinity for agonists was not due to agonist-induced desensitization, and correlated well with the corresponding IC50 values for the inhibition of cyclic AMP accumulation by isoprenaline. It is suggested that only a low affinity state of A1 receptors can be detected in intact rat myocytes due to the presence of high concentrations of guanine nucleotides in intact cells.

  8. cAMP-dependent protein kinase activation decreases cytokine release in bronchial epithelial cells

    PubMed Central

    Poole, Jill A.; Nordgren, Tara M.; DeVasure, Jane M.; Heires, Art J.; Bailey, Kristina L.; Romberger, Debra J.

    2014-01-01

    Lung injury caused by inhalation of dust from swine-concentrated animal-feeding operations (CAFO) involves the release of inflammatory cytokine interleukin 8 (IL-8), which is mediated by protein kinase C-ε (PKC-ε) in airway epithelial cells. Once activated by CAFO dust, PKC-ε is responsible for slowing cilia beating and reducing cell migration for wound repair. Conversely, the cAMP-dependent protein kinase (PKA) stimulates contrasting effects, such as increased cilia beating and an acceleration of cell migration for wound repair. We hypothesized that a bidirectional mechanism involving PKA and PKC regulates epithelial airway inflammatory responses. To test this hypothesis, primary human bronchial epithelial cells and BEAS-2B cells were treated with hog dust extract (HDE) in the presence or absence of cAMP. PKC-ε activity was significantly reduced in cells that were pretreated for 1 h with 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) before exposure to HDE (P < 0.05). HDE-induced IL-6, and IL-8 release was significantly lower in cells that were pretreated with 8-Br-cAMP (P < 0.05). To exclude exchange protein activated by cAMP (EPAC) involvement, cells were pretreated with either 8-Br-cAMP or 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-CPT-2Me-cAMP) (EPAC agonist). 8-CPT-2Me-cAMP did not activate PKA and did not reduce HDE-stimulated IL-6 release. In contrast, 8-Br-cAMP decreased HDE-stimulated tumor necrosis factor (TNF)-α-converting enzyme (TACE; ADAM-17) activity and subsequent TNF-α release (P < 0.001). 8-Br-cAMP also blocked HDE-stimulated IL-6 and keratinocyte-derived chemokine release in precision-cut mouse lung slices (P < 0.05). These data show bidirectional regulation of PKC-ε via a PKA-mediated inhibition of TACE activity resulting in reduced PKC-ε-mediated release of IL-6 and IL-8. PMID:25150062

  9. cAMP-dependent protein kinase activation decreases cytokine release in bronchial epithelial cells.

    PubMed

    Wyatt, Todd A; Poole, Jill A; Nordgren, Tara M; DeVasure, Jane M; Heires, Art J; Bailey, Kristina L; Romberger, Debra J

    2014-10-15

    Lung injury caused by inhalation of dust from swine-concentrated animal-feeding operations (CAFO) involves the release of inflammatory cytokine interleukin 8 (IL-8), which is mediated by protein kinase C-ε (PKC-ε) in airway epithelial cells. Once activated by CAFO dust, PKC-ε is responsible for slowing cilia beating and reducing cell migration for wound repair. Conversely, the cAMP-dependent protein kinase (PKA) stimulates contrasting effects, such as increased cilia beating and an acceleration of cell migration for wound repair. We hypothesized that a bidirectional mechanism involving PKA and PKC regulates epithelial airway inflammatory responses. To test this hypothesis, primary human bronchial epithelial cells and BEAS-2B cells were treated with hog dust extract (HDE) in the presence or absence of cAMP. PKC-ε activity was significantly reduced in cells that were pretreated for 1 h with 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) before exposure to HDE (P < 0.05). HDE-induced IL-6, and IL-8 release was significantly lower in cells that were pretreated with 8-Br-cAMP (P < 0.05). To exclude exchange protein activated by cAMP (EPAC) involvement, cells were pretreated with either 8-Br-cAMP or 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-CPT-2Me-cAMP) (EPAC agonist). 8-CPT-2Me-cAMP did not activate PKA and did not reduce HDE-stimulated IL-6 release. In contrast, 8-Br-cAMP decreased HDE-stimulated tumor necrosis factor (TNF)-α-converting enzyme (TACE; ADAM-17) activity and subsequent TNF-α release (P < 0.001). 8-Br-cAMP also blocked HDE-stimulated IL-6 and keratinocyte-derived chemokine release in precision-cut mouse lung slices (P < 0.05). These data show bidirectional regulation of PKC-ε via a PKA-mediated inhibition of TACE activity resulting in reduced PKC-ε-mediated release of IL-6 and IL-8. PMID:25150062

  10. Adenosine modulation of [Ca2+]i in cerebellar granular cells: multiple adenosine receptors involved.

    PubMed

    Vacas, Javier; Fernández, Mercedes; Ros, Manuel; Blanco, Pablo

    2003-12-01

    Elimination of adenosine by addition of adenosine deaminase (ADA) to the media leads to alterations in intracellular free calcium concentration ([Ca(2+)](i)) in cerebellar granular cells. Adenosine deaminase brings about increases or decreases in [Ca(2+)](i) depending on the previous activation state of the cell. These effects are dependent on the catalytic activity of adenosine deaminase, since its previous catalytic inactivation with Hg(2+) prevents the above-mentioned changes in intracellular calcium. Extracellular calcium is required for the increase in [Ca(2+)](i) promoted by ADA. This rise is insensitive to thapsigargin, but sensitive to micromolar concentrations of Ni(2+). Toxins specific for L, N and P/Q calcium channels do not overtly reduce this effect. N(6)-Cyclopentyl adenosine (CPA), an A(1) receptor agonist, produces a partial reversion of ADA effects, while CGS21680, A(2A)/A(2B) receptor agonist, slightly enhances them. Expression of A(1), A(2A), A(2B) and A(3) adenosine receptor mRNAs was detected in cerebellar granular cell cultures. These results suggest that adenosine modulate [Ca(2+)](i) in cerebellar granule cells through different adenosine receptor subtypes which, at least in part, seem to act through R-type calcium channels.

  11. Nitric oxide and cyclic guanosine monophosphate signaling in the eye.

    PubMed

    Murad, Ferid

    2008-06-01

    This brief review describes the components and pathways utilized in nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) signaling. Since the discovery of the effects of NO and cGMP on smooth muscle relaxation about 30 years ago, the field has expanded in many directions such that many, but not all, biochemical and biological effects seem to be regulated by these unique signaling molecules. While many of the effects of NO are due to activation of soluble guanylyl cyclase (sGC) that can be considered the receptor for NO, cGMP, in turn, can activate a cGMP-dependent protein kinase (PKG) to phosphorylate an array of proteins. Some of the effects of cGMP can be independent of PKG and are due to effects on ion channels or cyclic nucleotide phosphodiesterases. Also, some of the effects of NO can be independent of sGC activation. The isoenzymes and macromolecules that participate in these signaling pathways can serve as molecular targets to identify compounds that increase or decrease their activation and thus serve as chemical leads for discovering novel drugs for a variety of diseases. Some examples are given. However, with about 90,000 publications in the field since our first reports in 1977, this brief review can only give the readers a sample of the excitement and opportunities we have found in this cell signaling system.

  12. [Identification of thiamine monophosphate hydrolyzing enzymes in chicken liver].

    PubMed

    Kolos, I K; Makarchikov, A F

    2014-01-01

    In animals, thiamine monophosphate (TMP) is an intermediate on the path of thiamine diphosphate, the coenzyme form of vitamin B1, degradation. The enzymes involved in TMP metabolism in animal tissues are not identified hitherto. The aim of this work was to study TMP hydrolysis in chicken liver. Two phosphatases have been found to contribute to TMP hydrolysis in liver homogenate. The first one, possessing a maximal activity at pH 6.0, is soluble, whereas the second one represents a membrane-bound enzyme with a pH optimum of 9.0. Membrane-bound TMPase activity was enhanced 1.7-fold by 5 mM Mg2+ ions and strongly inhibited by levamisole in uncompetitive manner with K1 of 53 μM, indicating the involvement of alkaline phosphatase. An apparent Km of alkaline phosphatase for TMP was calculated from the Hanes plot to be 0.6 mM. The soluble TMPase has an apparent Km of 0.7 mM; this enzyme is Mg2+ independent and insensitive to levamisole. As estimated by gel filtration on a Toyopearl HW-55 column, the soluble enzyme has a molecular mass of 17.8 kDa, TMPase activity being eluted simultaneously with peaks of flavinmononucleotide and p-nitrophenyl phosphatase activity. Thus, TMP appears to be a physiological substrate for a low-molecular weight acid phosphatase, also known as low-molecular-weight protein phosphotyrosine phosphatase.

  13. Phosphatidylinositol 3-monophosphate is involved in toxoplasma apicoplast biogenesis.

    PubMed

    Tawk, Lina; Dubremetz, Jean-François; Montcourrier, Philippe; Chicanne, Gaëtan; Merezegue, Fabrice; Richard, Véronique; Payrastre, Bernard; Meissner, Markus; Vial, Henri J; Roy, Christian; Wengelnik, Kai; Lebrun, Maryse

    2011-02-01

    Apicomplexan parasites cause devastating diseases including malaria and toxoplasmosis. They harbour a plastid-like, non-photosynthetic organelle of algal origin, the apicoplast, which fulfils critical functions for parasite survival. Because of its essential and original metabolic pathways, the apicoplast has become a target for the development of new anti-apicomplexan drugs. Here we show that the lipid phosphatidylinositol 3-monophosphate (PI3P) is involved in apicoplast biogenesis in Toxoplasma gondii. In yeast and mammalian cells, PI3P is concentrated on early endosomes and regulates trafficking of endosomal compartments. Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles. Interference with regular PI3P function by over-expression of a PI3P specific binding module in the parasite led to the accumulation of vesicles containing apicoplast peripheral membrane proteins around the apicoplast and, ultimately, to the loss of the organelle. Accordingly, inhibition of the PI3P-synthesising kinase interfered with apicoplast biogenesis. These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast. This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs. PMID:21379336

  14. Bioconjugation of zirconium uridine monophosphate: application to myoglobin direct electrochemistry.

    PubMed

    Qiao, Yuanbiao; Jian, Fangfang; Bai, Qian

    2008-03-14

    Porous nano-granule of zirconium uridine monophosphate, Zr(UMP)2.H2O is, for the first time, synthesized under mild experimental conditions and applied to the bioconjugation of myoglobin (Mb) to realize its direct electron transfer. UV-vis and resonance Raman spectroscopies prove that Mb in the Zr(UMP)2.H2O film maintains its secondary structure similar to the native state. The conjugation film of the Mb-Zr(UMP)2.H2O on the glassy carbon (GC) electrode gives a well-defined and quasi-reversible cyclic voltammogram, which reflects the direct electron transfer of the heme Fe III/Fe II couple of Mb. On the basis of the satisfying bioelectrocatalysis of the nano-conjugation of Mb and genetic substrate, a kind of mediator-free biosensor for H2O2 is developed. The linear range for H2O2 detection is estimated to be 3.92-180.14 microM. The apparent Michaelis-Menten constant (Km) and the detection limit based on the signal-to-noise ratio of 3 are found to be 196.1 microM and 1.52 microM, respectively. Both the apparent Michaelis-Menten constant and the detection limit herein are much lower than currently reported values from other Mb films. This kind of sensor possesses excellent stability, long-term life (more than 20 days) and good reproducibility. PMID:18180152

  15. Excretion of intracorporeal cadmium with S-benzoylthiamin monophosphate

    SciTech Connect

    Yamamoto, J.; Kaneda, Y.

    1995-05-01

    Examination was made of the excretion of intracorporeal methylmercury into body hair by the administration of thiamin tetrahydrofurfuryl disulfide (TTFD) which caused significant increase in mercury content in human mustache. The thiamin derivative, S-benzoylthiamin monophosphate (BTMP) failed to have such effect. The mechanism of mercury excretion is thus based on the side chain structure of TTFD, mercaptan. At the start of the 20th century in Japan, many multiparae were found to have itai-itai disease, the main symptom of which is general pain. The cause of this disease was considered cadmium deposition on the bone from sourced such as soil and river water which contaminated rice and cereals. The previous system for mercury was applied based on cadmium content variation in human mustache. Although TTFD worked well in the mercury excretion system, the usual dosage of TTFD administered orally did not cause significant increase in cadmium in the mustache. BTMP appeared to exert effect by generating thiol-type thiamin. Experiments in vitro should be conducted to demonstrate the chelating effects of thiol-type thiamin. It is quite important to find evidence for cadmium excretion from human body. 3 refs., 2 figs., 2 tabs.

  16. Bioconjugation of zirconium uridine monophosphate: application to myoglobin direct electrochemistry.

    PubMed

    Qiao, Yuanbiao; Jian, Fangfang; Bai, Qian

    2008-03-14

    Porous nano-granule of zirconium uridine monophosphate, Zr(UMP)2.H2O is, for the first time, synthesized under mild experimental conditions and applied to the bioconjugation of myoglobin (Mb) to realize its direct electron transfer. UV-vis and resonance Raman spectroscopies prove that Mb in the Zr(UMP)2.H2O film maintains its secondary structure similar to the native state. The conjugation film of the Mb-Zr(UMP)2.H2O on the glassy carbon (GC) electrode gives a well-defined and quasi-reversible cyclic voltammogram, which reflects the direct electron transfer of the heme Fe III/Fe II couple of Mb. On the basis of the satisfying bioelectrocatalysis of the nano-conjugation of Mb and genetic substrate, a kind of mediator-free biosensor for H2O2 is developed. The linear range for H2O2 detection is estimated to be 3.92-180.14 microM. The apparent Michaelis-Menten constant (Km) and the detection limit based on the signal-to-noise ratio of 3 are found to be 196.1 microM and 1.52 microM, respectively. Both the apparent Michaelis-Menten constant and the detection limit herein are much lower than currently reported values from other Mb films. This kind of sensor possesses excellent stability, long-term life (more than 20 days) and good reproducibility.

  17. Ag(+)-mediated assembly of 5'-guanosine monophosphate.

    PubMed

    Loo, Kristine; Degtyareva, Natalya; Park, Jihae; Sengupta, Bidisha; Reddish, Michaeal; Rogers, Christopher C; Bryant, Andrea; Petty, Jeffrey T

    2010-04-01

    Polymorphic forms of nucleic acids provide platforms for new nanomaterials, and transition metal cations give access to alternative arrangements of nucleobases by coordinating with electron-rich functional groups. Interaction of Ag(+) with 5'-guanosine monophosphate (5'-GMP) is considered in this work. Ag(+) promotes nucleotide stacking and aggregation, as indicated by the increased viscosity of 5'-GMP solutions with Ag(+), magnification of the circular dichroism response of guanine by Ag(+), and exothermic reactions between Ag(+) and guanine derivatives. Isothermal titration calorimetry studies show that the reaction is favored starting at 10 microM 5'-GMP. Utilizing the exothermic heat change associated with reaction of Ag(+) with 5'-GMP, local structure within the aggregate was assessed. On the basis of the salt dependence of the reaction and comparison with the corresponding nucleoside, the dianionic phosphate of 5'-GMP is one binding site for Ag(+), although this electrostatic interaction is not a dominant contribution to the overall heat change. Another binding site is the N7 on the nucleobase, as determined via studies with 7-deazaguanosine. Besides this binding site, Ag(+) also associates with the O6, as earlier studies deduced from the shift in the carbonyl stretching frequency associated with adduct formation. With these two binding sites on the nucleobase, the empirical stoichiometry of approximately 1 Ag(+):nucleobase derived from the calorimetry studies indicates that Ag(+) coordinates two nucleobases. The proposed structural model is a Ag(+)-mediated guanine dimer within a base stacked aggregate. PMID:20205377

  18. (−)-Epicatechin-3-O-β-d-allopyranoside from Davallia formosana, Prevents Diabetes and Hyperlipidemia by Regulation of Glucose Transporter 4 and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

    PubMed Central

    Shih, Chun-Ching; Wu, Jin-Bin; Jian, Jia-Ying; Lin, Cheng-Hsiu; Ho, Hui-Ya

    2015-01-01

    The purpose of this experiment was to determine the antidiabetic and lipid-lowering effects of (−)-epicatechin-3-O-β-d-allopyranoside (BB) from the roots and stems of Davallia formosana in mice. Animal treatment was induced by high-fat diet (HFD) or low-fat diet (control diet, CD). After eight weeks of HFD or CD exposure, the HFD mice were treating with BB or rosiglitazone (Rosi) or fenofibrate (Feno) or water through gavage for another four weeks. However, at 12 weeks, the HFD-fed group had enhanced blood levels of glucose, triglyceride (TG), and insulin. BB treatment significantly decreased blood glucose, TG, and insulin levels. Moreover, visceral fat weights were enhanced in HFD-fed mice, accompanied by increased blood leptin concentrations and decreased adiponectin levels, which were reversed by treatment with BB. Muscular membrane protein levels of glucose transporter 4 (GLUT4) were reduced in HFD-fed mice and significantly enhanced upon administration of BB, Rosi, and Feno. Moreover, BB treatment markedly increased hepatic and skeletal muscular expression levels of phosphorylation of AMP-activated (adenosine monophosphate) protein kinase (phospho-AMPK). BB also decreased hepatic mRNA levels of phosphenolpyruvate carboxykinase (PEPCK), which are associated with a decrease in hepatic glucose production. BB-exerted hypotriglyceridemic activity may be partly associated with increased mRNA levels of peroxisome proliferator activated receptor α (PPARα), and with reduced hepatic glycerol-3-phosphate acyltransferase (GPAT) mRNA levels in the liver, which decreased triacylglycerol synthesis. Nevertheless, we demonstrated BB was a useful approach for the management of type 2 diabetes and dyslipidemia in this animal model. PMID:26492243

  19. Effect of Thyrocalcitonin on Adenosine 3′:5′-Cyclic Phosphate Formation by Rat Kidney and Bone

    PubMed Central

    Murad, Ferid; Brewer, H. Bryan; Vaughan, Martha

    1970-01-01

    Thyrocalcitonin (TCT) increased the rate of accumulation of adenosine 3′:5′-cyclic phosphate (cyclic AMP) when added to incubations containing washed particles from whole rat kidney, adenosine triphosphate (ATP), MgSO4, and caffeine. The maximum stimulatory effect of TCT, 44 ± 6.7 per cent, was always less than the 150 to 250 per cent increase produced by parathyroid hormone (PTH). The effect of both hormones together was no greater than that of PTH alone when each was present at a maximally effective concentration. Since neither TCT nor PTH altered the rate of degradation of cyclic AMP by the kidney preparation, it may be inferred that their effects on cyclic AMP accumulation are the result of increased formation of cyclic AMP. Adenyl cyclase activity in homogenates of renal cortex was stimulated to a greater extent by TCT and PTH than was that of medulla, whereas, as reported earlier, the effect of vasopressin was much larger with homogenates of medulla. The accumulation of cyclic AMP in incubations of rat kidney cortex slices was increased 20 to 60 per cent by TCT and 50 to 140 per cent by PTH. The accumulation of cyclic AMP in incubations of rat calvaria was increased about threefold with TCT and nine to tenfold with PTH, while reduced and alkylated TCT had less than 10 per cent of the activity of TCT. These observations are consistent with the view that the physiological effects of TCT and PTH in kidney and bone are secondary to the enhanced formation of cyclic AMP. PMID:4313199

  20. Evaluation of neuronal phosphoproteins as effectors of caffeine and mediators of striatal adenosine A2A receptor signaling

    PubMed Central

    Sahin, Bogachan; Galdi, Stacey; Hendrick, Joseph; Greene, Robert W.; Snyder, Gretchen L.; Bibb, James A.

    2007-01-01

    Adenosine A2A receptors are predominantly expressed in the dendrites of enkephalin-positive γ-aminobutyric acidergic medium spiny neurons in the striatum. Evidence indicates that these receptors modulate striatal dopaminergic neurotransmission and regulate motor control, vigilance, alertness, and arousal. Although the physiological and behavioral correlates of adenosine A2A receptor signaling have been extensively studied using a combination of pharmacological and genetic tools, relatively little is known about the signal transduction pathways that mediate the diverse biological functions attributed to this adenosine receptor subtype. Using a candidate approach based on the coupling of these receptors to adenylate cyclase-activating G proteins, a number of membranal, cytosolic, and nuclear phosphoproteins regulated by PKA were evaluated as potential mediators of adenosine A2A receptor signaling in the striatum. Specifically, the adenosine A2A receptor agonist, CGS 21680, was used to determine whether the phosphorylation state of each of the following PKA targets is responsive to adenosine A2A receptor stimulation in this tissue: Ser40 of tyrosine hydroxylase, Ser9 of synapsin, Ser897 of the NR1 subunit of the N-methyl-D-aspartate-type glutamate receptor, Ser845 of the GluR1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptor, Ser94 of spinophilin, Thr34 of the dopamine- and cAMP-regulated phosphoprotein, Mr32,000, Ser133 of the cAMP-response element-binding protein, Thr286 of Ca2+/calmodulin-dependent protein kinase II, and Thr202/Tyr204 and Thr183/Tyr185 of the p44 and p42 isoforms, respectively, of mitogen-activated protein kinase. Although the substrates studied differed considerably in their responsiveness to selective adenosine A2A receptor activation, the phosphorylation state of all postsynaptic PKA targets was up-regulated in a time- and dose-dependent manner by treatment with CGS 21680, whereas presynaptic PKA

  1. Evaluation of neuronal phosphoproteins as effectors of caffeine and mediators of striatal adenosine A2A receptor signaling.

    PubMed

    Sahin, Bogachan; Galdi, Stacey; Hendrick, Joseph; Greene, Robert W; Snyder, Gretchen L; Bibb, James A

    2007-01-19

    Adenosine A(2A) receptors are predominantly expressed in the dendrites of enkephalin-positive gamma-aminobutyric acidergic medium spiny neurons in the striatum. Evidence indicates that these receptors modulate striatal dopaminergic neurotransmission and regulate motor control, vigilance, alertness, and arousal. Although the physiological and behavioral correlates of adenosine A(2A) receptor signaling have been extensively studied using a combination of pharmacological and genetic tools, relatively little is known about the signal transduction pathways that mediate the diverse biological functions attributed to this adenosine receptor subtype. Using a candidate approach based on the coupling of these receptors to adenylate cyclase-activating G proteins, a number of membranal, cytosolic, and nuclear phosphoproteins regulated by PKA were evaluated as potential mediators of adenosine A(2A) receptor signaling in the striatum. Specifically, the adenosine A(2A) receptor agonist, CGS 21680, was used to determine whether the phosphorylation state of each of the following PKA targets is responsive to adenosine A(2A) receptor stimulation in this tissue: Ser40 of tyrosine hydroxylase, Ser9 of synapsin, Ser897 of the NR1 subunit of the N-methyl-d-aspartate-type glutamate receptor, Ser845 of the GluR1 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptor, Ser94 of spinophilin, Thr34 of the dopamine- and cAMP-regulated phosphoprotein, M(r) 32,000, Ser133 of the cAMP-response element-binding protein, Thr286 of Ca(2+)/calmodulin-dependent protein kinase II, and Thr202/Tyr204 and Thr183/Tyr185 of the p44 and p42 isoforms, respectively, of mitogen-activated protein kinase. Although the substrates studied differed considerably in their responsiveness to selective adenosine A(2A) receptor activation, the phosphorylation state of all postsynaptic PKA targets was up-regulated in a time- and dose-dependent manner by treatment with CGS 21680

  2. MOLECULAR PROBES FOR EXTRACELLULAR ADENOSINE RECEPTORS

    PubMed Central

    Jacobson, Kenneth A.; Ukena, Dieter; Padgett, William; Kirk, Kenneth L.; Daly, John W.

    2012-01-01

    Derivatives of adenosine receptor agonists (N6-phenyladenosines) and antagonists (1,3-dialkyl-8-phenylxanthines) bearing functionalized chains suitable for attachment to other molecules have been reported [Jacobson et al., J. med. Chem. 28, 1334 and 1341 (1985)]. The “functionalized congener” approach has been extended to the synthesis of spectroscopic and other probes for adenosine receptors that retain high affinity (Ki ~ 10−9 −10−8 M) in A1-receptor binding. The probes have been synthesized from an antagonist xanthine amine congener (XAC) and an adenosine amine congener (ADAC). [3H]ADAC has been synthesized and found to bind highly specifically to A1-adenosine receptors of rat and calf cerebral cortical membranes with KD values of 1.4 and 0.34 nM respectively. The higher affinity in the bovine brain, seen also with many of the probes derived from ADAC and XAC, is associated with phenyl substituents. The spectroscopic probes contain a reporter group attached at a distal site of the functionalized chain. These bifunctional ligands may contain a spin label (e.g. the nitroxyl radical TEMPO) for electron spin resonance spectroscopy, or a fluorescent dye, including fluorescein and 4-nitrobenz-2-oxa-1,3-diazole (NBD), or labels for 19F nuclear magnetic resonance spectroscopy. Potential applications of the spectroscopic probes in characterization of adenosine receptors are discussed. PMID:3036153

  3. Determination of adenosine effects and adenosine receptors in murine corpus cavernosum.

    PubMed

    Tostes, Rita C; Giachini, Fernanda R C; Carneiro, Fernando S; Leite, Romulo; Inscho, Edward W; Webb, R Clinton

    2007-08-01

    This study tested the hypothesis that adenosine, in murine corpora cavernosa, produces direct relaxation of smooth muscle cells and inhibition of contractile responses mediated by sympathetic nerve stimulation. Penes were excised from anesthetized male C57BL/6 mice, dissected, and cavernosal strips were mounted to record isometric force. Adenosine, 2-chloroadenosine (stable analog of adenosine), and 2-phenylaminoadenosine (CV1808) (A2(A)/A2(B) agonist) produced concentration-dependent relaxations of phenylephrine-contracted tissues. Relaxation to 2-chloroadenosine was inhibited, in a concentration-dependent manner, by 2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH58261; A2(A) antagonist; 10(-9)-10(-6) M) and N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamida (MRS1706; A2(B) antagonist; 10(-8)-10(-6) M). The combination of both antagonists abrogated 2-chloroadenosine-induced relaxation. Electrical field stimulation (EFS; 1-32 Hz) of adrenergic nerves produced frequency-dependent contractions that were inhibited by compounds that increase adenosine levels, such as 5'-iodotubercidin (adenosine kinase inhibitor), erythro-9-(2-hydroxy-3-nonyl)adenine (adenosine deaminase inhibitor), and dipyridamole (inhibitor of adenosine transport). The adenosine A1 receptor agonist N(6)-cyclopentyladenosine (C8031) right-shifted contractile responses to EFS, with a significant inhibitory effect at 10(-6) M. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine (C101) (10(-7) M) enhanced contractile responses to EFS and eliminated the inhibitory effects of 5'-iodotubercidin. Dipyridamole and 5'-iodotubercidin had no effect on adenosine-mediated relaxation. In summary, adenosine directly relaxes cavernosal smooth muscle cells, by the activation of A2(A)/A2(B) receptor subtypes. In addition, adenosine negatively modulates sympathetic neurotransmission, by A1 receptor

  4. A New Activity of Anti-HIV and Anti-tumor Protein GAP31: DNA Adenosine Glycosidase – Structural and Modeling Insight into its Functions

    SciTech Connect

    Li, H.; Huang, P; Zhang, D; Sun, Y; Chen, H; Zhang, J; Huang, P; Kong, X; Lee-Huang, S

    2010-01-01

    We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.

  5. A new activity of anti-HIV and anti-tumor protein GAP31: DNA adenosine glycosidase - Structural and modeling insight into its functions

    SciTech Connect

    Li, Hui-Guang; Huang, Philip L.; Zhang, Dawei; Sun, Yongtao; Chen, Hao-Chia; Zhang, John; Huang, Paul L.; Kong, Xiang-Peng; Lee-Huang, Sylvia

    2010-01-01

    We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.

  6. Functional expression of adenosine A2A and A3 receptors in the mouse dendritic cell line XS-106.

    PubMed

    Dickenson, John M; Reeder, Steve; Rees, Bob; Alexander, Steve; Kendall, Dave

    2003-08-01

    There is increasing evidence to suggest that adenosine receptors can modulate the function of cells involved in the immune system. For example, human dendritic cells derived from blood monocytes have recently been described to express functional adenosine A1, A2A and A3 receptors. Therefore, in the present study, we have investigated whether the recently established murine dendritic cell line XS-106 expresses functional adenosine receptors. The selective adenosine A3 receptor agonist 1-[2-chloro-6[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-beta-D-ribofuranuronamide (2-Cl-IB-MECA) inhibited forskolin-mediated [3H]cyclic AMP accumulation and stimulated concentration-dependent increases in p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation. The selective adenosine A2A receptor agonist 4-[2-[[-6-amino-9-(N-ethyl-beta-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzene-propanoic acid (CGS 21680) stimulated a robust increase in [3H]cyclic AMP accumulation and p42/p44 MAPK phosphorylation. In contrast, the selective adenosine A1 receptor agonist CPA (N6-cyclopentyladenosine) did not inhibit forskolin-mediated [3H]cyclic AMP accumulation or stimulate increases in p42/p44 MAPK phosphorylation. These observations suggest that XS-106 cells express functional adenosine A2A and A3 receptors. The non-selective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) inhibited lipopolysaccharide-induced tumour necrosis factor-alpha (TNF-alpha) release from XS-106 cells in a concentration-dependent fashion. Furthermore, treatment with Cl-IB-MECA (1 microM) or CGS 21680 (1 microM) alone produced a partial inhibition of lipopolysaccharide-induced TNF-alpha release (when compared to NECA), whereas a combination of both agonists resulted in the inhibition of TNF-alpha release comparable to that observed with NECA alone. Treatment of cells with the adenosine A2A receptor selective antagonists 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a

  7. Adenosine A2B receptor stimulates angiogenesis by inducing VEGF and eNOS in human microvascular endothelial cells

    PubMed Central

    Du, Xiaolong; Ou, Xuehai; Song, Tao; Zhang, Wentao; Cong, Fei; Zhang, Shihui

    2015-01-01

    Angiogenesis is critical to wound repair due to its role in providing oxygen and nutrients that are required to support the growth and function of reparative cells in damaged tissues. Adenosine receptors are claimed to be of paramount importance in driving wound angiogenesis by inducing VEGF. However, the underlying mechanisms for the regulation of adenosine receptors in VEGF as well as eNOS remain poorly understood. In the present study, we found that adenosine and the non-selective adenosine receptor agonists (NECA) induced tube formation in HMEC-1 in a dose-dependent manner. Adenosine or NECA (10 µmol/L) significantly augmented the number and length of the segments in comparison with the control. Simultaneously, VEGF and eNOS were significantly upregulated following the administration of 10 µmol/L NECA, while they were suppressed after A2B AR genetic silencing and pharmacological inhibition by MRS1754. In addition, VEGF expression and eNOS bioavailability elimination significantly reduced the formation of capillary-like structures. Furthermore, the activation of A2B AR by NECA significantly increased the intracellular cAMP levels and concomitant CREB phosphorylation, eventually leading to the production of VEGF in HMEC-1. However, the activated PKA-CREB pathway seemed to be invalidated in the induction of eNOS. Moreover, we found that the elicited PI3K/AKT signaling in response to the induction of NECA assisted in regulating eNOS but failed to impact on VEGF generation. In conclusion, the A2B AR activation-driven angiogenesis via cAMP-PKA-CREB mediated VEGF production and PI3K/AKT-dependent upregulation of eNOS in HMEC-1. PMID:25966978

  8. Detection of adenosine triphosphate through polymerization-induced aggregation of actin-conjugated gold/silver nanorods

    NASA Astrophysics Data System (ADS)

    Liao, Yu-Ju; Shiang, Yen-Chun; Chen, Li-Yi; Hsu, Chia-Lun; Huang, Chih-Ching; Chang, Huan-Tsung

    2013-11-01

    We have developed a simple and selective nanosensor for the optical detection of adenosine triphosphate (ATP) using globular actin-conjugated gold/silver nanorods (G-actin-Au/Ag NRs). By simply mixing G-actin and Au/Ag NRs (length ˜56 nm and diameter ˜12 nm), G-actin-Au/Ag NRs were prepared which were stable in physiological solutions (25 mM Tris-HCl, 150 mM NaCl, 5.0 mM KCl, 3.0 mM MgCl2 and 1.0 mM CaCl2; pH 7.4). Introduction of ATP into the G-actin-Au/Ag NR solutions in the presence of excess G-actin induced the formation of filamentous actin-conjugated Au/Ag NR aggregates through ATP-induced polymerization of G-actin. When compared to G-actin-modified spherical Au nanoparticles having a size of 13 nm or 56 nm, G-actin-Au/Ag NRs provided better sensitivity for ATP, mainly because the longitudinal surface plasmon absorbance of the Au/Ag NR has a more sensitive response to aggregation. This G-actin-Au/Ag NR probe provided high sensitivity (limit of detection 25 nM) for ATP with remarkable selectivity (>10-fold) over other adenine nucleotides (adenosine, adenosine monophosphate and adenosine diphosphate) and nucleoside triphosphates (guanosine triphosphate, cytidine triphosphate and uridine triphosphate). It also allowed the determination of ATP concentrations in plasma samples without conducting tedious sample pretreatments; the only necessary step was simple dilution. Our experimental results are in good agreement with those obtained from a commercial luciferin-luciferase bioluminescence assay. Our simple, sensitive and selective approach appears to have a practical potential for the clinical diagnosis of diseases (e.g. cystic fibrosis) associated with changes in ATP concentrations.

  9. Alterations in the Cerebellar (Phospho)Proteome of a Cyclic Guanosine Monophosphate (cGMP)-dependent Protein Kinase Knockout Mouse*

    PubMed Central

    Corradini, Eleonora; Vallur, Raghavan; Raaijmakers, Linsey M.; Feil, Susanne; Feil, Robert; Heck, Albert J. R.; Scholten, Arjen

    2014-01-01

    The cyclic nucleotide cyclic guanosine monophosphate (cGMP) plays an important role in learning and memory, but its signaling mechanisms in the mammalian brain are not fully understood. Using mass-spectrometry-based proteomics, we evaluated how the cerebellum adapts its (phospho)proteome in a knockout mouse model of cGMP-dependent protein kinase type I (cGKI). Our data reveal that a small subset of proteins in the cerebellum (∼3% of the quantified proteins) became substantially differentially expressed in the absence of cGKI. More changes were observed at the phosphoproteome level, with hundreds of sites being differentially phosphorylated between wild-type and knockout cerebellum. Most of these phosphorylated sites do not represent known cGKI substrates. An integrative computational network analysis of the data indicated that the differentially expressed proteins and proteins harboring differentially phosphorylated sites largely belong to a tight network in the Purkinje cells of the cerebellum involving important cGMP/cAMP signaling nodes (e.g. PDE5 and PKARIIβ) and Ca2+ signaling (e.g. SERCA3). In this way, removal of cGKI could be linked to impaired cerebellar long-term depression at Purkinje cell synapses. In addition, we were able to identify a set of novel putative (phospho)proteins to be considered in this network. Overall, our data improve our understanding of cerebellar cGKI signaling and suggest novel players in cGKI-regulated synaptic plasticity. PMID:24925903

  10. Working memory and the homeostatic control of brain adenosine by adenosine kinase.

    PubMed

    Singer, P; McGarrity, S; Shen, H-Y; Boison, D; Yee, B K

    2012-06-28

    The neuromodulator adenosine maintains brain homeostasis and regulates complex behaviour via activation of inhibitory and excitatory adenosine receptors (ARs) in a brain region-specific manner. AR antagonists such as caffeine have been shown to ameliorate cognitive impairments in animal disease models but their effects on learning and memory in normal animals are equivocal. An alternative approach to reduce AR activation is to lower the extracellular tone of adenosine, which can be achieved by up-regulating adenosine kinase (ADK), the key enzyme of metabolic adenosine clearance. However, mice that globally over-express an Adk transgene ('Adk-tg' mice) were devoid of a caffeine-like pro-cognitive profile; they instead exhibited severe spatial memory deficits. This may be mechanistically linked to cortical/hippocampal N-methyl-d-aspartate receptor (NMDAR) hypofunction because the motor response to acute MK-801 was also potentiated in Adk-tg mice. Here, we evaluated the extent to which the behavioural phenotypes of Adk-tg mice might be modifiable by up-regulating adenosine levels in the cortex/hippocampus. To this end, we investigated mutant 'fb-Adk-def' mice in which ADK expression was specifically reduced in the telencephalon leading to a selective increase in cortical/hippocampal adenosine, while the rest of the brain remained as adenosine-deficient as in Adk-tg mice. The fb-Adk-def mice showed an even greater impairment in spatial working memory and a more pronounced motor response to NMDAR blockade than Adk-tg mice. These outcomes suggest that maintenance of cortical/hippocampal adenosine homeostasis is essential for effective spatial memory and deviation in either direction is detrimental with increased expression seemingly more disruptive than decreased expression.

  11. Pain-relieving prospects for adenosine receptors and ectonucleotidases

    PubMed Central

    Zylka, Mark J.

    2010-01-01

    Adenosine receptor agonists have potent antinociceptive effects in diverse preclinical models of chronic pain. In contrast, the efficacy of adenosine or adenosine receptor agonists at treating pain in humans is unclear. Two ectonucleotidases that generate adenosine in nociceptive neurons were recently identified. When injected spinally, these enzymes have long-lasting adenosine A1 receptor (A1R)-dependent antinociceptive effects in inflammatory and neuropathic pain models. Furthermore, recent findings indicate that spinal adenosine A2A receptor activation can enduringly inhibit neuropathic pain symptoms. Collectively, these studies suggest the possibility of treating chronic pain in humans by targeting specific adenosine receptor subtypes in anatomically defined regions with agonists or with ectonucleotidases that generate adenosine. PMID:21236731

  12. Cyclic AMP Represents a Crucial Component of Treg Cell-Mediated Immune Regulation

    PubMed Central

    Klein, Matthias; Bopp, Tobias

    2016-01-01

    T regulatory (Treg) cells are one of the key players in the immune tolerance network, and a plethora of manuscripts have described their development and function in the course of the last two decades. Nevertheless, it is still a matter of debate as to which mechanisms and agents are employed by Treg cells, providing the basis of their suppressive potency. One of the important candidates is cyclic AMP (cAMP), which is long known as a potent suppressor at least of T cell activation and function. While this suppressive function by itself is widely accepted, the source and the mechanism of action of cAMP are less clear, and a multitude of seemingly contradictory data allow for, in principle, two different scenarios of cAMP-mediated suppression. In one scenario, Treg cells contain high amounts of cAMP and convey this small molecule via gap junction intercellular communication directly to the effector T cells (Teff) leading to their suppression. Alternatively, it was shown that Treg cells represent the origin of considerable amounts of adenosine, which trigger the adenylate cyclases in Teff cells via A2A and A2B receptors, thus strongly increasing intracellular cAMP. This review will present and discuss initial findings and recent developments concerning the function of cAMP for Treg cells and its impact on immune regulation. PMID:27621729

  13. Cyclic AMP Represents a Crucial Component of Treg Cell-Mediated Immune Regulation.

    PubMed

    Klein, Matthias; Bopp, Tobias

    2016-01-01

    T regulatory (Treg) cells are one of the key players in the immune tolerance network, and a plethora of manuscripts have described their development and function in the course of the last two decades. Nevertheless, it is still a matter of debate as to which mechanisms and agents are employed by Treg cells, providing the basis of their suppressive potency. One of the important candidates is cyclic AMP (cAMP), which is long known as a potent suppressor at least of T cell activation and function. While this suppressive function by itself is widely accepted, the source and the mechanism of action of cAMP are less clear, and a multitude of seemingly contradictory data allow for, in principle, two different scenarios of cAMP-mediated suppression. In one scenario, Treg cells contain high amounts of cAMP and convey this small molecule via gap junction intercellular communication directly to the effector T cells (Teff) leading to their suppression. Alternatively, it was shown that Treg cells represent the origin of considerable amounts of adenosine, which trigger the adenylate cyclases in Teff cells via A2A and A2B receptors, thus strongly increasing intracellular cAMP. This review will present and discuss initial findings and recent developments concerning the function of cAMP for Treg cells and its impact on immune regulation.

  14. Cyclic AMP Represents a Crucial Component of Treg Cell-Mediated Immune Regulation

    PubMed Central

    Klein, Matthias; Bopp, Tobias

    2016-01-01

    T regulatory (Treg) cells are one of the key players in the immune tolerance network, and a plethora of manuscripts have described their development and function in the course of the last two decades. Nevertheless, it is still a matter of debate as to which mechanisms and agents are employed by Treg cells, providing the basis of their suppressive potency. One of the important candidates is cyclic AMP (cAMP), which is long known as a potent suppressor at least of T cell activation and function. While this suppressive function by itself is widely accepted, the source and the mechanism of action of cAMP are less clear, and a multitude of seemingly contradictory data allow for, in principle, two different scenarios of cAMP-mediated suppression. In one scenario, Treg cells contain high amounts of cAMP and convey this small molecule via gap junction intercellular communication directly to the effector T cells (Teff) leading to their suppression. Alternatively, it was shown that Treg cells represent the origin of considerable amounts of adenosine, which trigger the adenylate cyclases in Teff cells via A2A and A2B receptors, thus strongly increasing intracellular cAMP. This review will present and discuss initial findings and recent developments concerning the function of cAMP for Treg cells and its impact on immune regulation.

  15. Cyclic AMP Represents a Crucial Component of Treg Cell-Mediated Immune Regulation.

    PubMed

    Klein, Matthias; Bopp, Tobias

    2016-01-01

    T regulatory (Treg) cells are one of the key players in the immune tolerance network, and a plethora of manuscripts have described their development and function in the course of the last two decades. Nevertheless, it is still a matter of debate as to which mechanisms and agents are employed by Treg cells, providing the basis of their suppressive potency. One of the important candidates is cyclic AMP (cAMP), which is long known as a potent suppressor at least of T cell activation and function. While this suppressive function by itself is widely accepted, the source and the mechanism of action of cAMP are less clear, and a multitude of seemingly contradictory data allow for, in principle, two different scenarios of cAMP-mediated suppression. In one scenario, Treg cells contain high amounts of cAMP and convey this small molecule via gap junction intercellular communication directly to the effector T cells (Teff) leading to their suppression. Alternatively, it was shown that Treg cells represent the origin of considerable amounts of adenosine, which trigger the adenylate cyclases in Teff cells via A2A and A2B receptors, thus strongly increasing intracellular cAMP. This review will present and discuss initial findings and recent developments concerning the function of cAMP for Treg cells and its impact on immune regulation. PMID:27621729

  16. Vascular CD39/ENTPD1 Directly Promotes Tumor Cell Growth by Scavenging Extracellular Adenosine Triphosphate12

    PubMed Central

    Feng, Lili; Sun, Xiaofeng; Csizmadia, Eva; Han, Lihui; Bian, Shu; Murakami, Takashi; Wang, Xin; Robson, Simon C; Wu, Yan

    2011-01-01

    Extracellular adenosine triphosphate (ATP) is known to boost immune responses in the tumor microenvironment but might also contribute directly to cancer cell death. CD39/ENTPD1 is the dominant ectonucleotidase expressed by endothelial cells and regulatory T cells and catalyzes the sequential hydrolysis of ATP to AMP that is further degraded to adenosine by CD73/ecto-5′-nucleotidase. We have previously shown that deletion of Cd39 results in decreased growth of transplanted tumors in mice, as a result of both defective angiogenesis and heightened innate immune responses (secondary to loss of adenosinergic immune suppression). Whether alterations in local extracellular ATP and adenosine levels as a result of CD39 bioactivity directly affect tumor growth and cytotoxicity has not been investigated to date. We show here that extracellular ATP exerts antitumor activity by directly inhibiting cell proliferation and promoting cancer cell death. ATP-induced antiproliferative effects and cell death are, in large part, mediated through P2X7 receptor signaling. Tumors in Cd39 null mice exhibit increased necrosis in association with P2X7 expression. We further demonstrate that exogenous soluble NTPDase, or CD39 expression by cocultured liver sinusoidal endothelial cells, stimulates tumor cell proliferation and limits cell death triggered by extracellular ATP. Collectively, our findings indicate that local expression of CD39 directly promotes tumor cell growth by scavenging extracellular ATP. Pharmacological or targeted inhibition of CD39 enzymatic activity may find utility as an adjunct therapy in cancer management. PMID:21390184

  17. Studies on the roles of ATP, adenosine and nitric oxide in mediating muscle vasodilatation induced in the rat by acute systemic hypoxia.

    PubMed

    Skinner, M R; Marshall, J M

    1996-09-01

    1. In Saffan-anaesthetized rats, we have further investigated the mechanisms underlying the vasodilatation induced by adenosine in skeletal muscle by acute systemic hypoxia (breathing 8% O2 for 5 min). 2. In eleven rats the nitric oxide (NO) synthesis inhibitor nitro-L-arginine methyl ester (L-NAME, 10 mg kg-1, i.v.) reduced the increase in femoral vascular conductance (FVC) induced by hypoxia by approximately 50%. L-NAME had similar effects on the increase in FVC induced by intra-arterial (I.A.) infusion of adenosine (at 1.2 mg kg-1 min-1 for 5 min via the tail artery) and by ATP (I.A., 1 mg kg-1 min-1 for 5 min). Subsequent administration of the adenosine receptor antagonist 8-sulphophenyl theophylline (8-SPT, 20 mg kg-1, i.v.) virtually abolished the adenosine- and ATP-induced increase in FVC. 3. In a further nine rats, 8-SPT reduced the increase in FVC induced by hypoxia by approximately 50%. This remaining increase in FVC was substantially reduced by L-NAME. 4. In an additional nine rats, alpha,beta-methyleneADP (160 micrograms kg-1, i.v.) which inhibits the 5'-ectonucleotidase that degrades AMP to adenosine, reduced the peripheral vasodilatation (fall in arterial blood pressure, ABP) induced by ATP infusion, but had no effect on the increase in FVC or decrease in ABP evoked by systemic hypoxia. 5. These results provide the first evidence that the muscle vasodilatation induced by adenosine during systemic hypoxia is mainly dependent on NO synthesis. They also suggest that adenosine is released as such rather than being formed extracellularly from AMP. Given evidence that extraluminal adenosine acts in an NO-independent fashion we propose that hypoxia releases adenosine from the endothelium. Our results also indicate that hypoxia induces muscle vasodilatation that is adenosine independent but NO dependent: they allow the possibility that this is partly mediated by ATP released from the endothelium. PMID:8887765

  18. Adenosine induced coronary spasm – A rare presentation

    PubMed Central

    Arora, P.; Bhatia, V.; Arora, M.; Kaul, U.

    2014-01-01

    Adenosine is commonly used as a pharmacological agent in myocardial perfusion imaging, as an antiarrhythmic agent, and in Cath Lab. during PCI for treating no reflow phenomenon. Coronary spasm has been reported following adenosine injection during stress imaging. We report a rare complication with ST segment elevation, following adenosine injection, given for treatment of supraventricular tachycardia. PMID:24581102

  19. Renal Epithelial Cyst Formation and Enlargement in vitro: Dependence on cAMP

    NASA Astrophysics Data System (ADS)

    Mangoo-Karim, Roberto; Uchic, Marie; Lechene, Claude; Grantham, Jared J.

    1989-08-01

    Cysts, a common abnormality of kidneys, are collections of urine-like fluid enclosed by a continuous layer of epithelial cells. Renal cysts derive from nephrons and collecting ducts and progressively enlarge as a consequence of epithelial proliferation and transepithelial fluid secretion. The initiation of cyst formation and the factors that control cyst enlargement are unknown. We used an in vitro model of renal cysts to explore the role of the cAMP signal transduction system in the formation and expansion of cysts. MDCK cells, cultured in hydrated-collagen gel, produced polarized monolayered epithelial cysts when intracellular cAMP was increased by prostaglandin E1, arginine vasopressin, cholera toxin, forskolin, or 8-bromoadenosine 3',5'-cyclic monophosphate. All agonists were potentiated by 3-isobutyl-1-methylxanthine, a nucleotide phosphodiesterase inhibitor. The cell proliferation component of cyst enlargement was accelerated by cAMP agonists, as shown by the increased growth of MDCK cells in subconfluent monolayers. The fluid secretion component, reflected by the transepithelial movement of fluid across polarized monolayers of MDCK cells grown on permeable supports, was stimulated by cAMP agonists in the basolateral medium. Chloride levels were higher in the cyst fluid and the secreted fluid than in the bathing medium. We conclude that the development of MDCK cysts is dependent on cAMP. This signal transduction system may be an important modulator of epithelial cell proliferation and transepithelial fluid secretion in the kidney.

  20. Purification and characterization of (-)(/sup 125/I)hydroxyphenylisopropyladenosine, an adenosine R-site agonist radioligand and theoretical analysis of mixed stereoisomer radioligand binding

    SciTech Connect

    Linden, J.

    1984-11-01

    (-)-N6-(R-4-Hydroxyphenylisopropyl)adenosine (HPIA) was iodinated with NaI and trace /sup 125/I. Mono- and diiodinated reaction products and the starting material were separated by high pressure liquid chromatography and the structures of the reaction products were verified by NMR. (-)-N6-(R-Phenylisopropyl)adenosine (PIA), IHPIA, and I2HPIA decreased rat atrial contractility with ED50 values of 24, 28, and 33 nM, respectively. The contractile effects of these compounds were competitively blocked by theophylline (KI . 7.9 microM), but were not affected by adenosine deaminase. IHPIA also inhibited (-)isoproterenol-stimulated cyclic AMP accumulation in adipocytes with an ED50 (10 nM) and to an extent (83%) nearly identical to PIA. (/sup 125/I)HPIA prepared using carrier-free /sup 125/I bound to adenosine receptors on membranes from rat cerebral cortex, adipocyte ghosts, and heart ventricles. Binding was inhibited stereospecifically by PIA and by other adenosine analogues and alkylxanthines. The KD of (/sup 125/I)HPIA determined kinetically using brain membranes at 21 degrees was 0.94 nM in good agreement with the equilibrium determination of 1.94 nM. The density of adenosine receptors in brain membranes was found to be 871 fmol/mg of protein. When normalized to protein, the density of receptors in heart membranes and adipocyte ghosts, respectively, was found to be 39- and 2.3-fold less than in brain membranes. It was concluded that (/sup 125/I)HPIA can be rapidly synthesized and purified, binds to adenosine R-sites and is an agonist radioligand resistant to adenosine deaminase. Computer modeling of the equilibrium binding resulting from the use of mixed stereoisomers of a radioligand indicates that the combined use of (-)(/sup 125/I)HPIA and (+)(/sup 125/I)HPIA would result in the generation of nonlinear Scatchard plots.

  1. Design of inhibitors of orotidine monophosphate decarboxylase using bioisosteric replacement and determination of inhibition kinetics.

    PubMed

    Poduch, Ewa; Bello, Angelica M; Tang, Sishi; Fujihashi, Masahiro; Pai, Emil F; Kotra, Lakshmi P

    2006-08-10

    Inhibitors of orotidine monophosphate decarboxylase (ODCase) have applications in RNA viral, parasitic, and other infectious diseases. ODCase catalyzes the decarboxylation of orotidine monophosphate (OMP), producing uridine monophosphate (UMP). Novel inhibitors 6-amino-UMP and 6-cyano-UMP were designed on the basis of the substructure volumes in the substrate OMP and in an inhibitor of ODCase, barbituric acid monophosphate, BMP. A new enzyme assay method using isothermal titration calorimetry (ITC) was developed to investigate the inhibition kinetics of ODCase. The reaction rates were measured by monitoring the heat generated during the decarboxylation reaction of orotidine monophosphate. Kinetic parameters (k(cat) = 21 s(-1) and KM = 5 microM) and the molar enthalpy (DeltaH(app) = 5 kcal/mol) were determined for the decarboxylation of the substrate by ODCase. Competitive inhibition of the enzyme was observed and the inhibition constants (Ki) were determined to be 12.4 microM and 29 microM for 6-aza-UMP and 6-cyano-UMP, respectively. 6-Amino-UMP was found to be among the potent inhibitors of ODCase, having an inhibition constant of 840 nM. We reveal here the first inhibitors of ODCase designed by the principles of bioisosterism and a novel method of using isothermal calorimetry for enzyme inhibition studies.

  2. Extracellular purine metabolism and signaling of CD73-derived adenosine in murine Treg and Teff cells.

    PubMed

    Romio, Michael; Reinbeck, Benjamin; Bongardt, Sabine; Hüls, Sandra; Burghoff, Sandra; Schrader, Jürgen

    2011-08-01

    CD73-derived adenosine acts as potent inhibitor of inflammation, and regulatory T cells (Treg) have been shown to express CD73 as a novel marker. This study explored the role of endogenously formed adenosine in modulating NF-κB activity and cytokine/chemokine release from murine Treg and effector T cells (Teff) including key enzymes/purinergic receptors of extracellular ATP catabolism. Stimulating murine splenocytes and CD4(+) T cells with anti-CD3/anti-CD28 significantly upregulated activated NF-κB in CD73(-/-) T cells (wild type: 4.36 ± 0.21; CD73(-/-): 6.58 ± 0.75; n = 4; P = 0.029). This was associated with an augmented release of proinflammatory cytokines IL-2, TNF-α, and IFN-γ. Similar changes were observed with the CD73 inhibitor APCP (50 μM) on NF-κB and IFN-γ in wild-type CD4(+) T-cells. Treatment of stimulated CD4(+) T-cells with adenosine (25 μM) potently reduced IFN-γ release which is mediated by adenosine A2a receptors (A2aR). AMP (50 μM) also reduced cytokine release which was not inhibited by APCP. In Teff, A2aR activation (CGS21680) potently inhibited the release of IL-1, IL-2, IL-3, IL-4, IL-12, IL-13, IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), CCL3, and CCL4. However, in Treg, CGS21680 did not alter cytokine/chemokine release. In summary, CD73-derived adenosine tonically inhibits active NF-κB in CD4(+) T-cells, thereby modulating the release of a broad spectrum of proinflammatory cytokines and chemokines. Downregulation of P2X7 and upregulation of CD73 in Treg after antigenic stimulation may be an important mechanism to maintain the ability of Treg to generate immunosuppressive adenosine.

  3. Adenosine A2A receptor stimulation decreases GAT-1-mediated GABA uptake in the globus pallidus of the rat.

    PubMed

    Gonzalez, Brenda; Paz, Francisco; Florán, Leonor; Aceves, Jorge; Erlij, David; Florán, Benjamín

    2006-07-01

    We examined modulation of [(3)H]GABA uptake in slices of the rat globus pallidus because stimulation of adenosine A(2A) receptors increases extracellular GABA in this structure. Pharmacological analysis showed that GAT-1 is the main transporter present in these slices. Both adenosine and the A(2A) agonist CGS 21680 reduced GABA uptake. Antagonist ZM 241385 prevented these effects. Agents that increase protein kinase A activity like forskolin and 8-bromo-cAMP also inhibited GABA uptake. The inhibition of uptake produced by these substances and by CGS 21680 was prevented by the protein kinase A blocker H-89. The protein phosphatase blocker okadaic acid reduced uptake; this effect and the response to CGS 21680 were not additive. The effective concentrations of adenosine (EC(50)=15.2microM) are within the range measured in the interstitial fluid under some physiological conditions. Thus, inhibition of uptake may be important in increasing interstitial GABA during endogenous adenosine release.

  4. Studies on adenosine triphosphate transphosphorylases. Amino acid sequence of rabbit muscle ATP-AMP transphosphorylase.

    PubMed

    Kuby, S A; Palmieri, R H; Frischat, A; Fischer, A H; Wu, L H; Maland, L; Manship, M

    1984-05-22

    The total amino acid sequence of rabbit muscle adenylate kinase has been determined, and the single polypeptide chain of 194 amino acid residues starts with N-acetylmethionine and ends with leucyllysine at its carboxyl terminus, in agreement with the earlier data on its amino acid composition [Mahowald, T. A., Noltmann, E. A., & Kuby, S. A. (1962) J. Biol. Chem. 237, 1138-1145] and its carboxyl-terminus sequence [Olson, O. E., & Kuby, S. A. (1964) J. Biol. Chem. 239, 460-467]. Elucidation of the primary structure was based on tryptic and chymotryptic cleavages of the performic acid oxidized protein, cyanogen bromide cleavages of the 14C-labeled S-carboxymethylated protein at its five methionine sites (followed by maleylation of peptide fragments), and tryptic cleavages at its 12 arginine sites of the maleylated 14C-labeled S-carboxymethylated protein. Calf muscle myokinase, whose sequence has also been established, differs primarily from the rabbit muscle myokinase's sequence in the following: His-30 is replaced by Gln-30; Lys-56 is replaced by Met-56; Ala-84 and Asp 85 are replaced by Val-84 and Asn-85. A comparison of the four muscle-type adenylate kinases, whose covalent structures have now been determined, viz., rabbit, calf, porcine, and human [for the latter two sequences see Heil, A., Müller, G., Noda, L., Pinder, T., Schirmer, H., Schirmer, I., & Von Zabern, I. (1974) Eur. J. Biochem. 43, 131-144, and Von Zabern, I., Wittmann-Liebold, B., Untucht-Grau, R., Schirmer, R. H., & Pai, E. F. (1976) Eur. J. Biochem. 68, 281-290], demonstrates an extraordinary degree of homology.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Effects of adenosine infusion into renal interstitium on renal hemodynamics

    SciTech Connect

    Pawlowska, D.; Granger, J.P.; Knox, F.G.

    1987-04-01

    This study was designed to investigate the hemodynamic effects of exogenous adenosine in the interstitium of the rat kidney. Adenosine or its analogues were infused into the renal interstitium by means of chronically implanted capsules. In fusion of adenosine decreased glomerular filtration rate (GFR) from 0.81 +/- 0.06 to 0.37 +/- 0.06 ml/min while having no effect on renal blood flow (RBF). The metabolically stable analogue, 2-chloradenosine (2-ClAdo), decreased GFR from 0.73 +/- 0.07 to 021 +/- 0.06 ml/min. Interstitial infusion of theophylline, an adenosine receptor antagonist, completely abolished the effects of adenosine and 2-ClAdo on GFR. The distribution of adenosine, when infused into the renal interstitium, was determined using radiolabeled 5'-(N-ethyl)-carboxamidoadenosine (NECA), a metabolically stable adenosine agonist. After continuous infusion, (/sup 3/H)NECA was distributed throughout the kidney. The effects of NECA to reduce GFR were similar to those of adenosine and 2-ClAdo. They conclude that increased levels of adenosine in the renal interstitium markedly decrease GFR without affecting RBF in steady-state conditions. The marked effects of adenosine agonists during their infusion into the renal interstitium and the complete blockade of these effects by theophylline suggest an extracellular action of adenosine.

  6. Xanthine effects on renal proximal tubular function and cyclic AMP metabolism.

    PubMed

    Coulson, R; Scheinman, S J

    1989-02-01

    We evaluated the renal effects of xanthines using two in vitro models: the isolated perfused rat kidney (IPRK) and cultured opossum kidney (OK) cells, a continuous cell line that resembles proximal tubule and responds to parathyroid hormone (PTH). 1,3-Diethyl-8-phenylxanthine (DPX) a potent adenosine receptor antagonist, increased urine volume, glomerular filtration rate, vascular resistance and the fractional excretions of Na, K, Ca and Pi in the IPRK. DPX lowered the Na-dependent uptake of Pi by OK cells. By comparison enprofylline, 3-propylxanthine (ENP), a weak adenosine receptor antagonist, produced a slight elevation in glomerular filtration rate but no changes in electrolyte excretion by IPRK or Pi uptake by OK cells. Both DPX and ENP produced negligible elevations in basal IPRK cAMP. A 1-nM bolus of PTH elevated urinary